Vasculopathies: Behavioral, Chemical, Environmental, and Genetic Factors [1st ed.] 978-3-319-89314-3, 978-3-319-89315-0

Table of contents :
Front Matter ....Pages i-xxxiii
Cardiovascular Disease: An Introduction (Marc Thiriet)....Pages 1-90
Cardiovascular Risk Factors and Markers (Marc Thiriet)....Pages 91-198
Hypertension (Marc Thiriet)....Pages 199-300
Hyperglycemia and Diabetes (Marc Thiriet)....Pages 301-330
Hyperlipidemias and Obesity (Marc Thiriet)....Pages 331-548
Behavioral Risk Factors (Marc Thiriet)....Pages 549-594
Genetic Risk Factors (Marc Thiriet)....Pages 595-676
Back Matter ....Pages 677-888

Citation preview

Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8

Marc Thiriet

Vasculopathies Behavioral, Chemical, Environmental, and Genetic Factors

Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems Volume 8

More information about this series at http://www.springer.com/series/10155

Marc Thiriet

Vasculopathies Behavioral, Chemical, Environmental, and Genetic Factors

123

Marc Thiriet INRIA project team REO Université Pierre et Marie Curie Laboratoire Jacques-Louis Lions Paris, France

ISSN 2193-1682 ISSN 2193-1690 (electronic) Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems ISBN 978-3-319-89314-3 ISBN 978-3-319-89315-0 (eBook) https://doi.org/10.1007/978-3-319-89315-0 Library of Congress Control Number: 2018943695 © Springer International Publishing AG, part of Springer Nature 2018 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Sed ultimus gradus in quem potest artis complementum, cum omni naturae potestate, est prolongatio vitae humanae in magnum tempus. Quod autem hoc sit possibile, multa experimenta docuerunt. [But the highest degree of the art that the power of nature can reach is the extension of human life for a long period. That this might be possible can be taught by many experiments.] (J.D. van Hoven [1705–1793] [1])

Precision cardiovascular medicine incorporates differences between individuals to optimize screening, diagnosis, monitoring, prognosis, and therapeutic decisions. Individualized medicine is based on patient features, integrating risk factors, lifestyle, genetic variants, familial context, medical history, and circulating molecular markers (e.g., secreted proteins, microRNAs, long nonprotein-coding RNAs, and released microvesicles) in addition to structural and functional data obtained from clinical examinations, accurate and precise biological measurements (e.g., information related to genomics, epigenomics, proteomics, transcriptomics, and metabolomics), functional exploration, and imaging, along with modeling and simulations. Important Mendelian diseases are related to single nucleotide variations. Insertions and deletions are also responsible for inherited diseases. Analysis of genotype and phenotype enables mapping of gene variants to disease. Genome sequencing optimized by proper technologies and analysis algorithms, which maximize the sensitivity and specificity, is aimed at enhancing diagnostic sensitivity and medical decision-making, allowing early intervention and precise individualized therapy according to the unique patient genome. The genome comprises more than 51,000 genes and pseudogenes, about 20,000 of which are protein-coding genes, the size ranging from less than 10 (8 for a transfer RNA) to an order of magnitude of 106 base pairs and containing from 1 exon to an order of magnitude of 102 exons. Variability is the law of life, and as no two faces are the same, so no two bodies are alike, and no two individuals react alike and behave alike under the abnormal conditions which we know as disease. (W. Osler (1849–1919) [2])

v

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For example, a loss-of-function polymorphism in the CYP2C19 gene, which is observed in 35% of people of European and African ancestry and 60% of individuals of Asian ancestry, the product of which, a drug processor, reduces the conversion of clopidogrel, an antiplatelet agent used to prevent thrombosis after stent placement, to its active metabolite [3]. Medical strategies are aimed not only at promoting early diagnosis and improving prognosis, but also at avoiding unwanted drug effects. Pharmacogenomics assesses the response of individuals to drugs and identifies patients at risk for adverse reactions to given medications using genetic markers. In addition, regenerative medicine can use cells of the patient derived from induced pluripotent stem cells, which are transplanted to replace a damaged organ. Medical and surgical gestures (e.g., catheter-based procedures and minimally invasive treatment of a beating heart) require precision. Surgical planning and design gain from modeling implicated in virtual reality tools and mechanical exploration. These preliminary stages enable us to select the most appropriate surgical path and the most suitable repair technique. Navigation systems guide the surgeon’s gestures using imaging. Operating robots receive images of a target organ, analyze its motions, and assist surgical gesture according to these movements. Telesurgery consists of controlling a remote slave manipulator robot, which operates surgical instruments using a master control console. This console measures displacements of fictitious instruments driven by the surgeon and transmits them to the robot. Personalized medicine relies on a multidisciplinary research. However, the definition of a given noun may vary between disciplines. For example, the use of the terms “oxidative and reductive stress” are not handled in a similar fashion by biologists and chemists. In biology, oxidative stress implies that the production of reactive oxygen and nitrogen species exceeds that of anti-oxidants, an imprecise concept that mentions neither the types of oxidants and reductants involved nor the reactivity [4]. However, reactive oxygen species (ROS) sources, sinks, and fluxes are most often only partly known, and chemical reactions are frequently quite fast. Reductive stress is related to an excess of reducing equivalents that cannot be adequately accommodated by oxidoreductases. On the other hand, the objective of chemists is to describe redox reactions precisely. From a chemical point of view, ROS can oxidize molecules with a more negative redox potential, thereby exerting an oxidative stress, and reduce other molecules with a more positive redox potential, hence imposing a reductive stress.1 Therefore, the term “redox stress” is preferred. In a multidisciplinary context, a given name can even describe opposing features. For example, in neuroscience, plasticity is referred to as the adaptation of neuronal circuits to the history of stimuli experienced. At the nanoscopic level, created communication domains disappear when environmental conditions change and reappear once excitations similar to previous stimuli reappear. On the other hand,

1 Superoxide

anion can reduce disulfides and oxidize tocopherol. Hydrogen peroxide can reduce ferryl hemoglobin and oxidize methionine [4].

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in rheology, plasticity defines the propensity of a material to undergo permanent deformation under any load, the magnitude of which is greater than the plasticity threshold (also named yield strength and yield point). At the nanoscopic level, the molecular assembly displays irreversible structural changes and cannot return to the initial structure and morphology. In the former case, plasticity is linked to adaptive reversible remodeling, whereas in the latter case, plasticity designates an irreversible state that can lead to material rupture, although at very small strain and stress, the body undergoes an elastic (reversible) deformation (elastoplasticity).

Organ and Physiological Apparatus The body is made up of many several physiological apparatuses, which have a particular relation to their environment inside the organism, are connected to varying degrees, play a distinct role, and have great importance for the body’s survival. Certain organs are more vulnerable to a given type of disorders than others. This susceptibility is traditionally attributed to intrinsic causes, such as the density of stem cells and cellular turnover rate, and extrinsic factors, such as pollution and lifestyle (e.g., eating habits, cigarette smoking, and alcohol consumption), which do not affect all organs to the same extent. Large and paired organs with their specific and connected ecosystem may tolerate disorders more easily than small organs, which are critical to organismal survival, especially throughout the reproductive period, the functioning of organs being governed by compromises. For example, anti-cancer protection varies among organs [5]. Cancer is common in the colon, but rare in the small bowel. Among physiological apparatuses of the human body, the integument limits and protects the organism and serves as a sensory interface with the body’s environment. The skeletal (bones and cartilages) and muscular systems (skeletal muscles, tendons, and ligaments) are implicated in the body’s structure and motions, the larger bones containing the bone marrow, the production site of blood cells. The nervous and endocrine systems are involved in remote control. The circulatory circuit and respiratory and digestive tracts are responsible for nutrient processing and delivery. The urinary system (kidneys, ureters, the bladder, and urethra) contributes to waste removal, regulates the electrolyte balance and blood volume, and maintains the pH homeostasis and blood pressure. Immunity distinguishes the bodily cells from foreign elements, which are neutralized or destroyed. The reproductive system produces haploid gametes that fuse to form diploid zygotes, engendering a unique new combination of alleles, thus increasing genetic variation among offspring on which natural selection can operate. Human sexual reproduction employs internal fertilization using sexual structures of the male and female reproductive tracts. Bodily organs, which are mineralized (e.g., skeleton), solid and soft (e.g., brain, kidney, and liver), or hollow (e.g., heart and blood and lymph vessels in addition to the respiratory, digestive, and urinary tract), have a complicated morphology and structure made up of composite materials.

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Any physiological apparatus is characterized by a set of major properties. 1. Diversity, i.e., a huge between-subject variability in architecture (anatomy), which explains the need for image-based 3D reconstruction for precision medicine (i.e., subject-specific investigation). 2. Variability, i.e., permanent adaptation to environmental conditions, means that images acquired at a given time represented not only a model of the reality, but also a frozen structure that neglects the influences acting on a living organ. 3. Complicated structure and function are controlled both remotely by the fastoperating nervous and endocrine systems acting in the longer term and locally by a locoregional control exerted by hemodynamic stresses, secreted autacoids, and metabolism of perfused organs. Bodily organs, including transport circuits such as the vasculature, are coupled to information processing systems (the nervous and endocrine systems). Regulatory mechanisms are targets of mathematical modeling. 4. Complexity is illustrated by the heart pump, which has a chaotic behavior in the deterministic framework, enabling it to rapidly respond to environmental stimuli. A constant cardiac frequency yields a bad prognosis similar to strong anomalies in the generation and propagation of the cardiac action potential through the nodal tissue, which can engender an anarchic behavior of cardiomyocytes. This property, which is beneficial with respect to the system functioning, becomes a drawback in signal and image processing that involves averaging to enhance the signal-to-noise ratio. Like any physiological system, the cardiovascular apparatus functions with various length and time scales. Length scales are related to mechanisms that govern the function of the cardiovascular system and its response to a changing environment, from: (1) Cell signaling implicating messengers, receptors, and effectors (nm), such as molecules involved in the excitation–contraction coupling in cardiomyocytes or in the regulation of the vasomotor tone by the couple formed by the endotheliocyte and smooth myocyte. (2) Adapting cells and their organelles (μm). (3) Cell clusters organized in a tissue within an extracellular matrix (mm). (4) Organ, i.e., the cardiac pump or a vascular compartment (cm). Multiscale modeling is aimed at coupling these different length scales, whereas the major objective of multilevel modeling is to model the entire vascular circuit coupling models of the bloodstream considering three or fewer spatial dimensions. Whereas three-dimensional models describe the field of the hemodynamic variables (flow velocity and stress) in a set of nodes within a mesh obtained from discretization of a continuum, the distributed (one-dimensional) and lumped parameter models (i.e., electrical analogs) are strongly simplified versions of reality.2 2 In

a distributed parameter model, a vascular segment is assumed to be a succession of infinitesimally long slices, in which hemodynamic variables are only computed at the vessel axis, the axial tension between slices being neglected. In a lumped parameter model (zero-dimensional model), the simplification increases a step further, as hemodynamic variables are computed in a single node assumed to represent a given vessel between two branching points.

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The time scales range from the fast adaptation relying on signaling axes associated with stored molecule release (milliseconds to minutes) up to gene transcription (hours) and adaptive and adverse wall remodeling (e.g., adaptive and adverse cardiac responses to regular exercise and hypertension respectively; i.e., days, weeks, and months). For example, hemodynamic stress-gated ion channel opens in a time of the magnitude order O(ms) and cytosolic calcium (Ca2+ ) concentration and nitric oxide (NO) release happens in a time O(s). In addition, the cardiovascular apparatus is subjected to the circadian cycle (i.e., a major day–night rhythm related to the sleep– wake cycle and accessory signals, such as food intake and body temperature). As hemodynamic factors are implicated in vascular diseases via the local stress field (pressure and wall shear stress), impingement force, and residence time of conveyed molecules, mechanical simulations can complement the medical check-up.

Blood Flow The cardiovascular apparatus comprises two major compartments, the cardiac pump and vasculature. Structurally, the closed cardiovascular circuit includes two subcircuits in a series, the high-pressure systemic and low-pressure pulmonary circulation, blood being propelled in these subcircuits by two apposed pumps enclosed in a single organ, the heart. Blood is conveyed from the right ventricle to the left atrium through the pulmonary circulation, and from the left ventricle to the right atrium through the systemic circulation. The right and left cardiac pumps expel identical volumetric flow rate at each time of the systole, i.e., during the ejection phase of the cardiac cycle, according to the mass conservation principle. Blood flows mainly in a single direction from one cardiac pump to the organs and then to the second cardiac pump, despite relatively strong flow reversals in proximal elastic arteries during diastole. Once ejected from the ventricles, blood circulates down to the capillaries situated within diffusion distances (≤10 μm) of parenchymal cells. Blood vessels are endowed mainly with capacitance and flow resistance. The capacitance of proximal arteries (close to the cardiac pump) maintains blood flow during diastole. The capacitance of veins adjusts blood volume to the body’s needs. Resistance of small distal arteries and arterioles is linked to autoregulatory control that allows the organ to keep perfusion of downstream tissues constant. Blood pressure is determined by peripheral arterial resistance, stroke volume, and cardiac frequency. Blood pressure changes are sensed by the baroreceptors that influence cardiac frequency via the arterial baroreflex control loop. Conversely, cardiac frequency variations influence blood pressure. Blood pressure and cardiac frequency variability and baroreflex sensitivity are influenced by genetic factors, in addition to environmental parameters [6]. However, the genotype (genetic defects) may not be related to the phenotype (manifestations of a disorder), as a patient can have a positive genotype for a given trait (a genetic

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Preface

mutation) without the pathological condition. The transcriptome does not entirely reflect the proteome (i.e., cell- and circumstance-specific output of the genome, especially post-translational modifications). Among environmental factors, deficiencies or excesses in the content or availability of trace elements (e.g., degree of mineralization of local water supplies) can be involved in chronic cardiovascular diseases [7]. Trace metals (cadmium and lead) contribute to hypertension and atherosclerosis [8].

Gas Transport The cardiovascular and respiratory circuits are functionally coupled, as they achieve an adequate continuous supply of oxygen and nutrients and adjusted clearance of carbon dioxide and other metabolic wastes produced by the body’s cells. Oxygen and carbon dioxide are carried by blood from the lungs to bodily organs, hence by convection. Both gas species combine with chemical compounds, which increases the transfer amount. Oxygen and carbon dioxide diffusivity and solubility are important parameters of gas transfer in biological tissues. Diffusion of gas species occurs in gas (e.g., air in pulmonary alveoli), water, gels, and solids, and hence in biological tissues (e.g., alveolocapillary membrane and blood). It occurs because of the gradients of concentrations in air and partial pressures (total mixture pressure multiplied by the gas species fraction) through a biological tissue. The diffusion coefficient (D) is usually about 104 times greater in air than in water. It depends on temperature and pressure (D(T , p)); it increases with rising temperature, molecules moving faster, and decreases with augmenting pressure, which compacts molecules and reduces their motion (Table 1). The diffusion coefficient is most often given for a binary mixture, and hence not air, in which the composition of gases varies owing to the added water vapor in airways to the major gas components (nitrogen, oxygen, and carbon dioxide). The diffusivity is generally related to given pairs of gas species in a multispecies mixture. In fluids at a given temperature, the gas diffusion rate (Dg ) depends on the partial pressure difference between the fluid compartments (Δp), cross-sectional area of the diffusion pathway (A), gas solubility (sg ), diffusion distance (d), and gas molecular mass (mg ) [12]: Dg =

sg AΔp 1/2

,

(1)

dmg

the gas diffusion coefficient (D) being proportional to the ratio sg /m1g /2.3 3 The

molecular mass of a substance is the mass of one molecule of the chemical species (air molecular mass of air 28.97, carbon dioxide 44.01, nitrogen 28.02, oxygen 32.0, and water vapor 18.02 kg/kmol).

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Table 1 Diffusivities of oxygen and carbon dioxide (Sources: [9–11])

Diffusivity (m2 /s) Gas Oxygen diffusivity in blood pO2 = 5.3 kPa 1.51 × 10−9 [9] ◦ (40 mmHg) at 37.5 C pO2 = 13.3 kPa 1.33 × 10−9 ◦ (100 mmHg) at 37.5 C 16 gHb/dl at 38 ◦ C 1.6 × 10−9 [10] Dissolved carbon dioxide In water at 25 ◦ C 2.02 × 10−9 [11] (dissolved bicarbonate ion: 1.17 × 10−9 )

Table 2 Blood gas partial pressures (kPa [mmHg]) in various compartments

pO2 pCO2

Atmospheric air 21.3 (160) 0.03 (23)

Inspired humidified air 20.0 (150) 0.04 (30)

Alveolar air 13.3 (100) 4.8–5.3 (36–40)

Pulmonary arterial blood 13.3 (100) 5.3 (40) [48 ml/dl]

Mixed venous blood 5.3 (40) 6.1 (46) [52 ml/dl]

Expired air 2.1 (16) 3.6–4.3 (27–32)

The gas transfer rate through a biological tissue is related to Krogh’s diffusion. Krogh’s gas diffusion constant at a given temperature in a given biological tissue (Krg ) corresponds to the product of the gas diffusion coefficient and its capacitance (ratio of its concentration to its partial pressure). Fick’s law of diffusion, which provides the rate of gas mass transfer per unit time (m), ˙ is then given by: m ˙ = Krg AΔp/ h,

(2)

where h is the thickness of the diffusion barrier. At 37 ◦ C, Krogh’s diffusion coefficients for oxygen and carbon dioxide in rat skeletal muscle are 1.31 and 28.0 10−9 mmol/cm/mn/mmHg, respectively [13]. Oxygen level cascade, i.e., pO2 decrease from inhaled air to the mitochondrion comprises uptake in the lungs, the carrying capacity of blood, delivery to the capillaries, interstitium, and then cells, and the cellular use of oxygen (Table 2; Vol. 6, Chap. 4. Physiology of Ventilation). Large pulmonary veins receive both oxygenated blood from the pulmonary circuit and deoxygenated blood from the systemic bronchial veins (shunt flow). This venous admixture of blood reduces the O2 partial pressure to 12.6 kPa (95 mmHg) [12]. In the lung, oxygen is added in blood, its partial pressure rising from 5.3 kPa (40 mmHg) to 13.3 kPa (100 mmHg) and simultaneously carbon dioxide is removed, its partial pressure lowering from 6.1 kPa (46 mmHg) to 5.3 kPa (40 mmHg). The transfer rate of carbon dioxide is then greater than the rate necessary to balance the oxygen transfer.

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Table 3 Blood gas solubility at 37 ◦ C, solubility rising as the temperature falls Gas O2 CO2

Solubility (ml/dl/kPa) 0.023 0.52

Solubility (ml/dl/mmHg) 0.003 0.069

Solubility (mmol/l/kPa) 0.01 0.231

Solubility (mmol/l/mmHg) 0.0013 0.0308

The arterial partial pressure of oxygen (paO2 ) depends on inspired O2 concentration, atmospheric pressure, alveolar ventilation, ventilation/perfusion distribution in the lungs, and O2 diffusion from the alveoli to the pulmonary capillaries. Over the surface of the pulmonary alveolus, hemoglobin in a solution in red blood capsules binds oxygen (9.2 mmol O2 /l). Oxygen is mainly carried in blood by red blood capsules (97–98%), a small fraction dissolved according to Henry’s law4 (HO2 × pO2 ; HO2 : O2 Henry solubility; Table 3). • Oxygen saturation (SaO2 ) is the ratio of oxygen linked to hemoglobin with respect to the oxygen capacity (maximal binding amount). • Oxygen content in arterial blood (CaO2 ) is the sum of O2 carried on Hb and dissolved in plasma: CaO2 = SaO2 × [H b] × 1.34 + pO2 × 0.003 = 19.4 + 0.3 ∼ 20 ml/dl.

(3)

The amount of oxygen bound to completely saturated hemoglobin, i.e., the theoretical maximal oxygen carrying capacity, is 1.39 ml/g Hb, but measurement gives a capacity of 1.34 ml O2 /g Hb. • Oxygen delivery (DO2 [ml/mn]), which depends on the blood flow rate (q) DO2 = q × CaO2 ,

(4)

ranges from 0.9 to 1.1 l/mn. ˙ O2 [ml/mn]) is the amount of oxygen extracted by cells • Oxygen consumption (V during one minute: ˙ O2 = q(CaO − CvO ). V 2 2

(5)

It ranges from 200 to 300 ml/mn. • Oxygen extraction ratio, an index of efficiency of O2 transport, is the amount of oxygen extracted by cells divided by the amount delivered to cells. Oxygen is delivered from lungs to cells, blood flowing preferentially to organs where the metabolic activity and hence oxygen demand are greater owing to hypoxic vasodilation.

4 The

concentration of a solute gas in a solution is directly proportional to its partial pressure above the solution, when concentration and partial pressure are relatively low (i.e., ideal dilute solution).

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Carbon dioxide is produced by cell metabolism in mitochondria. The amount produced depends on the rate of metabolism and relative amounts of metabolized carbohydrates, lipids, and proteins (∼200 ml/mn at rest and eating a mixed diet for a respiratory quotient of 0.8) [14].5 Carbon dioxide eliminated from cells is dissolved in plasma, as it is more soluble (20-times) than oxygen (∼5–7%; sCO2 0.231 mmol/l/kPa [0.0308 mmol/l/mmHg; corresponding to 0.5 ml/kPa CO2 in 1 dl blood at 37 ◦ C (sCO2 = 0.069 ml/dl/mmHg)] at 37 ◦ C), binds to plasmatic proteins, particularly hemoglobin as carbamate (carbaminoHb; ∼10%), and is carried as bicarbonate ion, the bicarbonate buffer being the most efficient carrier in blood.6 Approximately 75% of carbon dioxide is carried in red blood capsules and 25% in plasma. Carbon dioxide then returns to the pulmonary alveolus, where it is released and exhaled. The percentage of the total carbon dioxide carried in each form and percentage exhaled from them differ: 5% of dissolved CO2 in solution and 10% tethered to proteins supplies 10 and 30% of exhaled CO2 amount respectively [14]. Chemoreceptors transduce a chemical signal into an action potential. The peripheral chemoreceptors, i.e., sensors of the peripheral nervous system in arterial walls, the carotid and aortic chemosensory bodies, and central ones, the chemosensory medullary neurons, primarily regulate breathing to maintain the partial pressures of gases (oxygen and carbon dioxide [paO2 and paCO2 ]) in addition to hydrogen ion concentration (pH) in the arterial blood within normal ranges, adapting the chemoreceptor firing rate. Chemoreceptors also influence the cardiovascular apparatus directly via medullary vasomotor centers and indirectly via pulmonary stretch receptors, triggering sympathetic signaling to the heart and vasculature to adapt breathing and vascular resistance and hence blood pressure. Carotid bodies detect a primarily decreased O2 level and, to a lesser extent, an elevated CO2 level and lowered arterial pH; aortic bodies sense arterial blood oxygen and carbon dioxide levels. In response to hypoxia, the oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation, release neurotransmitters that activate the carotid sinus nerve to increase breathing frequency within seconds. The chemoreceptor reflex that is activated by hypoxemia (paO2 < 10.6 kPa [80 mmHg]), hypercapnia (paCO2 > 5.3 kPa [40 mmHg]), and acidosis (pH < 7.4) increases the breathing frequency and tidal volume amplitude. The carotid body chemoreceptor discharge has a respiratory rhythm. In cats, the carotid chemoreceptor is extremely sensitive to small abrupt changes in pCO2 , 5 The

respiratory quotient is the rate of carbon dioxide production divided by the rate of oxygen consumption. A carbohydrate diet gives a quotient of 1 and a fat diet of 0.7 [14]. 6 Carbon dioxide diffuses into the red blood capsule (RBC), where carbonic anhydrase quickly converts it into carbonic acid (H2 CO3 ), an unstable intermediate molecule that immediately + + dissociates into bicarbonate (HCO− 3 ) and hydrogen ions (H ). Hemoglobin binds to H , limiting pH shift. The newly synthesized bicarbonate ion is exported from the RBC into the plasma in exchange for a chloride ion.

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these changes affecting breathing when they happen at an appropriate point in the respiratory cycle [15]. Both elevated pCO2 and lowered hydrogen carbonate concentration (bicarbonate [HCO− 3 ]) change impulse frequency, the chemoreceptor response produced by an elevated pCO2 occurring approximately twice as fast as that due to a decayed [HCO− 3 ]. In transient changes in pCO2 or pH, the effect of pCO2 may dominate. In cats, the response curve of carotid body chemoreceptors to lowered paO2 is hyperbolic, with a frequency of nerve impulses that first decreases rapidly when paO2 rises and then more slowly from a variable value according to the chemoreceptor unit and hence the nerve fibers (mean25.3 ± 5.3 kPa [190 ± 40 mmHg) [16]. The discharge of a single chemoreceptor afferent fiber augments both with increasing paCO2 at constant paO2 and pH and with elevating arterial H+ concentration at constant paO2 and paCO2 . In rodents, the acute response to hypoxia is linked to the olfactory G-proteincoupled receptor, OlfR78, which is highly and selectively expressed in oxygensensitive glomus cells and acts as an oxygen sensor [17]. The metabolite lactate, concentrations of which rapidly rise in blood during hypoxia, provokes hyperventilation. It binds to and activates OlfR78, thereby enabling glomus cells to detect hypoxia and stimulate breathing. Activated OlfR78 releases calcium from intracellular stores and causes calcium transients in glomus cells. Blood gas and hydrogen ion control, and hence control of breathing, relies primarily on the ability of the brain to sense CO2 and/or H+ levels, rather than oxygen sensing. Central chemosensitivity refers to a change in ventilation attributable to changes in levels of CO2 and H+ detected within the brain. The central chemoreceptors of the ventral medulla oblongata monitor CO2 level in the cerebrospinal fluid, as chemoreceptor neurons contain processes that can provide access to the cerebrospinal fluid circuit more efficiently than to penetrating branches of the brain vasculature. In addition, glial cells participate in the pH regulation of interstitial and cerebrospinal fluid. In the cerebrospinal fluid, pH is regulated by transport mechanisms through the capillary endothelium in the choroid plexus and blood–brain barrier, and by pCO2 changes associated with breathing and cerebral blood flow. Neuronal and glial processes close to blood vessels may detect changes in blood pH, hence minimizing the influence of the blood–brain barrier, which controls ion transfer. The central chemoreceptors in the brainstem protect against rapid changes in pH in the blood. In fact, central chemoreceptors are responsive to interstitial fluid pH in the brain. Many regions participate in central chemoreception, such as the brainstem, cerebellum, hypothalamus, and midbrain [18]. They sense H+ concentration in the cerebral interstitial fluid and detect and integrate information from: (1) Alveolar ventilation via arterial paCO2 (2) Cerebral blood flow and metabolism (3) Hydrogen ion control They then influence breathing, airway resistance, and blood pressure via the sympathetic nervous system.

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Chemoreception, sleep, and wakefulness depend on various cerebral regions that include Tac1 +7 Phox2b+ neurons in the retrotrapezoid nucleus, serotonergic neurons of the medullary raphe, neurons of the caudal ventrolateral medulla, neurons of the dorsal respiratory group in the nucleus tractus solitarius, hypothalamic orexin+ neurons, in addition to neurons of the locus ceruleus in the dorsal pons; of the rostral region of the fastigial nucleus, a deep cerebellar nucleus; and of the rostral ventral respiratory group, the pre-Bötzinger complex [18]. The retrotrapezoid nucleus and medullary raphe in the ventral medulla are two interacting chemoreceptor areas, the caudal medullary raphe amplifying the response to CO2 at the retrotrapezoid nucleus. In wakefulness (but not during NREM sleep), orexinergic neurons signal to the retrotrapezoid nucleus and rostral medullary raphe, thereby enhancing the responses from the carotid body, retrotrapezoid nucleus, caudal nucleus tractus solitarius, caudal ventral medulla, and likely the locus ceruleus [18]. In cats, medullary extracellular fluid pH changes within seconds following an acute modification of alveolar and arterial pCO2 [19]. The respiratory response inversely matches changes in the pH of the extracellular medium, but not in the cerebrospinal fluid. The relation between increasing end-tidal pCO2 upon airway occlusion and medullary hydrogen ion concentration is linear, but not that relating [H+ ] in the extracellular space to the respiratory response. The central respiratory chemosensitivity depends on two types of plasmalemmal proteins, the pH-sensitive TASK2 channel and proton-activated G-protein-coupled receptor GPR4 on chemosensory neurons of the mouse retrotrapezoid nucleus [20]. Regulation of breathing by CO2 relies on GPR4 (or GPR19), which senses the blood level of protons generated from carbonic acid. The ventilatory response to CO2 involves the K+ TASK2 channel (K2P 5). Hence, the control of breathing depends on the GPR4 that works with TASK2 on chemosensory neurons. Central and peripheral chemoreceptors cooperate. At least in rats, carotid afferents synapse at nucleus tractus solitarius neurons, which communicate directly with those of the retrotrapezoid nucleus [18]. Moreover, the carotid bodies raise the ventilatory response to central pCO2 , demonstrating the potent interaction between central and peripheral chemoreceptors. Central and peripheral chemoreceptors also participate in maintaining pH homeostasis. Metabolic acidosis stimulates ventilation via peripheral and central chemoreceptors to lower paCO2 and [H+ ]. The sympathetic nervous system also regulates activity of the heart as well as small arteries and arterioles responsible for the systemic vascular resistance, thereby controlling blood pressure. The sympathetic output is governed by many central and peripheral mechanisms, especially the baroreflex (baroreceptor-triggered reflex), the fastest mechanism that regulates acute arterial pressure changes via the cardiac

7 Tachykinin

receptor-1.

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frequency and contractility in addition to vasomotor tone and hence peripheral resistance.8 Yet, increased ventilatory output raises sympathetic nerve activity via presympathetic neurons of the rostral ventrolateral medulla and likely GABAergic neurons of the caudal ventrolateral medulla (CO2 -sensitive respirophasic change in sympathetic nerve activity) [18]. In addition, hypercapnia engenders tonic CO2 sensitive activity independently of respiratory events. With increased CO2 levels, sympathetic discharge, phrenic nerve activity, and blood pressure heighten. The paCO2 threshold for the pressor sympathetic response equals approximately 4.8 kPa (36 mmHg) and that for the phrenic nerve is about 5.8 kPa (44 mmHg) [18]. Catecholaminergic neurons of the brainstem project in the forebrain, hindbrain, and spinal cord to many regions implicated in the control of respiratory and cardiovascular function, hence modulating these functions [21]. Excitatory noradrenergic neurons of the A6 group maintain breathing frequency, whereas A5 neurons slow breathing and cardiac frequencies. Lesions of A2, A3, A5, and A6 neurons attenuate blood pressure. Orexin neurons that innervate the pre-sympathetic neurons of the rostral ventrolateral medulla can increase sympathetic nerve activity. In mice, orexin depletion diminishes blood pressure in wakefulness [18]. Carbon dioxide combines with water to form carbonic acid (H2 CO3 ), which + + dissociates in water into HCO− 3 and H ions. An increase in blood H concentration of only 100 nmol/l (i.e., a decrease of 0.1 unit of pH) can be fatal. Hence, regulated excretion of CO2 by breathing is an essential life-preserving process. In rodents, hypercapnia-primed release of ATP from the chemosensory areas at the ventral surface of the medulla is crucial for breathing regulation. In humans, paCO2 is regulated around a set point of 5.3 kPa (40 mmHg). Connexin-26, which constitutes hemichannels and gap junctions, is a CO2 sensor; CO2 binds to the Cx26-formed hemichannel, which then opens and releases ATP, thereby contributing to the CO2 dependent regulation of breathing [22]. In humans, a mutation that removes CO2 sensitivity of Cx26 reduces respiratory drive and causes periods of central apnea. At the cellular level, oxygen is the final electron acceptor during oxidative phosphorylation. Immediately, hypoxia suppresses ATP consumption and hence major ATP sinks in the cell, i.e., protein translation and ion channel activity. During a rescue phase, transcription of hypoxia-responsive genes increases; these

8 The

cardiopulmonary region of the body is innervated by multiple types of sensors and mechanoand chemosensitive nerves. Baroreceptors are mechanoreceptors located in the carotid sinus and aortic arch that sense arterial pressure changes and tension in the arterial wall and react. Impulses sent from activated aortic and carotid baroreceptors are transmitted to afferent vagal and glossopharyngeal nerves and then to the nucleus of the tractus solitarius and vasomotor center of the brainstem. Acute hypertension stretches the baroreceptors, which increase their impulse firing rate, decreasing sympathetic signaling and increasing parasympathetic vagal tone. Conversely, a sudden hypotension diminishes wall tension and hence the firing rate, stimulating sympathetic activity in the heart (cardiomyocytes and nodal cells, especially those of the sinusal node), and blood vessels and inhibiting vagal tone.

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genes encode proteins involved in glucose transport, glycolysis, erythropoiesis, angiogenesis, vasodilation, and respiratory rate to minimize the effects of hypoxia. Adaptation to hypoxia relies on the stabilization of hypoxia-inducible factors (HIF). Hypoxia-inducible factors are oxygen-sensitive heterodimeric transcription factors of the basic helix–loop–helix (bHLH) superfamily that control the long-term response to hypoxia. The HIF dimer consists of an oxygen sensor (HIF1α–HIF3α) and a constitutively expressed subunit HIF1β. Its activity is regulated by posttranslational modifications. Hypoxia-inducible factors trigger transcription of genes that facilitate adaptation to hypoxia and survival of cells in cooperation with transcriptional coactivators. Hypoxia-inducible factor-1 enhances glycolysis and attenuates oxidative phosphorylation and hence metabolic dependence on molecular oxygen for ATP synthesis. In hypoxia, the shift from oxidative phosphorylation to glycolysis decreases the generation of ROS. Hemoglobin functions not only as a carrier of proper amounts of oxygen to cells, but also as a sensor and transducer that detect the local oxygen content and convey oxygen-responsive NO, thereby regulating the vasomotor tone.

Modeling The golden number (also dubbed golden ratio, proportion, mean, or section) equals 1.618034, i.e., (51/2 + 1)/2. This irrational number can be defined by a special geometric construction, for example dividing a line into two segments, the ratio of the longer (a) to the smaller segment (b) equaling that of the whole length to the longer segment (a/b = (a + b)/a).9 It is denoted by the Greek letter ϕ, honoring the name of the Greek sculptor, painter, and architect Phidias (−480 to −430), who employed it extensively. In Leonardo da Vinci’s drawings of the human body (Vitruvian Man [∼1490]), depicting a body in a standing position inscribed in a square and then with feet and arms outspread inscribed in a circle, the ratio of the foot–umbilicus (navel) distance to umbilicus–head top is approximately the golden ratio. The ratio of two successive Fibonacci numbers (xi /xi−1 ) is very close to the golden ratio, especially after the couple (21, 13).10 Let us consider the following quantities: arterial diastolic (DBP), systolic (SBP), and pulse pressure (PP), in addition to cardiac period (T) and systolic (SD ∼ 0.38 T) and diastolic duration (DD ∼ 0.62 T). The cardiac cycle phase duration ratios

ratio of the circumference of a circle to its diameter, π = 3.141593, is another irrational number. 10 The Fibonacci sequence is the series of numbers starting from 0 and 1 in which the next number is calculated by the sum of its two previous numbers (xi+1 = xi + xi−1 ; 0, 1, 1, 2, 3, 5, 8, 13, 21, 34, etc.), every ith Fibonacci number being a multiple of xi . 9 The

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SD/DD and T/DD are nearly equal to the golden number. With the arterial pressure value triplet (16.0, 10.6, and 5.3 kPa [120, 80, and 40 mmHg]), the ratios DBP/ PP and SBP/DBP are equal to 2 and 1.5 respectively, whereas with the triplet (16.0, 9.8, and 6.1 kPa [120, 74, and 46 mmHg]), these ratios are nearly equal to the golden number [23]. The golden number, which describes two measures of any kind, can be used for simple modeling, which cannot usually be applied in natural sciences. Modeling in biology, physiology, and pathophysiology is associated with ordinary (ODEs) and/or partial differential equations (PDEs), PDEs usually being linked to a higher degree of abstraction. For example, active energy-consuming materials immersed in a fluid or a gel move and alter the properties of their environment. They are endowed with selfpropulsion, converting chemical energy into mechanical energy. Cell motility is represented by a hierarchy of PDE-based models. The classic equation set was proposed by C. S. Patlak in 1953 and E. F. Keller and L. A. Segel in 1970 [24]. Cell chemotaxis is modeled by a set of parabolic and elliptic equations that take into account only the cell density n(x, t) and concentration c(x, t) of chemoattractant released by cells, which immediately diffuses, using a set of parameters including a constant chemotactic sensitivity (Sca ) and cell (Dc ) and chemoattractant diffusivity (Dca ), cell proliferation rate (P), and chemoattractant production (Pca ) and degradation rate (D): ∂n = Dc ∇ 2 n − ∇ · (nSca ∇c) + Pn, ∂t ∂c = Dca ∇ 2 c + Pca n − Dc, ∂t

(6) (7)

According tothe Blanchet–Dolbeault–Perthame theorem in R2 [25], given the initial mass (m0 = R2 n0 (x) dx),  1. If R2 |x|2 n0 (x)dx < ∞ 8π , then the Keller–Segel system blows-up in finite time 2. When m0 > Sca  8π and R2 n0 (x)| log(n0 (x))|dx < ∞, then a weak solution exists 3. When m0 < Sca Modeling is aimed at enhancing knowledge and an understanding of natural processes, predicting, assessing quantities and parameters that cannot be directly measured, creating new hypotheses and paradigms, and redesigning models and theories according to outcomes, thereby driving rather than complementing investigations. However, this simplification of the reality is a falsification. Verification is aimed at proving that the equations of the mathematical model are solved correctly. Validation is aimed at comparing the solution obtained using the selected model with measurements to check the validity of numerical tests. Furthermore, systematic truncation error testing and accuracy estimation should be addressed.

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Il y a un imbécile en moi, et il faut que je profite de ses fautes... C’est une éternelle bataille contre les lacunes, les oublis, les dispersions, les coups de vent.[I host a dunderhead, and I need to benefit from his mistakes... This is an eternal battle against gaps, omissions, dispersions, and gales.](Paul Valéry [1871–1945]) [26]

Physiological and pathological processes can be explored using simple physical, mathematical, and numerical models to better comprehend and explain the nature and its patterns. All involved elements must be carefully pondered according to their function and the spatial and temporal scales. The biological complexity is then omitted and the process modeled in a given theoretical context, knowing the sources of errors and handling definitions, concepts, paradigms, and theories underlying the explored process. Usually, the model design needs to be adjusted and measurements and/or the solving procedure to be optimized and adapted to the evolving process. Natural complex processes operate at different length and time scales. Multiscale modeling is aimed at representing physiological and pathological phenomena across the biological continuum, i.e., from atoms, molecules, and molecular complexes (nanoscale), to subcellular compartments, cells, and cell clusters (microscale), to tissues (mesoscale), and organs and physiological apparatus (macroscale). Its objective is to describe mechanisms and, coupled with bioinformatics, to predict the occurrence of certain diseases in a given fraction of the population. Cellular and tissular organization, homeostasis, adaptation, control, and remodeling are not described by mathematical theories of optimal structure–function relations, as general quantities measuring the complex cell behavior are lacking. Adaptive and regulated cellular and tissular components cannot be isolated without missing the integrated function of the entire structure. In addition, chemical activities underlying the structure–function relations obey various rhythms governed by biological clocks. Hence, even in the absence of major changes in cell fate (e.g., growth, division, differentiation, and death), molecules are not only subjected to a constant turnover with given synthesis and degradation rates, but also their concentrations fluctuate in time. Nevertheless, mathematical modeling based on ODEs or PDEs is used to predict and redesign experiments for a deeper understanding of regulated cellular and tissular processes, such as angio- and organogenesis (implicating cell migration, proliferation, and death) and tissular growth that rely on chemical, physical, and mechanical agents, in addition to remodeling, healing, and repair, keeping in mind that mathematical descriptions afford neither quantitative analyses nor complete solutions and explanations. Ce qui est simple est toujours faux. Ce qui ne l’est pas est inutilisable. [What is simple is always false. What is not is unusable.(Paul Valéry) [27]

Any volume in the series “Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems” is aimed at describing physiological and pathophysiological processes to focus on proper mechanisms and carry out adequate modeling, highlighting the major signaling cascades, when they are known. Cells sense any change in their environment, integrate diverse signal sources, and respond to them by the release of material, gene transcription, metabolic adjustment, and adaptive fate.

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Signaling pathways can be targets of mathematical representations. They often comprise ubiquitous building blocks, such as the MAPK module, monomeric (small GTPases) and trimeric GTPases (G proteins) linked to their regulators, processed plasmalemmal lipids such as phosphatidylinositols that can engender second messengers. Once they are liganded or, for some, activated by mechanical stress, receptors change their conformation from inactive to susceptible and then active form and transfer signal, actuating intracellular pathways using a set of adaptors and recruiters. Most models of reaction sets use a deterministic continuous approach based on rate equations for concentrations of involved substances and complexes described by chemical kinetic models and represented by a set of ODEs, the spatial distribution of compounds being described using compartments. The rate of each chemical reaction or interaction is based on concentrations of the reacting or interacting chemical species (rate law), which is associated with a positive or negative term in the conservation equations, according to the conserved species generated or consumed, elementary reactions being formulated mathematically by the law of mass action. On the other hand, dynamics in a continuous space can be linked to relatively large concentration gradients. Concentrations are then considered to be functions of both time and space and the process is governed by PDEs. Indeed, outcome depends on magnitude, timing, and spatial localization of signals. Fluxes of the chemical species involved then depend on convection and diffusion in the presence of a pressure and concentration gradient respectively. Metabolic activities incorporate molecular concentrations and rates of synthesis, transport, molecular interactions, enzymatic transformations involving donors, acceptors, and products, and clearance, in addition to relaxation times (time required for a quantity to reach a new steady state after a disturbance) and regulation by negative and positive feedbacks. Cellular metabolism can be described by time and space scales according to displacements of the molecular species involved, and fluctuations of their concentrations owing to biological rhythms, weak and strong molecular coupling, referring to nonspecific and specific molecular interactions and control. Cell growth and proliferation depend on tissue perfusion and hence capillary density, uptake of nutrients, and controlled production of the required molecules (sugars, lipids, and proteins) in addition to regulators. The rate of change in cell density is given by birth (division and differentiation), transfer (convection, diffusion, and migration [taxis]), and loss (death) terms, which hinge on various types of stimuli, including growth factors and mechanical stresses, along with matrix properties (composition, organization, component density, and rheology). It is coupled with feeding, i.e., the rate of change in concentrations of nutrients (i.e., production, flux, reaction, and decay). Mathematical modeling targets maladaptive tissue growth, which causes mural pathological conditions, especially in the heart (hypertension-initiated cardiac hypertrophy) and arteries (atherosclerosis), or results from medical device implantation. Modeling is aimed at improving the clinical strategy.

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Tissular remodeling can be investigated on a macroscopic scale and all microand mesoscopic scale phenomena lumped in parameters, which are incorporated into a system of nonlinear, coupled, parametric, partial differential equations. This type of equation set is used in continuous-type models that rely on mixture theory. However, this solving procedure necessitates an efficient identification stage to estimate the parameters involved. Vascular wall remodeling is usually supposed to depend on the wall shear stress sensed by endotheliocytes on their wetted (luminal) surface and intramural circumferential and axial tension detected by smooth myocytes and fibroblasts, stretch and parietal shear playing a minor role on endotheliocytes and smooth myocytes respectively. Wall shear stress affects cell proliferation, apoptosis, and migration in addition to matrix deposition and re-organization via gene expression. Numerical simulations can be aimed at optimizing a drug dose for a given patient and administration time using pharmacokinetics–pharmacodynamics (PK– PD) models. These models describe the evolution of the drug concentration that circulates in the blood for a given dose and administration mode and link between the blood drug concentration and the magnitude of the drug’s effect. The objective is to predict the optimal time of drug re-administration and dose, hence guaranteeing the drug’s desired action, but minimizing side effects. This problem is related to optimal control. The model must take into account interindividual variability.

Mechanical Investigations The main components of biological tissues, cells, and matrix can be considered as a material that experiences fluidization–gelification cycles according to the stress field applied. The cytoplasm and extracellular medium contain filaments and fibers, actin and myosin, microtubules, and intermediate filaments in the intracellular medium, collagen, and elastin fibers in the matrix, in addition to relatively large particles, cellular organelles, and cells respectively. Biomechanics, i.e., continuum mechanics applied to physiology, deals with the mechanical behavior of biological fluids (e.g., air and blood) and solids (conduits and organs). Biomechanical methodology is based on computational models and experimental circuits that integrate the structure and function of the explored organ, the physical properties of which can be defined by continuous functions. Modeling obeys geometrical and dynamical similarity, i.e., keeps the values of ratios between calibers and lengths along with dimensionless parameters (e.g., Reynolds and Strouhal numbers), which are ratios of forces, and governs length and time scales. The objective of biomechanical works is to assess the function of a selected compartment of the cardiovascular circuit, such as the cardiac pump or a vascular segment, with or without branchings. This compartment is characterized by its morphology (shape); geometry (size); structure (tissue composition); rheology (mechanical properties), with given pre-stresses (axial and circumferential residual

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tension); and values of the flow governing parameters, under normal and pathological conditions. For example, in hypertension, which is associated with altered cell and matrix mechanics and dysregulated mechanotransduction, the vascular wall stiffens. Physical phenomena employ a set of quantities (e.g., mass, temperature, pressure, velocity, and energy). The goal of biomechanical modeling is to predict the fields of the physical variables involved by solving well-posed boundary value problems associated with the balance laws of mechanics (e.g., the conservation of mass, momentum, and energy) in a given context. Moreover, once the numerical procedure is verified and the solution validated, the role played by a given parameter can be easily tested, the others remaining constant. Multilevel models couple three-dimensional compartments (3D) of the cardiovascular circuit to one-dimensional (1D or distributed parameter models) and lumped parameter models (0D models or electrical analogs) of other compartments of the circuit. Coupling of 3D, 1D, and 0D models enables the vascular network with its upstream and downstream impedances to be incorporated. The unsteady 3D developing flow of viscous incompressible blood through a segment of the vascular circuit under pathological conditions can be described by the Navier–Stokes equations derived from the theory of continuum mechanics. This equation set predicts flow behavior for given initial and boundary conditions (i.e., input and output impedances) and the values of flow governing dimensionless parameters, which control the local flow dynamics, in a given rigid or deformable computational domain derived from medical images. The Navier–Stokes equations describe the flows of fluid particles that can be conveyed through deformable curved conduits of complicated configuration. Threedimensional models provide the entire flow field (intraluminal pressure and shear stress, mural stress, and velocity, in addition to its derived variables such as vorticity), more precisely in a set of nodes separated by a discretization scale (space step). The higher the node number, the closer the computational domain is to the continuum. However, solving is computationally expensive, especially when dealing with time-dependent blood–vessel wall interaction. They are then used to explore a segment of the anatomical circuit and are sensitive to boundary conditions. The simplest lumped parameter models assume that all the properties (mainly resistance and compliance) of the blood vessel can be concentrated in a single point. Each duct of a network is then represented by a single node. This approximation amounts to using electrical analogs, i.e., considering linearization. Hence, many effects, nonlinear convective acceleration, kinetic energy effects due to duct geometry changes and branchings, wave propagation, and large displacements are neglected. Modeling of pulse wave propagation generally relies on distributed parameter models that are based on mass and momentum conservation integrated over the cross-sectional area of the explored vascular segments. The underlying hypothesis is that all flow features (transmural pressure, luminal cross-sectional area, and fluid velocity) in any vessel segment of infinitesimal length can be assumed to be uniform; they are integrated in a single point at the vessel axis. Therefore, the

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vessel is assumed to be a continuous set of infinitesimal thickness slices, which interact via the flow dynamics coupled with the wall mechanics, but without mural connection between the selected sections (or stations), as axial tension exerted by the deformation of a cross section by a traveling wave to its apposed stations is omitted. Each conduit is then represented by a set of nodes separated by an infinitesimal length. Modeling pulse wave propagation in the arterial tree in diseases requires the vessel law, which relates the cross-sectional area (A) to the transmural pressure (p). The simplest numerical procedure is based on finite differences and the method of characteristics, when the flow remains subcritical. However, in diseases, the vessel law must be investigated. Adequate simulations of multiphysics problems rely on solver-coupling platforms. Physiological ducts have deformable walls with a given rheology under given dynamical conditions. Solid media are most often heterogeneous, multidomain-containing viscoelastic continua. They are described by the constitutive equation that relates stress (i.e., force per unit area), strain (a dimensionless change in configuration), and rate of deformation. These quantities are mathematically defined by symmetrical tensors, i.e., nine component elements (Ti,j , i, j = 1, 2, 3; i, row index corresponding to the plane on which the stress is exerted; j , column index corresponding to the direction of stress according to coordinate axes) in the three-dimensional space; they can be represented by a 3 × 3 symmetrical matrices in the absence of external moments. The fundamental behavior of material, which is determined by time-independent and -dependent tests (e.g., cyclic and multi-axial loading and test at constant stress [creep test] and at constant strain [relaxation test]), is commonly represented by elementary mechanical features and models. • An elastic body is modeled by a spring. Most materials can be assumed to obey the elasticity law at a low level of strain. The stress (applied load) is related to the strain (deformation) using the stiffness coefficient (elasticity modulus) for axial loading (compression and tension in a given direction, i.e., in general, one of the main axes of the body) and the shear modulus for shear (torsion and bending, i.e., forces acting in a tangential or transverse direction with respect to the surface and major axis of the body respectively). These moduli expresses the resistance to deformation. Compliance is the inverse of the elasticity modulus. Stress and strain are in phase. The body’s configuration returns to its original shape when the applied stress is removed. • Viscosity is represented by a dashpot. The stress is related to the speed of strain (deformation rate) using the viscosity coefficient, which also expresses the resistance to deformation. The faster the strain rate, the greater the stress. Stress and strain are shifted from each other. The resistance to deformation is the strongest when the deformation rate is the smallest for a given load. • Plasticity corresponds to the behavior of a friction element. • In addition, intermediate behaviors can be observed (e.g., viscoelasticity [i.e., materials such as biological tissues behaving as a combination of viscous and elastic components] and viscoplasticity).

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The strain energy function relates stress to strain in a hyperelastic material such as vessel walls. The partial derivatives of the strain energy function with respect to Green’s strain components are related to the second Piola–Kirchhoff stresses. The strain energy function can be determined by solving an inverse problem based on experimental data. The influence of perivascular support from the surrounding tissue is generally neglected. However, some vessels are more constrained than others (e.g., distinct environments for epicardial and intramural coronary arteries). A radial constraint increases the radial stress, but decreases the longitudinal and circumferential stresses [28]. The nonlinear pressure–cross-sectional area relation, which couples blood dynamics to the vascular wall mechanics, yields the compliance of the explored vessel (slope at a given pressure C[p]). In an unsteady flow, other important quantities include the mean circumferential stress cθ = pRh /2h (p: blood pressure; Rh : hydraulic radius; h: wall thickness) for a thin-walled (h  R) cylindrical pressurized vessel (Laplace–Young equation) and characteristic impedance ρc/A (ρ: blood density; c wave speed in the blood vessel; and A: cross-sectional area). Four pre-requisites of any problem in biomechanics include: (1) Achievement of the computational domain, i.e., personalized geometry, with its given structure based on anatomical and histological data (2) Determination of the material constants of the body of interest or of each subdomain in the case of a composite material according to available rheological results, if possible, obtained properly in vivo (3) Selection of the equation set associated with the problem based on the governing physical laws, depending on assumptions (4) Definition of the appropriate initial and boundary conditions that incorporate the constraints of the neighborhood Medical signal and image processing provide the three-dimensional domain of interest for numerical simulations. Image data are characterized by image quality (contrast, edge quality, artifacts) with a given temporal and spatial resolution and noise level. Segmentation of organs and vessels refers to their separation from each other and the background. Three-dimensional reconstruction of their surfaces is followed by adapted meshing with coarsening and refinement of some regions (e.g., in the core flow area centered around the local vessel axis and in the layer close to the wall respectively). A mesh must be adaptive when coping with evolving processes. Meshes are then used for computational mechanics, in particular, fluid dynamics. Numerical results are influenced by geometry accuracy, i.e., the quality of image acquisition and processing, mesh design (numbers of mesh nodes and computational cell size, near-wall refined mesh, etc.), boundary conditions associated with the set of differential equations to solve (e.g., simple traction-free conditions [without tangential component of the local stress tensor [T]) at outlets11 or loose or strong

11 The traction vector t is defined as the scalar product of T and the unit normal n to the surface of interest. The surface is said to be tractionless if t = T · n = 0. In a Newtonian fluid, the stress tensor is given by: T = −pI + 2μD, where D is the rate of deformation tensor and I the unit tensor.

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coupling with distributed and/or lumped parameter models of the remaining part of the circulatory circuit), control parameters (time and space steps), and simulation factors (fluid properties, flow governing parameters, and blood and vascular wall rheology). Computed values have an intrinsic uncertainty due to assumptions, approximations, and errors linked to the geometrical reconstruction, meshing procedure, physical modeling, and mathematical and computational methods. However, some features (e.g., material constants and, in the case of cardiac mechanics, the subject-dependent orientation of myofibers within the different myocardial layers across the heart wall), pressure and/or velocity boundary conditions, and variables (impedance of various involved vascular segments) of lumped parameter models, even when they are obtained in vivo, are often taken from different individuals, as proper measurements are tedious and hence cannot be carried out systematically in every subject. Medical devices are substitutes for defective organs. They include sensors that gather information on the patient state, physiological variables following a circadian rhythm, and, depending on the subject, an actuator that acts on the body, and a control algorithm that enables the decision of the action to be achieved. Another goal of implantable devices is to restitute the caliber of a narrowed conduit and to obviate a secondary narrowing. The shape of implantable devices can be adapted to the patient’s anatomy using shape optimization-based modeling.

Book Series Whereas volume 7 of the series “Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems” was devoted to the cardiac pump and genesis of cardiopathies from a modeling perspective rather than a clinical point of view, volumes 8 to 13 primarily focus on diseases of the vasculature. They are aimed at presenting the processes and agents involved to adequately model some aspects of vasculopathies. Volumes 8 and 12 set the stage, i.e., yield pathophysiological elements implicated from the intracellular to the tissular level. Volume 13 describes major vasculopathies that are studied by mathematical and mechanical approaches. Volume 8 presents the clinical field of vasculopathies, i.e., cardiovascular markers and risk factors, focusing on hypertension, hyperglycemia and diabetes, hyperlipidemias and obesity, behavioral risk factors, and the genetic framework. Volume 9 presents the individual context, i.e., medical history with the favoring circumstances (aging, ciliopathies, and anomalies of the respiratory tract). It explores cell responses to various types of stressors such as hypoxia, how its cellular organelles involved in quality control manage stress more or less efficiently, and describes cell autophagy and different types of cellular death, which are normal phenomena that can be exacerbated under some circumstances. Volume 10 updates the data on vascular wall structure and architecture and reviews the genesis of new circuits in the arterial tree of the human adult and

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arteriogenesis, i.e., the development of collaterals. It also describes processes implicated in adverse wall remodeling. Volume 11 gives the molecular context within the cell, i.e., epigenomic factors, regulatory RNAs, lipids and lipoproteins, reactive oxygen and nitrogen species, gaseous messengers, as their deficiencies, excessive production, and dysregulated activity disturb the body’s homeostasis. Endothelial dysfunction and chronic vascular inflammation, which generally yield the framework of cardiovascular disease, in addition to the metabolic syndrome, the consequence of an imbalance between caloric intake and energy consumption, thrombosis, and lymphedema, are studied in volume 12. Volume 13 focuses on major diseases of the vasculature that are targets of mechanical and mathematical investigations, especially atherosclerosis and aneurysms. Lung diseases constitute the content of volume 14. It includes respiratory infections and allergies and obstructive and restrictive disorders. The main mechanical concepts and parameters, starting from a brief introduction of historical findings for a fast understanding are introduced in volume 15. Physical principles govern living organs and physiological apparatuses. Physical phenomena employ a set of quantities (e.g., mass, temperature, pressure, velocity, and energy). Mechanical concepts are used not only in mechanical modeling, but also in theoretical aspects of biology and medical practice. In particular, the Navier– Stokes equations describe flows of fluid particles. In general, physiological fluids are conveyed through deformable curved conduits of complicated configuration. Mechanical modeling is aimed not only at predicting the fields of the physical variables and testing the effect of the parameters involved, keeping all other quantities constant, but also at solving inverse problems, thereby enabling physiological quantities that cannot be directly measured to be assessed. Volume 15 contains chapters devoted to hemodynamics, air transport, aerosols, and rheology, as walls of the vascular circuit and respiratory tract are deformable and blood is a composite material, this flowing biological tissue carrying gas, nutrients, wastes, and cells to maintain life. It also introduces the methodology, i.e., measurements using physical models and, when ethically possible, in vivo, and numerical simulations, which are becoming a mandatory step in the development of medical devices.

Book Organization The present book includes seven chapters for handling the biological and clinical framework, which is mandatory for adequate modeling. Chapter 1 briefly introduces major cardiovascular diseases that are potential targets of mathematical and mechanical investigations. Chapter 2 presents cardiovascular risk factors and markers, the search for new criteria being aimed at improving the early detection of chronic diseases. The following chapters focus on: (1) Hypertension (Chap. 3), which involved the kidney among other organs in addition to many agents (2) Hyperglycemia

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and diabetes (Chap. 4) (3) Hyperlipidemias and obesity (Chap. 5) (4) Behavior, i.e., altered circadian rhythm, tobacco and alcohol consumption, physical inactivity, and an unhealthy diet; Chap. 6). Chapter 7 is related to the genetic framework of vasculopathies. Among personal and environmental conditions, the genetic ground explains certain pathophysiological processes involved in vasculopathies, such as dyslipidemia and mutations affecting the vasomotor tone. This treatise is aimed at serving as a vade mecum for scientists who are not specialized in biology, but model physiological and pathological processes. It does not avoid the pitfall of pleasureless concatenation of observations. Collecting data and key relations is yet a mandatory work done during the preliminary stage of any modeling to handle and accurately represent all important mechanisms involved in the explored process before selecting proper parameters, obviating any redundancies and eliminating accessory information. The main text contains the essential information needed to steer the explored process and to estimate the level of influence of the implicated agents before modeling. Some biological aspects, which are not mandatory for achieving proper modeling, are not incorporated in these books. Further details and proper citations to original works can be found, especially in review articles. Any information bears interest only when it is inscribed in the continuity of development. Footnotes are immoderately used throughout the text, as they are aimed at affording a deeper understanding and details, bringing complementary, but accessory information, which nevertheless enriches and illustrates the main text. Another objective is disambiguation, especially for molecules with multiple names and aliases. In the present text, footnotes are not intended to authorize a break, where what is not handled is hidden, although the knowledge of the process of interest often remains limited.

Acknowledgments The author acknowledges the patience of his wife Anne, daughter Maud, sons Alrik and Damien, and their respective French (Julien, Jean, and Louis), American (Raphaëlle, Matthieu, and Alexandre), and Polish (Joanna and Frédéric) families. Paris, France

Marc Thiriet

Contents

1 Cardiovascular Disease: An Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Vasculopathies and Vasculitides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Ethnic Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2 Gender Influence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.3 Vasculitis (Angiitis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.4 Vascular Wall Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.5 Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Vasculopathies and Cardiac Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Cardiac Wall Remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2 Cardiomyocyte Remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.3 Altered Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.4 Interrelation Between the Heart and Kidney. . . . . . . . . . . . . . . . 1.2.5 Ectopic Calcification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Autoimmune Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Congenital Vascular Malformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.1 Classification of Congenital Vascular Malformations . . . . . . 1.4.2 Venous Malformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.3 Capillary Malformations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.4 Lymphatic Malformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.5 Endothelial Signaling in Vasculo- and Angiogenesis . . . . . . 1.4.6 Hereditary Hemorrhagic Telangiectasia . . . . . . . . . . . . . . . . . . . . 1.4.7 Cerebral Cavernous Malformations . . . . . . . . . . . . . . . . . . . . . . . . . 2

Cardiovascular Risk Factors and Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Environmental Stressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Air Pollution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2 Noise. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Circular RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 3 3 14 15 42 45 47 53 58 64 66 67 68 68 72 73 75 77 88 90 91 101 101 114 115 120 163

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Contents Y RNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 2.2.4 Long Nonprotein-Coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5 Ribosomal RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Types of Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165 165 176 176 176 178 194

3

Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Hypertensive Heart Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Cardiac Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Pregnancy Maternal Hypertensive Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Renovascular Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Arterial Wall Stiffening and Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Arterial Pressure Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Kidney and Blood Pressure Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1 Pressure-Induced Natriuresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2 Nephron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.3 Renal Control of Water and Ion Balance. . . . . . . . . . . . . . . . . . . . 3.7.4 Hydrogen Ion Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.5 Compartments of the Nephron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Intestinal Flora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.1 Genetic and Epigenetic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.2 Sympathetic Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.3 Nuclear Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.4 Renin–Angiotensin Axis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.5 Aldosterone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.6 Endothelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.7 Galectin-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.8 Prolactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.9 20-Hydroxyeicosatetraenoic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9.10 Membrane Depolarization-Limited Vasoconstriction . . . . . . 3.10 Hypertension, Cerebrovascular Disease, and Therapy . . . . . . . . . . . . . . .

199 202 203 204 204 205 207 208 208 210 210 233 244 269 270 271 271 273 274 289 296 297 297 298 299 299

4

Hyperglycemia and Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Diabetes Mellitus and Epigenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Vascular Complications in Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 Insulin Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2 Insulin Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 MicroRNAs and Insulin Sensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Kidney and Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Heart and Glucose Tolerance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

301 302 303 305 307 308 309 309 322 323 324

2.3

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4.9 4.10

Insulin Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Redox Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

5

6

Hyperlipidemias and Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Classification and Etiology of Dyslipoproteinemia . . . . . . . . . . . . . . . . . . 5.1.1 Hypercholesterolemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2 Hypertriglyceridemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3 Dyslipoproteinemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.4 Ceramides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Gluco- and Lipotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Overweight and Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1 Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2 Cardiovascular Effects of Obesity. . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3 Obesity-Associated Chronic Inflammation and Insulin Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.4 Non-Alcoholic Fatty Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Adipose Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1 Structural and Functional Types of Adipose Depots . . . . . . . 5.4.2 Adipogenesis: A Brief Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3 Adipose Tissue Remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4 Lipid Droplets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.5 Adipose Tissue Secretome: Adipokines . . . . . . . . . . . . . . . . . . . . 5.5 Nuclear Receptors FXRs, LXRs, PXR, and RXRs . . . . . . . . . . . . . . . . . . 5.5.1 FXRs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2 LXRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.3 PXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.4 RXRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Control of Body Weight and Energy Homeostasis . . . . . . . . . . . . . . . . . . . 5.6.1 Signal Integration by the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.2 Brain–Gut Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.3 Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.4 Peptidic Messengers: Maturation by Cleavage . . . . . . . . . . . . . 5.6.5 Gastrointestinal Epithelial Barrier. . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.6 Endocrine Cells of the Gastrointestinal Tract Mucosae and the Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.7 Satiation Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.8 Feeding Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

331 335 337 339 343 360 361 365 368 369

528 530 543

Behavioral Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Circadian Rhythm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1 Circadian Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2 Seasonal Variations of the Circadian Rhythm . . . . . . . . . . . . . . 6.1.3 Cell Division Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.4 Cellular Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

549 550 552 561 562 563

382 398 401 404 429 433 434 440 478 482 484 486 486 486 488 497 515 517 522

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6.1.5

Circadian Rhythm of Mitochondrial Activity in Hepatocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.6 Oxidation–Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.7 Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.8 Desynchrony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tobacco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Prenatal Environmental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . Physical Inactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detrimental Use of Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unhealthy Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1 Dietary Sodium Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.2 Dietary Lipid Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.3 Dietary Carbohydrate Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.4 Dietary Protein Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.5 Healthy Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.6 Diet and Gut Flora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of the Gut Microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.1 Gut Flora and Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.2 Gut Flora and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.3 Gut Flora and Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.4 Gut Flora and Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

566 568 568 569 571 573 574 574 575 575 575 577 578 580 584 586 588 592 593 593

Genetic Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Messenger RNA Synthesis and Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1 Gene Accessibility by Chromatin Relaxation . . . . . . . . . . . . . . 7.1.2 Gene Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.3 Formation Modes of Messenger RNA Subtypes . . . . . . . . . . . 7.1.4 RNA Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Smooth Myocyte Contractility and Vasomotor Tone . . . . . . . . . . . . . . . . 7.3 Blood Pressure and Hypertension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Genetic Determinants of Dyslipidemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Atherosclerosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Chromosomal 9p21 Risk Locus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Aortopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.8 Aneurysms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.9 Stroke. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

595 601 609 616 617 621 622 625 640 656 663 665 668 672

6.2 6.3 6.4 6.5

6.6

7

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681 Notation Rules: Abbreviations, Aliases, and Symbols . . . . . . . . . . . . . . . . . . . . . . . 763 List of Molecule Shortened Abbreviations and Chemical Symbols . . . . . . . . 797

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List of Shortened Aliases of Anatomical and Histologic Terms . . . . . . . . . . . . . 847 List of Shortened Aliases of Physiological and Medical Terms . . . . . . . . . . . . . 853 List of Chemical, Mathematical, and Physical Symbols . . . . . . . . . . . . . . . . . . . . . 863 Subscripts and Superscripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 875

Chapter 1

Cardiovascular Disease: An Introduction

Wo aber Gefahr ist, wächst das Rettende auch! [Where there is danger, that which will save us also grows] (F. Hölderlin, Patmos [1803])

Cardiovascular disease (CVD) is a collective term designating all types of affliction affecting the blood circulatory system, including the heart and vasculature, which, respectively, displaces and conveys the blood. This multifactorial disorder encompasses numerous congenital and acquired maladies. CVD represents the leading noncommunicable cause of death in Europe (∼50% of all deaths; ∼30% of all deaths worldwide) [29]. In 2008, nine million people died of noncommunicable diseases prematurely before the age of 60 years; approximately eight million of these premature deaths occurred in low- and middle-income countries [30]. Cardiovascular disease encompasses atherosclerosis with its subtypes (coronary [CoAD], cerebral [CeAD], and peripheral artery disease [PAD]) with two major complications, myocardial infarction and ischemic stroke (more common than hemorrhagic stroke; Sect. 1.1.5 and Vol. 13, Chap. 5. Atherosclerosis), heart failure (HF), cardiac valvulopathies and arrhythmias, rheumatic heart disease (damage of the myocardium and cardiac valves caused by streptococci bacteria), congenital heart disease, and deep vein thrombosis with its own complication, pulmonary embolism. Rare cardiovascular maladies are classified into [31]: • • • • • • •

Rare afflictions of the systemic (class I ) and pulmonary circulation (class I I ) Rare cardiomyopathies (class I I I ) Rare congenital cardiovascular disorders (class I V ) Rare cardiac arrhythmias (class V ) Cardiac tumors and cardiovascular affections related to cancer (class V I ) Cardiovascular sickness in pregnancy (class V I I ) Other types of rare cardiovascular illness (class V I I I )

© Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0_1

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Etiology1 of a given malady refers to the cause, set of causes, or manner of causation of a disease. CVD is multicausal, with clinical (dyslipoproteinemia and hypertension) and behavioral factors (sedentarity, overnutrition, smoking, and a stressful life). Deficiency or excess of trace elements in soil may contribute to CVD [32]. A major cause of CVD is atherosclerosis. Diagnosis2 of many diseases, in addition to assessment of prognosis, is facilitated by the utilization of specific markers that can be proteins and nucleic acids, such as short microRNAs and long a priori nonprotein-coding RNA implicated in the regulation of metabolism, control of blood circulation, and inflammation. Epidemiological studies are aimed at extracting individual,3 environmental (indoor and outdoor air pollution, second-hand smoke), and societal risk factors, ranking them to the determined predominant factors according to their impact, morbidity, and mortality rate. These studies also aimed to propose strategies to reduce CVD burden and for the prevention of early adverse events. In a health system that brings research into practice, the aim is to reduce risk factors and determine appropriate treatments [33].

1.1 Vasculopathies and Vasculitides Vasculopathy corresponds to any disease affecting the blood vessels that can be caused by degenerative, metabolic (e.g., diabetic vasculopathy), and inflammatory disorders in addition to thromboembolic maladies. Vasculitis, or angiitis, is more specific, as it is defined by a focal or widespread inflammation of the vascular wall, whatever the blood vessel type (i.e., arteries, arterioles, capillaries, venules, and veins), size (i.e., large, medium, or small), number, and location. Ethnicity and gender can affect vascular physiological and pathophysiological mechanisms. Data obtained in a general population, such as correlation between a risk factor and a given disease or complication associated with a given gene mutation, may not be representative of features of a homogeneous subpopulation. Sex differences in incidence, prevalence, morbidity, and mortality from CVD, which include sex-specific disorders and sex-dependent symptom presentation and evolution of pathophysiological processes common to both genders (e.g., hypertension and atherosclerosis), represent a source of health disparity [34]. Sex and racial differences in pharmacodynamics and -kinetics affect therapy efficiency [35].

1 αιτιoλoγ ω:

inquire into causes, reason, account for; αιτιoλoγια: giving the cause. know one from the other, distinguish, discern. 3 For example, abdominal obesity, diabetes mellitus, hypertension, smoking, unhealthy diet, regular alcohol consumption, lack of physical activity, and psychosocial factors, in addition to biological indices such as concentrations of total cholesterol and low-density lipoproteins, total cholesterol/ high-density lipoprotein, and apolipoprotein ApoB/ApoA1 ratios. 2 διαγιγνoσκω:

1.1 Vasculopathies and Vasculitides

3

1.1.1 Ethnic Differences Ethnicity is a source of health inequalities. People from certain ethnicities suffer from premature CVD. In particular, ethnic heritage influences the occurrence rates of hypertension and diabetes. According to the American Heart Association and National Institute of Health, 40% of African American men and women have a coronary disease (versus 30 and 24% of European-American men and women, respectively) due to genetic differences between ethnic groups rather than by life conditions and diet. Moreover, African American women with coronary disease are at a twofold higher risk for myocardial infarction than European American women. In the USA: • • • •

African Americans are at a higher risk for hypertension. North American Latinos have higher rates of obesity and diabetes. The coronary artery disease rate is highest in the South Asian population. The stroke rate is the highest in individuals of African-Caribbean descent, the prevalence of diabetes in these two ethnic groups being much higher than in the White population. In the UK [36]:

• The incidence of myocardial infarction is higher in South Asians than in nonSouth Asians for both sexes. • The incidence of stroke in black people is higher than in White people, whatever the sex. • The prevalence of CoAD is highest in Indian and Pakistani men. • The revascularization rate is higher in the White ethnic group than in the black and Asian ethnic groups. • Individuals from different ethnic groups tend to store fat in different regions of the body. • The prevalence of overweight and obesity in young children is highest in the Black ethnic group. • The prevalence of diabetes is much higher in Black Caribbean, Indian, Pakistani, and Bangladeshi men. However, these inequalities can be related not only to genetic differences but also to distinct cultural and social practices. The influence of ethnicity can be difficult to distinguish from that of the socioeconomic status. Moreover, a genetic diversity can exist within racial and ethnic groups.

1.1.2 Gender Influence Hormonal in addition to genetic and environmental factors contribute to sex differences in CVD. The organism environment influences hormonal secretion, which, in

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turn, affects the brain function, hormones acting on the genome of neural cells that contain their cognate receptors. For example, the ventromedial and ventrolateral and arcuate nuclei contain estrogen-sensitive neurons, the ventromedial nucleus responding more rapidly than the arcuate nucleus [37]. In addition, hormonal secretion by the adrenal gland and gonads (testis and ovary) is controlled not only by temperature (heat or cold), threat, and sexual excitement, which prime adaptive responses, but also by circadian oscillators entrained by environmental signals (light and dark). Repeated psychosocial stress can provoke neuronal loss in the hippocampus, which receives heavy input from the dentate gyrus mossy fiber system [37]. Adrenal steroids, which protect in the short term, operate in conjunction with neural excitatory amino acids, causing damage and allostatic load in the long term, when the adaptive response is not managed efficiently and persists. Gonadal hormones can also produce both protection and damage according to their concentration and duration of exposure. Sex is mainly determined by the X and Y chromosomes, which create a sexspecific expression pattern. Men possess a single copy of each type (XY genotype) and women two copies of the X chromosome (XX genotype), one of the X chromosome being silenced during embryogenesis (X-chromosome inactivation). In general, premenopausal women are at a lower CVD risk than men of a similar age. Men develop hypertension at younger ages than women. The sexdetermining region Y (SRY locus) of the Y chromosome regulates the transcription of tyrosine hydroxylase (TH; or Tyr 3-monooxygenase), the rate-limiting enzyme in the synthesis of catecholamines such as noradrenaline, and yields a genderdependent difference in sympathetic activity, predisposing men to hypertension to a greater extent than women [34]. The Y chromosome also includes genes involved in inflammation and innate immunity linked to macrophage activation [34]. On the other hand, the X chromosome affects expression of genes associated with apoptosis, lipid oxidation, and generation of reactive oxygen species (ROS) by the mitochondrion. In individuals from European countries, some chromosomal loci related to lipid metabolism exhibit sex-specific effects, in particular the HMGCR and NCAN genes encoding 3-hydroxy 3-methylglutaryl coenzyme-A reductase and neurocan (or chondroitin sulfate proteoglycan CSPG3), respectively [34]. A sex-specific single-nucleotide polymorphism in the locus of the CPS1 gene encoding mitochondrial carbamoyl–phosphate synthase-1, which is involved in hepatic nitrogen urea metabolism and synthesis of arginine, a precursor of nitric oxide (NO), has a greater effect in women than in men [34]. The gene encoding the androgen receptor resides on the X chromosome and displays a polymorphism linked to a highly variable number of CAG repeats. Variants in this gene have a greater impact in men than in women [34]. Genetic variants also affect enzymes involved in synthesis, conversion, and degradation of sex steroids.

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Estrogens (e.g., estradiol [E2 ]) and androgens (e.g., testosterone and dihydrotestosterone [DHT; or androstanolone]) govern multiple processes in both women and men [38]. 1.1.2.1

Estrogen Signaling

Estradiol is synthesized primarily in the granulosa cells of ovaries and Sertoli cells in males. It tethers to various types of cytosolic ERα and ERβ (i.e., ligand-activated transcription factors NR3a1–NR3a2) in cardiomyocytes (CMCs) and vascular smooth muscle (vSMCs) and endothelial cells (ECs) in addition to plasmalemmal estrogen receptor GPER1 (or GPR30) in vascular endotheliocytes and smooth myocytes, renal intercalated and tubular cells, and cells of the hypothalamic– pituitary–adrenal axis [38]. Membrane-initiated rapid (nongenomic) signaling launched by sex steroid hormones produced by the adrenal cortex, ovary, and testis involves estrogen receptors in the plasma membrane. In addition to the membrane estrogen receptor GPER1, sex steroid receptors such as NR3a1 and NR3a2 can localize to the plasma membrane; NR3a1 can be identified in caveolae associated with proteic complexes), its palmitoylation (Cys447) being required for its translocation to the plasma membrane [39]. NR3a1 and NR3a2 are necessary and sufficient for rapid estrogentriggered signaling. At the plasma membrane, caveolin-1 serves as a scaffold for other signaling molecules (e.g., trimeric G protein, Src, PI3K, GFR, and MNAR)4 that are activated by the E2 –NR3a1 couple in caveolae, which facilitates the fast generation of early signals (e.g., Ca2+ influx).5 Other NR3a1 move to the nucleus chaperoned by heat shock protein HSP90 owing to a nuclear localization sequence or to mitochondria. Nuclear NR3a1 is mandatory for the development of the female reproductive tract and mammary gland. NR3a2 prevents adverse cardiac remodeling (hypertrophy and fibrosis). The E2 –NR3a2 couple stimulates PI3K and primes transcription of the RCAN gene that encodes the regulator of calcineurin (PP3) [39].6 In addition, the E2 –NR3a2 4 GFR:

growth factor receptor; MNAR: modulator of nongenomic action of the estrogen receptor. The coactivator of estrogen receptor-mediated transcription and corepressor of other nuclear hormone receptors (transcription factors) that facilitates NR3a1 nongenomic signaling via Src and PI3K is also called proline-, glutamate-, and leucine-rich protein PELP1. The plasmalemmal NR3a signalosome comprises G-protein subunits, receptor and nonreceptor protein Tyr kinases (e.g., Src), protein Ser/Thr kinases (PKB), lipid kinases (PI3K and PDK1), and scaffold proteins (MNAR, SHC, striatin) [39]. It interacts with several growth factor signaling components (e.g., EGFR and HGF-regulated protein Tyr kinase substrate (HRS)). It interacts with androgen (AR or NR3c4) and glucocorticoid receptor (GR or NR3c1) and thus influences nuclear receptor (NR) signaling. 5 Both NR3a1 and NR3a2 can activate Gα and Gβγ. Plasmalemmal NR3a monomers in the absence of sex steroids rapidly homodimerize upon estrogen exposure. These dimers can then associate with Gα and Gβγ subunits. Estradiol rapidly stimulates calcium entry via the TRPV6 channel [39]. 6 Regulators of calcineurin RCan1, RCan2, and RCan3 are also termed calcipressin-1 to -3 in addition to modulatory (or myocyte-enriched) calcineurin-interacting proteins MCIP1 to MCIP3.

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couple launches synthesis of the natriuretic peptides ANP and BNP, which hampers adverse cardiac hypertrophy via ERK kinases in the CMC. On the other hand, atherogenic 27-hydroxycholesterol serves as an endogenous selective estrogen receptor modulator (SERM), which abounds in the diseased arterial wall. It competitively precludes E2 –NR3a binding and hence both the rapid (i.e., NOmediated vasodilation) and delayed transcriptional E2 actions [39]. Estrogens are also synthesized in the central nervous system from cholesterol or converted from aromatizable androgens in presynaptic terminals [38]. Estrogens can then diffuse. Both NR3a1 and NR3a2 are produced in nuclei in the forebrain and brainstem that regulate cardiac frequency and blood pressure (solitary tract [NTS] and parabrachial nuclei [PBN] and rostral ventrolateral medulla [RVLM]), enhancing sympathetic nervous system-mediated baroreflex. They regulate the local renin–angiotensin axis (RAA), these brain nuclei possessing renin, angiotensinogen, angiotensin convertases ACE1 and ACE2, and angiotensin Agt2 receptors (e.g., AT1 and AT2 ). Angiotensin-2 and aldosterone stimulate ROS production in the brain by NAD(P)H oxidase, thereby raising sympathetic nerve activity. In the subfornical organ (SFO), estrogens via NR3a1 and NR3a2 prevent intracellular ROS formation. Estradiol reduces both Agt2- and aldosterone-induced hypertension in male and ovariectomized female rodents [38]. However, NR3a1 and NR3a2 regulate blood pressure differently. In male rats, injection of E2 into the paraventricular nucleus (PVN) does not affect cardiac frequency and blood pressure. In female mice, activated CNS NR3a1 protects against Agt2-induced hypertension, whereas PVN NR3a2 and RVLM NR3a2 protect against aldosterone-induced hypertension. In female and male mice, activated CNS NR3a2 preserves resting blood pressure via RVLM CaV channels [38]. Therefore, at least in female and male rodents, specific NR3a subtypes mediate E2 -mediated protection in different nuclei. Nitric oxide synthase NOS1 is produced to a greater extent in the SFO and PVN of female mice than in these nuclei in male mice [38]. In addition, estrogens rapidly stimulate NO production by NOS3 via NR3a1 in the vascular endothelium, whatever the gender. Estrogens signal to the kidney when salt sensitivity increases in menopausal women, likely because estrogens support NO action and lower the AT1 /AT2 ratio, hence preserving renal Na+ handling [38]. In premenopausal women, salt loading during estrogen peaks alleviates filtration fraction and causes a sustained renal vasodilation. In postmenopausal women, salt loading raises the filtration fraction. In addition, NR3a1 mediates regulation of the renal ACE1/ACE2 ratio by estrogens, ACE2 converting vasoconstrictive, prohypertrophic, and proproliferative angiotensin-2 into Agt(1–7) .

The RCan protein binds to protein phosphatase PP3 in the cytoplasm, blocking its activity triggered by angiotensin-2 and other hypertrophic factors. Indeed, it prevents dephosphorylation of transcription factors of the NFAT family, sequestering them in the cytoplasm and impeding their nuclear translocation [39].

1.1 Vasculopathies and Vasculitides

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GPER1 on arterial and venous endotheliocytes and smooth myocytes counters endothelin-1– and prostanoid-primed vasoconstriction and lessens superoxide production, hence protecting against hypertension [38]. Furthermore, GPER1 activates NOS3 in the vascular endothelium. It also attenuates vascular smooth muscle cell (vSMC) proliferation and vascular inflammation. Estrogens operate on low-density lipoproteins (LDLs) and the LDL receptor (LDLR) to improve lipidemia. Estradiol upregulates LDLR production and stimulates sterol 27-hydroxylase CyP27a1 activity, which hampers LDL formation. In addition, estrogens promote synthesis of the apolipoprotein ApoA1 in the liver and of ApoE [38]. They also boost ABCa1 production, facilitating reverse cholesterol transport, but hinder ScaRb1 expression, hence prolonging the duration of circulating high-density lipoproteins (HDLs). Many leukocytes infiltrating atherosclerotic plaques, such as macrophages, B and T lymphocytes, and mastocytes possess sex hormone receptors; estrogens can thus influence inflammation [38]. Estrogens exert the anti-inflammatory M2 phenotype in macrophages; reduce LDL oxidation, endothelial activation, and adhesion of neutrophils and monocytes to the endothelium; and impede NOx activity and hence ROS production. Estradiol that dampens inflammation can reduce formation of tumor-necrosis factor superfamily member TNFSF1 and prevent its secretion [38]. Moreover, it activates NR3a2 and subsequently Iκ Bα which represses NFκB-boosted inflammation. Estrogen supports angiogenesis in PAD owing to NOS3 [38]. Proangiogenic estrogens favor mobilization of endothelial progenitor cells and incorporation into neovascularization sites owing to NOS3 stimulation and MMP9 activity in the bone marrow. Relatively high concentrations of circulating female sex hormones protect against abdominal aortic aneurysm (AAA) development, as these hormones reduce inflammation and matrix metallopeptidase activity in the aortic wall [38]. In female animals, a higher concentration of plasminogen activator inhibitor PAI1, which precludes MMP2 and MMP9 production, protects against AAA development. Estrogens also lower MMP2 and MMP9 concentrations in addition to immunocyte infiltration in AAA and hence slow dilation rate with respect to ovariectomized rodents. In addition, NO production stimulated by estrogens protect against AAA. Aortas from female mice contain larger NR3a1 amounts and lower matrix metalloproteinase (MMP) activity. On the other hand, in humans, AAA samples contain larger concentrations of 3-hydroxyanthranilic acid (3HAA), indoleamine (2,3)-dioxygenase (IDO), and kynureninase than adjacent aortic segments. Indoleamine dioxygenase is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism that creates 3HAA. Acute infusion of angiotensin-2 favors abdominal aortic aneurysm development in APOE−/− mice, but not in APOE−/− and Ido−/− mice, in which elastic lamina degradation and aortic expansion decay [40]. Angiotensin-2 activates interferon-γ which launches expression of IDO and kynureninase, thereby raising

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production in medial smooth myocytes and subsequently its concentration in the aortic wall and plasma of 3HAA, which upregulates synthesis of MMP2 via NFκB. The risk of cerebral aneurysms is augmented in postmenopausal women, whereas estrogen replacement protects against intracranial aneurysms, protection ensured by estrogens being mediated by NR3a2 and cerebral vascular NO production [38].7 1.1.2.2

Androgen Signaling

Testosterone is synthesized in testicular Leydig cells and ovarian theca cells. It is converted to the more potent 5α-DHT by 5α-reductase. Both androgen types connect to another ligand-activated transcription factor, androgen receptor (AR or NR3c4), which is detected in endotheliocytes and smooth myocytes, platelets, and macrophages. Two variants (ARa–ARb) lodge in most organs with varying expression levels according to the tissue type [38]. Testosterone is also converted to E2 by aromatase, in particular in the brain. Androgen signaling is linked to metabolism, cell proliferation, differentiation, and apoptosis, and protein secretion, whatever the gender. Androgens can trigger alternative rapid (nongenomic) signaling after binding to membrane-associated or cytosolic AR that releases intracellular Ca2+ and activates kinases (e.g., MAPK, PKA, PKB, and PKC) [38]. Membrane-associated ARs in aortic endotheliocytes interact with Src and caveolin-1. Postmenopausal women experience more rapid age-related hypertension than age-matched men [38]. Hypoandrogenism may be linked to hypertension in older men, suggesting that a normal androgen concentration is antihypertensive. Testosterone rapidly activates NOS3 in vascular endotheliocytes via the PI3K–PKB pathway. On the other hand, in young, obese, hypoandrogenic male rats, 10-week testosterone supplementation improves body weight and lipid profiles but increases blood pressure. Androgens can elevate blood pressure via ruptured abdominal aortic aneurysm (RAAA) constituents in the kidney. They can contribute to Agt2-induced hypertension in male animals via renal inflammation, renal lymphocyte infiltration being greater in male than in female mice [38]. Although NR3a diminishes the formation of adhesion molecules in endotheliocytes exposed to atherogenic factors, NR3c4 stimulates vcam1 production in male-derived endotheliocytes due to a higher NR3c4 concentration (not in cells of female origin). In men, androgen deficiency is linked to endothelial dysfunction. On the other hand, in women, hyperandrogenemia favors atherogenesis and arterial calcification [38]. 7 Connexin-37

forms gap junctions in myoendothelial communication between microvascular endothelial and smooth muscle cells. Its phosphorylated Tyr332 controls the gap junctiondependent spread of calcium signals. Nitric oxide precludes Tyr332 dephosphorylation by PTPn11 and hence Ca2+ transfer induced by mechanical stimulation of endotheliocytes, but enhances Ca2+ spreading within the endothelium, thereby boosting endothelium-dependent vasodilation in response to acetylcholine, even despite inhibition of soluble guanylate cyclase [41].

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In both sexes experiencing hypoxia caused by occlusive peripheral artery disease, androgens promote angiogenesis via NR3c4, the upregulated formation of vascular endothelial growth factor (VEGF) and its receptors in endotheliocytes, and VEGF-primed phosphorylation of PKB and NOS3 [38]. Administration of DHT augments male-derived (but not female) endotheliocyte migration, proliferation, and tubulogenesis. In male animals, relatively high concentrations of androgens, which upregulate expression of RAAA components, favor macrophage recruitment and extracellular matrix degradation via MMPs, and hence AAA formation, the castration of male mice limiting Agt2-induced AAA genesis and expansion to levels observed in intact female mice [38]. Male rodents can also be predisposed to AAA by androgens via elevated MMP activity.

1.1.2.3

Enzymes of Steroid Hormone Metabolism

Steroids are lipophilic, low-molecular-weight compounds derived from cholesterol. In fluids, they are usually found in either a conjugated form (i.e., linked to a hydrophilic moiety, such as sulfate or glucuronide derivatives) or bound to proteins. In the plasma, unconjugated steroids are mainly bound to carrier proteins, albumin (20–50%), serpin-A6 (or corticosteroid-binding globulin [CBG]), and sex hormonebinding globulin (SHBG) [42]. Mitochondria of steroidogenic cells of the adrenal gland, gonads, placenta during gestation, and brain are essential sites for steroid hormone synthesis. The adrenal gland synthesizes androgens and corticosteroids (mineralo- and glucocorticoids), ovary estrogens and progestins, and testis (mainly androgens). In men, the adipose tissue contains aromatase, a source of androgen-derived estrogens. Once they are released into the bloodstream, these endocrine messengers act on target cells, including those of the central nervous system. The latter also form neurosteroids with auto- and paracrine effects. They diffuse easily through the plasma membrane. Circulating steroids are processed in target cells, which can form active metabolites. Removal of part of the cholesterol side chain generates C21-steroids of the pregnane series (progestins and corticosteroids), total removal of C19-steroids of the androstane series (e.g., androgens), and loss of the 19-methyl group the estrane series (e.g., estrogens) [42]. Steroids are characterized by the presence or absence of functional groups (mainly hydroxy, keto(oxo), and aldehyde) at certain positions of the carbon skeleton (particularly at positions 3, 5, 11, 17, 18, 20, and 21) [42]. These functional groups characterized by their type, number, position, and orientation engender a large number of stereoisomers (i.e., molecules having the same chemical formula but distinct three-dimensional conformation). Enzymes involved in steroid synthesis include (Tables 1.1 and 1.2) [42]:

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Table 1.1 Enzymes of steroid synthesis (Part 1; CyPiXj : cytochrome-P450 family i, subfamily x, polypeptide j [i, j: integers, x: letter]) Type CyP11a1 CyP17a1

CyP21a2 (CyP21, CyP21b)

CyP11b1

CyP11b2

CyP19a1

Other name Action Mitochondrial cholesterol side-chain cleavage enzyme Converts cholesterol to pregnenolone Steroid 17α-hydroxylase and (17,20)-lyase Converts pregnenolone and progesterone into their 17α-hydroxylated products and subsequently to dehydroepiandrosterone (DHEA) and androstenedione Steroid 21-hydroxylase Catalyzes 21-hydroxylation of steroids Converts 17-hydroxyprogesterone and progesterone into 11 β-deoxycortisol and deoxycorticosterone, respectively Required for adrenal synthesis of mineralocorticoids and glucocorticoids Mitochondrial steroid 11β-hydroxylase 11β-, 18-, and 19-hydroxylation of steroids, aromatization of androstenedione to estrone Production of cortisol and corticosterone from 11β-deoxycortisol and deoxycorticosterone, respectively Mitochondrial aldosterone synthase; steroid 18-hydroxylase Catalyzes the conversion of corticosterone and then 18-hydroxycorticosterone into aldosterone Aromatase, estrogen synthase Converts C19 androgens to C18 estrogens

Androstenediol and androstenedione are weak androgen and estrogen steroid hormones and intermediates in the synthesis of estrone and testosterone. Androstenedione is converted to testosterone and estrone by the 17β-hydroxysteroid dehydrogenases (HSDH17β1–HSDH17β3 and HSDH17β7) and aromatase, respectively. Androstenediol is processed by the 3β-hydroxysteroid dehydrogenases HSDH3β1 and HSDH3β2 into testosterone. The three estrogens, estrone (E1 ), a weakly active E2 precursor, which is also named the estrogen of the menopause; estradiol (E2 ), the most active estrogen; and estriol (E3 ), a weakly active E2 metabolite, which is also called the estrogen of pregnancy, are produced by aromatase. Both estriol and estrone can interact with the estrogen receptor and thus antagonize estradiol action Table 1.2 Enzymes of steroid synthesis (Part 2; AKR aldo–keto reductase) Type SRd5a1/2/3 SRd5a1/2 AKR1c3

Other name Action Steroid 5α-reductases, 3-oxo 5α-steroid 4-dehydrogenases Convert testosterone into more potent dihydrotestosterone and progesterone or corticosterone into their corresponding 5α 3-oxosteroids 17β HSDH5, brain 3α HSDH2 Preferentially transforms androstenedione to testosterone

1.1 Vasculopathies and Vasculitides

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1. Mitochondrial desmolases (or lyases), which remove parts of the cholesterol side chain via sequential hydroxylation of adjacent carbon atoms using molecular oxygen, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and cytochrome P450 2. Membrane-bound mitochondrial or microsomal hydroxylases, which also require cytochrome-P450, O2 , and NADPH 3. Cytosolic and microsomal hydroxysteroid dehydrogenase, these oxidoreductases depending on NADP(H) or NAD(H) 4. Membrane-bound aromatase that involves a sequence of hydroxylation and loss of the C19 methyl group, its substrate being 4-androstenedione or testosterone Mitochondria contain the cholesterol desmolase CyP11a1, which catalyzes cholesterol side-chain cleavage to yield pregnenolone, a C21 compound, in addition to its electron transfer partners, ferredoxin and ferredoxin reductase [43]. Pregnenolone can be converted either to progesterone, which leads to glucocorticoids, androgens, and estrogens, or to 17α-hydroxypregnenolone, which also forms androgens and estrogens. In the adrenal gland, androgen formation is limited to dehydroepiandrosterone and androstenedione, whereas in Leydig cells of the testis, 17β-hydroxysteroid dehydrogenase (17HSDH) produces testosterone [43]. In granulosa cells of the ovary, estrogen synthesis requires the aromatase complex that uses the substrate androstenedione and testosterone to create estrone and estradiol, respectively. Hydroxylation of progesterone at carbon 21 yields 11-deoxycorticosterone (DOC) and additional hydroxylation at carbon 11 corticosterone, a major glucocorticoid in mammalian species that do not produce cortisol. The main glucocorticoid secreted by human adrenal glands, cortisol, is formed of 17α-hydroxyprogesterone via the intermediate 11-deoxycortisol. Further hydroxylation and redox at carbon 18 give rise to aldosterone. Several other steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase, 11β-hydroxylase, and aldosterone synthase, also reside in mitochondria. Cholesterol ingress into the mitochondrion is regulated by the steroidogenic acute regulatory protein, StAR, the action of which requires the machinery of the outer mitochondrial membrane, which comprises translocator protein (Tspo),8

8 18-kDa

Translocator protein, previously called peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import through the OMM upon hormonal stimulation from the cytosol to the aqueous intermembrane space of the mitochondrial envelope (intermembrane space [IMS]) and steroid production. The importing of Tspo into steroidogenic cell mitochondria is regulated by cAMP. The translocase of the outer mitochondria membrane complex (TOMM), which recognizes mitochondrial proteins for importing, does not interact with Tspo and thus is not required for Tspo importing and insertion into the OMM. Initial targeting of Tspo to mitochondria depends on the cytosolic chaperones interacting with the import receptor TOMM70, which is loosely associated with the TOMM complex, and its integration into the OMM on metaxin-1 [44]. Translocator protein interacts with voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT), comprising the mitochondrial permeability transition pore

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voltage-dependent anion channel VDAC1, Tspo-associated acylCoA-binding domain-containing protein ACBD3, and protein kinase-A regulatory subunit PKAr1α [43]. The main site of catabolism is the liver. It involves various reaction types: 1. Reduction of a double bond at C4 and a reduction of an oxo(keto) group at C3 to a secondary alcoholic group 2. Reduction of an oxo group at C20 to a secondary alcoholic group 3. Oxidation of a 17β-hydroxyl group 4. Further hydroxylations at various positions 9 and/or glucuronosyl groups 5. Conjugation of a sulfate group (SO2− 4 ) (glucuronidation), which yields steroid sulfates and glucuronides, by Mg2+ dependent steroid sulfokinases and glucuronyl transferase, respectively, thereby forming hydrophilic molecules that can be more easily excreted by the kidney Corticosteroid 11β-dehydrogenase HSD11β2 converts cortisol into its inactive metabolite cortisone. Four human aldo–keto reductases (AKR1c1–AKR1c4), also named hydroxysteroid (HSDHs) and dihydrodiol dehydrogenases (DHDHs), are involved in the metabolism of steroids in addition to drugs. These enzymes catalyze the conversion of aldehydes and ketones into their corresponding alcohols using NADH and/or NADPH as cofactors (Table 1.3) [45]. In humans, the aldo–keto reductase superfamily also includes aldehyde (AKR1a1) and aldose reductase (AKR1b1) and aldose reductase-like proteins AKR1b10 of the small intestine and AKR1b15, δ(4–3)-ketosteroid 5β-reductase AKR1d1, aldo–keto reductase-1C-like proteins AKR1cL1 and AKR1cL2 (or testis-specific AKR1e2), and aflatoxin aldehyde reductases AKR7a2 and AKR7a3 [46].

(MPTP), which lodges at contact sites between the OMM and the inner mitochondrial membrane (IMM) [44]. Tspo thus participates in mitochondrial cholesterol and protein import, cell proliferation, and apoptosis. Translocator protein clusters owing to ATP and the cytosolic chaperone HSP90 and is imported as 66-kDa heteropolymers with metaxin-1, VDAC1, and nonspecific lipid-transfer protein [44]. At the OMM–IMM interface, Tspo forms 800-kDa mitochondrial complexes with VDAC1, VDAC3, ANT, ApoA1, ApoA2, fatty acid synthase, annexin-A2, and mitofilin. The transduceosome is composed of the cytosolic proteins star, PKA, and acylCoA-binding domain-containing protein ACBD3 (Golgi complex-associated protein GoCAP11, Golgi bodyresident protein GCP60, or peripheral benzodiazepine receptor [PBR]- and PKA-associated protein PAP7), and OMM proteins Tspo and VDAC1. 9 A sulfonate group (sulfur trioxide moiety [SO− ]) can be transferred rather than a sulfate group 3 (sulfation). Sulfation means that esters or salts of sulfuric acid (sulfates) are formed. Sulfonation refers to attachment of the sulfonic acid group (–SO3 H) to a carbon in an organic compound by sulfotransferases using phosphoadenosine phosphosulfate as donor. However, sulfation is also defined either by the replacement of a hydrogen atom of an organic compound with a sulfate group (–OSO2 OH) and sulfonation by the replacement of a hydrogen atom of an organic compound with a sulfonic acid group (–SO3 H).

1.1 Vasculopathies and Vasculitides

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Table 1.3 Aldo–keto reductases (AKRs) in steroid hormone metabolism (Source: [47]) Gene Chromosomal locus AKR1C1 10p14–p15

AKR1C2 10p14–p15 AKR1C3 (17β HSDH5) 10p14–p15

AKR1C4 10p14–p15 AKR1D1 7q32–q33

Protein Reaction AKR1c1, 20α-hydroxysteroid dehydrogenase Progesterone → 20α-hydroxyprogesterone; 5α-dihydrotestosterone → 3β-androstanediol AKR1c2, type-3 3α-hydroxysteroid dehydrogenase 5α-Dihydrotestosterone → 3α-androstanediol AKR1c3, type-2 3α-hydroxysteroid dehydrogenase, type-5 17β-hydroxysteroid dehydrogenase, prostaglandin-F synthase Δ4 -Androstenedione → testosterone; PGh2 → PGf1α ; PGd2 → 11β PGf2 AKR1c4, type-1 3α-hydroxysteroid dehydrogenase 3-ketosteroid → 3α-hydroxysteroid AKR1d1, steroid 5β-reductase Δ4 -3-ketosteroid → 5β-dihydrosteroid

Enzymes of the AKR set are soluble, monomeric, NAD(P)(H)-dependent oxidoreductases that interconvert carbonyl groups with alcohols. In humans, 13 isoforms exist, among which AKR1c1 to AKR1c4 and AKR1d1 regulate the local concentration of steroid hormones. The members of the AKR1C subset (AKR1c1–AKR1c4) reduce ketosteroids to hydroxysteroids

AKR1c1 converts progesterone to inactive 20α-dihydroxyprogesterone, AKR1c2, and liver-specific AKR1c4 dihydrotestosterone to less active 3α-diol, and AKR1c3 catalyzes reduction of prostaglandins PGd2 and PGh2 , and oxidation of (9α, 11β)PGf2 to PGd2 in addition to preferentially transforming androstenedione to testosterone [45]. AKR1c1, AKR1c2, and AKR1c3 reduce cytotoxic aldehydes derived from lipid peroxidation into less toxic metabolites. The liver is the primary site of metabolism of steroid hormones containing a Δ4 -3 functionality, such as testosterone and progesterone, which are converted into tetrahydrosteroids that are then eliminated. Steroid hormones are conjugated in twophase reactions, reduction by 5α- or 5β-steroid reductases to form the respective dihydrosteroids, and, in the subsequent step, the 3-oxo group of dihydrosteroids is reduced by ketosteroid reductases to form tetrahydrosteroids. In humans, the four members of the AKR1C subset (AKR1c1–AKR1c4) reduce 5α- and 5βdihydrosteroids [48]. The AKR1C isozymes are thus involved in the metabolism of testosterone and progesterone. They are pluripotent, but with a cell-specific expression pattern and distinct substrate preference. All four isozymes are produced in the liver. AKR1c1 to AKR1c3 are highly expressed in the mammary gland and prostate but distinctly expressed in the lung, mammary gland, prostate, and testis, whereas AKR1c4 is specific to the liver [48]. In particular, AKR1c3 is detected in stromal, endothelial, and uroepithelial cells, in addition to adenocarcinoma cells in the prostate [47]. Prostate epitheliocytes produce higher concentrations of AKR1c1 to AKR1c3 than stromal cells, the

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1 Cardiovascular Disease: An Introduction

synthesis rate augmenting in prostate cancers. In the mammary gland, AKR1c3 creates a pro-estrogenic state, as it converts androstenedione to testosterone, which, upon aromatization by CyP19 aromatase, yields 17β-estradiol, and transforms active progesterone to inactive 20α-hydroxyprogesterone, thereby altering the estrogen/ progesterone ratio [47]. The AKR1C enzymes catalyze ketosteroid reduction at the 3-, 17-, or 20-position to varying degrees according to the substrate. The 5β-pathway is linked to 5βsteroid reductase AKR1d1. 5β-Pregnane (3,20)-dione is a potent ligand for the pregnane X receptor (PXR or NR1i2) and constitutive androstane receptor (CAR or NR1i3). Activated hepatic NR1i3 stimulates cytochrome-P450 CyP3a4, which processes approximately 50% of consumed drugs [48]. In addition, 5β-reduced pregnanes are neuroactive steroids (synthesized in the brain) that are implicated in vasodilation [49]. They are implicated not only in the regulation of steroid receptors, exerting their action on gene expression via nuclear steroid hormone receptors, but also of ligand-gated ion channels, thereby influencing neuronal excitability. They inhibit or stimulate neurotransmission, as they act as allosteric modulators of the GABAA receptor. Unsaturated fatty acids (FAs) are potent competitive inhibitors of the AKR enzymes. The sensitivity of AKRs for FAs varies, and the most potent inhibitors for AKR1c1, AKR1c2, and AKR1c4 are docosahexaenoic, palmitoleic, and linoleic acid, respectively [45]. FAs have the strongest inhibitory potency for 3αhydroxysteroid dehydrogenase AKR1c3. Sulfate conjugation is involved in the transformation of steroid and thyroid hormones, catecholamines, cholesterol, and bile acids, in addition to the detoxification of dietary and environmental xenobiotics. Cytosolic sulfotransferases (SulTs) transfer the sulfonate group from phosphoadenosine phosphosulfate (PAPS) to acceptor substrates. In humans, 13 SulT isoforms constitute four subsets [50]. The SULT1 subfamily encompasses SulT1a1 to SulT1a3, SulT1b1, SulT1c2 to SulT1c4, and SulT1e1, and the SULT2 subfamily SulT2a1, SulT2b1a, and SulT2b1b, whereas other subfamilies contain a single element, SulT4a1 and SulT6b1, respectively. Splice variants encode distinct SulT1c3 isoforms (SulT1c3a, SulT1c3c, and SulT1C3d) [50]. Although SulT1c3a has a weaker activity and is specific for hydroxyl-chlorinated biphenyls, SulT1c3d has a broader substrate specificity, sulfating bile acids, thyroid hormones, pyrenes, and hydroxyl biphenyls.

1.1.3 Vasculitis (Angiitis) Vasculitides are defined by the presence of inflammatory leukocytes in vascular walls caused by various immunological processes and possibly triggered by infectious agents. Vasculitis targets arterial and venous walls of any size in any organ, but frequently in the skin. Vasculitides can be classified according to the size of blood vessels or histological examination (e.g., lymphocytic, leukocytoclastic, and granulomatous [nodular]).

1.1 Vasculopathies and Vasculitides

15

Giant cell (GGA) and young women (Takayasu [TA]) arteritis10 and autoinflammatory Behçet disease (BD)11 affect large vessels. Complications include inflammatory obstructions and aneurysms. Livedoid vasculitis, also named segmental hyalinizing vasculopathy and livedo reticularis, most commonly affects women with thromboses and ulcerations of the lower extremities. Eosinophilic granulomatosis with polyangiitis (EGPA), previously called Churg– Strauss syndrome, mainly affects small and medium-sized blood vessels of men and women between 30 and 45 years of age. It commonly targets the lung and skin but also the heart, kidney, bowel, and nerves. Granulomatosis with polyangiitis, previously named Wegener’s granulomatosis, mainly affects blood vessels in the nose, sinuses, ears, lungs, and kidneys of middleaged or elderly individuals.

1.1.4 Vascular Wall Disorders Wall disorders in large arteries and veins appear not only in the presence of risk factors, such as smoking, long periods without bodily motion, hypertension (Chap. 3), diabetes (Chap. 4),12 obesity (Chap. 5), and a family history of vasculopathies, but also most often in a context that encompasses aging (Vol. 9, Chap. 3. Aging), one of the most important cardiovascular event predictors, injury, ciliopathies (Vol. 9, Chap. 1. Ciliopathies), replication stress, air pollution, and sleep disorders (Vol. 9, Chap. 4. Anomalies of the Respiratory Tract), among other factors.

1.1.4.1

Hemostasis and Thrombosis

The intact and healthy vascular endothelium maintains an anticoagulant surface. Thrombomodulin is an integral membrane protein on the wetted surface of endotheliocytes that serves as a cofactor for thrombin. Once it is bound to thrombin, the anticoagulant serine peptidase protein-C is rapidly activated [52]. Activation of protein-C by the thrombin–thrombomodulin complex depends on Ca2+ ion. This 10 Takayasu

arteritis affects arteries exiting from the heart and their main branches.

11 Behçet disease is characterized by ulcers of the mouth and genital organs, skin lesions, and ocular

anomalies. 12 Diabetes

mellitus is characterized by an altered production or response to insulin, provoking abnormal metabolism of carbohydrates and hyperglycemia. 1. In type-1 diabetes mellitus (T1DM), the body lacks pancreatic insulin-producing β cells. Patients with T1DM have a three- to five-fold elevated CVD risk [51]. 2. In type-2 diabetes mellitus (T2DM), which is more common and often develops later in life, cells fail to respond to insulin. Diabetes insipidus is characterized by an impaired secretion of or response to vasopressin.

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1 Cardiovascular Disease: An Introduction

complex also prevents thrombin activation of the clotting factor-V . Thrombomodulin thus has two distinct anticoagulant functions: (1) to impede the ability of thrombin to clot fibrinogen and to activate FV and (2) to accelerate activation of the anticoagulant protein-C [52]. Heparan sulfate proteoglycans on the endotheliocyte surface stimulate activation of the serine peptidase inhibitor antithrombin, or serpinC1, which inactivates thrombin and factor-Xa [53]. In addition, the endothelium produces the antiplatelet aggregation factors prostacyclin and nitric oxide. Some types of activated platelets also generate NO, thereby stimulating the sGC–PKG axis and limiting their own adhesion and aggregation [54]. PKG phosphorylates vasodilator-stimulated phosphoprotein (VASP), preventing integrin-α2B β3 activation, which stabilizes initial platelet rolling, adhesion, and recruitment to the site of the injury. PKG also represses integrin-α2B β3 activation via IP3 R-associated cyclic guanosine monophosphate (cGMP) kinase substrate (IRAG) and inhibition of thromboxane receptor activation. When any segment of the vasculature is damaged, the subendothelial matrix is exposed to blood. Matrix components launch hemostasis, initiating formation of a blood clot composed primarily of platelets and fibrin within seconds. Hemostasis stops bleeding from a damaged blood vessel, thereby avoiding hemorrhage, a normal blood flow being maintained elsewhere in the circulatory circuit. This first stage of wound healing involves blood coagulation, blood, a suspension of cells in plasma, in which molecules are suspended, changing from a liquid to gel, that is, by the local formation of a hemostatic plug. Primary hemostasis refers to aggregation of activated platelets, which are small anuclear cell fragments derived from megakaryocytes, and subsequent platelet plug production. In humans, platelets form subpopulations according to the presence and absence of NOS3, which produces NO, an endogenous platelet inhibitor [54]. Approximately 20% of platelets lack NOS3 and thus fail to produce NO and have defective sGC–PKG signaling. NOS3− platelets primarily initiate adhesion to collagen or von Willebrand factor; activate integrin-α2B β3 , which elicits between-platelet aggregation; and secrete MMP2, which elicits recruitment of NOS3+ platelets to the forming aggregate. Conversely, platelets with intact NOS3–sGC–PKG signaling form the bulk of the aggregate (thrombus) owing to their higher PGhS1 (COx1) content and greater thromboxane-A2 generation, the platelet aggregate being amplified by thromboxane-A2 synthesis, and ultimately limit the aggregate size via NO. Secondary hemostasis designates the simultaneous deposition of insoluble fibrin generated by the proteolytic coagulation cascade that forms a meshwork into and around the platelet plug, which strengthens and stabilizes the blood clot.

1.1 Vasculopathies and Vasculitides

17

Hemostasis relies on the balance between procoagulant (platelets and coagulation cascade components) and anticoagulant elements (protein-C and -S, fibrinolysis, serpins). As a blood clot in hemostasis, a pathological thrombus is the final product of blood coagulation in the absence of vascular contusion (but not intrinsic vascular injury). An elevated ratio of NOS3+/NOS3− platelets may contribute to thrombosis. Thromboembolism results from thrombus breakage and shedding followed by embolus carriage in the bloodstream, and subsequent obstruction of a distal vessel. • Venous walls can be injured and lose their strength, thereby being the source of thrombi that are shorn and generate emboli (Vols. 12, Chap. 4. Thrombosis and Lymphedema, and 13, Chap. 8. Venous Pathologies). Venous thrombus consists mostly of fibrin with entrapped red blood capsules. Venous thrombi can cause pulmonary embolism. Venous thromboembolism (VTE) is a collective name incorporating deep vein thrombosis (DVT) and pulmonary embolism. • Arterial thrombus subjected to a higher flow rate and shear is mainly composed of aggregated platelets. Arterial emboli most often provoke ischemia and infarction of the heart, brain (stroke), gastrointestinal tract, kidney, or leg. Obesity and dyslipidemia are risk factors for both arterial and venous thrombosis. The classical acquired risk factors for venous thrombosis include cancer, immobilization, surgery, fractures, and pregnancy. Neutrophils contribute to host defense, not only as they process pathogens via phagocytosis and produce toxic chemicals to kill intruders directly but also as dying neutrophils mix their DNA with toxic components from their cytosolic granules and release them in the form of neutrophil extracellular traps (NETs) that trap and neutralize microbes. Neutrophil extracellular traps are lattices of processed chromatin (i.e., neutrophil DNA and histones) linked to secreted and cytoplasmic proteins released by neutrophils during inflammation. However, inappropriate NETosis is harmful, favoring sustained and excessive inflammation and thrombosis. NETs released into the vasculature can cause platelet adhesion and activation of the extrinsic and intrinsic coagulation cascade. They also damage pulmonary epithelia and endothelia. On the other hand, two deoxyribonucleases, Dnase1 and Dnase1L3, degrade extracellular (cell-free) nuclear and mitochondrial DNA, hence circulating NETs in a partly redundant manner [55]. However, Dnase1 disrupts NETs, but does not dissolve them. The receptor tumor necrosis factor receptor superfamily (TNFRSF)-interacting protein kinase RIPK3 is involved not only in inflammation in addition to apoptosis and necroptosis but also in hemostasis, as it amplifies platelet activation. Upon vessel injury, platelets are recruited by adenosine diphosphate (ADP), thrombin, and thromboxane-A2 , which connect to their cognate G-protein-coupled receptors and activate integrin inside–out signaling mediated by extracellular signal-related kinases (ERKs) and launch granule secretion. RIPK3 produced in platelets interacting with G13 activates PKB and supports platelet aggregation and spreading on fibrino-

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1 Cardiovascular Disease: An Introduction

gen via PKB1 or PKB2 in addition to the second wave of dense granule content secretion in response to thrombin, thromboxane-A2 , and clot retraction [56]. The G13 subtype selectively enables thrombin- and TxA2 -induced platelet aggregation, but does not influence ADP-primed aggregation. RIPK3 operates independently of its substrate used in cell necrosis and clearance, mixed lineage kinase domain-like pseudokinase (MLKL). Therefore, RIPK3 favors arterial thrombus formation. On the other hand, heparin, a sulfated polysaccharide, prevents blood coagulation, as it connects to antithrombin (serpin-C1) and then accelerates the interaction of antithrombin with thrombin (FI I a), and activated clotting factors FV I I a and FI Xa to FXI I a, thereby preventing completion of the coagulation cascade.

1.1.4.2

Inflammation and Angiogenesis

Inflammation of the vascular wall is initiated in response to injury, infection, and lipid peroxidation. Moreover, hypertension (Chap. 3), obesity (Sect. 5.3.3), and diabetes (Chap. 4) are associated with chronic inflammation. Elevated concentrations of inflammatory markers predict future cardiovascular events [57]. Hypertension is linked to both macro- and microvascular disease. It alters endothelial integrity and hence vascular permeability, facilitating inflammatory leukocyte recruitment. In addition to vasoconstriction, angiotensin-2 causes redox stress, inflammation, endothelial dysfunction, and vascular remodeling with fibrosis. It provokes accumulation of PTPRc+ leukocytes in aortic perivascular adipose tissue and upregulates MMP2 expression in these leukocytes, MMP2 favoring Agt2-primed vascular inflammation and injury [58]. Agt2 augments the generation of ROS in the aortic media and perivascular medium and of vcam1 and CCL2, thereby eliciting perivascular infiltration of monocytes, macrophages, and T lymphocytes. It also increases the density of monocytes and activated CD4+ helper and CD8+ cytotoxic T cells in the spleen in the presence of MMP2. MMP2 is synthesized in higher amounts in CD4+ effector TH1 cells than in TH2 or naive TH0 cells. In addition, TH1 cells can stimulate MMP2 synthesis in macrophages. Within the cell, Agt2 promotes phosphorylation of EGFR in addition to ERK1 and ERK2 via heparin-binding epidermal growth factor (HBEGF) shedding in vascular smooth myocytes [58]. Matrix metallopeptidases synthesized in vascular smooth muscle and endothelial cells not only modify and remodel the extracellular matrix, degrading matrix constituents (e.g., collagen, elastin, and fibronectin), but also shed growth factors (e.g., HBEGF and matrix-bound latent transforming growth factor-β), cytokines, and chemokines, hence favoring inflammation, in addition to autacoids (e.g., big endothelin-1 and other vasoactive peptides) [58]. In particular, MMP2 cleaves (activates) CCL7 and CXCL12 and processes S100 [59]. The MMPs are regulated by tissue inhibitors of metallopeptidases (TIMPs), which impede their activity, as they bind to their catalytic site. Among the four TIMPs (TIMP1–TIMP4), TIMP2 can inhibit or activate MMPs; TIMP2 is

1.1 Vasculopathies and Vasculitides

19

required with MMP14 for proMMP2 activation [58]. In addition to MMP2, other MMP types may participate in Agt2 action. On the other hand, MMP2 deficiency reduces Agt2-induced redox stress, inflammation, endothelial dysfunction, medial hypertrophy, and vascular stiffness, but not SBP elevation [58]. Both vascular and immune cell-derived MMP2 contribute to impaired vascular relaxation to acetylcholine and endothelial dysfunction. Immunocytes contribute to Agt2-induced hypertension, as Mmp2 deletion in immunocytes reduces BP [58]. Atherosclerosis (Sect. 1.1.5 and Vol. 13, Chap. 5. Atherosclerosis—Biological Aspects) can be considered as a diffuse inflammatory disease of the vasculature. Inflammation is indeed observed at all stages of atherogenesis, from initial lesions to fatty streaks, evolved plaques, and end-stage complications, that is, thromboembolism after unstable plaque rupture linked to an excess inflammatory episode [60]. Atherosclerosis is triggered by oxidized LDLs conveying cholesterol. Activated endotheliocytes express adhesion molecules for the diapedesis of circulating leukocytes, activated macrophages, lymphocytes, and smooth myocytes releasing cytokines and chemokines. The procoagulant cytokine increases the synthesis and secretion of fibrinogen, plasminogen activator inhibitor PAI1 (serpinE1), and acute phase proteins such as C-reactive protein (CRP), thereby amplifying the inflammatory and procoagulant response [61]. Inflammatory cytokines (e.g., IL1, TNFSF1, and CRP) induce the formation of adhesion molecules, provoking a vicious cycle. C-reactive protein supports the production of tissue factor by monocytes and represses that of NO, hence contributing to the creation of a proinflammatory and prothrombotic milieu. Anti-inflammatory drugs can reduce cardiovascular risk [62]. Furthermore, systemic autoimmune rheumatic diseases (SARDs),13 that is, a group of disorders that share chronic inflammation causing connective tissue and organ damage (rheumatoid arthritis [RA], systemic lupus erythematosus [SLE], ankylosing spondylitis, gout, psoriatic arthritis, systemic sclerosis [SSc; or scleroderma], polymyositis [PM], dermatomyositis [DM], Sjögren’s syndrome [SjS],14 mixed connective tissue disease [MCTD],15 and systemic vasculitis), can be associated with medium- and large-vessel vasculitides (granulomatous and microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis, and giant cell arteritis)

13 In

autoimmune diseases, immunological tolerance of the body’s cells is lost and hence the immune system, which is aimed at identifying and destroying foreign invaders attacking target cells. A genetic susceptibility predisposes the immune system to defective immunological tolerance. The genetic marker HLAdr4 increases the risk for developing rheumatoid arthritis. An environmental trigger (e.g., viruses, smoking) initiates the disease. Angiogenesis participates in the genesis of rheumatoid arthritis and other inflammatory diseases. 14 Sjögren’s syndrome affects the lachrymal and salivary glands, thereby drying the mouth and eyes. 15 A rheumatic overlap syndrome with anti-RNP antibodies (i.e., abnormally high concentrations of antibodies against U1 small nuclear ribonucleoprotein) and characterized by arthritis and often myositis, pulmonary hypertension, and interstitial lung disease.

20

1 Cardiovascular Disease: An Introduction

and an increased risk of premature cardiovascular disease, in particular coronary arteritis and premature atherosclerosis [63]. Angiogenesis is not only involved in organogenesis and repair but also in inflammatory diseases (at least in RA, SLE, SSc, and vasculitides) [64]. This programmed cascade of events relies on cellular (monocytes, macrophages, and endotheliocytes) and molecular mediators and inhibitors (angiostatin, endostatin, osteonectin [SPARC], thrombospondin, and, under some circumstances, TGFβ; cytokines IL1, IL4, IL6, Ifnα, and Ifnβ; and chemokines CXCL4, CXCL9, and CXCL10). Angiogenic factors, such as growth factors (EGF, FGF1, FGF2, HGF, IGF1, PDGF, TGFβ, and VEGF), cytokines (TNFSF1, IL1, IL6, IL13, IL15, and IL18), chemokines (CCL2, CXCL1, CXCL5, CXCL7, CXCL8, CXCL12, and CX3 CL1), cell adhesion molecules (endoglin, integrins, selectins, pecam1, and vcam1), matrix components (collagen-1, fibronectin, laminin, and heparan sulfate proteoglycans), and other factors (angiogenin, platelet-activating factor, substance-P, prostaglandinE2 , and prolactin]) activate endotheliocytes. Endotheliocytes then produce matrix metallopeptidases and plasminogen activators to degrade their basement membrane and the perivascular extracellular matrix. These cells proliferate and migrate, forming a sprout that grows, tubulates, matures, and anchors onto another vessel or builds a capillary network, the endotheliocytes producing further generations of sprouts from the primary sprout. Mastocytes are involved in innate and acquired immunity, inflammation, allergy, and autoimmunity. They release histamine, tumor-necrosis factor TNFSF1, interleukins IL1β and IL6, chemokine CXCL8, and VEGF. Proinflammatory substanceP and IL33, two major agents of diseases, cooperate to enhance TNFSF1 synthesis and secretion from mastocytes via activation of the tachykinin (neurokinin/ substance-P) receptor TacR1 (NK1R or SPR) and IL1RL1 (IL33R) [65]. Owing to this mutual excitation, IL33 potentiates SP-primed TNFSF1 production more than 100-fold in mastocytes. Mastocyte-derived tryptase can cleave extracellular IL33 into its mature active form, which then activates mastocytes, which, in turn, can release soluble IL1RL1 that modulates the effects of IL33. Substance-P also stimulates histamine secretion from mastocytes. Moreover, IL33 and SP upregulate synthesis of both TacR1 and IL1RL1 receptors. In addition, IL3 enhances SPtriggered VEGF release by mastocytes [66]. IL33 also augments the frequency and magnitude of mastocyte degranulation and chemokine production, worsening chronic inflammation, even at low concentrations. The receptors TacR1 and IL1RL1 interfere; TacR1 complexes with IL1RL1 and its coreceptor IL1RAP (IL1R accessory protein); IL33 may participate in complexing TacR1 and IL1RL1 [65]. The stem cell factor receptor (SCFR) also complexes with IL1RL1 and IL1RAP in mastocytes for cross-activation. The natural flavonoid tetramethoxyluteolin inhibits mastocytes stimulated by IL33, SP, or their combination, thereby reducing chronic inflammation.

1.1 Vasculopathies and Vasculitides

1.1.4.3

21

Oxidative and Nitrosative Stresses

Accrual amounts of ROS, which are toxic by-products of aerobic metabolism, cause redox stress and alleviate the fitness level and ability to maintain homeostasis. The term oxidative stress was coined by H. Sies as “a disturbance in the prooxidant– antioxidant balance in favor of the former.” The rate of ROS production increases with aging, ROS being responsible for the accumulation of cellular and tissular deterioration over time in the postreproductive phase of life [67]. Injurious oxidative stress is characterized by a shift in the oxidative–reductive balance to a more oxidative state because of augmented ROS production by prooxidant enzymes and reduced antioxidant defense mechanisms that scavenge excess ROS. Deleterious reductive stress is characterized by an aberrant increase in reducing equivalents, such as reduced glutathione and reduced NADPH, increased activation of antioxidant enzymes, and reduced prooxidant capacity, shifting the redox balance from an oxidative to a reduced state. Exercise is an oxidant stimulus used in redox biology studies; free radicals produced during exercise modulated muscular and systemic adaptation to physical activity. However, exercise induces oxidative or reductive stress according to the individual [68]. Using redox markers (e.g., glutathione, F2-isoprostanes, and protein carbonyls) in plasma, red blood capsules, and urine samples before and 2 days after exercise, concentrations of the oxidant markers, F2-isoprostanes and protein carbonyls, increase or decrease, whereas the amount of glutathione amount declines or rises, respectively.16 The term redox stress, which is associated with the oxidation–reduction reaction disorder, combines oxidative and reductive stress. Both contribute to the pathogenesis of CVD; hence, redox stress is the preferred term. Loss of function of glutathione peroxidase GPOx1 causes both oxidative and reductive stress. Reductive stress provokes S glutathionylation of the cytoplasmic protein Tyr phosphatase PTPn1 (SHP2) and vascular remodeling [69].

Reactive Oxygen and Nitrogen Species All layers of the vascular wall produce ROS and reactive nitrogen species (RNS; Vol. 11, Chap. 7. Reactive Oxygen and Nitrogen Species) that include superoxide • anion radical (O•− 2 ), hydrogen peroxide (H2 O2 ), hydroxyl radical (OH ), nitric • − oxide (NO ), and peroxynitrite (ONOO ). Superoxide can be converted by superoxide dismutase (SOD) into hydrogen peroxide. Hydroxyl radical is formed from oxidation of glutathione, ascorbic acid,

16 The

reduced (GSH ) and oxidized glutathione (glutathione disulfide [GSS G]) redox couple is the traditional marker of oxidative stress. F2-isoprostanes serve as the reference marker of oxidative damage.

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1 Cardiovascular Disease: An Introduction

NADPH, hydroquinone, catechol, and riboflavin by hydrogen peroxide and H2 O2 catalysis [70]; glutathione can scavenge it [71]. Oxy- and methemoglobin can generate hydroxyl radicals from hydrogen peroxide [72]. Superoxide reacts rapidly with anti-inflammatory, anticoagulant, and vasodilatory NO, forming the oxidant peroxynitrite. The latter oxidizes tetrahydrobiopterin, a NOS cofactor, lowering NO availability and provoking endothelial dysfunction. Among ROS and RNS, hydroxyl radical and peroxynitrite are not considered signaling molecules; these highly reactive agents contribute to redox stress and tissular damage.

ROS and RNS Sources The main sources of vascular ROS comprise: 1. NAD(P)H oxidases (NOx1–NOx2 and NOx4–NOx5) 2. Mitochondrial electron transport chain (ETC) involved in oxidative phosphorylation, mainly ETC complex-I and -I I I (i.e., NADH–ubiquinone and ubiquinone– cytochrome-C reductase) 3. Uncoupled nitric oxide synthase and to a lesser extent 4. Xanthine oxidase 5. Prostaglandin-G/H synthases (cyclooxygenases) 6. Lipoxygenases 7. The endothelial cytochrome-P450 epoxygenase CyP2c9, which produces vasodilatory epoxyeicosatrienoic acids (11,12)EETs [73] 8. Myeloperoxidase17 Mitochondrial Electron Transport Chain In the heart, ROS are produced primarily by mitochondrial ETC and mitochondrial and extramitochondrial enzymes, such as NOxs. Superoxide produced in the mitochondrial matrix is rapidly dismutated by SOD2 to hydrogen peroxide, which diffuses out of the mitochondrion. NAD(P)H Oxidases Enzymes of the NOX set synthesize O•− 2 , except NOx4, which predominantly produces H2 O2 . They localize to caveolae and membrane rafts, endoplasmic reticulum, endosomes, and mitochondria. Constitutively active canonical NOx1 in addition to NOx2 function in a complex formed by their regulators and binding partners: 17 Myeloperoxidase

abounds in granules of activated neutrophils, monocytes, and macrophages. It converts hydrogen peroxide to hydroxyl radical (OH• OH), peroxynitrite, hypochlorous acid (HOCl), and nitrogen dioxide (NO•2 ).

1.1 Vasculopathies and Vasculitides

23

• NOx1: P22PhOx–NoxO1–NoxA1–Rac1/2 • NOx2: P22PhOx–NOxO2–NOxA2–P40PhOx–Rac1/2 NOx4 remains constitutively active in the presence of oxygen. Calcium-dependent NOx5 is regulated by cytosolic calcium concentration. Although NOx2 and NOx4 produce ROS in CMCs and fibroblasts, NOx1, NOx4, and NOx5 operate in the vascular smooth myocytes [71]. The NOx complexes produce O•− 2 on the extracytoplasmic face of cellular membranes, that is, plasma membrane-bound NOxs outside the cell and intracellular NOxs in the lumen of organelles [74]. The NOx4 subtypes in the outer mitochondrial membrane and mitochondrial ETC are major ROS sources in diabetes. NOx4 is involved in migration and differentiation of vascular smooth myocytes, cardiac cells, fibroblasts, and stem cells. Upon TGFβ exposure, NOx4 oxidizes (inhibits) the phosphatase DUSP1 (MAPK phosphatase MKP1), inactivating P38MAPK, which phosphorylates SRF, which binds to MRTF, activating smooth muscle α-actin (Actα2) and promoting vSMC differentiation [71]. In addition, NOx4 activates RhoA. Xanthine Oxidoreductase (Dehydrogenase/Oxidase) Xanthine dehydrogenase (XDH) and oxidase (XOx) are interconvertible forms encoded by a single gene, the XDH (XOR) gene. Whereas XOx uses hypoxanthine or xanthine as substrate and O2 as cofactor (electron acceptor) to produce superoxide and uric acid, XDH acts on the same substrates but utilizes NAD+ as cofactor (electron receptor) to produce NADH [75]. Hypoxia, inflammation, apoptosis, and ROS generation from other sources cause XDH conversion into XOx [71]. Xanthine oxidase participates in the cellular redox status. It is a source of oxygen radicals in granulocytes and endothelial, epithelial, and connective tissue cells. It is involved in detoxification of aldehydes. It serves as a messenger in the activation of neutrophils and T lymphocytes and the triggering of defense mechanisms rather than as a free radical generator. However, it can be implicated in cytotoxicity and tissue injury, especially in inflammation and ischemia. Lipoxygenases Arachidonate lipoxygenases ALOx5, ALOx12, and ALOx15 are implicated in CVD genesis. Arachidonic acid (AA) is oxidized by ALOxs into hydroperoxides, which are further reduced into hydroxides and leukotrienes. Each ALOx subtype generates different metabolites according to the target AA carbons. Arachidonate lipoxygenases mediate Agt2-primed NOx activity in vSMCs. Synthesis of ALOx5 is upregulated by redox stress following excess ROS formation by NOx or mitochondrial ETC. It generates 5HETE and LTa4 from AA, which serves as a substrate for several enzymes producing proinflammatory molecules LTb4 , LTc4 , LTd4 , and LTe4 , which activate endotheliocytes, macrophages, neutrophils, mastocytes, T lymphocytes, in addition to foam cells [71].

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1 Cardiovascular Disease: An Introduction

Both ALOx12 and ALOx15 are involved in inflammation and redox stress. Generated AA metabolites 12HPETE and 15HPETE and their reduced 12HETE and 15HETE are pro- and anti-inflammatory. They oxidize LDLs [71]. The metabolite 15HETE favors ROS creation by mitochondrial ETC and NOx4. Under hypoxia, 15HETE provokes endotheliocyte migration and pulmonary arterial smooth myocyte proliferation via P38MAPK activation, hence favoring pulmonary vascular remodeling and pulmonary hypertension. NOx4 is associated with ALOx12 and ALOx15 activity in diabetic hearts. Myeloperoxidase Myeloperoxidase (MPOx), encoded by the MPO gene, which produces a singlechain precursor, subsequently cleaved into a light and heavy chain that tetramerize. It is stored in large quantities in neutrophils, constituting a major component of neutrophil azurophilic granules, and, to a lesser extent, in monocytes and macrophages. This heme protein is synthesized during myeloid differentiation. This microbicide is an element of the host immune defense. It also influences endothelial function. Myeloperoxidase catalyzes H2 O2 and halide or semihalide ions reactions that produce hypohalous acids,18 such as hypochlorous acid (HOCl− ) and hypothiocyanous acid (HOSCN), a potent microbicide [71]. HOCl− reacts mainly with nitrogen and sulfur atoms in cysteine residues, especially glutathione, Cys oxidation inactivating or activating cellular molecules. Myeloperoxidase is involved in redox stress and inflammation; it can serve as a marker of atherosclerotic plaque instability [76]. Glutathione sulfonamide, the product of GSH oxidation primarily by HOCL− , can serve as a marker of MPOx damage. Smoking engenders high amounts of thiocyanate (SCN− ),19 a small 18 Hypohalous

acids are oxoacids of halogens (e.g., bromine [Br], chlorine [Cl], fluorine [F], and iodine [I]), such as hypobromous, hypochlorous, hypofluorous, and hypoiodous acid (general formula HOX, where X is the halogen atom). Hypohalites are any salts of hypohalous acids (general formula M(OX)N ). 19 Many types of peroxidases utilize sodium voltage-gated channels (SCNs), such as eosinophil (EPOx), gastric (GaPOx), salivary (SPOx; or secreted lactoperoxidase [LPOx]), and thyroid peroxidase (TPOx), in addition to MPOx. These enzymes generate HOSCN via a two-electron halogenation. Thiocyanate is detected at various concentrations (0.01–3 mmol/l) in extracellular fluids (plasma, saliva, airway surface fluid, milk, tears, and gastric juice) [77]. Airway SCN is concentrated from the plasma pool via its active transport through the basolateral sodium–iodide symporter (SLC5a5 or NIS) and apical anion channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR) in addition to cytokine-regulated channels SLC26a4 (pendrin), an electroneutral halide exchanger, and anoctamin-1 (transmembrane protein TMem16a), a Ca2+ dependent Cl− channel and halide transporter [77]. The SCN originates primarily from the diet, especially from glucosidic cyanogen-rich plants (e.g., cassava, linseed, maize, sorghum, sugar cane, and yam). It is also a product of glucosinolate metabolism in addition to N-conjugated thiocyanates and structurally related isothiocyanates (e.g., sulforaphane) [77].

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25

ubiquitous acidic pseudohalide thiolate that reduces H2 O2 by MPOx and increases quantities of hypothiocyanous acid [71]. The POx–SCN–H2 O2 axis is an element of host defense. Thiocyanate can act as an antioxidant, as it interacts with peroxidases and can protect cells against injurious redox damage via hypohalous acids such as HOCl and HOBr [77]. It ablates toxicity yielded by the MPOx–Cl− –H2 O2 axis at concentrations of 100–400 μmol/l in the nervous system and lungs, among other organs, in addition to endotheliocytes. It also detoxifies H2 O2 formed by the LPOx–SCN–glucose oxidase (GluOx) axis [77]. On the other hand, SCN can play a cytotoxic role. However, diseases associated with increased SCN and HOSCN amounts are also related to exposure to other toxic agents (e.g., cyanide, tobacco smoke, and cyanogenic glucosides), which can contribute to pathogenesis. Hypothiocyanous acid reacts with thiols, oxidizing Trp and damaging protein Tyr phosphatases, causing a hyperphosphorylation state within the cell, altering MAPK signaling, and launching apoptosis. It can also oxidize LDLs and HDLs, in addition to NO [71]. Cytochrome-P450 Enzymes of the cytochrome-P450 superfamily are involved in the oxidative metabolism of various xenobiotics using molecular oxygen and electrons supplied by CyP450 oxidoreductase (POR), also called NADPH–CyP450 reductase (CPR) [78].20 They insert an oxygen atom into a substrate. Processing by CyP450 enzymes is inefficient as the oxidation of substrates is associated with the production of varying proportions of superoxide and/or hydrogen peroxide. Three types of NADPH-dependent oxidations by microsomal CyP450 monooxygenases comprise [79, 80]: 1. Regio- and stereo-selective olefin epoxidation of arachidonic acid (epoxygenase reaction), which produces (5,6)-, (8,9)-, (11,12)-, and (14,15)-EETs by the cytochrome-P450 epoxygenases (HETEs)21 2. Arachidonic acid allylic oxidation (lipoxygenase-like reaction), which generates 5-, 8-, 9-, 11-, 12-, 15-hydroxyeicosatetraenoic acids (HETEs) 3. ω- and (ω-1)-Hydroxylation (at or near the terminal carbons [C16–C20]), which forms 16- (ω-4) to 20HETEs (ω) by AA ω- and (ω-1)-hydroxylases CyP1a1, CyP1a2, CyP4a11, and CyP4a22 Four EET regioisomers, (5,6)-, (8,9)-, (11,12)-, and (14,15)-EETs, operate as auto- and paracrine messengers. The prime vasodilation of EETs is via smooth myocytic large-conductance Ca2+ -activated K+ channel (big potassium

20 Catalytic

turnover requires electron transfer from NADPH to the P450 heme iron, a reaction catalyzed by the membrane-bound flavoprotein, POR. 21 In particular, membrane-bound, heme-containing cytochrome-P450 epoxygenases metabolize polyunsaturated FAs such as arachidonic acid to epoxide products such as (14,15)EET.

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1 Cardiovascular Disease: An Introduction

[BK]) [81].22 Activation by EETs of endothelial TRP channels and resulting Ca2+ influx is an alternative endothelial-derived hyperpolarizing factor. They also have an anti-inflammatory effect on blood vessels and promote angiogenesis via an EPHb4-coupled PI3K–PKB pathway or sphingosine kinase SphK1 [81]. They convert eicosapentaenoic acid into vasoactive epoxy derivatives and endocannabinoids, whereas soluble epoxide hydrolase (sEH) transforms EETs to dihydroxyeicosatrienoic acids (DHETs), attenuating many EET effects. Heme Oxygenases Membrane-bound heme oxygenases HOx1 and HOx2 catalyze the rate-limiting step of heme catabolism using molecular oxygen and electrons supplied by CyP450 oxidoreductase, converting heme to CO, biliverdin, and ferrous iron. Heme is a potent hydrophobic prooxidant that intercalates in membranes and mediates peroxidation of membrane phospholipids [78]. The HOx1 subtype is constitutively expressed in the liver, spleen, and bone marrow and is inducible in most organs by redox stress, heat shock, nutrient depletion, disrupted intracellular calcium homeostasis, exposure to cytotoxins, and proinflammatory stimuli [78]. It synthesizes the second messenger CO, a gaseous vasodilator, thereby protecting hepatic microcirculation subjected to redox stress, among other vascular beds. The HOx2 isoform resides in the brain, liver, spleen, and testis. Heme oxygenase HOx1 protects against redox stress, as it competes with CyP450 for binding to their common redox partner, CyP450 oxidoreductase, diminishing CyP450 action and associated ROS production [78]. Induction of HOx1 slows down the microsomal production rate by CyP1a2 of hydrogen peroxide and hydroxyl radical. In addition, oxidative injury caused by CyP2e1 is partly prevented by HOx1. Crosstalk Crosstalk exists among ROS sources. Hydrogen peroxide can activate NOx and induce xanthine dehydrogenase transformation into xanthine oxidase. Peroxynitrite induces superoxide production [71]. In addition, mitochondrial ETC and NOx can interact for mutual induction, elaborating an oxidative cycle. Hyperglycemia favors this interference.

22 Endothelial

cytochrome-P450 monooxygenases, such as CyP1a, CyP2b6, CyP2c, and CyP2j, oxidize arachidonic acid, enzymatic cleavage of molecular oxygen being followed by insertion of a single atom of oxygen into the substrate, whereas the remainder is released as water. These enzymes regulate the vasomotor tone via produced epoxy FAs such as vasodilatory (11,12)EET [82].

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27

Production of ROS partners depends on vessel location. Vascular smooth muscle and endothelial, immune, and other hematopoietic types of cells have different expression patterns for ROS-related proteins.

Redox Signaling At low concentrations, certain ROS, such as superoxide and hydrogen peroxide, are signaling mediators involved in redox signaling (or redox control). Intracellular signaling effectors stimulated by ROS encompass the MAPK module with ERK1, ERK2, and ERK4, protein Tyr kinases Src and Syk, and different redox-sensitive isoenzymes of the PKC set in addition to redox-sensitive transcription factors, such as AP1, ETS, HIF1, NFκB, and P53 [74, 83]. Hydrogen peroxide has a longer half-life than superoxide, and unlike superoxide, it can cross lipidic membranes by diffusion or transfer through aquaporins to initiate intracellular signaling [89]. Superoxide penetrates the cell through anion chloride channel ClC3 [74]. Superoxide and hydrogen peroxide can provoke cell growth, proliferation, and via oxidative activation of signaling molecules (e.g., PKB, Src, PLC, and MAPK) or inactivation of protein Tyr phosphatases [74]. At low concentrations, ROS regulate vascular smooth myocyte proliferation in addition to its contraction–relaxation state [84].

Antioxidant Defense Organisms use enzymatic and non-enzymatic antioxidant defense to prevent overload of highly reactive very short half-life free radicals. Redox-sensitive proteins are confined to signaling nanodomains in cells of the cardiovascular apparatus. Antioxidant protection consists of four sequential levels: preventive, chain-breaking, repairing, and adaptive [83]. (1) The first level of antioxidant defense involves enzymes, such as superoxide dismutases (SOD1–SOD3), glutathione peroxidases (GPOx1–GPOx8), and catalase. Extracellular SOD is produced by vSMCs (but not ECs). (2) The second level of defense, which involves vitamins C and E and probably carotenoids, prevents accumulation of secondary radicals produced in chain reactions such as lipid peroxidation. (3) The third level of defense corresponds to enzymatic prevention of the formation and removal of secondary radicals. Adaptation to stress relies on stress response linked to protein cysteine reduction– oxidation and launched by the transcription factors NFκB NFE2L2. ROS upregulate the formation of NFE2L2, which increases synthesis of numerous antioxidant enzymes. Upon redox stress, MAP3K5 operates in a ROS-induced cellular response. ROS mediate angiotensin-2-induced MAP3K5 activation. In unstressed cells, MAP3K5 homo-oligomerizes and forms the inactive MAP3K5–TRdx signalosome. Upon

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Table 1.4 Catabolism of reaction oxygen species (ROS) (Source: [71]; Cat catalase [encoded by the CAT gene], GPOxi type-i glutathione peroxidase [encoded by the GPX1–GPX8 genes], GsR glutathione reductase [encoded by the GSR gene], PRdxi type-i peroxiredoxin [encoded by the PRDX1–PRDX6 genes], TRdxRdi type-i thioredoxin reductase [encoded by the TXNRD1– TXNRD3 genes]) Enzyme GsR

Reactants GSS G NADPH GSH H2 O2 H2 O2 TRdxSH H2 O2 TRdxSS TRdx NADPH

GPOx1 Cat PRdx2 TRdxRd1

Products GSH NADP+ GSS G H2 O O2 , H2 O TRdxSS TRdx H2 O TRdxSH NADP+

Disulfide bridge characterizes the oxidized form (SS or S2 )

ROS stimulation, this signalosome liberates its inhibitor TRdx and forms a fully activated complex with TRAF2 and TRAF6 [85]. Antioxidants include superoxide dismutases, catalase, glutathione peroxidases (GPOxs), and the thioredoxin–thioredoxin reductase couple, which counterbalance ROS production (Table 1.4). Glutathione peroxidase, catalase, and peroxiredoxins catabolize hydrogen peroxide. Removal of hydrogen peroxide prevents formation of the highly reactive hydroxyl radical, which can be formed by the reaction of hydrogen peroxide with Fe2+ (Fenton’s reaction). In various intracellular antioxidant reactions such as H2 O2 removal, the reduced form of glutathione (GSH ) is oxidized into glutathione disulfide (GSS G), which can then be excreted from cells or reconverted to GSH by NADPHdependent glutathione disulfide reductase. Superoxide Dismutases Extracellular (SOD3), cytosolic copper- and zinc-(SOD1), and mitochondrial manganese-containing superoxide dismutase (SOD2) process O•− 2 into the messenger hydrogen peroxide and molecular oxygen, thereby preventing peroxynitrite formation (Table 1.5). Dismutation of O•− 2 into H2 O2 by SOD involves the reduction and re-oxidation of a redox active transition catalytic metallic ion, such as copper ox red 2+ (SOD with its oxidized [SODMI ] and reduced metal ion [SODMI ]: SODCu and + 3+ 2+ SODCu , respectively) and manganese (SODMn and SODMn ) [74].23

23 Oxidation

and reduction correspond to a loss and gain of electrons, respectively.

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29

Table 1.5 Superoxide dismutases in vascular walls (Source: [74]; Atox copper chaperone antioxidant, ATP7α copper-transporting ATPase-7α, CCS copper carrier and chaperone for superoxide dismutase, IMS intermembrane space of the mitochondrial envelope) Isoform SOD1 (homodimer) SOD2 (homotetramer) SOD3 (homotetramer)

Catalytic metallic ion Cu2+ Zn2+ Mn3+ Cu2+ Zn2+

Metal delivery CCS, GSH ND Atox1, ATP7α

Location Nucleus, cytosol, endosomes, lysosomes, peroxisomes, IMS Mitochondria matrix Cell surface, extracellular matrix and fluids

The SOD3 isozyme is anchored in the extracellular matrix via heparan sulfate proteoglycans, collagen, and fibulin-5

Catalase Catalase lodges principally in peroxisomes, H2 O2 being generated by peroxisomal β-oxidation of long-chain FAs. Heme-containing homotetrameric catalase does not usually lodge in mitochondria, except in the heart, where it resides in the mitochondrial matrix. Red blood capsules, in addition to the liver and kidney, have the highest catalase activity, the brain, heart, and skeletal muscle having a low catalase activity. It neutralizes hydrogen peroxide, thereby preventing accumulation of hydroxyl radicals. Catalase degrades H2 O2 using two different mechanisms [86]. In dismutation, the oxyferryl heme is reduced back to the ferric form by another H2 O2 molecule, H2 O2 being both oxidant and reductant (catalatic reaction). Alternatively, catalase can use other electron donors (peroxidatic mechanism). Peroxiredoxins Ubiquitous homodimeric peroxiredoxins are nonheme peroxidases that detoxify low- and high-molecular-mass peroxides (ROOH, where R can be a hydrogen atom or a complex phospholipid). Most PRdxs use thioredoxin as a donor of reducing equivalents (of hydrogen), although PRdx6 functions as a reduced glutathionedependent peroxidase. Glutaredoxins and cyclophilins are additional electron donors for peroxiredoxins. Peroxiredoxins lodge in different subcellular compartments, such as the mitochondrion (e.g., PRdx3 and PRdx5) and cytosol (e.g., PRdx1, PRdx2, and PRdx6), PRdx4 residing predominantly in the endoplasmic reticulum and PRdx5 also in the cytosol and peroxisomes [87]. Peroxiredoxins are regulated by phosphorylation in response to extracellular signals, redox state, and oligomerization. They contain one or a pair of active cysteines sensitive to oxidation by H2 O2 , which reacts with the thiolate deprotonated form of cysteine. Peroxiredoxins are classified into three sets: typical (PRdx1–PRdx4) and atypical 2-Cys (PRdx5) and 1-Cys forms (PRdx6). They also reduce ONOO− and lipid peroxides.

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Antioxidant sestrins can regenerate oxidized peroxiredoxins, scavenge ROS, and hamper expression of NOx4, especially in glomerular mesangiocytes, and TORC1induced ROS [88]. Glutathione Peroxidases Glutathione peroxidase GPOx1 is one of the most abundant members of the GPOX family, which includes epithelial GPOx2, highly expressed in the intestine, and secreted GPOx3, among other subtypes. GPOx1 lodges in the cytosol, mitochondrion, and peroxisome. The intracellular antioxidant selenocysteine-containing enzyme GPOx1 reduces hydrogen peroxide to water, thereby limiting its accumulation and subsequent harmful oxidative effect on nucleic acids, proteins, and membrane lipids and preventing carcinogenesis and the development of cardiovascular disease [89]. GPOx1 can also reduce lipid hydroperoxides and other soluble hydroperoxides after their release from membrane lipids [89]. It also reduces phospholipid and monoacylglycerol hydroperoxides, such as linoleoyl lysophosphatidylcholine hydroperoxide, but not tri- or diacylglycerol hydroperoxides. These other types of membrane-associated phospholipids are reduced by GPOx4 [89]. GPOx1 may also act as a peroxynitrite reductase. Expression of GPOx1 is regulated by transcriptional, post-transcriptional, translational, and post-translational mechanisms [89]. Estradiol and ROS contribute to GPOx1 transcription control. Selenium stabilizes mRNA, avoiding nonsensemediated decay. Translation involves Sec insertion sequence (SecIS)-binding proteins such as SBP2. At the post-translational level, GPOx1 can be oxidatively inactivated by excess ROS or NO, whereas the kinase Abl phosphorylates (activates) GPOx1.

Redox Stress At excessive and sustained concentrations, ROS have deleterious effects. Reversible and irreversible oxidations of cellular proteins, lipids, carbohydrates, RNA, and DNA have an impact on cellular functions. Mitochondrial DNA is particularly vulnerable to ROS and RNS. Generalized oxidation causes cell dysfunction, apoptosis, or necrosis [90]. Reactive oxygen species operate in inflammation. In particular, macrophages release glutathionated peroxiredoxin-2, which acts as an alarmin (or damageassociated molecular pattern molecules [DAMPs]), which triggers innate immune response and production of TNFSF1 [91]. Reactive oxygen species function in the initiation and progression of CVD. They are involved in proinflammatory signaling within vascular endothelial cells (vECs) and vSMCs, which then synthesize cell adhesion molecules and chemokines. They also activate MMPs.

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31

Major vascular risk factors (hypertension, dyslipidemia, diabetes, and smoking) are associated with augmented vascular ROS production. A chronic metabolic disturbance favors inflammation and redox stress, an imbalance between pro- and antioxidants and their sources and sinks. Obese sedentary individuals have greater NOx activity in skeletal muscles and blood ROS concentrations than lean active subjects [92]. Adequate diet that attenuates redox stress prevents obesity-associated disorders [84]. Mitochondrial superoxide production corresponds to 1–2% of the molecular oxygen consumed. However, excess mitochondrial O•− 2 influences the perivascular neutrophil niche [92]. Lysophosphatidylcholine is implicated in mitochondrial ROS production and in endotheliocyte activation likely because of electron leakage across the mitochondrial membrane. Hydrogen peroxide derived from mitochondrial O•− 2 alters the caliber of the coronary resistance artery. Chronic production of inflammatory and vasoconstrictive prostaglandins exacerbates hypertension via both inflammation and vasoconstriction. Oxidation and glycation of LDLs engender proinflammatory and proatherogenic adducts. On the other hand, high-density lipoproteins lessen lipoprotein oxidation and hence generation of oxidized LDLs (oxLDLs), the antioxidant effect relying on HDL-associated paraoxonase [90]. In atherosclerotic lesions, ROS stabilize HIF1α, which is produced in hypoxic regions of plaques and favors M1 macrophage phenotype and hence atherogenesis [92]. Migration of macrophages primed by oxLDLs depends on FAK, PTPN11, NOx, ROS, and ScaRb3 [92]. On the other hand, ScaRb3 activation by ROS in extracellular vesicles precludes the migration of endotheliocytes. Growth factors (e.g., platelet-derived growth factor [PDGF] and TGFβ), cytokines (e.g., TNFSF1 and IL1β), and hemodynamic stress (shear and stretch) regulate expression and/or activity of vascular NOxs [83]. The autacoids angiotensin-2, endothelin-1, and thrombin activate NOx. Agt2 not only stimulates NOx but also upregulates expression of its subunits, provoking ROS generation by ECs, vSMCs, and adventitial fibroblasts via its AT1 receptor. Thrombin, in addition to PDGF, TGFβ, and TNFSF1, also activates NOx in vSMCs. Endothelin-1 increases NOx activity in ECs via its ETA receptor. Angiotensin-2 causes mitochondrial dysfunction via endothelial NOx, PKC, and ONOO− , elevates mitochondrial H2 O2 production, and reduces endothelial NO availability [93]. On the other hand, the amount of mitochondrial ROS is lowered by manganese-containing superoxide dismutase SOD2, and/or peroxiredoxins PRdx3, and/or PRdx5, which protects against mitochondrial oxidative damage. In vascular smooth myocytes and endotheliocytes, the mitochondrial ATP-sensitive potassium channel is implicated in Agt2-induced mitochondrial ROS production, as it increases K+ influx and alkalinizes the mitochondrial matrix. Mitochondrial permeability transition pore-opening also contributes to Agt2-mediated ROS production. In some diseases, NOx1 expression is upregulated in vascular endotheliocytes and smooth myocytes. Interaction between thrombospondin-1 with neurophilin

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(CD47) activates NOx1 [92]. Cyclic stretch applied on vessels induces formation of myocyte-enhancing factor MEF2b, which launches NOx1 production and vSMC phenotype switching to a proliferative state. Both NOx1 and NOx4 syntheses are upregulated by hyperglycemia, leading to ROS-induced PKC-dependent downregulation of PKG production and hence repression of the NO–sGC–cGMP–PKG signaling. Inducible NOx2, which is produced to a greater extent in fibroblasts and immunocytes, participates in recruiting macrophages to inflammation sites to remove infectious pathogens. However, NOx2 overexpression in the endothelium favors sustained leukocyte infiltration in the vasculature and thrombosis. Hydrogen peroxide (H2 O2 ) formed by constitutively active brown adipocytic NOx4 protects the vasculature via PKG [92]. In addition, adipocytic NOx4 slows obesity-linked inflammation in addition to T2DM progression. On the other hand, endoplasmic reticular stress stimulates NOx4, which produces both superoxide and hydrogen peroxide. Intermedin1−−53 , a N-terminal fragment of adrenomedullin-2, reduces NOx4 production. Reactive nitrogen species, that is, NO• , an endothelial function marker, and its derivatives, participate in vasculopathies. In healthy vessels, NO prevents circulating leukocyte adhesion to the wetted endothelial surface and triggers vasodilation. In addition to endotheliocytes, circulating hematopoietic cells are important sources of NO in blood [92]. Excess NO production, often due to the hyperactivity of NOS2, has harmful effects on the vasculature. In obese mice, the perivascular adipose tissue causes NOS3 uncoupling, converting it from NO producer to O•− 2 generator, which exacerbates the underlying pathological condition. In endothelial and smooth muscle cells, the oxidation state (ferric versus ferrous) of hemoproteins modulates NO signaling. In particular, the redox state of hemoglobin Hbα at the myoendothelial junction regulates NO activity. In the ferric state, Hbα has a reduced binding affinity for NO, which then diffuses between endotheliocytes and smooth myocytes [92]. In the reduced ferrous Hbα state, NO is sequestered, and the NO–sGC–cGMP–PKG axis and subsequent vasodilation of resistance arteries in both the systemic and pulmonary circulation are repressed. The flavoprotein methemoglobin reductase24 also inhibits NO signaling via the myoendothelial junction. On the other hand, in vSMCs, methemoglobin reductase reduces soluble guanylate cyclase (sGC) heme iron from the ferric to the ferrous state, thereby enabling NO sensing and subsequent arterial dilation. In addition, cGMP is not only degraded by phosphodiesterases PDE3 and PDE5 but is also exported from the cell [92].

24 Also

known as NADH cytochrome-B5 reductase-3.

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33

Receptor for Advanced Glycation End Products Proinflammatory multiligand receptor for advanced glycation end products (RAGE) generates ROS via NOx activation and mitochondrial production amplification and thus operates via redox stress [94]. The RAGE resides on diverse cell types (e.g., endothelial progenitor cells, cardiac endotheliocytes, vascular smooth myocytes, cells of the nervous system, pancreatic β cells, renal mesangiocytes, osteoblasts, and inflammatory leukocytes). Its cytoplasmic domain binds to the formin diaphanous-1 that activates Rac1 and NOx in aortic smooth myocytes exposed to the RAGE ligand S100b. The RAGE connects to proinflammatory members of the S100–calgranulin set (S100a8, S100a9, and S100a12 [calgranulin-A–calgranulin-C]), which are predominantly expressed by neutrophils, monocytes, and activated macrophages, S100a8 being a potent antioxidant, in addition to high-mobility group box-containing protein HMGB1, amyloid β-peptide and β-sheet fibrils, lysophosphatidic acid, αM β2 -integrin (CR3), and complement component C1q [94]. Advanced glycation end products (AGEs) are products of non-enzymatic glycation and oxidation of proteins and lipids formed in vascular cells, CMCs, neurons of the central and peripheral nervous systems, alveolar pneumocytes, podocytes, and inflammatory leukocytes, among other cell types [94]. In the heart, the detoxifier glyoxalase-1 of glycation precursors such as 3-deoxyglucosone of the AGE N 1-carboxymethyllysine prevents diabetes-induced redox damage, inflammation, fibrosis, and diabetic cardiomyopathy [95].

Lipid Peroxidation Lipids, with their reactive double bonds, are targets of oxidation. Lipid peroxidation generates isoprostanes and malondialdehyde (MDA; Table 1.6). Isoprostanes are stable prostaglandin-like compounds engendered from arachidonic acid peroxidation and subsequently released from cellular membranes into the bloodstream by phospholipases. Isoprostane concentrations in plasma and urine samples correlate with cigarette smoking, hypercholesterolemia, obesity, T2DM, and hyperhomocysteinemia [90].

Table 1.6 Lipid peroxidation (Source: [71]) Reagent Free radical Lipid radical Lipid radical

Reactant Lipid GSH O•− 2

Product Lipid radical (L• ) Lipid hydroxide (LH) Lipid peroxyradical (LOO• )

Lipid peroxyradical Lipid hydroxyperoxide

GSH GSH

Lipid hydroxyperoxide (LOOH) Lipid alcohol (LOH)

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1 Cardiovascular Disease: An Introduction

Malondialdehyde is formed from peroxidation of polyunsaturated FAs. It interacts with proteins, particularly with lysine residues, building Lys–Lys crosslinks, such as in ApoB of oxLDLs [90].

Protein Tyrosine Nitration Proteins are also oxidized and then associated with pathophysiological processes in addition to aging. Protein tyrosine nitration, which consists of adding a nitro group (–NO2 ), is mediated by RNS such as peroxynitrite (ONOO− ) and nitrogen dioxide (NO•2 ). This reaction involves two steps: the oxidation of the phenolic ring of tyrosine to tyrosyl radical (Tyr• ) and the addition of NO•2 to the Tyr by a nitrating agent. Myeloperoxidase, with its transition metal center, can react with ONOO− and hence facilitate nitration [90]. Nitrotyrosine formation on enzymes, such as sarcoplasmic reticulum Ca2+ ATPase (serca2a), manganese-containing superoxide dismutase (Mn SOD or SOD2), prostacyclin synthase, tyrosine hydroxylase, and aldolase-A, inhibits their activity. On the other hand, nitrotyrosine in fibrinogen raises its activity and accelerates clot formation.

Protein Glutathionation S Glutathionation,

that is, formation of a disulfide bridge between a reactive cysteine residue and the tripeptide glutathione, mediates redox regulation of numerous cellular proteins (e.g., NOS3, ryanodine receptor, SERCA, and Na+ –K+ ATPase, thereby affecting their function and intracellular Na+ and Ca2+ handling [90]). S Glutathionation of hemoglobin can serve as a marker of redox stress.

1.1.4.4

Aging

Aging is associated with declining organ functioning and metabolism. It is related to chronic inflammation, the so-called inflammaging, redox stress, and arterial stiffening. Arterial redox stress contributes to arterial stiffening, as it favors elastin degradation and collagen overproduction, in addition to inflammation. Many lipids are synthesized from precursors within the body, but some essential FAs must be ingested with food intake. For example, fish contains the essential longchain ω3 FAs eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). Vegans do not eat animal products and vegetarians neither meat nor fish; semi-vegetarians consume fish, seafood, and sometimes even poultry; lacto-vegetarians add dairy products and lacto-ovo-vegetarians eggs to their diet. Lipids can be divided into eight categories: FAs, glycerolipids, glycerophospholipids, sphingolipids, sterol and prenol lipids, saccharolipids, and polyketides). Their concentration ranges from the attomolar to the micromolar level. Lipidomics is aimed at investigating lipid fate and signaling in addition to the effects of nutritional supplementation and its role in immune and inflammatory responses

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35

and cardiovascular and pulmonary diseases. Signaling lipids include oxylipins, especially the initiation and termination of inflammation. Oxylipins are lipophilic messengers generated by oxygenation by cyclooxygenases (COxs; or prostaglandin-G/H synthases [PGhS]), cytochrome-P450 enzymes (CyPs), and lipoxygenases (LOxs), in addition to the non-enzymatic auto-oxidation of polyunsaturated fatty acids (PUFAs), such as the ω6-fatty acids arachidonic (AA) and linoleic acid (LA) in addition to ω3-fatty acid α-linolenic acid (α LA), the plasmatic concentrations of which change not only with diet but also during aging [96]. Oxylipins are involved in immunity and hence inflammation in addition to vasomotor tone and blood coagulation. They can also be bactericides. Eicosanoids are oxylipins derived from AA, a component of cellular membrane phospholipids. Cyclooxygenases generate class-I I (2 double bonds) prostaglandins (PGs) and thromboxanes (Txs) from arachidonic acid. Class-I and-I I I PGs and Txs are formed from dihomo-γ-linolenic acid (DGLA) and EPA, respectively. Lipoxygenase products encompass: 1. HETEs from AA, which mediate neutrophil chemotaxis and degranulation 2. Hydroxyoctadecadienoic acids (HODEs) from LA 3. Hydroxyeicosapentaenoic acids (HEPEs) from EPA Lipoxygenase LOx5 synthesizes leukotrienes and other metabolites, such as proinflammatory 5HETE, 5oxoETE, and LTb4 , from AA, and 9HODE and trihydroxyoctadecenoic acids (triHOMEs) from LA. ALOx12 and ALOx15 produce 12HETE, 12oxoHETE, and proinflammatory 9HETE from AA. The CyP enzymes generate epoxides, such as EETs from AA and epoxyoctadecamonoenic acids (EOMEs) from LA, which dilate arteries. These products are converted to dihydroxyoctadecenoic (diHOMEs) and DHETs by soluble epoxide hydrolase (sEH).

Aging, Inflammation, and Redox Stress Inflammaging partly results from increased concentrations of alarmins, which activate pattern recognition receptors (PRRs). Toll-like (TLRs) and nucleotidebinding oligomerization domain-like receptors (NLRs) are expressed not only on or in innate immunocytes but also on or in cells of the neurovascular unit and blood–brain barrier [97]. Among these PRRs, TLR2, TLR4, NLRP1, and NLRP3 are activated during aging in neurons, astrocytes, microgliocytes, and possibly endotheliocytes and pericytes. Cardiovascular disease is linked to chronic obstructive pulmonary disorder (COPD) via chronic inflammation and aging with reduced sirtuin activity and exposure to cigarette smoke [98]. Desmosine and isodesmosine are involved in elastin crosslinking and can serve as indicators of elevated elastin fiber turnover and degradation, such as in COPD and atherosclerosis complications.

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Sirtuins (SIRT1–SIRT7) are NAD+ -dependent protein (histone) deacetylases implicated in lifespan and health regulation. Sirtuin-1 regulates endothelial function as it deacetylates NOS3 [98]. In addition, it counters senescence, as it deacetylates P53 and STK11 (LKB1), and angiogenesis, as it deacetylates FoxO1 and notch1 [98]. It also activates liver X receptor (NR1h2/3), which is involved in reverse cholesterol transport, hence promoting cholesterol efflux. It has an antioxidant effect. Furthermore, it inhibits NFκB [98]. On the other hand, sirtuin-1 precludes vascular smooth myocyte proliferation and atherothrombosis, as it downregulates endothelial formation of tissue factor and upregulates that of tissue inhibitor of metallopeptidase TIMP3 [98]. Prolonged moderate exercise training enhances FoxO3a expression, reduces redox stress, and raises SIRT1 activity in the heart and adipose tissue of aged rats. Sirtuin-3 hampers cardiac hypertrophy as it controls ROS concentrations. Sirtuin-6 in endotheliocytes protects against telomere and gene damage, and Sirtuin7 interacts with P53 and protects CMCs against apoptosis and redox and genotoxic stresses [98]. Reactive oxygen species participate in aging. However, dietary antioxidants, such as vitamins C and E, do not slow aging [99]. Mitochondria are a major ROS source and thus mediate adverse processes in aging. Supplementation with the orally active mitochondrial antioxidant MitoQ ([dimethoxy methyl dioxo-cyclohexadien decyl] triphenyl methanesulfonate), a derivative of the potent antioxidant ubiquinone conjugated to triphenylphosphonium, which accumulates within mitochondria, prevents mitochondrial redox damage. It thus attenuates the production of proliferative, proinflammatory, and profibrogenic mediators (e.g., tumor growth factor [TGFβ], connective tissue growth factor [CTGF], and PDGF) by neighboring and infiltrating cells in the liver (i.e., activated hepatic stellate cells, which form a collagen-rich matrix, Kupffer cells, and cholangiocytes, in addition to injured hepatocytes, platelets, and leukocytes), and hence redox stress, hepatocyte death, and hepatic inflammation, together with liver fibrosis and cirrhosis in mice [100]. Its administration for 4 weeks limits the reduction of elastin content and decreases aortic stiffness in 27month-old mice, affects neither young mice nor age-related collagen synthesis and deposition, and increases proinflammatory cytokine formation [101].

Aging and Altered Proteostasis Aging and age-related diseases are associated with disturbed balance of protein production, folding, and degradation, in addition to subsequent accumulation of misfolded proteins and proteic aggregates. In CMCs, altered proteostasis can participate in the development of cardiac hypertrophy, cardiomyopathies, and heart failure. Hydrotropes are small molecules, typically amphiphilic agents, that solubilize hydrophobic molecules in aqueous solutions. The canonical energy carrier and autacoid, adenosine triphosphate (ATP), an energy source for chemical reactions

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37

at micromolar concentrations, including muscular contraction, possesses at physiological millimolar concentrations (5–10 mmol/l) the properties of a hydrotrope, as it maintains protein solubility and prevents molecular aggregation [102]. At relatively high concentrations, ATP enhances the solubility of solutes, such as nonpolar lipophilic proteins (unlike polar hydrophilic proteins) and organic substances, which are nearly insoluble in the usual aqueous solutions. Amphiphilic hydrotrope molecules have shorter hydrophobic regions and therefore do not spontaneously self-aggregate in the aqueous phase [103]. Adenosine triphosphate precludes not only formation of protein aggregates, hampering aggregation of prion and amyloid fibers from amyloid-β4 protein, but also contributes to dissolving a previously formed agglutinated mass, as it can dissolve liquid–liquid phase-separated droplets, keeping the RNA-binding protein fused in sarcoma (FUS) in a water-soluble state and preventing its accumulation into separate liquid drops [102]. For most ATP users such as ATP-dependent enzymes, the Michaelis–Menten constant of cardiac actin-based nanomotor myosin evolves in the micromolar range (β-myosin encoded by the MYH7 gene [cardiac myosin heavy chain-7]: 40 ± 6μmol/l), whereas the nucleotide ATP is typically present in millimolar concentrations in the cytoplasm of CMCs [103]. Therefore, the discrepancy between ATP concentration needed by ATP-consuming enzymes and its intracellular level can be explained, at least partly, by its hydrotrope function. The intracellular ATP amount declines with aging in addition to impaired mitochondrial oxidative phosphorylation. Variant proteins containing expansions of glutamine repeats (polyQ repeats), that is, with increased polyglutamine motif length, which is encoded by the DNA nucleotide sequence CAG, can misfold and form aggregates, which can sequester proteins. Expansion of polyQ domains in huntingtin and the deubiquitinase ataxin3 causes Huntington’s disease characterized by loss of striatal neurons and hence changes in mood and personality, defective motor coordination, and involuntary movements and type-3 spinocerebellar ataxia (SCA3), a form of neurodegeneration in the striatum and cerebellum, respectively [104]. PolyQ expansions in addition to soluble N-terminal huntingtin fragment comprising exon 1 are toxic. Autophagy that is aimed at attenuating protein toxicity removes polyQ-expanded proteins, such as abnormal huntingtin and ataxin-3. On the other hand, some polyQ-containing proteins regulate degradation of misfolded proteins and autophagy. Ataxin-3 deubiquitinates beclin-1 that then escapes proteasomal destruction and triggers starvation-induced autophagy. Short polyQ domain enables interaction between ataxin-3 and beclin-1 [105]. On the other hand, a mutated form of huntingtin that contains an expanded polyQ region competes with ataxin-3 for beclin-1 binding, thereby increasing beclin-1 degradation and dysregulating autophagy. Huntingtin also participates in stressactivated autophagy, as it competes for binding to another autophagic regulator. Expansion of the polyglutamine stretch in ataxin-1 (Atxn1) causes the hereditary neurodegenerative disease type-1 spinocerebellar ataxia (SCA1), hence its other

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Table 1.7 Ataxins (Atxns; Source: [107, 108]; boat brother of ataxin-1, MJDL Machado–Joseph disease protein-like protein) Type Atxn1 (SCA1) Atxn1L (boat) Atxn2 (SCA2) Atxn2L Atxn3 (SCA3) (MJD1) Atnx3L (MJD1L) Atxn7 (SCA7)

Function Chromatin-binding factor repressing notch signaling (CBF1 corepressor) Also named chromatin-binding repressor of notch cooperating with Atxn1 Inhibition of EGFR internalization Regulation of stress granule and P-body formation Deubiquitinase of proteostasis maintenance Involved in gene transcription, cytoskeleton regulation, and myogenesis Deubiquitinase Component of the deubiquitination module of the SAGA histone acetyltransferase and deubiquitinase complex Histone remodeling for transcriptional regulation Atxn7L1 (Atxn7L4), Atxn7L2: undetermined role Atxn7L3 Component of the transcriptional regulatory SAGA complex Atxn8 ND (SCA8, P1c2) Atxn10 (SCA10) Survival of cerebellar neurons Expansion of the polyglutamine sequence in ataxins provokes spinocerebellar ataxia (SCA)

alias SCA1. The ATXN family includes proteins characterized by the presence of an AXH domain implicated in protein–protein interactions.25 The deubiquitinase Atxn3L targets the zinc finger-containing transcription factor KLF5, which promotes cell survival and proliferation in addition to tumoral growth, partly as it upregulates synthesis of fibroblast growth factor-binding protein FGFBP126 and microsomal prostaglandin-E synthase PtgES1 [106]. It belongs to the DUB subset of Machado–Joseph disease (MJD) proteic domain-containing peptidases with Atxn3 (a.k.a. MJD1 and SCA3), encoded by the gene mutated in MJD, also termed type-3 spinocerebellar ataxia (SCA3), and Josephin domaincontaining DUbs JosD1 and JosD2 (Table 1.7).27 Aggregates formed by polyglutamine-expanded ataxin-7 sequester ubiquitinspecific peptidase USP22 that cannot then fulfill its deubiquitinating function in the SAGA complex, causing cytotoxicity and neurodegeneration [109].

25 The

alias AXH stands for Ataxin-1 and HBP1 (HBP1: high-mobility–group box transcriptional repressor-1). 26 Also known as 17-kDa heparin-binding growth factor-binding protein HBP17. 27 Deubiquitinases can be categorized into six subsets: ubiquitin-specific peptidases (USPs); ubiquitin carboxy-terminal hydrolases (UCHs); ovarian tumor peptidases (OTus); MJD peptidases; JAMM/MPN domain-associated metallopeptidases (JAMMs); and monocyte chemotactic proteininduced protein (MCPIP).

1.1 Vasculopathies and Vasculitides

1.1.4.5

39

Sleep Disorders

Sleep disorders and short sleep duration (≤ 5 h/night) alter neurohormonal regulation and the circadian rhythm of blood pressure with its nocturnal decrease, blunting nocturnal surge in melatonin secretion and favoring hypertension. Sleep deprivation is related not only to hypertension but also diabetes mellitus and coronary artery disease [110]. Cardiovascular and metabolic disease (i.e., hypertension, atherosclerosis, heart failure, cardiac arrhythmias, obesity, and metabolic syndrome) are linked to sleep anomalies (sleep curtailment, shift work, and sleep-disordered breathing) [111]. Sleep affects the autonomic nervous system, hemodynamics, endothelial and myocardial function, and blood coagulation. Central sleep apnea (CSA) is caused by a lack of neural input for breathing. Breathing effort is attenuated or absent during airflow cessation, typically for 10– 30 s, either intermittently or in cycles. Breathing is controlled by central and peripheral chemoreceptors. Medullary neurons respond to CO2 content via shifts in H+ concentration and chemoreceptors of the carotid body to arterial blood O2 and CO2 content. Elevated chemoresponsiveness along with blunted chemosensitivity can destabilize the breathing pattern. In addition, several other homeostatic feedback mechanisms regulate breathing amplitude and frequency to maintain gas exchange, such as afferent input from Golgi tendon organs and muscle spindles from respiratory muscles [112]. Ventilatory response to hypoxia and hypercapnia and respiratory load compensation are reduced during sleep, particularly during the rapid eye movement stage. Several CSA manifestations encompass high-altitude-induced periodic breathing, idiopathic CSA, narcotic-induced central apnea, obesity hypoventilation syndrome, and Cheyne–Stokes breathing [112]. Nighttime breathing disturbances increase the risk for adverse cardiovascular outcomes. Obstructive sleep apnea (OSA) with breathing pauses 5–30 times per hour during sleep because of upper airway hindrance is associated with respiratory efforts. It can be linked to hypertension, arrhythmia, stroke, and heart failure. Obstructive sleep apnea is associated with obesity; the resulting sleep deprivation can favor obesity, forming a vicious cycle.

1.1.4.6

Vascular Tumors and Malformations

Vascular anomalies encompass tumors and malformations (direct connections between arteries and veins), in addition to infection, trauma, and adverse remodeling. • Vascular congenital tumors comprise infantile congenital hemangioma, especially in girls, which is usually solitary, but can be multiple, along with tufted angioma, infantile fibrosarcoma, myofibromatosis, and kaposiform hemangioepithelioma.

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• Vascular malformations that bypass the capillary bed generally result from embryogenic errors. However, most arteriovenous malformations are idiopathic. They arise spontaneously. They differ from those engendered by gene mutations by their location and evolution. A classification of vascular anomalies was proposed by the International Society for the Study of Vascular Anomalies (ISSVA) that categorizes benign vascular lesions into two groups according to the predominant type of vascular channel affected and flow magnitude: (1) vascular tumors, the most common form being infantile hemangioma, and (2) vascular malformations, which are created by errors of vasculo- and angiogenesis [113]. Vascular malformations usually develop gradually, but their growth is faster than that of the body, with peak growth occurring during puberty. Birth defects can be independent of genetic cause but rely on environmental factors. For example, cardiac and craniofacial birth defects can result from maternal fever during the first trimester of pregnancy. Neural crest cells are precursors of cells forming tissues of the heart and head (face). Hyperthermia-activated TRPV1 and TRPV4 channels28 in neural crest cells of chick embryos provoke cardiac and craniofacial birth defects [114]. Vascular tumors encompass non-involuting (NICH) and rapidly involuting congenital (RICH) and infantile hemangiomas, tufted angiomas, kaposiform, spindle cell, and other rare hemangioendotheliomas, in addition to dermatologically acquired vascular tumors (e.g., pyogenic granuloma, targetoid, glomeruloid, and microvenular hemangioma). Slow-flow vascular malformations include venous (e.g., blue rubber bleb nevus syndrome, familial cutaneous and mucosal venous malformation, glomuvenous malformation), capillary (e.g., telangiectasia and angiokeratoma), and lymphatic malformations (primary lymphedema, and micro- and macrocystic lymphatic malformations in addition to combined vascular malformations [capillary (C), venous (V), and/or lymphatic (L) malformations (M), that is, CVMs, CLMs, LVMs, and CLVMs]). Telangiectasias29 are small, permanently dilated blood vessels that engender small cutaneous red dots or linear or stellate lesions. They often progress to form papules, particularly on the face [113]. Angiokeratomas constitute a heterogeneous group of red–violaceous to black papules due to vascular dilation in the papillary dermis with epidermal hyperplasia and hyperkeratosis [113]. Angiokeratoma corporis diffusum represents a diffuse form of angiokeratoma. They are associated with deficiencies in:

28 TRP:

transient receptor potential. telangiectasis (plural telangiectases). From Greek τ λιωμα: completion; τ λoς: end, term, achievement; αγγ ιoν: (hollow) vessel, vein; κτασις: extension, dilation.

29 Also

1.1 Vasculopathies and Vasculitides

41

1. Lysosomal αN acetylgalactosaminidase (NAGα), which is encoded by the NAGA gene, mutations of which causes aspartylglucosaminuria and type-I (infantile) and -I I (adulthood) Schindler disease 2. Lysosomal α-galactosidase-A (Glα), mutations in the GLA gene engendering Fabry disease 3. α-Fucosidase (Fucα), mutations in the FUCA1 (or FUCA) gene causing severe infantile type-I and milder type-I I fucosidosis 4. β-Galactosidase (Glβ) and neuraminidase (Neu1–Neu4 or sialidase-1 to -4), which provokes early and late infantile and juvenile/adult galactosialidosis, which results from mutations in the CTSA gene that encodes lysosomal cathepsin-A, which cooperates and complexes with neuraminidase-1 and βgalactosidase (hence the other CtsA name, protective protein for β-galactosidase [PPGβ]) 5. Lysosomal β-mannosidase (Manβ), mutations in the MANBA gene causing βmannosidosis (mutations in the MAN2B1 gene that encodes lysosomal acid αmannosidase class 2B member 1 provoking α-mannosidosis) 6. Lysosomal monosialotetrahexosylganglioside GM1, mutations in the GLB1 gene that encodes acid galactosidase-β1 (Glβ1), generating GM1 gangliosidosis, GM1 ganglioside that cannot be catabolized accumulating to toxic levels 7. Lysosomal sialidase-1, or neuraminidase Neu1, mutations in the NEU1 gene engendering type-I (partial Neu1 deficiency) and more severe type-I I sialidosis (severe reduction or even elimination of Neu1 activity) Verrucous hemangioma is a separate entity with respect to angiokeratoma. These generally deep lesions are often linked to hyperkeratosis [113]. Fast-flow vascular malformations comprise arterial and arteriovenous malformations, arteriovenous fistulas (AVFs), and combined vascular malformations (e.g., arterial [A], venous, and lymphatic (AVMLMs) and CMAVMs).

1.1.4.7

Ectopic Vascular Calcification

Arteries are not only sites of abnormal caliber changes, either narrowing (stenosis; Vol. 13, Chap. 7. Arterial Stenosis—Mechanical and Clinical Aspects) or enlarging (aneurysm; Vol. 13, Chaps. 3. Aortopathies and 4. Aneurysms), but also of ectopic calcifications. Vascular calcification (Vol. 10, Chap. 3. Adverse Wall Remodeling) relies on bone morphogenetic proteins (BMPs; Sect. 1.4.5.6), the Wnt pathway (Sect. 1.4.5.2), tumor-necrosis factor superfamily member TNFSF11, and receptors TNFRSF11a and TNFRSF11b, in addition to various other calcification regulators, such as inflammatory factors and oxidized lipids.

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Lipoprotein-A (LPa) carries proinflammatory and procalcific phosphocholinecontaining oxidized phospholipids (OxPLs) [115]. In fact, various lipoproteins contribute to the progression from sclerosis to stenosis, although LPa is the preferential OxPL carrier.

1.1.5 Atherosclerosis Atherosclerotic cardiovascular disease (ASCVD), or simply atherosclerosis,30 is characterized by the subendothelial retention of modified lipoproteins, immunocyte infiltration, maladaptive chronic inflammation of the arterial wall, and vSMCmediated fibrous cap formation. Atherosclerosis progression is linked to cell death, fibrous cap thinning, plaque rupture, and thrombosis. Accumulated intracellular cholesterol can be removed using the reverse cholesterol transport that begins from cholesterol egress from cells and subsequent elimination from the body, thereby protecting against the development and progression of atherosclerosis. On the other hand, an imbalance between the uptake of cholesterol from oxidized or aggregated LDLs through scavenger receptors and the efflux of cholesterol to apolipoprotein-A and HDLs through ABC transporters leads to atherogenesis. Low-density lipoproteins can be oxidized, glycated, acetylated, ethylated, and methylated. Oxidized and glycated LDLs in arterial walls initiate atherogenesis. Modifications target LDL components such as their surface protein ApoB, which mediates LDL binding to its receptor. The early stage of atherogenesis is linked to oxidized LDLs that accumulate in the subendothelial space, where they activate endotheliocytes, which then produce adhesion molecules and chemokines, recruiting inflammatory leukocytes. Attracted monocytes differentiate into macrophages that internalize oxLDLs and release cytokines and ROS, further oxidizing LDLs and attracting medial smooth myocytes into the intima. These smooth myocytes contribute to atherogenesis via apoptosis and foam cell formation. Non-enzymatic glycation of lysine residues of ApoB diminishes LDL affinity for its receptor, thereby augmenting its plasmatic lifetime and uptake of glycated LDLs (glLDLs) by vascular cells and macrophages. Furthermore, LDL glycation renders them more susceptible to oxidation (gl–oxLDLs). Upon uptake of modified LDLs via scavenger receptors and pinocytosis, macrophages in the arterial intima differentiate into foam cells. Mitochondria produce ATP and are involved in ion transfer, ROS generation, and apoptotic signaling. Mitochondrial DNA contains 37 genes that encode subunits of

30 αθηρωμα:

tumor full of gruel-like matter; σκληρo: hard. The Swiss scientist A. von Haller (1708–1777) described atherosclerosis in his book “Opuscula Pathologica” published in 1755. The German physician F. Marchand (1846–1928) introduced the term atherosclerosis in 1904.

1.1 Vasculopathies and Vasculitides ETC complex-I ,

43

-I I I , and -I V and ATP synthase (i.e., ETC complex-V ), in addition to corresponding ribosomal and transfer RNAs. Mitochondrial ROS damage mitochondrial DNA, a circular molecule linked to the inner mitochondrial membrane; mitochondrial dysfunction and subsequent mitophagy precede lesion development [116]. Mitochondrial DNA damage lessens mitochondrial oxidative phosphorylation. Decreased mitochondrial oxidative phosphorylation causes thinning of the fibrous cap via vascular smooth myocyte dysfunction and apoptosis, and increased necrotic core formation due to macrophage activation. Mitochondrial DNA is replicated by the Mt DNA replisome, which comprises the twinkle helicase, Mt DNA polymerase, and mitochondrial single-stranded DNAbinding protein. Reduced Mt DNA number and oxidative phosphorylation increase mitophagy in plaque vSMCs, whereas overexpression of the mitochondrial DNA helicase twinkle reduces Mt DNA damage but does not affect Mt DNA copy number [117]. Twinkle protects vascular smooth myocytes and macrophages against redox stress-primed apoptosis. In macrophages, overexpression of twinkle increases Mt DNA copy number without affecting Mt DNA damage. In both cell types, possibly via increased ETC subunit synthesis, twinkle overexpression enhances oxidative phosphorylation, thickening the fibrous cap via increased vSMC proliferation and reduced apoptosis and attenuating necrotic core formation via macrophage inactivation. Atherosclerosis is a chronic disease of the arterial wall that involves both innate and adaptive immunity, inflammation being implicated at all stages of the disease. This inflammatory disease involves accumulation of lipids in the arterial intima, infiltration and proliferation of monocytes, and their differentiation into macrophages, among other leukocytes, recruitment of medial smooth myocytes, and production and degradation of the extracellular matrix. It is characterized by hardened arterial segments with narrowed or enlarged lumens (i.e., stenoses and fusiform aneurysms). Arteriosclerosis, the hardening (or stiffening) of normally distensible arteries was described by the German-born French pathologist J.G.C.F.M. Lobstein (1777– 1835). It encompasses atherosclerosis, medial thickening, and medial and intimal calcifications (e.g., Mönckeberg medial sclerosis, the most common form of medial calcifications in the arteries of the extremities) [118]. In 2013, atherosclerosis, particularly angina pectoris and myocardial and cerebral infarction (stroke), and other types of cardiovascular affections (e.g., arrhythmias, heart failure, and cardiac valvulopathies) caused 51% and 42% of deaths among women and men, respectively [29]. In many countries, they provoke more than twice the number of deaths as cancer. However, in at least ten countries (Belgium, Denmark, France, Israel, Luxembourg, Netherlands, Portugal, Slovenia, Spain, and San Marino), cancer engenders more deaths than CVD among men and in one country (Denmark), among women [29].

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Coronary atherosclerosis, also currently named coronary artery and heart disease and ischemic heart disease,31 and cerebrovascular disease(another collective term standing for all diseases of arteries irrigating the brain), which are the first and second leading contributors to CVD burden, account for 20 and 12% of all deaths in Europe annually, respectively [29]. Inequalities exist among countries (e.g., Russia and Ukraine versus France). Approximately 63% of ischemic and 80% of hemorrhagic strokes now occur in low- and mid-income countries [121]. Acute coronary syndrome is a collective term incorporating unstable angina, ST-elevation myocardial infarction, and non-ST-elevation myocardial infarction due to atherosclerotic plaque erosion that can evolve into rupture. The resulting intraluminal thrombosis engenders sustained myocardial ischemia and infarction owing to local partial or complete vascular occlusion or, most often, embolization and subsequent obstruction of downstream arterial segments upon shedding of platelet aggregates. Ischemia causes simultaneous massive cell death, releasing alarmins that activate NFκB, thereby producing proinflammatory cytokines. Debris from dead cells are taken up by macrophages, which then launch inflammation, relying on interferon regulatory factor IRF3 and type-I interferons, which protect against infection and cancer, IRF3 initiating a specific gene expression program. However, excessive IRF3 activation and type-I Ifn production are deleterious. Myocardial infarction stimulates IRF3 in a distinct population of interferon-inducible cardiac macrophages [122]. Secreted type-I interferons target the IfnAR receptor in an auto- and paracrine manner. In mice, deficiency in the cytosolic DNA sensor cyclic GMP–AMP synthase (cGAS), its adaptor, STING, the cGAS–STING axis activating IRF3 via TBK1, IRF3, type-I Ifns, or IfnAR, improves cell survival. In Irf3−/− mice, myocardial infarction-induced type-I Ifn response is nearly completely abrogated. Therefore, a transient inhibition of the interferon-dependent innate immune response in ischemia can reduce inflammation and limit the adverse ventricular remodeling. Limbs subjected to brief periods of ischemia protect multiple organs, in particular the lung, from ischemia–reperfusion damage. Limb remote ischemic preconditioning results from the release into the bloodstream of irisin, a myokine derived from

31 Although

coronary computed angiography provides the anatomy of stenoses and evaluates the extent of the lesion, it fails to assess ischemia and thus to guide the clinical management of coronary atherosclerosis. Nevertheless, some morphological features of atherosclerotic plaques, such as low-density plaque and expansion, in addition to fractional flow reserve derived from modeling, are employed to evaluate perfusion quality downstream from lesions and the infarction risk. Myocardial perfusion single-photon emission computed tomography is used to detect myocardial ischemia [119]. Plaques with lipid-rich necrotic cores are one of the main causes of myocardial infarction. Low-density noncalcified plaque is the most relevant feature associated with ischemia in arteries with 30–69% stenoses [120]. Contrast density difference, which is defined as the maximum percentage difference in contrast attenuation between the stenosed lumen and proximal normal reference segment is used to predict ischemia, is the most relevant plaque feature associated with ischemia in stenoses equal to or larger than 70%.

1.2 Vasculopathies and Cardiac Dysfunction

45

the extracellular portion of fibronectin domain-containing protein FnDC5 in skeletal muscle, which targets mitochondria and prevents some of the deleterious effects of redox stress [123]. Interaction between irisin and mitochondrial uncoupling protein UCP2 hampers ischemia–reperfusion event-induced redox stress and preserves mitochondrial function.

1.2 Vasculopathies and Cardiac Dysfunction Heart failure, a complication of coronary atherosclerosis, hypertension, cardiomyopathies, myocarditis, heart defects, and valvular heart disease (or heart valve disease), has an estimated prevalence in North America and Europe of up to 2%. Eighty percent of new cases occur in people older than 65 years, contributing to about 11% of deaths [121]. Rheumatic heart disease (RHD), which is most frequently detected in lowincome countries (Oceania; Central, South, and Southeast Asia; sub-Saharan Africa; the Caribbean; and Middle East [e.g., Yemen]), is the fifth and sixth leading cause of CVD-related mortality and disability, respectively [121]. Cardiomyopathies are categorized into various disease spectra according to their etiology and natural history, and these determine their medical management (Sect. 7.1). They can result from (1) left ventricular stiffening associated with adverse wall remodeling, (2) impaired sensitivity to β-agonists and insulin, (3) depressed autonomic function with altered myocardial catecholamine concentrations, (4) endothelial dysfunction, (5) abnormal ionic currents, and (6) disturbed flow in the coronary macro- and microcirculation. The most common forms are dilated and ischemic cardiomyopathies. Dilated cardiomyopathy (DCM) is currently defined by left ventricular or biventricular dilation and systolic dysfunction (i.e., abnormal ejection fraction) in the absence of abnormal loading conditions (e.g., hypertension and valvulopathies) or coronary atherosclerosis. It comprises a set of time-varying electrochemical and functional anomalies and can be engendered by genetic and acquired disorders. Genetic predisposition can be combined with environmental factors [124]. Inherited DCM can be transmitted by an autosomal dominant or recessive, Xlinked, or matrilinear mode. The main genes implicated in DCM encompass BAG3 (BCL2-associated athanogene-3), LMNA (lamin-A/C), MYBPC3 (cardiac myosinbinding protein-C), MYH7 (myosin heavy chain), MYPN (myopalladin), PLN (phospholamban), RBM20 (RNA-binding motif protein-20), SCN5A (voltage-gated sodium channel α subunit NaV 1.5), TNNT2 (troponin-T), and TTN (titin). DCM can also have a genetic origin within the framework of neuromuscular disorders, such as Becker and Duchenne muscular dystrophy and myotonic dystrophy, may be linked to mitochondrial diseases and tafazzin [124]. On the other hand, DCM can derive from viral, bacterial, fungal, and parasitic infections in addition to systemic diseases (e.g., polymyositis, sarcoidosis, and systemic lupus erythematosus) [124]. It can arise as a complication of acromegaly,

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1 Cardiovascular Disease: An Introduction

diabetes mellitus, hyper- and hypothyroidism, Addison and Cushing disease,32 and pheochromocytoma. Dilated cardiomyopathy can be induced by excess alcohol consumption and chemotherapeutic and psychiatric drugs and by electrolyte disturbances (hypocalcemia and hypophosphatemia), overload (iron) or deficiency (carnitine, copper, selenium, thiamine, and zinc), anti-heart antibodies, and toxics (e.g., arsenic and cobalt). Peri- and postpartum cardiomyopathy (PPCM) is caused by autoimmunity, fetal microchimerism, viral infection, stress-activated cytokines, and toxic cleavage product of prolactin [124]. Hypokinetic nondilated cardiomyopathy (HNDC) is defined by left ventricular or biventricular systolic dysfunction (ejection fraction < 45%) without dilation [124]. Ischemic cardiomyopathy results from altered flow in large epicardial coronary arteries that are stenosed and/or parietal microcirculation associated with chronic inflammation. Rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis yield an important risk background for myocardial ischemia. Hypertrophic cardiomyopathy (HCM) is another form with abnormal and often asymmetric myocardial thickening, preserved left ventricular function, phenotypic heterogeneity, and incomplete penetrance. This autosomal dominant inherited disease can be caused by mutations of genes (>50 genes, mainly those encoding sarcomeric constituents [e.g., MYL2]). The presence of additional risk factors, especially hypertension, exacerbates the disease penetrance and severity, as MYL2 E22K mutation does not exhibit clinical symptoms in most carriers [125]. Diabetic cardiomyopathy is characterized by reduced diastolic function and left ventricular hypertrophy. Its clinical management relies on appropriate glucose and HbA1c monitoring. However, in individuals of African ancestry, a specific variant that shortens the lifespan of red blood capsules reduces HbA1c concentration [126]. Diabetic cardiomyopathy is mainly linked to a shift to exclusive FAs as an energetic substrate for CMCs, instead of the usual sources (amino acids, carbohydrates, FAs, ketones, and lactate), due to AMPK at an early stage and then

32 Addison

disease is a chronic primary adrenal insufficiency, or hypocortisolism, cortisol being a glucocorticoid synthesized in the adrenal gland. It is most often caused by an autoimmune disorder that gradually destroys the adrenal cortex. This rare hormonal disorder affects about 1 in 100,000 individuals. Cortisol participates in maintaining blood pressure and controlling inflammatory response and the metabolism of carbohydrates, lipids, and proteins, especially the effect of insulin in carbohydrate catabolism. The pituitary gland secretes adrenocorticotropic hormone (ACTH; a.k.a. adrenocorticotropin and simply corticotropin), a component of the hypothalamic–pituitary– adrenal axis, which stimulates the adrenal gland. ACTH is secreted from corticotropes in the anterior lobe of the pituitary gland (or adenohypophysis) upon stimulation by corticotropinreleasing hormone (CRH) released by the hypothalamus. Conversely, glucocorticoid hormones block release of both CRH and ACTH (negative feedback). Secondary adrenal insufficiency results from a lack of ACTH. Chronically elevated ACTH concentration results from primary adrenal insufficiency such as Addison disease. Cushing disease results from benign ACTH-producing tumors of the pituitary gland that augments ACTH concentration, subsequently causing hypercortisolism. Surgical removal of ACTH-producing tumors of the pituitary gland engenders secondary adrenal insufficiency.

1.2 Vasculopathies and Cardiac Dysfunction

47

NR1c1 (PPARα) [127].33 On the other hand, endotheliocytes use preferentially glucose (∼85%) for ATP synthesis, the faster rate of glycolysis compensating for the greater amounts of ATP per mole of glucose yielded by mitochondrial oxidative phosphorylation and sparing oxygen for CMCs [127]. However, diabetic endotheliocytes have an aberrant metabolism. Production of GluT1 is not sensitive to hyperglycemia, and glucose egress to the CMC is not adequate. Moreover, high intracellular glucose concentration creates ROS and prevents glycolysis, glycolytic intermediates accumulating and being processed by the polyol, hexosamine, and methylglyoxal pathways, which form ROS and RNS and AGEs. Arrhythmogenic cardiomyopathy is mainly caused by mutations in genes encoding desmosomal elements. It is characterized by progressive fibroadipose replacement of the myocardium, arrhythmias, and sudden death. Cardiac mesenchymal stromal cells have a lower expression of plakophilin, contain more lipid droplets, and differentiate into adipocytes, contributing to the adipogenic substitution in arrhythmogenic cardiomyopathy. Arrhythmogenic atrial fibrosis characterized by excess extracellular matrix deposition and fibroblast proliferation and differentiation into collagen-secreting myofibroblasts favors atrial fibrillation (AF), the most common persistent arrhythmia. This cardiac rhythm disorder is the sixth and eighth leading cause of CVD-related mortality and disability among other CVD causes, respectively, the highest prevalence being observed in North America and lowest in the Asia-Pacific region [121]. Excess collagen can disrupt atriomyocyte bundle continuity, lessen intercellular coupling, and engender longitudinal anisotropy. Moreover, fibroblasts and myofibroblasts are electrochemically connected to CMCs, thereby modulating their electrical activity and promoting re-entry [128]. Persistent AF is maintained by re-entrant drivers (or rotors) related to extensive atrial remodeling, slowing action potential propagation, reducing cell excitability, and causing unidirectional block [129]. These rotors are confined in regions characterized with high fibrosis density.

1.2.1 Cardiac Wall Remodeling Cardiac walls remodel after pressure and volume overload or myocardial injury. This can result in heart failure.

33 Glucose

and lactate generate about 30% ATP and fatty acid oxidation about 70% [127]. Adaptive activation of AMPK ensures adequate cardiac energy supply, as it raises fatty acid delivery via its activation of lipoprotein lipase (LPL), repositioning of the fatty acid transporter ScaRb3 to the plasma membrane, and inactivating phosphorylation of acetylCoA carboxylase, inhibiting carnitine palmitoyltransferase CPT1, which carries fatty acylCoA into the mitochondrion. Activated cardiac NR1c1 elicits transcription of genes involved in various steps of fatty acid oxidation.

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Heart failure is associated with impaired signaling and pathological cardiac (or ventricular) wall remodeling (Vol. 7, Chap. 3. Adverse Cardiac Remodeling). Maladaptive cardiac remodeling is characterized by structural changes in dimensions, mass, and shape and metabolic remodeling with functional alterations due to molecular, cellular, and interstitial changes in response to abnormal hemodynamic load and/or damage linked to neurohormonal activation. Congestive heart failure is marked by atrial and ventricular wall enlargement and reduced cardiac contractility and adrenergic responsiveness. The sympathetic nervous system and renin–angiotensin–aldosterone axis are activated to compensate for reduced cardiac output but further favor heart failure progression via maladaptive wall remodeling. Cardiac modifications comprise cell death, redox stress, inflammation, hypertrophy and/or atrophy, fibrosis, and occurrence of arrhythmias. In particular, cardiac fibrosis causes electrical and mechanical dysfunction. Ion carriers (channels, pumps, and transporters), such as plasmalemmal (sarcolemmal) NaV 1.5 and CaV 1.2 channels, Na+ –Ca2+ and Na+ –H+ exchangers, KATP channel, sarco(endo)plasmic reticulum ryanodine-sensitive Ca2+ channel, and SERCA pump, in addition to their regulators, in particular kinases and phosphatases, are implicated in heart failure [130]. In heart failure, regulation of intracellular sodium and activity of K+ channels and Ca2+ cycling are defective.

1.2.1.1

Cardiac Wall Hypertrophy

Adverse left ventricular and arterial and arteriolar wall hypertrophy, along with associated stiffness, results from sustained hypertension (Vol. 7, Chap. 3. “Adverse Cardiac Remodeling”). However, hypertension-induced arterial wall hypertrophy of large- and medium-caliber arteries is not necessarily associated with a decreased arterial distensibility [131]. On the other hand, aging alters distensibility independently of blood pressure. Na+ –K+ ATPase (Na+ pump) is a plasmalemmal αβ dimer [132]. The catalytic ouabain-resistant α1 isoform is expressed in all cell types; most cells produce a second α isoform (ouabain-sensitive α2–α3, α4 being detected in the sperm). The catalytic α subunit contains the Na+ , K+ , ATP, and cardiotonic steroid-binding sites. Sodium ATPase β subunit exists in three isoforms (β1–β3) that support catalytic activity of chaperoned α subunit. β1 Subunit is the most important isoform in cardiac and vascular smooth muscle cells, where it forms both α1β1 and α2β1 protomers.34 Arterial smooth myocytes also manufacture α2 isoform, which localizes to endoplasmic reticulum–plasma membrane contact sites, the so-called plasmerosomes, and controls myogenic tone. On the other hand, α1 subunit is more uniformly distributed.

34 Astrocytes

synthesize α1 and α2 subunits and most neurons α1 and α3 subunits.

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49

Sodium pumps are regulated by multiple factors, such as hormones (e.g., aldosterone, insulin, and catecholamines) and protein phosphorylation [132]. The cardiotonic steroids, ouabain, digoxin, and bufalin, block cation transport by Na+ pump. Both Na+ and K+ affinities are modulated by the transmembrane regulator phospholemman (Plm) encoded by the FXYD1 gene (FXYD domain-containing ion transport regulator-1), which also regulates activity of Na+ –Ca2+ exchanger NCX1 [132]. Unphosphorylated Plm binds to the α2β dimer and reduces affinity of α2 subunit for intracellular Na+ and extracellular K+ ion. Phosphorylation of cardiac and arterial Plm by PKA or PKC relieves Na+ ATPase inhibition and restores high Na+ affinity. Hence, in arterial smooth myocytes, Na+ pump α2 subunit is structurally and functionally linked to NCX1. This crosstalk may be influenced by other adjacent channels, pumps, and transporters, such as TRPC6 and serca2 [132]. Activation of the renin–angiotensin–aldosterone axis stimulates ROS generation, causing glutathionation of β1 subunit and Na+ pump inhibition. On the other hand, Plm promotes Na+ pump deglutathionation and protects against oxidation (inhibition) of Na+ pump in arteries and the heart [132]. Reduced expression of smooth muscle-specific Na+ pump α2-subunit elevates blood pressure and sensitivity to angiotensin-2 and dietary salt, whereas its overexpression lowers basal BP and Agt2 and NaCl sensitivity [132]. Chronic salt retention augments endogenous ouabain-like compound (EOLC), a cardio- and vasotonic steroid synthesized and secreted by the adrenal cortex and Na+ pump inhibitor, thereby causing salt-dependent hypertension mediated by Na+ –Ca2+ exchanger. Ouabain triggers signaling that relies on various effectors, such as ERK1, ERK2, PI3Kc1α , PKB, and Src, in addition to NFκB [132]. In addition, another cardiotonic steroid, marinobufagenin, can be detected in human plasma and urine [132]. Prolonged exposure to ouabain or marinobufagenin causes hypertension in normal rats (but neither digoxin nor digitoxin). Sodium ion and water retention raises blood volume and subsequently plasmatic EOLC concentration, thereby inhibiting Na+ pump. Resulting elevated cytosolic Na+ concentration elevates cytosolic Ca2+ concentration due to Ca2+ entry through NCX, hence increasing myogenic tone and total peripheral systemic vascular resistance to blood flow. Transaortic constriction-induced hypertrophy in mice is impeded by immunoneutralizing circulating endogenous ouabain [133]. Endogenous ouabain and its receptor, Na+ pump α2-subunit, are involved in hypertension-induced cardiac hypertrophy. In many forms of hypertension, the brain RAAA is activated via circumventricular organs such as the subfornical organ, increasing arterial sympathetic nerve activity by the central nervous system and α-adrenoceptor-mediated arterial constriction [132]. The hypothalamic component of this neurohumoral pathway involves local aldosterone production, mineralocorticoid receptor, ENaCs, local endogenous ouabain release, and Na+ pump.

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The cardioprotective deacetylase sirtuin-2 operates via STK11 (LKb1) and AMPK in aging-related and angiotensin-2-induced adverse cardiac hypertrophy [134]. Sirtuin-2 deacetylates STK11 (Lys48), thereby eliciting STK11 phosphorylation and subsequently launching the STK11–AMPK axis. In Sirt2−/− aged (24-month-old) mice and Agt2-treated mice, cardiac hypertrophy and fibrosis are magnified. Conversely, cardiac-specific SIRT2 overexpression protects against Agt2-primed cardiac hypertrophy and fibrosis and rescues cardiac function.

1.2.1.2

Cardiac Wall Fibrosis

Fibrosis is a complication of chronic inflammatory diseases. Initiation of fibrogenesis involves activation of monocytes and differentiation into profibrotic macrophages. On the other hand, TGFβ provokes proliferation of myofibroblasts. Fibrosis is assessed by its regulators and markers MMP1–MMP3 and MMP7– MMP28 and TIMP1–TIMP4. In particular, TIMP1 inhibits MMP9 [135]. TIMP1 can be a strong predictor of death from CVD, at least in some populations, such as Iceland. Fibroblasts transdifferentiate into activated myofibroblasts, which synthesize α-smooth muscle actin (Actα2) and secrete matrix constituents such as typeI α1-procollagen (encoded by the COL1A1 gene). Persistent myofibroblast activation distinguishes pathological fibrosis from wound healing. Myofibroblasts integrate a feedback loop that perpetuates fibrosis and extracellular matrix stiffening. Fibroblast-to-myofibroblast differentiation driven by matrix stiffness provokes mitochondrial priming in activated myofibroblasts (but not in quiescent fibroblasts). Activity of proapoptotic proteins such as BCL2L11 thus increases in myofibroblasts that become particularly susceptible to apoptosis; these agents can reverse fibrosis [136]. On the other hand, myofibroblasts depend on antiapoptotic proteins such as BCL2L1 to prevent their death. Cardiac fibrosis is characterized by an uncontrolled accumulation of extracellular matrix by cardiofibroblasts in the interstitial and perivascular spaces. Transcription of typical fibrosis genes, such as Comp and NOX4, which encode cartilage oligomeric matrix protein and NADPH oxidase subtype NOx4, respectively, is upregulated. Hepatic fibrosis is characterized by the accumulation of matrix proteins, mainly fibrillar collagen-1, which confers mechanical stability. Cartilage oligomeric matrix protein (COMP),35 or thrombospondin-5, provokes collagen-1 formation via ScaRb3 and MAP2K1/2–ERK1/2 pathway, in addition to its deposition. Also, COMP supports matrix metallopeptidases MMP2, MMP9, and MMP13, but does not prevent collagen-1 cleavage by MMP1 [137].

35 Cartilage oligomeric matrix protein is an abundant component in the extracellular matrix of load-

bearing organs, such as tendons, cartilage, and pericartilage tissues. It interacts with other matrix proteins, such as collagens and fibronectin, thereby stabilizing the matrix.

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51

Cartilage oligomeric matrix protein binds with high affinity to collagen-1 and to collagen-12 at the surface of collagen-1 fibrils [138]. In fact, it tethers to the fibrilforming collagens Col1 and Col2, the nonfibrillar fibril-associated collagens with interrupted triple helices (FACIT) collagens (Col9, Col12, and Col14), along with other types of matrix proteins, such as fibronectin and matrilins, and proteoglycans. It also assists secretion of collagens [138]. Therefore, in the extracellular matrix, COMP helps the organization of a collagen fibril meshwork that yields the organ rheology, whereas within the cell, COMP enables efficient secretion of collagens into the extracellular space. This fibrillar collagen assembly regulator is implicated in fibrosis in various organs and can thus serve as a fibrosis marker [139, 140]. Cartilage oligomeric matrix protein is indeed synthesized in fibrotic regions, where it colocalizes with vimentin around SMAD3+ cells. Stimulation of fibroblasts with TGFβ1 increases COMP production. Reactive oxygen species produced by NADPH oxidases regulate cell differentiation. The isozyme NOx4 is implicated in cardiac and pulmonary myofibroblast differentiation. For example, in idiopathic pulmonary fibrosis, NOx4 expression rises in fibrogenic lung fibroblasts, which contain high concentrations of the hyaluronan receptor, epican variant containing exon 6 (CD44v6), thereby mediating TGFβ1-induced fibroblast differentiation into myofibroblasts [141]. Synthesis of hyaluronan and epican is augmented in numerous fibrotic organs. The TGFβ1– CD44v6 pathway is implicated in collagen-1 and Actα synthesis in pulmonary myofibroblasts [142]. It raises early growth response EGR1 formation. Production of CD44v6 is triggered by TGFβ1 via EGR1 and activator protein AP1. The ERK–EGR1 axis promotes CD44v6 splicing. Conversely, CD44v6 sustains ERK signaling, which supports AP1 activity in pulmonary fibroblasts. Hyaluronan produced by hyaluronan synthase HAS2 is required for colocalization of CD44v6 and Tβ R1 and subsequent TGFβ1–CD44v6–ERK1–EGR1 signaling, which constitutes a positive feedback loop that links TGFβ1 to the myofibroblast phenotype [142]. Transforming growth factor (TGFβ1), a major profibrotic factor, upregulates synthesis of IL11, which serves as its profibrotic effector. Interleukin-11 produced by activated fibroblasts does indeed cause cardiac fibrosis [143]. Its receptor IL11Rα is expressed at its highest concentration in fibroblasts. In these cells, the IL11–IL11Rα couple launches alternative ERK-dependent autocrine signaling used in fibrogenic protein synthesis. Production of IL11 is also upregulated in fibroblasts from patients with idiopathic pulmonary fibrosis (100-fold). After an acute myocardial injury, cardiofibroblasts release proinflammatory cytokines that trigger their proliferation (feedforward loop) and differentiation into myofibroblasts, which secrete high amounts of proinflammatory and fibrotic agents and matrix constituents. Adaptive collagen-based fibrotic scarring preserves myocardial structure, but prolonged activation of cardiofibroblasts causes fibrosis (Vol. 10, Chap. 3. Adverse Wall Remodeling). Fibroblasts extend filopodia into the T-tubular lumen. Heterotrimeric collagen6 (Col6α1–Col6α2–Col6α3 with other possible chains homologous to Col6α3 [Col6α4–Col6α6]) tetramerizes and, once it is secreted, forms microfibrils. Into

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T-tubules, collagen colocalizes with the dystrophin complex, which links the extracellular matrix to actin microfilaments and microtubules of the cytoskeleton, and transmits stress and strain between these two compartments [144]. Dystrophin localizes to the T-tubule periphery and serves as a mechanosensor at the Z disc. Deposition of fibrillar Col1 and Col3 stiffen, T-tubule membranes, whereas nonfibrillar Col4 and Col6 may anchor fibrillar collagens to the basement membrane of CMCs [145]. The endothelium-controlled paracrine couple constituted by neuregulin-1 and receptor protein Tyr kinase human epidermal growth factor receptor (HER) modulates cardiac performance and adaptation [146]. Neuregulin-1 operates on cardiofibroblasts and has an antifibrotic effect in the left ventricle. It attenuates myocardial hypertrophy and fibrosis in a mouse model of angiotensin-2-induced myocardial remodeling in addition to pulmonary fibrosis. Moreover, the Nrg1–HER axis also regulates the function of macrophages. Neuregulin-1 at least partly inhibits macrophages, alleviates myocardial macrophage infiltration and cytokine expression, and improves ventricular stiffness. On the other hand, in mice with myeloid cellspecific deletion of the Her4 gene, myocardial fibrosis in response to Agt2 increases. Neuregulin-1 activates HER4 on macrophages and inhibits the PI3K–PKB pathway in addition to STAT3, lessening inflammatory cytokine release. Myofibroblasts, which are derived from the differentiation of fibroblasts, fibrocytes, and epitheliocytes, are the principal effectors of fibrosis. Establishment and maintenance of myofibroblasts rely on TGFβ1-primed promotion of a hyaluronanrich pericellular matrix, the hyaluronan coat [147]. The heparan sulfate proteoglycan epican (CD44) is a receptor for the extracellular matrix constituent hyaluronan, which mediates cell–cell and cell–matrix interactions. It is encoded by the Cd44 gene, which consists of 19 exons. Its other ligands include collagens, osteopontin (or secreted phosphoprotein SPP1), soluble galactoside-binding lectin LGalS9, and matrix metallopeptidases. Hyaluronan has various isoforms due to a variable pattern of N- and O-linked glycosylation and the existence of multiple splice variants. Exons 1 to 5, 15 to 17, and 19, which encode the extracellular N-terminus, transmembrane domain, and the cytoplasmic region, are present in all alternatively spliced Cd44 mRNA species [147]. The presence of exons 6 to 14 varies between isoforms. Exon 18 is removed before translation in most isoforms owing to an early stop codon. In mice, exons v4 to v6 in splice variants (CD44v4, CD44v5, and CD44v6) facilitate migration of Langerhans cells (dendrocytes of the skin and mucosa) to lymph nodes [148]. In rats, exons v3 and v6 are involved in FGF-mediated mesenchymal cell proliferation during limb bud development. The standard epican isoform enhances myofibroblast differentiation, thereby favoring fibrosis [147]. On the other hand, its alternatively spliced isoform containing variant exons v7–v8, CD44v7v8, prevents myofibroblast differentiation. Hyaluronan is degraded by hyaluronoglucosaminidases or hyaluronidases, encoded by the HYAL1 to HYAL4 genes, Hyal1 and Hyal2 being the most abundant species. Hyaluronidase-2 supports CD44v7/8 production [147]. Hyaluronidase-2 lodges in lysosomes, the acidic milieu being optimal for Hyal activity. However,

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53

Hyal2 is also a plasma membrane-anchored protein with weak enzymatic activity. Hyaluronidase-2 can be enzymatically inactive. It plays a non-enzymatic role, as it participates in regulating epican splicing, promoting CD44v7v8 production. On the other hand, when pulmonary fibroblasts are stimulated by BMP7, which prevents or reverses differentiation of cells into myofibroblasts, Hyal2 translocates to the nucleus, where it displaces components of the splicing machinery from the spliceosome, enabling Hyal2, the spliceosomal components U1 and U2 small nuclear ribonucleoproteins, and Cd44 pre-mRNA to complex, whereas arginineand serine-rich (RS) proteins, which mediate exon exclusion, promote profibrotic standard CD44 synthesis. Both SRSF2 and SRSF5 control Cd44 pre-mRNA splicing relevant to fibrosis.36 Splicing regulators, argonaute-mediated histone modifications, KHDRBS1,37 and RS-rich splicing factor SFRS10 regulate Cd44 splicing [147]. Hyal2 facilitates the inclusion of Cd44 exons 11 and 12, which support expression of the antifibrotic CD44v7v8 isoform at the cell surface [147].

1.2.2 Cardiomyocyte Remodeling Markers of cardiac remodeling have either an increased expression, such as αmyosin heavy chain isoform (MyH6), GluT1, α-actin (ActC1), natriuretic peptide, galectin, caveolin, nitric oxide synthase (NOS1), angiotensin convertase or decreased production, such as β-MHC (MyH7), GluT4, and serca2a [149].

1.2.2.1

Energy Metabolism

Cardiac energetics is impaired because of mitochondrial dysfunction in addition to calcium handling, disturbing myocardial contractility. In the heart, the rates of ATP production and turnover are very high owing to contraction–relaxation cycles. Under normoxia, more than 95% of ATP generated in the heart is created by oxidative phosphorylation in mitochondria and the remaining

36 SRSF5

binds to intronic splicing sites within introns 10 and 11 of Cd44 pre-mRNA and recruits small nuclear ribonucleoproteins, forming mature spliceosomes (binary U1–U2 and ternary U4– U5–U6 complexes), which prime variant exon exclusion via double-exon skipping alternative splicing [147]. SRSF2 does not bind to the intron-10 and -11 region of Cd44 pre-mRNA but connects to the U1–U2 snRNP complex, promoting U1–U2 splicing initiation and mature trisnRNP U4–U5–U6 complex binding, thereby promoting the synthesis of standard Cd44 transcripts, which are translated into CD44s protein [147]. In the nucleus, Hyal2 counteracts SRSF5 action, displacing it from the early U1–U2 spliceosome and precluding the SRSF5-mediated formation of mature spliceosome and SRSF5 binding to intron 12 of the Cd44 pre-mRNA. In addition, Hyal2 may inhibit SRSF2 production and its interaction with Cd44 pre-mRNA. 37 KH domain-containing, RNA-binding, signal transduction-associated protein-1 is also termed 68-kDa Src-associated in mitosis protein (SAM68). It is activated by Ras and its effectors ERK1 and ERK2 [147], and favors profibrotic CD44v5 expression.

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1 Cardiovascular Disease: An Introduction

mainly from glycolysis and, to a lesser extent, from the tricarboxylic acid cycle (TCAC; also named citric acid and Krebs cycle) [150]. Approximately 70–90% of cardiac ATP is produced by fatty acid oxidation and the remaining from the oxidation of glucose and lactate, small amounts deriving from ketone bodies and certain types of amino acids. About two-thirds of the ATP generated is used by the sarcomere and the remaining by ion pumps such as endoplasmic reticulum Ca2+ ATPase (SERCA), which determines lusitropy. Substrates are transported across the plasma membrane into the cytosol, where they are metabolized. In oxidative pathways, the metabolic intermediates, such as pyruvate or acylCoA from glycolysis and β-oxidation, are transported across the inner mitochondrial membrane by specific carriers. Inside the mitochondrial matrix, these substrates are oxidized or carboxylated (anaplerosis) and enter the TCAC, thereby generating reducing equivalents, such as FADH2 and NADH, which are used by the ETC to generate a proton gradient, which, in turn, is used for ATP production. The energy generated is immediately used or stored in the form of phosphocreatine. Metabolic intermediates regulate many pathways in addition to ATP production, serving as messengers (Table 1.8).

Table 1.8 Metabolic intermediates regulators (Source: [150]; AMPK AMP-activated protein kinase, BCAA branched-chain amino acids, CoA coenzyme-A, ER endoplasmic reticulum, PGC peroxisome proliferator-activated receptor PPATγ coactivator, PKC protein kinase-C, PP protein phosphatase, ROS reactive oxygen species, TOR target of rapamycin) Metabolic regulators Acylcarnitines AcetylCoA AMP Ceramides Fatty acids Hexosamine NAD(P)+ /NAD(P)H Pyruvate ROS BCAA

Effects Activate Ca2+ channels Induces insulin resistance Primes cell growth and proliferation, as it promotes histone acetylation Influences metabolism via AMPK Activate PP2, PKCζ (insulin inhibition) Trigger mitochondrial and ER stress and apoptosis Activate NR1cs (PPARs) Modulate ion channel activity via palmitoylation Operates via O-GlcNAcylation of regulators Modulate activity of metabolic enzymes Regulate redox state and sirtuin action Stimulates mitochondrial genesis Regulates PGC1α expression Determine redox state and enzyme activity At high levels, induce apoptosis and inflammation Stimulates protein synthesis Activates TOR Inhibits autophagy

O-GlcNAcylation is the O-linked attachment of monosaccharide βN acetylglucosamine (O GlcNAc )

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55

The myocardial energy pool includes ATP and phosphocreatine (PCr), the latter being an ATP transporter and buffer. In the mitochondrion, the high-energy phosphate bond in ATP can be transferred to creatine by creatine kinase to form PCr, which can easily diffuse through the mitochondrial membrane into the cytosol, where it can generate ATP from ADP using cytosolic creatine kinase [150]. Glucose metabolism comprises glycolysis and accessory pathways, that is, glycogen synthesis and pentose phosphate (PPP) and hexosamine synthetic pathway (HSP). The PPP, which relies on glucose 6-phosphate dehydrogenase (G6PDH), is an NADPH source used in lipid synthesis and anaplerosis (i.e., replenishment of the TCAC intermediate pool through pathways independent of acetylCoA, as these intermediates are constantly removed from the TCAC for synthesis of amino and nucleic acids and thus need to be replaced), in addition to redox stress. The hexosamine synthetic pathway (HSP), which forms UDPN acetylglucosamine (GlcNAc), a monosaccharide donor for the O-GlcNAcylation of proteins, requires glucose together with acetylCoA and glutamine. These accessory pathways can play a greater role in heart disease genesis. Fatty acid oxidation is impaired in cardiac hypertrophy and failure, leading to reduced ATP production. Glucose oxidation can remain unchanged in compensated hypertrophy but can decrease in heart failure [150]. Non-ATP-generating pathways of glucose metabolism (HSP, PPP, and anaplerosis) are boosted. Metabolic remodeling in HF is characterized by a declined energy production linked to progressive impaired substrate use and mitochondrial genesis and function. 1.2.2.2

Ion Carriers

Cardiomyocytic T tubules contribute to regulating ion fluxes. Bridging integrator BIn1 (or amphiphysin Amph2) is a T-tubule protein residing in the inner membrane folds that is linked to calcium motion. The cardiac-specific splice variant BIn1v13v17 (including exons 3 and 17) promotes actin polymerization. It then generates and stabilizes dense T-tubule membrane folds that create a diffusion barrier to extracellular ions [151]. Its expression is downregulated in heart failure. Cardiofibroblasts influence myocardial function by their chemical, electrical, and mechanical interactions with CMCs. Multiple ion channels regulate their fate and activity (e.g., CaV 1, CaV 3, BK, SOCE, TRPA1, TRPC1, TRPC6, TRPM7, NaV 1.5, KV 4.3, KIR 2.1, and volume-sensitive [Clvol ] and Ca2+ -gated Cl− channel [ClCa or anoctamin-1]) [152]. They participate in myofibroblast differentiation. Defective ion handling by Ca2+ , K+ , and Na+ channels, pumps, and transporters in addition to connexins and nonselective channels causes cardiac arrhythmias. Defective Calcium Handling In the membrane of the endoplasmic (sarcoplasmic) reticulum, ryanodine-sensitive Ca2+ channel and SERCA pump enable Ca2+ release from and reuptake within this organelle. Released Ca2+ binds to the actin-connected troponin-C and allows actin–myosin interaction.

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Homotetrameric ryanodine receptor at junctions between the endoplasmic reticulum and transverse tubule is an essential component of excitation–contraction coupling via Ca2+ -induced Ca2+ release (CICR).38 It is linked to regulators, such as FK506-binding protein FKBP1b, a member of the immunophilin family of cis-trans peptidyl prolyl isomerases, cAMP-dependent protein kinase PKA and its anchoring protein, AKAP6, and protein phosphatases, PP1 and PP2 [130]. Muscle-selective AKAP6 coordinates a cAMP-sensitive negative feedback loop that comprises PKA and the cAMP-selective phosphodiesterase PDE4d3, PKA phosphorylating PDE4D3 and increasing its affinity for AKAP6, enhancing recruitment of PDE4D3 and hence faster signal termination [155]. Upon depletion of ER Ca2+ content and arrest of CaV 1.2 gating, that is, at the end of the systole, diastole begins, RyRs are inactivated and Ca2+ is pumped back into the ER by serca2a regulated by phospholamban and out of the cell by sarcolemmal NCX, which governs lusitropy. Stimulation by β-adrenoceptor increases inotropy. Calcium flux regulators are phosphorylated (activated) by PKA and CamK2, both targeting CaV 1.2 and RyR in addition to phospholamban, the latter facilitating the process. Failing ventriculomyocytes have an impaired contractility. The Ca2+ transient amplitude lessens, excitation–contraction coupling declines, and the rate of diastolic Ca2+ transient decay slows down. The SR Ca2+ content drops, serca2a activity is diminishing, and Ca2+ extrusion is caused by NCX rising. Neurohormonal stimulation causes RyR hyperphosphorylation by PKA and RyR dephosphorylation decays due to defective association of PP1 and PP2 and presence of PDE4d3 in the RyR complex [130]. In addition, increased RyR2 phosphorylation (Ser2808) favors its dissociation from its regulator FKBP1b, which increases Ca2+ sensitivity and reduced RyR closing, and hence diastolic ER Ca2+ leak. Dephosphorylation of phospholamban by PP1 inhibits serca2a; this inhibition is relieved upon its phosphorylation by PKA (Ser16) and CamK2 (Thr17). In

38 The

South American plant Ryania speciosa contains an insecticidal alkaloid, ryanodine. Ryanodine binds RyRs preferentially in the open state [153]. At nanomolar concentrations, it locks the channel in a subconductance state; at micromolar concentrations (>100 μ mol/l), it inhibits Ca2+ release. Among the three isoforms (RyR1–RyR3), RyR1 is widely expressed in the skeletal muscle, RyR2 is identified primarily in the heart, and RyR3 in the brain, although each isoform is found in many different cell types. The primary trigger for RyR opening is Ca2+ ion. Calsequestrin is a major Ca2+ buffer in the ER lumen that oligomerizes and interacts with the membrane-associated proteins junctin and triadin to control RyR activity [153]. Calmodulin associates with RyR at its cytoplasmic face; at high Ca2+ concentrations, it inhibits both RyR1 and RyR2; at low Ca2+ concentrations, it activates RyR1 but inhibits RyR2 [153]. According to [154], at nanomolar free Ca2+ concentrations, although apoCam inhibits RyR2, it potentiates RyR1 and RyR3 activity; at micromolar ones, Ca2+ –Cam inhibits all RyR isoforms. apoCam and Ca2+ –CaM inhibit RyR2. The adaptor homer-1C can activate RyR1 and inhibit RyR2 [153]. In addition, RyR is inhibited by Mg2+ and activated by ATP, cytosolic dimeric Ca2+ -binding S100a1, and NO, thereby potentiating Ca2+ release.

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heart failure, expression of both serca2a and Pln is altered [130]. In addition, Pln phosphorylation is reduced, repressing serca2a activity.

Altered Sodium Control In heart failure, intracellular Na+ concentration rises, in particular because of the late Na+ influx, a fraction of NaV 1.5 channels failing to enter an inactivated state [130]. Cardiac-specific sodium–hydrogen exchanger SLC9a1 (NHE1) electroneutrally exchanges intracellular H+ for extracellular Na+ to regulate intracellular H+ and intracellular Na+ concentrations, the inward gradient produced by the Na+ –K+ ATPase providing a driving force for SLC9a1-mediated H+ extrusion and Na+ influx [130]. Its activity increases upon exposure to ROS, intracellular acidosis, angiotensin-2, endothelin, and α1-adrenoceptor. Increased SLC9a1 activity causes Ca2+ overload through Na+ –Ca2+ exchanger. The Na+ –K+ ATPase39 actively transports Na+ out and K+ into the CMC. Phospholemman regulates the function of this enzyme. It is also the receptor of cardiac glycosides (e.g., digoxin and ouabain) and exerts a positive inotropic effect, as they inhibit pump activity, thereby decreasing the driving force for Na+ –Ca2+ exchange and increasing cellular content and release of Ca2+ during depolarization [156].

Impaired Potassium Control The transient outward current (iK,to ) through KV 4.2 and KV 4.3 is involved in early repolarization. It decays in heart failure, extending action potential duration [130]. Mitochondrial KATP channel (KIR 6.2) serves as a metabolic sensor, adjusting membrane excitability to match cellular energetic demand. It opens in response to ischemia, physical exercise, and stress hormone exposure, shortening the action potential. It can protect the heart against hypertension.

39 This heteromeric pump consists of α and β subunits. Several cell-specific isoforms of these subunits exist (α1–α4 and β1–β3). In the human heart, α1 to α3 are expressed together with β1 and, to a lesser extent, β2 in a region-specific manner [156]. The α1 isoform is ubiquitous and participates in pumping and signaling and in cell survival, ROS generation, and cardiac hypertrophy and fibrosis [157]. The α2 isoform contributes to regulating intracellular Ca2+ signaling and contractility in addition to adverse hypertrophy. The α3 isoform may be involved in cardiac hypertrophy. The expression of the α subunit is often altered in cardiac hypertrophy and failure.

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1.2.2.3

1 Cardiovascular Disease: An Introduction

Transverse Tubules

In heart failure, loss and defects of transverse tubules (T-tubules), that is, adverse reorganization of the T-tubular structure (e.g., dilation of the tubular lumen and the occurrence of sheet-like structures), affect Ca2+ signaling [145]. In ventriculomyocytes, excitation–contraction coupling depends on these deep invaginations of the plasma membrane at Z-line levels, where apposed cellular structures and proteic complexes reside, aimed at synchronizing Ca2+ release from the endoplasmic reticulum and hence contraction. Disrupted Ca2+ release reduces contractility in heart failure. Enlargement of the T-tubular lumen augments the diffusion space of extracellular ions, in particular Ca2+ and K+ , thereby favoring arrhythmia. In addition, loss of the T-tubular anchor junctophilin-2 decreases T-tubule density. Moreover, several collagen isoforms (Col1, Col3–Col4, and Col6) are involved in T-tubule expansion, whereas modest amounts of collagen normally exist within the interior of T-tubules [144]. 1.2.2.4

Mitochondrion

Integral outer mitochondrial membrane FUN14 domain-containing protein FunDC1 provokes hypoxia-induced mitophagy. It interacts with kinesin light chain KLC1 (but with neither the motor subunit of kinesin-1 KIF5b nor the motor subunit of kinesin-2 KIF3a) [158]. In addition, FunDC1 binds to endoplasmic reticulumresident inositol trisphosphate receptor IP3 R2 and localizes to mitochondrionassociated endoplasmic reticulum membranes, which participate in apoptosis and autophagy, hence promoting communication between mitochondria and the endoplasmic reticulum, modulating Ca2+ release, and maintaining MAERMs and mitochondrial morphology and function [159]. Overexpression of FunDC1 increases mitochondrial concentrations of IP3 R2 and Ca2+ ion. Ablation of the FUNDC1 gene reduces intracellular Ca2+ concentration and suppresses formation of mitochondrial fission protein Fis1 and hence mitochondrial fission, as it impedes the binding of CREB to the Fis1 promoter. The FunDC1–CREB–Fis1 axis is repressed in patients with heart failure.

1.2.3 Altered Signaling In normal conditions, CMCs and endotheliocytes interfere and mutually control their metabolism. Proper functioning of the cardiovascular system relies at least partly on interactions between CMCs and endotheliocytes of the cardiac capillaries and endocardium, dysregulated communication between these two cell types being implicated in the development of cardiac structural and functional anomalies and disturbed endothelium-related signaling based on NO and neuregulin being involved in heart failure [160].

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Table 1.9 Endothelial modulators of CMC activity (Source: [160]; cGMP cyclic guanosine monophosphate, CTGF connective tissue growth factor, Dkk dickkopf, ET endothelin, Fst follistatin, FstL follistatin-like protein, HER human epidermal growth factor receptor, miR microRNA, Nrg neuregulin, NO nitric oxide, PGi2 prostacyclin, PtgIR prostacyclin receptor, sGC soluble guanylate cyclase, TGF transforming growth factor, Tsp thrombospondin) Factor Apelin

Signaling AplnR

CTGF Dkk3 ET1

Integrin Wnt axis ETA(B)

Fst, FstL

TGFβ axis

MiR146a NO

Prolactin axis sGC–cGMP

Nrg1

HER4

Periostin PGi2

Integrin PtgIR

Tsp1/2/4

Adhesion molecule

Effect Positive inotropy Antihypertrophic Prohypertrophic Antihypertrophic and -fibrotic Positive inotropy and lusitropy Prohypertrophic and -fibrotic Antiapoptotic (FstL1) Antihypertrophic (FstL1/3) Induces peripartum cardiomyopathy Dose-dependent inotropic effect Positive lusitropy Attenuates cardiac remodeling Reduces contractility Attenuates heart failure progression Profibrotic Positive or negative inotropy Antihypertrophic Stretch-mediated contractility (Tsp4) Antihypertrophic and -fibrotic

Endothelial cardioactive factors encompass angiopoietins, angiotensin-2, apelin, dickkopf-3, endothelin-1, follistatin, neuregulin-1, NO, periostin, prostaglandins such as prostacyclin, thrombospondin-1, connective tissue, fibroblast, vascular endothelial growth factor, and endothelial microRNAs (Table 1.9) [160]. They operate briefly or have a sustained action, and they can cooperate. Nitric oxide and natriuretic peptides launch the synthesis of cyclic guanosine monophosphate using different effectors, sGC and particulate guanylate cyclase (pGC), respectively, and spatially distinct pools, sGC and pGC lodging in the cytosol and cortex, and hence different responses. Among phosphodiesterases hydrolyzing cGMP, PDE2 limits the subsarcolemmal cGMP pool and PDE5 the cytosolic cGMP pool [160]. Whereas cGMP produced by pGC and sGC has a positive lusitropic effect, cGMP produced by sGC blunts myocardial response to β-adrenoceptor. At low NO concentrations, the NO–sGC–cGMP axis can have a positive inotropic effect via activation of PKG and PKA, which increases Ca2+ concentration [160]. Higher NO amounts have a negative inotropic effect due to the blockage of sarcolemmal Ca2+ channels and reduction in the sensitivity of troponinC to Ca2+ ion. Moreover, NO has a positive lusitropic effect. Phosphorylation

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Table 1.10 Features of NOS isozymes (Source: [161]) Isozyme Typical site synthesis Other major sites of production

NO production magnitude Function

NOS1 Neuron Smooth muscle Skeletal muscle Cardiomyocyte Moderate (n–μ mol/l) Neurotransmission Inotropy − Lusitropy +

NOS2 Macrophage Smooth muscle Liver High (μ mol/l) Immunity

NOS3 Endotheliocyte Smooth muscle Cardiomyocyte Platelet Low (p–nmol/l) Vasodilation Vascular homeostasis Cardioprotection

of troponin-I reduces sarcomeric sensitivity to Ca2+ and promotes cross-bridge detachment. Phosphorylation of titin by PKA and/or PKG also improves lusitropy. Furthermore, NO provokes vasodilation and hence reduces afterload. The chronotropic effect of NO depends on its site of action, being positive upon stimulation by cGMP of a hyperpolarization-activated pacemaker current and negative at the postsynaptic level [160]. Nitric oxide is synthesized by nitric oxide synthases (NOSs), constitutive NOS1 and NOS3 binding their cofactors (FAD, FMN, and BH4 ), dimerizing, and being stimulated by Ca2+ –calmodulin (Table 1.10) [161]. NOS3 also requires proper localization to caveolae using HSP90 and caveolin and phosphorylation. In addition to constitutive NOS1 and NOS3 and inducible NOS2, constitutively active Mt NOS localizes to the inner mitochondrial membrane, where it participates in modulating the transmembrane potential. Nitric oxide mediates parasympathetic endothelium-dependent vasodilation in the vasculature in addition to parasympathetic control of cardiac function, guanylate cyclase supporting muscarinic agonists on the cardiac frequency, atrioventricular conduction, and myocardial contractility. Endotheliocytes secrete nitric oxide, which not only relaxes vascular smooth myocytes and prevents platelet aggregation, leukocyte–endotheliocyte adhesion, and vascular smooth myocyte proliferation but also influences CMC contractility via β-adrenergic and muscarinic acetylcholine receptors and control cardiac substrate utilization, NOS3 concentration being much higher in endotheliocytes than CMCs [127].40 The chronotropic and inotropic response to β-adrenergic and muscarinic agonists is preserved in isolated cardiac tissue preparations from Nos3−/− mice in addition

40 Nitric

oxide synthesized by vascular and endocardial NOS3 participates in controlling myocardial contractility via the NO–cGMP–PDE3–cAMP–PKA–Ca2+ channel axis [162]. On the other hand, NO reduces contractile response to adrenergic stimulation in heart failure, limits post-infarction remodeling, and protects against ischemia, at least partly via the NO–AC– cGMP–PKG–KATP pathway [162].

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to β-adrenergic stimulation and muscarinic inhibition of CaV 1.2a current [163]. However, NO formed in CMCs attenuates inotropic and lusitropic response to stimulation in addition to basal conditions [161]. In CMCs, NOS3 links to caveolin3 at the plasma membrane, where it can interact with CaV 1.2a and β AR, thereby blunting inotropic response to isoproterenol stimulation. On the other hand, NO produced by NOS1 at the CMC endoplasmic reticulum nitrosylates (activates) the ryanodine receptor [161]. In addition, NOS1 associated with NOS1-activating protein (NOS1AP) regulates cardiac frequency. In young Nos3−/− and Nos3+/+ mice, cardiac contractibility does not differ [164]. However, CMCs from old Nos3−/− mice exhibit a reduced inotropic response to isoproterenol with respect to age-matched Nos3+/+ mice. On the other hand, CMCs of Nos1−/− mice display a greater contraction and slower relaxation. Therefore, constitutive NOS3 in murine ventriculomyocytes does not markedly affect the muscarinic-mediated inhibition of β-adrenergic signaling and controls neither basal nor β-adrenoceptor stimulated CMC contraction. The myocardial constitutive NOS1 isozyme is responsible for the NO-mediated autocrine regulation of myocardial inotropy and lusitropy [164]. In addition, NO contributes to the metabolism regulation. It inhibits the ETC complex-I , -I I , and -I V of the mitochondrial electron transport chain [165]. Acute NOS inhibition reversibly affects cardiac substrate utilization. Cardiac uptake of lactate and glucose increases whereas that of free fatty acids decreases owing to a shift to carbohydrate oxidation, acute administration of a NO donor canceling cardiac metabolic changes [165]. Hence, NO hinders glucose uptake and supports free fatty acid consumption. Neuregulin-1, a member of the EGF superfamily,41 operates in the cardiovascular apparatus genesis and in the postnatal heart to regulate cardiac adaptation to stress. Nrg1 is released by the endocardial and microvascular endothelia. It binds to the receptor protein Tyr kinases HER3 and HER4 receptors, which are expressed in ventriculomyocytes [166], HER4 being the most important in the heart [160]. Nrg1 activates ERK1 and ERK2 (sarcomeric organization and protein synthesis), the Src– FAK couple (focal adhesion formation), NOS (cardiac function), and the PI3K– PKB axis (CMC survival), thereby attenuating adrenergic stimulation and hence its positive inotropic effect and enhancing lusitropy [160]. The Nrg1–HER couple also influences myocardial metabolism, provoking glucose uptake via PI3K by CMCs, and excess saturated fatty acid exposure causing Nrg resistance [127, 166]. In response to hyperglycemia and subsequent notch activation, ECs secrete inactive latent and lysosomal-stored active forms of heparanase using ATP, both

41 Four

related genes encode neuregulins (NRG1–NRG4), NRg1 being the most abundant member in the cardiovascular system. Alternative splicing at the C-terminus of the EGF domain of NRG1 leads to Nrg1α and Nrg1β variants, with distinct receptor affinity. Neuregulin-1 can be further subdivided into three types. Type-I Nrg1 is a type-I transmembrane protein, its active form being released after cleavage by adam17, adam19, or memapsin; type-I I Nrg1 is also cleaved, generating an active ligand on secretion; and type-I I I Nrg1 is almost exclusively produced in neurons and binds to membranes.

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heparanase forms liberating VEGFa and VEGFb bound to heparan sulfate proteoglycans (HSPG) of the CMC surface, which represent a rapidly accessible auxiliary reservoir, to facilitate fatty acid transfer by FABP4 (Sect. 2.3.2.2)42 and FATPs43

42 Fatty

acid-binding proteins (FABP1–FABP9) are intracellular lipid chaperones that can bind various types of hydrophobic ligands, such as saturated and unsaturated long-chain FAs and eicosanoids (e.g., leukotrienes and prostaglandins). Fatty acid-binding proteins facilitate FA transport in the cell for lipid oxidation in the mitochondrion or peroxisome, transcriptional regulation in the nucleus, membrane synthesis and trafficking in the endoplasmic reticulum, regulation of enzyme activity, and storage as lipid droplets in the cytoplasm [167]. The plasmalemmal PM FABP belongs to a distinct family of fatty acid-handling proteins. It is detected on the extracellular surface of cardiac and skeletal myocytes, hepatocytes, adipocytes, and endotheliocytes [168]. It also lodges in the mitochondrial membrane, acting as the glutamate oxaloacetate transaminase-2 (GOT2) and aspartate aminotransferase (AspAT). Fatty acid-binding protein FABP4, both a nuclear and cytoplasmic protein, contributes to maintaining glucose and lipid homeostasis. FABP4 is not only produced in adipocytes and macrophages but also in endotheliocytes, in which VEGFa via VEGFR2 (but not VEGFR1) and FGF2 upregulate its synthesis [169]. Inhibition of FABP4 blocks most of the VEGFa effects [170]. the DLL4–notch couple triggers FABP4 synthesis using the transcription factor FoxO1, independently of VEGFa [170]. Hence, FoxO1 is needed for the basal expression of FABP4, whereas its upregulated proangiogenic formation relies on VEGFa or notch. In fact, three FABPs are expressed in endotheliocytes (FABP3–FABP5). FABP3 is also synthesized in CMCs, renal epitheliocytes, and neurons of the brain; FABP4 in adipocytes and macrophages; and FABP5 in the heart, skeletal muscle, lung, and skin [168]. Adipocytic, macrophagic, and dendrocytic FABPs include FABP4 and FABP5 [167]. In adipocytes, FABP4 is a carrier protein for the transport of FAs generated by lipolysis from lipid droplets. β AR–AC–PKA and NPRa/GC–PKG pathways activate (trigger phosphorylation) of hormone-sensitive lipase (HSL or lipase-E), thereby priming lipolysis; FABP4 interacts with HSL [167]. In addition, FABP4 is secreted in association with lipolysis. Its plasmatic concentration decays after a meal with a high fat content, when the insulin concentration rises [167]. Insulininduced antilipolytic signaling does indeed suppress FABP4 secretion. Furthermore, FABP4 serves as an adipokine that promotes hepatic glucose production, reduces CMC contraction in addition to NOS3 activity in vascular endotheliocytes, and supports the proliferation and migration of vascular smooth myocytes in addition to glucose-stimulated insulin secretion in pancreatic β cells. In endotheliocytes, FABP4 promotes angiogenesis. Intermittent hypoxia increases FABP4 formation in endotheliocytes [167]. Conversely, angiopoietin-1 impedes FoxO1-mediated FABP4 synthesis. On the other hand, FABP4 and FABP5 may be involved in endotheliocyte senescence. In the kidney, FABP4 is expressed in endotheliocytes of the peritubular capillaries and veins in both the cortex and medulla, but not in glomerular or arterial endotheliocytes [167]. Ectopic FABP4 expression in the glomerulus is associated with renal dysfunction. In the lung, FABP4 is detected in endotheliocytes of peribronchial blood vessels and a subset of macrophages [167]. Interleukins IL4 and IL13 raise FABP4 production in bronchial epitheliocytes, whereas interferon-γ hampers it. 43 Diet-derived circulating lipids comprise mostly long- (lcFAs; 12–20 carbon atoms) and, to a lesser extent, medium- (mcFAs; 6–12 carbons) and short-chain FAs (scFAs; 1 cm), consists of multiple dilated feeding arteries and draining veins, which have a tortuous shape, hence with multiple arteriovenous connections [197]. It is adjacent to the collecting circuit. 2. Angiomatous AVM (size 1 cm), which resembles an acquired fistula. Yakes’ AVM classification defines: • Type-I anomalies, which connect a single artery to a single vein without a vascular nidus • Type-I I lesions, which link many arteries directly to veins and indirectly via arterioles and venules, which form a relatively simple network • Type-I I I malformations, which associate many arteries via arterioles to a dilated segment, which gives birth to a single or many veins • Type-I V anomalies that connect arteries and veins via a complicated arteriolovenular network [198] Coronary arteries can communicate with the cardiac chambers (coronary–cameral fistulas) or veins (coronary arteriovenous malformations). An arteriovenous fistula is an abnormal single direct passage from artery to vein. In addition to congenital fistulas, those acquired after birth are caused by infections, degeneration, trauma, or iatrogenic interventions (e.g., during angiography, biopsy, bypass grafting, and pacemaker implantation). Congenital arteriovenous malformation and acquired AVFs are rare causes of secondary hypertension.

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1.4.2 Venous Malformations Venous malformations (VMs) includes sporadic and cutaneomucosal (or mucocutaneous) VMs (CMVMs), and glomuvenous malformations (GVM) [190]. Multiple inherited venous lesions are identified in venous, glomuvenous, and cerebral cavernous malformations. Sporadic venous malformations are bluish or violaceous, solitary or multiple, localized or diffuse, superficial or deep lesions most often on the head and neck [113]. Venous malformations evolve slowly and progressively in the absence of bleeding. Thrombi and calcifications (phleboliths) can occur in tortuous veins. Venous nevus, or nevus venosus, is a variant of sporadic venous malformations. Blue rubber bleb nevus51 is a sporadic syndrome (BRBNS; also called Bean syndrome)52 characterized by cutaneous and gastrointestinal VMs of various numbers, sizes, and locations. It can be caused by mutations in the TEK (Tie2) gene. Nevi in the intestine can bleed spontaneously, provoking anemia. Families follow autosomal dominant inheritance but in fact have other multifocal venous malformations. Hemangiomatosis chondrodystrophica, also termed dyschondrodysplasia with hemangiomas, enchondromatosis with multiple cavernous hemangiomas, and Maffucci syndrome, primarily affects the bone and skin. It is characterized by multiple enchondromas (cartilage enlargements), bone deformities, and hemangiomas (tangles of abnormal blood vessels [benign tumors]). IDH is caused by mutations in the Idh1 or Idh2 gene, which encode NADP+ -dependent IDH1 and IDH2, respectively. Klippel–Trenaunay–Weber syndrome (KTWS) is a disorder pertaining to the PIK3CA gene-related overgrowth spectrum (PROS), which also includes megalencephaly capillary malformation and polymicrogyria syndrome (MCAP) and congenital lipomatous overgrowth, vascular malformations, epidermal nevi, and skeletal/spinal abnormalities (CLOVES) syndrome, hemimegalencephaly, fibroadipose hyperplasia, and epidermal nevus. It affects the development of blood vessels, engendering varicose veins and malformations of deep veins in the limbs, and causes overgrowth of soft tissues and bones. It results from mutations in the PIK3CA gene, which encodes PI3Kc1α .

1.4.2.1

Cutaneomucosal Venous Malformations

Cutaneomucosal venous malformations (in the skin and mucosae) commonly infiltrate underlying muscle and joints [190]. Although mostly sporadic (∼98% cases), CMVMs obey autosomal dominant inheritance. They are caused by mutations in the TEK gene located in the VMCM1 locus on chromosomal locus 9p21.22 (e.g., single-nucleotide polymorphisms R849W 51 A

nevus is an abnormal benign tissular patch caused by a cellular overgrowth. 1958, W. B. Bean coined the term blue rubber nevus syndrome for its color and consistency [199], although nevi of the viscera were discovered in 1860 by M. Gascoyen [200].

52 In

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73

and Y897S), which encodes the endothelial-specific receptor protein Tyr kinase TIE2 [190].53 These gain-of-function (GOF) mutations (e.g., C2545T in exon 15, A2690G, G2744A, C2752T, G2755T, and G2773T in exon 17, and G3300C in exon 22 [201]) increase ligand-independent autophosphorylation of TIE2 without launching endotheliocyte proliferation. Three TIE2 ligands include angiopoietins AngPt1, AngPt2, and AngPt4 (the latter corresponding to mouse AngPt3). AngPt1 has a stronger effect than competitive AngPt2, which is considered to be a AngPt1 inhibitor. Once it is liganded, TIE2 dimerizes and cross-phosphorylates, triggering mainly the MAPK module and PI3K pathway, which activates PKB and inhibits apoptosis.

1.4.2.2

Glomuvenous Malformations

Glomuvenous malformations are usually nodular multifocal lesions located on the extremities that involve the skin and subcutis, occasionally the mucosa [190]. They are characterized by abnormally differentiated vSMCs (glomus cells) in the walls of distended veins. These autosomal dominant disorders are caused by loss-of-function (LOF) mutations in the GLMN gene on chromosomal locus 1p21-22, which encodes glomulin, an essential protein for vasculature development. Glomulin is a ligand of the immunophilins FKBP1a and FKBP4, hence its other name, FKBP-associated protein. Glomulin synthesis is restricted to vSMCs; it is involved in their differentiation [190]. Differentiation of vSMCs also depends on TGFβ, which competes with glomulin to bind Tβ R1; glomulin thus precludes TGFβ signaling. Conversely, lack of glomulin provokes TGFβ hyperactivity. Glomulin also interacts with HGFR; upon HGF binding, glomulin is phosphorylated and released and triggers phosphorylation of S6K, thereby influencing protein synthesis. As it also interacts with Cul7, glomulin can also control protein degradation via ubiquitination by the CRL7 complex.

1.4.3 Capillary Malformations Capillary malformations (CapMs) form cutaneous lesions most frequently located in the head and neck. These slow-flow vascular malformations can comprise arterioles and postcapillary venules. Except for birthmarks, capillary malformations do not have a predilection for gender [113]. They are generally sporadic, but familial cases can be observed.

53 TIE:

protein Tyr kinase with immunoglobulin and epidermal growth factor homology domains.

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Megalencephaly capillary malformation syndrome, or macrocephaly (megalocephaly) cutis marmorata telangiectatica congenita, a rare, sporadic congenital capillary malformation, associates overgrowth of organs (megalencephaly) and cutaneous capillary malformations. These malformations are most often unilateral on the lower limbs. Enlarged capillaries augment blood flow near the skin surface. They can disappear spontaneously after several months or years, but they occasionally persist throughout life. This disorder is caused by mutations in the PIK3CA gene on chromosomal locus 3q26. PTEN hamartoma tumor syndrome (PHTS) refers to a spectrum of disorders characterized by multiple hamartomas,54 which are often intramuscular, multifocal, and associated with ectopic lipid depots. This spectrum includes (1) Cowden and Cowden-like syndrome involving mutations in the PTEN, SDHB, SDHD, and KLLN genes, which encode PTen, succinate dehydrogenase subunits B and D, and killin, respectively; (2) Bannayan–Riley–Ruvalcaba syndrome, characterized by macrocephaly and hamartomas of the intestine (hamartomatous intestinal polyps) resulting from mutations in the PTEN gene or partial or complete deletion of this gene; and (3) Proteus and Proteus-like syndrome,55 which is characterized by usually asymmetrical overgrowth of the bones, skin, and other organs and results from a mutation in the AKT1 (Pkb1) gene on chromosomal locus 14q32.3. Familial multiple nevi flammei is caused by mutations in the GNAQ gene, which encodes guanine nucleotide-binding (G) protein subunit Gαq . Nevi flammei (nevus flammeus neonatorum) correspond to birthmarks. These non-elevated, sharply circumscribed patches fade progressively. Salmon patches on the forehead, eyelids, and neck, in addition to the back, legs, and arms, also termed angel kisses, when erythematous macules typically affect the glabella, but also eyelids, nose, upper lip, and sacral region, and stork bites, when observed in the back of the neck, are picturesque names that depict very common birthmarks. A port wine stain (nevus flammeus) is a cutaneous firemark due to an abnormal aggregation of capillaries, the color of which (pink to purple macules) resembles port wine, the most common location being the face.

54 αμαρτας:

error; αμαρτημα and αμαρτια: failure, fault; the suffix “-oma” from -ωμα in medical terms meaning morbid growth, tumor (καρκινωμκ: cancer, chancre, sore, ulcer). A hamartoma is commonly a benign, focal malformation linked to disorganized tissular growth, which is made up of an abnormal mixture of cells normally found in the organ where it resides. For example, in the lung, hamartomas are composed of adipose, epithelial, and fibrous tissue and cartilage; pulmonary hamartomas are the most common benign tumors of the lung detected as solitary pulmonary nodules on medical images. 55 Πρωτ υς: Proteus, the old God of the sea, Poseidon’s eldest son; πρωτιoν: chief rank, first place; πρωτιoς: of the first quality. The Greek god Proteus, the ancient polymorphous creature, who can change his shape at will via manifold transformations. Proteus syndrome is an extremely variable condition involving atypical growth of the skin and skull observed in unrelated children.

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Nevus comedonicus is a rare type of epidermal nevus with predilection for the face and neck caused by mutations in the NEK9 gene56 on chromosomal locus 14q24. Nevus anemicus is a nonhereditary congenital disorder characterized by irregular hypopigmented macules that coalesce to form plaques, which are generally present at birth or develop in the first postpartum days. They localize especially on the chest. This disorder results from sustained vasoconstriction due to vascular hypersensitivity to catecholamines and not to partial aplasia of dermal blood vessels [113]. Nevus roseus is characterized by a pale red or even pink color; hence, its other name “rosé wine stain." It remains unchanged during life [113]. Phacomatosis pigmentovascularis associates a vascular nevus and extensive pigmentary nevus. It is categorized into five groups according to the pigmentary anomaly [113]: type I corresponds to nevus flammeus and pigmented linear epidermal nevi; type I I to nevus flammeus, Mongolian spots, and/or nevus anemicus; type I I I to nevus flammeus and spilus and/or anemicus; type-I V to nevus flammeus, Mongolian spots, and nevus spilus and/or anemicus; and type V to cutis marmorata telangiectatica congenita and Mongolian spots. This classification was later simplified into phacomatosis cesioflammea (i.e., nevus cesius [blue spot] and flammeus) and spilorosea (i.e., nevus spilus and roseus). Capillary malformation–arteriovenous malformation syndrome (CMAVM) results from mutations in the RASA1 gene, which encodes the Ras GTPase-activating protein (RasGAP) RasA1 (also aliased as CMAVM) [190]. Sporadic and autosomal dominant angioma serpiginosum (AS) is a benign cutaneous disease characterized by a progressive dilation of the subepidermal vessels manifesting as clusters of punctate erythematous lesions, usually on the lower limbs. It can be considered a type of capillary nevus. It occurs almost exclusively in women. It results from mutations in the chromosomal locus Xp11.3– Xq12.

1.4.4 Lymphatic Malformations Two categories of lymphatic malformations (LMs) affect the skin: lymphedema and congenital, superficial or deep, solitary or multiple lymphatic malformations. Lymphatic malformations are localized dilated lymphatic channels or pseudovesicles (lymphangiectasias) that are not connected to the lymphatic circuit. Cystic lesions are macro- (formerly called cystic hygromas) or microcystic, or mixed. Microcystic lymphatic malformations are also termed lymphangioma, lymphangioma circumscriptum or simplex, verrucous hemangioma, and angiokeratoma circumscriptum [113]. Macrocystic lymphatic malformations lodge in the neck, axillas, or lateral edges of the trunk. They can be solitary or multiple, and can be interconnected. 56 NeK:

never in mitosis gene-A (NIMA)-related kinase.

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Lymphedema, that is, chronic swelling in the body, usually in the lower extremities, due to abnormal lymphatic vessels, can be primary or secondary. Primary lymphedema comprises various types (Lmph1A–Lmph1D), which are linked to the chromosomal region 5q35, 6q16.2–q22.1, 1q42, and 4q34 [194]. Type-I A hereditary lymphedema (Lmph1A), also named primary congenital lymphedema (PCL) and Milroy disease, most commonly affects the inferior limbs, from the feet up to the knees. This autosomal dominant disorder is caused by missense mutations in the FLT4 gene, which encodes VEGFR3 [190]. Type-I B hereditary lymphedema (Lmph1B) is caused by anatomical or functional defects in the lymphatic circuit. It usually appears at birth or in early childhood but can occur later [194]. Type-I C hereditary lymphedema (Lmph1C) can be governed by autosomal dominant inheritance of heterozygous mutations in the GJC2 gene that encodes gap junction protein-γ2, or connexin Cx46.6 or Cx47 [194]. Type-I D hereditary lymphedema (Lmph1D) is engendered by heterozygous mutations in the VEGFC gene, the transmission pattern being consistent with autosomal dominant inheritance [194]. Type-I I late-onset lymphedema (Lmph2), also called Meige’s disease and lymphedema praecox, develops around puberty. It involves the upper and lower limbs, face, and larynx and can provoke a persistent pleural effusion. It can result from truncating and some missense mutations in the FOXC2 gene situated in the chromosomal locus 16q24.3 [190]. Hypotrichosis–lymphedema–telangiectasia syndrome (HLTS) is characterized by lymphedema and cutaneous telangiectasias. Both autosomal dominant and recessive inheritance can be observed [190]. Dominant and recessive forms are caused by mutations in the SOX18 gene, which encodes the transcription factor Sox18, an early marker of lymphatic differentiation. Sox18 interacts with MEF2c and regulates synthesis of vcam1. Osteoporosis lymphedema anhydrotic ectodermal dysplasia with immunodeficiency syndrome (OLEDAID) is engendered by mutations (e.g., X420W) in the IKBKG gene that encodes Iκ BKγ, which reduces NFκB activation [190].57 Lymphedema–cholestasis syndrome (LCS), also termed Aagenaes syndrome, is most often an autosomal recessive disorder, although an autosomal dominant mutation may be involved [190].

BKγ: inhibitor of NFκB (nuclear factor κ light chain enhancer of activated B cells) subunit-γ. The Iκ B kinase (IKK) complex is composed of three subunits IKKα, IKKβ, and IKKγ, which are encoded by conserved helix–loop–helix ubiquitous kinase (CHUK; also abbreviated as IKK1, IKKA, and IKBKA), IKBKB, and IKBKG gene, respectively.

57 Iκ

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1.4.5 Endothelial Signaling in Vasculo- and Angiogenesis Vasculo- (i.e., de novo blood vessel formation involving differentiation and migration of endothelial precursors), angio- (i.e., development of new blood vessels by capillary sprouting from preexisting vessels), and lymphangiogenesis construct (1) the vascular closed circuit, which is formed from arteries downstream from the heart, capillaries perfusing the body’s tissues, and veins upstream from the heart and (2) vascular walls, which are composed of vECs and mural cells (vMCs). The proper structure of blood and lymph vessels consists of a single layer of endotheliocytes surrounded by pericytes or a variable number of layers of vascular smooth myocytes separated by elastic laminae in the micro- (i.e., capillaries and upstream lymphatics) and macrovasculature, respectively. Correct organization of the vascular circuit requires the controlled activities of multiple types of messengers that regulate vessel formation, vascular branching, elongation and pruning, capillary fusion, vascular stability and anastomosis, and arterial and venous differentiation of endotheliocytes, which segregates arteries from veins. Vascular development and maintenance are controlled by a transcriptional program that integrates both extra- and intracellular signals in endotheliocytes. Vasculo- and angiogenesis are controlled by numerous signaling cascades in addition to hemodynamic stress. The initiation and formation of new blood vessels, that is, sprouting angiogenesis, is mainly regulated by the messengers VEGFa and notch. Angiogenesis is orchestrated by endothelial tip cells that form the vascular front and are followed by proliferating stalk cells. Tip cells sense multiple extracellular pro- and antiangiogenic signals and migrate toward the hypoxic region. In mice and cardiac organ culture, coronary vessels arise from angiogenic sprouts of the sinus venosus, that is, the vein returning blood to the embryonic heart [202]. Sprouting venous endotheliocytes thus dedifferentiate as they migrate over and invade the myocardium. Intramyocardial ECs then redifferentiate into arterial and capillary cells, whereas epicardial ECs redifferentiate into venous cells. Endotheliocyte differentiation into arterial and venous cells is genetically controlled for both vessel types. It precedes the onset of blood circulation. Arterial and venous angioblasts segregate from the beginning of vasculogenesis [203]. Acquisition of arterial identity is governed by a set of messengers (e.g., notch [Sect. 1.4.5.1], SHh [Sect. 1.4.5.3], and VEGF [Sect. 1.4.5.4]). Acquisition of venous phenotype relies on the nuclear receptor NR2f2 that suppresses notch signaling [203]. Ephrins and their receptors (e.g., the transmembrane ligand ephrin-B2 and its cognate receptor EPHb4 [Sect. 1.4.5.5]) are also involved in the establishment of arterial and venous identity. Susceptibility to certain vasculopathies differs between arteries and veins. The intracellular receptor NR2f2 is involved in regulating pathophysiological processes in adult blood vessels [204]. It acts as an antiatherogenic and -osteogenic agent that downregulates formation of inflammatory factors, upregulates that of antithrombotic agents, and represses osteogenic transcriptional program and endothelial-

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to-mesenchymal transition. It also regulates the TGFβ pathway, as it controls production of TGFβ2 and BMP4, which support conversion of endotheliocytes into mesenchymal stem cell-like cells and undergo osteogenic differentiation.

1.4.5.1

Notch, FoxC, SoxF, and NR2f2

The notch receptor (notch-1–notch-4) binds one of its ligands, jagged proteins (Jag1–Jag2) and delta-like ligands (DLL1–DLL3). In mouse early embryo at least, Jag1, Jag2, and DLL4 are specifically expressed in arterial endotheliocytes. Notch cleavage releases the notch intracellular domain (notchICD ) into the cytosol. NotchICD associates with RBPJκ,58 a DNA-binding protein and transcriptional repressor in the absence of notch signaling, and Mastermind (Mam), a transcriptional coactivator. NotchICD translocates to the nucleus, where it interacts with RBPJκ and converts it to a transcriptional activator, priming synthesis of basic helix–loop–helix (bHLH) transcription factors HESs59 and HRTs.60 In zebrafish at least, notch signaling acts downstream of the SHh and VEGF pathways in arterial specification. Notch-1 and notch-4 are essential for maintaining vessel identity. Notch signaling overcomes activin receptor-like kinase (ALK1) loss, as it restores EfnB2 expression in endotheliocytes. Notch signaling is also implicated in tip-to-stalk cell conversion (Vol. 5, Chap. 10. Vasculature Growth, and Vol. 10, Chap. 2. Vascular Growth and Remodeling). In endotheliocytes of mouse and zebrafish embryos, SOXF61 proteins act in synergy with RBPJκ [207]. They function upstream from notch signaling. Sox17 activates notch signaling, as it tethers to promoters of multiple genes involved in the notch pathway. Three SOXF genes, SOX7, SOX17, and SOX18, encode transcription factors of the SOXF group, which are expressed in vascular endotheliocytes during blood circulation development, whereas only Sox18 is involved in lymphangiogenesis [208]. Sox7 and Sox18 cooperate in the specification of arterial and venous identity.

58 RBPJκ:

recombination signal-binding protein for immunoglobulin-κ J region, that is, suppressor of hairless (SuH) homolog. It is also called C promoter-binding factor CBF1, SuH, and LAG1 and is abbreviated CSL. 59 HES: hairy and enhancer of split (HES1–HES7 [bHLHb37–bHLHb43]). 60 HRT: HES-related transcription factor (HRT1–HRT3 [bHLHb31–bHLHb33]). 61 Sox: sex-determining region Y (SRY)-related high mobility group (HMG) homeobox-derived (DNA-binding domain)-containing transcription factor. In other words, Sox transcription factors contain an SRY-related HMG homeodomain that is a DNA-binding sequence. In humans, 20 SOX genes are categorized into several groups: SOXA (SRY); SOXB1 (Sox1– Sox3); SOXB2 (Sox14 and Sox21); SOXC (Sox4 and Sox11–Sox12); SOXD (Sox5–Sox6 and Sox13); SOXE (Sox8–Sox10); SOXF (Sox7 and Sox17–Sox18); SOXG (Sox15); and SOXH (Sox30) [205, 206]. They are expressed by multiple types of progenitor and stem cells.

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Single nucleotide polymorphisms at the SOX17 chromosomal locus can engender intracranial aneurysms. High concentrations of VEGF stimulate production of delta-like ligand DLL4 by tip cells, which activates notch on adjacent endotheliocytes to confer stalk cell identity. In adult venous endotheliocytes, VEGFa inhibits formation of EPHb4, a venous marker, and stimulates that of DLL4, an arterial marker. Whereas DLL4 is involved in initiating the arterial program, DLL1 is required for the maintenance of arterial identity. In mouse embryos, DLL1 formation is restricted to arterial endotheliocytes after embryonic day 13 [208]. VEGF controls notch signaling, which is activated by DLL1, notch regulating neuropilin-1 (Nrp1) synthesis. Among transcription factors of the forkhead box group implicated in cardiovascular system development, members of the FOXC subgroup, FoxC1 and FoxC2, which are expressed in both arteries and veins of the mouse embryo, play an overlapping role. They contribute to regulating the formation of arterial-specific genes (e.g., Dll4 and Hrt2) and vascular remodeling of primitive blood vessels. They directly activate the Dll4 gene transcription using a FoxC-binding element (FBE), upstream of notch signaling [208]. In FOXC1+/− and FOXC2−/− mice, AVMs form and their endotheliocytes fail to express DLL4, and expression of other arterial markers (notch-1, notch-4, Jag1, HRT2, and EfnB2) declines [209]. Although FoxC1 and FoxC2 are required for DLL4 synthesis, deletion of the forkhead-binding element on the Dll4 promoter does not attenuate Dll4 gene transcription by the FoxC factors, notch1ICD and notch4ICD using the RBPJ-binding site. As FoxC2 and notchICD act synergistically on the Hrt2 gene promoter, FoxC and notchICD may also cooperate on the Dll4 gene promoter [209]. In endotheliocytes, FoxC1 and FoxC2 control expression of HRT2, the Hrt2 promoter containing two FBEs [208]. In addition, Foxc2, but not FoxC1, binds to the RBPJ (or Csl) gene promoter. Foxc2 complexes with CSL and notchICD to launch HRT2 synthesis. Production of DLL4 and HRT2 by FoxC is enhanced by VEGF in endotheliocytes. The genetic determinant of venous specification, NR2f2, is specifically expressed in venous endotheliocytes and acts upstream from EPHb4 in mice, impeding Nrp1 and notch formation. However, it cooperates with other factors for venous cell fate determination [208]. Furthermore, it interacts with Prox1 to launch lymphatic gene expression. Notch regulates responsiveness of endotheliocytes to BMP2 and BMP6 via inhibitory SMAD6, which is involved in neovessel branching formation [210]. The notch- and ALK1-mediated signaling cascades interact and can partly compensate for each other. Sequestration of BMP9 and BMP10 and subsequent ALK1 inhibition and notch blockage engender a hyperfused and hypersprouting vascular plexus in a neonatal mouse retina model [209]. SMAD1/5/8 binding sites exist in the regulatory region of many notch-targeted genes (e.g., HES1 and HRT1– HRT2).

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Wnt and Sox

Wnt signaling regulates multiple biological processes, such as angiogenesis, inflammation, and tumorigenesis. Wnt morphogens are secreted by cysteine-rich palmitoylated glycoproteins that play an essential role in cell fate determination, tissue homeostasis, and embryo- and fetogenesis. Canonical Wnt signaling elicits vascular invasion into the central nervous system [207]. Messengers Wnt and norrin target the receptor frizzled, coreceptors LRP5 and LRP6, coactivators tetraspanin-12 and GPR124, and effector β-catenin. In the absence of any of these Wnt signaling mediators, vascular cerebral network formation aborts, despite the high VEGF concentration produced by the hypoxic organ. Norrin (or Norrie disease protein), a homodimeric secreted cysteine-rich and cystine knot-like62 growth factor produced from a precursor encoded by the NDP gene, is an atypical Wnt ligand. It activates the canonical Wnt signaling pathway via Fzd4 and LRP5, acting in cooperation with TSpan12 to activate Fzd4, independently of Wnt [207]. Norrin mimics Wnt, as it can tether and activate frizzled via assembly of a molecular platform consisting of Fzd4, its LRP5–LRP6 coreceptor complex, auxiliary TSpan12, and associated HSPG [211, 212]. It then launches the Ctnnβ1— LEF/TCF axis. Norrin maintains the blood–retina and blood–brain barriers and regulates angiogenesis in the eye, ear (cochlea), brain, and female reproductive organs (uterus) [211]. In addition, norrin connects to secreted frizzled-related proteins (sFRPs). In retinal arterioles, capillaries, and veins, Sox17 production depends on frizzled4 and norrin [207]. Members of the SOXF group participate in regulating the development of the blood and lymph vasculature in addition to arterial and venous identity (i.e., vascular differentiation), remodeling, and maintenance in a functionally redundant fashion (strong, but partial redundancy), compensating for defective activity of any SOXF factor. Each SOXF factor exhibits a distinct pattern of production among the different classes of retinal blood vessels [207]. Sox7 and Sox18 have a similar temporal expression pattern. They are mainly produced in endothelia at the very early stages of endothelial differentiation, but their synthesis is differently regulated [203]. They also localize to distinct vessels in zebrafishes [213]. Both Sox7 and Sox18 are dispensable for the initial specification and positioning of the major trunk vessels. On the other hand, Sox17 is mainly formed during gastrulation [203]. All three SOXF group members are coexpressed in vascular endotheliocytes. Members of the SOXF group are reciprocally regulated in the developing blood vasculature.

62 The

cystine knot structural motif is contained in various types of peptides and proteins, such as ion channel blockers, hemolytic agents, and antiviral and antibacterial molecules. Three types of cystine knots exist: the growth factor cystine knot (GFCK), inhibitor cystine knot (ICK), and cyclic cystine knot (CCK). Norrin belongs to the GFCK category.

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In mice, Sox17 and Sox18 act redundantly in postnatal angiogenesis; Sox7 and Sox18 present an overlapping expression pattern [213]. Sox7, Sox17, and Sox18 are functionally redundant in the developing mouse retinal vasculature (cell differentiation and vessel growth) and maintenance of the mature vasculature [207]. Vascular endothelial-specific deletion of a single SOXF member gene has little or no effect on vascular architecture or differentiation because of the overlapping function of Sox7 and Sox17 and the reciprocal regulation of gene expression. Combined deletion of Sox7, Sox17, and Sox18 at the onset of retinal angiogenesis leads to a dense capillary plexus, with a nearly complete loss of radial arteries and veins, whereas the presence of a single Sox17 allele largely restores arterial identity with vSMC coverage. Indeed, Sox17 plays a major role in vSMC coverage of radial retinal arteries. In the developing retina, expression of all three SOXF genes is reduced in the absence of canonical Wnt signaling mediated by norrin and frizzled4 but remains unaffected by reduced VEGF signaling after deletion of the NRP1 gene. In adulthood, Sox7, Sox17, and Sox18 also have redundant functions in blood vessel maintenance. At adulthood onset, vascular endothelial-specific deletion of all three SOXF genes causes massive edema, despite nearly normal vascular architecture. The production of the endothelial adhesion G-protein-coupled receptor GPR124, also named tumor endothelial marker TEM5,63 is upregulated in endotheliocytes during physiological and tumoral angiogenesis. Its synthesis is induced by Rac during capillary network formation [214], and it prevents endotheliocyte proliferation.

1.4.5.3

Hedgehog

Secreted sonic hedgehog signals via the transmembrane receptor patched (Ptc) and G-protein-coupled receptor smoothened (Smo) on recipient cells. It can induce arterial cell fate in zebrafish angioblasts. Zebrafish embryos lacking SHh lose arterial expression of ephrin-B2, as it generates formation of VEGF, which, in turn, activates notch [208]. In mice, defective Shh signaling does not cause severe vascular defects, although vascularization is attenuated in the developing lung and formation of the dorsal aorta and remodeling of the yolk sac vasculature are altered. Murine SHh signaling may be dispensable for arterial and venous specification.

1.4.5.4

Vascular Endothelial Growth Factor

In angiogenesis, the specification of tip and stalk cells relies on VEGF. Relatively high VEGF concentrations provoke DLL4 synthesis in tip cells, which activates

63 Transmembrane tumor endothelial markers TEM1, TEM5, TEM7, and TEM8 abound in tumoral

vessels.

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notch signaling on adjacent endotheliocytes, thereby conferring stalk cell identity (Vol. 10, Chap. 2. Vascular Growth and Remodeling). In mice, lower-molecular-weight isoforms, diffusible VEGFa120 and intermediate VEGFa164 are required for arterial development in the retina, rather than VEGFa188 [208]. Neuropilin-1, a VEGFa164 coreceptor, cooperates with VEGFR2 to trigger signaling. In mice, when Nrp1 activity is defective, arterial differentiation is impaired. Vascular endothelial growth factor triggers the PI3K pathway and induces synthesis of notch-1 and DLL4, the VEGF–DLL4–notch–HRT2 cascade promoting arterial cell determination [208]. It modulates FoxC activity, and this modulation depends on the balance between PI3K and ERK activity. Relatively high VEGF concentrations (∼50 ng/ml) induce arterial marker genes, whereas lower VEGF concentrations (≤10 ng/ml) upregulate expression of the venous marker NR2f2; according to its level, VEGF signaling may preferentially activate either the PI3K or ERK pathway [208]. The protein Tyr phosphatase receptor, PTPRJ, is involved in arterial specification. It interacts with VEGFR2-primed signaling in endotheliocytes [208]. The calcitonin receptor-like receptor (CalRLR) is a G-protein-coupled receptor for adrenomedullin, which is coordinated with the VEGF and notch pathways in arterial differentiation in mouse embryos. CalRLR is expressed in the somite and arterial progenitors of zebrafish upon VEGF exposure, VEGF activity being regulated by SHh [208]. CalRLR supports arterial gene expression such as ephrinB2 and notch-5. Neuropilin-2 is expressed in venous and lymphatic endotheliocytes [208]. VEGFR3, which is initially detected in blood vessels of the early embryo, later becomes restricted to venous and then lymphatic endotheliocytes. The VEGFR3 ligand, VEGFc, is mainly expressed in mesenchymal cells surrounding embryonic veins [208]. Prox1+ VEFR3+ lymphatic endothelial progenitors subsequently bud and migrate from veins using paracrine VEGFc– VEGFR3 signaling, initiating developmental lymphangiogenesis. Hence, a subpopulation of venous endotheliocytes progressively synthesize the transcription factors Sox18 and Prox1 and acquire a lymphatic endothelial phenotype. Sox18 is first detected in a subpopulation of the cardinal vein and precedes the onset of Prox1 synthesis [208]. Sox18 induces Prox1 expression using two Sox18-binding sites on the Prox1 promoter. Sox18 is indispensable for induction of lymphatic differentiation but dispensable for lymphatic phenotype maintenance. Prox1 is a master regulator of lymphatic endothelial identity that elicits expression of lymphatic markers, such as VEGFR3 and lymphatic vessel endothelial hyaluronan receptor LyVE1. Moreover, Prox1 controls migration of lymphatic endotheliocytes triggered by VEGFc, as it cooperates with NR2f2 to prime synthesis of FGFR3, VEGFR3, and integrin-α9 [208]. FoxC1 and FoxC2 may contribute to regulating lymphatic vessel development, pericyte recruitment to lymphatic vessels, and lymphatic valve formation in a paracrine manner [208].

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Table 1.14 Regulators of arterial, venous, and lymphatic specification (Source: [208]; CalCRL calcitonin receptor-like receptor, Efn ephrin [EPH receptor interactor], EPH erythropoietinproducing hepatocyte receptor kinase, Fox forkhead box-containing transcription factor, NR nuclear receptor [transcription factor], Nrp neuropilin [VEGFR coreceptor], Prox Prospero homeodomain-containing transcription factor, PTPRJ protein Tyr phosphatase receptor type J, SHh sonic hedgehog, Sox sex determining region-Y box (SOX) homeodomain-containing transcription factor, VEGF vascular endothelial growth factor) Factor Arterial identity CalCRL EfnB2 FoxC1/2 Notch (notch-4) Nrp1 PTPRJ SHh Sox7/17/18 VEGF Venous identity EPHb4 NR2f2 Sox7/18 Lymphatic identity EfnB2 FoxC1/2 NR2f2 Prox1 Sox18

1.4.5.5

Effects Synthesized under control of the SHh–VEGF axis Segregates arteries from veins Regulate DLL4 and HRT2 expression SHh and VEGF effector DLL1 maintains arterial identity DLL4 elicits arterial specification Involved in a positive feedback loop of VEGF signaling Acts upstream from PI3K in arterial specification VEGF affector Control arterial and venous identity SHh effector Activates notch via the PLCγ–ERK pathway Segregates arteries from veins Suppresses arterial fate in endotheliocytes Inhibits Nrp1 and notch Confer arterial identity Lymphatic remodeling and maturation Lymphangiogenesis Interacts with Prox1 to regulate lymphatic gene expression Maintains lymphatic endotheliocyte identity Induces Prox1 expression

Ephrin-B2 and Its EPHb4 Receptor

The protein Tyr kinase receptor EPHb4 and its primary transmembrane ligand ephrin-B2 (EfnB2) are exclusively expressed on venous and arterial endotheliocytes, respectively (Table 1.14). They support but are not mandatory for arterial and venous specification. Expression of EfnB2 and EPHb4 is distinctively detected in the primary vascular plexus before the onset of circulation in the developing embryo [208]. Arterial–venous identity is genetically predetermined, although it is influenced by hemodynamic forces that enable remodeling and EC phenotype change.

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Bidirectional signals mediated by both proteins play an important role in vascular development. EfnB2 and EPHb4 are differentially expressed in arterial and venous endotheliocytes of the mouse embryo and thus considered to be markers of arterial and venous identity during embryogenesis. EfnB2 forward signaling via EPHb4 (EfnB2–EPHb4 axis) prevents cell adhesion and migration and suppresses cell proliferation, whereas EPHb4 reverse signaling via EfnB2 (EPHB4–EfnB2 axis) elicits cell attachment and migration [215]. The EfnB2–EPHb4 is involved in embryonic vascular circuit development, vascular remodeling, in addition to neovascularization, arteriovenous differentiation, and tumoral angiogenesis in adults. In hemorrhagic (hCeAVMs) and nonhemorrhagic cerebral arteriovenous malformations (nhCeAVMs), veins and arteries are coated by EPHb4+ and EfnB2+ endotheliocytes, respectively, EPHb4 and EfnB2 content being larger in hCeAVMs than in nhCeAVMs, whereas endotheliocytes of the normal superficial temporal artery express neither EPHb4 nor EfnB2 [216]. Arterial specification relies on VEGF that induces expression of notch and DLL4, the transcription factors FoxC1 and FoxC2 regulating DLL4 synthesis [208]. Notch stimulates HRT1 and HRT2, promoting arterial differentiation. On the other hand, the nuclear receptor NR2f2 is a determinant for venous specification, as it hampers expression of arterial specification genes, such as Nrp1 and notch [208]. A subpopulation of venous endotheliocytes progressively express the transcription factors Sox18 and Prox1, thereby acquiring lymphatic fate and differentiating into lymphatic endotheliocytes [208]. A mutual coordination of size between developing arteries and veins establishes a functional vasculature. The size of the developing dorsal aorta and cardinal vein is reciprocally balanced in mouse embryos. Gain-of-function notch mutations engender enlarged aortas and small cardinal veins, whereas LOF mutations show small aortas and large cardinal veins [217]. The dorsal aorta emerges before the cardinal vein via the assembly of endotheliocytes into the dorsal aorta primordium, a transient capillary plexus. Remodeling of this primitive structure generates the dorsal aorta. The cardinal vein appears slightly later, at a stage during which transient capillaries develop between the dorsal aorta and cardinal vein. Ephrin-B2 is specifically expressed in arterial endotheliocytes before the onset of blood circulation, but does not determine arterial specification of endotheliocytes [217]. Notch controls the proportion of endotheliocytes in the dorsal aorta and cardinal vein, as it promotes arterial specification and regulates both artery and vein size. Interdependence between arterial and venous size relies on a balanced allocation of endotheliocytes between these vessel types. Notch regulates endotheliocyte allocation, because it determines arterial specification and hence the ratio of arterial to venous endotheliocytes. Loss of EfnB2 or EPHb4 also leads to enlarged aortas and small cardinal veins. However, endotheliocytes with venous identity mislocalize in the aorta. EfnB2– EPHb4 signaling may operate distinctly from notch, sorting arterial and venous endotheliocytes into their respective vessels.

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Arterioles and venules are covered by EPHb4+ endotheliocytes. EPHb4+ capillaries of sprouts contain a significantly higher EphB4 amount than capillaries connecting arterioles and venules [218]. Hence, EPHb4 is not an arterial- or venousspecific marker in adult rat microvasculature but rather an indicator of capillary sprouting.

1.4.5.6

Transforming Growth Factor-β Group

Members of the transforming growth factor-β superfamily and among them, bone morphogenetic proteins, play an essential role in embryo- and fetogenesis and in the maintenance of organ function. Altered signaling in endotheliocytes by members of the TGFβ and BMP group causes diffuse malformations. Aortic aneurysms also arise from deregulated TGFβ/BMP signaling. The TGFβ signaling cascades involve: 1. Numerous messengers, three TGFβ subtypes (TGFβ1–TGFβ3), BMPs, growth differentiation factors (GDFs), activins, nodal, and inhibins. 2. 7 type-I receptors (ALK1–ALK7), ALK5 corresponding to Tβ R1, ALK3 and ALK6 to BMPR1a and BMPR1b, and ALK2, ALK4, ALK7, and ALK164 to AcvR1a to AcvR1c and AcvRL1, respectively. 3. 5 type-I I receptors (Tβ R2, BMPR2, AcvR2a–AcvR2b, and AMHR2). 4. Coreceptors, endoglin, which resides predominantly on endotheliocytes, cryptic, and β-glycan (Tβ R3); they modulate the activity of type-I and -I I receptors. Numerous ligand–receptor combinations trigger distinct TGFβ/BMP signaling. For example, ALK2, which primarily propagates BMP6 signal, can also function as a BMP9 receptor. Heterotetrameric receptors made up from type-I and -I I receptors, which, according to targeted type-I receptor type in the endothelium, predominantly endothelial ALK1 and ubiquitous ALK5, stimulate a given SMAD signaling cascade; ALK1 signals via SMAD1, SMAD5, and SMAD8 and ALK5 via SMAD2 and SMAD3. The transmembrane receptor ALK1 is activated by BMP9 (GDF2) and BMP10 and by TGFβ1, but weakly. Once ALK1 is liganded, cytosolic SMAD1, SMAD5, and SMAD8 are phosphorylated by the ALK1–Eng–Tβ R2 complex. Intracellular ALK1 signaling is implicated in diseases. Endothelium-specific ALK1 promotes arterial endothelial maturation and quiescence. In mice, deletion of genes encoding ALK1, endoglin, and MAP3K7 causes embryonic lethality associated with altered morphogenesis of the vascular circuit resulting from impaired arterial endothelium differentiation [219]. Phosphorylated SMADs complex with common SMAD4 and translocates to the nucleus, where they activate or repress transcription of specific target genes.

64 Type-1

activin receptor-like kinase ALK1 is also abbreviated AcvRL1 and HHT2, as LOF mutations in the ACVRL1 gene cause type-2 hereditary hemorrhagic telangiectasia.

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Inhibitory SMADs, Smad6 and Smad7, are linked to ALK1 and ALK5, respectively (self-regulatory loop). They compete for type-I receptor binding or recruitment of specific ubiquitin ligases or phosphatases for proteasomal degradation or dephosphorylation of receptors. In the early stages of blood vessel formation, proangiogenic BMP2 and BMP6 prevail; they signal via ALK2 or ALK3 [210]. On the other hand, antiangiogenic BMP9 and BMP10, two major ALK1 ligands, signal via ALK1 predominantly during vascular remodeling and maturation. Both BMP9 and BMP10 impede EC proliferation and migration. Endoglin and ALK1 are active in sites of vasculoand angiogenesis during embryogenesis. During mouse postnatal development, except in the lung endothelium, ALK1 synthesis decreases, but at a high enough concentration to keep the adult vasculature quiescent via BMP9–ALK1 signaling. In wound healing and during tumorigenesis, ALK1 production linked to angiogenesis increases. On the one hand, the BMP9–ALK1 couple upregulates the formation of notchrelated mediators, HES1 (bHLHb39), HRT1 (bHLHb31), HRT2 (bHLHb32), and Jag1, in addition to endoglin, ephrin-B2, transmembrane protein TMem100, and endothelin-1 [210]. On the other hand, it downregulates the formation of E-selectin, CXCR4, and apelin. Intracellular transmembrane protein TMem100, which localized mainly to the endoplasmic reticulum (but not to the plasma membrane), is an embryonic endothelium-enriched protein, synthesis of which is activated by BMP9 and BMP10 via the ALK1 receptor [219]. TMem100 may assist in post-translational protein modification or intracellular sorting. In neurons, TMem100 controls the interaction between ankyrin-like and vanilloid TRP channels TRPA1 and TRPV1, disconnecting them and promoting Ca2+ influx [209]. Both TRPA1 and TRPV1 reside on endotheliocytes; TRPV1 contributes to regulating the vasomotor tone, whereas TRPV4 facilitates arteriogenesis. Calcium signaling upstream from NFATc1 is defective in TMEM100−/− embryos [209]. In Bmp10−/− mice, cardiac growth is impaired without defects of angiogenesis [219]. Among ALK1 targets, ablation of TMEM100 gives rise to a phenotype similar to Alk1 mutants (but not identical) [209]. Both Alk1−/− and TMEM100−/− mice have heart defects, failed vascular remodeling, and abnormal dilation and narrowing of the dorsal aorta, in addition to detachment of the endoand mesodermal layers in the yolk sac. When TMEM100 is ablated postnatally, AVMs form in the lung and intestine, but not injury-induced cutaneous AVMs, as in Alk1−/− mice. Both TMEM100−/− and EC TMEM100−/− mice die in utero because signaling from ALK1, notch, and PKB decays or is even suppressed and hence differentiation of arterial endothelium and vascular morphogenesis are defective [219]. The notch heterodimer forms owing to calcium; hence, the transplasmalemmal gradient in Ca2+ concentration participates in notch activation in endotheliocytes [209]. When extracellular calcium concentration is low, notch subunits dissociate, promoting its cleavage.

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Notch and its ligands abound in arterial (but not venous) endothelium of mouse embryos. Notch-1, notch-4, DLL4, CSL65 (or RBPJκ), HRT1, and HRT2 elicit arterial cell fate [219]. Altered signaling from ALK1, notch, and TMem100 affects vascular smooth myocyte recruitment or differentiation during arterial maturation. In addition, activity of PKB and presenilin-1, which interacts with the notch pathway, are repressed in TMEM100−/− and Alk1−/− mouse embryos. The PKB kinase enhances PS1-mediated notch cleavage. Reciprocally, the PS1 peptidase provokes PKB activation. Altered presenilin-1 causes apoptosis via impaired PKB activity. Synthesis of ALK1 depends on blood flow, which promotes the association of endoglin with ALK1, thus sensitizing endotheliocytes to low BMP9 concentrations [210]. In ALK1-deficient mice, arteriovenous malformation, enlarged veins, and hyperbranching of the capillary plexus in the retina are observed. Retinal arteriovenous malformations occur predominantly in regions of higher blood flow. In addition, endotheliocytes have a migratory phenotype. Although the pericyte coverage is normal at the migration front of the retina, there is less pericyte coverage in capillaries in the central region of the capillary plexus. Endotheliocytes of AVMs express the venous marker EPHb4, but loss of the arterial marker Jag1. Endoglin-defective endotheliocytes are unable to sense and adapt to applied wall shear stress. This homodimeric glycoprotein of the vascular endothelium binds TGFβ1 with high affinity. It contributes to the regulation of angiogenesis, which involves tip cell selection, endotheliocyte proliferation and migration, mural cell recruitment, lumen formation, anastomosis, neovessel growth, and pruning. Endoglin is linked to Tβ R3 encoded by the TGFBR3 gene that retains TGFβ for presentation to the signaling receptors. It acts as a TGFβ coreceptor, which is particularly implicated in BMP9 signaling in endotheliocytes. Endoglin participates in the regulation of VEGFR2 signaling. Endoglin and VEGFR2 colocalize in intracellular vesicles. Endoglin affects VEGFR2 transfer and recycling and hence the balance between endotheliocyte proliferation and migration after VEGFa stimulation, favoring PKB activation. Furthermore, PKB phosphorylation promotes venous differentiation at the expense of arteriogenesis. Hereditary hemorrhagic telangiectasia (HHT; or Osler–Weber–Rendu syndrome) and cerebral cavernous malformation (CCM) result from lowered and elevated signaling from the TGFβ/BMP receptor complexes and sensitivity to messengers, respectively [210]. Endoglin cooperates with the component of the CCM pathway, which inhibits angiogenesis KRIT166 (or CCM1), and ALK1 (or HHT2). The CCM complex includes CCM1 (KRIT1), CCM2 (aka OSM67 and malcavernin), and CCM3 (or PdCD10).68

65 CSL:

C promoter-binding factor CBF1, suppressor of Hairless [SuH], and LAG1. Kirsten sarcoma virus Ras-revertant [KRev]-interaction trapped protein-1 [Krev1 being

66 KRIT1:

Rap1a]. 67 OSM:

osmosensing scaffold for MAP3K3. programmed cell death protein-10.

68 PdCD10:

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Loss-of-function mutations in the ENG and ACVRL1 genes provoke a defective regulation of processes involved in angiogenesis. Although LOF ENG mutations are linked to a mild hyperbranching phenotype, LOF ACVRL1 mutations favor tip cell potential and branching. Cells with LOF ENG mutations fail to adequately respond to migratory signals provided by the direction of blood flow [220].

1.4.6 Hereditary Hemorrhagic Telangiectasia Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder characterized by AVMs, capillary overgrowth, and fragile vessels in less than 2 in 10,000 individuals. In AVMs, flow bypasses capillaries; blood flows directly from some arteries directly to veins; the latter then undergo higher stress and strain and thus enlarge (enlarged shunts). Near the skin, they form telangiectasias, that is, focal dilations of postcapillary venules with excessive layers of vSMCs. Cutaneomucosal telangiectasias cause bleeding (epistaxis). AVMs occur in the lung, liver, and brain [190]. Several forms of HHT are distinguished mainly by their genetic cause rather than by differences in symptoms. Patients with type-I (HHT1) have symptoms earlier than those with type-I I (HHT2) and more frequently present vascular malformations in the brain and lung. The prevalence of pulmonary AVMs is greater in HHT1 than in HHT2 [209]. Juvenile polyposis combined with HHT, that is, a syndrome characterized by both AVMs, which grow and regress during life, and polyps in the gastrointestinal tract, is caused by mutations in the Smad4 gene [190]. Two additional chromosomal loci, 5q31 and 7p14, are linked to other types, HHT3 and HHT4 [190]. Heterozygous LOF autosomal dominant mutations in the ENG69 and Alk1 genes, which encodes two receptors of the TGFβ pathway that predominantly lodge on endotheliocytes, engender HHT1 and HHT2, respectively. Arteriovenous malformations are observed in the brain, spinal cord, lung, gastrointestinal tract, and liver. In endotheliocytes, LOF mutations of the ENG gene, which encodes endoglin cause type-1 HHT (HHT1) favored by VEGFa, and arteriolar endotheliocytes acquire venous characteristics. Endotheliocytes overexpressing endoglin serve as tip cells, preferentially in the arterial compartment [220]. Deletion of the ENG gene alters VEGFa–VEGFR2 signaling but primes the PI3K–PKB axis. Mutations in the ACVRL1 gene that encodes ALK1 are responsible for HHT2. Arterial endotheliocytes produce EfnB2, which participates in vascular development. Its concentration decreases in ACVRL1−/− mice. The ALK1 ligand BMP9 induces EfnB2 production in endotheliocytes via ALK1 and its coreceptors BMPR2 and AcvR2 [221]. 69 Eng:

endoglin. The ENG gene resides in chromosomal locus 9q34.

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89

BMP9 activates inhibitors of DNA binding ID1 and ID3 (bHLHb24–bHLHb25), both being required for EfnB2 formation [221]. Inhibitors of DNA binding heterodimerize with other ubiquitous or cell type-specific bHLH transcription factors, especially the class-1 bHLH transcriptional activators, E-proteins (TcFE2α [E2A or bHLHb21], TcF4 [E2-2 or bHLHb19], and TcF12 [HEB or bHLHb20]), but also effectors of ALK1 and notch signaling, HES1 (bHLHb39) and HRT1 (bHLHb31), thereby inhibiting their DNA binding.70 Both ID1 and ID3 repress cell differentiation, but support cell proliferation [209]. In addition, IDs downstream from SMAD1 and SMAD5 promote stalk cell phenotype during angiogenesis, avoiding excessive tip cell formation. Loss of ALK1 or EfnB2, which targets EPHb4 receptor involved in venous specification, causes arteriovenous anastomosis, whereas loss of ALK1 (but not EfnB2) upregulates VEGFR2 production and capillary sprouting. Conversely, BMP9 blocks endothelial sprouting via the ALK1–BMPR2–AcvR2 receptor complex in addition to ID1 and ID3 [221]. Several HHT markers encompass VEGF, TGFβ, soluble endoglin, angiopoietin2, clotting factor FV I I I , and von Willebrand factor, in addition to microRNAs, such as miR27a, a proangiogenic microRNA, miR205, which reduces EC proliferation, migration, and tubulogenesis and inhibits SMAD1 and SMAD4, and miR210 [210]. The GJA5 gene that encodes the gap junction protein connexin-40 is targeted by the BMP9–ALK1 pathway in human aortic endotheliocytes and can explain heterogeneity and the severity of HHT2 [222]. In ACVRL1+/− mice that develop AVMs similar to those in HHT2 patients, GJA5 haploinsufficiency causes arterial vasodilation and rarefaction of the capillary bed. Reduced Cx40 concentration also provokes ROS production and hence vessel remodeling. Capillaries form transient arteriovenous shunts that can develop into large malformations upon stressor exposure. Although some idiopathic AVMs are linked to elevated notch signaling (notch-1 and notch-4 in addition to Jag1 and DLL4), in HHT notch activity declines [209]. Both GOF and LOF notch signaling cause abnormal arterial and venous specification and hence fusion of arteries and veins. In adult mice, constitutively active notch-4 causes AVMs in the brain, liver, skin, and uterus, which can shrink upon removal of constitutively active notch-4. Endotheliocyte-specific constitutively active notch-1 also provokes AVMs. In HHT patients, AVMs grow because of endotheliocyte proliferation, which enlarges the arteriovenous shunt, whereas idiopathic AVMs result from endotheliocyte hypertrophy [209]. A decayed notch signaling increases endotheliocyte proliferation and may at least partly explain vascular enlargement in HHT-related AVMs.

70 HES1

binds to its own promoter, thereby preventing its synthesis and enabling proper loss of arterial identity in endotheliocytes. On the other hand, IDs preclude HES1 autoinhibition, but do not affect regulation of other HES1 target genes [209].

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1.4.7 Cerebral Cavernous Malformations Cerebral cavernous (or capillary venous) malformations consist of dilated capillarylike vessels (cavernomas) mixed with large saccular vessels with thickened walls in the cerebral parenchyma. Endotheliocytes lack tight junctions and are thus separated by gaps [190]. These disorders occur in a sporadic and familiar form with, in general, single and multiple lesions, respectively. They obey autosomal dominant inheritance; four chromosomal loci are implicated: (1) 7q11.22 with mutations in KRIT1 gene (Ccm1; ∼40% cases), (2) 7p13 with mutations in the Ccm2 gene (encoding malcavernin, a stabilizer of endotheliocyte junctions, also abbreviated CCM2), (3) 3q26.1 with mutations in the PDCD10 gene (Ccm3), and (4) 3q26.3–27.2 [190]. These disorders are pseudodominant diseases. Although patients are heterozygous for mutation in the Ccm genes, biallelic mutation of the Ccm genes is observed locally in lesions [210]. Biallelic Ccm mutation is also observed in lesions of patients with sporadic CCM. Loss-of-function mutations of the KRIT1 gene, which encodes KrIT1 produced in neurons, astrocytes, various types of epitheliocytes, and in capillary and arteriolar endotheliocytes, engender hyperkeratotic cutaneous capillary venous malformations in addition to CCMs [190]. KrIT1 links to microtubules and interacts with integrin-β1 -binding protein Itgβ1BP1, which participates in regulating cell adhesion and migration, thereby controlling EC fate. Conversely, Itgβ1BP1 can sequester KrIT1 in the nucleus. Malcavernin is able to sequester KrIT1 in the cytoplasm. PdCD10 may also be involved in the same pathway [190]. Gain-of-function mutations in any of the CCM genes exacerbate TGFβ/BMP signaling, endothelial-to-mesenchymal transition, and ultimately cerebral cavernomas. Sustained exposure to TGFβ and subsequent KLF4-induced augmented formation of BMP2 and BMP6 dismantle cellular junctions, increase vascular permeability, and provoke hyperproliferation and acquisition of mesenchymal markers, creating AVMs and multilumen cavernomas [210]. The transcription factor KLF4 enables endothelial-to-mesenchymal transition. Both KLF2 and KLF4 are overexpressed early after ablation of any Ccm genes. Stimulation by BMPs in cultured human umbilical vein ECs upregulates KLF4 synthesis [210]. However, KLF4 can also be activated by the MAP3K3–MAP2K5– ERK5 pathway, which also stimulates the transcription factors MEF2a and MEF2c, upregulating KLF4 production. In addition, KLF4 can launch BMP6 formation, thereby establishing a positive feedback loop.

Chapter 2

Cardiovascular Risk Factors and Markers

Quamdiu vivimus, necesse habemus semper quærere [As long as we live, we must always investigate] (H. de Lubac, [1896–1991]) [223]

Cardiovascular risk is assessed for the prediction and appropriate management of patients using collections of identified risk markers obtained from clinical questionnaire information, concentrations of certain blood molecules (e.g., N-terminal proB-type natriuretic peptide fragment and soluble receptors of tumor-necrosis factor-α and interleukin-2 [IL-2]), imaging data using various modalities, and electrocardiographic variables, in addition to traditional risk factors. These risk markers and factors can be combined to form standard cardiovascular risk scores (e.g., the Framingham risk score). These risk markers and factors can also be ranked according to their efficiency in predicting each cardiovascular outcome. The term biomarker has been defined as an indicator of physiological or pathophysiological processes and hence used for diagnostic, prognostic,1 and/or therapeutic purposes. Biomarker is defined by the National Institutes of Health as an entity “that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.” In a pathophysiological context, the useless prefix bio is omitted. Markers of redox stress encompass molecules modified by interactions with reactive oxygen species (ROS) (i.e., proteins, lipids, carbohydrates, and DNA) and molecules of the antioxidant defense [90]. Examples of circulating markers that have a value in addition to traditional cardiovascular risk factors include glycated hemoglobin (HbA1c) for glycemic control in diabetes, high-sensitivity troponin-I

1 Prognosis,

in general, refers to the expected course of a disease without treatment (natural prognosis) or after treatment (clinical prognosis). Prognosis is determined by certain endpoints (e.g., absence of complication recurrence). Prognosis is ameliorated by the technical improvement of medical and surgical interventions. © Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0_2

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(hsTnnI) and C-reactive protein (hsCRP) for cardiovascular risk prediction, and Nterminal proB-type natriuretic peptide for heart failure (HF). A risk marker is not necessarily involved in the cause or set of causes of a cardiovascular disease (CVD), like a risk factor. Markers of pathological conditions serve to: 1. Detect and sort individuals at risk for CVDs 2. Monitor disease progression 3. Select therapy and assess treatment efficacy Hence, features that determine the clinical utility of a marker include (1) the ease and cost of measurement, (2) its sensitivity and specificity, and (3) evidence for guiding management and improving patient outcome. A competing risk or cause precludes the occurrence of another risk or cause. For example, an adverse event is a competing cause with respect to death attributable to any cardiovascular event. The health of individuals can be defined, at least partly, by low levels of adverse markers of CVD. Cardiovascular health also relies on sets of risk factors that determine long-term survival and quality of life (Vol. 7, Chap. 2. Context of Cardiac Diseases). Clinical markers and factors, in particular, those related to inflammation, adverse remodeling, and thrombosis, assess the risk for CVD, which is refined using biological constants and imaging data. The risk factors comprise behavioral habits, such as smoking, physical inactivity, eating pattern (food quantity, meal composition and frequency, and use of processed, canned, and frozen food), which have an impact on the body weight, along with blood concentrations of cholesterol and glucose, and secondarily on arterial pressure [224]. In addition to clinical markers that are used for early detection, prevention of acute cardiovascular events targets modifiable risk factors, especially changes in habits (e.g., smoking cessation and supervised exercise) and therapy. Most CVD is indeed related to risk factors that can be controlled or treated, such as hypertension (systolic and diastolic arterial pressure greater than 18.6 kPa [SBP >140 mmHg] and 12.0 kPa [DBP >90 mmHg], respectively), dyslipoproteinemia, obesity, tobacco smoking, physical inactivity, and diabetes. The leading CVD risk factors comprise hypertension (∼13% deaths), followed by tobacco use (∼9%), hyperglycemia (∼6%), lack of physical activity (∼6%), and obesity (∼5%) [30]. Cardiovascular risk factors are similar for men and women. • The total risk of a person can be estimated by summing the risk imparted by each of the major risk factors. • The absolute risk is defined as the probability of developing CVD over a given time period. • The relative risk is the ratio of the absolute risk of a given patient to that of a low-risk group, that is:

2 Cardiovascular Risk Factors and Markers

93

– Systolic and diastolic blood pressure lower than 130 and 80 mmHg – Total cholesterol concentration ranging from 160 to 199 mg/dl – Low-density lipoprotein (LDL)CS ranging from 100 to 129 mg/dl2 and a 1-mmol reduction in LDLCS in middle-aged individuals for 5 years being estimated to yield a 20% decrease in CVD risk [121] – High-density lipoproteins (HDLs)CS greater than or equal to 45 mg/dl for men or 55 mg/dl for women – Nonsmoker – Absence of diabetes mellitus [226] The major independent cardiovascular risk factors include cigarette smoking, physical inactivity, aging, hypertension, obesity, diabetes, and dyslipoproteinemia with hypercholesterolemia and elevated LDLCS concentration but decreased HDLCS concentration [226]. Behavioral risk factors are shared by atherosclerosis complications (myocardial infarction [MI] and stroke) and diabetes, cancer, and respiratory disease [30]. Hypertension is a primary risk factor for stroke, especially long-term large variations of both systolic and diastolic arterial pressure observed in a series of medical examinations (either measured or recorded by ambulatory blood pressure monitoring), such as early morning (arousal) arterial pressure surges [227]. Large variations in arterial pressure can result from abnormal baroreceptor functioning, excessive sympathetic activity, elevated sensitivity to the renin–angiotensin axis, transient high-salt diet, acute fluctuations in body weight, sleep disorders, and ingestion of nonsteroidal anti-inflammatory drugs [228]. In addition, excessive arterial pressure reductions during treatment (SBP A>F. NKCC2a operates better under high perfusion rates. A second splicing mechanism involves an alternative polyadenylation site in exon 16 that produces two distinct C-termini: a long (NKCC2L ) and a short isoform (NKCC2S ), which lacks the last 329 residues of NKCC2L but contains 55 residues at its end that are absent in NKCC2L (NKCC2S has the property of a cotransporter and a regulator of NKCC2L but acts independently of K+ (Na+ –2Cl− cotransporter), which is inhibited by cAMP). Because these two splicing mechanisms are independent, six isoforms can be created (NKCC2L a, NKCC2L , and NKCC2L f and NKCC2S a, NKCC2S , and NKCC2S f).

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which have both renal and extrarenal effects, as they downregulate the expression of epithelial sodium transporters, cause vasodilation, suppress angiotensin-2 formation, and reduce Ald secretion [516]. Storage of sodium chloride in the skin attracts macrophages and engenders hyperplasia of the local lymphatic circuit via the NFAT5–VEGFc axis to guard against hypertension. However, local concentration of NaCl also activates proinflammatory helper T cells via NFAT5 and SGK1 [516]. In the kidney of individuals with essential hypertension, HSP70 can cause a proliferation of blood lymphocytes.

3.7.2 Nephron The nephron, the functional unit of the kidney, is organized into serial segments from the glomerulus to the cortical (CCD) and medullary collecting ducts (MCD). It successively comprises the proximal convoluted (PCT) and straight tubules (PST), the thin descending (tDL) and thin (tAL) and thick ascending limbs (TAL) of the loop of Henle, distal convoluted tubule (DCT), and connecting tubule (CnT; Fig. 3.1).

3.7.3 Renal Control of Water and Ion Balance The kidneys filter blood in their corpuscles take up in their tubules and ducts a fraction of filtered molecules, which return to the bloodstream and excrete the remaining with urine, which is collected by the ureter and stored in the bladder, before being removed through the urethra. Micturition need results from activated stretch receptors upon bladder distention and generate a reflex that triggers contraction of the detrusor muscle via its parasympathetic nerves and relaxation of the external urethral sphincter via inhibition of its motor nerves, both nerve types being under voluntary control. Kidneys maintain a proper water amount and salt concentration in the body. An elevated plasmatic osmolality (normal range 285–295 mosm/kg)6 is detected by hypothalamic osmoreceptors, which are neurons that sense changes in the

6 Osmolarity

describes the solute content in a solution, being the concentration of an osmotic solution; it corresponds to the number of milliosmoles per liter of the solution. It thus measures the bodily water–electrolyte balance (electrolytes: ionized or ionizable constituents of a living cell or biological tissues, e.g., blood). Osmolality is the number of milliosmoles per kilogram of solvent. Because the total solvent weight excludes the weight of any solutes, whereas the total solution volume includes solute content, osmolarity is slightly less than osmolality, the osmolar gap being the difference between the measured osmolality and calculated osmolarity. The equation used to estimate plasmatic osmolality is 1.86(Na+K)+1.15(Glu/18)+(Urea/6)+14 [519].

3.7 Kidney and Blood Pressure Control

CT

CT

211

DCT

G

S1 PCT

JGB DCT

S1

S2

PCT

G

JGB EGM MD

CDD cTAL

S3 cortex

S2 S3

mTAL

PST

tDL

TAL

PST outer stripe of outer medulla

OMCD

inner stripe of outer medulla tDL (HL)

VRR

tAL

IMCD

inner medulla

VRR

Fig. 3.1 Anatomy of short- and long-loop nephrons (CCD cortical collecting duct, CT connecting tubule, cTAL cortical thick ascending limb, DCT distal convoluted tubule, EGM extraglomerular mesangium, G glomerulus, IMCD inner medullary collecting duct, JGB juxtaglomerular body, MD macula densa, mTAL medullary thick ascending limb, OMCD outer medullary collecting duct, PCT proximal convoluted tubule, PST proximal straight tubule, tAL thin ascending limb, TAL thick ascending limb, tDL thin descending limb, VRR vasa recta renis). The proximal tubule can be divided into two serial segments, pars convoluta (i.e., PCT) and pars recta (i.e., PST)

osmolarity of the ECF. When ECF osmolarity rises, these osmoreceptors increase the action potential firing frequency to the neurohypophysis. Vasopressin is produced by neurosecretory cells of the hypothalamus and secreted from the neurohypophysis (or posterior pituitary gland). Elevated osmolality liberates ADH, causing insertion of aquaporins into the apical membrane of CD epitheliocytes and hence water reabsorption in the kidney and urine concentration. Aldosterone, the production of which in the adrenal cortex is triggered by the RAA, promotes K+ excretion and Na+ reabsorption, which provokes water reuptake and hence increases blood volume and pressure.

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Conversely, atrial natriuretic peptide (ANP), which is released by the stretched atriomyocytes subjected to an elevated blood volume, prevents renin secretion by the juxtaglomerular cells and elicits Na+ excretion and hence water removal, thereby reducing blood volume and pressure. Urine can be very dilute. Production of a concentrated or dilute urine relies on water reabsorption regulation in the CD immersed in a medullary hyperosmotic interstitial fluid. The loop of Henle generates a hyperosmotic environment in the medulla, because its upstream descending and downstream ascending limbs have different salt and water permeabilities. The water-permeable and ion-impermeable descending limb wall concentrate the filtrate by osmosis in the streamwise direction. The waterimpermeable TAL actively reabsorbs ions, thereby engendering a hyperosmotic interstitial fluid with respect to the tubular filtrate. The hypertonic interstitium drives a countercurrent flux in the loop of Henle that creates an osmolarity gradient into the medulla. Blood flow to the medulla through the parallel arteriolar and venular segments of the vasa recta is sluggish, plasma adjusting to the surrounding interstitial fluid without eliminating the osmotic gradient. An integrated endocrine system maintains calcium, phosphate, and magnesium homeostasis. It involves the calciotropic hormones, PTH,7 calcitonin (Calc),8 and (1,25)-dihydroxycholecalciferol,9 and other hormones (e.g., adrenaline, cortisol,

7 Parathyroid

hormone, or parathyrin, is synthesized and secreted by chief cells of the parathyroid glands as preproparathyroid hormone (proPTH). The Pth transcript (mRNA) encodes not only PTH, but also a 25-amino acid pre-peptide and a pro-hexapeptide. Furin (PCSK3), which localizes to the trans-Golgi network and functions in the constitutive secretory pathway, processes proPTH [520]. The 106-amino acid proPTH is rapidly converted into the storage (glandular) 84-amino acid PTH, which, shortly after entering the bloodstream, is cleaved into small inert fragments. Both PTH and parathyroid hormone-related protein (PTHRP), which act as an endo-, intra-, auto-, and paracrine messenger and also derive from a prohormone, are encoded by distinct genes that arise from gene duplication, Pth and Pthrp on chromosomes 11 and 12, respectively. Transcription of the PTH gene is activated by hypocalcemia, glucocorticoids, and estrogen [522]. It is released in response to hypocalcemia, adrenergic agonists, dopamine, and prostaglandin-E2 . On the other hand, its release is hindered by G-protein (Gq)-coupled calcium-sensing receptor (CaSR), which abounds in the parathyroid glands and kidney. In the kidney, CaSR hampers reabsorption of calcium, potassium, sodium, and water according to the activated nephron segment. Parathyroid hormone is involved in bone remodeling. 8 Calcitonin, or thyrocalcitonin, is synthesized and secreted by parafollicular (C) cells of the thyroid. It is cleaved from a high-molecular-weight precursor, procalcitonin (concatenated NT Calc–Calc–CnT Calc) peptide), which also engenders katacalcin and calcitonin gene-related peptide (CGRP). In addition to the free active mature Calc, small amounts of ProCalc, NT proCalc, and calcitonin C-terminus peptide CCP1 (CnT proCalc), the concatenated Calc–CCP1 peptide, and immature Calc (Imm Calc) circulate [521]. Calcitonin maintains bodily calcium stores, especially during growth, pregnancy, and lactation. It is also a neuroendocrine peptide produced by neuroendocrine cells throughout the body. 9 Also known as (1,25)-dihydroxyvitamin-D [(1,25)(OH)2 D ]). Vitamin D (cholecalciferol) 3 3 3 is a liposoluble steroid and a dietary component or cutaneous precursor synthesized from 7dehydrocholestrol by ultraviolet light. Hepatic 25-hydroxylase hydroxylates vitamin D to form 25-hydroxyvitamin-D (calcidiol), which enters the bloodstream and travels to the kidney, bound to its binding protein. In the kidney, tubular cells contain 1α- and 24α-hydroxylase, which can further

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Table 3.4 Renal effects of calciotropic hormones (↑ increase, ↓ decrease, PTH parathyroid hormone) Type PTH

Calcitonin Vitamin D

Table 3.5 Renal fractional reabsorption of calcium, inorganic phosphate, and magnesium (Source: [522])

Effect ↑ calcemia ↑ reabsorption of calcium and magnesium ↓ reabsorption of phosphate and bicarbonate (↑ phosphaturesis and bicarbonaturesis) ↑ excretion of cAMP ↑ conversion of 25OH D3 to (1,25)(OH)2 D3 1,25(OH)2D3 ↓ calcemia (PTH antagonist) ↓ reabsorption of calcium and phosphate ↑ calcemia ↓ calcium reabsorption Ion Ca2+ Pi Mg2+

PCT 60–70% 80–85% 10–30%

TAL 20% 10% 40–70%

DCT CD 10% 3–10% 3–5% 2% 5–10%

estrogen, growth hormone, insulin, somatomedin, testosterone, and thyroxin) regulate fluxes of minerals into and out of the ECF compartment via their effects on the intestine, kidney, and bone (Table 3.4). Imbalance between calcium, phosphorus, and magnesium causes arrhythmias and breathing anomalies, among other pathological effects [522]. Concentrations of calcium, phosphate, and magnesium ions depend partly on their regulation in different nephron segments, which involves numerous types of ion channels and transporters (Table 3.5). Differences in ion concentrations on opposite sides of the plasma membrane cause a difference in electrical potential (or voltage) between the inside and the outside of the cell, the transmembrane potential (TMP). The concentration of potassium ions is higher in the intracellular medium, and hence at the edge of the plasma membrane facing the matrix, than in the extracellular space and thus at the edge of the plasma membrane facing the cytosol. Conversely, there are higher concentrations of sodium and chloride ions in the extracellular region. The Na+ – K+ ATPase, which establishes potassium and sodium gradients between intra- and extracellular fluids, is a major determinant of the resting transmembrane potential (RTMP). The electrical charge separation across the membrane creates the membrane voltage, the electrical potential difference being physically situated in the immediate

hydroxylate calcidiol to form (1,25)(OH)2 D3 , the most active form of vitamin D3 (concentration 30 pg/ml), and (24,25)-dihydroxyvitamin-D3 , an inactive metabolite, respectively. PTH boosts production of (1,25)(OH)2 D3 , but blocks that of (24,25)(OH)2 D3 . (1,25)(OH)2 D3 exerts a negative feedback, as it simultaneously inhibits 1α-hydroxylase and stimulates 24α-hydroxylase.

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vicinity of the plasma membrane. A negative voltage in the cell interior exists with respect to the cell exterior, ranging at rest from −40 to −80 mV, and results from intracellular anions (mostly proteins). By convention, the zero potential value is assigned to the outside of the cell and thus the resting TMP is negative. Whereas passive ion channels are leaky pores that are always open, active channels are gated channels that open and close in response to stimuli (voltage-, chemical ligand-, and mechano-gated ion channels). Ions cross the plasma membrane driven by a chemical potential (i.e., moves down its concentration gradient) or electrical potential (i.e., moves away from ions of the same charge). A net inward movement (influx) of cations (Na+ ) causes depolarization, the TMP becoming less negative than the RTMP; a net outward movement of cations (K+ ) hyperpolarizes the cell, the TMP becoming more negative. Activity of certain gated ion channels depends on time. Rapidly inactivated ion channels can close a fraction of second after opening and cannot reopen again until the TMP is back to its resting level, thereby preventing further ionic flux. The change in TMP (u(t)) due to ionic fluxes (i(t)) can be described by an ordinary differential equation such as: dt u = ii (u, gi ) + io (u, go ) + ii (u) + io (u) + S,

(3.1)

where ii and io are inward and outward ionic currents, respectively, which can be gated [ii (u, gi ) and io (u, go )] or ungated [ii (u) and io (u)] (g(t): activation or inactivation gate of ionic flux), and S a source term related to a chemical, electrochemical, or mechanical stimulus.10 Any gated ionic flux can be given by: ii(o) (u, gi(o) ) =

gi(o) f (u) , τi(o)

(3.3)

where the function f (u) can be cubic (e.g., u2 (u − 1)), especially in the presence of slow–fast dynamics, and τ a time constant of selected in(out)ward ion flux that controls membrane polarization (i.e., strength of ionic current). Gating can be defined by:

10 In

the presence of an action potential propagation, a partial differential equation describes the evolving transmembrane potential:  i ∂t u = ∇ · (D∇u) − , (3.2) Cm where D = Gel /(RSV Cm ) is the anisotropic diffusion tensor linked to a preferential propagation direction, which is a function of the tissular electrical conductivity (Gel ), membrane capacitance per unit area (Cm ), and surface-to-volume ratio (RSV ).

3.7 Kidney and Blood Pressure Control

dt gi(o) =

215

f (gi(o) ) , τopen(close)

(3.4)

where the function f (gi(o) ) depends on whether the gate is open or closed.

3.7.3.1

Water Homeostasis

Water balance results from controlled urine output, in addition to less regulated loss of water through the lung, skin, and gastrointestinal tract, and water intake, feedback mechanisms controlling thirst and diuresis. Sodium and chloride are the major solutes in ECF and determinants of body water content and circulating volume [523]. Water channels of the aquaporin family (Aqp1–Aqp7) ensure water transport across the plasma membrane such as that of tubular epitheliocytes in the kidney. Among ten aquaporin isoforms, at least seven reside in the kidney at distinct sites along the nephron and CD (Table 3.6) [524]. Aqp1, Aqp2, Aqp3, and Aqp5 tetramerize in the membrane, whereas Aqp4 oligomerizes. The “orthodox set” of aquaporins have a relative selectivity for water, whereas the “cocktail set” of aquaglyceroporins also convey glycerol and other small solutes in addition to water. The Aqp6 subtype, which exclusively lodges only on intracellular vesicles, also conducts anions, hence functioning as an anion channel. The Aqp1 pore has the capacity to participate in ionic signaling upon cGMP activation, but without marked effect owing to a limited permeation (a single ion channel per 107 Aqp1) [524]. Nevertheless, Aqp1 can be permeable to small gases such as CO2 , but also without significant effect. These water channels can be gated upon activation of

Table 3.6 Aquaporins (Aqps) in the nephron (Source: [524]; CD collecting duct, C(OM[IM])CD cortical (outer [inner] medullary) CD, CnT connecting tubule, ICC intercalated cell of the distal nephron, PC principal cell of the distal nephron, PT proximal tubule, tDL thin descending limb of the loop of Henle) Isoform Aqp1 Aqp2 Aqp3/4 Aqp3 Aqp4 Aqp6 Aqp7 Aqp8

Location PT (S1/2/3), tDL Descending vasa recta CnT, CD (PCs; apical and subapical regions) (predominant ADH-regulated channel for urine concentration) CD (PCs; basolateral plasma membrane) (water reabsorption in cooperation with Aqp2) Abounds in CnT, CCD, OMCD, IMCD Abounds in the inner medulla; also in PT (S3) CD (CCD, OMCD, IMCD; ICCA s; intravesicular) PST (i.e., S3; brush border) PT, CD (PCs) [intracellular]

Osmotic water transport across the tubular epithelium relies mainly on aquaporins. Vasopressin stimulates transfer of Aqp2 from intracellular vesicles to the apical plasma membrane, thereby acutely promoting water permeability of the collecting duct

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adenylate cyclase. Concentration of cAMP in CD principal cells (PCs) rises upon binding of vasopressin to its Gs-coupled V2 receptor; cAMP activates PKA, which phosphorylates Aqp2 that can then be carried to the plasma membrane. On the other hand, PGe2 reduces the cAMP level and thus prevents elevation in water permeability induced by vasopressin. The Aqp1 isoform also localizes to the descending vasa recta (arteriolar segment), at least in rat kidneys, but not in the ascending vasa recta (venular segment) [524]. Water equilibration across the vasa recta may also avoid disruption of the osmotic gradient established by the countercurrent exchanger. Collecting duct water permeability is modulated by the vasopressin–aquaporin-2 pathway using an acute regulation of Aqp2 membrane insertion from subapical storage vesicles (short-term regulation linked to vasopressin-regulated Aqp2 translocation) and delayed adaptation to water restriction (long-term regulation resulting from augmented synthesis of Aqp2 in the PCs of the CCD and inner medullary collecting duct [IMCD]) [525].

3.7.3.2

Sodium Homeostasis

The kidney is a main actor in the long-term control of BP. High salt intake is a major risk factor for hypertension. The distal nephron under control of aldosterone determines the final quantity of salt that is excreted and hence supports the maintenance of salt balance, which controls the ECF volume and BP. Inhibitors of Na+ carriers in the aldosterone-sensitive distal nephron (ASDN; i.e., DCT2, CnT, and CD), such as ENaC and Na+ –Cl− cotransporter (NCC), permit BP control. Repressors either prevent insertion in the plasma membrane or promote retrieval of the channel from the plasma membrane (e.g., NEDD4-2). The proximal tubule reabsorbs 50–60% of water contained in the glomerular ultrafiltrate and 40–50% of filtered salt. On the one hand, NaC1 reabsorption in the PST relies on an active transcellular Na+ transport generating a lumen-negative transepithelial potential difference with passive paracellular Cl− transfer down its electrochemical gradient [526]. On the other, Na+ conductance is coupled with organic solute transport in the PCT. Sodium absorption is coupled with the uptake of solutes (e.g., glucose, amino acids, phosphate, sulfate, and lactate) by cotransporters [527]. Excreted fractions of glycine, histidine, and taurine are small (3.5, 6, and 6%, respectively), as amino acids are reabsorbed predominantly in the PCT and to a small extent in the PST through carriers using a Na+ electrochemical gradient across the luminal membrane [528]. The high-affinity electrogenic sodium–glucose cotransporter SGlT1 (or SLC5a1) in the apical membrane of epitheliocytes of proximal tubules (PT) uses the Na+ electrochemical gradient to drive the uphill glucose influx (ion–substrate coupling stoichiometry: two Na+ and one glucose molecule). In the ASDN, WNK1 and WNK4 control the activity of apical Na+ –Cl− cotransporter (DCT NCC or SLC12a3) [529]. WNK4 hampers SLC12a3 insertion

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217

from cytoplasmic vesicles into the plasma membrane, whereas WNK1 abolishes inhibition of WNK4 [530]. Type-I I pseudohypoaldosteronism (PHA2) is an autosomal dominant disorder characterized by hyperkalemia and hypertension caused by mutations in the genes encoding WNK1 and WNK4; gain-of-function Wnk1 mutations favor NCC activation, contributing to PHA2, the opposite of the Gitelman syndrome, an autosomal recessive disease with metabolic alkalosis and hypokalemia, in which SLC12A3 gene mutations disrupt NCC function. Salt reabsorption and its control are described, at least in some compartments of the nephron in sections devoted to these tubular subdomains (e.g., Sect. 3.7.5.4). Flux of Na+ is related to those of water and K+ (Sect. 3.7.3.3).

Dopamine-Mediated Natriuresis The picomolar concentration of circulating dopamine is too low to efficiently activate dopamine receptors, but nanomolar concentrations can be attained in dopamine-producing tissues, such as renal proximal tubules and the jejunum. Proximal tubule epitheliocytes synthesize dopamine using aromatic amino acid decarboxylase, or DOPA decarboxylase. Dopamine is not converted to noradrenaline, as it is in the nervous system. In addition, dopamine is synthesized in the kidney independently of renal nerves [531]. Dopamine is degraded to 3-methoxytyramine by catechol O methyl transferase. Dietary sodium is the major determinant for dopamine synthesis in the proximal tubule and release preferentially into the tubular lumen [506]. Dopamine, an auto- and paracrine regulator, acts as an intrarenal natriuretic hormone, which is responsible for more than 50% of incremental sodium excretion, especially when sodium intake augments. It hinders activity of renal Na+ transporters and − pumps (ENaC, NCC, NHE1, NHE3, NaPi2a, Na+ –HCO− 3 cotransporter, Cl – − + + HCO3 exchanger, Na –K ATPase) in the short term via their internalization and downregulates the formation of several renal Na+ transporters in the long term.11 Moreover, dopamine regulates fluid and sodium intake via the appetite center in the brain and gastrointestinal transport [506]. In addition, dopamine controls the secretion of hormones and humoral agents involved in the sodium balance regulation, atrial natriuretic peptide and prolactin increasing and angiotensin-2 and insulin decreasing dopamine inhibition on sodium reabsorption. Dopamine receptors of the central nervous system and kidney include two subsets: (1) Gs-coupled D1 -like subtypes D1 and D5 12 and (2) Gi/o-coupled D2 -like

stimulates NKCC2 in the mTAL, but because Na+ –K+ ATPase is inhibited, overall transport declines [506]. 12 D (D 1 1A in rodents), but not D5 (D1B in rodents), couples with Go, whereas D5 , but not D1 , couples with Gz and G12/13 [506]. The D1 -like receptors are also linked to Gq in a tissue-specific manner. D1 stimulates adenylate cyclases and causes renal vasodilation and electrolyte excretion via cAMP [532]. D5 inhibits PLC and PLD in proximal tubular cells [506]. 11 Dopamine

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isoforms,13 long (D2L ) and short D2 isoforms (D2S ),14 D3 ,15 and D4 [506].16 The dopamine receptors mutually interact, creating signaling pathways that are probably specific to some cell types.17 Both dopamine receptor subsets link to MAPK activation using different pathways. All dopamine receptor subtypes are expressed in the nephron, from the proximal tubule to the CD, and renal vasculature [506]. The D1 -like receptors (D1 and D5 ) reside in the apical and basolateral membranes of the cells of the proximal tubule, the mTAL of the loop of Henle, the DCT, and the CCD, but not in the glomerulus [531].18 Both D1 and D5 localize to large and small intrarenal arteries in humans [531]. The D2 -like receptors (D2 –D4 ) are all synthesized in the kidney [531]. The renal D2 -like receptors lodge in the renal cortex and inner medulla; they stimulate prostaglandin-E2 production rather than D1 -like receptors [532]. In humans, D2 localizes to the proximal tubule, the TAL, the DCT, and the cortical and outer MCD (OMCD, but not IMCD) and in podocytes D2L , rather than D2S , being produced in the renal tubule [531]. In humans, D3 is expressed in proximal tubule cells. In the human kidney, D4 mRNA can be detected. • The PT contains all dopamine receptors, D1 being responsible for about 80% of D1 -like activity [506]. • The mTAL possesses D1 R, D3 , and D5 , whereas cTAL expresses D3 only • The DCT produces D1 and D3 . • The CD contains all subtypes except D2 . The D2 -like receptors are vasodilators or -constrictors and natriuretic or antinatriuretic according to renal nerve activity or the sodium balance. Both D1 and D3 preclude Na+ reabsorption in several nephron segments. Dopamine receptor desensitization (i.e., loss of responsiveness), which damps short-term signaling, is caused by phosphorylation, sequestration, and degradation.

13 D , 2

D3 , and D4 inhibit both adenylate cyclase and calcium channels and modulate potassium channel activity [506]. D3 can also couple with Gq in proximal tubular cells. D2 represses PKB activity. 14 In the central nervous system, pre- and postsynaptic D effects are mediated by the short and long 2 isoform, respectively [506]. In fibroblasts, D1 is coupled with Gq and PLC. In the hippocampus, cortex, and striatum, D5 is also coupled with PLC; in the striatum, D1 stimulation of PLC requires D2 , whereas D5 mobilizes calcium, an event prevented by D2 [506]. 15 Seven alternatively spliced D variants exist. Whereas the full-length (D ) and shorter D 3 3L 3 isoform (D3S ) bind to dopamine, the five other alternatively spliced variants do not bind to dopamine but regulate receptor dimerization [506]. 16 Several D isoforms can also be identified. 4 17 In neurons, the D –D heterodimer stimulates PLC, whereas D 1 2 2S activates PLC independently of D1 [506]. In renal cortical cells, D1 , independently of D2 , can stimulate PLCβ1 [531]. In fibroblasts, D1 activates PLCγ. 18 Both D and D are yet to be identified in cultured mouse podocytes. In addition, D , but not 1 5 1 D5 , is detected in the macula densa and juxtaglomerular cells in rats.

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At least three types of regulators intervene: second messenger-dependent and G-protein-coupled receptor kinases (GRK1–GRK7), which prime hetero- and homologous desensitization, respectively, and arrestins, which uncouple the receptor from the trimeric G-protein [506].19 The phosphorylated GPCR–β Arr complex is endocytosed; GPCR is dephosphorylated and recycled back to the plasma membrane. In particular, D1 re-sensitization relies on its dephosphorylation by PP2. Sorting nexins assist in the GPCR recycling to the plasma membrane. On the other hand, unrecycled GPCRs are degraded in proteasomes and/or lysosomes. In humans, D1 is regulated by GRK2 and, to a greater extent, by GRK4 [506].20 The GRK4 subtype participates in regulating dopamine-mediated natriuresis. Aberrantly activated renal dopaminergic axis contributes to some forms of hypertension. The GRK4 gene in the human chromosomal locus 4p16.3 is linked to elevated BP from childhood to adulthood and hypertension in adults [506]. Variants of the GRK4 gene, alone or in conjunction with variants of other genes, are associated with salt-sensitive essential hypertension. Constitutively activated GRK4 gene variants (R65L, A142V, and A486V), which hyperphosphorylate, desensitize, and internalize D1 and D3 and upregulate the formation of AT1 , directly or via other factors involved in BP regulation, are associated with essential and/or saltsensitive hypertension in several ethnic groups (Caucasians, Chinese, Ghanaians, and Japanese) [506]. Other GRK subtypes are implicated in hypertension. In vascular smooth myocytes of mice, GRK2 overexpression engenders hypertension and impairs vasodilation mediated by β-ARs [506]. Vasoconstriction upon Agt2 exposure is impaired in these mice. Moreover, GRK2 phosphorylates (activates) ENaC, which then becomes insensitive to inactivation of ubiquitin–protein ligases NEDD2 and NEDD4. Renal GRK2 expression is upregulated in aging, metabolic syndrome, obesity, and oxidative stress. 19 GRK1

and GRK7 lodge in rods and cones, respectively; GRK2, GRK3, GRK5, and GRK6 are ubiquitous; constitutively active GRK4 resides in the testis and myometrium and, to a lesser extent, in specific brain regions, the intestine, and kidney. Four GRK4 splice variants (GRK4α–GRK4δ) have been identified in humans [506]. 20 The GRK4α variant desensitizes D and calcium-sensing (CaSR), some metabotropic glutamate 1 (mGluRs) and GABAB , follicle-stimulating hormone receptor, and shared receptor LHCGR of luteinizing hormone (LH) and human chorionic gonadotropin (hCG) of the hypothalamic– pituitary–gonadal axis, but not AT1 , formyl peptide, mGlu4, mGlu5, PTHR, β2-adrenergic (β2AR), and M1–M5 muscarinic receptors [506]. GRK4β desensitizes LHCGR and possibly vasopressin V2 receptor. GRK4γ desensitizes D1 and D3 in addition to AT1 in the kidney, inhibiting the antinatriuretic effect of AT1 to further promote natriuresis mediated by D1 and D3 . GRK4δ does not desensitize D1 . D2 is regulated by GRK2, GRK3, GRK5, and GRK6, D2S to a greater extent than D2L [506]. GRK2 or GRK3, but not GRK5 or GRK6, is involved in the desensitization of calcium signaling mediated by the D1 –D2 heterodimer. D3R is regulated by GRK2, GRK3, and GRK4 (GRK4γ > GRK4α) [506].

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The receptors D1 , D2 , and D5 contribute to the maintenance of the redox balance [531].21 In the kidney, the antioxidant effect of these receptors results from inhibition of NOx and stimulation of antioxidant enzymes. • D1 impedes NOx activity via PKA–PKC crosstalk. It stimulates superoxide dismutase, glutathione system enzymes, and heme oxygenase HOx1. • D2 promotes formation of the Parkinson disease protein Park7, paraoxonase PON2, and HOx2. It can lead to free radical scavenging and suppresses lipid peroxidation.22 • D5 attenuates NOx activity via inhibition of PLD2 and raises HOx1 expression and activity of SOD, HOx1, in addition to glutathione system enzymes.23 Therefore, D1 -like receptors prevent ROS production.24 Conversely, ROS preclude D1 -like receptor formation and function [531].

21 Balance

between oxidation and reduction reactions interferes with cell proliferation, apoptosis, and senescence, in addition to inflammation. 22 D increases the activity of glutathione, catalase, and SOD in the striatum. It protects striatal 2 dopaminergic neurons against 6-hydroxydopamine injury in mice. 23 Glutathione carries out many of its functions in cooperation with glutathione enzymes, which constitute the glutathione system; these enzymes produce or recycle glutathione, such as glutamate–cysteine ligase (GCL) and glutathione synthetase, γ-glutamyl transpeptidase yielding an additional pathway for glutathione synthesis, as it catabolizes extracellular glutathione for intracellular formation or supports its function, such as glutathione reductase, peroxidases, and S transferases. Reduced glutathione (GSH ), a major cellular redox buffer, is a tripeptide (γglutamylcysteinylglycine) composed of glutamic acid, cysteine, and glycine. It is sequentially synthesized by glutamylcysteine (GCS, now called GCL, the rate-limiting enzyme) and glutathione synthetase. The heterodimeric holoenzyme γ-GCS ligase possesses a heavy catalytic and light modifier (regulatory) subunit. Defective GCL activity is involved in multiple diseases, such as diabetes, chronic obstructive pulmonary disease, and cancer. Homodimeric glutathione synthase, the second enzyme of GSH synthesis and a potent antioxidant, catalyzes condensation of γGCS and glycine. Glutathione protects against ROS via conjugation with ROS, glutathione peroxidase neutralizing ROS, and is involved in the detoxification of xenobiotics using glutathione S transferases. The ratio of oxidized glutathione (glutathione disulfide [GSS G]) to GSH measures the intracellular redox environment [533]. Plasmalemmal γ-glutamyl transpeptidase (or transferase) GGT1 on the apical surface of ducts and glands, with its highest activity in the kidney, liver, pancreas, and lung, cleaves the γ-glutamyl bond of extracellular reduced and oxidized glutathione (in addition to glutathione conjugates and other γ-glutamyl compounds), releasing free glutamate and the dipeptide cysteinyl–glycine, which is hydrolyzed to cysteine and glycine by dipeptidases, thereby providing cells with the amino acids necessary for intracellular GSH synthesis. It is thus a component of the cellular antioxidant defense. 24 ROS include oxygen-centered radicals, such as singlet oxygen (1 O ), hydroxyl radical (OH• ), 2 • superoxide anion (O•− 2 ), and peroxyl radical (RO2 ), along with hydrogen peroxide (H2 O2 , ROO• /RO•2 ) and ozone (O3 ). In general, intracellular H2 O2 concentration is controlled by catalase, which has a high turnover rate, glutathione peroxidases (GPOx1–GPOx4), and peroxiredoxins [534].

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3.7.3.3

221

Potassium Homeostasis

Potassium is the most abundant cation in the intracellular fluid. Almost all cell types possess an Na+ –K+ ATPase that pumps Na+ out and K+ into the cell, establishing a K+ gradient across the cell membrane, which is partly responsible for the potential difference across the plasma membrane. Concentration of K+ in the extra- (low) and intracellular space (high [120–140 mmol/l]) not only determines the membrane resting potential and maintains the electrical properties of excitable and non-excitable cells but also contributes to the intracellular osmolarity, a determinant of cell volume. Storage and release of K+ from internal stores depend on renal reuptake and excretion of K+ ions [535]. Maintenance of the total body K+ content is achieved by adjusting renal K+ excretion to its intake. As this matching takes several hours, change in extracellular K+ concentration is initially buffered by K+ fluxes in the skeletal muscle [536]. The most important regulators under normal conditions include insulin (Ins– InsR–IRS1–PI3K–PDK1–PKB–aPKC–Na+ –K+ ATPase axis) and catecholamines (β2AR–AC–cAMP–PKA–ATP axis), atypical protein kinase-C, eliciting insertion of the Na+ –K+ pump into the plasma membrane and activity of the Na+ –K+ ATPase enabling K+ uptake [536].25 Dietary K+ intake is related to hypertension, as a low K+ diet increases arterial pressure in hypertensive subjects, which is associated with elevated renal Na+ reabsorption [536]. Two WNK1 isozymes, ubiquitous long isoform (WNK1L ) and kidney-specific subtype (Ki WNK1), are generated by the Wnk1 gene. The former is associated with increased retrieval of the renal outer medullary K+ channel (ROMK), thereby limiting K+ secretion. However, WNK1L also stimulates the epithelial Na+ channel (ENaC) and relieves inhibition of WNK4 on Na+ reabsorption through Na+ –Cl− cotransporter (NCC) in the distal convoluted tubule of the nephron. Deficiency in K+ raises the WNK1L /Ki WNK1 ratio, reducing K+ secretion occurring at the expense of increased Na+ retention [536]. The cellular functions of K+ channels of renal epitheliocytes include [537] (1) generation and maintenance of the negative transmembrane potential; (2) hyperpolarization, which yields a driving force for electrogenic transepithelial ion and solute transport (e.g., Na+ –glucose and Na+ reabsorption); (3) K+ recycling across the basolateral membrane to maintain electroneutrality (e.g., coupling of the basolateral K+ channel with Na+ –K+ ATPase to balance the charge movement across the apical membrane); (4) K+ recycling across the apical membrane of the TAL cells for continuous activity of Na+ –K+ –2Cl− cotransporter NKCC2; (5) K+ secretion across the apical membrane; and (6) participation in tubular cell volume regulation. The basolateral K+ channels of renal epithelia are members of the KCNE (KV ), KCNJ (KIR ), KCNK (K2P ), KCNQ (KV ), and SLO (KCa ) families [537].

25 Whereas

β-AR promotes K+ influx via activation of the Na+ –K+ ATPase, α AR impairs it.

222 Table 3.7 Potassium channels of the nephron

3 Hypertension Gene KCNJ1 KCNJ2 KCNJ4 KCNJ8 KCNJ10 KCNJ13 KCNJ15 KCNJ16

Protein KIR 1.1, ROMK KIR 2.1, IRK1 KIR 2.3, IRK3 KIR 6.1, KATP KIR 4.1, KIR 1.2 KIR 7.1, KIR 1.4 KIR 4.2, KIR 1.3 KIR 5.1, β-cell inward rectifier BIR9

(Part 1) KCNJ set (Source: [537]). Inwardrectifying K+ channels (KIR ) are encoded by 15 KCNJ genes. All KIR species are characterized by a larger inward current than outward flux. They also share a common structure, with two transmembrane domains, a K+ selectivity filter, and cytoplasmic N- and C-termini

The KCNE family are ancillary proteins that assemble as a β subunit with a pore-forming α subunit to form a voltage-gated K+ channel complex, modulate the gating kinetics, and enhance stability of the channel complex. In the nephron, they comprise minimal potassium channel (MinK) and minimum K+ channel-related peptide MiRP2 encoded by the KCNE1 and KCNE3 genes, respectively [537]. They are delayed rectifier K+ channel subunits. Members of the KCNJ gene family encode inward-rectifying K+ channels in the nephron (Table 3.7). They are characterized by two membrane-spanning domains linked by a pore-forming region. They encompass epithelial K+ transport, classical KIR , G-protein-gated K+ , and ATP-sensitive K+ channels [537]. • The KCNJ1 gene that encodes the ATP-dependent inward rectifier ROMK, a predominant apical membrane K+ channel in various segments of the nephron, engenders a transcript with three splice variants: ROMK1 to ROMK3 (KIR 1.1a– KIR 1.1c). These isoforms differ in their N-termini and localization along the nephron. The basolateral K+ channels contribute to the resting transmembrane potential in addition to K+ recycling across the basolateral membrane of renal epitheliocytes. • KIR 2.1 is an inward rectifier K+ channel (IRK1), the inward rectification being mainly due to the blockage of outward flux by internal magnesium. • KIR 2.3 (IRK3) resides in the basolateral membrane of CD cells, where it may be a small-conductance K+ channel of cortical CD PCs. • KIR 4.1 exists as a homomeric channel, but it can heteromerize with KIR 5.1. • KIR 4.2, which is also produced in the lung, brain, and pancreas. • KIR 5.1, which is identified in the basolateral membrane of DCT and CD cells, can form heteromeric channels with other members of the KCNJ family. At least in mice, it is a basolateral K+ channel in the TAL. It maintains pH in proximal

3.7 Kidney and Blood Pressure Control Table 3.8 Potassium channels of the nephron

223 Gene KCNK1 KCNK3 KCNK5 KCNK6 KCNK10 KCNK12 KCNK13

Protein K2P 1.1, TWIK1 K2P 3.1, TASK1 K2P 5.1, TASK2 K2P 6.1, TWIK2 K2P 10.1, TREK2 K2P 12.1, THIK2 K2P 13.1, THIK1

(Part 2) Basolateral K+ channels of the KCNK set (Source: [537]; TALK TWIKrelated alkaline pH-activated K+ channel) TASK TWIKrelated acid-sensitive K+ channel, THIK tandem pore domain-containing halothaneinhibited K+ channel, TREK TWIK-related K+ channel, TWIK tandem weak inward rectifying K+ channel

tubular cells. It can thus be involved in the regulation of fluid and pH balance. In cooperation with KIR 4.1, it mediates basolateral K+ recycling in distal tubules for Na+ reabsorption. • ATP-dependent KIR 6.1 lodges in the basolateral membrane of DCT and CD cells; it is also weakly expressed in PT cells. It may play a role in volume regulation. • KIR 7.1 is observed in human PT cells and in the basolateral membrane of rat DCT, CNT, and CD cells. It maintains a high K+ efflux at low interstitial K+ concentrations. Potassium channels of the KCNK group are two-pore leak K+ channels. In humans, the nephron possesses (Table 3.8): • Weakly inward rectifying background rapidly inactivating channel with variable ion selectivity K2P 1.1. • wWeakly rectifying pH-sensitive K2P 3.1, which can contribute to a prominent background K+ current. • Non-inactivating, outward-rectifying, acid-sensitive K2P 5.1, the gating of which is modulated by extracellular pH; it belongs to the TALK subgroup and localizes to the PT, DT, and CD, where it may contribute to cell volume regulation and modulates K+ conductance with respect to the rate of basolateral HCO− 3 transport. • Mechanosensitive, Gs-, Gi-, and Gq-coupled receptors and polyunsaturated fatty acid-stimulated outward rectifier K2P 10.1. • K2P 12.1, which lodges in the CD, where it contributes to recycling of K+ across the basolateral membrane. • K2P 13.1, which resides in the PT, TAL, and CD.

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Both K2P 12.1 and K2P 13.1 may modulate Na+ and Cl− transport during hypoxia. The glomerulus is endowed with K2P 3.1, K2P 6.1, and K2P 10.1 [537]. Members of the KCNQ family are voltage-gated K+ channels. KV 7.1 can complex with a β subunit MinK to form a slow outward-rectifying cAMP-dependent KV 7.1–MinK channel along with MiRP2. Both KV 7.1 and MinK are expressed in the apical membrane of PT cells and KV 7.1 in the basolateral membrane of TAL, DCT, CNT, and CD cells [537]. The member of the SLO family member, KCa 4.1 (a.k.a. Slo2.2 and sequencelike-A calcium-activated K+ channel [SLACK]) localizes to the basolateral membrane of TAL cells. It is also activated by Na+ and Cl− ions. It may couple basolateral K+ conductance to apical Na+ influx [537]. Potassium that is filtered by the glomerulus is mainly reabsorbed in the proximal tubule and loop of Henle, with less than 10% reaching the distal nephron. In the PT, K+ intake is linked to Na+ and water reabsorption. In the TAL, K+ reabsorption uses both para- and transcellular routes (via Na+ –K+ –2Cl− cotransporter) [536]. In the PCs of the CCD, apical electroneutral K+ and Cl− cotransport ensures K+ reabsorption upon K+ depletion, owing to the upregulated expression of apical H+ – K+ ATPase on α intercalated cells (ICCs; or type-A ICCs; Sect. 3.7.5.7). In the PT, K+ moves mainly through the paracellular gap. Active Na+ reabsorption drives a net fluid reabsorption, which enables K+ reabsorption using a solvent drag mechanism [536]. The shift in transepithelial voltage from slightly negative to slightly positive as filtrate flows yields an additional driving force for K+ diffusion through the low-resistance paracellular space. An apical K+ channel stabilizes the cell’s negative potential, particularly during activity of Na+ -coupled cotransport of glucose and amino acids, which causes depolarization. In the TAL, K+ is reabsorbed through both para- and transcellular routes. The basolateral Na+ –K+ ATPase provides a favorable gradient for apical Na+ –K+ – 2Cl− cotransporter. On the other hand, apical ROMK channel recycles K+ from the cell to the tubular lumen. The ClCKB channel permits Cl− efflux across the basolateral membrane. Potassium ion can exit the cell through a K+ channel and K+ –Cl− cotransporter [536]. Under normal conditions, K+ delivery to the distal nephron is usually weak and constant, but the rate of K+ secretion in the distal nephron varies according to needs. In the PCs, K+ secretion is determined by intracellular and luminal K+ concentrations, the potential difference across the luminal membrane, and K+ permeability of the luminal membrane, the K+ secretion rate rising with elevated intracellular K+ concentration, decreased luminal K+ concentration, or depolarized plasma membrane. In the DCT upstream segment (DCT1), luminal Na+ is taken up through Na+ – − Cl cotransporter owing to a favorable gradient produced by the basolateral Na+ – K+ ATPase [536]. The epithelial Na+ channel enables Na+ absorption in the DCT downstream segment (DCT2), the entrance ASDN segment. Electrogenic K+ flux begins in the DCT2 with the combined action of ENaC and ROMK, the latter being observed throughout the DCT [536]. Electroneutral K+ –Cl− cotransport functions

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225

in the DCT and CD; delivery of poorly reabsorbable anions (e.g., sulfate, phosphate, or bicarbonate) promotes K+ excretion through KCC. In the initial cortical collecting duct (CCD), PCs are responsible for K+ secretion. Basolateral Na+ –K+ ATPase actively imports K+ ; the resultant high intracellular K+ concentration provides a favorable diffusion gradient for K+ export through ROMK and BK channels toward the tubular lumen [536]. The Na+ –K+ pump also lowers intracellular Na+ concentration, thus promoting through ENaC Na+ import from the tubular lumen. Apical electroneutral H+ –K+ ATPase secrete H+ into the lumen and reabsorb K+ ; its activity increases in K+ depletion. Principal determinants of K+ secretion in the CCD include aldosterone, which stimulates K+ secretion,26 and distal delivery of Na+ and water.27 An increased filtrate flow rate also raises K+ secretion.28 The ROMK channel, which has a low conductance and a high opening probability, is the major K+ secretory route [536]. The Ca2+ -activated BK channel, which has a large conductance, contributes to the flow-dependent K+ secretion, elevated flow raising Ca2+ concentrations within PCs via the primary cilium and TRPP2 (or polycystin-2). Increased tubular flow also stimulates Na+ reabsorption through mechanosensitive ENaC in the CD. Upon volume depletion, activation of the RAA stimulates release of aldosterone that facilitates renal Na+ retention to restore ECF volume, without affecting renal K+ secretion. Salt and water absorption augments in the PT, diminishing delivery of Na+ and water to the distal nephron, which counterbalances the effect of aldosterone and does not markedly modify K+ excretion. On the other hand, hyperkalemia acts on zona glomerulosa cells that then release aldosterone, which stimulates renal K+ secretion to reestablish the kalemia to its normal level, without concomitant renal Na+ retention. This ability of aldosterone to stimulate salt retention in the kidney without K+ secretion under volume depletion and K+ secretion without salt retention in hyperkalemia is designated the aldosterone paradox [536]. Vasopressin stimulates renal K+ secretion. This kaliuretic effect counters K+ retention linked to a low filtrate flow rate [536]. Angiotensin-2 modulates this switch. Fluid volume depletion augments Agt2 and Ald concentrations. Angiotensin-2 not only enhances NaCl reabsorption in the proximal tubule but also activates Na+ –Cl− cotransporter in the DCT1 and activates basolateral Na+ –K+ ATPase, thereby increasing intracellular K+ concentration and Na+ export to the interstitium and hyperpolarizing the plasma membrane, an event favoring K+ secretion to repolarize the plasma membrane at its resting level. Indeed, it increases membrane Na+ permeability via elevated Na+ permeability of the apical membrane and activation of enzymes involved in intermediary metabolism (e.g., citrate synthase), in addition to Na+ –K+ ATPase stimulation, and the turnover rate of the Na+ –K+ pump. It also raises K+ permeability (passive K+ conductance) [538]. 27 Increased Na+ delivery to the distal nephron stimulates Na+ reabsorption, which depolarizes the plasma membrane, thereby promoting K+ secretion for a return to the RTMP. 28 At high filtrate flow rates, secreted K+ are washed out more rapidly, thereby depolarizing the plasma membrane. 26 Aldosterone

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ENaC in the ASDN compartment. Angiotensin-2 and aldosterone cooperate in Na+ retention. Angiotensin-2 inhibits ROMK, enabling Na+ conservation without K+ wasting [536]. The WNK4 kinase may act as a molecular switch that determines the balance between renal NaCl reabsorption and K+ secretion. It reduces surface expression of the Na+ –Cl− cotransporter, stimulates ROMK endocytosis in the CD, and phosphorylates claudins, hence enhancing paracellular Cl− permeability, hyperpolarizing the cell, and impeding K+ secretion [536]. Although WNK1 is ubiquitous, its renal shorter spliced variant Ki WNK1 (WNK1S ) is restricted to the DCT and a part of the CnT; it antagonizes WNK1L , which stimulates ROMK endocytosis and prevents WNK4-mediated inhibition of NCC in the DCT, thereby increasing NaCl reabsorption. Furthermore, Ki WNK1 stimulates ENaC. Therefore, increased activity of Ki WNK1 in response to dietary K+ loading facilitates K+ secretion through the combined effects of augmented Na+ delivery upon NCC inhibition, electrogenic Na+ reabsorption through ENaC, and ROMK amount [536]. Urinary K+ excretion undergoes a circadian rhythm, being lower during the night and early morning and higher in the afternoon [536]. In the mouse distal nephron, ROMK production is greater during periods of activity, whereas that of the H+ –K+ ATPase is higher during rest, which correspond to periods of greater and smaller renal K+ excretion, respectively.

3.7.3.4

Phosphate Homeostasis

After processing of phosphorus-rich food, phosphorus is transported across the cell membrane as phosphate (31 mg/l elemental phosphorus engendering 1 mmol/l phosphate) [522]. Phosphate in plasma or extracellular fluid is carried into cells, deposited in bone, or eliminated predominantly by the kidney. About 15% of plasmatic phosphate (2.5–4.5 mg/dl in adults) is bound to proteins; the − − remainder consists of free HPO2− 4 and NaHPO4 (∼85%) and free H2 PO4 (∼15%). Phosphorus is mainly stored in the bone. Phosphorus engenders ubiquitously distributed common anions. Inorganic free 2− phosphate ions (H2 PO− anions; abbreviated as Pi ) are involved 4 and HPO4 in calcification, generation of high-energy bonds (e.g., ATP), metabolism, and regulation of cellular functions, in addition to being components of membranes and nucleic acids. Their concentrations influence ammoniagenesis, glycolysis, gluconeogenesis, and phosphate reabsorption, along with PTH secretion and formation of (1,25)-dihydroxycholecalciferol from 25-hydroxycholecalciferol [539]. Only about 1% of total bodily Pi Pi content is present in ECFs (plasmatic concentration in adults 0.8–1.4 mmol/l [3–4.5 mg/dl] at pH 7.4, mostly as HPO2− 4 , with diurnal variations of approximately 0.2 mmol/l [540]. A small fraction of plasmatic Pi is bound to proteins or complexed with the major plasma cations (mainly Ca2+ , Mg2+ , and Na+ ).

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In addition to being a constituent of phospholipids and nucleic acids, phosphate serves in energy metabolism, signal transduction, and bone formation. Phosphate handling is regulated by three organs, parathyroid, kidney, and bone, via three feedback loops. These counter-regulatory loops also regulate intestinal absorption [539]. Parathyroid hormone, fibroblast growth factor FGF23, vitamin D, and klotho coreceptor29 are key regulators of phosphate balance. Parathyroid glands produce PTH, which stimulates FGF23 and phosphate release from the bone; FGF23 inhibits PTH secretion, but phosphate tends to trigger PTH production. In the kidney, PTH elicits phosphate excretion and calcitriol synthesis, calcitriol boosting FGF23 production by bone cells; low phosphate and calcitriol levels prevent PTH formation. Under normal conditions, absorption of dietary Pi in the small intestine balances renal and intestinal losses. Renal Pi excretion allows adjustment of plasmatic Pi concentration. Renal Pi reabsorption occurs in the proximal tubule by Na+ –Pi cotransporters of the apical brush border membrane. Most of the plasmatic Pi (90– 95%) is ultrafiltrated in the renal glomerulus, 80–90% being reabsorbed by the renal tubule (60–70% in the PT) and the remainder excreted in the urine [539]. Phosphate transport and reabsorption in the CCD are independent of PTH. Both PTH and FGF23 control Pi excretion via Na+ –Pi cotransporters (Table 3.9). Sodium–phosphate cotransporters (NaPi1 [SLC17a1], NaPi2b–NaPi2c [SLC34a2– SLC34a3], and NaPi3 [a.k.a. NaPi2a and SLC34a1]) in the apical membrane of PCT epitheliocytes use the Na+ electrochemical gradient for uphill P import (inward i flux of three Na+ ions and one divalent phosphate [HPO2− ] for electrogenic NaPi 4

29 Klotho

was originally identified as an aging suppressor gene in mice. In Greek mythology, Kλωτ ς (Spinner) was one of the three Fates (Moιραι) who determine human lifespan with Λαχ σις (Allotter or Dispenser), the measurer of thread length, and Aτρoπoς (Inflexible), who cut the thread, thereby determining the moment of death of the individual. These three Greek goddesses of fate were responsible for spinning the thread of life (κλωτω: spin) and correspond to the Roman Parcae (Nona, Decuma, and Morta) and the Norse Norns. Klotho markedly increases the affinity of FGF23 for FGFRs that launch signaling, which involves ERK1, ERK2, and FGFR substrates (FRS2/3) phosphorylation. The cofactors α-, β, and γ-klotho (the latter, which is encoded by the LCTL gene, is also named klotho and lactase-phlorizin hydrolase-related protein [KLPH] and lactase-like protein [LctL]) are required for receptor binding [541]. α-Klotho (or simply klotho), which is encoded by the KL gene, is especially expressed in the kidney and parathyroid glands, where it forms constitutive binary complexes with FGFR1c, FGFR3c, and FGFR4 and serves as the high-affinity receptor for FGF23 [542]. β-Klotho, which is expressed in the liver and adipose tissue, complexes with FGFR1c and FGFR4 and supports FGF19 and FGF21 signaling. γ-Klotho complexes with FGFR1b, FGFR1c, FGFR2c, and FGFR4; it is expressed in the kidney, adipose tissue, and the eye [542]. Additional FGF signaling regulation is provided by a fifth protein nontyrosine kinase FGFRL1, which can bind FGFs and possibly function as a decoy receptor, dimerization-induced inhibitor of FGFRs, or modulator of receptor turnover or signaling [541]. Klotho expression is restricted to the kidney (mainly in the distal tubule), parathyroid glands, brain, and skeletal muscle. Both adam10 and adam17 can cleave and release Klotho from the plasma membrane. Its soluble form, which is detected in plasma and urine, derives from shedding of full-length klotho. A smaller circulating form arises from alternative RNA splicing [539].

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Table 3.9 Three families of NaPi cotransporters (Sources: [539, 543]) Type SLC17a1 SLC17a2 SLC17a3 SLC17a4 SLC20a2 SLC34a1 SLC34a2 SLC34a3

Other alias NPT1, NaPi1 NPT3 NPT4 NPT5 PiT2 NaPi2a, NPT2 NaPi2b NaPi2c

Location PT (brush border) Kidney, liver, muscle Apical wetted surface of renal tubules Kidney, liver, stomach, intestine Ubiquitous PT Lung, small intestine PT

Cotransporters of the subfamily I of the SLC17 family (SLC17a1–SLC17a4) are renal Na+ dependent inorganic phosphate transporters (NPTs), which reside in the plasma membrane. Both SLC17a1 and SLC17a3 convey organic anions, in particular, urate and para-aminohippurate. The lysosomal acidic sugar transporter SLC17a5, another sodium–phosphate cotransporter, also called sialin, lodges on lysosomes and transports glucuronic acid and free sialic acid out of the lysosome after cleavage from sialoglycoconjugates that are degraded. In the central nervous system, it serves as a vesicular excitatory amino acid transporter in synaptic vesicles, where it mediates membrane potential-dependent uptake into synaptic vesicles for vesicular storage and subsequent exocytosis + of aspartate and glutamate [194]. It also acts as an electrogenic 2NO− 3 –H cotransporter in the plasma membrane of salivary gland acinar cells, enabling nitrate efflux (25% of circulating nitrate ions is secreted in saliva) [108]. The three vesicular glutamate (VGluT1–VGluT3 [SLC17a7/6/8], respectively) and nucleotide transporter (VNuT [SLC17a9]) localize to synaptic vesicles

cotransporter [NaPi2a–NaPi2b (SLC34a1–SLC34a2)] and two Na+ and one HPO2− 4 for electroneutral NaPi cotransporter [NaPi2c (SLC34a3)]). Sodium-dependent phosphate transporter PiT2, which is electrogenic, preferentially carries monovalent phosphate [522]. Whereas the transport rate NaPi2a and NaPi2c rises from pH 6.5 to 8, PiT2 remains constant over this range. Activity of Na+ –Pi cotransporters and hence Pi absorption are regulated in a coordinate manner by PTH and (1,25)(OH)2 D3 .30 In animals fed with a lowPi diet, plasmatic Pi concentration lowers, and circulating Ca2+ concentration rises, which prevents PTH release, thereby reducing renal Pi excretion [539]. In addition, synthesis of (1,25)(OH)2 D3 is upregulated, thereby promoting intestinal (via NaPi2b) and renal uptake. Conversely, in animals fed with a high-Pi diet, plasmatic Ca2+ concentration in addition to (1,25)(OH)2 D3 synthesis decays, but PTH release increases.

30 (1,25)(OH)2 D : 3

dihydroxycholecalciferol or dihydroxyvitamin-D3 .

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Several phosphaturic peptides, such as FGF7, FGF23,31 secreted frizzled-related protein sFRP4, and secreted calcium-binding matrix extracellular phosphoglycoprotein (mepe),32 inhibit NaPi2a in renal epitheliocytes [539]. Parathyroid hormone decreases renal reabsorption of phosphate, as it reduces rapidly the content of NaPi2a (within minutes) and slowly those of NaPi2c and PiT2 (within hours) in the proximal tubule brush border membrane using PKA, PKC, ERK1, and ERK2 in addition to myosin-6 [522]. In the PT [522]: • Glucocorticoids downregulate NaPi2a and changes lipid composition in the brush border and thus may modulate NaPi activity. • Estrogens cause phosphaturia, as they lower the quantity of PT NaPi2a, but not that of NaPi2c. Estrogens also upregulate FGF23. • Thyroid hormones increase PT NaPi2a synthesis. • Dopamine, a catecholamine that modulates behavior, movement, nerve conduction, hormone synthesis and release, BP, and ion fluxes, especially regulating sodium balance, provokes phosphaturia, as it primes internalization of NaPi2a using SLC9a3R1 (or NHERF1).

31 FGF23

is a circulating peptide secreted by osteocytes, osteoblasts, and osteoclasts in response to hyperphosphatemia and vitamin D [539]. The osteogenic endocrine messenger FGF23 binds to and activates a receptor complex formed by FGFR1, FGFR3, and/or FGFR4 with klotho [539]. FGF23 participates in the control of phosphate balance. It favors urinary phosphate excretion. It downregulates NaPi2a and NaPi2c expression, thereby attenuating renal phosphate reabsorption. It also diminishes calcitriol, as it precludes 1α-hydroxylase and stimulates its catabolizing enzyme (24,25)-hydroxylase [539]. A reduced calcitriol amount downregulates via VDR (NR1i1) synthesis of intestinal NaPi2b. 32 Mepe is a member of the small integrin-binding ligand N linked glycoprotein (sibling) family, which are components of the extracellular matrix of bone and dentin that regulate mineralization. Siblings are phosphorylated by family with sequence similarity member Fam20c (or dentin matrix protein DMP4), Fam20c is an atypical protein kinase that localizes within the Golgi body. It is the Golgi body casein kinase that phosphorylates secretory pathway proteins implicated in mineralization such as siblings. Substrates of Fam20c encompass casein, apolipoproteins ApoA2, ApoA5, ApoB, ApoE, and ApoL1, PCSK9, IGFBP1 and IGFBP3, FGF23, BMP4, secretogranin-1, cystatin family members, many serpins, ADAM10, cadherin-2, glypican-3, syndecan-2, fibronectin, some laminin subunits, testican, versican, matrillin-3, complement factors C3 and C4a, blood coagulation FV , fibrinogen, protein-C, protein disulfide isomerases PDIa1 and PDIa6, among others [544]. These casein kinase-like enzymes are cytosolic and nuclear proteins along with secreted glycoproteins. Many secreted proteins are indeed phosphorylated by kinases of the secretory pathway and/or in the extracellular space. The FAM20 family of kinases (Fam20a–Fam20c) phosphorylate secreted proteins and proteoglycans. The GASK set of secretory Golgi body-associated kinases (GasKs) comprises members of the FAM198 subfamily (Fam198a– Fam198b). Mepe promotes renal phosphate excretion in adult human kidney, predominantly in the PCT [194]. It is also called osteoblast and osteocyte factor OF45. It shares molecular similarities with several dentin and bone extracellular matrix RGD (tripeptide Arg–Gly–Asp) cell-attachment motif-containing phosphoglycoproteins, such as dentin sialophosphoprotein (DSPP), osteopontin (a.k.a. uropontin, bone sialoprotein BSP1, and secreted phosphoprotein SPP1), and dentin matrix acidic phosphoprotein DMP1.

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An acute increase in arterial pressure decreases phosphate reabsorption owing to the removal of NaPi2a from the proximal tubule brush border membrane microvilli to subapical endosomes [522].

3.7.3.5

Calcium Homeostasis

Three main organs, the intestine, kidney, and bone, ensure Ca2+ homeostasis. Calcium balance results from coordinated Ca2+ absorption in the intestine, reabsorption in the kidney, and exchange from bone under the control of the calciotropic hormones, which are released upon a Ca2+ demand. Approximately 99% of bodily calcium resides in the skeleton; the remainder lodges in the extra- and intracellular media [522]. Calcium ion serves in nerve impulse transmission, muscular contraction, blood coagulation, hormone secretion, and intercellular adhesion. Blood Ca2+ concentration is detected by Ca2+ -sensing receptors and regulated by several messengers, such as PTH, calcitonin, and vitamin D [545]. Aging interferes with Ca2+ metabolism from calcium intake and absorption, vitamin D intake and absorption to vitamin D production, hydrolyzation and action, in addition to secretion and action of calciotropic hormones (e.g., PTH, calcitonin, and gonadal steroids) [546]. Three major pools of calcium include active Ca2+ ion, inert protein-bound calcium fraction, and complexed, that is, bound to molecules such as phosphate and citrate (approximately 48, 46, and 7%, respectively) [522]. Calcium bound to albumin and globulin is a source of available Ca2+ ion. Normal concentration of serum calcium ranges 8.9–10.1 mg/dl (2.2–2.5 mmol/l) [522]. Calcium filtered in the glomerulus is reabsorbed (60–70% in the PT, ∼20% in TAL, 5–10% in the DCT, and 3–10% in the CnT) [522].33 Calcium reabsorption in the PCT is linked to that of sodium and water mainly using diffusion and solvent drag in the paracellular route but also a small contribution of active transport across the apical membrane and exit through the basolateral membrane (10–15%). In the TAL, apical Na+ –K+ –2Cl− cotransporter and ROMK channel, together with basolateral Na+ –K+ ATPase and Cl− channel, generate a lumen-positive transepithelial potential difference and the driving force for paracellular cation transport. Calcium transport is also influenced by the basolateral calcium-sensing receptor that increases the paracellular permeability to calcium. Calcium-sensing receptor (CaSR) upregulates formation of claudin-14, which lowers the permeability of paracellular cation, and downregulates that of claudin-16, which stimulates paracellular permeability [547]. Rare loss-of-function mutations in the CASR gene reduce urinary Ca2+ excretion in PTH-dependent hypercalcemia.

33 Approximately

85% of phosphate is reabsorbed in the PCT, 10% in the loop of Henle, 3% in the DCT, and 2% in the CD [522]. Approximately 10–30% of the filtered magnesium is absorbed in the PT, 40–70% in the TAL, and the remaining 5–10% in the DCT.

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Calcium-sensing receptor is synthesized throughout the nephron, but at various locations according to the segment. In the PT, CaSR lodges at the apical membrane of the brush border base, concentration decaying from S1 to S3. In the TAL, CaSR resides on the basolateral membrane, where it monitors extracellular Ca2+ concentration in the peritubular interstitium. Once it is activated, CaSR regulates NaCl absorption directly through NKCC2 (SLC12a1) and indirectly through ROMK22 (KIR 1.1b), which recycles K+ to the TAL luminal fluid [518]. Hypercalcemia hampers calcium absorption in the TAL; activated CaSR suppresses paracellular Ca2+ flux. In fact, elevated peritubular Ca2+ concentration (but not luminal Ca2+ and Mg2+ concentrations) reduces absorption of both Ca2+ and Mg2+ ions. In the DCT, once it is stimulated, Mg2+ transfer decays. In the CCD and IMCD, CaSR localizes to apical plasma membrane. It is expressed in only some of the α ICCs of the CCD [518]. Calcium-sensing receptor is coupled with Gi2 and Gi3 subunits along with those of the Gq/11 set. Upon calcium binding, CaSR launches the Gq/11– PLC–IP3 pathway and hence rapid transient release of Ca2+ from its intracellular store. Another CaSR signaling uses effectors PLA2, PLD, MAPK, PI3K, and PI4K [518]. Whereas CaSR signals via PLC in most cell types, in the TAL, cytosolic Ca2+ , which is imported from the extracellular space rather than from cellular stores, suppresses cAMP synthesis by AC6 and increases cAMP catabolism by Ca2+ -activated phosphodiesterase. TAL CaSR signals via PLA2, increasing the cytosolic level of arachidonic acid, rapidly metabolized by CyP4 to 20HETE rather than to prostaglandins by PGhS2, which is produced to a lesser extent. However, activated CaSR supports stimulation by TNFSF1 of PGhS2, thereby generating PGe2 [518]. Metabolites produced by members of the cytochrome-P450 superfamily and eicosanoids in the short and long term, respectively, regulate TAL K+ channels; 20HETE inhibits NKCC2, ROMK, and basolateral Na+ –K+ ATPase; PGe2 further inhibits NKCC2-mediated Na+ uptake. In mTAL and cTAL, hypercalcemia and elevated extracellular Ca2+ prevents cAMP production in response to vasopressin and PTH, respectively (but not PTHinduced cAMP formation in the PCT) [518]. In addition to paracellular Ca2+ flux in the TAL, transcellular Ca2+ motion is regulated by hormones calcitonin and PTH in the mTAL and cTAL, respectively. Renal Ca2+ reabsorption via trans- and paracellular routes is regulated by the Gs-coupled parathyroid hormone receptor PTH1R for PTH and its related protein PTHRP,34 in the TAL of the loop of Henle and distal convoluted tubules.35 It directly and indirectly controls claudin-14, a tight junction protein and hence inhibitor

34 Also

known as parathyroid hormone-like hormone (PTHLH). This neuroendocrine peptide regulates cell differentiation, proliferation, migration, and survival in addition to epithelial Ca2+ transport. The PTH family includes PTH, PTHRP, and PTH2, also dubbed tuberoinfundibular peptide TIP39, a potent and selective agonist of the PTH2 receptor [548]. 35 Two PTH receptors exist (PTH1R–PTH2R, or PTH –PTH ). The classical PTH receptor is 1 2 1 produced in bones and kidneys, where it regulates Ca2+ homeostasis. PTH1 is activated by both PTH and PTHRP. The Gs-coupled PTH2 receptor localizes mainly to the central nervous

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of the paracellular Ca2+ transport in the TAL [550]. In mice with hyperthermia, whatever the calcemia (high or low), PTH–PTH1R signaling suppresses claudin-14 expression and hence can cause hypercalciuria and hypocalcemia. The distal tubule reabsorbs Ca2+ exclusively through the transcellular route against a chemical and electrical gradient using apical TRPV5, cytosolic calbindin1, and basolateral Na+ –Ca2+ exchanger and Ca2+ ATPase PMCA1b [522].

3.7.3.6

Magnesium Homeostasis

Magnesium is the second most abundant intracellular divalent cation. It is a cofactor for protein and DNA synthesis and contributes to oxidative phosphorylation, vascular tone, neuromuscular excitability, and bone formation [522]. Although magnesium is mainly stored in the intracellular medium, its blood concentration (0.7–1.1 mmol/l, i.e., 1.4–2.2 mEq/l or 1.7–2.6 mg/dl) is regulated and urinary magnesium excretion can rapidly drop when its extracellular concentration falls [551]. Serum magnesium exists in the ionized, free, and active form (∼60%), complexed to serum anions (∼10%), and bound to albumin (∼30%) [522]. Intestinal magnesium absorption uses a saturable transcellular path using TRPM6 and TRPM736 (∼30%) in addition to the nonsaturable, paracellular, passive route [522]. Magnesium is reabsorbed (∼96% of filtered magnesium) in the PT, TAL, and DCT [551]. It is conveyed through the paracellular route in the PT and TAL owing to a chemical gradient generated by Na+ gradient-driven water flux and a lumenpositive transepithelial voltage established by the apical NKCC2–ROMK couple, respectively [522]. In the TAL, the ClCKB channel and Na+ –K+ ATPase allow the basolateral exit of Cl− and Na+ , respectively. The tight junction protein, claudin-10, determines paracellular permeability not only to Na+ but also to Mg2+ ; a defective function and loss causes hy-

system, pancreas, testis, and placenta. It interacts strongly with PTH and PTH2 but weakly with PTHRP [549]. They trigger AC–cAMP–PKA and PLC–PKc–Ca2+ pathways. 36 Transient receptor potential melastatin TRPM6 and TRPM7 are divalent cation channel kinases (chanzymes) controlling Mg2+ homeostasis. Ionic fluxes through TRPM6 and TRPM7 are related to a highly nonlinear current–voltage relationship with pronounced outward rectification. The small inward current at physiologically relevant negative membrane voltages are carried by divalent cations (Mg2+ and Ca2+ ), whereas the large outward current at positive voltages results from the efflux of monovalent cations. Channel kinase heteromerization slightly affects permeation, pH sensitivity, and single-channel conductance. ATP inhibits TRPM7, but neither TRPM6 nor heterodimeric TRPM6–TRPM7 channel [552]. In fact, TRPM7 is also inhibited by both ATPMg and intracellular free Mg2+ . Homodimeric TRPM6 is highly sensitive to intracellular free Mg2+ . Heterodimeric TRPM6–TRPM7 channel has an altered sensitivity to intracellular ATPMg compared with the TRPM7 homodimer. Activity of TRPM6–TRPM7 is independent of intracellular ATPMg concentration, TRPM6 uncoupling channel activity from the cellular energy level.

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permagnesemia [522]. Mutations in the CLDN16 or CLDN19 genes provoke hypermagnesuria and hence hypomagnesemia. In the DCT, Mg2+ moves through the transcellular path, that is, through apical TRPM6 owing to KV 1.1, which yields a favorable luminal potential and basolateral Mg2+ –Na+ exchanger SLC41a1 assisted by KIR 4.1 and Na+ –K+ ATPase, which generate a Na+ gradient [522]. Cyclin-M2, which predominantly lodges in the basolateral membrane of distal tubules, is implicated in renal Mg2+ reabsorption. On the other hand, EGF increases Mg2+ transfer through the TRPM6 channel. Parathyroid hormone, calcitonin, vasopressin, epidermal growth factor, and β-AR agonists increase renal magnesium reabsorption. On the other hand, prostaglandin-E2 decreases magnesium reabsorption. Other factors influence magnesium reabsorption, which decreases when blood concentrations of calcium and magnesium are elevated and in metabolic acidosis [551].

3.7.4 Hydrogen Ion Control The organs involved in the regulation of acid–base balance are the lung and kidney.37 According to the Arrhenius theory, acids and bases are substances that produce hydrogen (H+ ) and hydroxide ions (OH− ) in solution. Hydrogen ions from acids react with hydroxide ions from bases. Neutralization results from a − 38 that produces water. Hydrogen chloride in reaction between H+ aq and OHaq water (HClaq ), which is found in gastric acid, is neutralized by ammonia solution (NH3aq ), which is a base according to the Arrhenius definition. Ammonia does − indeed reversibly react with water to generate NH+ 4aq and OHaq , although, in a dilute ammonia solution, most of the ammonia molecules remain. Hence, the Arrhenius theory of acids and bases has limitations, because most of the reaction is a direct reaction between ammonia molecules and hydrogen ions, which does not match the Arrhenius definition. Instead, the Bronsted–Lowry theory of acids and bases states that an acid is a hydrogen ion (proton) donor and a base is a hydrogen ion 37 The

term “acid–base balance” is a misnomer, as the physiological process does not deal with acids and bases but with anions and cations. The body’s fluids comprise weak acids and bases because of the presence or absence of hydrogen ions. The fundamental process in acid–base reactions is the transfer of hydrogen ions. Acids and bases are H+ donators and acceptors, respectively. The dissociation of a weak acid (H+ A− ) is the dissociation of a hydrogen ion and its anion (A− ). The activity of H+ ions in a solution determines the acidity. In the blood, the activity coefficient ∼ 1. Therefore, pH = − ln[H+ ], as in an infinitely dilute solution. An acid (AH) that transfers its H+ can be transformed into its conjugated base anion (A− ) and H+ cation, and conversely according to its [H+ ] value. Once one H+ has been released, any acid becomes a base buffer able to bind free H+ . The mass action law relates the concentration of the three reactants: [H+ ] = k[AH]/[A− ]. pK is the point at which the concentrations of the two conjugate substances are equal. 38 The subscript • aq that stands for aqueous solution is used to distinguish matter states (e.g., hydrogen chloride in water (HClaq ) versus hydrogen chloride gas (HClg )).

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acceptor. Hence, ammonia is a base from an acid (H+ donor) because it accepts a H+ , hydrogen atom being attached to the lone pair on the nitrogen atom of the ammonium molecule (NH+ 4aq ). The lung excretes carbon dioxide (the respiratory acid), concentrations of CO2 − and HCO− 3 being interdependent. Carbonic anhydrase renders dissolved HCO3 available for rapid conversion to CO2 , thereby increasing the amount of transported CO2 by the amount of HCO− 3 present in the solution. The kidney removes acid anion and associated H+ that cannot be eliminated by the lung, certainly in smaller amounts. Furthermore, the kidney reabsorbs filtered − + bicarbonate (HCO− 3 ). Both HCO3 reabsorption implicates H excretion through the renal tubular lumen. Therefore, the kidney has two major functions in the maintenance of pH homeostasis, reabsorption of filtered bicarbonate and generation of new bicarbonate linked to renal ammonia metabolism, ammonia commonly referring to both NH3 (ammonia) and NH+ 4 (ammonium) molecules. 3.7.4.1

Carbonic Anhydrases

The ubiquitous zinc-containing metalloenzymes carbonic anhydrases include 15 isoforms (CA1–CA4, mitochondrial CA5a–CA5b, and CA6–CA14), that is, three cytosolic (CA1–CA3), five membrane-bound (CA4, CA7, CA9, CA12, and CA14), and a secreted salivary isozyme (CA6).39 These carbonate dehydratases catalyze reversible carbon dioxide hydration and bicarbonate dehydration; CO2 can freely diffuse in and out of the cell across lipidic membranes, whereas HCO− 3 must be carried. They are involved in H+ secretion into the tubular fluid and hence HCO− 3 reabsorption. Carbonic anhydrases often cluster along cellular membranes or localize to the extracellular space, thereby facilitating intracellular transfer of CO2 and H+ , dissipating intracellular pH gradients, and maintaining a uniform cellular H+ concentration. Carbonic anhydrases are also implicated in gluconeo-, lipo-, and ureagenesis. The soluble isoform CA2 resides in the cytoplasm (∼95% of CA activity) and the membrane-bound subtype CA4 in the brush border membrane (∼5% of CA activity) of cells of nephron segments such as the proximal tubular cells, the cytosolic CA2 subtype governing the HCO− 3 reabsorption rate. Luminal membrane CA4 facilitates dehydration of carbonic acid (H2 CO3 ) formed when secreted H+ combine with filtered HCO− 3 . Cytosolic CA2 favors − HCO3 efflux via H2 CO3 dehydration. Both CA2 and CA4 associate with HCO− 3 transporters (e.g., SLC4a1, SLC4a4, SLC4a8, and SCL26a6), and proton antiporter NHE1 in a membrane complex, the transport metabolon [553]. Carbonic anhydrase facilitates acid–base transport in the proximal tubule, TAL, and distal nephron segments (Table 3.10). 39 Three classes of carbonic anhydrases encompass α ACs in mammals, β ACs mainly in plants, and γ ACs in prokaryotes, which possess all three AC classes.

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Table 3.10 Location of carbonic anhydrases (CAs) isoforms along the nephron (Source: [553]) Isozyme CA2 CA4 CA12

Sites PCT, tDL, TAL, DCT, CnT, CCD, MCD PCT, TAL, CCD, MCD PCT, TAL, DCT, CnT, CCD, MCD

Ions H+ and HCO− 3 are formed from H2 CO3 dehydration, which derives from combination of H2 O and CO2 , in a reaction catalyzed by carbonic anhydrase. Whereas CA2 resides in the cytoplasm of cells in almost all nephron segments, except for the loop of Henle and tAL, CA4 lodges both apically and basolaterally in the proximal tubule (PCT and PST) and TAL, and, in the distal nephron, exclusively on the apical surface of type-A (α) ICCs of the CCD and the acidsecreting cells of the medullary collecting duct (MCD). The other isozyme CA12 is detected in the basolateral membrane of PCT and PST and TAL and, in the distal nephron, in the DCT, CCD PCs, and MCD Table 3.11 Carbonic anhydrases (CAs) and carriers of the solute carrier superclass SLCs; Source: [554])

SLC type SLC4a1 SLC4a2 SLC4a3 SLC4a4 SLC4a7 SLC26a3

CA interactors CA2, CA4 CA9 CA2, CA14 CA1, CA2, CA4 CA2 CA2

The SLC4a1 carrier interacts with CA2 and CA4, forming the intra- and extracellular component of a bicarbonate transport metabolon, respectively

Carbonic anhydrases both produce and consume the substrate HCO− 3 of bicarbonate transporters. They are thus incorporated in the bicarbonate transport metabolon,40 especially CA2, which is the most widespread cytosolic isoform, and the extracellular membrane-associated isoforms that include CA4, CA9, CA12, and CA14 (Table 3.11) [554].

3.7.4.2

Renal Ammoniagenesis and NH3 and NH+ Transport 4

Renal ammonia metabolism, transport, and excretion is a major player in hydrogen ion control and the predominant axis of net renal acid clearance. The majority of renal ammonia excretion derives from intrarenal ammoniagenesis (i.e., not from glomerular filtration) and depends on its regulated transfer of NH3 or NH+ 4 ion through the epitheliocyte membrane in nephron segments. 40 A

metabolon is a sequence of molecules acting in a metabolic pathway that maximizes flux through the pathway and may thus accelerate the transport rate through a carrier.

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Renal ammoniagenesis predominantly results from glutamine metabolism, − which produces two NH+ 4 and two HCO3 for each glutamine metabolized by mitochondrial phosphate-dependent glutaminase41 and glutamate dehydrogenase42 and cytosolic phosphoenolpyruvate carboxykinase43 during metabolic acidosis and hypokalemia [555].44 The proximal tubule is the primary site for ammoniagenesis, especially in response to metabolic acidosis, but ammonia is formed in most renal epitheliocytes [555]. Ammonium generated by the PT is reabsorbed by the TAL and is concentrated by countercurrent multiplication within the medullary interstitium, the corticomedullary NH+ 4 gradient rising during acidosis, and then carried down its concentration gradient via apical NH3 carriers in the CD [578]. Ammonia can be transported by paracellular transport and across the plasma membrane due to NH3 permeability and through NH3 and NH+ 4 carriers (e.g., NKCC2 and NHE4) in the epitheliocyte membrane of the nephron. The HCO− 3 produced is preferentially transported across the basolateral plasma membrane and into peritubular capillaries, then returns to the systemic circulation [555]. Ammonia produced in the kidney and eliminated into urine favors acid excretion. It can also return to the systemic circulation through the renal veins to be metabolized in the liver via the HCO− 3 -consuming urea cycle [555].

41 Phosphate-dependent

glutaminase is the initial enzyme in renal ammoniagenesis. It localizes to the proximal straight and convoluted tubules and to the tDL of the loop of Henle, mTAL, DCT, and CD [555]. 42 Glutamate dehydrogenase is also a mitochondrial enzyme that exists with two isoforms encoded by two genes: GLUD1, which is widely expressed, and GLUD2, which is a neural and testicularspecific subtype. 43 Phosphoenolpyruvate carboxykinase is encoded by the PCK1 gene. In the kidney and other organs (e.g., liver, adipose tissue, and small intestine), it is implicated in gluconeogenesis. 44 Phosphate-dependent glutaminase processes glutamine to glutamate, producing NH+ ion. 4 Glutamate is predominantly metabolized by glutamate dehydrogenase, yielding α-ketoglutarate + and NH4 [555]. α-Ketoglutarate is metabolized by α KG and succinate dehydrogenase into oxaloacetic acid, a substrate for phosphoenolpyruvate carboxykinase to form phosphoenolpyruvate used for gluconeogenesis. α-Ketoglutarate metabolism generates HCO− 3 ion. Although transamination of glutamate via glutamic–oxaloacetic transaminase does not release NH+ 4 , the aspartate produced can be processed via the purine nucleotide cycle, forming fumarate and releasing NH+ 4 ion. This pathway plays a minor role in overall ammoniagenesis. Glutamate can also be metabolized through glutamate decarboxylase into γ-aminobutyric acid (GABA; 25% of glutamate oxidation in the renal cortex), but GABA metabolism to succinate semialdehyde by γ-aminobutyrate transaminase regenerates glutamate. Glutamate can also be converted back to glutamine by glutamine synthetase, utilizing NH+ 4 as a cosubstrate [555]. Glutamine can also be processed by γ-glutamyl transpeptidase (or phosphate-independent glutaminase) into glutamate, which is carried into the cell, where it can be further metabolized by glutamate dehydrogenase, further producing NH+ 4 ion. Glutamine ketoacid aminotransferase and ω-amidase also form αketoglutarate and NH+ 4 [555].

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Under basal conditions, renal ammonia excretion comprises 50–70% of bulk acid excretion. During metabolic acidosis, increased renal ammonia excretion comprises 80–90% of the overall acid excretion in humans. Ammonia production and transport are regulated by extracellular H+ and K+ concentrations along with hormones, such as mineralo- and glucocorticoids and angiotensin-2 [555]. Ammonium cation (NH+ 4 ) is formed by the protonation of ammonia (NH3 ) and vice versa. The former represents most of these twin molecules; at pH 7.4, NH3 , a small uncharged molecule, corresponds to less than 2% of the integrated amount [555]. The relative amounts of NH3 and NH+ 4 are governed by the quasiinstantaneous reaction: NH3 + H+ ←→ NH+ 4.

(3.5)

At pH 7.4, slightly more than 98% of total ammonia is present as NH+ 4 [556]. In most biological fluids, small changes in pH cause exponential changes in NH3 concentration, but do not substantially modify NH+ 4 concentration (Table 3.12). The cellular membrane with its lipid bilayer has a limited permeability for NH+ 4, + have in the absence of specific transporters. In aqueous solutions, NH+ and K 4 nearly identical biophysical characteristics (Table 3.13), which enables NH+ 4 motion through K+ carriers [556]. In addition, some Na+ –H+ exchanger isoforms can function in Na+ –NH+ 4 exchange mode. Table 3.12 Influence of pH on NH3 and NH+ 4 concentrations in a solution with 1 mmol/l total ammonia and a measure of the strength of an acid as a proton donor (or the weakness of its conjugate proton acceptor, acids being H+ donors and bases H+ acceptors, acidity rising + with increasing positive charge [i.e., from NH− 2 to NH3 and from NH3 to NH4 ]), pKa of 9.15 (Source: [556]) pH 5.0 6.0 6.5 7.0 7.2 7.4 7.6

[NH3 ] (μmol/l) 0.07 0.71 2.22 7.03 11.1 17.5 27.4

Change (%) −99.6 −96 −87 −60 −36 0 57

[NH+ 4] (μmol/l) 999.9 999.3 997.8 993.0 988.9 982.5 972.6

Change from pH 7.4 (%) 1.8 1.7 1.6 1.1 0.6 0 −1.0

Table 3.13 Some biophysical features of K+ and NH+ 4 cation (Source: [556]) Cation NH+ 4 K+

Ionic radius nm 0.0133 0.0143

Mobility in H2 O 10−4 cm2 /s/V 7.60 7.62

Transference number in H2 O 0.49 0.49

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Ammonium cation is produced predominantly within the proximal tubular cells, although ammoniagenesis takes place in most renal epitheliocytes (glomerulus cells and in epitheliocytes of S1, S2, and S3 segments of the PT, tDL, mTAL and cTAL, DCT, CCD, OMCD, and IMCD). The major source is glutamine, which enters the cell from peritubular capillaries (∼80%) and filtrate (∼20%) [555]. Plasmatic glutamine is entirely filtered by the glomerulus, and filtered glutamine is almost completely reabsorbed by the PCT. Renal glutamine uptake involves both apical and basolateral transfer using glutamine transporters (apical SLC6a18 and SLC6a19 and basolateral SLC7a7 and SLC38a3). Glutamine processing by glutaminase engenders two NH+ 4 and + two HCO− per metabolized glutamine. Further NH is formed when glutamate 3 4 is metabolized to α-ketoglutarate (footnote 44). Ammonia production and transport are regulated by extracellular H+ and K+ concentrations and mineralocorticoid and glucocorticoid hormones, insulin, and growth hormone, along with angiotensin-2 [555]. Ammoniagenesis is also regulated by tricarboxylic acid cycle intermediates and prostaglandin-F2α . In response to metabolic acidosis, urinary ammonia excretion increases, being almost entirely responsible for the net acid excretion. Chronic metabolic acidosis is associated with increased formation and activity in phosphate-dependent glutaminase, glutamate dehydrogenase, and phosphoenolpyruvate carboxykinase, which contribute to elevated ammoniagenesis [555]. Because of competition between K+ and NH+ 4 for the same membrane carriers, hyperkalemia impedes NH+ transmembrane transport and can provoke acido4 sis [578]. In hypokalemia, urinary ammonia excretion rises despite concomitant metabolic alkalosis. Chronic hypokalemia increases ammoniagenesis using mechanisms similar to those for chronic metabolic acidosis [555]. Ammonia stimulates H+ secretion through H+ –K+ ATPase, independently of intracellular pH change. Both NH3 and NH+ 4 transporters in specific renal epitheliocytes allow transfer of these chemical species across plasma membranes into the tubular lumen [555]. • In the proximal tubule, the apical Na+ –H+ exchanger NHE3 is a major carrier + functioning in Na+ –NH+ 4 exchange for NH4 secretion [556]. + + − • In the TAL, the apical Na –K –2Cl cotransporter NKCC2 is a major contributor to ammonia reabsorption, and basolateral Na+ –H+ exchanger NHE4 enables NH+ 4 exit. • The CD is a major site for NH3 and H+ secretion. Glycoproteins of the Rhesus blood group category, basolateral RhBG (SLC42a2) and apical and basolateral RhCG (SLC42a3), are ammonium transporters in the distal tubule and CD [556]. • In the IMCD, basolateral Na+ –K+ ATPase enables active basolateral NH+ 4 uptake. About 75% of ammonium is removed from the tubular fluid in the medulla within the TAL. A certain amount of interstitial ammonium returns to the PST and then reenters the medulla, thereby keeping ammonium at a high concentration in the medullary interstitium. The lower the urine pH, the higher the amount of ammonium excreted.

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Ammonia produced in the kidney is not only excreted into urine but also enters the blood circulation through the renal veins and is then metabolized in the liver using a HCO− 3 -consuming process. Regulated ammonia transport by renal epitheliocytes determines the proportion of ammonia excreted in urine and fraction returned to the systemic circulation.

3.7.4.3

and NH+ Transport of HCO− 4 3

Ion carriers involved in HCO− 3 flux belong to the SLC4 set, which includes (Table 3.14) [559]: 45 to 1. Sodium-independent electroneutral Cl− –HCO− 3 exchangers SLC4a1 SLC4a3 (AE1–AE3) and SLC4a9 (AE4) 2. Electrogenic (SLC4a4–SLC4a5 [NBCe1–NBCe2]) and electroneutral Na+ – HCO− 3 cotransporters (SLC4a7 [NBCn1 or NBC3]) 3. Na+ -dependent Cl− –HCO− 3 (or NaHCO3 –HCl) exchangers SLC4a8 and SLC4a10 4. Electrogenic Na+ –borate cotransporter SLC4a11 (a.k.a. NaBC1 and bicarbonate transporter-related protein BTR1) − Plasmalemmal Cl− –HCO− 3 exchangers regulate intracellular pH and [Cl ] and cell volume and contribute to transepithelial secretion and reabsorption of acid–base equivalents and of chloride. Members that facilitate bicarbonate transport in humans include SLC26a3, SLC26a4, SLC26a6, SLC26a7, and SLC26a9 (Table 3.15) [554]. Apical, carbonic anhydrase-dependent HCO− 3 reabsorption is accomplished owing to Na+ –H+ exchanger, primarily NHE3, apical H+ exit being accompanied by basolateral HCO− 3 egress [578]. + + Basolateral HCO− 3 outflux associated with Na –H exchanger can take several − − routes, such as (1) Cl –HCO3 exchangers (i.e., anion exchangers AE1–AE2) and + − (2) K+ –HCO− 3 cotransporter, likely K –Cl cotransporter KCC4 [578]. On the − other hand, Na+ –HCO3 cotransporter (symporter; NBCn1 or SLC4a7), which abounds in the basolateral membrane of TAL cells, primarily functions in HCO− 3 influx rather than outflux (as can operate an Na+ –HCO− 3 antiporter). Bicarbonate reabsorption by the TAL is regulated by acid–base status. It increases in acidosis and decreases in metabolic alkalosis.

45 Also

known as erythrocytic membrane band-3 (Diego blood group) anion transport protein (EMPB3, EPB3, or Bnd3), Diego antigen (in reference to the first patient in which it was discovered), and anion exchanger AE1. Twenty-one Diego antigens exist, including mainly Dia and Dib , the most common Diego phenotype being Di(a−–b+). The Dia and Dib antigens result from a single nucleotide polymorphism of the SLC4A1 gene.

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Table 3.14 Bicarbonate carriers of the SLC4A set (Sources: [554, 557, 558]; AE anion exchanger, BTR bicarbonate transporter-related protein, NaBC1 Na+ –borate cotransporter, (k)NBC (kidney) sodium–bicarbonate cotransporter, NBCe electrogenic sodium–bicarbonate cotransporter, NBCn electroneutral sodium–bicarbonate cotransporter, NCBE sodium-driven chloride–bicarbonate exchanger, NDCBE electroneutral Na+ -driven Cl− –HCO− 3 exchanger) Type Other aliases Distribution Na+ -independent electroneutral Cl− –HCO− 3 exchangers SLC4a1 AE1 RBC, heart, colon, kidney (CD ICC) (basolateral) SLC4a2 AE2 Widespread (basolateral in most epitheliocytes) SLC4a3 AE3 Brain, retina, heart, smooth muscle, kidney, pituitary and adrenal glands, digestive tract Electrogenic Na+ –HCO− 3 cotransporters SLC4a4 NBCe1, NBC1 Brain, kidney, heart, stomach, colon, pancreas, thyroid, prostate (basolateral) NBCe1a: proximal tubule, eye NBCe1b: widespread NBCe1c: brain SLC4a5 NBCe2, NBC4 Brain, myocardium, smooth muscle, kidney, liver, spleen, testes, choroid plexus (apical) Electroneutral Na+ –HCO− 3 cotransporters SLC4a7 NBCn1, NBC2/3, Widespread SLC4a6 (basolateral) SLC4a10 NBCn2, NBCE Brain, choroid plexus, kidney, adrenal cortex, uterus, cardiomyocytes (basolateral) − exchanger Electroneutral Na+ –2 HCO− –Cl 3 SLC4a8 NDCBE, kNBC3 Brain, kidney, ovary, testis, cardiomyocytes Others SLC4a9 AE4 Kidney (apical) SLC4a11 BTR1, NaBC1 Kidney, salivary gland, testis, thyroid, trachea

+ The NH+ 4 ion has a similar ionic radius to that of K and can be exported by api+ + cal NKCC2 and other K transporters. The NH4 ion exits the TAL predominantly through basolateral NHE4, which operates in the Na+ –NH+ 4 exchange mode [578]. + channels and the intercellular gap plays a through apical K Transfer of NH+ 4 smaller role under physiological conditions. The corticomedullary NH+ 4 gradient increases during acidosis.

3.7.4.4

Proximal Tubule and Hydrogen Ion Control

The proximal tubule contributes to H+ control via reabsorption of bicarbonate filtered in the glomerulus (85–90%) and production of ammonium ions. Ammonia produced in the proximal tubule is secreted preferentially into the tubular lumen.

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Table 3.15 Bicarbonate carriers of the SLC26 set (Source: [554]) Transporter SLC26a3

SLC26a4 (pendrin)

SLC26a6

SLC26a7

SLC26a9

Distribution Ileum, colon, epididymis, eccrine sweat gland, cardiomyocytes, enterocytes, (apical) Kidney, airway epithelium, thyroid, inner ear, prostate (apical) Kidney, heart, intestine, bronchial epithelium, pancreas, thymus, prostate, (apical) Type-A intercalated cells, thyroid, retina, stomach, olfactory epithelium (basolateral) Brain, heart, kidney, salivary gland, stomach, trachea, bronchiolar and alveolar epithelia (apical)

Transport Cl− –HCO− 3 exchange, also SO2− 4 (electroneutral) Cl− –HCO− 3 exchange, − I− , NO− 3 , SCN , and formate transport (electroneutral) Cl− –HCO− 3 exchange, 2− OH− , NO− 3 , SO4 , formate, oxalate transport (electroneutral) Cl− –HCO− 3 exchange, Cl− channel, sulfate (SO2− 4 ) transport oxalate transport (electrogenic) Cl− –HCO− 3 exchange, NaCl cotransport (electrogenic)

Conversely, The proximal tubule, primarily its downstream segment, can also reabsorb luminal ammonia [555]. Metabolic acidosis converts the distal proximal tubule from a reabsorber into a net secretor. The H+ ion exits the PCT cells and enters the PCT lumen through Na+ –H+ antiporter, the major route, and proton pump (H+ ATPase). Filtered HCO− 3 cannot cross the apical membrane of the PCT cell and combines with secreted H+ using CA4 to produce CO2 and H2 O. Lipid soluble CO2 crosses the plasma membrane and enters the cytosol, where it combines with OH− to produce bicarbonate. The latter crosses the basolateral membrane through an electrogenic Na+ –HCO− 3 symporter, +. for a single Na which transfers 3 HCO− 3 The basolateral membrane has a Na+ –K+ ATPase that exports three Na+ and imports two K+ ions. This pump is electrogenic in a direction opposite to that of + the Na+ –HCO− 3 symporter and sets up a Na concentration gradient required for + + the apical Na –H antiporter. + The resulting effect is reabsorption of a HCO− 3 and Na molecule from the + tubular lumen to the bloodstream per secreted H molecule. + excretion: extracellular Many factors control HCO− 3 reabsorption and H − volume, tubular flow rate, tubular HCO3 concentration, blood pCO2 , and action of autacoids such as angiotensin-2. Bicarbonate reabsorption augments upon volume expansion and when any of these other factors increases. In addition, a low

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intracellular K+ concentration elevates HCO− 3 reabsorption. Chloride deficiency − that limits HCO3 excretion provokes a metabolic alkalosis, HCO− 3 being reabsorbed instead of Cl− ion. Hypercapnia causes intracellular acidosis and raises H+ secretion. The amount of phosphate in the distal tubule does not vary markedly and thus does not have a significant regulatory effect. Aldosterone elicits Na+ reabsorption and urinary excretion of H+ and K+ ions. In the proximal tubule, ammonia is preferentially secreted into the luminal fluid through NHE3 but also Ba2+ - and acid-sensitive K+ channel. In addition, NH3 crosses the apical plasma membrane by passive lipid-phase diffusion and/or through an apical NH3 carrier [556]. Basolateral K+ channels and Na+ –K+ ATPase take up interstitial NH+ 4. 3.7.4.5

Thick Ascending Limb and Hydrogen Ion Control

+ The TAL reabsorbs luminal HCO− 3 and excretes NH4 ions. A significant amount of + HCO− 3 filtered in the glomerulus (∼15%) is exchanged by H using the NHE2 and NHE3 exchangers. Newly synthesized NHE1 localizes to both apical and basolateral plasma membranes, but mature NHE1 lodges almost exclusively in the basolateral membrane of all nephron segments, except in the macula densa and CCD ICCs [584]. In the tubular lumen, secreted H+ and luminal HCO− 3 are converted to CO2 and H2 O by apical membrane-associated carbonic anhydrase AC4, CO2 and H2 O rapidly entering the cell. In the cytosol, CO2 and H2 O are converted back to HCO− 3 + and H+ by CA2, HCO− 3 exiting the cell with Na across the basolateral membrane via electrogenic Na+ –HCO− 3 cotransporter SLC4a4 (NBCe1) into the interstitial space and then blood. Carbonic anhydrase CA2 binds to NHE1, thereby increasing its activity. Nitric oxide (NO), which can preclude Na+ transport in the proximal tubule and in TAL in addition to Cl− transport in TAL, influences Na+ –H+ exchange. It inhibits NHE3 (but not NHE2) via the sGC–cGMP–PKG pathway [560]. The majority of luminal ammonia, which is generated by the proximal tubule, especially in response to metabolic acidosis, is reabsorbed in the TAL through NKCC2, total ammonia delivered to the distal nephron ranging from 20 to 40% of the total excreted ammonia [556]. Diffusion of NH3 across the apical plasma membrane is not quantitatively significant. Cytosolic NH+ 4 can exit the cell through basolateral NHE4 and can dissociate into H+ and NH3 , which leaves the cell likely − + via diffusion, whereas intracellular H+ is buffered by HCO− 3 owing to Na –HCO3 cotransporter NBCn1 (SLC4a7). A fraction of ammonia that is reabsorbed by the TAL is recycled in the tDL and contributes to generation of an axial interstitial ammonia concentration gradient.

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Ammonium is concentrated by countercurrent multiplication within the medullary interstitium [578]. Although in the TAL a significant amount of luminal NH+ 4 is reabsorbed, it is secreted again in the CD down its concentration gradient through the apical NH3 carrier.

3.7.4.6

Distal Nephron and Hydrogen Ion Control

+ In the distal nephron, reabsorption of HCO− 3 is mediated by apical H secretion by + + + + α ICCs. Two transporters secrete H , a vacuolar H and an H –K ATPase. Distal segments secrete ammonia, the CD being the site of most ammonia secretion, which involves parallel H+ and NH3 ouflux. Net ammonia secretion occurs between the early and late segments of the DCT and accounts for 10–15% of total urinary ammonia excretion under basal conditions [556]. In the CD, ammonia secretion involves mainly parallel NH3 and H+ transport. Transporters that play an important role are basolateral Na+ –K+ ATPase in the IMCD for NH+ 4 uptake and Rhesus glycoproteins, RhBG and RhCG, which pertain to the SLC42 set (SLC42a1–SLC42a3), for NH3 transport (Table 3.16).46 Intracellular NH3 is secreted across the apical membrane by apical RhCG and H+ by H+ and H+ –K+ ATPases. Intracellular H+ secreted by these pumps is generated after CO2 hydration by carbonic anhydrase CA2, which forms carbonic acid, which − − − dissociates to H+ and HCO− 3 . Basolateral Cl –HCO3 exchanger carries HCO3 + + across the basolateral membrane, which combines with H released from NH4 to form carbonic acid, which dissociates to CO2 and water. Na+ –K+ –2Cl− cotransporter NKCC1, which resides in the basolateral region of ICCs in the OMCD and IMCD and of IMCD PCs, neither markedly influences OMCD ammonia secretion nor has an impact on significantly peritubular NH+ 4 uptake in the IMCD [556].

46 Three

mammalian Rhesus glycoproteins exist (RhAG–RhCG). They include the heterooligomeric erythroid Rhesus glycoprotein RhAG and two epithelial membrane molecules RhBG and RhCG [561]. Erythroid Rhesus-A glycoprotein is specific to red blood capsules. The Rhesus complex consists of RhAG linked to the nonglycosylated Rhesus proteins RhD and RhCE in humans [556]. It mediates electroneutral NH3 transport. The RhAG–SLC4a1–ankyrin complex can also modulate HCO− 3 transport. RhAG may also reside in human esophageal epithelia and mouse brain [561]. RhBG is produced in organs involved in ammonia metabolism, such as the kidney, liver, lung, stomach, gastrointestinal tract, and skin. In the kidney, it lodges in the DCT, CnT, CCD, OMCD, and IMCD, as a basolateral carrier, in both α ICCs (albeit in a greater amount) and PCs [556]. It lodges in all ICC types of the OMCD and IMCD [561]. RhCG is widespread; it is detected in the kidney, central nervous system, lung, liver, throughout the gastrointestinal tract, and testes [556]. In the kidney, RhCG has the same distribution as RhBG, with the exception of the IMCD, in which only intercalated cells synthesize RhCG. It localizes to both apical and basolateral membranes of mouse CD cells [561].

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Table 3.16 Human Rhesus glycoproteic ammonium transporters (Source: [561])

Gene SLC42A1

Protein RHAG

SLC42A2

RHBG

SLC42A3

RHCG

Predominant substrate NH+ 4, NH3 NH+ 4, NH3 NH+ 4, NH3

Transport features Coupling ions H+ Electrogenic No coupled ions Electroneutral possibly H+ +

Distribution Red blood capsule Kidney, liver, digestive tract, skin, sweat glands, ovaries (basolateral membrane) Brain, pancreas, kidney (apical membrane), prostate, testis, placenta

Whereas Na+ –K+ ATPase does not significantly contribute in the CCD to ammonia secretion, it mediates basolateral NH+ 4 uptake from the interstitium in the IMCD, favoring ammonia and acid secretion [556]. Various subtypes of H+ –K+ ATPases lodge in the apical region of CD cells. They can carry NH+ 4 [556]. As H2 O and NH3 have similar molecular sizes and charge distribution, some aquaporin species (e.g., Aqp1 and Aqp3) can transport ammonia.

3.7.5 Compartments of the Nephron The nephron consists of the renal corpuscle and tubule, itself composed of a PCT and a straight tubule, loop of Henle with its tDL and tAL and TAL, DCT, and CD.

3.7.5.1

Renal Corpuscle

The renal corpuscle consists of the glomerulus and its surrounding Bowman’s capsule (Fig. 3.2). 1. The renal glomerulus is the capillary circuit inside Bowman’s capsule. 2. Bowman’s capsule is a cup-like sack at the entrance of the tubular component of the nephron that encloses the renal glomerulus. Glomerular filtrate from blood is collected in BS to be processed along the nephron and form urine. The kidney contains about 2·106 glomeruli that produce approximately 180 l per day of primary filtrate. Downstream tubules reabsorb most of the water, salt, and low-molecular-weight substances, excreting 1–2 l per day of urine containing unwanted waste.

3.7 Kidney and Blood Pressure Control

245 EpC

BS PC

PCT

N

EC

AA IGMC

JGC EGMC MDC

JGC

EpC EA

Fig. 3.2 Renal corpuscle and juxtaglomerular apparatus. The renal corpuscle comprises the glomerulus (i.e., glomerular capillary loops and connecting branches) and its surrounding Bowman’s capsule composed of the Bowman’s space (BS) limited by epitheliocytes (EpC) and podocytes (PC) at its outer and inner edge, respectively. It engenders the proximal convoluted tubule (PCT). Intraglomerular mesangiocytes (IGMC) lodge between glomerular capillaries, thus contacting endotheliocytes (EC) and possibly PCs. The capillary circuit conveys blood from the afferent arteriole (AA) to the efferent arteriole (EA). Smooth myocytes of the glomerular ends of these arterioles differentiate into renin-producing juxtaglomerular cells (JGCs) that form the juxtaglomerular apparatus with extraglomerular mesangiocytes (EGMC) and macula densa cells, which are specialized epitheliocytes at the edge facing the glomerulus at the junction between the thick ascending limb of the loop of Henle and the distal convoluted tubule. The juxtaglomerular apparatus is innervated by sympathetic fibers. Mesangiocytes, podocytes, and endotheliocytes cooperate in this structural and functional unit via juxta- and paracrine signaling and crosstalk

Nephrons can be categorized according to the position of their glomeruli in the cortex: superficial, midcortical, and juxtamedullary. Superficial and midcortical glomeruli are smaller than juxtamedullary glomeruli [562].

Bowman’s Capsule: Podocytes Bowman’s capsule is composed from its outside to its inside surface of a single parietal layer of epitheliocytes, BS, and a visceral layer of podocytes in contact with the glomerular capillaries. Podocytes wrap glomerular capillary endotheliocytes. Foot processes of podocytes form the filtration barrier through which water, salts, and waste products pass into the urine. Their long regularly spaced pedicels interdigitate. Thin (40 nm) betweenpodocyte filtration gaps, or slits, are covered by slit diaphragms. The slit diaphragm is a specialized cell junction and signaling site. It is made up of the transmembrane

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proteins nephrin47 and nephrin-like protein Neph1, podocalyxin,48 podocin,49 CD2AP,50 and the mechanosensory stretch-activated ion channel TRPC6, along with adherens (e.g., P-cadherin, cadherin family member CdhF7 [Fat1], and catenins) and tight junction proteins (e.g., occludin, cingulin, JAMa, and ZO1), which restrict the passage of plasma proteins and other macromolecules [563].

Glomerular Mesangiocytes Intraglomerular (or simply glomerular) mesangiocytes located along glomerular capillaries are specialized pericytes (30–40% of the total glomerular cell population). They form with their matrix the vascular pole of the renal glomerulus. They constitute the framework of the glomerulus, which maintains capillary circuit organization. Mesangiocytes build with endotheliocytes and podocytes a structural and functional unit. For example, podocyte-derived VEGF ensures glomerular endotheliocyte survival and functioning. Interdigitations between endotheliocytes and mesangiocytes favor signaling between these two cell types. Mesangiocytes produce matrix constituents, such as α1 and α2 chains of collagen-4, collagen-5, and -6; fibronectin; laminins LamA, LamB1, and LamB2; 47 Nephrin

(Nphn) is also called renal glomerulus-specific cell adhesion receptor and type-1 nephrosis (or nephrotic syndrome) protein Nphs1. Nephrosis is characterized by severe proteinuria, hypoalbuminemia, hyperlipidemia, and edema, due to increased glomerular permeability. Type1 nephrosis is a congenital nephrotic syndrome of the Finnish type caused by mutations in the NPHS1 gene. 48 Podocalyxin (Podxl) maintains an open filtration passage between neighboring foot processes of podocytes by charge repulsion. 49 Podocin (Pdcn) is also called type-2 nephrosis (or nephrotic syndrome) protein Nphs2. At the plasma membrane, podocin homo-oligomerizes. Podocin localizes to membrane rafts, where it recruits nephrin into these rafts that are involved with carriers in the renal filtration barrier. Type2 nephrosis caused by mutations in the NPHS2 gene is an autosomal recessive steroid-resistant nephrotic syndrome (SRN), hence the additional alias SRN1 for this protein. Mutations of the NPHS1 and NPHS2 genes provoke an early onset of heavy proteinuria and rapid progression to end-stage renal disease. 50 CD2-associated protein, also named CRK-associated substrate (CAS) ligand with multiple SH3 domains (CMS), is a binding partner of the CAS docking protein, which colocalizes with F actin, especially at membrane ruffles. Like CAS, it is phosphorylated by protein Tyr kinases. This adaptor (scaffold) protein facilitates intercellular junctions. It lodges between membrane proteins and the actin cytoskeleton at the insertion of the podocyte slit diaphragm. It thus anchors the slit diaphragm to the actin cytoskeleton in the renal glomerulus, among other functions in other cell types, such as neurons and immunocytes. The adhesion molecule CD2 (a.k.a. sheep erythrocyte receptor [SRBC] and T-cell surface antigen) initiates protein segregation, CD2 clustering, and cytoskeletal polarization [194]. It interacts with its receptor lymphocyte function-associated antigen LFA3 (CD58) and its ligand B-cell activation marker BCM1 (plasmalemmal glycoprotein expressed primarily in mitogen-stimulated lymphocytes CD48; a.k.a., B-lymphocyte activation marker Blast1 and SLAM [signaling lymphocytic activation molecule] family member SLAMF2), a member of the immunoglobulin superclass, to mediate adhesion between T cells and other cell types.

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heparan (e.g., perlecan)51 and chondroitin sulfate proteoglycans; small leucinerich proteoglycans decorin52 and biglycan;53 and sulfated monomeric glycoproteins nidogens [565].54 They also control the turnover of the mesangial matrix. They yield a structural support for glomerular capillary loops and regulate blood flow via their contractility, but this feature may play a weak role. They contribute to the regulation of the glomerular filtration surface and ultrafiltration. Mesangiocytes may sense capillary tension generated by glomerular pressure. They respond to stretch, as they release CTGF, TGFβ1, VEGF and activate signaling cascades implicating PKC, ERKs, JNKs, P38MAPKs, and the PI3K–PKB axis [565]. Conversely, angiotensin-2, endothelin-1, and platelet-activating factor affect mesangiocyte growth. The growth factors CTGF, EGF, FGF, HGF, PDGFa to PDGFc, and TGFβ influence mesangiocyte proliferation and mesangial matrix production [565]. Although VEGF is a survival factor generated by podocytes for endotheliocytes, PDGF produced by endotheliocytes supports the survival of mesangiocytes. Mesangiocytes also serve as local modulators of innate and adaptive immunity [565]. They are able to phagocytize components of the glomerular basement membrane, removing trapped and aggregated proteins from it to keep the filter free of debris, and support clearance by neutrophils of dying cells. Mesangiocytes possess the glycan recognition receptors C-type lectins and Siglecs. In addition to these receptors for glycoproteins, mesangiocytes are endowed with TLR2 to TLR4.

Filtration Barrier The filtration barrier permits the passage of water, ions, and small molecules from the bloodstream into BS but prevents the flux of large proteins and negatively charged molecules, slits spanned by a diaphragm acting as a low-porosity molecular sieve.

51 Perlecan

(pearl-like structure) is a large proteoglycan that is primarily confined to basement membranes. 52 Decorin “decorates” collagen fibrils. It is involved in cell growth, survival, metastasis, and angiogenesis. It neutralizes diverse growth factors. It interacts with thrombospondin, collagen, and fibronectin and connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)β, and epidermal growth factor receptor (EGFR) and HER1 to HER4, in addition to myostatin [564]. Multiple peptidases produced by inflammatory cells counter its anti-inflammatory effect. 53 Biglycan is involved in collagen fibril assembly, cell proliferation, migration, and apoptosis, along with inflammation and innate immunity. 54 Nidogens Nod1 (formerly known as entactin) and Nid2 are ubiquitous components of the basement membrane.

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The basement membrane of endotheliocytes, which forms a fenestrated endothelium, and that of podocytes are fused. Glomerular endothelial fenestrae can be spanned by a diaphragm and are covered by a dense glycocalyx thats limit barrier permeability. The glomerular basement membrane (GBM) consists of, from the endotheliocyte to the podocyte, (1) the lamina rara interna, (2) the lamina densa, and (3) the lamina rara externa. The lamina rara interna and externa contain heparan sulfate and the lamina densa collagen-4 and laminin. Molecular size-dependent permeation into the GBM and the gel-like coat covering the slits, in addition to saturable tubular reabsorption, determine the molecular species that are conveyed by urine [566]. Spreading throughout the GBM of macromolecules is inversely related to their hydrodynamic size. Proteins and neutral dextran also permeate through the GBM. Molecules, such as parvalbumin and ovalbumin, which can pass through the glomerulus, are captured by proximal tubule cells and do not reach the urine unless this capture is saturated. On the other hand, filtered albumin and IgG are fully captured and are not excreted with urine. Nanoparticles, the size of which is similar to that of IgG dimers, do not permeate through the lamina densa; IgG monomer-sized particles permeate slightly; and albumin-sized particles permeate extensively [566]. Particles that traverse the lamina densa tend to accumulate in front of the podocyte glycocalyx, which spans the slit, but not upstream of the slit diaphragm. At low concentrations, ovalbuminsized nanoparticles reach the primary filtrate, are then captured by proximal tubular cells, and endocytosed. At higher concentrations, the tubular capture is saturated, and they reach the urine. The Ogston model describes the motion of a spherical particle through a random, inert, isotropic meshwork of straight, rigid, randomly oriented fibers of infinitesimal thickness assumed to be infinitely long rods according to the fractional volume, which enables the particle displacement, the pore space being randomly distributed [567]. The distribution of pore size has a greater influence than the fiber distribution. The position of a fiber is defined by that of its center. The sizedependent permeation of macromolecules (e.g., albumin [caliber 3.5 nm], carbonic anhydrase [caliber 2.2 nm], cytochrome-C [caliber 1.7 nm], and myoglobin [caliber 1.9 nm]) into a gel, that is, the fraction (FV ) of the total volume of the fiber suspension (uniform fiber density ρf ; radius Rf ) that is available for a spherical particle (radius Rp ; half-length ) is given by: FV = exp{−(2πρf Rp2 + 4/3π Rp3 )},

(3.6)

the first term corresponding to a tangential contact between the particle and the fiber and the second term to a point contact between the particle and fiber end. Expression was also obtained for a soluble solute (radius rsol ) immersed in a gel (void volume VV ) made up of moderately flexible rods [568]: FV = exp{(VV − 1)(1 + rsol /rf )p },

(3.7)

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249

where p is a scaling parameter related to the fiber stiffness. Using a statistical approach, another formula was proposed [569]: FV = exp{1 − VV } exp{

  r + r 1 sol f 2 (1 − VV ) 1 − ) }, VV rf

(3.8)

Proteinuria results from damage to podocyte processes. In a rat model of chemically induced glomerular disease and acute nephrosis, the transcriptional coactivator YAP is activated and transcription of YAP target genes precedes proteinuria [570]. Upon YAP stimulation, the amount of extracellular matrix proteins augments and can contribute to fibrosis. Concentration of the YAP target gene product CTGF in renal biopsies from glomerular disease patients increases. Activity of YAP rises in mouse models of diabetic nephropathy. Activity of YAP and Taz is stimulated by mechanical stress in most cell types; mechano-transduction activates YAP in podocytes. Injury reduces YAP and Taz activity in podocytes cultured in stiff matrix, whereas soft matrix in addition to inhibition of stress fiber formation mimics the effects of damage-induced YAP upregulation [570]. When interaction between YAP and TEAD is prevented in rats, damage-induced mechano-signaling in the renal glomerulus lowers and glomerular disease ameliorates.

3.7.5.2

Juxtaglomerular Apparatus

The juxtaglomerular apparatus, an anatomical and functional unit situated at the hilum of the glomerulus, consists of juxtaglomerular, extraglomerular mesangial, and MDCs (Fig. 3.3). Therefore, the juxtaglomerular apparatus consists of a tubular component, the macula densa, attached to a vascular component, JGCs of the afferent and efferent arterioles, that both contact the extraglomerular mesangium. 1. Juxtaglomerular cells are specialized smooth myocytes in the wall of the afferent and efferent arterioles endowed with β1-ARs that synthesize, store, and secrete renin. 2. Macula densa cells, which constitute a cluster of 15–20 cells, at the TAL– DCT junction detect salt (sodium chloride) concentration to control activity of JGCs. These specialized epitheliocytes of the terminal segment of the TAL and DCT entry segment have a basolateral membrane that contacts extraglomerular mesangiocytes, which are contiguous with granular cells in the juxtaglomerular apparatus. Therefore, MDCs have an apical membrane exposed to the tubular fluid, whereas their basilar aspects are connected to cells of the mesangium and the afferent arteriole (Table 3.17). 3. Extraglomerular mesangiocytes reside in the region between the afferent and efferent arterioles.

250

3 Hypertension aldosterone Ca V

ATn2

AT2 AT 1

AC5/6 MR

Ca 2+

ET1

PDE3a

ETA/B

cAMP

1AR

N

PKA preprorenin

NAd

prorenin

PGi PGe 2 NO

cGMP NOS3

storage

DAG−−PKC IP3 −−Ca2+

PKG2 Gi

cAMP Gi

sGC

1AR

exocytosis

A1

EGMC

ROS

(+)

PGeR2/4 sGC

renin

PLC

JGC

(+)

PGiR

gene transcription

PGeR2/4

renin adenosine

EC

Gs Gs NPR1

D 1/5 AdCyAP1R1

D

2/4

ANP ATNg

D 2/3 (−) 1AR

MDC

NO

ATP

NOS1

redox stress inflammation hypertension

(+) Na

+

PGe 2

PGhS2, PGeS1

ATn1

ACE1 ATn ATn 1−9 ACE2 ACE1 ATn2 ACE2

ATn

1−5

ACE1 1−7

ERK1/2 D

1/2

NKCC2 NHE2 tubular salt sensors

Fig. 3.3 Juxtaglomerular apparatus (JGA) constituted by juxtaglomerular (JGC), extraglomerular mesangial (EGMC), and macula densa cells (MDC). It is the site of crosstalk between various types of cells including not only the constituent JGA cell types, but also adjacent endotheliocytes (EC) and nerve endings (N). Renin is released primarily from juxtaglomerular cells of afferent arterioles of the glomerulus. Circulating prorenin and renin are secreted mainly from the kidney, although other organs liberate prorenin into the circulation. Prorenin can be converted to renin by proteolysis catalyzed, for example, by trypsin in the circulation. Circulating prorenin can be taken up by cells. Most of the circulating dopamine derives from the kidney. Dopamine impedes ROS synthesis by NOx and hence redox stress, inflammation, and subsequently hypertension Table 3.17 Examples of proteins synthesized in epitheliocytes of the thick ascending limb (TAL) of the loop of Henle and macula densa (MD) cells (Source: [571]; EGF epidermal growth factor, G6PDH glucose 6-phosphate dehydrogenase, PGhS prostaglandin-G/H synthase [COx], V 2 type-2 vasopressin (or antidiuretic hormone [ADH]) receptor) MD cell G6PDH NOS1 PGhS2 NKCC2 Maxi anion channel V2

TAL epitheliocyte Na+ –K+ ATPase Uromodulin PGhS2 NKCC2 EGF V2

In the macula densa, the Na+ –K+ –2Cl− cotransporter NKCC2 regulates renin secretion

Macula densa cells express on their apical surface chemosensory components of olfactory signaling, such as olfactory G protein and adenylate cyclase isoform AC3 [572]. AC3 supports glomerular filtration and renin secretion.

3.7 Kidney and Blood Pressure Control

251

The negative tubuloglomerular feedback relies mainly on the macula densa, which senses changes in luminal NaCl delivery via the apical cotransporter NKCC2 and adjusts the vascular tone of the afferent arteriole. A high salt concentration ([NaCl]) in the tubular fluid linked to an elevated glomerular filtration rate (GFR) and reduced reabsorption of sodium and water by the PCT triggers high distal tubular NaCl-induced afferent arteriolar vasoconstriction. Conversely, a small NaCl concentration related to a reduced GFR primes a low tubular NaCl-induced renin release. Extracellular adenosine, which derives from released cellular adenosine or extracellular breakdown of ATP, AMP, or cAMP, is generated at an elevated rate when tubular NaCl reabsorption increases [573]. Elevated salt concentration raises osmolarity in macula densa cells, as the amount of basolateral Na+ –K+ ATPase is insufficient, and causes cell swelling. Resulting ATP liberation through ATP-releasing stretch-activated nonselective maxi anion channel [574]55 and subsequent processing into adenosine that binds to its A1 receptor,56 leads to constriction of the adjacent afferent arteriole and lower blood flow to the glomerular capillaries and hence the GFR. A decrease in sodium concentration reduces Na+ reabsorption in macula densa cells that augment release of renin and production of NO to dilate the afferent arteriole. Signaling causes synthesis and release of PGe2 that connects to its PGeR2 and PGeR4 receptors in JGCs that then secrete renin, which leads to a GFR increase [572].57 In addition, NOS1 in MDCs operates in the renin signaling. anion channel belongs to the set of volume-regulated Cl− channels. This ubiquitous nonselective channel is sensitive to arachidonic acid and permeable to anionic metabolites, such as glutamate and ATP, thereby operating in nucleotidic and glutamatergic signaling [575]. It frees anionic signaling molecules ATP and excitatory amino acids from cells subjected to osmotic perturbation, ischemia, or hypoxia. Maxi anion channel is another swelling-induced ATP release route, in addition to hemichannels constituted by pannexin-1 and -2 and connexin-43. 56 The AC–cAMP pathway is stimulated by the adenosine A , but inhibited by A and A 2 1 3 receptors [573]. Both A1 and A2 lodge in the afferent arteriole in the kidney, constricting and dilating the afferent arteriole, respectively. Angiotensin-2, nitric oxide, and superoxide modulate the response of the adenosine–A1 couple. ATP provokes vasoconstriction of preglomerular arterioles via the P2X1 receptor, but also acts via its metabolite adenosine. The A2 receptor counteracts A1 action and hence modulates the TGF [573]. 57 Renin release primed by the macula densa involves a signaling cascade with NKCC2 that activates P38MAPK, ERK1, and ERK2, PGhS2, and PGeS [572]. Three prostaglandin-E synthase isozymes include PGeS1 to PGeS3, which are also called microsomal membrane-associated prostaglandin-E synthases mPGeS1 and mPGeS2 and cytosolic PGeS (cPGeS), respectively, which are encoded by three genes (PTGES1–PTGES3). The PGeS1 subtype, which is highly inducible by cytokines, participates in BP regulation within the kidney. It predominates in the distal nephron, where its synthesis is induced by salt loading [576]. It may facilitate renal salt excretion and modulate the response to angiotensin-2. It predominantly localizes in the CD. In the macula densa, the synthesis of PGhS2 and PGeS1 is primed in response to salt depletion and Agt convertase inhibition. The PGeS2 isozyme is linked to Golgi body membrane. It is constitutively expressed. The PGeS3 enzyme, which is also constitutively expressed, resides in the cytosol. Concentration of PGeS2 is higher in the renal cortex than in the medulla in mouse kidneys, but it is ubiquitous along the nephron, albeit at a much lower concentration in the glomerulus. PGeS3 is also 55 Maxi

252

3.7.5.3

3 Hypertension

Proximal Tubule

The proximal tubule of superficial and juxtamedullary nephrons can be decomposed into S1 to S3 segments according to their histological features [577]. Epitheliocytes of the S1 segment have the most extensive cellular interdigitation and dense brush border membranes. • In superficial nephrons, the S1 segment begins at the urinary pole of the renal corpuscle and transforms gradually to the S2 segment that generates at different medullary levels the S3 segment, which terminates at the border between the outer and inner stripe of the outer medulla. • In juxtamedullary nephrons, the short S3 segment also terminates at the border between the outer and inner stripe of the outer medulla. Proximal tubules are functionally heterogeneous (Table 3.18). The GFR is relatively higher in juxtamedullary nephrons than that of superficial nephrons [577]. Proximal tubular reabsorption of sodium, bicarbonate, and fluid in juxtamedullary nephrons exceeds that in superficial nephrons. In addition, the sodium/chloride permeability ratio differs along the PCT length of superficial nephrons, with Na+ and Cl− permeability higher in the upstream and downstream segments, respectively [577]. On the other hand, this heterogeneity in relative sodium and chloride permeability is not observed in the PCT of juxtamedullary nephrons. − The HCO− 3 /Cl permselectivity ratio is greater in the PCT of juxtamedullary nephrons than in that of superficial nephrons. In the proximal tubule, the electroneutral Na+ –H+ exchanger links Na+ reabsorption to that of bicarbonate. A lumen-negative electrical potential difference persists in the PCT of juxtamedullary nephrons, but not in those of superficial nephrons. Expression and distribution of enzymes (e.g., APs), ion carriers (e.g., Na+ –H+ exchanger NHE3, Na+ –K+ ATPase, and Na+ –HCO− 3 cotransporter), and G-protein-coupled receptors are also heterogeneous [577]. Molecules reabsorbed in the PT include not only ions (Na+ , K+ , Cl− , bicarbonate, phosphate, and sulfate), but also solutes (glucose, amino acids, and some organic acids, among other molecules) [537]. Reabsorption of Na+ and glucose

formed in all nephron segments [576]. The renal medulla predominantly produces PGs and NO, the two important regulators of distal tubular fluid reabsorption, which have a diuretic effect to stabilize BP during salt loading; PGhS2, and NOS1 to NOS3 are all induced by a high-salt diet. Nitric oxide activates PGhS2 and hence PGe2 release via the MAPK module; conversely, PGe2 stimulates NOS1 probably via cAMP and hence NO formation. This mutual activation between PGe2 and NO in the kidney that happens at least during chronic salt loading may be restricted to the CD [576]. In addition to NKCC2, the apical Na+ –H+ exchanger SLC9a2 (NHE2) not only participates in Na+ transport, contributing to the regulation of cell volume and intracellular pH, but also in macula densa salt sensing and renin control. SLC9a2 inhibits ERK1 and ERK2 [572]. This polarized SLC9a2–SLC9a4 (NHE2–NHE4) configuration in the macula densa differs from the usual SLC9a1–SLC9a3 (NHE1–NHE3) arrangement in other nephron segments.

3.7 Kidney and Blood Pressure Control

253

Table 3.18 Functional heterogeneity within and between proximal tubules in superficial and juxtamedullary nephrons (Source: [577]; NBC Na+ –HCO− 3 cotransporter, NaPiC sodium–phosphate co-transporter, PT proximal tubule) Feature Na+ –HCO− 3 reabsorption Na+ permselectivity Cl− permselectivity − HCO− 3 -to-Cl selectivity Lumen-negative electrical potential difference NHE3 expression NaPiC expression NBC expression Reabsorption in response to renal perfusion pressure Reabsorption in response to atrial natriuretic peptide

Superficial nephron Smaller Higher upstream Higher downstream Smaller Absent

Juxtamedullary nephron Greater Uniform Uniform Greater Persistant

Abundant Greater Lower Decreased

Prominent Lower Higher Decreased

Unaltered

Increased

from the filtrate occurs via apical Na+ –glucose cotransporters SGlT1 (SLC5a1) and SGlT2 (SLC5a2) according to the PT segment, which is driven by the Na+ gradient.

3.7.5.4

Loop of Henle

The loop of Henle of long-loop nephrons comprises four parts: (1) a possible thick descending limb in the outer stripe of the outer renal medulla; (2) a tDL in the inner layer of the outer renal medulla and inner medulla, which has a low permeability to ions and urea but is highly permeable to water and forms a sharp bend; (3) a tAL in the inner medulla, which is impermeable to water but permeable to ions; and (4) a TAL in the outer and inner layers of the outer medulla and cortex, where it ends in front of the glomerulus to engender the distal convoluted tubule. About 30% of filtered salt is reabsorbed in this segment. Nephrons are also classified according to the length of the loop of Henle. The long-loop nephron has a loop of Henle in the inner medulla, where the osmotic pressure is the highest, whereas the loop of Henle of other nephron types resides either in the outer medulla or is confined to the cortex (cortical nephron). Shortloop nephrons have longer cTALs than long-loop nephrons (Fig. 3.1) [562]. The mTALs of short- and long-loop nephrons generally have a similar length. Longloop nephrons have larger glomeruli, longer PCT, shorter PST, and longer thin descending and thin ascending limbs; the latter are not found in short loops of Henle. The short- to long-loop nephron ratio is estimated to be approximately 5 in humans [562].

254

3 Hypertension

The classification of nephrons by glomerulus position is not mapped to categorization according to the length of the loop of Henle [562], although (not as a general rule), short-loop nephrons that originate from superficial and midcortical nephrons have a short descending limb within the inner stripe of the outer medulla, which, close to the hairpin-shaped turn of the loop, is followed by the TAL, and long-loop nephrons originating from juxtamedullary glomeruli have a long ascending thin limb [578]. Efferent arterioles of superficial glomeruli ascend directly to the surface; those of midcortical glomeruli are relatively short; and those of juxtamedullary glomeruli descend directly into the outer medulla to form descending (arteriolar) VRR [562]. Vasa recta renis58 are relatively straight capillaries in the renal medulla paralleling the loop of Henle between the proximal and DCTs. Vasa recta renis comprise arteriolae and venulae rectae renis. Straight arterioles branch off efferent arterioles of juxtamedullary nephrons, enter the renal medulla, and travel along the loop of Henle. They give rise to peritubular capillaries, which are permeable to sodium, chloride, urea, and water. Straight arterioles convey blood at a very slow rate for countercurrent exchange that engenders the high osmolality of the medulla with a high urea concentration deep in the medulla. In their descending arteriolar segment, salt (NaCl) and urea are reabsorbed into blood, whereas water is secreted into the renal interstitium; hence, blood in these straight capillaries loses water and gains NaCl and urea progressively as it descends in the medulla. In their ascending venular segment, which goes in the reverse direction back to the cortex, NaCl is secreted into the interstitium, whereas water is reabsorbed; hence blood progressively gains water and loses NaCl and urea. Medullary blood flow through the vasa recta circuit is weakly autoregulated.

Paracellular Transport Apical ionic influxes through NKCC2 does not suffice to balance basolateral Na+ and Cl− outfluxes. Additional Na+ is transported across the epithelium via the paracellular route. In fact, the paracellular route enables reabsorption partly of Na+ and entirely of Ca2+ and Mg2+ in the TAL. Tight junctions in the TAL are cation-selective, with a ratio of relative permeability of Na+ to that of Cl− (PNa /PCl ) ranging from 2 to 5 [578]. The TAL reabsorbs 50–60% of filtered magnesium and approximately 20% of filtered calcium exclusively via the paracellular pathway.

58 Vasa

recta are also called straight seminiferous tubules (tubuli recti) and intestinal vasa recta (in the ileum and jejunum). From Latin vas rectum (plural vasa recta); vasum (plural vasa), or vasus (plural vasi), in fact, vas (plural vasa): vessel, dish, utensil, instrument, and, in anatomy, a duct (vessel) transporting any bodily fluid, such as blood, lymph, chyle, or semen (plural nominative vasa and genitive vasorum, vasa vasorum being blood vessels irrigating the large blood vessels); recta: straightway, directly.

3.7 Kidney and Blood Pressure Control

255

Mouse TAL cells coexpress claudins Cldn3, Cldn10, Cldn11, Cldn14, Cldn16, and Cldn19, which ensure the charge and size selectivity of tight junctions [578]. Claudins Cldn16 and Cldn19, which interact, are major agents of the cation selectivity of TAL tight junctions. Other claudins expressed in the TAL either modulate activity of the Cldn16–Cldn19 heterodimer or have independent effects on paracellular transport. Transmembrane Ion Fluxes in the TAL The TAL of the loop of Henle participates in the maintenance of ECF volume, the urinary concentrating mechanism, calcium and magnesium homeostasis, bicarbonate and ammonium balance, and urinary protein composition [578]. The TAL exerts its function using coupled proteins. In particular, apical Na+ – + K –2Cl− cotransporter and inward rectifier K+ channel ROMK and basolateral Cl− channel ClCKB coordinate their ion fluxes to mediate salt absorption. Potassium ions entering the cell through NKCC2 return to the tubular lumen through ROMK; this recycling ensures a proper NKCC2 function. In addition, the lumen-positive voltage in the TAL due to K+ recycling enables absorption of Na+ , Ca2+ , and Mg2+ through the paracellular pathway [518]. The TAL is an important site of Ca2+ and Mg2+ absorption. Salt absorption is also regulated by the G-protein-coupled calcium-sensing receptor, which constitutively homodimerizes [518]. Its activity is modulated by ionic strength and by pH. Activated CaSR prevents Ca2+ absorption induced by PTH in addition to passive paracellular Ca2+ flux. Aquaporin-1 is a marker of the tDL. The apical membrane of TAL cells is impermeable to water [578]. However, the basolateral membrane of TAL cells possesses aquaporin-1, allowing water flux across the basolateral membrane adaptive to changes in cell volume in response to changes in interstitial osmolality (but not linked to a transcellular flux). The TAL contains a rough-surface cell type (R TAL cells) with prominent apical microvilli and smooth-surface cell type (S TAL cells) with abundant subapical vesicles [578]. They differ by their expression pattern of EGF and NKCC2, the latter being nevertheless expressed in both cell types. Three types of K+ channels at the apical membrane (urinary surface) of TAL epitheliocytes include (Table 3.19 Fig. 3.4): (1) high-conductance, Ca2+ -activated, voltage-gated BK channel, (2) ATP-dependent inward-rectifying ROMK (ROMK [KIR 1.1]), and (3) electroneutral Na+ –K+ –2Cl− cotransporter NKCC2 (SLC12a1). Sodium and chloride ions traversing the apical cell surface through NKCC2 leave the cell at the basolateral membrane through the Na+ –K+ ATPase and ClCKB channel. The Na+ gradient generated by Na+ –K+ pump drives the apical entry of Na+ , K+ , and Cl− through NKCC2 [578]. The basolateral exit of Cl− from TAL cells is primarily, but not exclusively electrogenic and mainly mediated by Cl− channels. At least two ClC channels, ClCKA and dominant ClCKB , are coexpressed on the TAL cells. The ClCKA subtype resides in both apical and basolateral membranes of the tAL, whereas

256 Table 3.19 [Ion carriers of epitheliocytes lining the thick ascending limb (TAL) of the loop of Henle (Source: [579])

3 Hypertension Type Ion flux type Apical membrane (urine pole) NKCC2 Influx of Na+ , K+ , Cl− (SLC12a1) Efflux of NH+ 4 ROMK Efflux of K+ (KIR 1.1) Influx of NH+ 4 BK (KCa 1) Efflux of K+ Na+ –H+ exchanger Influx of Na+ (NHE2 or SLC9a2) Efflux of H+ (NHE3 or SLC9a3) Basolateral membrane (blood pole) Na+ –K+ ATPase Efflux of Na+ Influx of K+ ClCKB Efflux of Cl− KCC4 (SLC12a7) Efflux of K+ , Cl− NHE1 Influx of Na+ (SLC9a1) Efflux of H+ NHE4 Influx of Na+ (SLC9a4) Efflux of NH+ 4 − + Na –HCO3 symporter Influx of Na+ , HCO− 3 (SLC4a4 [NBCe1]) Cl− –HCO− Influx of Cl− 3 exchanger (SLC4a1/2 [AE1/2]) Efflux of HCO− 3 A paracellular flux conveys Na+ , Ca2+ , and Mg2+ ions from the tubular lumen to the blood. Two major transfer pathways contribute to Na+ absorption in the TAL, the electroneutral Na+ –K+ –2Cl− cotransporter and Na+ –H+ exchanger. Apical electroneutral cotransport of Na+ , K+ , and 2 Cl− or Na+ , NH+ 4, and 2 Cl− through NKCC2 is complemented by K+ recycling through apical ROMK channel, Cl− efflux through basolateral Cl− channels, and basolateral extrusion of Na+ by Na+ –K+ ATPase. Ionic fluxes are aimed at ensuring the ion–water homeostasis and hence proper regulation of bodily fluid volume in addition to hydrogen ion control, that is, maintenance of pH homeostasis (AE: anion exchanger)

ClCKB lodges in the basolateral membrane of the TAL, in addition to in the DCT, CnT, and α ICCs of the CnT, CCD, and MCD [578]. Electroneutral K+ –Cl− cotransporter KCC4 mediates K+ -dependent Cl− exit at the basolateral membrane of medullary and cortical TAL cells. Transepithelial salt transport through the TAL is regulated by multiple competing neural and hormonal factors, particularly those that increase cytosolic cAMP concentration (stimulatory calcitonin, glucagon, parathyroid hormone, and vasopressin, in addition to angiotensin-2 and β-AR) [578]. Inhibitors of transepithelial salt

3.7 Kidney and Blood Pressure Control 2 AR

urine pole Na + K+

+ K

Cl NKCC2

−

( NH +4 )

257

ADH, PTH, Calc, Gcg

CaSR G

DAG−−PKC, IP2−−Ca2+ H

+

+

HCO 3−

20HETE

ClCKb

K+ NHE1 H 2 CO 3

+ NH 4 K−NH4 X

Na+ NBCn1 Na +

AC2

Na+ NHE2 NHE3

H 2O

+

HCO − 3 AE1/2

CO 2

Cl − NHE4 + Na

NH +4 AC4 apical

NO NOS3

ET1 AR

blood pole + K + ( NH 4 ) − − Cl ( HCO 3 ) CO 2 + K NH 3 Cl

Na−K ATPase

BK

+

KCC4

MAPK

+ K

H

cPLA2

cAMP

ROMK

+ K

Gq PLC

−

Na + H

+

HCO 3− + NH 4

CaSR PTH

2 Ca + basal Na+ Mg 2 + interstitium

Fig. 3.4 Ion carriers and fluxes in the TAL of the loop of Henle. The TAL is an important segment of the nephron for luminal bicarbonate reabsorption. Ammonia is also reabsorbed in + the TAL. NKCC2, which can convey NH+ 4 instead of K , is the primary carrier responsible for + + ammonium reabsorption. Apical electroneutral K –NH4 exchange and conductive NH+ 4 transport are less significant. Ammonium exits across the basolateral plasma membrane through Na+ –NH+ 4 exchanger NHE4 (but not NHE1), in addition to outflux through KCC4 and basolateral diffusive − NH3 egress. Basolateral HCO3 uptake, which buffers intracellular H+ release is mediated by the NBCn1 cotransporter

transport include mainly prostaglandin-E2 and extracellular Ca2+ concentration in addition to messengers signaling via cGMP such as nitric oxide. Apical K+ Channels The ROMK channel (KIR 1.1; Table 3.20) serves as small-conductance K+ channels in the TAL and the apical membrane of the CD. Their function is regulated by an ATP-binding cassette protein cofactor (ABCc7 [CFTR]) [580]. It connects to scaffold proteins SLC9a3R1 and SLC9a3R2,59 SLC9a3R2 being coexpressed with ROMK in the TAL [578]. Regulators of ROMK are recruited because of A-kinase anchor proteins and SLC9a3R2, which facilitates the assembly of the ROMK–CFTR–SGK1 complex. The SLC9a3R1/2–ROMK couple localizes ROMK near CFTR, thereby yielding ATP sensitivity. Both apical ROMK2 (KIR 1.1b) and ROMK3 (KIR 1.1c) channels recycle K+ to the luminal solution to maintain the local K+ concentration for proper functioning of NKCC2 (SLC12a1) [537]. On the other hand, basolateral K+ channels of the KCNJ, KCNK, KCNQ, and SLO families carrier family-9 isoform-A3 regulatory factors are also called Na+ –H+ exchanger regulatory factor NHERF1 and NHERF2.

59 Solute

258

3 Hypertension

Table 3.20 Renal outer medullary potassium channel (ROMK or KIR 1.1) isoforms, which are major K+ secretory channels in the kidney with identical biophysical properties (Source: [580]; CCD cortical collecting duct, CnT connecting tubule, cTAL, cortical thick ascending limb of the loop of Henle, DCT distal convoluted tubule, mTAL medullary thick ascending limb, OMCD outer medullary collecting duct) Subtype ROMK1 ROMK2 ROMK3

Location CnT, CCD, OMCD mTAl, cTAL, DCT, CnT, CCD mTAl, cTAL, DCT

(K2P 12.1 and 13.1, KIR 5.1 and 7.1, KV 7.1, and KCa 4.1) recycle K+ back to the interstitium for proper Na+ –K+ ATPase functioning. The TAL reabsorbs 25–30% of NaCl filtered by the glomeruli through NKCC2.60 Splicing of Nkcc2 mRNA forms alternative transcripts. Full-length isoforms (1095 amino acids) NKCC2L a, NKCC2L b, and NKCC2L f, which derive from the alternative splicing of the variable 96-bp exon 4 of the SLC12A1 transcript, differ in their localization along the TAL and affinity for Na+ , K+ , and Cl− ions [581].61 A second splicing produces three short NKCC2 isoforms (770 amino acids) with a truncated C-terminus, at least in mice (NKCC2S a, NKCC2S b, and NKCC2S f). Various mechanisms govern NKCC2 activity: transfer to the apical plasma membrane upon glycosylation (maturation) and from it, that is, the balance between exo- and endocytosis leading to recycling or turnover, phosphorylation by AMPK,62 PKA, OSR1, and STK39 (SPAK),63 and between-protein interactions [579].64

60 Throughout

the TAL, NaCl concentration decreases gradually from nearly 140 mmol/l in the inner stripe of the outer medulla to a value ranging from 30 to 60 mmol/l at the macula densa [579]. The ions Na+ , K+, and Cl− enter the cell across the apical membrane through NKCC2, owing to an electrochemical gradient generated by Na+ –K+ ATPase, which mediate Na+ extrusion across the basolateral membrane, whereas Cl− exits via the basolateral K+–Cl− cotransporter KCC4 or ClCKB and a fraction of K+ recycles through the apical membrane via the ROMK channel. 61 The NKCC2a isoform has an intermediate affinity for chloride. It is located in the medullary and cortical TAL and is more abundant in the cortex. The NKCC2b isoform has the highest affinity for chloride. It is primarily located in the MDCs and cortical TAL segment. The NKCC2f isoform has the lowest affinity for transported ions. It lodges mainly in the mTAL [579, 581]. The mouse pool of NKCC2f, NKCC2a, and NKCC2b is about 70:20:10 [581]. The order of apparent affinity for Cl− as a transport-limiting ion in the TAL tubular fluid is NKCC2b > NKCC2a > NKCC2f. 62 AMPK phosphorylates (activates) NKCC2 (Ser126) [518]. 63 OSR1: oxidative stress-responsive kinase-1; SPAK: Ste20/SPS1-related Pro/Ala-rich kinase. Both kinases pertain to the germinal center kinase subfamily 6. In the kidney, SPAK is controlled by members of the family of WNK (with-no-lysine) kinases (WNK1 and WNK3–WNK4) [579]. This modification may stimulate exocytosis and/or recycling of internalized NKCC2, impede endocytosis, or combine both actions. WNK3 phosphorylates (activates) NKCC2 (Thr96 and Thr101). 64 NKCC2 interacts with the glycolytic enzyme aldolase-B, which may control membrane insertion of NKCC2; secretory carrier membrane protein SCAMP2, which may retain NKCC2 in the recycling compartment; and raft-associated protein MAL (myelin and lymphocyte protein) [579].

3.7 Kidney and Blood Pressure Control

259

Vasopressin and growth hormone induce phosphorylation of NKCC2 at the same Thr residues. In humans, loss-of-function homozygous or compound heterozygous mutations in the Nkcc2 (SLC12A1) gene in the chromosome locus 15q21 cause antenatal type1 hypokalemic alkalosis with hypercalciuria hyperprostaglandin-E syndrome, or type-1 Bartter syndrome (BartS1), characterized by severe salt and volume loss and decayed BP.65 On the other hand, gain-of-function Nkcc2 mutations and resulting augmented NaCl absorption by the TAL engender salt-sensitive hypertension. In the TAL, noradrenaline released by β1- to β3-AR enhances NaCl reabsorption. In fact, β-ARs participate in hydrogen ion control in addition to regulation of glomerular filtration and renin secretion, along with NaCl reabsorption [582]. β1-Adrenoceptor lodges in mesangial and juxtaglomerular granular cells, macula densa epitheliocytes, in addition to proximal and distal tubules, and acid-secreting α ICCs of the CCDs and MCDs [582]. β2-Adrenoceptor predominantly localizes to the apical and subapical compartment of proximal and, to a lesser extent, distal tubular epithelia. Both β1AR and β2AR are observed on smooth myocytes of renal arteries. β3-Adrenoceptor resides at the basolateral membrane of mouse TAL cells and in other vasopressin-sensitive segments [583]. It stimulates NKCC2 activity via phosphorylation. Apical and Basolateral Na+ –H+ Exchangers The set of Na+ –H+ exchangers (or antiporters [NHAs]) comprises ten isoforms (NHE1–NHE10 [SLC9a1–SLC9a10]). These secondary active transporters exchange one sodium for one proton ion across the plasma membrane, the electrochemical gradient for one of the solutes driving the countertransport of the other. Plasmalemmal electroneutral NHEs function as homodimers [584]. The widespread NHE1 glycoprotein acts as a scaffold regulating cell survival and actin cytoskeleton organization via its signaling partners. Ezrin, radixin, and moesin tether to NHE1 for its proper localization to the plasma membrane. In the kidney, it resides in the basolateral membrane of all nephron segments, except macula densa and CCD ICCs [584].

65 Bartter

syndrome refers to a group of disorders due to autosomal recessive transmission of impaired salt reabsorption in the TAL of the loop of Henle. • Antenatal type-2 Bartter syndrome (BartS2) is caused by loss-of-function mutations in the KCNJ1 gene that encodes ATP-sensitive K+ channel ROMK (KIR 1.1). • Type-3 Bartter syndrome (BartS3) is caused by mutations in the CLCNKB gene in the chromosomal locus 1p36, which encodes the renal Cl− channel ClCKB . • Type-4A Bartter syndrome is caused by mutations in the BSND gene that encodes barttin, the β subunit for the chloride channels ClCKA and ClCKB . • Neonatal type-4B Bartter syndrome results from simultaneous mutations in both the CLCNKA and CLCNKB genes.

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NHE1 participates in cell volume regulation and stress response, such as hypoxia and acidification, and reacts to cellular stretch. It can mediate Na+ influx, which primes osmotic movement of water through water channels such as aquaporin-1, hence countering cell shrinkage (compensatory cell swelling or regulatory volume increase) [584].66 NHE1 requires ATP and may act as an ATP-binding transporter. Calmodulin blocks its autoinhibitory site. Calcineurin homologous proteins CHP1 to CHP3 bind to NHE1, stabilizing it. Phosphatidylinositol (4,5)-bisphosphate connects to NHE1 for optimal activity. Carbonic anhydrase CA2 links to NHE1 and raises its activity. The NHE1 isoform participates in multiple signaling cascades triggered by protein Tyr kinase and G-protein-coupled receptors and integrins. It is activated by aldosterone via ERK1 and ERK2 [584]. Upon phosphorylation (Ser703), 14-3-3 binds to NHE1 and protects it from dephosphorylation. Intracellular acidosis is the major factor that quickly activates NHE1 [584]. It is phosphorylated by rock, hence regulating the cytoskeleton dynamics in addition to PKC and components of the MAPK module, such as JNK and P38MAPK, in the cell shrinkage response [584]. Conversely, NHE1 is implicated in the regulation of MAPK activity. The sodium–hydrogen exchanger NHE2 lodges in the apical membrane of the medullary TAL.67 The NHE3 isoform resides at high levels in apical membranes of epitheliocytes in the proximal tubule and loop of Henle.68 PT Cells secrete H+ across the apical membrane into the lumen via NHE3 and vacuolar H+ ATPase, an electrogenic pump.69 In the proximal tubule, secreted H+ and luminal HCO− 3 are converted to CO2 and H2 O by apical membrane-associated carbonic anhydrase CA4; CO2 and H2 O

66 Regulatory

volume increase is a process relying on NHE1 and, according to the cell type, + + − symporter [584]. Restoration of on Cl− –HCO− 3 exchanger SLC4a2 and/or Na –K –2Cl intracellular volume can stop apoptosis. Neither SLC4a2 (AE2), SLC12a2 (NKCC1), nor SLC12A1 (NKCC2) are formed in PT cells; hence, their survival depends on NHE1 [584]. 67 It also resides in the apical membrane of intestinal epitheliocytes. 68 Also in small intestinal and colonic cells. 69 The H+ content of cellular organelles varies widely; mitochondria are alkaline, whereas lysosomes are acidic. Vacuolar H+ ATPase pumps cytosolic H+ into the lumen of transfer vesicles and maintains the acidic pH (4.5 < pH ≤ 7) [585]. An acidified endosomal lumen is required for the dissociation and targeting of receptors and their ligands. In some cell types (e.g., renal ICCs, osteoclasts, and phagocytes [neutrophils and macrophages]), vATPase also localizes to the plasma membrane, where it extrudes H+ to the extracellular space. Neurotransmitter uptake into synaptic vesicles also relies on vATPase. The electrochemical gradient generated by vATPase powers ion flux through various carrier types. The endocytic antiporter CLC7 exchanges two Cl− for 1 H+ , accumulating Cl− in the vesicular lumen. Proton-coupled amino acid transporter SLC36a2 (PAT2) in the endoplasmic reticulum and recycling endosomes exchanges a small neutral amino acid for a proton.

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Table 3.21 Control of NaCl reabsorption in the TAL of the loop of Henle by hormones and autacoids (Source: [579]; ANP atrial natriuretic peptide, PDE2 cGMP-stimulated phosphodiesterase-2, PTH parathyroid hormone) Second messenger cAMP

Target NKCC2

cGMP

NHE2/3 PDE2

Stimulators effect ADH, calcitonin, glucagon, PTH, β AR (noradrenaline) ANP, ET1, NO α AR Inhibition of cAMP

rapidly reenter the cell across the apical membrane; in the cytosol, CO2 and H2 O are converted back into H+ and HCO− 3 by CA2. In the medullary TAL, basolateral NHE1 regulates apical NHE3 and + + HCO− 3 absorption, the latter process being supported by basolateral Na –H exchanger [584]. In the TAL, a significant amount of luminal ammonium is also reabsorbed. Both NHE1 and NHE4 subtypes70 may mediate NH+ 4 extrusion at the basolateral membrane [584]. Whereas NHE5 is expressed in the brain, NHE6 to NHE9 are ubiquitous. On the other hand, NHE8 abounds in proximal tubules and in the renal outer medulla and cortex [584].71

Control of Salt Reabsorption Salt reabsorption in the TAL is controlled by adrenergic agonists (noradrenaline and adrenaline) and hormones (e.g., glucagon, PTH, and vasopressin)72 that stimulate NaCl reabsorption, whereas atrial natriuretic peptide and autacoids, such as nitric oxide and some prostaglandin species, inhibit NaCl reabsorption (Table 3.21) [579]. In addition, 20-hydroxyeicosatetraenoic acid (20HETE) and prostaglandin-E2 preclude NaCl reabsorption in the TAL [579].

70 The

highest NHE4 amont is detected in the stomach; it is produced at intermediate levels in the small intestine and colon; lesser amounts are found in TAL cells, the brain, skeletal muscles, and uterus. 71 The NHE5 subtype also localizes to the skeletal muscle, spleen, and testis. Ubiquitous intracellular NHE6 is an endosomal isoform. Ubiquitous intracellular NHE7 is an isoform of the trans-Golgi network. Ubiquitous intracellular NHE8 is identified at relatively high levels in skeletal muscles and the kidney. Ubiquitous intracellular NHE9 localizes to late recycling endosomes. NHE10 is expressed in osteoclasts. 72 Vasopressin, or arginine vasopressin, has a direct antidiuretic action on the kidney, hence its other name, ADH. It also causes vasoconstriction of the peripheral vessels. Vasopressin enhances NKCC2 activity via cAMP.

262 Table 3.22 Types of ATP anions and their size with respect to other molecule types (Source: [586])

3 Hypertension Anion ADP3− ATP4− H ATP3− Mg ATP2− Cl− HPO2− 4 NO− 3 UTP4− Aspartate Glutamate Proline Betaine Gluconate Glycerophosphocholine Myoinositol Taurine

Effective radius (nm) 0.53–0.56 0.57–0.58 0.56–0.58 0.59–0.61 0.181 0.275 0.212 0.53–0.54 0.339 0.345 0.28 0.285 0.349 0.367 0.306 0.263

The LRRC8 (VRAC/VSOR) channel conducts iodide (I− ) better than chloride and also organic osmolytes such as taurine

Extracellular ATP, an auto- and paracrine messenger, is another regulator of renal tubular transport. Once released, extracellular ATP binds to its ubiquitous P2 receptors, which include seven ionotropic P2X and eight G-protein-coupled P2Y receptor subtypes. The messenger ATP is mainly produced by mitochondrial oxidative phosphorylation and cytosolic glycolysis. Most ATP molecules exist in anionic forms, that is, in the absence of Mg2+ , ATP4− , and protonated ATP H ATP3− , and in the presence of an equivalent concentration of Mg2+ , Mg ATP2− , and Mg−−H ATP− (Table 3.22); ATP and its Mg2+ and/or H+ salts, which exist in anionic forms at physiological pH, exit cells via anion channels, such as CFTR (a.k.a. ABC35 and ABCc7),73 a cAMP-activated chloride channel; the LRRC8a–LRRC8b(c/d/e) dimer,74 which may be permeable to ATP− 4 ; and maxi anion channel, a large-conductance anion channel,75 which releases ATP in response to hypoxic and osmotic stresses [586].

73 CFTR:

cystic fibrosis transmembrane conductance regulator. leucine-rich repeat-containing protein-8A. It is also termed volume-regulated anion channel (VRAC), volume-sensitive outward-rectifying chloride channel (VSOR), and volumesensitive organic osmolyte anion channel, which maintains a constant cell volume in response to extra- or intracellular osmotic changes. Its highest expression is found in adult brain, followed by kidney, ovary, lung, liver, and heart [194]. In fact, LRRC8a is a VRAC subunit that requires at least one other LRRC8 family member (LRRC8b–LRRC8e) to form a functional channel [108]. 75 Plasmalemmal maxi anion channel is similar to mitochondrial ATP-conductive voltagedependent anion channel (porin) [586]. 74 LRRC8a:

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The hydrophilic molecule ATP can also exit cells upon lysis, with released vesicles, and through connexin-built hemichannels. Ectonucleotidases hydrolyze ATP, ADP, and AMP to adenosine; ectoadenylate kinase converts 2 ADP to ATP and AMP; and ectonucleoside diphosphate kinase transforms ADP to ATP. Basolateral ATP inhibits Na+ and Cl− absorption in mouse medullary TAL through a P2X ligand-gated ion channel such as the P2X4 receptor via a reversible intracellular alkalization of mTAL epitheliocytes mediated by apical Na+ –H+ exchanger (NHE3) [587]. Hence, ATP triggers NHE3-dependent H+ secretion upon inhibition of tubular transport. On the other hand, the Gq-coupled receptor TAL P2Y2 causes acidification of tubular epitheliocytes. The intracellular pH-sensitive ROMK channel closes upon exposure to a minor cytosolic acidification. 3.7.5.5

Distal Nephron

The distal nephron is the tubular compartment that begins with the TAL of the loop of Henle and ends with the CCD [588], rather than the papillary CD. The distal convolution of superficial nephrons is a simple tube that merge to the most peripheral extensions of a CCD. On the other hand, distal tubules of deeper nephron generations open into arcades that drain into a CCD, the CnT including these arcades. The average number of nephrons drained by each CCD in the human kidney is 11 [588]. The cortical distal nephron is the site devoted to the regulation of salt and water excretion by peptidic and mineralocorticoid hormones. The distal nephron transports sodium, potassium, calcium, magnesium, chloride, and hydrogen ions. It adjusts renal ion excretion according to the tubular fluid composition and flow rate, in addition to exposure to various types of hormones. The transcellular transfer of Na+ and Ca2+ ions are inter-related [589]. Calcium reabsorption relies on the basolateral Na+ –Ca2+ exchanger (NCX), plasmalemmal Ca2+ ATPase (PMCA), and cytoplasmic calbindin-1, which abound in the distal nephron in particular, but not in other nephron compartments [589]. The constitutively active Ca2+ -selective cation channel TRPV5 is also required for Ca2+ reabsorption in the DCT.76 The entire human DCT and CCD produce calbindin-1, albeit in variable amounts [589]. The presence and amount of ion and water carrier species depend on the distal nephron segment (Table 3.23). Salt subtraction from the tubular fluid through NKCC2 in the water-impermeable TAL is the precondition for urinary concentration. Na+ –Cl− cotransporter (NCC [SLC12a3]) lodges in the DCT; it overlaps (30–35%) with ENaC (65–70%) in a short downstream segment of the DCT (DCT2) [588]. The major part of the CnT and CCD coexpress aquaporin-2 with ENaC. 76 Transient

receptor potential cation channel TRPV5 is activated by a low cytosolic Ca2+ concentration. Whereas TRPV5 corresponds to the epithelial calcium channel ECaC1, TRPV6 is also called ECaC2. They can assemble to form a voltage-gated TRPV5–TRPV6 heteromer [108].

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Table 3.23 Distribution of aquaporins and ion carriers in the distal nephron (Sources: [588, 589]; CCD cortical collecting duct, CnT connecting tubule (70% of the distal convolution), DCT distal convoluted tubule, TAL TAL of the loop of Henle, which has medullary (mTAL) and cortical (cTAL) segments, Aqp aquaporin, a vasopressin-sensitive water channel, ENaC epithelial Na+ channel, NCC Na+ –Cl− co-transporter, NCX Na+ –Ca2+ exchanger, NKCC Na+ –K+ – 2Cl− cotransporter, PMCA plasma membrane Ca2+ ATPase [basolateral calcium extruder], ROMK renal outer medullary potassium channel [KIR 1.1], TRPV5 transient receptor potential cation channel-5 [or epithelial calcium channel ECaC1]) Segments TAL DCT

CnT

CCD

Apical carriers NKCC2 NCC ENaC (exit part) TRPV5 (exit part) ROMK ENaC Aqp2 TRPV5 (EcaC1) ROMK ENaC Aqp2 TRPV5 (EcaC1) ROMK

Basolateral carriers Na+ –K+ ATPase PMCA NCX

PMCA NCX

PMCA NCX

Sodium Import and Its Regulation The distal nephron finely tunes urinary Na+ excretion. Apical salt influx in epitheliocytes of the distal nephron results mainly in activity of TAL NKCC2 (i.e., confined to the TAL), DCT NCC, and CnT/CCD ENaC. The Na+ uptake fraction in the distal nephron also depends on the Na+ –H+ exchanger NHE2; this compartment is thus implicated in H+ control. The major Na+ transporter, ENaC, operates in the ASDN part of the nephron. Mineralocorticoid receptors (MR or NR3c2) and 11β-hydroxysteroid dehydrogensase (11β HSDH2), which confers mineralocorticoid specificity to the MR, as it rapidly processes glucocorticoids, and resides in the distal nephron [588]. Whereas MR lodges all along the DCT and CnT, 11β HSDH2 is only detectable in the ENaC+ DCT2 segment and in the CnT and CD. Consequently, glucocorticoid hormones may stimulate NCC-mediated Na+ influx in the upstream DCT1 segment, whereas aldosterone may support Na+ import in the NCC+, ENaC+ DCT2 segment. Sodium uptake through ENaC is electrogenic; it favors potassium secretion most probably via the apical ROMK channel. Whereas upstream nephron regulation lowers renal Na+ excretion without raising the content of Na+ importers (e.g., PCT NHE3), in the downstream nephron, aldosterone-stimulated response upregulates NCC and ENaC [590]. Other messengers, such as angiotensin-2, catecholamines, nitric oxide, and vasopressin, participate in the regulation of renal tubule Na+ reabsorption. Receptors

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Table 3.24 Phosphorylation and ubiquitination of NCC and ENaC (Sources: [527, 588]) Carrier NCC

ENaC

Regulation WNK1 −→ WNK4 −→ NCC Aldosterone⊕ −→ SGK1 −→ NEDD4-2 SGK1⊕ −→ NCC WNK4⊕ −→ {SPAK, OSR1}⊕ −→ NCC (cascade launched by angiotensin-2 and aldosterone) Aldosterone⊕ −→ SGK1 −→ NEDD4-2 Aldosterone⊕ −→ ENaC transfer to the plasma membrane Aldosterone⊕ −→ ENaCα subunit synthesis Aldosterone −→ ERK⊕ −→ NEDD4-2 Activating proteolysis by chymotrypsin, elastase, kallikrein, trypsin, and channel-activating peptidases prostasin (CAP1 [PesS8]) and TMPesS4 (CAP2) Deubiquitination by ubiquitin-specific peptidase USP2-45 WNK1⊕ −→ SGK1 −→ NEDD4-2

Phosphorylation of NCC by Ste20/SPS1-related proline–alanine-rich protein kinase (SPAK; or STK39) and oxidative stress-responsive kinase OSR1, which are controlled by angiotensin-2, in addition to serum and glucocorticoid-regulated kinase SGK1, which is activated by aldosterone, increases its activity. On the other hand, with-no-K (Lys) kinase WNK4 reduces NCC transfer to the plasma membrane; WNK1 interacts with WNK4 and relieves its inhibition on NCC. Both NCC and ENaC are inhibited upon ubiquitination by NEDD4–2 (⊕ −→: stimulation; −→: inhibition). Hormonal regulation of ENaC involves various kinase types, such as stimulation of SGK1 and inhibition of extracellular signal-regulated kinase (ERK) by aldosterone, PKA by ADH and ANP, PI3K by insulin, and SRC family kinases by endothelin

for peptidic hormones (e.g., calcitonin and PTH) increase cAMP activity in the distal nephron. Angiotensin-2, which launches aldosterone secretion from the adrenal gland, stimulates Na+ reabsorption in the distal tubule. The AT1 receptor upregulates NCC formation in response to a low dietary Na+ intake. In addition to aldosterone, other hormones influence the ENaC synthesis rate, such as vasopressin and angiotensin-2 [588]. Activity of ENaC in the DCT2, CnT, and CD is stimulated upon phosphorylation by the aldosterone-induced kinase SGK1 and impeded upon ubiquitination by NEDD4–2 (Table 3.24) [588]. Extracellular peptidases, such as kallikrein synthesized in the upstream ASDN segment and prostasin, concentrations of which rise in urine when plasmatic aldosterone concentration increases, affect ENaC activity. The Wnk1 gene encodes two WNK1 isozymes, ubiquitous long isoform (WNK1L ) and a kidney-specific subtype Ki WNK1 [527]. The latter is formed predominantly in the DCT; it lacks most of the kinase domain and acts as a WNK1 inhibitor that thus enables the action of WNK4 on NCC. WNK4 localizes to the ASDN at the tight junctions and subapical membrane region of the DCT, in the cytoplasm of CnT and CCD [527].

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Calcium Influx Sodium and calcium fluxes in the distal nephron interact. The transcellular calcium transport occurs in the DCT, CnT, and CCD [588]. Whereas CaV 1.2 is involved in cell signaling, transcellular Ca2+ flux relies on apical Ca2+ -selective hyperpolarization-activated TRPV5 and basolateral PMCA and NCX carriers. Plasmalemmal hyperpolarization activates apical Ca2+ channels [588]. This hyperpolarization results from a lowered Cl− entry through NCC that enhances Cl− influx through chloride channels and reduced Na+ import through ENaC that attenuates intracellular Na+ concentration and subsequently raises the driving force for basolateral Na+ –Ca2+ exchange. Estrogens upregulate the production of NCC and TRPV5; synthesis of the latter is also augmented by vitamin D3 [588].

Chloride Ingress Chloride channels in the distal nephron enable NaCl reabsorption from the tubular fluid through cotransporters, Cl− channels facilitating transepithelial transport via Cl− exit across the basolateral membrane in the TAL and DCT, whereas in acidsecreting cells of the distal nephron, anion channels help to recycle imported Cl− in + − exchange for HCO− 3 [591]. In addition, K –Cl cotransporter functions in parallel with these channels, offering an alternative route for Cl− flux. Chloride fluxes are observed through basolateral ClCKB in cells of the TAL, CnT, and CCD, to a much larger extent in CCD ICCs than CCD PCs and in the TAL and CnT than in the CCD [591]. After CO2 hydration in the cytoplasm to form H+ − + + and HCO− 3 , H is exported into urine by H ATPase, whereas HCO3 leaves the − cell across the basolateral membrane in exchange for Cl ion through Cl− –HCO− 3 exchanger, K+ –Cl− cotransporter contributing to Cl− efflux.

Acid and Base Transfer Intercalated cells of the DCT2, CnT, and CD serve in acid and base transport and are thus involved in the final regulation of acid and base excretion [592]. Intercalated cells are regularly interspersed among the ENaC+ PCs. Intercalated cells regulate H+ donor–H+ acceptor handling by two main carriers, a V-type H+ ATPase and a Cl− –HCO− 3 exchanger. • Type-A (or type-α) ICCs possess an apical H+ ATPase and a basolateral Cl− – HCO− 3 exchanger (i.e., AE1). − − • Type-B (or type-β) ICCs secrete HCO− 3 through the apical Cl –HCO3 ex+ changer SLC26a4 (pendrin), which cooperates with the basolateral H ATPase, which transfers H+ into the interstitium.

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Both cell types also possess H+ –K+ ATPase. In the DCT, H+ is mainly secreted through the Na+ –H+ exchanger.

3.7.5.6

Distal Convoluted Tubule

The TAL epithelium abruptly transforms into the DCT epithelium at various distances downstream of the macula densa [588]. DCT Epitheliocytes have a lateral surface that forms interdigitated processes. In turn, DCT epitheliocytes are abruptly replaced by CnT epitheliocytes. The DCT can be subdivided into an upstream (NCC+ DCT1) and a downstream segment (NCC+ and ENaC+ DCT2). The latter coexpresses apical NCC, ENaC, and TRPV5 in addition to basolateral NCX and PMCA, produces relatively very large amounts of calbindin-1, and contains ICCs [588]. Electroneutral NaCl uptake into DCT epitheliocytes is mediated by Na+ –Cl− cotransporter (NCC). Both IGF1 and IGFBP3 are involved in DCT epitheliocyte hypertrophy [588]. The DCT reabsorbs nearly 5% of Na+ and Cl− from the filtrate. In both the DCT1 and DCT2 cells, Na+ and C− enter through apical NCC (SLC12a3) using the Na+ gradient. In addition, in the DCT2, apical ENaC cooperates with NCC. Magnesium enters the DCT1 and DCT2 cells through TRPM6. Reabsorbed Na+ exits the DCT through Na+ –K+ ATPase; K+ through basolateral K+ channels of the KCNK, KCNJ, KCNQ, and KCNE families (K2P 1.1, KIR 4.1, 4.2, 5.1, 6.1, AND 7.1, AND KV 7.1); and Cl− through ClCKB and ClCKA [537].

3.7.5.7

Connecting Tubule and Collecting Duct

CnT Epitheliocytes

have basolateral plasma membrane infoldings. The mitochondrion density is much smaller than that of DCT epitheliocytes. Principal cells possess the ENaC and ROMK channels. Intercalated cells begin to appear in the downstream transition (DCT–CnT) segment and are observed all along the CnT and CCD [588]. They appear in the downstream DCT segment or in the CnT according to the mammalian species [593]. In deep and intermediate nephrons, the change from the DCT to the CnT epithelium regularly occurs a few cells before fusion of two tubules. The transition from the CnT to the CCD is abrupt, principal CCD cells substituting to CnT epitheliocytes. Basolateral membrane infoldings in CCD epitheliocytes are restricted to the most basal cellular region. They are intermingled with ICCs. Intercalated cells are renal tubular epitheliocytes involved in salt (sodium and chloride) and water reabsorption in addition to potassium and hydrogen ion (and hence ammonia) control [593]. They abound in the CD from the cortical segment to the initial part in the inner medulla. They are interspersed among ENaC+ ROMK+ PCs, which are more numerous.

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Table 3.25 Major molecular carriers in principal and intercalated epitheliocytes (ICCs) of the rat collecting duct (Source: [593]) Cell type Principal cell Type-A ICC (α ICC)

Apical (luminal) membrane ENaC (Na+ in) ROMK (K+ out) H+ –K+ ATPase (H+ out, K+ in) H+ ATPase (H+ out) SLC26a11 (Cl− out)

Type-B ICC (β ICC)

nonA nonB ICC

SLC42a3 (RhCG) SLC4a8 (NDCBE) − (Na+ , HCO− 3 in, Cl out) SLC26a4 (pendrin) − (HCO− 3 out, Cl in) SLC42a3 (RhCG) H+ ATPase SLC26a4

Basolateral membrane Na+ –K+ ATPase (Na+ out, K+ in) SLC9A1 (NHE1) (Na+ in, H+ out) SLC12a2 (NKCC1) (Na+ , K+ , Cl− in) SLC4a1 (AE1) (Cl− in, HCO− 3 out) SLC42a2 (RhBG) H+ ATPase (H+ out) SLC4a9 (AE4) (Na+ , HCO− 3 out) SLC42a2 (RhBG)

Three types of ICCs are distinguished according to their structure and content in molecular carriers, being currently classified by the presence of SLC4a1 and SLC26a4 and subcellular localization of H+ ATPase (Table 3.25). These cells are categorized into type-A (or α), which functions in urinary acidification, and type-B (or β) and nonA and nonB ICCs, which operate in bicarbonate secretion [593]. In the CCD and OMCD, α ICCs express H+ and H+ –K+ ATPases at the apical (luminal) membrane and Cl− –HCO− 3 exchanger SLC4a1 at their basolateral membrane. The bicarbonate sensor soluble adenylate cyclase and protein kinase-A participate in the regulation of H+ ATPase [578]. Sodium-independent sulfate anion transporter SLC26a11, an electrogenic Cl− transporter, resides at the apical membrane of α ICCs. On the other hand, β ICC possesses an electroneutral salt carrier that − implicates apical SLC26a4 (pendrin) and Na+ –2 HCO− 3 –Cl exchanger SLC4a8a − − and basolateral Cl –HCO3 exchanger SLC4a9, reabsorption of NaCl from the tubular lumen being energized by the basolateral H+ ATPase [578]. In the kidney, ammonium transporters of the Rhesus blood group RhBG and RhCG lodge in the basolateral and both apical and basolateral membrane, respectively. All types of ICCs contain cytosolic carbonic anhydrase CA2, a zinc metalloenzyme that reversibly catalyzes the hydration of CO2 into bicarbonate, hence both producing and consuming the substrate HCO− 3 of BTRs. CA2 acts in tandem with H+ ATPase, facilitating the coordinated proton and bicarbonate secretion from ICCs [593].

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269

Downstream from the DCT, that is, from DCT2, epithelial Na+ channel is responsible for electrogenic Na+ absorption using a favorable electrochemical gradient. The resulting depolarization of the apical membrane provides the driving force for K+ secretion through ROMK, which colocalizes with ENaC at the apical membrane of PCs. Sodium reabsorption in the distal nephron and collecting tubule is controlled by the RAAA. Aldosterone increases concentration of both apical NCC and ENaC in PCs and hence Na+ import in the DCT, CnT, and CCD, whereas angiotensin-2 enhances Na+ absorption in the DCT [527]. In addition, vasopressin stimulates Na+ influx in the DCT, CnT, and CCD. Principal cells of the CnT and CD are responsible for about 10% of the reabsorption of Na+ and Cl− from the filtrate. The mechanism of reabsorption of Na+ in the CnT and CD is similar. Sodium ion enters through ENaC and exits through Na+ –K+ ATPase, whereas K+ is recycled back out of the cell via basolateral K+ channels of the KCNJ, KCNK, and KCNQ families (KIR 2.3, 4.1, 5.1, 6.1, and 7.1, K2P 12.1 and 13.1, and KV 7.1) [537]. Potassium ion that enters through Na+ –K+ ATPase can exit across the apical membrane through ROMK1 (KIR 1.1a) in the DCT and both ROMK1 and ROMK2 (KIR 1.1b) in the CCD, but not through ROMK2 in the OMCD [537].

3.8 Intestinal Flora The gut microbiota is involved in BP control. Bowel dysbiosis contributes to local and remote disturbances; it is observed in animal models of hypertension and in hypertensive patients [594]. An increased Firmicutes/Bacteroidetes ratio is associated with a decrease in acetate- and butyrate-producing bacteria and an increase in lactate-producing bacteria. High-fiber diet and acetate supplementation correct gut dysbiosis and lower arterial pressure. Diet and some environmental factors influence both intestinal flora and hypertension. Some medications, especially antibiotics, affect the gut microbiota. Certain antihypertensive drugs reduce gut contractility. Patients with constipation have significantly less lactate-producing bacteria and a higher intestinal permeability than controls. Conversely, the microbiota can affect drug pharmacokinetics. The gut flora can influence the efficacy and toxicity of antihypertensive drugs. Pre- and probiotics, which modulate cholesterol level, glycemia, inflammation, and activity of the RAA, affect gut microbiota and moderately lower arterial pressure. The response depends on gut microbial composition and hence ethnicity, as Asian and African populations carry Lactobacilli, which are rarely found in Western people.

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3.9 Regulators Blood pressure is rapidly regulated by the central nervous system, which controls cardiac frequency and output in addition to peripheral vascular resistance, and in the longer term by the kidney and adrenal gland via hormones that maintain salt and water balance and hence bodily fluid volume. In the distal nephron, transport of Na+ and K+ is regulated by many hormones, such as aldosterone, angiotensin, bradykinin, endothelin, insulin, and vasopressin [636]. Angiotensin-2 acts via its apical AT1 receptor in the nephron. The RAA is the primary regulator of aldosterone secretion in response to abnormal extracellular volume. Aldosterone operates via Na+ channels and MR, which are synthesized in the distal nephron in addition to vascular endotheliocytes and smooth myocytes. Hypertension-induced heart failure causes pulmonary edema, which provokes diaphragmatic remodeling. However, in stable heart failure, dyspnea results from diaphragmatic weakness, most often without pulmonary edema. The progressive decline in diaphragmatic strength during hypertension is engendered by a ventilatory overdrive generated by modified codependent signaling from β-AR and angiotensin-2 [595]. Production of the eIF2α K3 kinase and hence phosphorylation of eIF2α increase, eIF2α impeding protein synthesis and leading to diaphragmatic atrophy. Placental activation of the RAA favors preeclampsia, which is characterized by an acute onset of hypertension in late pregnancy. Oxidized angiotensinogen, which is more efficiently cleaved by renin, is frequently detected in preeclampsia patients. Heterodimerization of AT1 with bradykinin B2 receptor increases the responsiveness to Agt2 [500]. Activated AT1 stimulates NOx. Redox stress due to ROS accumulation activates inflammation but precludes angiogenesis in the placenta and increases the risk of preeclampsia. Proinflammatory cytokines, concentrations of which rise in preeclampsia, are potent inducers of ROS production. Cytokines and chemokines (e.g., CCL2 and CCL5) can induce angiogenesis and hence placental vascularization. Heme-oxygenase HOx1, an antioxidant enzyme, can, at least partly, protect against preeclampsia and may participate in placental angiogenesis. On the other hand, the antioxidant factor NFE2L2 impedes placental angiogenesis, whereas its deficiency, which augments ROS signaling, favors angiogenesis in preeclamptic mice. In addition, NFE2L2 suppresses the formation of inflammatory cytokines in activated macrophages [500]. According to the context and cell type, NFE2L2 and ROS stimulate or suppress angiogenesis. Systemic NFE2L2 deletion promotes angiogenesis in hindlimb ischemia [500]. On the other hand, endotheliocytespecific NFE2L2 deletion impairs retinal angiogenesis.

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3.9.1 Genetic and Epigenetic Factors Blood pressure is a quantitative physiological trait, the variability of which is partly determined by genetic or epigenetic factors (genetic predisposition to hypertension; Sect. 7.3). Epigenetic factors can affect concentrations of renal sodium transporters and the renal response by angiotensin-2 and/or renal sympathetic nerve activity. In mice, deletion of AT1 in the proximal tubule of the nephron, which elicits expression of Na+ –H+ exchanger SLC9a3 (NHE3), suffices to protect against angiotensin-2induced hypertension [516]. In humans, essential and genetic hypertension can be at least partly resolved by kidney transplantation with a graft from a normotensive donor. Monogenic alterations that affect renal Na+ handling in the loop of Henle, the DCT, and the CD raise or lower BP. In particular, mutations in the HSD11B2 gene encoding type-2 11β-hydroxysteroid dehydrogenase, which deactivates glucocorticoids and confers aldosterone specificity on the MR, engender unregulated sodium reabsorption in the distal nephron and hypertension [516]. Activation of the sympathetic nervous system can contribute both to the origin and maintenance of hypertension. This enzyme is also synthesized in the adult brain, where it promotes a preference for salt diet, salt appetite relying on the mineralocorticoid receptor, and causes salt-sensitive hypertension. On the other hand, mutations in the UMOD gene, which encodes the glycosyl phosphatidylinositol-anchored protein uromodulin, which is synthesized in the TAL (but in neither MDCs nor DCT [578]), can raise renal salt excretion and lower BP.77

3.9.2 Sympathetic Nervous System Most often, elevated sympathetic nerve activity contributes to the development of hypertension.

3.9.2.1

Renal Nerves

The renal sensory afferent and efferent sympathetic nerves cooperate to control ECF volume. Both the afferent and the efferent renal innervation contribute to the neural dysregulation of the kidney in hypertension. Sympathetic overactivity contributes to 77 A

high-salt diet increases uromodulin production. Uromodulin can be cleaved and released from the apical membrane and then excreted in urine (20–100 mg/d) [578]. Uromodulin promotes NKCC2 and ROMK activity. Autosomal dominant mutations in the UMOD gene cause type-2 medullary cystic disease and familial juvenile hyperuricemic nephropathy, or uromodulinassociated kidney disease. Uromodulin overexpression in transgenic mice causes distal tubular injury and salt-sensitive hypertension owing to the activation of STK39 (SPAK) and activating NKCC2 phosphorylation [578].

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hypertension, the kidney being the source of augmented afferent signaling. In renal disease or injury, activation of afferent sensory nerve is excitatory, raising peripheral sympathetic nerve activity and arterial pressure because of vasoconstriction [596]. Renal sympathetic nerve activity is determined by sensory information, which is integrated by nuclei in the hypothalamus and medulla. Pressure reduction at the carotid sinuses increases rSNA, which reduces diuresis and primes renin secretion. On the other hand, activation of cardiopulmonary receptors by stretch attenuates renal sympathetic nerve activity [597]. Activation of visceral mechanoand chemoreceptors in the gut, liver, and splenic regions decreases renal blood flow via sympathetic signaling. Urinary flow rate via mechanoreceptors and urinary electrolyte concentration via chemoreceptors within the kidney contribute to the regulation of sympathetic nerve activity. The sensory nerve ending contains CGRP and substance-P [597]. When the local pressure rises, bradykinin activates the B2 receptor, stimulating PKC and then PGhS2, generating PGe2 , which acts on the EP4 receptor. This triggers the AC– cAMP–PKA axis and releases substance-P, which depolarizes the afferent nerve ending and launches action potential to the central nervous system. On the other hand, angiotensin-2, excited by consumption of a low-sodium diet, represses the PGe2 –AC pathway, thereby resetting the sensitivity of the transduction [597]. Sensory innervation of the kidney uses nerve fibers passing into the spinal cord primarily at the T12–L3 level and synaptic connections in laminae I and I I I to V that project to the nucleus of the solitary tract (nucleus tractus solitarii [NTS]), rostral ventrolateral medulla (RVLM), and paraventricular nucleus, where afferent signals from the kidney and other organs in addition to baroreceptors are integrated [597]. Efferent preganglionic neurons originate from the medullary centers (e.g., NTS and RVLM). Postganglionic efferents synapse at the celiac, supramesenteric, and inferior mesenteric ganglia innervate the renal tubule, juxtaglomerular apparatus, and afferent and efferent arterioles [516]. Renal ARs encompass β1AR of granular renin-containing cells of the afferent arteriole and α1AR of vascular smooth myocytes of the afferent and efferent arterioles and epitheliocytes of the proximal and distal tubules and TAL of the loop of Henle [597]. Renal ARs decrease renal blood flow and the GFR and increase renal tubular sodium and water reabsorption and renin release. Increased efferent signaling does indeed trigger the renin–angiotensin–aldosterone cascade, not only augmenting renal reabsorption of sodium and vascular resistance but also inducing effects in the central nervous system via the dorsal root ganglia. A negative feedback loop consists of efferent rSNA increasing afferent renal nerve activity, which in turn inhibits efferent rSNA to avoid excess renal sodium retention. Afferent sensory nerves sense stretch and elicit an inhibitory renorenal reflex with a compensatory diuresis in the contralateral kidney due to diminished efferent rSNA. In the excitatory renorenal reflex, activation of afferent nerves of one kidney increases the efferent activity in the contralateral kidney. Infusion of adenosine or bradykinin in the renal artery raises BP via sensory receptors [597].

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Renal nerves are partly causal, but not totally responsible for hypertension. Nevertheless, afferent renal nerves play an important role in the long-term regulation of BP. 3.9.2.2

Cerebral Arteries and Sympathetic Nervous System

Walls of cerebral arteries and arterioles remodel in hypertension with increased resistance to blood flow. A high resistance in the vertebral arteries and consequently a decreased vertebral arterial flow correlate with hypertension [598]. In humans, cerebral vascular resistance increases before the onset of sympathetic hyperactivity and hypertension. A marked but weaker link exists between hypertension and decreased internal carotid arterial flow. A weaker relation also exists with other arteries, such as femoral and renal arteries. Elevated sympathetic nerve activity, especially on vertebral arteries that irrigate the vasomotor center of the RVLM in the brainstem, is linked to hypertension to ensure adequate perfusion to the brain, but its causal factors are not always identified (Cushing mechanism or the selfish brain hypothesis of hypertension). Frank hypertension is linked to both augmented sympathetic nerve activity and vascular resistance. Borderline hypertension without increased muscular sympathetic nerve activity (daytime SBP 17.3–18;0 kPa [130–135 mmHg]) is only associated with elevated vascular resistance. Vertebral artery hypoplasia increases vulnerability to hypertension.78

3.9.3 Nuclear Receptors Nuclear receptors NR1c1 to NR1c3 (PPARα, PPARβ [δ], and PPARγ) from the class of ligand-activated transcription factors, which are encoded by three genes (PPARA, PPARD, and PPARG), are therapeutic targets. Among them, NR1c2 has an antihypertensive effect in rats used as models of hypertension (spontaneous and induced hypertension in addition to dyslipemic and gestational models) [599]. Activated NR1c2 upregulates the expression of regulators of G-protein-coupled signaling, provokes acute vasodilation, improves endothelial function, and reduces vasoconstriction, vascular inflammation, and sympathetic signaling. Hypertension-induced heart failure is associated with a metabolic shift from fatty acid oxidation to glycolysis. On the other hand, NR1c1 maintains fatty acid oxidation. Activated NR1c1 preserves heart function in the early stage of heart failure using an inducible transgenic mouse model of hypertension-induced heart failure [600]. In addition, NR1c1 downregulates the expression of fibrosis-related genes. 78 In

humans, vertebral arterial caliber ranges from 2 to 5.0 mm [501]. Vertebral artery hypoplasia is defined by a vessel diameter lower than 2 mm.

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3.9.4 Renin–Angiotensin Axis The RAA relies on an enzymatic reaction cascade. Angiotensinogen (Agtg), the initial substrate, is cleaved by renin, an aspartyl peptidase that governs the ratelimiting step of RAA activation, to Agt1 (a decapeptide [Agt(1–10) ]), processed by angiotensin convertase ACE1 to Agt2 (an octapeptide [Agt(1–8) ]), which is cleaved by ACE2 to Agt(1–7) (a heptapeptide). Angiotensin-2 is the major peptidic RAA effector, as it is the most powerful RAA product, although other active components exist, such as Agt3 (another heptapeptide [Agt(2–8) ]), Agt4 (a hexapeptide [Agt(3–8) ]), and Agt(1–7) . The RAA participates in the control of BP via coordinated effects of blood and local RAAs in the cardiovascular apparatus, kidney, and central nervous system via intra-, auto-, para-, and endocrine effects. In the kidney, it exerts its main function, regulating bodily fluid balance; Agt2 controls intrarenal hemodynamics and glomerular filtration, as it elicits vasoconstriction, in addition to tubular water and Na+ reabsorption, regulating ECF volume and hence BP. In addition to vasoconstriction and its antidiuretic and antinatriuretic action, the activated ACE1–Agt2–AT1 axis triggers secretion of aldosterone and vasopressin (or ADH), which are synthesized in the adrenal cortex and pituitary gland, respectively. The former causes Na+ reabsorption and concomitant K+ and H+ excretion by the kidney; the latter raises water reabsorption in the CD. Angiotensin-2 raises sodium and water reabsorption not only through renal but also intestinal epitheliocytes to reestablish fluid balance and normal BP. Angiotensin-2 also enhances myocardial contractility, stimulates release of catecholamines from the adrenal medulla and sympathetic nerve endings, and increases sympathetic nerve activity [502]. It participates in the control of cell growth, proliferation, migration, differentiation, and apoptosis, in addition to inflammation and oxidative stress. On the other hand, increased ACE2 activity shifts the balance to the countering Agt(1–7) –Mas pathway rather than the Agt2–AT1 axis. The monocarboxypeptidase ACE2 also processes Agt1 to Agt(1–9) (a nonapeptide), hence launching another counter-regulatory pathway, the Agt(1–9) –AT2 axis. Innate antigen-presenting cells and adaptive immune T lymphocytes are implicated in the development of hypertension. Among innate-like T lymphocytes, γδ-T lymphocytes, which express the γδ-T-cell receptor (γδ TCR) rather than the αβ TCR, can participate in the initiation of the immune response in hypertension. Deficiency in γδ-T lymphocytes blunts angiotensin-2-induced hypertension. In WT mice, 7- to 14-day Agt2 infusion increases γδ-T lymphocyte density and their activation in the spleen and perivascular adipose tissue [601]. Infusion of Agt2 for 14 days raises SBP and lowers endothelial function at least in mesenteric arteries, these effects being abrogated in TCRD−/− mice. In humans, γδ-T lymphocyte density correlates with SBP. Therefore, γδ-T lymphocytes mediate Agt2-induced SBP elevation in mice and may contribute to the development of hypertension in humans.

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Hypertension caused by increased RAA activation is associated with elevated ROS production by NAD(P)H oxidases. Their catalytic subunit generates ROS via electron transfer from NADPH to molecular oxygen. Different NOx isoforms play distinct roles, even in the same cell type, as they can be coupled with different signaling pathways and produce different ROS (i.e., superoxide versus hydrogen peroxide). Angiotensin-2 is a potent activator of both NOx1 and NOx2. vSMC NOx1 magnifies Agt2-induced hypertension [602]. Production of ROS in the subfornical organ by NOx1, NOx2, and NOx4 is implicated in the vasoconstriction launched by Agt2. Both NOx1 and NOx2 in infiltrated NOxO2+ T lymphocytes and NOx2+ monocytes provoke constriction of renal afferent arterioles [602]. The NOx2 subtype is synthesized in multiple cell types, such as cardiomyocytes, endotheliocytes, fibroblasts, immunocytes, and microgliocytes. Deletion of the NOX2 gene in myeloid cells (but not in endotheliocytes) significantly reduces BP owing to decreased NO availability [602]. However, myeloid-specific NOX2 deletion does not have an impact on Agt2-induced hypertension. Nevertheless, NOx2 deficiency in both endothelial and myeloid cells attenuates Agt2-induced hypertension and preserves endothelium-dependent vasodilation upon Agt2 exposure. Therefore, arterial pressure depends on myeloid cell NOx2, whereas EC NOx2 regulates Agt2-induced hypertension [602]. Myelomonocytic cells reversibly affect basal arterial pressure. Cellular repressor of E1A-stimulated genes (CREG)79 is a ubiquitous endosomal and lysosomal glycoprotein implicated in cellular growth and differentiation and hence vascular remodeling engendered by hypertension [603]. CREG diminishes vSMC proliferation and dedifferentiation. It is processed by lysosomal cysteine peptidases. The transcription factor E2F1 and microRNA-31 bind to the Creg promoter and 3 UTR at chromosomal locus 1q24.9 to repress CREG formation, respectively. Downregulation of Creg gene transcription is independent from BP. Upon Agt2 stimulation, E26 transformation-specific protein ETS180 binds to the Creg promoter and prevents gene transcription in vascular smooth myocytes [603]. In heart failure, the RAA is inhibited using ACE1 inhibitors and/or angiotensin receptor AT1 blockers [604]. The ACE2 enzyme is an ACE1 counter-regulator. Agt(1–9) has antihypertensive and, in the myocardium, antihypertrophic and antifibrotic effects. Angiotensin(1–7) attenuates an Agt2-induced increase in the formation of mitochondrial ROS, cardiac hypertrophy, and myocardial perivascular fibrosis via the deacetylase sirtuin-3, which deacetylates FoxO3a and promotes SOD2 expression in cardiomyocytes [605]. In addition, activated antihypertensive

79 Transforming proteins of small-DNA tumoral viruses such as the adenovirus E1A protein, which

alters the transcriptional program of the host cell, both activating and repressing gene expression to promote cellular proliferation and inhibit differentiation. 80 Members of the ETS family are involved in the regulation of cell differentiation and proliferation in addition to inflammation. ETS1 is synthesized in ECs and vSMCs. It regulates Agt2-mediated vascular inflammation and remodeling. Its formation is induced in vSMCs in response not only to Agt2 but also PDGF, thrombin, TNFSF1, and IL1 [603]. It acts as both a transcriptional activator and repressor according to its association with specific cofactors.

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ACE2–Agt(1–7) –Mas1 axis stimulates cardiac contractility via the PI3K–PKB– NOS3 pathway [604].

3.9.4.1

Angiotensinogen

Angiotensinogen (or serpin-A8; plasmatic half-lifeO[1 h]), in the classical RAA, is synthesized in hepatocytes and secreted into the bloodstream after removal of the 33-amino acid signal peptide. It is successively processed by renal renin and ubiquitous endothelial ACE1 into Agt1 and Agt2, respectively. The liver is the major source of circulating angiotensinogen. The kidney also produces Agtg, which is synthesized in the proximal tubule. However, in the kidney, most Agtg and Agt2 derive from the liver, whereas urinary Agtg originates from proximal tubular epitheliocytes [606]. Angiotensinogen produced in PT epitheliocytes is secreted into the tubular lumen [502]. Its metabolites produced intracellularly are also released into the tubular lumen. Estrogens trigger transcription of the AGT gene in the liver. In normal individuals, the reduced–oxidized Agtg ratio is 40:60, oxidized Agtg being a more efficient substrate for renin [606]. In addition to the kidney, other organs synthesize Agtg, such as brain, spinal cord, retina, heart, blood vessels, lung, adrenal gland, adipose tissue, stomach, large intestine, pancreas, spleen, and ovaries [604, 606]. These local RAAs can operate independently of the circulatory (systemic) RAA.

3.9.4.2

Renin

Renin is synthesized and secreted by the juxtaglomerular apparatus in the media of afferent arterioles upstream from the branching that gives rise to the glomerular capillary network. Medial cells of efferent arterioles or extraglomerular mesangial cells rarely express renin. A low concentration of renin is detected in the proximal tubule and principal cells of the CnT and CD [606]. Removal of 23 amino acids from the C-terminus of preprorenin generates prorenin [606]. Cleavage of 43- to 47 amino acids from the N-terminus of prorenin engenders active renin. Circulating active renin and prorenin are released mainly from the kidney, but other tissues also secrete prorenin into the circulation [502]. Reduced ECF volume increases circulating and interstitial renin concentration. Major stimuli that control renal renin release thus include: 1. The renal perfusion pressure detected by baroreceptors, which, when it is low, primes a local baroreflex that causes renin liberation 2. Sympathetic innervation augmented sympathetic activity provoking renin release 3. NaCl concentration sensed by the macula densa in the boundary between TAL outlet and DCT inlet, which, when it drops, stimulates renin secretion 4. Concentrations of autacoids, such as Agt2 and atrial naturetic peptide

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The main signaling cascades that control renin secretion rely on various paracrine messengers: adenosine, Agt2, NO, and PGhS2-derived prostaglandins, mainly PGe2 and PGi2 . The autacoids PGe2 , PGi2 , and NO raise the production or preclude the degradation of cAMP in JGCs, which stimulates renin release. The source of macula densa-derived prostaglandins mediating renin synthesis and release, PGhS2, has a low activity under normal conditions. Its action is increased by a salt-deficient diet, ACE1 inhibition, and diuretic administration. Production of PGhS2 in the macula densa depends on positive and negative feedback loops. Agt2 inhibits it via AT1 , but stimulates it via AT2 [572]. The (pro)renin receptor increases PGhS2 formation. Dopamine also hinders PGhS2 expression. On the other hand, an elevated intracellular cAMP concentration in reninsecreting cells causes efflux of cAMP, which is converted to adenosine in the extracellular space, triggering a negative feedback on renin liberation. Renin synthesis and release is inhibited by augmented Ca2+ concentration in JGCs, although Ca2+ usually facilitates exocytosis, because of inhibition by Ca2+ of adenylate cyclase subtype AC5 (calcium paradox of renin release) [572]. Adenosine hampers renin release via its A1 receptor on renin-secreting cells [503]. β1-Adrenoceptor abounds in the juxtaglomerular apparatus. It stimulates renin release but plays a modulatory rather than a primary role. Renin production and secretion are regulated by the renal baroreceptor and sodium chloride delivery to the macula densa, a reduced chloride ion concentration in the filtrate of the distal tubule triggering renin release [606]. Both NKCC2 (SLC12a1) and NHE2 (SLC9a2) on the apical surface of the MDCs play a role in renin release. Several signaling cascades can link distal tubule solute concentration and renin secretion using adenosine, nitric oxide, and prostanoids [606]. Macula densa stimulation of renin involves PGhS2 activation. Prostaglandin-E2 activates PGe2 R4 on granular cells in the juxtaglomerular apparatus and renin release. Adenosine stimulates the A1 receptor in the macula densa when luminal NaCl concentration drops. Macula densa cells have high levels of nitric oxide synthase NOS2. However, in the macula densa, NO plays an accessory role in the control of renin release. Renin synthesis and release is controlled by three main intracellular second messengers: cAMP, cGMP, and Ca2+ ion [606]. 1. Stimulated Gs-coupled receptors (e.g., those of PGe2 , PGi2 , and NAd) activate adenylate cyclases that generate from ATP cAMP, the main regulator of renin release. The mediator cAMP activates PKA, which phosphorylates transcription factors of the CREB–ATF1 family, among other targets. It is degraded by the phosphodiesterases PDE3 and PDE4. 2. Nitric oxide and atrial natriuretic peptide that signal via cGMP inhibit via PKG2 or stimulate renin release, according to the circumstances. 3. Calcium modulates activity of enzymes of cAMP synthesis and degradation. Elevated cytosolic Ca2+ concentration inhibits renin secretion via PKC. Vasopressin, endothelin, and Agt2 that rise [Ca2+ ]i impedes renin release. In humans, miR181a and miR663 target the REN transcript [606].

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Chronic ischemia, prolonged adrenergic stimulation, and sodium depletion increase the number of renin+ cells along the afferent arteriole, in the interstitium, and inside the glomerulus upon dedifferentiation of arteriolar smooth myocytes, mesangiocytes, and interstitial cells from the renin cell lineage that re-acquire the renin phenotype under control of epigenetic and transcriptional mechanisms in addition to microRNAs [606]. Renin synthesis in the distal nephron increases in models of diabetes and chronic kidney disease via the succinate receptor SucnR1 (or GPR91) on the luminal membrane of MDCs of the juxtaglomerular apparatus, close to renin-producing granular cells, the cTAL, and CCD and IMCD. Acute stimulation triggers exocytosis of secretory granules that contain renin only. Chronic stimulation releases both prorenin and renin into the blood circulation [606]. Depolarization suppresses renin release, whereas hyperpolarization promotes renin exocytosis. Cells of the juxtaglomerular apparatus possess NKCC1 and Ca2+ -activated Cl− channel.

3.9.4.3

Angiotensin Convertase ACE1

Angiotensin convertase ACE1 is a dipeptidyl carboxypeptidase that removes a dipeptide from the C-terminus of substrates. It has many substrates in addition to Agt1, such as vasodilatory and natriuretic bradykinin (hence its previous name kininase-2). It degrades bradykinin into bradykinin(1–7) and bradykinin(1–5) , a peptide with thrombin inhibitory activity. Somatic ACE subtype is a monomeric glycoproteic ectoenzyme (exopeptidase) on the surface of endotheliocytes, which particularly abound in the lung, intestine, choroid plexi, and placenta, and on renal brush border membranes [606].81 A soluble ACE form cleaved from tissular ACE circulates in plasma. In addition, ACE1 also localizes to membranes of various other cell types. It abounds in the proximal tubule brush border, expression in renal vascular endotheliocytes being lower in humans. Its activity is higher in the initial segment of the proximal tubule and is present in tubular fluid throughout the nephron except in the terminal segment of the distal tubule, decreasing down to the distal nephron [502].

3.9.4.4

Angiotensin Convertase ACE2

The type-I transmembrane ectoenzyme ACE2, a zinc metallopeptidase, acts as a monocarboxypeptidase that removes a single amino acid. It hydrolyzes three peptides with high efficiency: Agt2, apelin-13, and dynorphin-A [604]. Although it processes Agt2 with high efficiency, it has a much lower activity on Agt1; the human

81 The

somatic and testicular ACE isoforms are both plasma membrane-anchored proteins, which are shed by a secretase. However, somatic ACE is shed less efficiently than testicular ACE.

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ACE2 catalytic efficiency is 400-fold higher with Agt2 than with Agt1, generating Agt(1–7) and the nonapeptide Agt(1–9) , respectively. A soluble active truncated form of ACE2 shed by ADAM17 can cleave Agt1 and Agt2, but not bradykinin [604]. Angiotensin-2 acts via AT1 and P38MAPK, which phosphorylates (activates) ADAM17, priming membrane-bound ACE2 shedding. On the other hand, calmodulin tethers to ACE2 and prevents its shedding. Angiotensin convertase 2 resides in the cardiovascular system, kidney, lung, brain, and testis. In the heart, it localizes to cardiac myocytes and fibroblasts and coronary endotheliocytes. It also lodges on the surface of certain endotheliocyte types, with the highest expression in the kidney, followed by the heart and testis [606]. Transcription of the Ace2 gene is activated by AMPK via sirtuin-1 [604]. Hypoxia and AMPK increase the cellular ratio of NAD+ to NADH. Apelin binds to the promoter region of the ACE2 gene and enhances its transcription, especially in heart failure. MicroRNA-421 controls the Ace2 mRNA level via post-translational repression rather than degradation.

3.9.4.5

Collectrin

The third member of the ACE family, collectrin, is homologous to the transmembrane ACE2 sequence and lacks the carboxypeptidase domain [606]. It contributes to the regulation of amino acid transfer in the kidney and intestine in addition to arginine uptake in endotheliocytes, which is then used for NO synthesis. As ACE2 has a restricted expression in the kidney, lodging in the endothelium and proximal tubular cells, collectrin localizes to collecting duct cells [502].

3.9.4.6

Alternative Routes of Angiotensin-Derived Peptide Synthesis

Angiotensin-2 can be hydrolyzed by more than 13 peptidases, such as amino(AP), carboxi- (CP), and endopeptidases (EP), ACE2, and membrane metalloendopeptidase (MME),82 generating Agt3, Agt4, Agt(1–7) , Agt(3–4) , AgtA, and alamandine, which bind to specific receptors (AT4 and Mas1) or share a given receptor (Tables 3.26 and 3.27) [607]. Cathepsins, kallikrein, and kallikrein-related peptidases can process Agtg to form Agt1 or Agt2 directly [502].

Aminopeptidases Angiotensinogen(1–7) is preferentially hydrolyzed by APs and MME, generating Agt(1–4) [607].

82 Also

known as neutral endopeptidase neprilysin, enkephalinase, and skin fibroblast elastase.

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Table 3.26 Alternative synthesis routes of the RAA (Source: [607]; ACE angiotensin convertase, AP aminopeptidase [APa aminopeptidase-A, APn aminopeptidase-N], CP carboxypeptidase [CPp carboxypeptidase-P, prCP prolyl carboxypeptidase], EP endopeptidase, MME membrane metalloendopeptidase [a.k.a. neutral endopeptidase and neprilysin NEP1], prEP prolyl endopeptidase [a.k.a. prolyl oligopeptidase (prOP)]) Synthesis routes Agtg–Agt1–Agt2 Agt1–Agt(1–9) –Agt(1–7) Agt1–Agt(1–7) Agt2–AgtA–alamandine Agt2–Agt3–Agt4–Agt(5–8) –Agt(5–7) Agt2–Agt(1–7) Agt(1–7) –Agt(2–7) –Agt(3–7) –Agt(3–4) Agt(1–7) –Agt(1–5) –Agt(1–4) –Agt(3–4)

Table 3.27 Enzymes of the RAA (ACE angiotensin convertase, AP aminopeptidase, CP carboxypeptidase, EP endopeptidase, MME membrane metalloendopeptidase, prEP prolyl endopeptidase)

Enzymes Renin, ACE1 ACE2, ACE1/MME/PrEP APa/MME/PrEP Decarboxylase, ACE2 APa, APn, AP, CP CPp/prCP/ACE2 AP, EP ACE1, CP/MME, AP

Enzyme ACE1

ACE2

AP

APa APn CP CPp prCP Chymase Decarboxylase EP MME prEP renin

Reactions Agt1 −→ Agt2 Agt(1–9) −→ Agt(1–7) Agt(1–7) −→ Agt(1–5) Agt1 −→ Agt(1–9) Agt2 −→ Agt(1–7) AgtA −→ alamandine Agt4 −→ Agt(5–8) Agt4 −→ Agt(3–7) Agt(1–7) −→ Agt(2–7) Agt(2–7) −→ Agt(3–7) Agt(1–4) −→ Agt(3–4) Agt2 −→ Agt3 Agt3 −→ Agt4 Agt(5–8) −→ Agt(5–7) Agt(1–5) −→ Agt(1–4) Agt2 −→ Agt(1–7) Agt1 −→ Agt2 Agt2 −→ Agt(1–7) Agt1 −→ Agt2 Agt2 −→ AgtA Agt(3–7) −→ Agt(3–4) Agt(1–9) −→ Agt(1–7) Agt(1–5) −→ Agt(1–4) Agt(1–9) −→ Agt(1–7) Agtg −→ Agt1

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Aminopeptidase-A (APa), a membrane-bound and soluble enzyme and component of the RAA, metabolizes Agt2 (Agt(1–8) ) into Agt3 (Agt(2–8) ), which activates renal AT2 receptor and induction of natriuresis (neither Agt2 nor Agt(1–7) causes natriuresis [608]). In fact, Agt3 binds to AT1 with greater affinity than to AT2 [607]. Nevertheless, in the proximal tubule, Agt3 provokes natriuresis via the AT2 –cGMP axis. In addition to Agt3, Agt(2–10) is another cleavage product of APa from Agt1 Agt(1–10) . In mouse plasma, concentrations of Agt1, Agt3, and Agt(2–10) are the highest of all the RAA peptide levels, whereas renal Agt2 concentration is higher than its plasmatic level [609]. The genetic obesity model, ob/ob mice, are normotensive with a weak activation of the sympathetic nervous system, lack of leptin,83 and a low ACE1 activity, but a strongly elevated circulating APa activity and hence plasmatic Agt3 concentration [608]. A high-fat diet (HFD) raises APa activity proportionally with the gain in body weight and adipose tissue. Inhibition of AT2 , the production of which rises in the kidney, and APa increases sodium excretion and BP. Circulating APa activity declines with weight loss independently of leptin. Aminopeptidase-N (APn) hydrolyzes Agt3 into Agt4 [607]. Aminopeptidases APa and APn abound in the kidney, especially in the proximal nephron. Agt4 is also formed in the glomerulus. It increases the renal blood flow and decreases Na+ transport in the proximal tubule, where it mobilizes Ca2+ via AT1 [607]. In the absence of AT4 , Agt4 provokes renal vasoconstriction via AT1 .

Carboxypeptidases Carboxypeptidase-N catalyzes the conversion of Agt2 to Agt(1–7) , which subsequently and successively generates Agt(1–5) , Agt(1–4) , and Agt(3–4) , a major route in the RAA using ACE1, carboxypeptidase or MME, and AP [607]. Angiotensinogen(3–4) is remarkably stable in human blood. It has an antihypertensive action in spontaneously hypertensive rats, as it inhibits Na+ –K+ ATPase via the AT2 –cAMP–PKA pathway. It may relieve inhibition of plasma membrane Ca2+ ATPase by nanomolar Agt2 concentrations, also via the AT2 –cAMP–PKA pathway. The basolateral membrane of proximal tubular epitheliocytes possesses all the peptidases required to produce Agt(3–4) in the vicinity of Ca2+ ATPase, this local RAA rapidly enabling Ca2+ flux, as Agt(3–4) counteracts inhibition of the Ca2+ ATPase by Agt2 [610]. Proline carboxypeptidase (prCP),84 can process both inert Agt1 and active Agt2 into Agt(1–7) , another active peptide that stimulates NO and PGi2 formation, induc-

83 Chronic leptin infusion causes hypertension via activation of the melanocortin receptor-4 and the

sympathetic nervous system. known as prolyl carboxypeptidase, Pro–X carboxypeptidase, lysosomal carboxypeptidaseC, and angiotensinase-C.

84 Also

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ing vasodilation and potentiating the effect of the vasodilator bradykinin [611].85 Proline carboxypeptidase is thus an Agt2 inhibitor and a prekallikrein activator. On the other hand, plasmatic kallikrein converts prorenin to renin, further demonstrating mutual interactions between the plasmatic kallikrein–kinin axis (KKA) and the RAA, the hypotensive KKA counterbalancing the hypertensive RAA [611].86 Kallikrein is thus a kinin generator and prorenin activator. Moreover, AT1 can heterodimerize with the bradykinin B2 receptor and Agt2 promotes synthesis of B1 and B2 receptors [612]. Both B1 and B2 participate in the regulation of the vasomotor tone and hence BP, one compensating for inhibition of the other [613]. Combined administration of B1 and B2 antagonists engenders a significant BP increase, which is partly prevented by the Agt antagonist losartan. When both bradykinin receptors are inhibited, upregulated transcription of the genes encoding NOS3, AT1 , PGe2 receptor, and Klk1 is more pronounced in the heart and kidney.87 Cathepsins Cathepsin-A acts as a component of the intrarenal RAA, as it can convert Agt1 to Agt2 [614]. Renal cathepsin-G generates Agt2 [615]. Neutrophil membranebound inducible CtsG converts both Agtg and Agt1 to Agt2, thereby causing vasoconstriction and chemotaxis at sites of inflammation [616]. Chymase Angiotensin-2 is produced not only by ACE1, but also by chymase, a serine peptidase. Rat vascular chymase, which is constitutively expressed in vascular smooth myocytes, converts Agt1 to Agt2 [502]. In the human heart, chymase is synthesized and stored in endothelial and mesenchymal cells; it is secreted into the interstitium. Mastocytes in the heart, kidney, and other organs produce chymase that, once released into the extracellular matrix (pH 7.4, i.e., in the optimal pH range of chymase activity), augments Agt2 production. Mastocytes can also form and secrete renin. 85 PrCP

also converts prekallikrein to kallikrein, which processes kininogens to bradykinin [611]. such as plasmatic and tissular kallikrein. Kinins are rapidly catabolized into inactive peptides and agonists of the B1 receptor by kininases, such as APs, carboxypeptidase CPm and CPn, ACE, MME, platelet cathepsin-A, and intracellular prolylendopeptidase. Bradykinin, an ACE1 substrate, stimulates tPA, NO, and PGi2 production, thereby countering the prothrombotic effect of Agt2, which stimulates release of plasminogen activator inhibitor PAI1 (serpin-E1) from endotheliocytes. In the cTAL, bradykinin counters Ca2+ influx primed by Agt2 via4 protein Tyr kinase and the MAPK module. Bradykinin also suppresses Agt2-induced Na+ transport. 87 The constitutive B receptor abounds in the heart, kidney, and vasculature, where it elicits the 2 action of vasodilatory prostaglandins and NO, whereas B1 is inducible and mostly involved in inflammation [613]. 86 Kinins are synthesized from kininogens in blood and organs by kininogenases,

3.9 Regulators Table 3.28 Cardiovascular effects of certain receptors of the RAA

283 Receptors AT1 AT2 Mas1

Effects Vasoconstriction Hypertrophy, fibrosis Vasodilation Anti-hypertrophy, anti-fibrosis Vasodilation Natriuresis Inhibition of adverse cardiac remodeling

Decarboxylase The octapeptide AgtA (Ala–Arg–Val–Tyr–Ile–His–Pro–Phe) derives from Agt2 decarboxylated at Asp1 in the presence of mononuclear leukocytes [607]. It has a higher affinity for AT2 than that of Agt2 and the same affinity for AT1 . It causes renal vasoconstriction in normo- and hypertensive rats. It is processed by ACE2 into alamandine (an heptapeptide). Alamandine has vasodilatory, antifibrotic, and antihypertensive effects. It acts via the Mas-related G-protein-coupled receptor-D (MRGd).

3.9.4.7

Receptors

The classical RAA comprises two types of receptors (AT1 –AT2 ), most cardiovascular effects of Agt2 (e.g., vasoconstriction, water and salt retention, aldosterone synthesis and release, tissular growth, and remodeling) being mediated by the AT1 receptor. The AT2 receptor counteracts the cardiovascular action of AT1 (Table 3.28).

(Pro)Renin Receptor Both prorenin and renin bind to and activate the specific (pro)renin receptor (PRR). The PRR can activate ERK1, ERK2, and P38MAPK [502]. In the heart and kidney, it increases the efficiency of Agt1 formation from angiotensinogen. In the human kidney, PRR lodges in the glomerular mesangial cells, the subendothelium of renal arteries, the distal nephron, the CDs, and mostly at the apical surface of ICCs, where it stimulates synthesis by PGhS2 of prostaglandins, which attenuate the antinatriuretic and vasoconstriction of Agt2 [607]. The prorenin–PRR couple leads to the development of nephropathy in type-2 diabetes [607]. The extracellular domain of the prorenin–renin receptor is cleaved by membranebound transcription factor peptidase site 1 (MBTPS1) and furin (or PCSK3), thereby generating a soluble PRR form (PRRS ), which can serve as a marker [617].

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The prorenin–renin receptor contributes to the regulation of hypothalamic, sympathetic, and neurosecretory outputs to the cardiovascular system such as release of vasopressin. It is produced by ADH+ neurons, its activation increasing neuronal activity [618]. It excites ADH+ neurons via inhibition of the inactivating A-type K+ current,88 restraining firing activity. It also augments the inactivation rate and hyperpolarization of the membrane potential, thereby lowering channel availability for activation at any given membrane potential. It fails to increase superoxide production within the supra-optic and paraventricular nuclei.

Angiotensin-2 Receptors Angiotensin-2 is the active peptide that acts via its receptors AT1 and AT2 on cells of the brain, heart, vasculature, kidney, and immune system. Angiotensin-2 receptors can tether to selective and AT1 /AT2 dual ligands [621]. In the kidney, transcript of AT1 can be detected in the glomerulus (e.g., mesangiocytes and podocytes), proximal tubule, TAL, macula densa, renal arteries (e.g., afferent and efferent arterioles), vasa recta, arcuate arteries, and JGCs [502, 607]. The AT2 receptor localizes to glomerular epitheliocytes, the proximal tubule, CDs, and renal vasculature, at least in adult rats. It can counteract the effect of AT1 , as it stimulates formation of bradykinin and NO [502]. Angiotensin-2 precludes renin release at the juxtaglomerular apparatus using a short feedback loop via Gq-coupled AT1 receptor, PKC, calmodulin, and increased cytosolic Ca2+ concentration, thereby precluding the circulating RAA [606]. Vascular and tubular Agt2 receptors respond differently when Agt2 concentration is high. In general, a high Agt2 level associated with a low-salt diet downregulates glomerular AT1 expression, but upregulates or does not significantly modify that of tubular AT1 [502]. On the apical membrane of the PCs in the CCD, luminal Agt2 stimulates AT1 and ENaC activity. In the CCD and OMCD, activated AT1 stimulates Na+ –H+ exchange, as it increases the concentration of vacuolar sodium–hydrogen ATPase in the apical membrane of the α ICC, increasing bicarbonate reabsorption [606]. The AT1 receptor also modulates the activity of pendrin, a Na+ -independent Cl− transporter on the β ICC, enhancing chloride reabsorption and raising BP. The AT1 receptor also participates in the regulation of tubuloglomerular feedback,89 release of aldosterone from the adrenal glomerulosa, SMC contraction, and

A-type K+ channels include KV 1.1, KV 1.2, KV 2.1, KV 3.1b, KV 3.4, and KV 4.3 [619]. This transient inactivating current has a fast activation and relatively fast inactivation. A-type K+ current is involved in repolarization in sympathetic neurons and spinal motor neurons. In the dendrites of hippocampal pyramidal neurons and cerebellar Purkinje cells, A-type K+ current acts as a highpass filter of incoming synaptic signals, damping synaptic excitatory signals [620]. 89 The tubuloglomerular feedback balances glomerular filtration and tubular reabsorption. Elevated concentrations of Na+ , Cl− , and K+ in the TAL fluid release ATP and AMP from the MDCs, which are catabolized into adenosine, adapting afferent arteriolar resistance (increase), glomerular 88 Six

3.9 Regulators

285

stimulation of the hypothalamic thirst sensor [606]. It signals via the Gq–PLC– IP3 –Ca2+ axis in addition to the JaK–STAT and β Arr–ERK pathways and EGFR transactivation. The AT2 receptor90 can protect against organ deterioration, especially during aging due to redox stress. Agt2 via AT1 activates NAD(P)H oxidase (e.g., NOx2), which generates superoxide anion [622]. The NOx–ROS axis favors cardiac hypertrophy and fibrosis. Excessive superoxide production uncouples NOS3, which then augments ROS production and causes aging. In addition, Agt2 accelerates cellular senescence via telomere shortening. Moreover, signaling primed by AT1 downregulates SIRT3 formation, which protects cells such as cardiomyocytes against mitochondrial dysfunction, redox stress, and apoptosis [622].91 The intramitochondrial angiotensin axis contributes to senescence. The AT2 receptor lodges on the inner mitochondrial membrane; its concentration decreases with aging, whereas that of AT2 increases [622].

Mas1 Receptor The counter-regulatory messenger of the RAA, Agt(1–7) , operates via the G-proteincoupled Mas1 receptor to oppose Agt2 effects. It provokes release of vasodilators, such as NO, PGe2 , and bradykinin. It causes natriuresis and prevents Agt2-induced adverse cardiac remodeling. Angiotensin(1–7) impedes Na+ reabsorption in the proximal tubule via different receptors [607]. In the proximal and distal tubule, it inhibits Na+ –K+ ATPase via the AT2 –Gi/o–cGMP–PKG and AT1 –PLC–PKC pathways. Angiotensin-2 is involved in BP control via its short-term effect on vascular resistance (vasoconstriction via SMC AT1 and NO-mediated vasodilation via EC AT2 ) and its long-term action on the water and electrolyte balance, the AT1 receptor promoting renal retention of sodium and water.92 The Agt2–ACE2–Agt(1–7) –

flow rate (decrease), and hence filtration rate (decrease). Constriction of the afferent arteriole relies on the A1 –Gi–PLC–IP3 –Ca2+ axis. The tubuloglomerular feedback additionally depends on Agt2, which regulates renal hemodynamics via vasoconstriction and the subsequent rise in renal vascular resistance. However, Agt2 does not directly mediate the tubuloglomerular feedback; instead, it modulates the sensitivity of vascular smooth muscle and mesangial cells, which respond to signals from MDCs along with the Na+ –H+ exchange of macula densa cells [502]. The calciumdependent chloride channel contributes to Agt2 vascular action. 90 The ubiquitous AT receptor is synthesized in the fetus and its production decreases after birth, 2 remaining at a low level in adulthood in vascular endothelia, adrenal medulla, and some brain regions. 91 Among mitochondrial sirtuins (SIRT3–SIRT5), SIRT3 is associated with longevity in humans [622]. It reduces ROS production and can promote activity of mitochondrial superoxide dismutase, SOD2. It deacetylates the acetylCoA synthase, ACS2, isocitrate dehydrogenase, IDH2, ornithine transcarbamoylase, long-chain acylCoA dehydrogenase, and glutamate dehydrogenase. 92 Human cells possess a single AT receptor, whereas rodents have two isoforms (AT –AT ) 1 1A 1B with similar affinities for Agt2 [622].

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Mas–NOS3 axis provokes vasodilation, which counteracts AT1 signaling. It also has antiproliferative and antifibrotic effects [622]. Endotheliocytes constitutively express Mas1; regulation of NOS3 by Agt(1–7) may occur via the PI3K–PKB axis [623]. At a low dose, Agt(1–7) not only primes vasodilation but also promotes angiogenesis via Mas1, GTPases of the RHO category, PI3K, PKD1, and ERK1/2 [623]. Expression of VEGFR1 and VEGFR2 results from the Ras–MAPK–ERK signaling. Equimolarly low doses of Agt2 or Agt(1–7) can have similar effects. Agt2 signals via AT1 coupled with heterotrimeric G proteins of the Gq/11, Gi/o, and G12/13 subclasses, which activate PLC and RHO group GTPases but inhibit adenylate cyclase. Physiological (subpressor) circulating concentrations of Agt2 do not affect endothelial function and hence vasodilation [623]. The Mas1 receptor signals through the RHO hyperfamily GTPases (Ras, Rho, Rac), the MAPK module (ERK1/2 and P38MAPK), and the PI3K–PKD1–PKB–TOR pathway to promote EC survival, angiogenesis, and vasodilation, in addition to raising TOR and lowering NFκB activity [623]. Both ERK1 and ERK2 are essential effectors of the Agt(1–7) –Mas1 pathway for vasodilation and angiogenesis. Although Agt(1–7) acts specifically via Mas1, Mas1 can functionally interact with AT1 and AT2 , as does EGFR, transactivation being crucial for AT1 -primed ERK1/2 signaling. In glomerular mesangiocytes, ERK1/2 activation via the Agt(1–7) –Mas1 axis is reduced by the cAMP–PKA pathway [623]. In humans, obesity is assumed to be a major factor of EHT. It provokes hemodynamic alterations and predisposes to heart failure. Obesity and hypertension are linked to the RAAA, which is activated in animals fed with an HFD that are hypertensive. In male obese hypertensive mice, plasmatic Agt2 concentration increases and adipose ACE2 activity decreases. On the other hand, in female obese mice, adipose ACE2 formation mediated by estrogens and plasmatic Agt(1–7) concentration augment, protecting against obesity-induced hypertension owing to the counterregulatory RAA arm, the ACE2–Agt(1–7) –Mas axis [624]. A 16-week HFD in male and female mice combined with MasR deficiency alters the cardiovascular function. In female mice, double deletion of Mas does not affect obesity but abolishes protection from obesity-linked hypertension. In obese Mas−/− males, DBP is lower, but the left ventricular wall is thicker than in Mas+/+ males because of decline in fibrosis and the ejection fraction. Infusion of Agt(1–7) restores obesityinduced cardiac dysfunction in WT (Mas+/+ , but not Mas−/− ) male mice [624]. Angiotensinogen(1–7) protects the heart against the effects of Agt2 in adverse cardiac remodeling. In normotensive rats, prolonged (4-week) subcutaneous infusion of Agt(1–7) alone or together with Agt2 affects neither basal arterial pressure nor Agt2-elicited hypertension but attenuates Agt2-primed cardiac hypertrophy and perivascular fibrosis via Mas1 and a drop in the Agt2-induced increase in mitochondrial superoxide formation due to elevated SOD2 production upon deacetylation of FoxO3a by sirtuin-3 in cardiomyocytes [605].

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AT4R The action of Agt4 is mediated by its high-affinity AT4 receptor, the insulinregulated aminopeptidase (IRAP), a type-I I integral membrane protein,93 which lodges in the central nervous system and kidney [607]. In fact, AT4 is widely distributed in organs and is also detected in the heart, aorta, adrenal glands, sympathetic ganglia, thymus, bladder, and hippocampus [625]. Multiple AT4 isoforms exist, which may be specific to organs [625]. Whereas the classical cerebral RAA regulates BP, the body’s sodium and water balance, the cyclicity of reproductive hormones, and the release of pituitary gland hormones, the Agt4–AT4 pathway participates in the control of blood flow and stress response and in learning and memory acquisition [626]. Agt4 facilitates cognitive processing and promotes the neuronal firing rate and long-term potentiation. In the brain, AT4 abounds in the cerebral and cerebellar cortex, hippocampus, cholinergic circuits, in addition to sensory and motor nervous systems. In several cerebral nuclei, Agt4 interact with AT4 to boost memory retrieval and induce Fos expression in the hippocampus [627]. In addition, the decapeptide LVV-hemorphin-7 (LVVYPWTQRF, i.e., Leu– Val–Val–Tyr–Pro–Trp–Thr–Gln–Arg–Phe), a globin fragment that has weak opioid activity (hence its name hemorphin), is another high-affinity AT4 ligand.94 The promnestic peptides Agt4 and LVV-hemorphin-7 enhance learning and memory and reverse memory deficits in amnesic animals. The AT4 agonists may prevent AT4 catalytic activity in addition to that of AP APn [628]. In the human proximal tubular and in CD epitheliocytes, activated AT4 triggers several signaling pathways, thereby rising cytosolic Ca2+ concentration along with that of Na+ and MAPK activity [629]. In addition, AT4 increases cortical blood flow and serpin-E1 (PAI1) expression in PT cells in addition to in vascular endotheliocytes [625]. It primes Ca2+ signaling in mesangial, proximal tubular, and CD cells [629]. Angiotensin-4 dilates pial and renal cortical arteries. In endotheliocytes, the Agt4–AT4 couple is internalized and then can be recycled to the plasma membrane [630]. Receptor and Signaling Mediator Angiotensin convertase, ACE1, renin, and prorenin can also activate intracellular signaling cascades, acting as an outside–in mediator or via PRR [607]. Stimulated ACE1 does indeed initiate a series of intracellular events. The prorenin and renin receptor generates its effects independently of angiotensin. 93 IRAP

belongs to the M1 family of zinc-dependent metallo-aminopeptidases. It localizes predominantly to GluT4+ vesicles in insulin-responsive cells. 94 This fragment results from the processing of hemoglobin by pepsin and a high-molecular-weight aspartic peptidase [627].

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The other ACE substrate, bradykinin, favors PGhS2 formation, ACE1 phosphorylation (Ser1270), and activation of JNK in endotheliocytes upon ACE1 dimerization [607].

3.9.4.8

Local Renin–Angiotensin Axes

Local RAAs in the brain, heart, and kidney, in addition to in immunocytes operate independently or in interaction with components of the blood RAA. The cerebral RAA regulates fluid balance and energy metabolism. In the mouse central nervous system, ACE2 reduces redox stress. In cardiomyocytes and coronary vessels, the RAA comprises synthesis of Agt1 from renin and conversion of Agt1 to Agt2 [631]. ACE2 synthesized in cardiac myocytes and fibroblasts and coronary endotheliocytes converts Agt2 into its antagonist Agt(1–7) . Chymase released from cardiac mastocytes yields a major source of cardiac Agt2 during aging. In aged rats, exercise normalizes concentration of superoxide concomitantly produced by NOx in the heart and promotes cardiac ACE2 production, hence hindering the chymase–ATN2–AT1 –NOx pathway and supporting the ACE2– Agt(1–7) –Mas axis [631]. Every RAA component is present in the kidney; intrarenal Agt2 is formed by independent multiple mechanisms, in particular via prorenin receptors and chymase [502]. Production of PT Agt, CCD renin, and tubular AT1 augments upon exposure to intrarenal Agt2; the latter exerts a positive feedback on PT Agt. Concentration of Agt2 is relatively high in some regions and compartments within the kidney, which is distinctly regulated with respect to circulating Agt2 concentration. In addition, circulating Agt2 is internalized into proximal tubular cells using a AT1 -mediated mechanism. Concentration of Agt2 in the interstitium is much higher than in the plasma [502]. An Agt2 fraction that binds to AT1 is internalized with its receptor. Endocytosis of Agt2–AT1 in PT cells uses the clathrin-dependent and preferentially microtubulelinked routes [607]. It is also internalized in vascular smooth myocytes owing to AT1 , Src, and clathrin adaptor protein-2. Megalin (or LRP2), a multiligand transmembrane endocytic receptor involved in the tubular uptake of proteins, mediates the internalization of Agt2 and Agt(1–7) in PT cells.95 Agt2 can then be recycled and secreted or exert intracellular effects. In Agt2-dependent hypertension, a higher fraction of the total renal Agt2 is endocytosed [502]. 95 Among

proteins normally filtered in the glomerulus, ligands of megalin include vitamin-binding proteins, lipoprotein lipase, cytochrome-C, ApoB, ApoE, ApoH, ApoJ, albumin, transthyretin, thyroglobulin, plasminogen, lactoferrin, PAI1, Ca2+ , insulin, prolactin, PTH, EGF, and α1- and β2-microglobulin, the latter two molecules being a lipocalin and an antigen-presenting molecule, respectively [632]. They are then degraded in lysosomes or transported back to the blood circulation. In the proximal tubule, proteins are also taken up by cubilin.

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Agt2 lowers glomerular blood flow and filtration rate and raises both afferent and efferent (i.e., pre- and postglomerular) arteriolar resistance. In the proximal tubule, locally produced Agt2 at physiological concentrations (pmol/l) increases transcellular Na+ reabsorption, which is coupled with bicarbonate reabsorption, independently from circulating Agt2, via activation of apical + + Na+ –H+ exchanger and basolateral Na+ –HCO− 3 cotransporter, Na –K ATPase, + and insertion of H ATPase into the apical membrane [502]. In addition, Agt2 provokes hypertrophy of proximal tubular cells. In the proximal and distal tubule, Na+ serves as a counterion for H+ secretion, which is then augmented. In the distal tubule, Agt2 favors bicarbonate and sodium reabsorption in the upstream and downstream segment, respectively [502]. It also stimulates Na+ –H+ exchanger in the entire compartment and vacuolar H+ ATPase in the downstream segment. In the CD, Agt2 directly stimulates apical ENaC activity, in addition to its effect via aldosterone, and increases water reabsorption [502]. Locally produced Agt2 can cause podocyte injury via AT1 and hence proteinuria. Inappropriate activation of the renal RAA contributes to hypertension. Chronic augmentation of Agt2 concentration blunts the autoregulatory responsiveness of the afferent arteriole in Agt2-dependent hypertension. Agt2 is a major regulator of aldosterone that triggers Na+ reabsorption and K+ secretion via the MR in the connecting tubule and CCD.

3.9.5 Aldosterone The mineralocorticoid hormone aldosterone controls potassium homeostasis and intravascular volume. It increases intestinal absorption and renal reabsorption of Na+ and Cl− ions. This salt-conserving hormone increases the concentration of ENaC and controls Na+ reabsorption and K+ secretion in the distal nephron and hence fluid volume and subsequently BP. Primary aldosteronism, the most common curable form of secondary hypertension, which is caused by bilateral adrenal hyperplasia and unilateral aldosteroneproducing adenomas, is characterized by hypervolemia, salt retention, hyperkaliuria, and hence hypokalemia, suppressed plasma renin activity, and hypertension. The main source of circulating aldosterone (concentration ∼100 pmol/l) is the zona glomerulosa cells, that is, in the outermost layer of the adrenal gland cortex. Aldosterone can be synthesized in other organs, such as the brain, heart, kidney, and vasculature (endothelial and smooth muscle cells). Its synthesis from cholesterol and secretion involves plasmalemmal depolarization, opening of CaV 1 and CaV 3 channels, and aldosterone synthase (CyP11b2) activation.

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Aldosterone synthesis is stimulated by increased plasmatic concentrations of Agt2, Agt3, adrenocorticotropic hormone (ACTH; or corticotropin),96 and serotonin via the Gs–cAMP–PKA–Ca2+ –CyP11b2 axis, in addition to sodium deprivation, hyperkalemia (via depolarization), acidosis, hypotension via atrial stretch receptors, and a lipid factor from the pineal gland (epiphysis cerebri) adrenoglomerulotropin. Secretion of aldosterone is controlled by an excitatory–inhibitory system based on pituitary corticotropin and pineal adrenoglomerulotropin and anticorticotropin [634]. These regulators interact to determine the secretory rate of aldosterone. The balance among renal perfusion pressure, glomerular filtration, and net renal Na+ reabsorption preserves bodily fluid volume. This balance is regulated by the sympathetic nervous system and hormones in addition to the intrinsic properties of vascular cells. Increased perfusion pressure, glomerular filtration, and interstitial pressure, in addition to decreased proximal reabsorption, deliver a greater Na+ amount to the aldosterone-sensitive distal nephron.

3.9.5.1

Aldosterone and Mineralocorticoid Receptor

The adrenocortical steroid hormones are classified into mineralocorticoids, glucocorticoids, and sex steroids, particularly androgens, which have an hypertensinogenic action.97 The plasmatic concentration of glucocorticosteroids normally exceeds that of aldosterone. Aldosterone, cortisol, and corticosterone, act via structurally similar mineralo- and glucocorticoid receptors, which mediate their hypertensinogenic activity. Mineralocorticoid receptor connects to both mineralo- and glucocorticoids with high affinity. Under redox stress, cortisol evolves from an MR antagonist to an MR agonist. This bivalent activity is used to prevent MR signaling in EHT [635]. Mineralocorticoid receptor (MR; NR3c2) is expressed in specific segments of the distal nephron, whereas glucocorticoid receptor (GR; NR3c1) is produced in the glomerulus and entire nephron; MR and GR are thus coexpressed in the distal nephron (i.e., distal part of the DCT, CnT, and CD) [636]. Aldosterone tethers to cytosolic MR, which then translocates to the cell nucleus and stimulates transcription of responsive genes. Receptor activation elicits Na+ transfer in aldosterone-responsive epitheliocytes of the kidney, colon, salivary glands, sweat ducts, and skin.

96 ACTH

can stimulate aldosterone secretion acutely and transiently via its specific melanocortin receptor MC2 , but to a lesser extent than Agt2 and K+ [633]. also stimulates steroidogenesis in the zona fasciculata and reticularis. 97 The main steroids of the adrenal cortex comprise the mineralocorticoid aldosterone, glucocorticoids cortisol and corticosterone, and androgens androstenedione, 11β-hydroxyandrostenedione, and dehydroepiandrosterone sulfate, mainly synthesized in the zona glomerulosa, fasciculata, and reticularis, respectively.

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Chronic activation of the MR provokes cardiac and renal fibrosis in addition to inflammation and vascular remodeling. Aldosterone acts in its target organs mainly via type-2 11β-hydroxysteroid dehydrogenase (11β HSDH2), which converts cortisol and corticosterone to their inactive metabolites cortisone and 11-dehydrocorticosterone, thereby preventing illicit occupation of MR by glucocorticoids. 1. The 11β HSDH1 isoform is a predominant reductase in most cells. In the adult brain, it operates on the hypothalamic–pituitary–adrenal (HPA) axis. It is also widespread in the liver, adipose tissue, muscle, lungs, pancreatic islets, and gonads, in addition to inflammatory cells. It regenerates active glucocorticoids from circulating inactive 11-keto forms [637]. It catalyzes the NADPH-dependent reduction of cortisone to active cortisol. Its concentration is elevated in adipose tissues of obese subjects, where it contributes to metabolic complications, and in the aging brain, where it exacerbates glucocorticoid-induced cognitive decline. 2. The 11β HSDH2 isozyme inactivates hydroxyglucocorticoids to inactive ketoglucocorticoids, hence conferring ligand selectivity on MR, especially ensuring that only aldosterone is an MR agonist in the ASDN, where MR and 11β HSDH2 are coproduced. On the other hand, in the kidney, the presence of GR and 11β HSDH2 is mutually exclusive [638]. 11β HSDH2 also abounds in the nucleus tractus solitarius, where it rapidly inactivates cortisol to cortisone, but not in the hippocampus, which also expresses MR [639]. Expression of 11β HSDH2 is widespread in the central nervous system during fetogenesis, but progressively disappears from midgestation. In adulthood, 11β HSDH2 location is restricted to subpopulations of neurons in brain regions that influence BP and salt appetite [640]. In rats, salt restriction augments plasmatic renin and aldosterone concentrations in addition to 11β HSDH2 synthesis and lowers that of MR in the distal colon, whereas production of MR, GR, and 11β HSDH2 remains constant in the renal cortex and medulla, left ventricle, and aorta [638]. In the colon, GR concentration declines and hence GR-stimulated Na+ –H+ exchanger NHE3 (SLC9a3). Mineralocorticoid selectivity is regulated by NaCl intake via 11β HSDH2 synthesis in the distal colon, but not in the kidney. Age-related deficiency in 11β HSDH2 favors low-renin hypertension, hypokalemia, and sodium retention linked to unregulated MR activation by cortisol. Hypertension can result from mutations in the HSD11B2 gene with inappropriate activation of MR by glucocorticoids and Na+ transport in the ASDN. Apparent mineralocorticoid excess is linked to an augmented salt appetite. Heterozygote HSD11B2−/− salt-sensitive mice have normal basal blood pressure, but cannot efficiently excrete a sodium load. The sympathetic nervous system is activated in HSD11B2−/− mice, maintaining hypertension. In the mouse brain, selective deletion of 11β HSDH2 causes salt appetite, attenuates the baroreflex, and triggers an elevated pressor response to α-adrenoreceptor activation [640].

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Aldosterone and Epithelial Na+ Channel

A complementary regulation is exerted by the mechanosensitive renal epithelial Na+ channel (ENaC) in the apical membrane of Na+ -reabsorbing epitheliocytes that carry Na+ in the distal nephron and the vascular endothelial Na+ channel (EnNaC). Whereas ENaC is inhibited by increased Na+ concentrations in bodily fluids, EnNaC is activated by an increased external Na+ concentration [636]. Activity of EnNaC and ENaC maintains blood volume and pressure, whereas angiotensin-2 maintains organ perfusion. Heteromeric ENaC (αβγ),98 a member of the ENaC and degenerin set of cationselective channels, is a major regulator of salt and water reabsorption in numerous types of epithelia, such as in airways, where it controls airway surface liquid clearance, and in the colon, salivary ducts, and sweat glands, in addition to the distal nephron. This reabsorption determines ECF volume. The ENaC channel also lodges on vascular endothelial and vascular smooth muscle cells. Activity of ENaC is regulated by its gating in addition to its concentration and residence time in the apical wetted membrane. This activity is controlled by proteolytic processing of its external domains by several channel-activating peptidases (CAPs)99 ; indeed, it is cleaved not only within the protein secretory pathway but also after apical membrane localization [643]. It is internalized via clathrin, the recycling rate being governed by hormonal regulation. The ENaC channel cooperates with Na+ –K+ ATPase in the basolateral membranes of epitheliocytes, achieving an osmotic gradient that enables water flux in the same direction. Apical residence of ENaC in membrane rafts and caveolae100 depends on its internalization and degradation, in particular hormone-regulated ubiquitination.101 α, β, and γ subunits of ENaC are encoded by the SCNN1A, SCNN1B, and SCNN1G genes, respectively. Mutations in the SCNN1B and SCNN1G genes cause channel activation and early onset hypertension. 99 Both α- and γ ENaC subunits are cleaved in a region of the extracellular loop that contains furin (PCSK3) consensus cleavage sites. Furin is a serine peptidase that resides primarily in the transGolgi network. It cleaves the α subunit to moderate channel activation twice and the γ subunit once, which must be cleaved by a second peptidase to further activate the channel [636]. Serine peptidase PesS8 (a.k.a. CAP1 and prostasin) is a glycosyl phosphatidylinositol-anchored enzyme on the surface of renal and respiratory epithelia [641]. Cleavage by PesS8 of the γ subunit at a site distal to the furin cleavage site further activates ENaC. Transmembrane serine peptidase TMPesS4 (or CAP2), elastase, and plasmin also cleave the γ subunit at sites distal to the furin site and activate ENaC. TMPesS14 (a.k.a. CAP3 and matriptase), an activator of CAP1, activates the channel via cleavage of the γ subunit [642]. Trypsin activates ENaC and enhances subunit cleavage at sites in the vicinity of the furin cleavage sites [641]. Kallikrein-1 can also process the γ subunit in basal conditions, whereas other peptidases activate ENaC upon volume depletion. Activity of PesS8 along with peptidase nexin-1, a PesS8 inhibitor, and other serine peptidases, may be regulated by aldosterone. Cysteine, alkaline, and metallopeptidases also activate ENaCs [636]. 100 Membrane rafts and caveolae are implicated in mass transfer and signaling at the plasma membrane. 101 The ENaC subunits are poly- or multi-monoubiquitinated at the cell surface. Both NEDD4 and NEDD4-2 inhibit ENaC, thereby preventing hypertension. Ubiquitin ligase NEDD4-2 98 The

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The GRK2 kinase phosphorylates and maintains ENaC in an active state, as it lowers ENaC sensitivity to NEDD4-2 [645] in addition to NEDD4 at multiple sites [646]. On the other hand, ERK1, ERK2, and CK2 phosphorylate ENaC, enhancing ENaC– NEDD4-2 interaction. PKC inhibits both ENaC gating and surface expression via ERK. On the other hand, CK1 or PKCδ favor ENaC insertion in the plasma membrane [647]. Action of ERK is countered by the adaptor TSC22D3 (or GILZ1), which represses the inhibitory cRaf–MAP2K1(2)–ERK1(2) pathway.102 The protein kinases SGK1103 and PKA phosphorylate NEDD4-2, a hub for aldosterone and vasopressin–cAMP signaling. In addition, IKKβ and PKB1 can also phosphorylate NEDD4-2 [644]. In addition, ENaC is phosphorylated and dephosphorylated. Epithelial Na+ transport is regulated by the volume-regulatory hormones aldosterone and vasopressin, atrial natriuretic peptide, insulin, and endothelin. These hormones regulate ENaC via kinases [647]: • • • •

Aldosterone stimulates SGK1 and inhibits ERK. Vasopressin and atrial natriuretic peptide operate via PKA. Insulin via PI3K. Endothelin via SRC family kinases.

In addition, elevated intracellular cAMP concentration favors dephosphorylation of the two ERK sites in ENaC, thereby reducing ENaC endocytosis, in addition to promoting its exocytosis from a recycling ENaC pool and plasmalemmal insertion [650]. Other kinases regulate ENaC activity; WNK1 activates SGK1; PKB also disrupts the ENaC–NEDD4-2 linkage in response to insulin, whereas AMPK promotes this binding; PKD promotes insertion of ENaC into the plasma membrane [647]. ubiquitinates Lys residues on the N-terminus of α- and γ ENaC subunits [644]. Serum and glucocorticoid kinase SGK1 counters NEDD4-2 action. 102 TSC22D3: TGFβ-stimulated clone 22 (TSC22) domain-containing protein-3. It protects T lymphocytes against IL2 deprivation-induced apoptosis via inhibition of FoxO3a transcriptional activity, thereby lowering expression of the proapoptotic factor BCL2L11 [108]. TSC22D3 regulates T-cell activation as it connects to cRaf and prevents its phosphorylation, hence suppressing MAP2K1/2–ERK1/2 signaling and AP1-dependent transcription [648]. In macrophages, it acts in the anti-inflammatory and immunosuppressive effects of IL10 and glucocorticoids. Isoforms TSC22D3-1 and TSC22D3-4 inhibit myogenic differentiation and mediate antimyogenic effects of glucocorticoids, as they bind to and regulate MyoD1 (bHLHc1) and HDAC1 transcriptional activity, reducing MyoG (bHLHc3) expression. Ubiquitous glucocorticoid-induced leucine zipper protein, which has splice variants (TSC22D3-1–TSC22D3-4, or GILZ1–GILZ4), interacts with numerous mediators of ion transport and cell differentiation, proliferation, transformation, and apoptosis [649]. It protects SGK1 from rapid ER-associated degradation, as ER-associated ubiquitin ligases STUB1 and synoviolin (Syvn1) quickly ubiquitinate SGK1 for subsequent proteasomal degradation. 103 Serum and glucocorticoid-induced kinase SGK1 modulates ENaC activity in the CCD, where it stimulates Na+ flux through ENaC. It phosphorylates various ENaC regulators that influence ENaC formation, transfer, and activity. It phosphorylates α subunit of ENaC and WNK4 [649]. Moreover, it regulates other carriers in the distal nephron such as sodium–chloride cotransporter.

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In the absence of aldosterone, ENaC activity is mainly limited by cRaf and NEDD4-2 [649]. Aldosterone acts via the MR and affects the transcription of specific genes that encode not only the α subunit of ENaC and Na+ –K+ ATPase but also SGK1, TSC22D3, and possibly Ki WNK1 and kRas, in addition to the TSC22D3 interactor CNKSR3 [649].104 The scaffold protein CNKSR3 may coordinate assembly of the ENaC regulatory complex, which promotes aldosterone signaling. Recruitment of SGK1 by TSC22D3 to the ENaC-regulatory complex protects SGK1 from rapid degradation. In this complex, SGK1 phosphorylates its substrates NEDD4-2 and cRaf and is protected against ubiquitin ligases STUB1 and synoviolin (Syvn1) [649]. Phosphatidylinositol (4,5)-bisphosphate and (3,4,5)-trisphosphate with basic cytoplasmic residues enhance channel activity. The P2Y receptors that activate phospholipase-C inhibit ENaC [636]. Channel palmitoylation activates it. Activity of ENaC is inhibited by elevated extracellular Na+ concentration (Na+ self-inhibition of ENaC) in addition to cytosolic Na+ concentrations (feedback inhibition of ENaC), which reduces the probability of channel opening and surface expression [636]. The ENaC channel exposed to tubular flow is mechanosensitive. It responds to increased tubular flow rate by an increase in Na+ absorption. Shear stress activates ENaC, whereas increases in distal tubular flow release cellular ATP, which can dampen ENaC activation via the P2Y2 receptor [636].

3.9.5.3

Aldosterone and Distal Nephron

Blood pressure depends on the vasomotor tone, renal perfusion, and fluid handling by the kidney. Reabsorption of filtered Na+ in the DCT and CnT and the CD of the nephron controls ECF volume and BP.

3.9.5.4

Aldosterone and Vasculature

Aldosterone targets MR in vascular endothelial and smooth muscle cells (Fig. 3.5). It renders endotheliocytes sensitive to augmented extracellular Na+ concentration via EnNaC, which differs from ENaC in its response to change in external Na+ concentrations. Endotheliocytes synthesize MR and 11β HSDH2 [636]. Synthesis and plasma membrane insertion of EnNaCs is boosted by aldosterone. Activity of EnNaC is controlled by an extracellular Na+ sensor. Sodium-induced EnNaC insertion in the plasma membrane is rapid owing to intracellular EnNaC-storage vesicles.

104 CNKSR:

connector enhancer of kinase suppressor of Ras. It is produced in large amounts in the CnT and CCD. Its synthesis is required for Na+ transport through ENaC in renal epithelioctes. This protein is involved in many hormone-induced scaffolding platforms.

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295 WSS

aldosterone mAChR Ca

actin

EC

NaC + Na

2+

DNA

MR

NOS3

IK, SK, NaC

N

EC

Ca NO

MEP

SK

IK

+ K hyperpolarization

2+

GJ K Ca V 1.2

MES BK

K

MEJ

+

IR

Na + + Na −K + ATPase Ca 2+

SMC

? MR sGC

DNA

Ca

V

1.2

Na

NaC

+

N

SMC

Fig. 3.5 Aldosterone and interacting vascular endothelial and smooth muscle cells (Source: [636]). Aldosterone binds to MR or NR3c2 in the cytosol, which then translocates to the nucleus. In endotheliocytes, MR induces the transcription of small- and intermediateconductance Ca2+ -activated K+ channels SK and IK, which enable an outward K+ current, creating hyperpolarization of endothelial plasma membrane. This hyperpolarization is transmitted to adjacent smooth myocytes through gap junctions (GJ) of the myoepithelial junction (MEJ) creating myoepithelial projections (MEP) of EC and SMC, separated by a tiny myoepithelial space (MES), and triggering relaxation. In addition, MR launches the production of amiloride-sensitive ENaC-like channel (EC NaC), which inserts in the apical plasma membrane. Sodium entry through EC NaC and the wall shear stress (WSS) exerted on it cause cortical stiffness, owing to actin polymerization. Aldosterone also causes inhibitory phosphorylation of nitric oxide synthase, NOS3. In vascular smooth myocytes, MR increases the transcription of the gene encoding the voltage-gated calcium channel (CaV 1.2) and decreases that of the gene encoding the bigconductance Ca2+ -activated channel (BK). The amiloride-sensitive ENaC-like channel SMC NaC participates in the myogenic tone and hence autoregulation. However, SMC NaC regulation by aldosterone is not yet proven

The endothelial glycocalyx, a negatively charged polymeric layer at the blood– endothelium interface, functions as an effective Na+ buffering compartment that limits access of Na+ into endotheliocytes and may modulate plasmalemmal EnNaC activity [636]. In fenestrated endothelia, capillary hydrostatic and osmotic pressures drive most of the net Na+ transport across the vascular wall via a paracellular route. EnNaC may also contribute, although it is involved in controlling the rheological properties of endotheliocytes (cortical stiffening) [636]. ENaC interacts with actin and other cytoskeletal proteins, such as spectrin or cortactin. In endotheliocytes, EnNaC activity may be related to its interaction with cortical actin filaments, thereby favoring gelification, which stiffens the cellular cortex, whereas fluidification (G actin) softens the cortex.

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Nitric oxide release declines when endotheliocytes stiffen upon aldosterone exposure [636]. In addition, Na+ entry through EnNaCs influences NOS3 activity, possibly via the PI3K–PKB pathway. Aldosterone participates in the regulation of the vasomotor tone and peripheral vascular resistance. It can prime vasoconstriction via endothelin-1, glucose 6-phosphate dehydrogenase, and rock kinase, and via NOS3 inhibitory phosphorylation. However, the acute response comprises not only vasoconstriction via some endothelial prostanoids and modified Ca2+ handling, but also short-term vasodilation via NO formation, in a given blood vessel, according to the dose, exposure time, and endothelium status [651]. Aldosterone acts on the endothelium, promoting NO release, and on the vascular smooth muscle, supporting vasoconstriction. Chronic response involves reactive oxygen radicals, endothelin, endothelial Na+ influx, and smooth myocytic calcium channel expression, in addition to transcriptional effects [651]. It engenders redox stress and endothelial dysfunction. Conversely, ET1 primes the secretion of aldosterone via ETA and ETB , both being coupled with the PLC–PKC pathway and ETB with PGhS, in addition to cortisol from adrenocortical cells [652]. In endotheliocytes, ET1 exposure and PKC inhibition downregulate NOS3 production. Vascular smooth myocytes that experience mechano-activated Na+ currents produce an ENaC-like channel that can be involved in mechano-sensing and control of peripheral vascular resistance [636]. However, a combination of ENaC and ASIC105 subunits that forms mechano-sensory channel complexes may be responsible for mechano-gated Na+ currents. Intracellular MR ligands 11β HSDH1 and 11β HSDH2 are also produced in vSMCs. In the small resistance arteries and arterioles of hypertensive rats, 11β HSDH activity diminishes. In human coronary artery smooth myocytes, glucocorticoids and mineralocorticoids upregulate the formation of vasoconstrictor receptors, such as that of angiotensin-2, increasing vasomotor tone [653]. Downregulated formation of ENaC β subunit provokes defective myogenic tone and hence renal blood flow autoregulation [636]. Moreover, baroreflex sensitivity is lost in ENaC β-subunit-deficient mice, loss of the myogenic constrictor response to augmented BP resulting from a loss of vSMC ENaC β activity.

3.9.6 Endothelin Hypertension is associated with impaired autoregulation of renal blood flow, to which contributes ENaC, and natriuresis. Salt-sensitive hypertension is linked to altered natriuresis and inflammatory infiltrates in the renal interstitium.

105 ASIC:

acid-sensing ion channel.

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297

Endothelin receptor ETA is proinflammatory and prohypertensive [636]. Angiotensin-2 counteracts the action of medullary ETB receptor, which increases natriuresis. Overactivity of the RAAA coupled with an ETA –ETB imbalance thus favors hypertension.

3.9.7 Galectin-3 In hypertension, galectin-3 is a marker for a bad prognosis (heart failure). In angiotensin-2-induced hypertension, galectin-3 favors cardiac inflammation, fibrosis, and left ventricular dysfunction [654]. In Agt2-induced hypertension, deletion of the GAL3 gene (or LGALS3)106 precludes left ventricular dysfunction, inflammation, and fibrosis but affects neither BP nor LVH.

3.9.8 Prolactin Prolactin is a proangiogenic hormone that is cleaved into a short antiangiogenic form (PrlS ) by cathepsin-D and matrix metallopeptidases. An elevated concentration of the cleaved form is linked to preeclampsia. Concentration of prolactin in plasma, urine, and amniotic fluids is markedly higher in patients with preeclampsia than in normally pregnant women [655]. In humans, a loss-of-function SNP in the PRLHR gene107 is associated with reduced BP. Prolactin, which is secreted by the pituitary gland, elicits lactogenesis. It is also implicated in water and salt balance, cell proliferation and differentiation, hematopoiesis, adipogenesis, functioning of pancreatic β and testicular Leydig cells, and adaptive immunity. Its secretion is hampered by dopamine and launched by prolactin-releasing peptide synthesized in the hypothalamus. Full-length prolactin (PrlFL ) increases NO production via PrlR receptor, as it raises cytosolic calcium concentration in mammary epitheliocytes, supports activatory phosphorylation by PKB (Ser1177) of NOS3, and forms carboxypeptidase-D, which releases the NOS substrate arginine from polypeptides, thereby causing vasodilation [655]. On the other hand, short prolactin (PrlS ), which operates via PAI1 and NOS2, counters NOS3. In mice that produce prolactin in the liver, the single-copy transgene being inserted at the Hprt locus108 and driven by the indole 3-carbinol-inducible rat cytochrome-P450-1A1 promoter, feeding with the indole 3-carbinol found in broccoli and similar vegetables, provokes hypertension [655]. Urinary excretion of nitrite and nitrate in addition to the amount of phosphorylated NOS3 (Ser1177) augment in normally fed modified mice, but not in those fed with indole 3-carbinol. These effects are mediated by prolactin short form. 106 LGalS3:

lectin, galactoside-binding, soluble, protein-3. prolactin-releasing hormone receptor (or GPR10). 108 HPRT: hypoxanthine–guanine phosphoribosyltransferase. 107 PrlHR:

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3.9.9 20-Hydroxyeicosatetraenoic Acid 20-Hydroxyeicosatetraenoic acid is a major metabolite of arachidonic acid produced by enzymes of CYP4A and CYP4F families in the heart, blood vessels, kidney, lung,109 among other tissues.110 It activates the G-protein-coupled receptor GPR75 and signals via the Gq/11–PLC–IP3 /DAG–PKC and Src–EFGR pathways. The GPR75 receptor is also targeted by the chemokine CCL5 [656].111 The intracellular second messenger 20HETE and hence GPR75 participate in the regulation of the vasomotor tone, natriuresis, cell proliferation, and angiogenesis, in addition to redox stress, inflammation, and vascular remodeling. The GPR75 receptor tethered to CCL5 also stimulates insulin secretion in pancreatic islet β cells [657].112 Its vasoconstrictory and natriuretic actions rely on the PLC– PKC axis and its effects on cell migration and proliferation, endothelial function, and inflammation on Src and the MAPK module. Increased concentration of the vasoconstrictor 20HETE is associated with vasospasm, hypertension, vascular restenosis, myocardial infarction, and stroke [656]. In the proximal tubule and thick ascending loop of Henle, 20HETE impedes sodium reabsorption. In the proximal tubule, 20HETE inhibits via the PKC sodium– hydrogen exchanger (antiporter) NHE3 (SLC9a3) and Na+ –K+ ATPase [656]. In the thick ascending loop, 20HETE inhibits via PKC NKCC2, ROMK, and basolateral Na+ –K+ ATPase via PKC (Sect. 3.7.3.5). On the other hand, GPR75 is synthesized at a relatively low level in the kidney. Hence, activation by Src of these channels is restricted. In human endotheliocytes, the 20HETE–GPR75 couple primes dissociation of the Gq/11 subunit and association with GRP75 of GIT1,113 thereby unbinding GIT1 from Src, which transactivates EGFR [657]. Once it is phosphorylated by Src, EGFR activates the MAPK–IKKβ–NFκB pathway, thereby upregulating the production of inflammatory cytokines and angiotensin convertase. 20HETE also uncouples NOS3 via HSP90.

109 20HETE,

one of the main eicosanoids produced by cytochrome-P450 (CyP) enzymes (the product of ω-hydroxylation of arachidonic acid by CyP4a and CyP4f isozymes), dilates both bronchiolar and pulmonary arterial smooth myocytes. On the other hand, it primes smooth myocyte contraction, migration, and proliferation, in addition to endotheliocyte inflammation and dysfunction. 110 Mutations in the CYP4A11 and CYP4F2 genes are associated with hypertension [656]. The CYP4A enzymes are observed in pial vessels, astrocytes, and hippocampal neurons. GPR75 abounds in the brain. 111 The CCL5–GPR75 couple ensures neuroprotection [656]. GPR75 is predominantly expressed in cells surrounding retinal arterioles and in other regions of the brain but is detected in most human organs (e.g., in the brain, heart, and kidney) [657]. The chemokine CCL5 may facilitate, amplify, or hinder the binding of 20HETE to GPR75 [657]. 112 Isoforms or splice variants of GPR75 may exist in different cell types [656]. The chemokine CCL5 fails to dissociate Gq/11 from GPR75 in ECs [657]. 113 G1T1: GPCR kinase-interacting protein-1.

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299

In rat aortic smooth myocytes, the 20HETE–GPR75 pair, which is also associated with Gq/11 and GIT1, elicits dissociation of the Gq/11 subunit, thereby activating the PLC–IP3 /DAG–Ca2+ /PKC axis and triggering vasoconstriction [657]. In addition, PKCα cooperates with Src and enhances phosphorylation (inhibition) of the calcium-activated potassium BK channel β subunit. Because 20HETE raises and lowers the activity of TRPC6 and BK, it depolarizes the plasma membrane and promotes calcium entry through voltage-gated Ca2+ channels.

3.9.10 Membrane Depolarization-Limited Vasoconstriction Smooth myocyte contraction in small arteries and arterioles regulates regional blood flow distribution. It is elicited by plasma membrane depolarization in arterial and arteriolar smooth myocytes that stimulate voltage-gated Ca2+ channel CaV 1.2b and hence the influx of Ca2+ ions. On the other hand, relaxation of arterial and arteriolar smooth myocytes from the constricted state is primed by Ca2+ -activated K+ channel BK (KCa 1.1), which exports K+ ions and repolarizes the plasma membrane. However, membrane depolarization triggers a signaling cascade that activates BK channel [658]. Membrane depolarization and subsequent calcium influx through CaV 1.2b channel activate the rock1 and rock2 kinases that activate Rab11a, thereby boosting delivery by Rab11a+ recycling endosomes of the β1 auxiliary subunit of the BK channel to the plasma membrane, where it connects to the pore-forming α subunit to increase the calcium sensitivity of the BK channel. Activation of the BK channel in contracted arterial and arteriolar smooth myocytes counters local vasoconstriction by vasodilation.

3.10 Hypertension, Cerebrovascular Disease, and Therapy Therapy is aimed at lowering SBP, especially in cerebrovascular disease. Untreated or uncontrolled hypertension leads to cerebrovascular accidents (stroke), myocardial ischemia and infarction, cardiac arrhythmias, and CHF. Early detection of hypertension and treatment reduce cardiovascular complications, especially preventing the occurrence of stroke and myocardial infarction. However, in hypertension linked to aging, current treatments are palliative, but not curative. In aged mice, increased expression of thioredoxin, which scavenges free radicals, reduces hypertension; this effect lasts for at least 20 days [659]. Overexpression of thioredoxin decreases arterial stiffness, promotes endotheliumdependent relaxation, increases NO production, and lowers superoxide anion formation. Cerebral vascular resistance is a significant predictor of hypertension [598]. Cerebral hypoperfusion in the posterior cerebral circulation due to congenital

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variants with anatomical incompleteness of the circle of Willis114 and vertebral artery hypoplasia can trigger EHT. Lowering BP can worsen cerebral perfusion in susceptible individuals. The brainstem of hypertensive rats is hypoxic and becomes even more hypoxic when hypertensive treatment is administered. Therapeutic adverse renal effects (e.g., acute renal injury and failure) can be more common in persons receiving an intensive treatment [660]. Incident chronic kidney disease (i.e., a decrease in estimated glomerular filtration rate ≥30%) occurs more frequently upon high-dose treatment, that is, a rapid decline in the renal function is observed in patients among the lower BP group compared with that of persons in the higher BP group, in the first year only [661]. Fast decrease is associated with a higher risk for stroke. Therefore, in patients with a previous lacunar stroke and relatively preserved kidney function, a strong antihypertensive treatment is related to a greater likelihood of renal dysfunction, especially during the first year of treatment.

114 Hypoplasia

of the posterior communicating arteries is frequent in men.

Chapter 4

Hyperglycemia and Diabetes

Defective control of glucose homeostasis by the brain, pancreas, liver, adipose tissue, and skeletal muscle engenders type-2 diabetes mellitus (T2DM).1 Impaired glucose tolerance and overnight fast hyperglycemia increase the risk for T2DM (Vol. 12, Chap. 3. Metabolic Syndrome). Prediabetes is defined by concentrations of glycated hemoglobin (HbA1c)2 ranging from 5.7 to less than 6.5% and fasting glucose from 100 to less than 126 mg/dl. It is associated with an increased risk for T2DM [662]. The highest prevalence rates are observed in aged people (≥65 years; 54.6%) and in men (39.1%) rather than women (33.8%). In addition to other complications of diabetes, the risk for adverse cardiovascular events is two- to threefold higher in people with diabetes [30]. The risk is higher in women. Diabetes causes vascular endothelial dysfunction. Early detection and treatment of T2DM prevent cerebro- and cardiovascular events.

1 From the Greek διαβαδιζω, go across, and διαβαινω, walk or stride (i.e., walk with long, decisive

steps), neither διαβ της (official) nor διαβητης (compass), as diabetic patients present polyuria (i.e., pass water such as in a siphon [from Latin sipho], a tube (σιϕων) immersed in a reservoir at a given level with the other end outside the tank below this level conveying a liquid upward from the reservoir after transiently forcing it into the tube [typically by suction], liquid flowing unaided, the atmospheric pressure driving the liquid through the pipe out of the tank), and from the Latin mellitus, of honey, sweet with honey. Diabetes insipidus (from Latin insipidus: insipid) is due to an impaired secretion of or response to the pituitary hormone vasopressin. Diabetes mellitus results from a defective production (type-1 diabetes mellitus [T1DM]) or response (type-2 diabetes mellitus [T2DM]) to the pancreatic hormone insulin. As the present text refers mainly to T2DM, the disease can be simply named diabetes. 2 Measurement of HbA1c enables estimation of the average glycemia over the duration of red blood capsule survival (8–12 weeks). The amount of glucose attached to hemoglobin is proportional to the concentration of glucose in the bloodstream. © Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0_4

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In the heart, prolonged hyperglycemia provokes chamber dilation with thinning of the left ventricular wall and myocyte loss [663]. In cardiomyocytes, Ca2+ handling relies on a set of cytosolic proteic buffers and membrane carriers (e.g., CaV 1.2, NCX, PMCA, SERCA, RyR, and Mt CU). Calcium ion is released from intracellular stores and is imported across the plasma membrane. Store-operated Ca2+ entry depends on stim1–orai1 and the cation transient receptor potential channels, TRPC1, which is activated by stim1, TRPC4, and TRPC5; diacylglycerol-activated TRPC3 and TRPC6 in addition to TRPC7 can also participate in SOCE [664].3 Cardiomyocytes have a defective Ca2+ signaling with an augmented spontaneous 2+ Ca release from the endoplasmic reticulum and reduced cytoplasmic Ca2+ clearance and ER Ca2+ load (negative ino- and lusitropic effects).

4.1 Epidemiology From 1980 to 2012, the prevalence of diabetes mellitus increased significantly and remained stable at 8.3 per 100 persons, whatever the age, sex, ethnic group, and education category [662, 665]. However, the proportion of diabetic patients increases mainly among overweight and obese subjects (85.2% with T2DM).

3 TRPC3, TRPC4, and TRPC6 contribute to SOCE involved in adverse cardiomyocyte hypertrophy.

Transient hypoxia, ischemia, and hence infarction damage cells, especially after reperfusion due to Ca2+ overload, which causes cell death. Calcium enters the mitochondrial matrix through Ca2+ uniporter (Mt CU) and TRPC3 in the inner mitochondrial membrane. Myocardial ischemia–reperfusion (I/R) injury is characterized by inflammation, reactive oxygen species (ROS) production, Ca2+ entry, at least partly through TRPCs, mainly TRPC3 and TRPC6 along with TRPC7, and apoptosis [664]. Reactive oxygen species formed by plasmalemmal NOx2, which can be activated by TRPC3 stretched upon cell swelling, can activate BAD, which then binds to BCL2, releasing the proapoptotic BAX protein, which oligomerize and insert into the mitochondrial outer membrane, where they form the BAK–BAX mitochondrial apoptosis channel (MAC). Therefore, ROS are synthesized in the mitochondrion by the uncoupling of the respiratory chain linked to Ca2+ overload and at the plasma membrane by NOx2 due to membrane stretching. In ischemic cardiomyocytes, aberrant intracellular Ca2+ concentration is taken up by mitochondria. Its accumulation in mitochondria uncouples the respiratory chain, generates more ROS by mitochondrial NOx4, and stimulates assembly of the mitochondrial permeability transition pore in the presence of matrix cyclophilin-D by aggregated VDACs in the OMM and ANTs in the IMM, thereby provoking necrosis. Moreover, a positive feedback loop is triggered by Ca2+ , which enters through TRPCs, as it activates the Cam–PP3–NFATc3 axis, which regulates transcription of the Trpc genes, further raising Ca2+ influx. On the other hand, activation of TRPC6 during hypoxia activates PI3K via Ca2+ –Cam. Stimulation of PI3K in I/R events is further enhanced by a fall in transcription of the PIK3IP1 gene encoding PI3K-interacting protein PI3KIP1, a glycosylated membrane inhibitor of PI3K [664]. PI3K phosphorylates antiapoptotic PKB, which phosphorylates BAD (Ser155), preventing it from sequestering BCL2 away from BAX, in addition to BAX (Ser184), increasing its affinity for BCL2, impeding BAX oligomerization and assembly into the MAC channel [664].

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303

Improved economic conditions, better living standards, and adoption of the adverse lifestyle habits of wealthier nations such as China raised the T2DM prevalence from 2.3% in 1994 to 9.7% in 2008 [665]. The same observations can be made in populations of West Africa and India. The highest T2DM prevalence is observed in Oceania, North Africa, the Middle East, and Caribbean (age-standardized prevalence 21–25% in men and 21–32% in women [665]). Pacific Islanders, South Asians, and Filipinos have the highest T2DM prevalence (18.3, 15.9, and 16.1%, respectively) among ethnic groups, including minorities at a high risk such as Blacks, Latinos, and Native Americans [662].

4.2 Diabetes Mellitus and Epigenome The genetic basis of T2DM is related to chromosomal sites carrying genetic variants that collectively explain about 10% of the variation in the predisposition for T2DM [666]. Epigenetic changes, transient or not, result from DNA methylation and histone positioning and post-translational modifications that affect chromatin structure and access to genes. DNA methylation modulates the binding capacity of DNA-binding regulators of gene transcription. Histone types and modifications regulate the binding of chromatin modifiers that relax or compact DNA. Epigenetic changes can influence metabolism and can be heritable, with possible transgenerational epigenetic inheritance; ancestral early life exposures to stressors can have a transgenerational effect on health in descendants.4 However, the patterns of DNA methylation and histone acetylation can change with aging [666]. This evolving state can explain variations in the onset of agingrelated diseases. MicroRNAs regulate mRNA translation and thus also govern gene expression but at the post-translational level.

4 Male

offspring of female rats exposed prenatally to dexamethasone, a model of fetal exposure to glucocorticoid stress hormones, have a small birth weight, glucose intolerance, and elevated hepatic phosphoenolpyruvate carboxykinase activity [666].

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Exercise5 and diet6 can modify the epigenome, and these changes can be inherited. Epigenetic modifications can also arise from undernutrition,7 obesity, and toxins.8 Tripartite motif-containing protein, TriM28, launches epigenetic changes that suppress expression of a cluster of genes that are strongly active in the hypothalamus and adipose tissue, hence favoring obesity [666]. Epigenetic modifications are implicated in insulin sensitivity and pancreatic βcell function and hence T2DM [666]. In insulin-sensitive organs, such as the liver, skeletal muscle, and adipose tissue, epigenetic modifications, especially those related to genes that control glucose and lipid metabolism, can contribute to T2DM genesis. Promoters of many genes are differentially methylated in subcutaneous adipose tissue (ADCY5, CAV1, CDKN2A/2B, CIDEC, DUSP9, HNF4A, IDE, IRS1, KCNQ1, MTNR1B, TSPAN8, and WFS1) and skeletal muscle (CDKN2A, DUSP9, HNF4A, HHEX, KCNQ1, KLF11, PPARGC1A, and SLC30A8) in human young and old T2DM-discordant twin pairs [666].

5 Epigenetic

factors participate in the regulation of gene expression both after acute and prolonged exercise. Acute exercise can activate the MAPK, AMPK, and CamK2 kinases that phosphorylate and dissociate specific HDAC isoforms from the transcription factor MEF2, thereby removing the transcriptional repression and launching GluT4 synthesis. Acute exercise in healthy men and women reduces DNA methylation in the skeletal muscle in addition to hypomethylation of the PDK4, PPARD, and PPARGC1A gene promoters, promoting transcription [666]. Moderate exercise during pregnancy reduces the risk of obesity in the progeny during childhood and preadolescence. In animals, regular exercise in the mother or father confers a health benefit on the first generation [666]. In female mice, a high-fat diet (HFD) hypermethylates the PPARGC1A promoter, thereby downregulating mRNA expression in skeletal muscle in offspring. The transcriptional coactivator PGC1α operates in mitochondrial genesis and oxidative metabolism. On the other hand, maternal exercise prevents PPARGC1A hypermethylation transmitted by HFDfed females. Exercise-trained males yield another source of metabolic trait inheritance. Sedentary HFD-fed male mice have offspring that are metabolically less efficient and are at a decreased risk for insulin resistance and obesity. 6 Progenies of rats exposed to a low-calorie diet have a higher susceptibility to diabetes than control rats and perturbed epigenetic markers in the promoter of the insulin gene [666]. In offspring of male mice fed a low-protein diet, hepatic expression of genes involved in lipid and cholesterol synthesis increases, and levels of cholesterol and cholesterol esters decrease. Maternal or paternal exposure to a HFD leads to insulin resistance in offspring [666]. 7 Malnutrition in utero and after birth (i.e., famine during life from embryo- and fetogenesis to early childhood and adolescence) leads to metabolic disorders and cardiovascular diseases in adulthood (thrifty phenotype). The timing of the famine, particularly when it occurs during late gestation, alters glucose tolerance in adulthood and in the progeny, as observed by studies of the Austrian (in 1918–1919, 1938, and 1946–1947), Ukrainian (in 1932–1933), Dutch (in 1944), and Chinese famines (in 1959–1961). 8 Ancestral exposure of female rats to the insecticide dichlorodiphenyltrichloroethane causes kidney, prostate, and ovary disease in addition to tumorigenesis in generation G1 adults and obesity in G3 adult male and female rats.

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On the other hand, hyperinsulinemia, -glycemia, -lipidemia, and -cytokinemia can affect the epigenome.9 In healthy men, a 5-day HFD changes the skeletal muscle epigenome, especially at the CDKN2A, CDKN2B, PDX1, PKB2, PPARG, and SLC30A8 genes. These modifications are partly reversible, as, after 6–8 weeks of a controlled diet, DNA methylation is partly removed.

4.3 Vascular Complications in Diabetes Mellitus Unlike microvascular complications specific to diabetes, which engenders retinopathy, nephropathy, and neuropathy10 (Vol. 12, Chap. 3. Metabolic Syndrome), cardiomacrovascular disorders in diabetic patients are similar to those in nondiabetic people. In fact, the risk for coronary atherosclerosis is about 40% lower in diabetic patients and without previous myocardial infarction than in patients with an antecedent of myocardial infarction, but without diabetes [665]. Diabetes worsens the stroke outcomes. Whereas T1DM is associated with an increased risk for both ischemic and hemorrhagic strokes, T2DM raises the risk of ischemic but not hemorrhagic stroke [665]. Diabetes increases two- to fourfold the risk of developing peripheral artery disease [665]. Diabetic patients have additional conditions that exacerbate complications of limb vascular insufficiency, such as neuropathy and altered foot mechanics. Diabetes may also increase the risk for venous thromboembolism via its effect on platelets and components of the coagulation cascade. Diabetes is not only associated with an expansion and activation of myeloid cells, particularly monocytes and neutrophils, thereby favoring atherosclerosis, but also with an elevated platelet count. Mean platelet volume is higher in diabetic than

9 Relatively

short (48 h) exposure of cultured human skeletal myocytes to elevated lipid concentrations or to tumor-necrosis factor, TNFSF1, but neither insulin nor glucose, provokes methylation of the PPARGC1A gene promoter [666]. Palmitate treatment (duration 48 h) alters the DNA methylation of genes implicated in T2DM (GLIS3, HNF1B, SLC30A8, and TCF7L2) in pancreatic islets. 10 Endothelial dysfunction and microangiopathy, which is characterized by vascular wall thickening and augmented vasoconstriction, contribute to the genesis of diabetic neuropathy, which is associated with elevated vasa nervorum density and permeability (defective blood–nerve barrier) in addition to inflammation. The morphogen desert hedgehog, which is mainly produced in peripheral nerves by myelinating Schwann cells, is implicated in the development of diabetic neuropathy [667]. DHh contributes to organization of nerve sheaths (i.e., epi-, peri-, and endoneurium) via signaling to perineurial cells. During organogenesis, DHh is synthesized in the endothelium of large vessels. In adults, the Hh pathway is reactivated after ischemic injury. In particular, DHh is involved in ischemia-induced angiogenesis. In endotheliocytes of endoneurial capillaries, DHh influences nerve functioning. Smoothened promotes claudin-5 expression, hence controlling vascular permeability.

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in nondiabetic subjects, signifying the existence of younger reticulated platelets. Immature reticulated platelets are more reactive than mature platelets.11 Hyperglycemia favors interaction of neutrophil-derived calcium-binding proteins S100a8 and S100a9 with receptor for advanced glycation end products (AGEs) on Kupffer cells,12 thereby raising interleukin-6 production,13 which is implicated in inflammatory thrombocytosis [668].14 In diabetic mice, the population of Kupffer cells increases, and a greater proportion of these hepatic macrophages produce IL6, which increases thrombopoietin production in hepatocytes.15 Thrombopoietin connects to its cognate receptor TPoR on megakaryocytes and bone marrow progenitor cells, thereby launching their proliferation and causing reticulated thrombocytosis. Lowering glycemia using a sodium–glucose cotransporter SGlT2 (SLC5a2) inhibitor, depleting neutrophils or Kupffer cells, or inhibiting S100a8/a9 binding to RAGE reduces diabetes-induced thrombocytosis. People with T2DM and peripheral vascular disease taking aspirin have higher densities of reticulated platelets and monocyte–platelet aggregates as well as concentrations of S100a8 and S100a9 than those in controls taking aspirin. Reticulated thrombocytosis correlates with glycated hemoglobin in addition to elevated plasmatic concentrations of S100a8 and S100a9 [668]. On the other hand, the arterial baroreflex control of muscular sympathetic nerve activity16 is preserved in diabetic patients with respect to obese (weight-matched

11 Reticulated

platelets due to elevated thrombopoiesis are engendered from fragmentation of long megakaryocytic protrusions. They are involved not only in thrombosis, but also in atherogenesis. Upon administration in mice of streptozotocin, a toxic for pancreatic β cells, the density of reticulated platelets, the number of platelet–leukocyte aggregates, and the rate of leukocyte activation and proliferation rise [668]. 12 RAGE is a promiscuous receptor that links to AGEs, HMGb1, S100a8, and S100a9. The RAGE ligands also signal via TLR4 and ScaRb3 [668]. Both S100a8 and S100a9 are potent RAGE ligands in hyperglycemia that tether to TLR4 in obesity. They are mainly produced by neutrophils, particularly in diabetes. Neutrophils rely on glycolysis and thus glycemia and glucose carriers. Normalizing glycemia with a sodium–glucose cotransporter SGlT2 (SLC5a2) inhibitor prevents accumulation of ROS by stressed neutrophils. 13 Obese mice have a higher IL6 production by Kupffer cells (hepatic macrophages) and density of reticulated platelets than lean mice. 14 Blockage of S100a8 and S100a9 attenuates the density of Kupffer cells in addition to atherosclerotic lesion size [668]. 15 Thrombopoietin is constitutively produced from bone marrow stromal and renal cells, but the predominant source is the liver. Diabetes-induced hepatic inflammation favors hepatic TPo production via IL6 synthesized in Kupffer cells, thus promoting thrombopoiesis within the bone marrow and hence reticulated thrombocytosis. 16 The muscle metabo-reflex and arterial baroreflex cooperate to regulate arterial pressure during exercise. Whereas the former mainly increases cardiac output via positive chronotropic and inotropic effects, the latter causes peripheral vasoconstriction including within active skeletal muscles during coactivation of these two reflexes, but to a lesser extent than in the other vasculature compartments, and hence a blood flow redistribution toward muscles, further elevating mean arterial pressure [669].

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controls) and lean age-matched individuals, whereas that of cardiac sympathetic nerve activity is impaired likely because of obesity [670]. Reactive oxygen species generated during hyperglycemia promote phosphorylation of VEGFR2 by SRC family kinases within the Golgi body, progressively diminishing VEGFR2 availability at the plasma membrane and thus limiting angiogenesis associated with diabetic endothelial dysfunction [671]. The widespread adaptor SHC1,17 a redox stress protein as a regulator of ROS formation, stimulates miR34a expression in the diabetic endothelium using an oxidantsensitive mechanism that causes endothelial dysfunction via SIRT1 [672]. In diabetic mice, production of miR34a is upregulated and sirtuin-1 is downregulated. Administration of miR34a inhibitor or endothelium-specific ablation of miR34a prevents downregulation of aortic SIRT1 and rescues impaired endothelium-dependent aortic vasodilation, which is altered by hyperglycemia. On the other hand, hyperglycemia and free palmitate provoke redox stress in endotheliocytes owing to an increased formation of hydrogen peroxide primed by SHC1, and upregulates miR34a and downregulates SIRT1 expression. The protein SHC1 is acetylated upon exposure to high glucose levels and deacetylated (Lys81) by sirtuin-1 [673]. Acetylated SHC1 favors redox stress and, in the vasculature, endothelial dysfunction. Its acetylation facilitates its phosphorylation (Ser36) and translocation to the mitochondrion, where it promotes hydrogen peroxide production via oxidizing cytochrome-C. In addition, SHC1 activates PKCβ2,18 which in turn phosphorylates SHC1, creating a positive feedback loop [673].

4.4 Pathophysiology Diabetes is linked to accelerated atherogenesis in addition to cardiomyopathy and diastolic and systolic cardiac failure with preserved and reduced ejection fraction, respectively [675]. As in arterial smooth myocytes (SMCs) from diabetic subjects, arterial myocytes exposed to sustained elevated extracellular glucose concentration provoke CaV 1.2 hyperactivity,19 elevated cytosolic Ca2+ concentration, and vasoconstriction [678].

17 SHC1:

66-kDa Src homology-2 domain-containing protein-1. cardiac dysfunction, activity of PKCa, PKCβ1, and/or PKCβ2 augments in response to pressure overload and ischemia. PKCα is the major classical isozyme in the adult mammalian heart, where it impedes contractility. On the other hand, activated PKCβ2 can preserve CMC contractility [674]. 19 Long-lasting (L-type) calcium channel belongs to the high-voltage activated group. The Ca 1.2 V subtype mediates Ca2+ influx into the cytosol in response to strong membrane depolarization. It is involved in cardiac and smooth muscle contractility and neuroendocrine regulation, among multiple other functions [676]. The calcium channel containing the α1c subunit encoded by the CACNA1C gene operates in excitation–contraction coupling in the heart. CaV 1.2 participates not 18 In

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Clusters of CaV 1.2 and PKA are observed in arterial myocytes. The PKA– AKAP5 axis induces phosphorylation of CaV 1.2 (Ser1928 of α1c subunit) in response to various stimuli such as β-adrenergic signaling, thereby enhancing its activity. Glucose may stimulate the Gs–AC–cAMP–PKA axis.20 In addition, calcium influx through CaV 1.2 activates AKAP5-anchored PP3 and hence the PP3– NFAT3 pathway, thereby reducing amounts of β1 subunit of BK and KV 2.1 and subsequently strengthening vasoconstriction in diabetic subjects.

4.5 Insulin Insulin21 is produced by β cells of the pancreatic islets.22 Its release is controlled by calcium ions and phosphoinositides. The pancreas secretes a basal amount of insulin during fasting. Eating triggers via glucose a rapid transient increase in insulin release.

only in excitation–contraction, but also excitation–secretion and excitation–transcription coupling linked to muscle contraction, hormone secretion, and neuronal transmission. In the brain, CaV 1.2 and CaV 1.3 are implicated in the synaptic transmission of auditory stimuli and synaptic remodeling linked to learning and memory [677]. The CaV 1.2 channel contains up to four subunits, which exist in various splice variants that affect the biophysical properties and biological functions of the channel. The four subunits include the α1 subunit (with isotypes encoded by the CACNA1A– CACNA1I and CACNA1S genes, α1s/c/d/f subunits making the CaV 1.1 to CaV 1.4 channels, α1a/b/e subunits the CaV 2.1 to CaV 2.3 channels, and α1g/h/i subunits the CaV 3.1 to CaV 3.3 channels), which shapes the Ca2+ selective pore and contains the voltage sensor and binding sites for most regulators, and the accessory subunits α2δ (with isotypes encoded by the CACNA2D1 to CACNA2D4 genes), β (with isotypes encoded by the CACNB1 to CACNB4 genes), and γ (with isotypes encoded by the CACNG1 and CACNG6 genes), which have anchorage, transfer, and regulatory functions [676]. CaV 1.2 is composed of α1, α2δ, and β subunits, whereas CaV 1.1 possesses the additional γ1 subunit. The human CACNA1C gene contains 50 exons; at least 20 exons undergo alternative splicing. Exon 1A (long N-terminus; CaV 1.2a) is expressed in cardiomyocytes, exon-1B (short N-terminus; CaV 1.2b) in smooth muscle and brain, and exon 1C (smallest N-terminus; CaV 1.2c) in resistance cerebral arteries [676]. Blood vessels experience both phasic and tonic contractions. Portal venous smooth myocytes (SMCs) are prototypical phasic SMCs, whereas small arteries exhibit a mixture of tonic and phasic contractions. 20 However, activated PKA also operates in arterial myocyte relaxation in response to vasodilators, but the action of PKA depends on its subcellular compartmentation and its recruitment by scaffold proteins such as AKAPs. Activation of a different PKA pool phosphorylates K+ channels in response to β-adrenoceptor excitation and causes membrane hyperpolarization and vasodilation. In addition, CaV 1.2 can also be phosphorylated at Ser1928 by PKC, increasing its activity in arterial myocytes. Angiotensin-2 binds to its Gq-coupled AT1 receptor, then stimulating PLC, generating DAG, and activating PKC. AKAP5 also connects to AT1 , enabling CaV 1.2 gating. 21 From Latin insula: island, isle. 22 Islets of Langerhans represent clusters of endocrine cells embedded in the exocrine pancreatic gland.

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4.5.1 Insulin Secretion Insulin is released in two phases, a first rapid burst being followed by a slow release in spurts. During the rapid release phase, insulin-containing granules docked at the plasma membrane release their material. In the second slow release phase, granules move to the plasma membrane and then secrete their content in a series of bursts, each being triggered by a spike cytosolic Ca2+ pulse. The second phase is impaired in type-2 diabetic patients. Transmembrane protein TMEM2423 regulates the pulsatility of cytosolic Ca2+ and phosphoinositide signaling and hence pulsatile insulin secretion during the slow release phase [679]. It is phosphorylated (inhibited) in response to elevated cytosolic Ca2+ concentration, thereby transiently dissociating from the plasma membrane, and then reassociates with this membrane upon dephosphorylation.

4.5.2 Insulin Effects Transiently elevated concentrations of insulin upregulate transcription of some genes and, less rapidly, downregulate that of other genes, but insulin concentration repressing gene expression is lower than that stimulating insulin-sensitive gene transcription [680]. Gene expression inhibition occurs before transcription, and the transcript degradation rate is higher than that of mRNA produced by insulinstimulated genes. Insulin controls carbohydrate and lipid metabolism and influences protein and mineral metabolism. It primes the assimilation of nutrients by cells. It participates in the regulation of nutrients storage and organizes their processing.

23 Also

known as C2 domain-containing protein C2CD2-like protein (C2CD2L). This type-I I transmembrane protein possesses a N-terminal short cytoplasmic region, single transmembrane domain, and C-terminal large extracellular domain. It abounds in neuroendocrine cells, in which it may be required for secretion. It is a lipid transport protein that concentrates at contact sites between the endoplasmic reticulum and plasma membrane, where it tethers both membranes. It helps to deliver phosphoinositides synthesized in the endoplasmic reticulum to the plasma membrane, thereby replenishing pools of this lipid converted to phosphatidylinositol (4,5)-bisphosphate during glucose-stimulated signaling and Ca2+ -dependent exocytosis. It also controls the activity of plasmalemmal Ca2+ channels. The other member of the C2CD2 set, C2CD2, corresponds to TMEM24L. C2CD3 is a component of the centriole that promotes centriole elongation in addition to assembly of centriolar distal appendage, which anchors the cilium. It is required not only for primary cilium formation, but also for sonic hedgehog signaling and Gli3 proteolysis [108]. C2CD4a and C2CD4b may regulate cell adhesion and be involved in inflammation. C2CD5 intervenes in insulin-stimulated glucose transport and SLC2a4 (GluT4) translocation from intracellular glucose storage vesicle to the plasma membrane in adipocytes.

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Table 4.1 Insulin effects (Source: [683]; ↑ increase, ↓ decrease, ⊕ −→ stimulation, −→ inhibition) Carbohydrate metabolism

Lipid metabolism

Protein metabolism

↑ glucose ingress ↑ glycolysis rate (↑ hexokinase and phosphofructokinase activity) ⊕ −→ glycogenesis −→ glycogenolysis −→ gluconeogenesis −→ lipolysis ⊕ −→ fatty acid and triacylglycerol synthesis ↑ uptake of triglycerides from blood ↓ rate of fatty acid oxidation ↑ rate of transport of some amino acids ⊕ −→ protein synthesis −→ proteolysis

• Insulin activates anabolism during the fed state (i.e., when nutrients are digested, absorbed, and delivered to organs from the intestinal tract). • Insulin manages nutrient distribution in the fasted state (i.e., when the intestinal tract stops being a source of nutrients), fatty acids and glycerol being released from the adipose tissue, amino acids from the skeletal muscle, and glucose from the liver [681]. Insulin regulates glucose metabolism, fatty acid uptake and synthesis, amino acid uptake and protein synthesis, and cholesterol synthesis. Insulin thus has two types of action [682]: (1) excitatory (autacoid),24 as it stimulates glucose uptake, especially in skeletal myocytes, hepatocytes, and adipocytes, and glycogenesis in addition to lipogenesis from glucose, and (2) inhibitory (chalone),25 as it inhibits lipolysis, proteolysis, glycogenolysis, gluconeogenesis, and ketogenesis (Table 4.1). Insulin signals primarily in the liver, muscle, and adipose tissue, where it precludes glucose production and elicits glucose uptake. The brain is another insulin-sensitive organ that regulates food intake in addition to glucose and lipid metabolism. 24 αυτoς:

same, self; ακoς: cure, remedy. Autacoid is a local regulator formed, acting, and processed locally. Once it is released from cells in response to various types of stimuli, it acts briefly near its site of synthesis. However, large amounts can be produced and carried by blood or lymph and can then have remote effects. E.A. Sharpey-Schäfer in his book “An Introduction to the Study of the Endocrine Glands and Internal Secretions,” published in 1914 by Stanford University Press, introduced the word autacoid to describe substances formed by cells and passed from them into the blood circulation, the action of which resembles that of drugs, excitatory autacoids being termed simply autacoids or hormones and restraining or inhibiting autacoids called chalones. 25 καλαϕoς: like the (hormone). Chalone is a secreted hormone-like substance that inhibits a physiological process.

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Insulin operates in the central nervous system, where it influences memory and appetite. Circulating insulin crosses the blood–brain barrier. After its uptake by and transport in microvascular endotheliocytes, insulin reaches the cerebral interstitial fluid via the subarachnoid cerebrospinal fluid and perivascular spaces (Virchow– Robin spaces) surrounding penetrating pial vessels that dive into the brain [684]. Insulin acts on its receptor in the hypothalamus, thereby countering food intake. In the hypothalamus, insulin activates ATP-sensitive potassium channel (KATP ), thereby impeding glucose production. Intranasal injection of insulin, which enables selective elevation in insulin concentration in the cerebrospinal fluid without altering the plasmatic concentration, hampers appetite in fasting men and affects brain activity in food-stimulated women [685]. This administration mode provokes a rapid transient and strong drop in glycemia.

4.5.2.1

Insulin Signaling

Insulin signaling is triggered by the binding of insulin to its plasmalemmal heterodimeric receptor on its target cells, which recruits and phosphorylates numerous substrate adaptors such as the insulin receptor substrates (IRS1–IRS4), SHC, DOK1, and GAB1, in addition to the Ub ligase CBL and CBL-associated protein (CAP). Once phosphorylated, these substrates act as dockers for SH2 domaincontaining proteins. Insulin receptor substrates differ according to organ distribution, subcellular localization, developmental expression, InsR binding, and interaction with SH2 domain-containing effectors. They act as docking proteins for signaling mediators. Whereas IRS1 plays a major role in skeletal muscles, IRS2 regulates hepatic insulin action in addition to β-cell development and survival [686]. On the other hand, IRS3 and IRS4 are redundant. Conversely, the insulin receptor and IRSs can be phosphorylated by IKKβ, ERK, JNK, and PKC, thereby inactivating InsR and priming the association of these dockers with the 14-3-3 proteins and arrestin signaling [687]. Among InsR residues, Ser1275, Ser1293, Ser1294, and Ser1309 are autophosphorylated, whereas Ser78, Ser82, Ser117, Ser1035, and Ser1037 are phosphorylated by PKC [688]. TyrPh IRSs contain binding sites for numerous signaling effectors, activating multiple kinases such as PI3K and its target PKB [689]. On the other hand, GRB2 activates the MAPK module to promote cell proliferation. The IRS proteins are phosphorylated (inhibited) by AMPK, in addition to GSK3β, JNK1, PKB, PKC, S6K, and salt-inducible kinase SIK2. Among IRS1 residues, Ser307 is phosphorylated by JNK1, IKKβ, and PKC, Ser636 and Ser639 by S6K, and Ser789 by SIK2 and AMPK [688]. Upon exposure to inflammatory cytokines, suppressors of cytokine signaling proteins tether to the insulin receptor and block its signaling [687]. Furthermore, phosphatases PTPn1, PTPn6, and PTen hamper insulin action.

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The major signaling cascades from activated IRSs for GluT4 regulation comprise the PI3K–PDK1–PKB and CAP–CBL–RhoQ pathways in addition to PKC and the MAPK module [688]. Heterodimeric PI3K is one of the key intermediates in the insulin pathway. It phosphorylates phosphoinositides to produce PI3P, PI(3,4)P2 , and PI(3,4,5)P3 that bind to other signaling mediators, which are then activated or re-localized, such as PDK1 and some of the atypical PKC isozymes [687]. Once it is phosphorylated by PI3K, PDK1 phosphorylates (activates) PKB, which, in turn, phosphorylates (deactivates) GSK3, which phosphorylates (inhibits) glycogen synthase. In addition, PKB phosphorylates FoxO1, which is expelled from the nucleus. Moreover, PKB promotes GluT4 residence at the plasma membrane. It also phosphorylates (inhibits) TSc2, thereby relieving TOR inhibition and favoring protein synthesis. PI3K activates PKCζ and PKCλ, thereby promoting translocation of GluT4 to the plasma membrane [687]. Insulin provokes phosphorylation of IRSs, GAB1, and SHC, which then connect to GRB2 [687]. The latter recruits SOS to the plasma membrane, where it activates Ras, which then induces the sequential phosphorylation (activation) of Raf, MAP2K, ERK1, and ERK2 kinases, which are responsible for cell proliferation launched by insulin. In particular, ERK1 is implicated in adipogenesis. Many insulin effects on lipogenesis are mediated via the transcription factor sterol regulatory element-binding protein SREBP1c, the dominant isoform in the liver and adipose tissue. Insulin increases SREBP1c transcription, maturation, and activity [687].

4.5.2.2

Insulin and Carbohydrate Metabolism

Insulin has two major effects on carbohydrate metabolism, although its action on glucose metabolism depends on the organ. 1. Insulin facilitates glucose entry into cells, especially adipocytes, hepatocytes, and skeletal myocytes. Skeletal muscles are the major site of glucose disposal in humans (80–90% insulin-stimulated glucose uptake [690] or 70–90% of an oral glucose load [687]). Insulin regulates redistribution to the plasma membrane of the major glucose transporter GluT4 to raise glucose influx. 2. Insulin elicits glucose storage in the form of glycogen in hepatocytes and skeletal myocytes, as it activates glycogen synthase (glycogenesis). It thus controls hepatic glycogen metabolism and hepatic glucose production. It not only activates glycogen synthesis, but also prevents glycogenolysis and gluconeogenesis, albeit less potently, using many mechanisms.

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Gluconeogenesis is the generation of glucose from noncarbohydrate substrates (glucogenic amino acids, glycerol, pyruvate, and lactate). Insulin inhibits [691]: 1. Synthesis and activity of hepatic gluconeogenic and glycogenolytic enzymes (e.g., phosphoenolpyruvate carboxykinase and glucose 6-phosphatase) via the transcription factor FoxO1 2. Secretion of glucagon, an gluconeogenesis activator 3. Lipolysis, hence reducing circulating concentrations of glycerol and nonesterified free fatty acids (FFAs), the latter providing energy for gluconeogenesis 4. Hepatic lactate uptake 5. Muscular proteolysis Hepatic glycogen stores are rapidly lost during fasting and gluconeogenesis, mainly from protein, maintains glucose supplies to the central nervous system, but proteic stores are limited. Hence, a glucose substitution mechanism is provided by lipids that are liberated by lipolysis, triglycerides being hydrolyzed to glycerol and non-esterified, that is, FFAs. Lipids substitute glucose as an energy substrate in many, but not all tissues. Skeletal muscles work without preference for glucose and FFAs. The nervous organs require only glucose, although they can utilize ketones for some (but not all) of their energy needs [682]. The FoxO transcription factors, which support hepatic glucose production via numerous mechanisms and thus allow adaptation to fasting and feeding in the liver, are insulin targets. In particular, FoxO1 is phosphorylated (inactivated) by insulinstimulated PKB in insulin-sensitive cells. In addition, FoxO1 operates in β cells. The InsR–PI3K–PKB–FoxO1 axis stimulates transcription of the genes involved in gluconeogenesis, glycerol transport, and amino acid catabolism and represses that of genes implicated in glycolysis, the pentose phosphate shunt, lipogenesis, and sterol synthesis [692]. On the other hand, activation of pro-lipogenic signaling pathways (PKB and atypical PKC) is not affected. In insulin resistance linked to obesity and diabetes, FoxO1 favors hyperglycemia and glucose intolerance. In addition, increase in pancreatic β-cell mass in response to hyperinsulinemia is blunted by FoxO1 [690]. However, FoxO1 can help the transcription of genes involved in combating redox stress and the switch from carbohydrate to fatty acid as the major energy source during starvation (Table 4.2).

4.5.2.3

Insulin and Lipid Metabolism

Lipids represent a major fuel for energy metabolism. Stored lipids in the form of triglyceride contain 9 kcal/g stored energy, whereas glycogen yields 2 kcal/g [682].

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Table 4.2 FoxO1 effects (Source: [690]; Apo apolipoprotein, Aqp9 aquaporin-9 [hepatic glycerol ingress], G6Pase glucose 6-phosphatase, PDK4 pyruvate dehydrogenase kinase-4 [phosphorylates the pyruvate dehydrogenase complex, thereby hindering conversion of pyruvate to acetylCoA and shifting pyruvate from the tricarboxylic acid cycle and fatty acid synthesis to gluconeogenesis], PDx1 pancreatic and duodenal homeobox gene product-1, PEPCK phosphoenolpyruvate carboxykinase, PGC PPARγ coactivator, SREBP sterol regulatory element-binding protein, ⊕ −→ stimulation, −→ inhibition) Adipose tissue Liver

Pancreas Skeletal muscle

Adipocyte differentiation Attenuates redox stress Gluconeogenesis via formation of PGC1α, PEPCK, and G6Pase and substrate supply (production of PDK4 and Aqp9) Lipid transport; lipid and sterol synthesis −→ SREBP1c expression −→ formation of fatty acid synthase and ATP–citrate lyase ⊕ −→ ApoC3 Promotion of insulin sensitivity β-cell proliferation −→ PDx1 production and activity Fusion of myocytes into myotubes Inhibition of TOR-mediated protein synthesis Muscle atrophy during starvation Switch from oxidation of carbohydrates to that of fatty acid Regulation of PGC1α

In the liver, insulin promotes synthesis of fatty acids. When the liver is saturated with glycogen (i.e., ∼5% liver mass), additional glucose taken up by hepatocytes is used for lipogenesis, synthesized fatty acids being then exported from the liver as lipoproteins. In adipose tissue, insulin prevents lipolysis, as it inhibits lipase, which hydrolyzes triglycerides into fatty acids. Insulin facilitates the entry of glucose into adipocytes, which can then be used to synthesize glycerol. The latter forms with fatty acids delivered from hepatic triglycerides.

4.5.2.4

Other Insulin Effects

Insulin stimulates the uptake of amino acids. It also increases entry in many cell types of potassium, magnesium, and phosphate ions. Insulin activates Na+ – K+ ATPase. Insulin activates the MAPK module that phosphorylates numerous substrates involved in cell growth, proliferation, and differentiation.

4.5 Insulin

4.5.2.5

315

AMPK and Insulin Sensitivity

The trimeric kinase AMPK,26 an epigenetic regulator, energy sensor, and master regulator of metabolism, promotes energy conservation and cell survival, as it suppresses anabolism and activates catabolism. Glucose deprivation primes phosphorylation of the insulin receptor and IRS1 by AMPK [688]. The enzyme AMPK is phosphorylated (activated) by the Ca2+ -dependent Cam2K227 and AMP-dependent LKb1 (STK11).28 In response to augmented cellular AMP/ATP ratio, AMPK is phosphorylated (Thr172) by LKb1. In some cell types (e.g., neurons, endotheliocytes, and lymphocytes), AMPK is phosphorylated (Thr172) by Cam2K2, Ca2+ entering cells upon K+ -induced depolarization [693].29 Other kinases such as MAP3K7 phosphorylate AMPKα (Thr172), activation of AMPK by MAP3K7, leading to autophagy upon exposure to TNFSF10, which induces apoptosis, hence ensuring cytoprotection [694]. In addition to histone H2b, chromatin-modifying protein ChMP1B,30 and DnaJc2,31 AMPK phosphorylates three other proteins involved in nucleosome remodeling [695], DNA methyltransferase DNMT1, histone acetyltransferase HAT1, and retinoblastoma-binding protein RBBP7, a DNMT1 inhibitor and HAT1 coactivator.32 This triggers nucleosome remodeling linked to augmented histone acetylation and reduced promoter methylation and favors transcription of the nuclear genes involved in mitochondrial genesis, that is, those encoding the transcriptional

possesses three subunits, α catalytic (α1–α2), β glycogen-sensing (β1–β2), and γ subunit (γ1–γ3), encoded by the PRKAA1, PRKAA2, PRKAB1, PRKAB2, and PRKAG1 to PRKAG3 genes. The γ subunit has two regulatory sites that bind the activating and inhibitory nucleotides AMP and ATP. 27 Cam2K: calcium–calmodulin-dependent protein kinase kinase. 28 LKb1: liver kinase-B1. 29 Gq-coupled receptors, which are linked to the PLC–IP –Ca2+ axis, such as PAFR for 3 platelet-activating factor, α1b-adrenergic receptor, bradykinin B2 , and thrombin receptor, trigger phosphorylation of AMPK by Cam2K2. Anorexigenic agents, such as insulin, leptin, and a melanocortin receptor agonist, inhibit AMPK, whereas orexigenic agents, such as ghrelin and cannabinoids, activate AMPK in the hypothalamus [693]. AMPK also appears to mediate many of the effects of adiponectin, especially in the liver and skeletal muscle. 30 The alias ChMP also stands for charged multivesicular body protein. This protein is also named vacuolar protein sorting-associated protein (VPS46-2). It is involved in chromatin structure maintenance [695]. 31 DnaJc2: HSP40 (DnaJ) homolog-C2. It is also called M-phase phosphoprotein MPP11 (or MPhosph11) and zuotin-related factor, ZRF1. It facilitates histone-2A Lys119 ubiquitination, which leads to chromatin remodeling for transcriptional activation [695]. 32 DNMT1 methylates DNA, thereby limiting the access of transcription factors to gene promoters. On the other hand, histone acetylation by HAT1 creates a relaxed chromatin–DNA structure that enables gene transcription. Cytosolic HAT1 acetylates newly synthesized free histones and participates in chaperoning histones into the nucleus for nucleosomal integration. It complexes with RBBP7 and H4. 26 AMPK

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coactivator PGC1α,33 the transcription factors Mt Tfa, NRF1, NRF2, and uncoupling proteins UCP2 and UCP3.34 Dysregulated mitochondrial genesis and function impair ATP production and Ca2+ signaling and raise ROS production, hence contributing to metabolic and cardiovascular disorders. Trimeric DNA-dependent protein kinase (DNAPK)35 is implicated not only in DNA double-strand break repair and V(D)J recombination, initiating DNA repair by the nonhomologous end joining (NHEJ) pathway, but also in metabolic regulation. DNAPK upregulates transcription of the genes involved in lipogenesis upon feeding and insulin signaling [698].36 During glucose deprivation, DNAPK activates AMPK, an energy sensor. In the skeletal muscles of old mice (but not in the lung), aging, which is linked to weight gain and loss of physical fitness in addition to a decline in the efficiency and accuracy of NHEJ repair, a fall in the level of NAD+ , the sirtuin-1 cofactor, augments DNA double-strand breaks, thereby leading to a constitutive DNAPK activation. This elevated activity alters mitochondrial function, energy metabolism, and physical fitness. DNA-dependent protein kinase phosphorylates

33 PGC1α:

peroxisome proliferator-activated receptor-γ coactivator-1α, which is encoded by the PPARGC1A gene. This coactivator responds to environmental factors. PGC1α is activated upon phosphorylation by AMPK and deacetylation by sirtuin-1. It controls many aspects of oxidative phosphorylation, as it coactivates the production of the nuclear respiratory factors NRF1 and NRF2. It is thus indirectly involved in Mt DNA transcription via the mitochondrial transcription factor-A (Mt Tfa) encoded by the TFAM gene, which is coactivated by NRF1. 34 Uncoupling proteins are implicated in circulatory, degenerative, and immunological diseases and aging. The UCP1 orthologs UCP2 and UCP3 (0.01–0.1% of inner mitochondrial membrane proteins) transport protons when they are specifically activated, for example, by reactive alkenals such as hydroxynonenal, which is produced by peroxidation of membrane phospholipids [696]. UCP1 (10% of IMM proteins), which is synthesized in brown adipose tissue via the synergistic action of noradrenaline and thyroid hormones, especially upon exposure to cold, acts in adaptive thermogenesis. It increases the proton conductance of the inner mitochondrial membrane. The leakage of protons through UCP1 uncouples substrate oxidation from phosphorylation of ADP to ATP, leading to fast oxygen consumption and heat production. The proton conductance of UCP1 is inhibited by purine nucleotides, such as ATP and GDP; this inhibition is relieved by fatty acids released from intracellular triacylglycerol stores upon adrenergic activation in response to cold [696]. Fatty acids increase transcription of the Ucp2 and Ucp3 genes. Both UCP2 and UCP3 support proton conductance, but only when activated by fatty acids and alkenals. Both UCP2 and UCP3 may export fatty acids and other anions. UCP2 attenuates mitochondrial ROS production in addition to insulin secretion from pancreatic β cells in response to glucose [696, 697]. UCP3, which lodges mainly in skeletal muscles and brown adipose tissue, influences energy metabolism, as it may be involved in ROS formation, mitochondrial fatty acid transport, and glucose metabolism in skeletal muscles [696, 697]. 35 The DNAPK trimer is composed of the catalytic subunit (DNAPKc) and the Ku70–Ku80 (XRCC5–XRCC6) heterodimer. 36 DNAPK phosphorylates the transcription factor USF1 and promotes fatty acid synthesis in response to insulin [699].

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(inactivates) HSP90α37 (Thr5 and Thr7), a chaperone supporting AMPK folding, hence inhibiting indirectly AMPK activity, impairing mitochondrial genesis, and contributing to metabolic and fitness decline during aging [699].38 Dysregulated AMPK is associated with aging-related obesity, insulin resistance, and diseases (T2DM, cardiovascular diseases, and cancer). In myocytes, AMPK activity, which increases upon glucose deprivation and exercise, correlates with elevated glucose uptake and glycolysis, independently of insulin. AMPK also enhances fatty acid transport and oxidation but arrests fatty acid, cholesterol, glycogen, and protein synthesis. The latter effects contribute to the insulin-sensitizing property of the AMPK. Several pathways lead to AMPK-enhanced insulin sensitivity and glucose transport [694]. In the major insulin-resistant organs (liver, muscle, and adipose tissue), glucose uptake and utilization strongly decrease and fatty acid oxidation is defective. In the liver and muscle, AMPK inhibits gluconeogenesis and lipogenesis, promotes mitochondrial fatty acid oxidation and glucose uptake, thereby ameliorating defects associated with the metabolic syndrome and type-2 diabetes. Numerous hormones and agents regulate AMPK activity. Full-length adiponectin activates AMPK in the liver and skeletal muscle. Leptin stimulates AMPKα2 in the skeletal muscle. Ciliary neurotrophic factor also activates AMPK in the skeletal muscle but reduces hypothalamic AMPKα2 activity [694]. The appetite stimulators endocannabinoids and ghrelin increase AMPK activity in the hypothalamus and heart but inhibit AMPK in the liver and adipose tissue. Interleukin-6 produced and released by skeletal myocytes in response to contraction rapidly and markedly increases AMPK activity in myotubes, enhancing fatty acid oxidation in addition to basal and insulin-stimulated glucose uptake [694]. Conversely, IL6 release from skeletal muscles declines upon AMPK activation. Numerous natural products, such as alkaloids (e.g., berberine from Rhizoma coptidis), bitter melon extracts, and epigallocatechin 3-gallate, a main catechin of green tea, supports AMPK activation by Cam2K [694]. Resveratrol from legumes and fruits increases sirtuin-1 deacetylase activity and STK11 (LKB1) phosphorylation (Ser428) and hence AMPK action. Aspalathin, a C glucosyl dihydrochalcone from rooibos (Aspalathus linearis), a South African herbal tea, attenuates fatty acid uptake and subsequently β-oxidation, as it impedes AMPK phosphorylation (Thr172) and carnitine palmitoyltransferase CPT1 function [700]. The CPT1 enzyme is a component of the shuttle between the cytosol and mitochondrial matrix of long-chain fatty acids such as palmitoylCoA, which, unlike short- and medium-chain fatty acids, cannot freely diffuse through

37 The

chaperone HSP90α, which is encoded by the HSP90AA1 gene, ensures maturation of specific target proteins involved in cell cycle control, signaling, and regulation of the transcriptional machinery. 38 AMPK stimulates glucose uptake, lipid oxidation, and decreasing visceral lipidic depots, in addition to energy production, scavenging of oxygen radicals, and mitochondrial genesis. AMPK promotes the synthesis of NAD+ via NAMPT, hence mitochondrial and metabolic functions.

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the inner mitochondrial membrane. Aspalathin increases acetylCoA carboxylase activity, which forms malonylCoA from acetylCoA, malonylCoA inhibiting CPT1. Aspalathin also upregulates GluT4 production and raises glucose oxidation, ameliorating a hyperglycemia-induced shift in substrate preference. Last, but not least, aspalathin can protect the myocardium against cell apoptosis, as it lowers the amount of ROS and augments those of glutathione, superoxide dismutase, and uncoupling protein UCP2, in addition to BCL2/BAX ratio. Aspalathin controls concentrations of regulators involved in lipid metabolism (adiponectin, NR1c3, SREBP1, SREBP2, ApoB, ScaRb3, VLDLR, CPT1, and SCD1), insulin resistance (IGF1, MAP2K1, PKB1, and PDE3), inflammation (LepR, SOCS3, JaK2, TNFSF13, IL3, and IL6), and apoptosis (BCL2 and IKK1). Aspalathin can protect cardiomyocytes and diabetic (db/db) mice against local high glucose concentration- and hyperglycemia-induced shifts in substrate preference, respectively, and against redox stress, apoptosis, and adverse cardiac remodeling (diabetic cardiomyopathy), as it upregulates expression of NFE2L2 [701].39 α-Lipoic acid, a cofactor of mitochondrial enzymes that catalyze decarboxylation of α-keto acids, diminishes hypothalamic AMPK activity but stimulates AMPK in skeletal muscles and endotheliocytes [694].

4.5.2.6

Energy Sources

Mitochondrial carbohydrate and fatty acid oxidation are the major sources of ATP production (90–95% at least in the heart) via generation of acetylCoA for the tricarboxylic acid. In the mitochondrial oxidative phosphorylation, reducing equivalents (protons and electrons) are transferred from energy substrates to the mitochondria by the reduced forms of flavin adenine dinucleotide and nicotinamide adenine dinucleotide (NADH) generated by dehydrogenase-mediated reactions in the fatty acid oxidation, tricarboxylic acid cycle, and glucose oxidation. In adult cardiomyocytes, mitochondria undergo much less frequent dynamic changes than in other cell types, despite the abundance of fission and fusion regulators [702]. Mitochondrial fusion, which creates filamentous and interconnected mitochondria, is mainly regulated by the outer mitochondrial membrane proteins mitofusins Mfn1 and Mfn2 and the dynamin-like inner mitochondrial membrane 39 NFE2L2

upregulates expression of antioxidant and detoxifying proteins, such as catalase, glutathione GSH -synthesizing catalytic (GCLc) and modifier subunits (GCLm), glutathione peroxidase GPOx2, thioredoxin reductase TRdxRd1, superoxide dismutase, and NADPH:quinone oxidoreductase (or NAD(P)H dehydrogenase quinone) NQO1. The antioxidant tripeptide glutathione GSH , which scavenges free radicals and serves as a cofactor for glutathione transferase used in the conjugation of many xenobiotics, is synthesized in two steps. The first rate-limiting step forms γ-glutamylcysteine using inducible heterodimeric glutamate cysteine ligase. Various dietary phytochemicals, such as sulforaphane, which is derived from a glucosinolate of broccoli, and quercetin, an aglycone from flavonol glycosides, can prevent redox stress, as they upregulate NFE2L2 expression [701]. A catechol group of flavonoids, such as that in shogaol derivatives, can contribute to NFE2L2 activation.

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protein optic atrophy OpA1. Furthermore, OpA1 is involved in the formation of mitochondrial cristae and mitofusins in mitochondrial–endoplasmic reticulum tethering [702]. Mitochondrial fission is mainly regulated by dynamin-related protein, DRP1, a cytosolic protein, which, upon phosphorylation and oxidation, translocates to the mitochondrion. Pharmacological and genetic inhibition of DRP1 in adult rodent cardiomyocytes, which do not change their mitochondrial morphology, significantly reduce maximal mitochondrial respiration via electron flow through an electron transport chain based on oxidative phosphorylation and hence oxygen consumption rate. However, the basal respiration increases because of elevated proton leakage in the inner mitochondrial membrane [702]. In the absence of DRP1, transient opening of mitochondrial permeability transition pore and mitochondrial ROS production decrease. Removal of cyclophilin-D, a Mt PTP regulator, abolishes the effect of DRP1 inhibition on mitochondrial respiration, which also depends on transient Mt PTP opening, intermediate metabolism, and ATP hydrolysis, in addition to Ca2+ and ROS [702]. The myocardial ATP pool experiences a nearly complete turnover every 10 s; thus, the heart uses various energy substrates (fatty acids, glucose, lactate, and ketone bodies) to generate ATP, the contribution of each energy substrates being tightly regulated [703]. In the normal adult heart, fatty acid β-oxidation accounts for 60–80% of ATP production; the remainder is primarily related to glucose and lactate oxidation. Nevertheless, oxidation of other energy substrates, such as ketones and branchedchain amino acids (BCAAs) such as leucine, can also contribute to energy production [704]. Both ketone bodies and BCAAs are minor providers of acetylCoA, but when circulating concentrations of ketone bodies or BCAAs are elevated, such as in diabetes, increased ketone body or BCAA oxidation can compete for acetylCoA formation. Both ketone bodies and BCAAs regulate various signaling pathways. They influence mitochondrial protein acetylation and TOR activity, which both affect insulin sensitivity. In particular, the ketone body β-hydroxybutyrate is a histone deacetylase inhibitor that can promote FoxO1 acetylation and activate the PP3– NFAT axis. In addition, β-hydroxybutyrate activates the G-protein-coupled receptor HCA2 .40

40 Also

known as GPR109a and nicotinic acid receptor NiAc1 . The hydroxycarboxylic acid receptors (HCAR1–HCAR3 or HCA1 –HCA3 ) were formerly called nicotinic acid receptors, although nicotinic acid has a weak affinity for HCA2 . These receptors respond to organic acids, such as short-chain fatty acids, butyric acid, and lactic acid, in addition to the lipid-lowering agent nicotinic acid (niacin). Lactate activates HCA1 (a.k.a. lactate receptor LaCR1, GPR81, and GPR104) on adipocytes. The receptor HCA3 (a.k.a. niacin receptor NiAc2 and GPR109b) is a low-affinity nicotinic acid receptor that tethers to 3-hydroxyoctanoic acid. All three Gicoupled receptors reside on adipocytes, where they mediate antilipolytic effects. Both HCA2 and HCA3 also lodge on immunocytes (e.g., monocytes, macrophages, neutrophils, and Langerhans dendrocytes), where they have anti-inflammatory effects.

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Considering the number of ATP molecules produced per oxygen atom reduced, that is, the phosphate/oxygen (P/O) ratio, the P/O ratio linked to palmitate, is lower than that linked to glucose; hence, palmitate is a less efficient substrate for ATP synthesis.41 β-Hydroxybutyrate processing is more efficient than that of fatty acids but less efficient than that of glucose [704]. Branched-chain amino acids can stimulate TOR that form TORC1, which acts via S6K, which phosphorylates (inhibits) the cytosolic adaptor IRS, thereby preventing insulin signaling. Fatty acids are also signaling molecules, as they act as ligands for the nuclear receptor NR1c1 and serve as precursors for the messengers diacylglycerol and ceramides [704]. Altered flux through fatty acid oxidation may thus have an impact on the corresponding signaling pathways. Glucose and glycolytic intermediates also have signaling functions. Impaired glucose metabolism can disturb these signaling pathways. In general, cancerous cells use aerobic glycolysis to generate ATP instead of more efficient mitochondrial oxidative phosphorylation (Warburg effect). Glucose is strongly taken up owing to the overexpression of glucose transporters, especially GluT1. Glucose is then irreversibly phosphorylated by hexokinases HK1 and HK2, which are also overproduced in cancers [707]. Glycolytic enzymes acting downstream from hexokinase include 6-phosphofructo 2-kinase and fructose

41 The

P/O ratio depends on the quantity of hydrogen atoms carried outward and the number of protons that return inward using ATP synthase. The P/O ratio represents the number of inorganic phosphate molecules utilized for ATP generation for every atom of oxygen reduced in addition to the number of molecules of ATP synthesized per pair of electrons passing from a particular substrate, typically NADH or succinate, through an electron transport chain to oxygen, two electrons being donated by reduction of an oxygen atom and traversing the electron transport chain. Four protons are transported inward from H2 O to NADP, and three protons move outward in ATP synthesis. Three sites of proton translocation generate a proton electrochemical gradient, or protonmotive force, which drives ATP synthesis using 2 H+ for each ATP made. In fact, the stoichiometry of H+ translocation is higher. The charge translocation stoichiometries are not equal for the three sites of proton translocation; ETC complex-I and -I V each translocate double the charge/2e relative to ETC complex-I I I and thus the ATP/2e for the latter must be one half that for the other two complexes [705]. ATP crosses the membrane with one more negative charge than ADP. Moreover, the entrance of inorganic phosphate with a proton is equivalent to the entrance of one proton and one positive charge during the transport of ADP and inorganic phosphate into the mitochondrion and ATP out during oxidative phosphorylation. The transport of ATP out of the mitochondrial matrix and uptake of ADP and inorganic phosphate into the matrix are coupled with the uptake of one proton. The coupling of nucleotide and inorganic phosphate transfer to proton motion implies that the P/O ratio for the synthesis and transport of ATP to a mitochondrion outside is lower than the P/O ratio for the synthesis of ATP inside the mitochondrion. The P/O ratio takes fractional values because the unit of energy linked to coupling sites of the electron transport chain is the proton rather than ATP. Values of about 2.5 (10/[3+1]) and 1.5 (6/[3+1]) with NADHlinked substrates and succinate, respectively, are compatible with H+ /O=10 or 6 with NADH or succinate, H+ /ATP=3 for ATP synthase, and H+ /ATP=1 for ATP transport to the cytoplasm. The fractional values result from the coupling ratios of proton transport. In fact, the H+ /ATP ratio is not an integer, equaling 10/3 and hence consistent with a P/O ratio of 2.3 and 1.4 with NADH and succinate, respectively [706].

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(2,6)-biphosphatase-3 (PFKFB3 or PFK2), phosphofructokinase PFK1, pyruvate dehydrogenase complex (PDH; containing pyruvate dehydrogenase [E1], dihydrolipoamide acetyltransferase [DLAT; E2], and dihydrolipoamide dehydrogenase [DLDH; E3]), and PDH kinase (PDHK1–PDHK4). The oncogenic PI3K–PKB, MyC, and HIF1 pathways influence the metabolic shift during carcinogenesis and support growth and proliferation of cancerous cells subjected to metabolic stress and hypoxia. On the other hand, P53 binds to the promoters of metabolic genes and represses their transcription, thereby precluding synthesis of glucose transporters GluT1 (SLC2a1) and GluT4 (SLC2a4) [707]. Furthermore, P53 inhibits the transcription factors MyC and HIF1α implicated in ATP production. Inhibition of MyC and HIF1α by P53 eliminates formation of glycolytic mediators in normoxia and hypoxia, respectively. The transcriptional cofactor SP1 cooperates with P53 in this process. The hippo pathway, which is controlled by estrogens via GPER, participates in organ size control and cancer prevention, as it represses its effector Yes-associated protein (YAP), which reprograms glucose metabolism, as it enhances glucose uptake and elicits glycolysis and lactate production, hence favoring carcinogenesis. During glucose starvation, YAP is phosphorylated (inactivated) by AMPK. The long nonprotein-coding RNA breast cancer antiestrogen resistance bcar4, a YAP effector, is involved in YAP-dependent glycolysis [708]. YAP promotes bcar4 expression. Subsequently, bcar4 associates with SMAD nuclear-interacting protein SNIP1 and protein Ser/Thr phosphatase-1 regulatory subunit PP1r10 , thereby relieving inhibition of SNIP1 on the histone acetyltransferase KAT3b and aberrantly activating the alternative hedgehog signaling, especially its effector Gli2, which primes transcription of the glycolytic enzymes HK2 and PFKFB3. Bcar4 thus signals via the two growth-related hippo and hedgehog pathways.

4.5.2.7

Insulin and NOS3 in Endotheliocytes

In healthy individuals, insulin is acutely released in response to food intake concomitantly with a decrease in vascular resistance due to the release of the signaling radical NO [689, 709]. In endotheliocytes, transient insulin stimulation induces NOS3 phosphorylation (Ser1177) by PKB (the InsR–IRS–PI3K–PKB– NOS3 axis), elevating NO formation. However, NO synthesis augments slowly and modestly with respect to the NO amount generated by Ca2+ influx upon exposure to acetylcholine or bradykinin. In fact, NOS3 regulation involves other stimulatory and inhibitory phosphorylations and dephosphorylation of Thr495 in its calmodulin-binding domain. Insulin, ATn2, H2 O2 , and hemodynamic stress provoke phosphorylation of NOS3 on Tyr657 by the redox-sensitive focal adhesion kinase FAK2, repressing NOS3 activity [689, 709]. However, FAK2 action decreases rapidly in cultured endotheliocytes, relieving its inhibition of NOS3. Therefore, insulin-triggered stimulatory Ser1177 phosphorylation of NOS3 by PKB is counterbalanced by insulin-primed inhibitory Tyr657 phosphorylation by FAK2, resulting in NO synthesis, thus depending on the degree of FAK2 activation.

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Elevated FAK2 activity caused by superoxide produced by NOx2, expression of which is upregulated by the PI3K–PKB axis and FAK2, raises phosphorylation of NOS3 at Tyr657, thereby contributing to endothelial dysfunction. The deleterious feedforward InsR–PI3K–PKB–NOx2–O•− 2 –FAK2–NOS3 axis, which primes excessive ROS production, leads to inhibitory phosphorylation of NOS3 at Tyr657 by PYK2, which abrogates the stimulatory phosphorylation of NOS3 at Ser1177 by PKB [709]. This feedforward loop impairs NO availability, as O•− 2 reacts with NO and FAK2 reduces NO production by NOS3. Angiotensin-2 can also activate FAK2. Therefore, endotheliocytes exposed to hyperinsulinemia, a strong predictor of atherogenesis, become dysfunctional and increase the risk for redox stress. In transgenic mice with endothelium-specific overexpression of the human InsR (EC hIRO mice), a model of hyperinsulinemia effect on the vascular endothelium, although sustained insulin signaling in endotheliocytes augments PKB activation and subsequent NOS3 phosphorylation at mouse stimulatory Ser1176 site, endotheliocytes do not respond properly to NO-dependent acetylcholine and insulin, in the absence of antioxidants [709]. Insulin-primed PKB activation is altered in endotheliocytes in obesity and diabetes. Ablation of all insulin signaling cascades in endotheliocytes affects atherogenesis. On the other hand, selective insulin resistance deals with an impaired insulin-stimulated PI3K–PKB pathway, whereas other signaling cascades such as those based on the MAPK module or SREBP1c remain sensitive to insulin [710]. Hyperinsulinemia, which is aimed at compensating for insulin resistance, provokes supranormal activation of intact insulin signaling axes. During supranormal insulin signaling, MAPK can also stimulate NOx2 [710]. Loss of insulin-initiated PKB–FoxO signaling accelerates atherosclerosis. In vascular endotheliocytes, activated PKCβ causes insulin resistance and depletion in insulin receptor, PKB1, or its effectors FoxO1, FoxO3, and FoxO4 favor atherosclerosis [710]. In addition, expression of the endothelin receptor ETB is upregulated by insulin signaling via PKB, which increases cytosolic Ca2+ concentration and activates NOS3 in endotheliocytes, insulin in cooperation with endothelin-1 causing higher NO production than endothelin-1 alone [710]. Hence, many pathways participate in insulin sensitivity of endotheliocytes and NO availability.

4.6 MicroRNAs and Insulin Sensitivity Among microRNAs that repress mRNA translation, miR155 enhances insulin sensitivity [711]. In diabetic patients, plasmatic concentration of miR155 is reduced. In mice, miR155 overexpression causes hypoglycemia and can improve glucose tolerance and insulin sensitivity, whereas its deletion provokes hyperglycemia and insulin resistance. Gain and loss of miR155 functions do not affect pancreatic βcell proliferation and function. On the other hand, miR155 is involved in adipocyte differentiation, adipogenesis, and lipid metabolism [711].

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MiR155 promotes glucose uptake in the examined cell types [711]. Its overproduction enhances glycolysis and insulin-stimulated PKB activity in the liver, adipose tissue, and skeletal muscle. MiR155 represses activity of inhibitors of insulin signaling, such as HDAC4, C/EPBβ, and SOCS1 [711]. Histone deacetylase, HDAC4, inhibits GluT4 formation. The factor SOCS1 provokes ubiquitin-mediated degradation of the IRS1 adaptor. In addition, SOCS1 suppresses production of CCAAT/enhancer-binding proteinC/EBPβ. The C/EPBβ factor launches transcription of the Pdk4 gene, which encodes pyruvate dehydrogenase kinase PDK4, inhibiting pyruvate dehydrogenase complex.42 The InsR–IRS–PI3K–PKB pathway hinders PDK4 activity, hence promoting glycogenesis via PKB and the pyruvate dehydrogenase complex. Glycolysis is coupled with lipogenesis in the liver. Glycolytic enzymes convert glucose to pyruvate and the pyruvate dehydrogenase complex, the activity of which is regulated by pyruvate dehydrogenase kinases and phosphatases, transforms pyruvate, coenzyme-A, and NAD+ into acetylCoA, NADH, and CO2 in mitochondria, thereby linking fatty acid and glucose metabolisms to the tricarboxylic acid cycle. In addition, miR155, although it does not target their transcripts, affects the synthesis of proteins themselves targeted by miR155 substrates, such as those involved in glycogen metabolism (hepatic glycogen synthase GyS2), glycolysis (glucokinase [GcK], PDK4, muscular pyruvate kinase PKM2, and lactate dehydrogenase LDHa), and glucose transporters (GluT1–GluT2, GluT4, SLC1a2 [EAAT2], and SLC3a2 [activator of dibasic and neutral amino acid transport]) [711]. Increased insulin sensitivity and improved glucose tolerance can result from enhanced glucose uptake upon phosphorylation of PKB and glycolysis upon increased activity of GcK and PKM2, which are important glycolytic enzymes that determine the rate of glycolytic flux, and decreased PDK4 action. Other microRNAs are involved in glucose homeostasis, insulin sensitivity, and pancreatic β-cell function. MiR34a and miR375 are implicated in pancreatic development [711]. MiR9 and miR375 intervene in insulin secretion. Whereas miR103–miR107, miR143, and miR802 counter insulin sensitivity, miR26a and miR130a-3p favor glucose tolerance and insulin sensitivity.

4.7 Kidney and Glucose The kidney contributes to glucose homeostasis via gluconeogenesis and glucose reabsorption in the proximal tubule [712]. In healthy humans, 160–180 g of glucose are filtered by the kidneys per day and then reabsorbed in the proximal tubule, the filtered glucose level remaining lower than the maximal renal glucose reabsorption

42 Isozymes

PDK2 and PDK4 are the most widespread PDK isoforms. They are abundantly produced in the heart, liver, and kidney in humans. PDK4 also abounds in pancreatic islets and skeletal muscles. On the other hand, PDK1 and PDK3 have a restricted tissular distribution.

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capacity (∼180 mg/dl). Glucosuria occurs when the filtered glucose amount exceeds the renal tubular glucose excretion threshold. Glucose is reabsorbed against a concentration gradient, glucose import being coupled with active sodium export through a sodium–potassium pump. Intracellular glucose then crosses the basolateral membrane of the tubule cell through GluT2 to enter blood. 1. About 90% of glucose filtered in glomeruli is reabsorbed in the proximal segment of the proximal tubule by the low-affinity and high-capacity sodium–glucose cotransporter SGLT2 (SLC5a2) in the apical membrane of renal epitheliocytes. 2. The remaining 10% is reabsorbed in the distal segment of the tubule by the highaffinity and low-capacity cotransporter SGLT1 (SLC5a1). In diabetic patients, the threshold increases to about 220 mg/dl, the maximal renal glucose reabsorption capacity rising owing to upregulated SLC5a2 expression in the proximal tubule [712]. Among antidiabetic (antihyperglycemic) medications, inhibitors of SLC5a2, which prevent glucose and sodium reabsorption in the proximal tubule, thereby increasing urinary glucose excretion and natriuresis and decreasing glycemia, have a cardioprotective effect in diabetic patients. They reduce the occurrence of major adverse cardiac events (myocardial infarction, stroke, and death) [712, 713]. Moreover, they exert many beneficial effects, as they lower blood pressure and change renal hemodynamics, attenuating intraglomerular hypertension, diminish the body’s mass, and support neurohormonal homeostasis. Inhibition of SLC5a2 is associated with increased glucagon secretion, glucagon having positive inotropic and chronotropic effects.

4.8 Heart and Glucose Tolerance Diabetes complications comprise diabetic cardiomyopathy. Activating transcription factor ATF3, an immediate early transcriptional regulator of the basic leucine zipper (bZip) superfamily, is rapidly, highly, and transiently upregulated upon exposure to multiple types of stressors (e.g., redox, ER, and metabolic stress). In particular, it senses metabolic stress in cardiomyocytes; it protects the heart against adverse remodeling [714]. It exerts both beneficial and detrimental effects; according to the context, ATF3 launches an adaptive or maladaptive response. In mice subjected to an HFD, ATF3 limits inflammation, lowering the production of cytokines (TGFβ, TNFSF1, and IL6) and TIMP1, TIMP3, and TIMP4, hence favoring MMP activity, but hampers adverse cardiac hypertrophy and fibrosis [715]. It connects to the cAMP response element and typically impedes transcription of genes that encode proinflammatory cytokines and chemokines. Therefore, in the absence of ATF3, a stressful environment associated with HFD causes maladaptive cardiac response.

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Cardiac ATF3 not only has beneficial effects in the heart, but also controls insulin sensitivity in other organs. Selective inhibition of ATF3 in cardiomyocytes not only reduces myocardial insulin sensitivity via downregulated expression of IRS1 and IRS2, but also exacerbates systemic insulin resistance and blood glucose intolerance in HFD-fed mice [715]. Elevated concentrations of the natriuretic peptides ANP and BNP, in cooperation with IL6 and TNFSF1, may mediate the crosstalk with organs, thereby provoking glucose intolerance in HFD-fed mice. In HFD-fed mice, specific cardiac deletion of mediator complex subunit Med13 also causes remote effects. Secretion of cardiokines may mediate a crosstalk between the heart and other organs.

4.9 Insulin Resistance Insulin resistance is defined by elevated glycemia after a fasting night (normal fasting glycemia 5 mmol/l). In the fasting state, the rate of glucose appearance into the blood circulation exclusively from the liver, that is, the hepatic glucose production rate, is normally approximately 12 μmol/kg/mn (2 mg/kg/mn) and matches the glucose utilization rate, that is, the rate of glucose disappearance in the brain (∼50%), lean bodily organs (∼30%), adipose tissue (∼10%), and red blood capsules (∼10%) [682]. Insulin resistance is the impaired ability of the liver, skeletal muscle, and adipose tissue to take up glucose, although pancreatic β cells continue secreting insulin to normalize glycemia, provoking hyperinsulinemia. Insulin resistance can be caused by diverse types of alterations, decreased production and kinase activity of InsR, expression and phosphorylation of IRS, altered recruitment of adaptors, or increased activity of protein Tyr phosphatases. Insulin resistance is a predictor of T2DM development. Because it correlates with the degree of abdominal obesity, it links diabetes to nutritional overload and obesity. Hyperglycemia and insulin resistance are two major consequences of diabetes responsible for cardiovascular disorders. Impaired insulin action in the adipose tissue augments the lipolysis rate and FFA release. The resulting hyperemia alters insulin secretion by pancreatic β cells and perturbs insulin signaling and glucose uptake in the liver and skeletal muscle. Insulin resistance is an element of the metabolic syndrome that is defined by a collection of metabolic anomalies (hyperglycemia, adaptive hyperinsulinemia, hypertriglyceridemia, and hypohdlemia). Metabolic syndrome is also strongly associated with endothelial dysfunction and atherosclerosis risk due to insulin resistance and hyperemia, which impairs NOmediated vasodilation and favors endothelial activation. Free fatty acids can increase hepatic gluconeogenesis and overproduction of triglyceride-rich very low-density lipoproteins that form atherogenic LDLs. Adipokines, concentrations of which correlate with adiposity, link obesity and metabolic syndrome. Metabolic disorders comprise, in particular, adipose

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tissue dysfunction and elevated concentration of tumor-necrosis factor-α (TNFα or TNFSF1), a proinflammatory adipokine. Production of TNFSF1 is augmented by hyperinsulinemia (Sect. 5.4.5.26). The cytokine TNFSF1 hinders carbohydrate metabolism and lipo-, adipo-, and thermogenesis but stimulates lipolysis [716]. Once it is attached by soluble or transmembrane TNFSF1, TNFR1 hampers adipogenesis. Many of the TNFR1-launched pathways mediate TNFSF1 actions in adipose tissue [716].43 Nevertheless, TNFR2 is implicated in some TNFSF1 effects on adipocytes. TNFSF1 impedes perilipin action, which coats lipid droplets, protecting them from hormone-sensitive lipase (LipE), thereby stimulating lipolysis. Activated TNFR1 stimulates AMPK, ERK1, ERK2, JNK, IKK, and PKA [716]. In addition, TNFSF1 causes phosphorylation of insulin receptor substrate-1, thereby precluding signaling from the insulin receptor.

4.10 Redox Stress Insulin resistance raises fatty acid flux and oxidation44 and engenders excessive ROS formation by NAD(P)H oxidase in arterial endotheliocytes and cardiomyocytes, thereby upregulating expression of nuclear receptor NR1c1 (PPARα)45 and favoring nuclear translocation of the FoxO1 factor, which causes cardiomyopathy [675].46

43 Once

TNFR1 is liganded, the adaptor TRADD (TNFR-associated death domain [DD]containing protein) recruits adaptors FADD (Fas-associated DD-containing protein), TRAF2 (TNFR-associated factor-2), receptor-interacting protein RIP1, and MADD (mitogen-activated protein kinase (MAPK)-activating DD-containing protein). These adapters then activate various signaling cascades (apoptosis, activation of NFκB, NFAT MAPK, and PKA). 44 Inhibition of FFA release from adipocytes or FFA oxidation in arterial endotheliocytes hampers excessive ROS production [675]. 45 Activated NR1c1 causes exchange of bound transcriptional repressors for transcriptional coactivators (primarily PGC1α and PGC1β), which form active transcriptional complexes with CBP (KAT3a), P300 (KAT3b), and NCoA1, among others. Transcriptional activity of NR1c1 is further increased upon phosphorylation by PKCβ2 [675]. In the heart, insulin resistance increases oxidation of FAAs and H2 O2 production than does elevated glucose oxidation because of augmented electron leakage from the electron transfer flavoprotein complex. ROS activate acylCoA:lysocardiolipin acyltransferase ALCAT1 transcription. This enzyme of the mitochondrial-associated endoplasmic reticulum membrane generates adverse remodeling of cardiolipin with highly unsaturated fatty acid side chains, thereby diminishing electron transport chain activity, ATP synthesis, and further increases ROS. 46 ROS engender JNK signaling and CamK2 activation in addition to FoxO O-GlcNAcylation, persistent activation, and translocation from the cytosol to the nucleus [675]. FoxO activation is associated with reduced activity of the IRS1–PKB axis.

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Cardiovascular disorders result not only from hyperglycemia and insulin resistance but also from prolonged elevated production of ROS by the mitochondrial electron transport chain within diabetic cardiovascular cells upon high intracellular glucose concentration [675]. In insulin resistance, mitochondria dynamics is altered with increased fission (elevated concentration of dynamin-related protein-1 in endotheliocytes; Vol. 9, Chap. 2. Hypoxia and Stress Response)47 and decreased fusion (diminished synthesis of mitofusin-1 in cardiomyocytes and optic atrophy protein-1 in endotheliocytes), thereby reducing the efficiency of the electron transport chain and hence ATP synthesis and augmenting ROS production [675]. Mitochondrial ROS overproduction can be amplified by activated NOxs and uncoupled NOS3. ROS diffuse into the nucleus, where they activate polyADP ribose polymerase (PARP), which parylates (inhibits) glyceraldehyde 3-dehydrogenase (GAPDH), hence causing accumulation of early glycolytic intermediates, which are diverted into four pathways [675]. 1. Diversion of glucose elicits the polyol pathway. 2. Diversion of fructose 6-phosphate promotes the hexosamine pathway. 3. Diversion of glyceraldehyde 3-phosphate to α-glycerol phosphate activates the diacylglycerol–PKC pathway. 4. Diversion of glyceraldehyde 3-phosphate to the highly reactive α-dicarbonyl methylglyoxal due to reduced GAPDH activity (methylglyoxal–AGE pathway). In mitochondria, superoxide releases Fe2+ from ferritin and iron–sulfur clustercontaining proteins, free iron interacting with hydrogen peroxide to form hydroxyl radicals [675]. Reactive oxygen species lead to inhibition of not only GAPDH but also sirtuins, PGC1α, and AMPK, thereby decreasing mitochondrial genesis, upregulating transcription driven by chronically activated NFκB of the neutrophil peptidylarginine deiminase, PADI4, which initiates NETosis,48 activating NLRP3 inflammasome,49 and disturbing synchronization of glucose and lipid metabolism by the circadian clock [675]. Under normal conditions, ROS produced at the appropriate time, in the appropriate duration, form, and concentration, function as signaling molecules (Vol. 4, Chap. 10. Other Major Types of Signaling Mediators and 11, Chap. 7. Reactive Oxygen and Nitrogen Species). Synthesis of ROS is coupled to the circadian

47 Increased

Ca2+ concentration activates calpain, which stimulates the phosphatase PP3, which dephosphorylates DRP1. DRP1 is then recruited to the outer mitochondrial membrane. 48 Neutrophils can stimulate macrophages to initiate inflammation, as they release neutrophil extracellular traps (NETs), that is, they cause NETosis. PADI4 citrullinates histones, hence priming formation and release of NETs. 49 Activated NLRP3 inflammasome causes cardiac inflammation, cell apoptosis, and fibrosis [675]. ROS engenders Ca2+ influx through TRPM2, thereby enabling oligomerization of inactive NLRP3. The RAGE–TLR4 heterodimer triggers via NFκB the formation of inactive NRLP3, proIL1β, and proIL18.

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clock and cellular metabolism (Sect. 6.1).50 Hydrogen peroxide hyperpolarizes and hence dilates arterioles via Ca2+ -activated K+ channels. In the heart, hydrogen peroxide induces proliferation of mouse embryonic stem cells in addition to neonatal cardiomyocytes [675]. Scavengers of ROS such as antioxidant enzymes (e.g., superoxide dismutases, catalases, glutathione peroxidases and reductase, thioredoxins, thioredoxin reductases, methionine sulfoxide reductases, and peroxiredoxins) limit the amount of RO. Synthesis of many of these antioxidant enzymes and that of glyoxalase Glo1, which prevents modification of proteins by methylglyoxal, the major AGE precursor, and transketolase, which controls the non-oxidative branch of the pentose phosphate pathway, is elicited by the transcription factor nuclear erythroid-derived factor NFE2-related factor, NFE2L2, which is constitutively formed [675]. Activation of transketolase precludes the DAG–PKC, methylglyoxal–AGE, and hexosamine pathways and hyperglycemia-induced NFκB activation. However, in diabetic patients, NFE2L2 production declines. Diabetes increases the conversion of aldose reductase substrates; the polyol pathway is implicated in several diabetic complications [675]. Cytosolic NADPHdependent aldose reductase catalyzes the reduction of hydrophobic and -philic carbonyl-containing compounds (e.g., glucose and several glycolytic intermediates) to their corresponding alcohols such as sorbitol. The latter is converted to fructose by NAD+ -dependent sorbitol dehydrogenase. In addition, aldose reductase accelerates atherogenesis, at least in mice. Diabetes favors protein O-GlcNAcylation [675]. Excessive amounts of intracellular glucose increase the level of fructose 6-phosphate (F6P), which is converted by glutamine:F6P amidotransferase to glucosamine 6-phosphate, which is further converted to N acetylglucosamine 6-phosphate and finally to UDPN acetylglucosamine. The latter is used by O GlcNAc transferase (OGT) to add N acetylglucosamine to 50 In

the liver, autonomous circadian clocks promote lipid catabolism, gluconeogenesis, and mitochondrial genesis during sleep and fasting, and lipogenesis, glycogen synthesis, and cholesterol and bile acid synthesis in the awake and feeding state. In the adipose tissue, they promote lipolysis during fasting and lipogenesis during feeding. In muscles, they promote oxidative metabolism during fasting and fatty acid uptake during feeding. PGC1α stimulates the transcription of clock genes [675]. Metabolic pathways are influenced by the circadian clock, and linked transcription factors reciprocally influence the circadian clock. Major metabolic coupling messengers include NAD+ , SIRT1, AMPK, and ROS. Sirtuin-1 deacetylates (inhibits) the CLOCK–BMAL1 complex and activates PGC1α and LKB1, the latter stimulating AMPKα2. AMPK controls proteolytic degradation of Per and Cry. Reduced deacetylation by SIRT1 lowers activity of LKB1, PGC1α, and AMPKα2, repressing mitochondrial genesis, raising ROS production, and disturbing synchronization by the circadian clock of glucose and lipid metabolism. The Krüppel-like factor KLF15 controls rhythmic expression of enzymes involved in glucose, lipid, and nitrogen metabolism. Declining KLF15 synthesis suppresses mitochondrial respiration and induces superoxide production. In addition, the circadian rhythmicity of KLF15 controls the cardiac expression of KV -interacting protein-2, a subunit required for the transient outward potassium current. Abnormal activity of KLF15 favors ventricular arrhythmias.

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proteins. Conversely, N acetylglucosaminidase (OGA) removes this modification. Alternative splicing of transcripts encoding OGT and OGA yields isoforms in the nucleus, cytoplasm, and mitochondria. The hexosamine pathway can lower mitochondrial function and autophagy in addition to myocardial contractility. Inhibition of NOS3, which is required for endothelium-dependent arterial vasodilation and mobilization of stem and progenitor cells from the bone marrow, results from its O-GlcNAcylation (Ser1177) and reduced activity of the PI3K–PKB axis, which is normally stimulated by insulin. O-GlcNAcylation of the NFκB inhibitor TNFα IP3 favors its ubiquitination and proteasomal degradation, especially in coronary endotheliocytes and SMCs. In addition, overactivity of OGT attenuates serca2 transcription. OGlcNAcylated (activated) CamK2 (Ser279) phosphorylates RyR2 and increases intracellular Ca2+ , thereby lessening myocardial contractility and engendering arrhythmias. Diabetes and associated hyperglycemia increase the formation of AGEs by glucose-derived dicarbonyls reacting with amino groups of unprotonated lysine and arginine residues of proteins [675]. The precursor methylglyoxal generated by the non-enzymatic fragmentation of the glycolytic intermediate triose phosphate upon exposure to hyperglycemia and excessive ROS amounts accounts for most AGE adducts in diabetic cells, as it reacts with unprotonated Arg residues to produce methylglyoxal hydroimidazolone-1.51 Precursors of AGEs damage cells, as they (1) modify intracellular proteins52 and extracellular matrix components and (2) increase binding of the transcription factors NFκB and activator protein AP1 to the promoters of AGE receptor (RAGE) and RAGE ligands, respectively, eliciting overexpression of RAGE and proinflammatory S100a8–S100a12 heterodimer (calgranulin-A/B). Diabetes stimulates the protein kinases PKCβ, PKCδ, and PKCθ, as elevated intracellular glucose concentration raises the diacylglycerol level and ROS can oxidize (activate) PKCs [675]. Stimulated PKCs affect the activity of the enzymes of the MAPK module and cytosolic phospholipase-A2 in addition to Na+ –K+ ATPase and several transcription factors. They cause endothelial dysfunction with increased vascular permeability and impaired angiogenesis along with apoptosis and vascular inflammation. In the myocardium of diabetic mice, PKCβ2 upregulates expression of the growth factors CTGF and TGFβ1, leading to fibrosis and cardiomyopathy. In diabetes, ROS in excess provokes an elevated intracellular calcium concentration and hence activates calcium-dependent NFAT isoforms (NFAT1–NFAT4), which are dephosphorylated by PP3, which is in turn stimulated by the Ca2+ 51 Intracellular methylglyoxal is detoxified by glyoxalase-1, which, in cooperation with glyoxalase-

2 and glutathione, reduces this highly reactive α-oxoaldehyde to D lactate [675]. Methylglyoxal hydroimidazolone-1 is linked to elevated concentrations of the chemokines CCL2 and CXCL8 and augmented MMP9 activity. 52 In the heart, methylglyoxal preferentially reacts with the calcium carriers RyR2 and serca2a [675]. Methylglyoxal also activates the unfolded protein response in cardiomyocytes, prolonged activation triggering apoptosis.

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activated neutral cysteine peptidase, calpain. Dephosphorylation enables NFAT nuclear translocation. In the nucleus, PARP1 adpribosylates NFATs, thereby launching cytokine gene transcription. Diabetes also provokes a sustained activation of EGR1, which primes the formation of vcam1 and tissue factor. In diabetes, inhibition of prostacyclin synthase shunts prostaglandin-H2 , the common precursor of prostacyclin and thromboxane, to thromboxane synthesis, thereby favoring vasoconstriction, platelet aggregation, expression of adhesion molecules, and apoptosis [675]. Diabetes is associated with a downregulation of serca2a transcription and increased phosphorylation by CamK2 of the ryanodine receptor [675]. Cardiac autonomic neuropathy in almost 50% of diabetes and coronary atherosclerosis is linked to an increased risk for cardiac arrhythmias. Reactive oxygen species oxidize certain microRNAs. Oxidized miR184 targets the antiapoptotic proteins BCLw and BCL2L1, thereby sensitizing cardiomyocytes to apoptosis [675]. ROS downregulate the formation of miR133a, miR373, and miR499 in diabetic cardiomyocytes. Insulin resistance upregulates miR1281 production. In diabetes, overexpression of miR451 reduces phosphorylation (activation) by AMPK of LKB1. Redox stress oxidizes nAchR α3 subunit, hence depressing synaptic transmission in the autonomic ganglion [675].

Chapter 5

Hyperlipidemias and Obesity

The organs require oxygen and other types of nutrients (amino acids, sugars, and lipids) to function, the heart consuming large amounts of fatty acids for oxidation and adenosine triphosphate (ATP) generation. In the postprandial period, adipocytes take up fatty acids delivered by chylomicrons and lipoproteins synthesized in the gut and liver, which then form triglycerides (TGs). TGs are stored in lipid droplets (LDs) in an inert healthy form. Adipocytes can also synthesize fatty acids from excess circulating glucose using acetyl-CoA carboxylase, although the liver is the major site of conversion of carbohydrates to lipids. In the postprandial period, insulin promotes glucose and fatty acid uptake in addition to lipogenesis and suppresses lipolysis. Conversely, secretion of adipokines and lipokines by adipocytes contributes to metabolism regulation. Regulators secreted by the adipose tissue (AT) have local effects, that is, target adipocytes and vascular cells, along with a global systemic impact. During periods of restricted nutrient supply and prolonged physical activity, organismal survival and muscular activity depend on the ability of adipocytes to process excess triacylglycerols temporarily stored in LDs. Lipolysis by adipose triglyceride lipase (ATGL; or patatin-like phospholipase PnPLA2), hormonesensitive lipase (lipase-E; HSL), and monoglyceride lipase releases fatty acids and glycerol. Fatty acids are conveyed in the bloodstream in an esterified form as TGs in lipoproteins and unesterified form bound to albumin. Esterified fatty acids are liberated by lipoprotein lipase on the luminal side of the vascular endothelium. Fatty acids are also carried by lymph. They are then used for ATP synthesis, whereas glycerol can serve as a substrate for gluconeogenesis in the liver [717]. Uptake of circulating lipids, lipogenesis, and lipolysis and hence adipocyte metabolism depend on the balance between anabolism and catabolism. These processes are regulated by endocrine messengers and the sympathetic nervous system. © Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0_5

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When nutrient intake exceeds energetic expenditure during a prolonged period, the AT, liver, and muscle store lipids and engender adipose tissue growth, insulin resistance, and persistent hyperglycemia. Adipose tissue grows by hyperplasia (cell number increase) and hypertrophy (cell size increase). Excess food intake and the modern Western diet, which is rich in carbohydrates (high-carbohydrate diet) and lipids (cholesterol and saturated fatty acids in addition to mono- [MUFA] and polyunsaturated fatty acids [PUFA] connected to hydrogen atoms; high-fat diet [HFD]), that is, high-carbohydrate, high-fat diet enriched in caloric drinks and alcohol overconsumption coupled with a sedentary lifestyle, engender obesity. On a microscopic scale, chronic intake of excess nutrients enlarges LDs. On a mesoscopic scale, it causes adipocyte hypertrophy and, on a macroscopic scale, a gradual increase in weight. Products of adipocytic lipid metabolism, lipokines, act as beneficial endocrine regulators. In particular, both palmitoleate (C16:1) and palmitic acid– hydroxystearic acid, formed in adipocytes during lipogenesis, can improve systemic glucose metabolism [717]. On the other hand, impaired TG storage in overloaded adipocytes is associated with constitutive fatty acid mobilization, reduced glucose uptake, and lipogenesis, in addition to generation of lipotoxic diacylglycerol and ceramides, which can accumulate in remote organs. Adipocyte hypertrophy correlates with dyslipidemia, inflammation, and impaired glucose homeostasis in humans, whereas adipocytes are smaller in metabolically healthy obese individuals (without metabolic disease) [717]. On the other hand, limited storage capacity and a concomitant increase in ectopic lipidic depots are major contributors to metabolic disease genesis. Various intermediates of fatty acid metabolism engender lipotoxicity in adipocytes in addition to myocytes, hepatocytes, and immunocytes [717]. In particular, diacylglycerol can activate some PKC subtypes, thereby hindering insulin signaling. In addition, saturated fatty acids connect to toll-like receptor TLR4, thereby favoring inflammation and insulin resistance (InsRce). Ceramides can interfere with insulin signaling and mitochondrial oxidation, provoking endoplasmic reticulum (ER) stress and apoptosis. Hyperlipidemia,1 the most common form of dyslipidemia,2 comprises: • Pure hypercholesterolemia • Isolated hypertriglyceridemia • Mixed hyperlipidemia (combined hypercholesterolemia and triglyceridemia) Obesity is often associated with insulin resistance and elevated insulin secretion from pancreatic β cells. Hypertrophic adipocytes have a reduced autonomous ability for insulin-stimulated glucose uptake. The lipid mediator leukotriene-B4 and macrophage-derived galectin-3 are implicated in obesity-linked systemic insulin

1 That 2 That

is, high blood concentrations of lipids and cholesterol. is, abnormal blood concentrations of lipids and lipoproteins.

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resistance [717]. Hyperglycemia is the consequence of the absence of compensation of insulin resistance by increased glucose-stimulated insulin secretion. The energy sensor AMPK promotes glucose uptake independently of insulin. This kinase constitutes 12 distinct complexes. A potent allosteric activator of all 12 AMPK complexes provokes a robust, sustained insulin-independent glucose uptake and glycogen synthesis in skeletal muscles but causes cardiac hypertrophy and an increased amount of cardiac glycogen [718]. The TORC1–S6K1 axis and its effectors, such as glutamyl prolyl tRNA synthetase (EPRS), are related to metabolism contributing to adiposity and aging [719].3 Upon phosphorylation (Ser999) by S6K1, EPRS is released from the aminoacyl tRNA multi-synthase complex for execution of tasks other than protein synthesis. In adipocytes, insulin stimulates EPRS phosphorylation. Phosphorylated EPRS binds to SLC27a1 (or fatty acid transport protein FATP1) and provokes its translocation to the plasma membrane for long-chain fatty acid uptake. Obesity raises the risk for metabolic and cardiovascular disease (CVD). However, excess AT in the trunk (android obesity), a compartment of TG-storage white adipose tissue (WAT), exerts more deleterious metabolic effects than fat depots in the limbs (gynoid obesity). Furthermore, among subcutaneous (scAT), subfascial, and visceral adipose tissue (vAT), which comprises omental, mesenteric, and periorgan adipose compartments, omental and mesenteric fat depots drained by the portal circulation have the strongest metabolic effects [720].4 In addition, local obesity around large blood vessels, perivascular adipose tissue (pvAT), including the perivascular compartment of epicardial adipose tissue (eAT), is involved in cardiovascular system regulation and pathogenesis. Obesity is associated with a shift from secretion by the AT of vasodilators to release of the vasoconstrictors and promoters of cell proliferation and migration and inflammation. These agents can cause endothelial dysfunction, which is reversible by weight loss, which also lowers AT-related inflammation and raises NO availability. In obesity, the endothelium loses its control of the vasomotor tone, vascular smooth myocyte (vSMC) proliferation, blood coagulation, and inflammation, thereby favoring adverse vascular remodeling.

3 Aminoacyl–tRNA

synthetases charge tRNAs with their cognate amino acids, a first step in mRNA translation. At least one synthetase exists for each amino acid. Glu/Pro-tRNA synthetase (GluProRS) is also named bifunctional glutamate– and proline–tRNA ligase and prolyl–tRNA synthetase (PARS). It catalyzes the attachment of the cognate amino acid to the corresponding tRNA. It is also a component of the γ-interferon-activated inhibitor of translation (GAIT) complex that represses Ifnγ-induced translation of diverse selective mRNAs in inflammation [108, 194]. 4 Adipocytes linked to the portal circulation are exquisitely sensitive to stimuli that mobilize free fatty acids (FFAs) owing to a high number of β-adrenoceptors and relatively weak inhibition by α-adrenoceptors. Exposure of the liver to elevated FFA concentrations increases synthesis and secretion of very low-density lipoproteins (VLDLs) and stimulates gluconeogenesis. It can also cause hepatic insulin resistance and reduce hepatic clearance of insulin [720].

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The grade of obesity is currently assessed according to the body mass index (BMI) in children and adults [226]: • Normal weight (BMI = 18.5–24.9 kg/m2 ; 5th– 40 kg/m2 ) This index is a height-normalized body mass (body mass [kg] divided by squared height [m]), that is, the sum of body fat and fat-free mass. Obese individuals have both high levels of fat and fat-free mass [721]. This index is thus not an index of adiposity, but instead an index of cardiometabolic risk. Higher fat-free mass is associated with a greater blood volume and hence cardiac load in obese individuals. Abdominal obesity is defined according to waist circumference (men >94 or 102 cm and women >80 or 88 cm according to national health agencies). Over the past few decades, abdominal obesity has augmented significantly in both sexes. In CVD and type-2 diabetes mellitus (T2DM), the BMI-related obesity paradox5 refers to overweight patients who have lower cardiovascular morbidity with respect to lean individuals, although obesity is a major risk factor of CVD. Abdominal (omental) adiposity assessed by waist circumference (in men >102 cm and women >88 cm) and waist-to-hip ratio (in men and women ≥0.90 and 0.85, respectively) usually weaken the survival rate with respect to overweight in other body compartments. Overweight and obese people have a higher rate of cardiac events than individuals with a normal weight, but overweight metabolically healthy (and not obese) people can have a higher survival rate than underweight subjects. An inverse correlation may be observed between mild adiposity and cardiorespiratory fitness in subjects with a higher exercise capacity who do not have other risk factors, such as insulin resistance and hypertension. A BMI–mortality plot is U- or J-shaped with a minimum close to a BMI of 25 kg/m2 . However, other types of diseases can explain this observation. Moreover, in aged patients (62–66 years), BMI is a better indicator of lean body mass than adiposity [723]. When data supporting the obesity paradox are adjusted by cardiorespiratory fitness, the paradoxical association between BMI and mortality is blunted [724]. The obesity paradox is questionable because of the insufficient control of cardiorespiratory fitness, inadequate determination of AT location, among other sources of bias. Low-risk adiposity may be related to a proper adiposecretome, regional AT distribution, and adipocyte turnover rate, in addition to the type of AT expansion (hyperplasia versus hypertrophy), matrix quality without adverse remodeling, angiogenic potential, adipocyte browning capacity, and macrophage density [722].

5 When

other obesity indices are used, the concept of obesity paradox disappears [722].

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In any case, weight reduction in CVD patients improves insulin sensitivity, blood pressure, lipidemia, reactivity of platelets, and endothelial function. In addition to weight loss, physical exercise and adequate diet enable prevention of CVDs in obesity. Exercise training provokes secretion of myokines from skeletal muscles and cardiomyokines from the myocardium, such as FGF21 and irisin, which act on pvAT and eAT.

5.1 Classification and Etiology of Dyslipoproteinemia Lipoproteins carry in the bloodstream water-insoluble TGs and cholesterol. They possess a core of hydrophobic cholesterol esters and TGs surrounded by a hydrophilic monolayer of phospholipids, free cholesterol, and apolipoproteins (Apos). The lipoprotein types include chylomicron; chylomicron remnant; very-low(VLDL), intermediate- (IDL), low- (LDL), and high-density lipoprotein (HDL); and lipoprotein-A (LPa), which are composed of distinct types of apolipoproteins. Their density depends on their content in TGs and cholesterol. Chylomicrons, chylomicron remnants, VLDLs, and IDLs are rich in both TGs and cholesterol, whereas LDL, HDL, and LPa mainly contain cholesterol and have a higher density. Lipoproteins are produced by exo- and endogenous pathways: 1. The exogenous pathway is related to chylomicrons produced by enterocytes. Chylomicrons are converted to chylomicron remnants by TG lipolysis in plasma. 2. The endogenous pathway is related to VLDLs assembled in hepatocytes. They are converted to IDLs and LDLs by TG lipolysis on the wetted surface of the capillary endothelium and in the vascular lumen and by exchange of lipids and apolipoproteins with other lipoproteins. After secretion of chylomicrons from the intestine and VLDLs from the liver, both types of lipoproteins are enriched in ApoE during their degradation to remnants [725]. Apolipoprotein-E, a ligand for receptors LDLR and LRPs, enables, at least partly, remnant uptake in the liver. Remnant lipoproteins can accumulate in the arterial wall and cause inflammation, especially in patients with familial dysbetalipoproteinemia who have a 7.7- to 14.3fold increased remnant cholesterol concentration [726]. Monocytes have a higher content of LDs and a greater surface concentration of integrins. On the other hand, HDLs have an anti-inflammatory, antioxidant, antithrombotic, and vasorelaxant action and promote the reverse cholesterol transport (RCT) from atherosclerotic lesions. According to the National Center for Biotechnology Information, dyslipidemias, a lipid overproduction or deficiency with abnormal serum lipid profiles including high concentrations of total cholesterol (CS), TGs, LDLCS , and low concentrations of HDLCS , include:

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Hyperlipidemias that comprise: 1. Hypercholesterolemia (blood cholesterol concentration exceeding the 95th percentile for the population 2. Familial combined hyperlipidemia (hypercholesterolemia and hypertriglyceridemia) engendered by multiple gene defects 3. Hyperlipoproteinemias 4. Hypertriglyceridemias, among which • Type-4 hyperlipoproteinemia • Type-5 hyperlipoproteinemia • Hypertriglyceridemic waist (elevated waist circumference and fasting blood concentrations of TGs) Hypolipoproteinemias (abnormally low blood concentrations of lipoproteins)6 with 1. Hypoalphalipoproteinemia (low blood HDL concentration), which can be associated with mutations in the genes encoding apolipoprotein-A1, lecithin– cholesterol acyltransferase (LCAT),7 and ATP-binding cassette transporters 2. Hypobetalipoproteinemia (low blood LDL concentration ≤ the 5th percentile for the population) consisting of autosomal dominant disorder linked to APOB gene mutations and autosomal recessive disorder resulting from mutation of the gene encoding microsomal TG transfer protein Smith–Lemli–Opitz syndrome, an autosomal recessive disorder of cholesterol metabolism caused by a deficient 7-dehydrocholesterol reductase Dyslipoproteinemias are inherited (primary) or acquired (secondary). Primary dyslipoproteinemias can be genetic or a result of a defect in lipoproteins (Sect. 7.4). Behavioral factors (e.g., diet, alcohol, and drugs) can contribute to primary dyslipoproteinemia. Secondary dyslipoproteinemias are provoked by sedentary lifestyle with excessive food intake. They can be caused by diseases such as diabetes, hypothyroidism, chronic kidney disease, pancreatitis, cholestatic liver disease, dysglobulinemia, and autoimmune hyperlipoproteinemia, in addition to some drugs. Low HDL concentrations can result from smoking, anabolic steroids, and

6 Whereas

the HDL1 subtype is not associated with CVD risk, concentrations of large and more buoyant HDL2 and small and dense HDL3 significantly decrease in coronary artery disease [727]. 7 Lecithin–cholesterol (phosphatidylcholine–sterol) acyltransferase is a major determinant of the metabolism of plasmatic lipoproteins. It is synthesized mainly in the liver and secreted into plasma, where it converts cholesterol and phosphatidylcholine (PC; lecithin) to cholesteryl esters and lysophosphatidylcholine (LPC) on the surface of HDLs and LDLs. The reciprocal transfer of cholesterol esters and TGs among lipoproteins relies on cholesterol ester transfer protein, which enables movement of cholesteryl ester from HDLs to TG-rich VLDLs and the equimolar motion of TGs from VLDLs to HDLs.

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nephrotic syndrome. On the other hand, intestinal postprandial overproduction of atherogenic chylomicrons is observed in diabetic dyslipidemia with insulin resistance. Acquired combined hyperlipidemia is common in patients with the metabolic syndrome. Treatment is aimed at correcting levels of LDLCS , TGs, and HDLCS to achieve a reduction in CVD risk. Combination therapy associating statins with niacin, fibrate, and ω3-fatty acids, among other drugs, has been proposed, especially in patients with mixed dyslipidemia and insulin resistance.

5.1.1 Hypercholesterolemia Hypercholesterolemia is a major cardiovascular risk factor. Atherosclerosis is initiated by retention and accumulation within the arterial intima of cholesterolrich ApoB+ lipoproteins, that is, LDLs (∼90% of circulating ApoB+ lipoproteins during fasting) and other species (diameter 10 mmol/l [>880 mg/dl]), proper drugs should be administered. It results from severely dysregulated diabetes, alcoholism, and rare cases of homozygous mutations in some genes (Sect. 7.4). The transcription factor CREB3L3,11 which may act during ER stress via activation of unfolded protein response target genes, is a determinant of TGs in humans. Rare loss-of-function mutations in the human CREB3L3 gene are associated with hypertriglyceridemia [286].

5.1.2.1

Apolipoprotein-C3

ApoC3 is a VLDL constituent and hence a regulator of TG and TGRL metabolism. In fact, ApoC3 resides on the surface of TGRLs, mainly VLDLs and, to a lesser extent, IDLs. In humans, ApoC3 is much smaller than the other major apolipoproteins regulating lipoprotein metabolism (ApoA1, ApoB100, and ApoE) [739]. In the human genome, the APOA1, APOC3, and APOA4 genes are tandemly organized in a short region on the chromosomal 11q23-q24 region. The APOA1 and APOA4 genes are transcribed from the same strand. The APOA1 and APOC3 are convergently transcribed. The lipid metabolism regulation relies on tissue-specific expression profiles of genes in the APOA1–APOC3–APOA4–APOA5 cluster and DNA demethylation [740]. ApoC3 inhibits lipoprotein (lipase-D) and hepatic lipase (lipase-C) in addition to hepatic lipoprotein receptors, thereby raising TG concentration, delaying clearance of ApoB+ lipoproteins, including large ApoB+ and ApoE+ LDLs and atherogenic remnants, and accelerating conversion of light to smaller dense proatherogenic LDLs [739]. ApoC3 not only inhibits hydrolysis by LipP of TGs in TRLs and counters conversion of VLDLs to IDLs and LDLs by LipC but also lowers hepatic uptake of TRLs through LDLR and LRP1 and, on light LDLs, enhances conversion to dense LDLs [741]. The smaller dense proatherogenic LDLs in addition to VLDL and remnant lipoproteins (small VLDLs and IDLs) enter the arterial intima, where they are oxidized and taken up by macrophages. Macrophages that reside

11 CREB3L3:

cAMP-responsive element-binding protein-3-like protein-3, also dubbed CREBH.

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in the arterial intima secrete LipD, which can process TRLs to remnants, which can then be taken up by macrophages, lipid accumulation generating foam cells. Enrichment of ApoC3 in ApoB+ lipoproteins (VLDLs and LDLs) increases the risk for atherosclerosis. Furthermore, ApoC3 favors inflammation, as it activates NFκB and thus elicits vcam1 production [739]. ApoC3 is independently associated with risk for chronic obstructive airway disease (CoAD), especially in subjects with hypertriglyceridemia [741]. ApoC3 concentration correlates positively with concentrations of TGs, VLDLs, IDLs, small dense LDLs, and C-reactive protein, and negatively with the concentration of large LDLs.

5.1.2.2

Very-Low-Density Lipoprotein

The liver stores about 100-fold less TGs than AT but secretes fatty acids and TGs lipidated in VLDLs at a similar rate [742]. A small pool of LDs is permanently and efficiently processed in hepatocytes to produce VLDLs. Kinesin nanomotors are recruited to LDs, which store cholesterol, TGs, and sphingolipids, by the small GTPase ARF1, which also activates lipolysis, upon insulin stimulation. Upon feeding, ARF1 and kinesin localize to TG-rich LDs and transfer them at the periphery of hepatocytes to the smooth endoplasmic reticulum (sER), which contains lipases that process LD content and is the VLDL assembly site [742]. In the fasting period, insulin action drops, ARF1 and kinesin are removed from LDs, and LD–sER contacts disappear, impeding lipid supply to the sER and hence TG availability for VLDL assembly. Adipose tissue-derived fatty acids reach the liver and are esterified into TG in hepatocytes leading to massive accumulation and protecting organs from lipotoxic FAs and TGs, whereas the plasmatic TG concentration remains nearly constant during the feeding–fasting cycle. Increased hepatic secretion of TG-rich VLDLs is a major determinant of hypertriglyceridemia. Hepatic VLDL overproduction is a common feature of insulin resistance. Disrupted subdiaphragmatic vagal signaling decreases circulating VLDL TG concentration, but raises concentration of glucagon-like peptide GLP1, thereby reducing the synthesis of sterol regulatory element-binding protein, SREBP1c, stearoylCoA desaturase, SCD1, and fatty acid synthase (FAS), but enhancing hepatic insulin sensitivity [743]. Whereas pre-VLDLs can be degraded, TG-poor VLDLs are converted to mature TG-rich VLDLs by the addition of TGs derived from LDs (Sect. 5.4.4). Lipid droplets are composed of a core surrounded by a phospholipid monolayer and 229 types of protein [744]. Ancient ubiquitous protein AUP1 is the first identified ER- and LD-associated protein. It is implicated in the degradation of ubiquitinated misfolded proteins in the ER (ER-associated protein degradation [ERAD]) of misfolded proteins, that is, in the quality control of proteins in the ER and LD clustering [744]. It is implicated in the retrotranslocation of misfolded proteins from the ER lumen to the cytosol

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for proteasomal degradation. Its C-terminus binds to ubiquitin conjugases such as UbE2g2 (UbC7 homolog). It contributes to ubiquitination and degradation of several regulators of lipid synthesis, such as 3-hydroxy 3-methylglutaryl coenzymeA reductase, thereby affecting the number and size of LDs [744]. It controls formation of apolipoprotein-B100, LD mobilization, and hepatic VLDL assembly and secretion. This determinant of hepatic VLDL metabolism interacts on the LD surface with ApoB100, the main structural protein of hepatic VLDLs, which connects to many chaperones (heat shock proteins) involved in the ERAD pathway.12 Lipidated ApoB100 translocates from the ER lumen to the LD surface. ApoB is lipidated to form pre-VLDL, whereas incorrectly folded and lipidated ApoB is destroyed. Accumulation of AUP1 on LDs hampers ApoB lipidation and VLDL assembly, independently of the MAP2K1/2–ERK pathway; the average size of the LDs declines; smaller underlipidated ApoB+ lipoproteins are secreted instead of fully lipidated VLDLs. Hepatic AUP1 production lowers in obese subjects and in those with non-alcoholic fatty liver disease (NAFLD; Sect. 5.3.4) and T2DM patients (diabetic dyslipidemia) [744].

5.1.2.3

Triglycerides and Protein-C

Triglycerides and LDLCS , but not HDLCS , are significantly associated with proteinC deficiency [745]. They may modulate protein-C synthesis or degradation. Protein-C is a vitamin-K-dependent glycoproteic zymogen produced by the liver. Once activated by the thrombin–thrombomodulin complex, protein-C is an anti-coagulant, anti-inflammatory, antiapoptotic, and cytoprotective molecule. Activated protein-C inactivates factor-V a and factor-V I I I a, thereby reducing thrombin generation. Autosomal dominant mutations in the PROC gene cause protein-C deficiency, thereby favoring venous thromboembolism. Single-nucleotide polymorphisms in the chromosomal regions linked to protein-C-influencing genes, that is, in Whites, the BAZ1B13 and GCKR loci14 in addition to, in both Whites and Blacks, the

12 The

chaperone HSPa5 of the ER lumen is involved in proteasomal degradation of misfolded apoB100 [744]. The MAP2K1/2–ERK pathway involved in crosstalk with ERAD impedes the synthesis, packaging, and secretion of mature VLDLs. On the other hand, adipose differentiationrelated protein, another LD-associated protein, which counters glucose tolerance and insulin sensitivity in the liver and skeletal muscle, can increase VLDL secretion in addition to LD size, but lowers their number. Cell death-inducing 45-kDa DNA-fragmentation factor, DFFα-like effector CIDEb, an apoptosis activator inhibited by DFFα, localizes to both the ER and LDs. It links to ApoB, facilitates LD clustering and fusion, and promotes VLDL assembly and secretion [744]. 13 BAZ1B: gene encoding bromodomain adjacent to zinc finger domain-containing protein-1B (BAZ1b), a kinase that phosphorylates H2a.x. 14 GCKR: gene encoding glucokinase regulator.

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PROC, PROCR–EDEM2,15 and the CELSR2–PSRC1–SORT1 region on chromosome 1 (1p13.3),16 especially rs12740374, a variant that influences LDLCS , disturb circulating protein-C concentration [745]. Among the three lipidic fractions, TGs influence protein-C concentration.

5.1.3 Dyslipoproteinemias Hyperlipidemia is also called hyperlipoproteinemia, as it usually results from altered lipoprotein metabolism. Lipoproteinemia thresholds are determined according to age and sex. Hyperlipoproteinemia is defined by abnormally elevated plasmatic concentrations of a given or many lipoprotein species. Dyslipoproteinemia is a more relevant term as anomalies of the lipidic checkup encompass not only high concentrations of LDLCS and TGs but also low HDLCS concentration. In general, dyslipoproteinemia also refers to abnormal plasmatic concentrations of lipoproteins and their associated apolipoproteins above the 90th percentile of the general population for total CS, LDL, TG, ApoB, and LPa or below the 10th percentile for HDL and ApoA (Vol. 11, Chap. 5. “Lipoproteins”). Dyslipoproteinemia is, in general, defined quantitatively by: 1. Elevated concentrations of: • Total cholesterol (≥239 mg/dl [6.2 mmol/l]) • LDLCS (≥3.36 mmol/l [≥130 mg/dl]) • TGs (≥1.69 mmol/l [≥150 mg/dl]) 2. Diminished concentration of HDLCS (≤1.03 mmol/l [≤40 mg/dl] for men and ≤1.29 mmol/l [≤50 mg/dl] for women). Concentration of LDLCS is most often measured in blood sampled during fasting, only when TG concentration is lower than 400 mg/dl. It is defined by the amount of cholesterol that is not contained in HDLs, VLDLs, and chylomicrons but incorporates the content of IDLs and LPa. It is calculated with an estimated cholesterol concentration in VLDLs being one-fifth of the total lipid content in VLDLs: [LDLCS ] = [CStot ] − ([HDLCS ] + [TG]/5).

15 PROCR:

(5.1)

gene encoding protein-C receptor; EDEM2: gene encoding ER degradation-enhancing α-mannosidase-like protein-2. EDEM2 initiates the ER-associated degradation that targets misfolded glycoproteins. 16 CELSR2: gene encoding cadherin EGF LAG seven-pass G-type receptor-2; PSRC1; gene encoding proline- and serine-rich coiled-coil protein-1; SORT1: gene encoding sortilin, a multiligand receptor.

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However, concentrations of total cholesterol and TGs, which are related to all types of circulating lipoproteins (chylomicron, VLDL, IDL, LDL, and HDL), in addition to HDLCS and LDL, can also be measured directly. Remnant cholesterol is defined as the cholesterol content of a subset of TGRL (i.e., VLDLs and IDLs) remnants, that is, chylomicron remnants, VLDLs, and IDLs in the feeding state in addition to VLDLs and IDLs in the fasting state. Remnant cholesterol concentration can be estimated as total cholesterol level minus LDLCS and HDLCS concentrations, concentrations of TGRLs in the fasting state and of chylomicron remnants in the feeding state being included [738]. In most individuals, chylomicrons are very rapidly degraded to chylomicron remnants due to TG hydrolysis by lipoprotein lipase. Remnant cholesterol concentration thus correlates with the TG level [725]. In plasma, TGs and cholesterol are exchanged between HDLs and remnants. Therefore, concentrations of HDLCS and remnant cholesterol are inversely correlated. An elevated concentration of remnant cholesterol causes cholesterol accumulation in the arterial wall, as does an augmented level of LDLCS . However, although an increased LDLCS level correlates with coronary artery disease, but not with low-grade inflammation, a heightened concentration of remnant cholesterol is associated with both low-grade inflammation and atherosclerosis. An increment of 1 mmol/l (39 mg/dl) in nonfasting remnant cholesterol concentration, which contributes to atherosclerosis, is associated with a 2.8-fold increase in the risk of cardiac ischemia [725]. Lipoproteins enriched in TGs (>2 mmol/l [176 mg/dl]) in addition to cholesterol or remnant cholesterol concentrations (>1 mmol/l [39 mg/dl]) are strong and independent predictors of atherosclerosis [738]. Triglyceride-rich lipoproteins contain both TGs and cholesterol, in addition to phospholipids and proteins. They are associated with low-grade inflammation and atherosclerosis. Circulating ApoB+ TGRLs include intestinal and hepatic lipoproteins (chylomicrons, VLDLs, and their remnants). Augmented TGRL concentration alters LDL and HDL composition and function. An elevated TGRL concentration in the postprandial period can engender endothelial inflammation [746]. Epidemiological studies cannot distinguish cause from mere correlation. On the other hand, genetic analyses and randomized controlled trials can buttress the causal role in atherogenesis of lipid markers, especially proinflammatory and proatherogenic plasmatic lipoproteins (i.e., ApoB+ LDL, LPA , and TGRLs; Vol. 11, Chap. 5. “Lipoproteins”). Elevated plasmatic concentrations of total cholesterol and LDLCS cause atherosclerosis, as LDLs enter the arterial intima. Mid-sized TGRLs (intermediate size between LDL and chylomicrons) can also penetrate the arterial intima. Once they are entrapped in the intima, lipoprotein lipase at the endothelial surface or in the arterial intima degrades TGs and liberates toxic FFAs and monoacylglycerols. Lipoprotein lipase is also synthesized in macrophages.

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345

High-Density Lipoprotein

Circulating HDLs comprise subpopulations according to their size, shape, charge, and lipid and protein composition. These particles can indeed be categorized according to their density and size, particularly in lipid-rich large HDL2 and proteinrich small HDL3 subpopulations. Constituents of HDLs enter the plasma separately and are assembled into HDLs within the plasma [747]. Multiple plasmatic factors remodel HDLs, removing HDL constituents separately from blood rather than as the cellular uptake of intact HDls. High-density lipoproteins protect against atherosclerosis, as they have antioxidant, anti-inflammatory, and anti-thrombotic effects, favor cholesterol egress from macrophages lodging in the artery wall, enhance endothelial function, promote endothelial repair, and support angiogenesis. Certain HDL constituents are responsible for their antioxidant and anti-inflammatory properties, whereas others serve in HDL transfer, especially in the RCT. Reverse cholesterol transport comprises cholesterol efflux from ABCa1+ macrophages to apolipoprotein-A1, which creates nascent HDLs followed by esterification of free cholesterol, hepatic extraction of HDLs, and hepatic conversion of HDLCS to bile salts, which are then excreted [748]. Cholesterol esterification by LCAT is a minor process in nascent HDL metabolism. Most free cholesterol of nascent HDLs and phospholipids is rapidly extracted by hepatocytes via ScaRb1, hepatic phospholipid uptake being promoted by phospholipid transfer protein; they are quickly transferred to HDLs and LDLs. On the other hand, ApoA1 is only transferred to HDLs and to the lipid-free form that can be recycled to nascent HDLs. Large human population studies have demonstrated that low HDL-emia predicts a high probability of a CVD event. On the other hand, in mice and rabbits, intravenous infusions of HDLs or increased synthesis of ApoA1, a major HDL apolipoprotein, markedly decreases susceptibility to atherogenesis. However, in humans, gene variants that raise plasmatic HDLCS concentration are not accompanied by a reduced risk of CVD events. Increased HDLCS concentration via inhibition of cholesteryl ester transfer protein (CETP) fails to reduce CVD events. In addition, the relation between HDLCS concentration and mortality is U-shaped, whatever the gender, both extremely high and low concentrations being associated with high mortality risk [729]. Low HDL-emia represents a strong independent cardiovascular risk marker in subjects with heterozygous familial hypercholesterolemia. Familial hypercholesterolemia is linked to mono- and polygenic defects. Low HDL-emia can be associated with environmental factors and lifestyle, and with primary hypoalphalipoproteinemia. A high [CStot ]/[HDLCS ] ratio is related to the extent and severity of atherosclerotic lesions.

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Reverse Cholesterol Transport In addition to other protective effects, HDL permits the elimination of excess cholesterol by RCT. This transfer mode corresponds to the return of an excessive amount of cholesterol from cells to the liver, where cholesterol is used to synthesize bile acids, which are excreted with the bile and partly with the feces. Bile acids facilitate the efficient solubilization and intestinal absorption of dietary lipids, cholesterol, and liposoluble vitamins. This protective metabolic pathway begins with cholesterol efflux from atherosclerotic plaque cells such as macrophagederived foam cells to HDLs. On the other hand, inflammation-responsive transcription factor C/EBPδ, which is detected in macrophages of atherosclerotic plaques, responds to modified LDLs via the P38MAPK–CREB pathway and favors lipid accumulation in M1 macrophages (but not M2 macrophages) [749]. It upregulates expression of pentraxin-3, which promotes LDL macropinocytosis, and downregulates expression of ABCa1, which impairs cholesterol efflux from M1 macrophages. After esterification of cholesterol in the plasma by LCAT, cholesteryl esters can be selectively delivered to the liver via the scavenger receptor ScaRb1. Alternatively, cholesterol ester transfer protein (CETP) can exchange cholesteryl esters for TGs on ApoB+ lipoproteins, which can deliver cholesterol to hepatocytes by receptor-mediated endocytosis. The biliary pathway consists of cholesterol conversion to primary bile acids and subsequent elimination from the liver that relies on transport across the canalicular surface by the ABCg5 and ABCg8 transporter or the bile salt export pump (BSEP, or ABCb11), the delivery of gallbladder bile to the intestinal lumen being intermittent and primed by food intake [750]. A fraction of cholesterol is reabsorbed in the proximal small intestine via Niemann–Pick-C1-like protein (NPc1L1) and bile acids in the distal small intestine through the combined actions of the apical sodium-dependent bile acid transporter and intestinal bile acid transporter, thereby limiting neutral and acidic sterol loss from the body [750]. The melanocortin 1 receptor (MC1R, or MC1 )17 resides on monocytes and macrophages and exerts anti-inflammatory actions, once it is linked to α-melanocytestimulating hormone, a peptidic hormone and neuropeptide of the melanocortin family.18 In atherosclerotic lesional macrophages, activated MC1 upregulates formation of ABCa1 and ABCg1 involved in initiating reverse cholesterol transport, thereby promoting cholesterol egress and countering foam cell generation [751]. In addition, MC1 lowers plasmalemmal ScaRb3 concentration and hence cholesterol uptake, further limiting lipid accumulation in macrophages.

17 A.k.a.

α-melanocyte-stimulating hormone receptor (MSHR).

18 MSH is an unselective ligand of the melanocortin receptors MC

which is exclusive for adrenocorticotropic hormone (ACTH).

1

and MC3 to MC5 , but not MC2 ,

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Transintestinal Cholesterol Excretion In addition to biliary cholesterol secretion, transintestinal cholesterol excretion (TICE) contributes to RCT. Liver-derived ApoB+ lipoproteins can deliver cholesterol to the basolateral surface of enterocytes, which is taken up by LDLR and other receptors. After transfer to the apical membrane, cholesterol exits into the intestinal lumen via ABCg5, ABCg8, and possibly ABCb1a and ABCb1b [752]. In mice, TICE (∼35% of fecal cholesterol removal in humans) is stimulated by liver X receptor (LXR) and farnesoid X receptor (FXR) agonists. However, most macrophage-derived cholesterol is excreted via the hepatobiliary route [752].

Endothelial Transcytosis of HDLs and LDLs The transmembrane class-B scavenger receptor ScaRb3 recognizes multiple ligand types. Its primary function is related to the capture and clearance of modified lipoproteins, advanced glycation products, apoptotic cells, microbial diacylglycerols, and Plasmodium-infected erythrocytes. In addition, it is involved in cellular uptake of long-chain fatty acids, hence its other name fatty acid translocase. Oxidized low-density lipoprotein (oxLDL) is a high-affinity ScaRb3 ligand. Diverse forms of oxidized lysophosphatidylcholine species (oxGPC) reside on the surface of lipoproteins and may serve as recognition elements by ScaRb3 [753]. Nutrients must traverse the endothelium to reach parenchymal cells. Fatty acid transport proteins FATP3 and FATP4 and ScaRb3 can carry fatty acids in endotheliocytes (ECs). Paracrine messengers secreted by parenchymal cells support nutrient transfer through the adjacent endothelium. For example, vascular endothelial growth factor b (VEGFb) is released from skeletal and cardiac myocytes to promote transendothelial lipid transport adapted to their metabolic need [754]. In ECs, the dynamics of LD genesis and degradation using diacylglycerol acyltransferase DGAT1 and adipocyte TG lipase, respectively, not only provides a fatty acid source for adjacent cells but also regulates endothelial glycolysis and protects ECs from lipotoxic stress [755]. Both LDLs and HDLs cross the vascular endothelium and exert their pro- and antiatherogenic activity, respectively, within the vascular wall. Accumulation of LDLs in the subendothelial layer of the intima causes atherosclerosis. Conversely, removal of cholesterol from the subendothelial space by RCT protects against atherosclerosis. In ECs, endocytosis of LDLs, which relies on the clathrin-dependent pathway and involves LDLR, leads to lysosomal degradation. The transendothelial LDL transfer implicates caveolae, ScaRb1, and activin-like kinase ALK1 [757]. Transcytosis of HDLs is carried out by ABCg1, ScaRb1, and endothelial lipase, in addition to the ectopic β-ATPase–nucleotide receptor axis [757]. Among modulators of transendothelial LDL and HDL transfer, VEGFa and VEGFR2 significantly lower the binding, uptake, and transcytosis of HDL, but

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not LDL, VEGFR1, and VEGFR3 having no impact on this transfer [757]. The VEGFa isoform elicits actin re-organization, hence contributing to HDL uptake. The VEGFa–VEGFR2 couple operates via the PI3K–PKB axis,19 P38MAPK (but not the Ras–Raf–MAP2K axis), and ScaRb1 in HDL uptake. Moreover, VEGFa promotes ScaRb1 localization in the plasma membrane of ECs; the VEGFa– VEGFR2-launched signaling acts as a rate-limiting factor for the plasmalemmal ScaRb1 concentration, thereby regulating HDL uptake by ECs.

CETP and S1P HDL particles are composed of apolipoproteins (e.g., ApoA1, ApoA2, and ApoM), antioxidant enzymes such as paraoxonase-1, LCAT, and diverse lipidic species (e.g., cholesterol esters, TGs, phospholipids, and sphingolipids such as sphingosine 1phosphate [S1P]) [759]. Endothelial function is restored by plasmatic HDLs. However, protection of the endothelium and vasodilation ensured by HDL depends on its S1P carrier. Sphingosine 1-phosphate is a vasoprotective lysophospholipid mediator, which is carried in blood by albumin (Alb) and ApoB+ lipoproteins; yet, plasmatic S1P travels mainly with ApoM+ HDLs, ApoM, a minor apolipoprotein type on HDLs, being a S1P carrier and modulator of its activity. The properties of S1P depend on its carrier, HDL or albumin. Effects of HDLS1P differ from those of AlbS1P in endothelial inflammation inhibition, barrier function,

19 PI3K

is involved in endosomal transfer. After a meal, insulin released from the pancreatic β cells triggers glucose uptake by target cells, such as skeletal myocytes and adipocytes. Insulin stimulates translocation of the glucose transporter GluT4 to the plasma membrane from specialized intracellular GluT4 storage vesicles via PI3K and a proteic octamer, the exocyst. This complex assembles at the site of exocytosis in response to insulin under the control of RhoJ and tethers GluT4+ vesicles to the plasma membrane via the PI3K–PKB axis and its effector RalA GTPase. The latter interacts with the nanomotor Myo1c complex, which facilitates recruitment of GluT4+ vesicles to the plasma membrane. Moreover, the Ral GAP complex (RGC) is implicated in RalA activation downstream from the PI3K–PKB pathway; it is composed of the RGC1 regulatory and RGC2 (or Akt [PKB] substrate of 250 kDa [AS250]) catalytic subunit [756]. Insulin inhibits the RGC1–RGC2 complex (RhoGAP4–RalGAPα2) via phosphorylation of RGC2 by PKB2, activated PKB, thus relieving inhibition of the RalGAP complex on RalA activity. Upon RalA activation due to inhibition of two GAP types, TBC1D4 (or Akt [PKB] substrate of 160 kDa [AS160]), a RabGAP, which inactivates Rab10 involved in insulin-stimulated GluT4 exocytosis and operates via the RalGEF RGL2, once it is phosphorylated, and the RalGAP complex RGC1–RGC2, RalA can interact with the exocyst subunits Sec5 and Exo84 [758]. Once Sec5 is phosphorylated by PKC, RalA dissociates from Sec5. The kinase TBK1, a member of the IKK family, which is involved in both inflammatory and insulin responses (TBK1 does not play an important role in NFκB activation, but acts in the regulation of type-I interferon production via interferon regulatory factor (IRF) phosphorylation and can counter overproduction of inflammatory mediators via IKK inhibition), phosphorylates the exocyst subunit Exo84, thereby reducing its affinity for RalA, enabling its release from the exocyst, and eliciting insulin-stimulated GluT4+ vesicle fusion with the plasma membrane in adipocytes [758]. In hepatocytes, insulin regulates via PI3K the cell surface expression of ScaRb1 and hence lipid ingress.

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and lymphopoiesis [760]. ApoM+ HDL promotes receptor activation. S1P carried on ApoM on HDL is more efficient in the maintenance of the endothelial barrier and insulin secretion from pancreatic β cells [762]. • Under normal conditions, HDL3 carries S1P, which interacts with endothelial S1P1 receptor to protect the endothelium [760]. • In type-1 diabetes mellitus (T1DM), the concentration of light lipid-rich, proteinpoor HDL2, which has a defective anti-inflammatory function, is higher than that of HDL3 particles. Hence, although plasmatic concentrations of ApoM and S1P in T1DM patients remain similar to those of controls, the ApoM–S1P complex transport shifts from dense to light HDLs [761]. At least in women, the ApoM–S1P complex in light HDLs is less efficient at inhibiting TNFSF1induced expression of vcam1 than that in denser HDLs. In addition, light HDLs cannot activate PKB, whereas all HDL subfractions are equally as efficient at activating ERK and receptor internalization. The HDL3-to-HDL2 change may lower S1P signaling to the endothelial S1P1 receptor [760]. Reduced HDLS1P level attenuates NOS3 activity. • In type-2 diabetes mellitus, glycation of HDL significantly lowers the S1P content of HDL, altering protection from redox stress [760]. The quantity of HDLs depends on CETP, which transfers cholesteryl ester from HDLs to ApoB+ lipoproteins and TGs from ApoB+ lipoproteins to HDLs. In addition, CETP modulates the distribution of S1P among lipoproteins [762]. Sphingosine 1-phosphate on VLDL, IDL, and LDL is cleared more rapidly than that on HDL [762]. The ApoE–LDLR couple is involved in the clearance of ApoM+ lipoproteins; LDLR removes S1P and ApoM carried on ApoE-rich HDLs, at least in mice. In addition, ApoM and S1P may be eliminated using the ApoB+ lipoprotein clearance pathway. Therefore, S1P, which abounds on HDL, can be cleared from blood circulation using two routes, the ApoE–LDLR pathway of ApoB+ lipoprotein removal and LDL clearance following transfer from HDL to LDL during CETP processing. CETP can also modulate the lipid composition of LDLs. The pathway involving ApoB+ lipoproteins may be a major clearance route of S1P bound to ApoM+ lipoproteins [762]. Sphingosine 1-phosphate on ApoB+ lipoproteins (i.e., VLDL and LDL) induces phosphorylation of PKB and NOS3 in ECs via S1P1 or S1P3 , to a greater extent, than S1P on HDLs [762]. Hence, in subjects with CETP deficiency, HDL activates NOS3 to a lesser degree. On ApoB+ lipoproteins, S1P provokes insulin secretion to a greater magnitude than S1P on HDL. Therefore, short-duration CETP overexpression (but not prolonged CETP overexpression) increases insulin secretion and sensitivity via S1P and S1P1 or S1P3 , thereby enhancing glucose tolerance in diabetes mellitus [762]. Cholesterol ester transfer protein does not control plasmatic concentrations of ApoM and S1P, although CETP lowers HDL concentration, but ApoM and S1P are transferred from HDLs to ApoB+ lipoproteins in CETP-overexpressing mice [762]. The quantity of LDL may be important in S1P transfer. Hence, CETP shifts the

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distribution of S1P and ApoM from HDL to ApoB+ lipoproteins, on which S1P exert some effects more potently, but is removed more rapidly. The receptor S1P1 abounds in ECs, where it contributes to the regulation of angiogenesis and maintenance of the microvascular barrier. The endothelial S1P1 receptor stabilizes developing vascular networks, prevents sprouting angiogenesis, and decreases vascular permeability. Endothelial S1P1 signaling augments in inflamed arterial segments [759]. In cultured human umbilical vein ECs, HDLApoM−−S1P , but not AlbS1P , upregulates formation of the plasmalemmal S1P1 –β Arr2 complex and attenuates activation of NFκB by TNFSF1 and hence the amount of ICAM1.20 Although S1P bound to either carrier stimulates the MAPK module, AlbS1P triggers greater Gi signaling and S1P1 endocytosis. Therefore, anti-inflammatory and endothelial protective functions of HDLs complement their role in cholesterol removal from cells and suppress endothelial dysfunction and inflammation characterized by impaired NO release and increased abundance of adhesion molecules for leukocytes, which are early events in CVD. However, a global increase in the amount of HDL does not repress EC inflammation. ApoM is unstable when it is not tethered to HDLs. An engineered soluble and stable form of ApoM bound to S1P can sustainably activate S1P1 on ECs and attenuates hypertension induced by angiotensin-2 [763].

5.1.3.2

Low-Density Lipoprotein

Elevated plasmatic LDLCS concentration alone does not suffice to provoke vascular lesions. Modifications of LDL such as oxidation in addition to small and dense LDLs are associated with atherosclerosis. Carbamylated LDL stimulates mitochondrial endonuclease G, which may be involved in DNA fragmentation during cell apoptosis. Plasmatic LDLs enter the endothelium and cross it to reach the subendothelial layer of the intima, where they are trapped. Transcytosis implicates both caveolamediated endocytosis and SNARE-mediated exocytosis. Caveolae are regulated by cavins.21 Endocytosis of LDLs depends on two routes [764]: 1. The first route relies on LDLR and favors LDL degradation. It declines at high LDL concentrations. Leukemia-inhibiting factor upregulates hepatic LDLR formation at least in rabbits, thereby increasing cholesterol clearance. 2. The second route diverts LDL from lysosomal degradation and promotes LDL transcytosis. It is enhanced with hypercholesterolemia. by ω3-polyunsaturated fatty acids of the receptor GPR120 (FFAR4) primes recruitment of β-arrestins, which prevents activation of the IKK complex and NFκB [759]. 21 A.k.a. polymerase-1 and transcript release factors (PTRFs). Redox stress can raise cavin-1 formation and thus the number of caveolae. 20 Activation

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Transmembrane protein TMem97, which under sterol depletion localizes to the endolysosomal compartment, binds to the LDLCS transport regulator, Niemann– Pick type-C protein NPc1, to control cholesterol level [765].22 Other regulators of cellular cholesterol homeostasis include sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and LRP6. Prosaposin (PSap) is a precursor of several non-enzymatic glycoproteins, sphingolipid activator proteins (SAPa–SAPd), related to four domains (A–D) from the N- to the C-terminus, which participate in the lysosomal degradation of sphingolipids.23 Reduced function of betaine–homocysteine methyltransferase BHMT2 decreases LDL uptake in ECs. C-reactive protein (pentraxin-1) increases LDL transcytosis, as it generates ROS, which increase endothelial permeability and activate PKC and Src,24 and translocation of caveolae or SNARE carriers [766].25 Endothelial activin-like kinase ALK1 connects to plasmatic LDLs in a noncompetitive manner with a lower affinity than LDLR [764]. This binding is inhibited neither by sterols, nor by PCSK9, a serine peptidase.26 The ALK1–LDL complex is then internalized and translocates from the apical to the basolateral surface of ECs to be released into the intima. Oxidized LDLs were originally defined as oxidatively modified LDLs containing protein components modified by aldehyde products creating net negative charges that enable interaction and uptake by macrophages. Formation of oxidation products depends on the oxidant type, the extent of oxidation, and the presence or absence of other agents such as redox metals [767]. Minimally oxidized LDLs possess lipid peroxides or their degradation products (e.g., oxovaleryl PC), without apolipoprotein modification. Some oxidation products, such as malondialdehyde

22 Cholesterol

derived from LDLs is transferred from the endosomal–lysosomal compartments to the endoplasmic reticulum under the control of NPC1 and NPC2. 23 Saposins SAPa and SAPc (SAP2) stimulate the hydrolysis of glucosylceramide by βglucosylceramidase and galactosylceramide by β-galactosylceramidase [108]. Saposin-B (SAP1) stimulates the hydrolysis of galactocerebroside sulfate by arylsulfatase-A, GM1 gangliosides by β-galactosidase, and globotriaosylceramide by α-galactosidase-A. Saposin-C is also an activator of β-glucosidase. Saposin-D is a specific sphingomyelin phosphodiesterase (PDE) activator. Prosaposin behaves as a myelino- and neurotrophic factor via its G-protein-coupled receptors, GPR37 and GPR37L1 [108]. It is internalized and phosphorylated by ERK. 24 The kinases PKC and Src phosphorylates Cav1 at Ser37 and Tyr14, respectively [766]. Dynamin Dnm2, which mainly localizes at the neck of the caveolae, executing the fission of the caveolae, is a target for PKC and Src; its translocation depends on activation of these kinases. C-reactive protein (CRP) may increase LDL transcytosis via formation of both the endocytotic Cav1–cavin-1–Dnm2 and exocytotic SNARE complexes. 25 The SNARE proteins (soluble N ethylmaleimide-sensitive factor-attachment protein receptors) support membrane docking and fusion via interactions with an ATPase, N ethylmaleimide-sensitive factor (NSF), and its SNAP “receptor” (α SNAP–γ SNAP). 26 PCSK9 is derived predominantly from the liver. It is secreted into the bloodstream and increases plasmatic LDLCS concentration, as it binds to LDLR both intra- and extracellularly and elicits its lysosomal degradation in hepatocytes.

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Table 5.1 Lipid and protein oxidation products in LDLs (Source: [767]; HODE hydroxylinoleic acid [octadecadienoic acid], HPODE hydroperoxylinoleic acid) Fatty acid Oxidation products

Lipid-derived products Protein Oxidation Products Other changes

Free and esterified fatty acid peroxides (e.g., 13HPODE) Free and esterified fatty acid hydroxides (e.g., 13HODE) Free and esterified isoprostanes Aldehydes (MDA, 4-hydroxy nonenal, and hexanal) Core aldehydes (e.g., oxovaleryl phosphatidylcholine) Pentane and other hydrocarbons Lysophosphatidylcholine Cholesterol oxidation products (e.g., 7-keto-cholesterol) Internally modified phosphatidylethanolamine/serine products Protein carbonyls Protein crosslinks Lipid–protein adducts Protein fragmentation Increased buoyant density Increased negative charge Loss of enzyme activities associated with LDL

(MDA), diffuse out of oxLDLs. Conversely, MDA-modified LDL (MDA LDL) can arise from MDA released by platelets or other sources. Polyunsaturated fatty acids favor LDL oxidation, but not monounsaturated fatty acids. Specific amino acids may propagate oxidation. Oxidized LDL is thus now defined as a particle derived from circulating LDLs that have peroxides or their degradation products. Oxidized LDLs contain unoxidized and oxidized fatty acid derivatives both in the ester and the free forms, their decomposition products, cholesterol and its oxidized products, proteins with oxidized amino acids and crosslinks, and polypeptides with varying extents of covalent modification with lipid oxidation products (Table 5.1) [767]. Low-density lipoproteins are heterogeneous in density, size, chemical composition, and net charge. The LDL spectrum can be characterized by a predominant peak of LDLs with large (pattern A) and small (pattern B) diameters. Small, dense LDLs have decreased levels of glycosylation of apolipoprotein-B and sialic acid content [768]. Any small LDL with a given valence (net negative charge) has a greater surface charge density than a larger LDL with the same valence. Mid-dense LDLs (1.030–1.039 g/ml) have a lower net negative electrical charge than the small, most buoyant, dense LDLs. On the other hand, mid-dense LDLs bind with a higher affinity to LDLR, thereby having greater rates of uptake and degradation than LDLs of lesser or greater density [768]. In humans, according to the net negative surface charge, plasmatic spherical LDLs are categorized into five subfractions from L1 to L5 with increasing electronegativity, which describes fast relative electrophoretic LDL mobility on agarose gel [769]. Whereas L1 LDL, the most abundant and least negatively

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Table 5.2 Composition of plasmatic L1 LDL and L5 LDL (low-density lipoprotein subfractions) from hypercholesterolemic humans (Sources: [769, 771]) L1 LDL

Core CE (38%), TG (4%) Periphery Proteins (25%) ApoB100 (99% LDL proteins) CS (8%), PLd (25%)

L5 LDL

CE (20%), TG (7%) Proteins (40%) ApoB100 (60% LDL proteins), ApoA1, ApoC3, ApoE, ApoA CS (8%), PLd (25%)

Spherical LDLs contain an apolipoproteic (Apo) framework, phospholipids (PLd) and free cholesterol (CS) on their surface, and triglycerides (TG) and cholesteryl esters (CE) at their core. Compared with L1 LDL, L5 LDL has a greater content of proteins and TGs, but a smaller amount of cholesteryl esters

charged LDL subtype, represents harmless normal LDL, the most negatively charged L5 LDL subfraction is atherogenic. The L5 LDL subfraction is isolated using anion-exchange chromatography from plasma of smokers and individuals with hypercholesterolemia, T2DM, and metabolic syndrome, whereas its concentration is negligible in healthy subjects. Its concentration is significantly elevated in STEMI patients [770].27 Its plasmatic concentration rises in patients with ischemic stroke, as it favors platelet aggregation and platelet–EC interaction. It can also induce endothelial dysfunction and impairs the differentiation of endothelial progenitor cells (EPCs).28 L5 LDL is neither smaller nor denser than L1 LDL, but has a higher aggregability. Whereas L1 LDL is composed mainly of apolipoprotein-B100 (99% proteic content), concentrations of ApoA1, ApoC3, ApoE, and ApoA progressively increase from L1 LDL to L5 LDL, the concentration of ApoB100 concomitantly decreasing (Table 5.2) [769]. Other minor proteins in L5 LDL (but not L1 LDL) include albumin, ApoJ, platelet-activating factor acetylhydrolase (PAFAH), paraoxonase POn1, the plasmatic apolipoprotein SAa4,29 and complement component C3. The L5 LDL particle does not tether to LDLR, but is endocytosed into vascular ECs via the scavenger receptor ScaRe1 (or CLec8a), the lectin-like oxLDL receptor,

27 STEMI:

ST segment elevation myocardial infarction. impedes expression of growth factor receptors and favors endothelial progenitor cell senescence, as it suppresses telomerase activity [771]. 29 SAa4: serum amyloid-A4. This major acute phase reactant is an apolipoprotein of HDLs. The group of HDL-associated apolipoproteins and cytokine-induced acute phase molecules is composed of: (1) major apolipoproteins on HDL produced in the liver (SAa1–SAa2, encoded by the SAA1 and SAA2 genes); (2) acute phase reactant SAa3, which is peripherally produced and is a minor HDL apolipoprotein (SAA3 is a pseudogene in humans); and (3) a constitutive acute phase reactant SAa4, which is a minor normal HDL apolipoprotein and is encoded by the SAA4 gene [772]. 28L5 LDL

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which has a high affinity for negatively charged ligands [769].30 It can also signal via the G-protein-coupled platelet-activating factor receptor (PAFR) on ECs and endothelial progenitor cells [771].31 L5 LDL precludes EC proliferation and favors EC apoptosis; L5 LDL and CuOxLDLs32 are equally potent at suppressing the Fgf2 gene transcription and inducing apoptosis in vascular ECs [771]. In addition, L5 LDL upregulates expression of adhesion molecules (e.g., vcam1), cytokines (e.g., IL1β), and chemokines (e.g., CCL2, CXCL1–CXCL3, CXCL5–CXCL6, and CXCL8), thereby favoring inflammation [774]. In macrophages, L5 LDL increases production of IL1β via ScaRe1, NFκB activation being required to produce proIL1β that is subsequently cleaved into IL1β by activated caspase-1, which lodges on the NLRP3 inflammasome, a molecular platform. Therefore, In addition to LDLCS plasmatic surplus, the proportion of L5 LDL that signals distinctly from usual LDLs such as L1 LDL, is an important factor for atherosclerosis. Atrial natriuretic peptide (ANP) is an endo-, auto-, and paracrine regulator. It acts via its guanylate cyclase receptor GC2a (NPR1)33 and the second messenger cGMP.34 It prevents NFκB-mediated proIL1β production and NFκB–NLRP3– Casp1-mediated IL1β release [775]. Natriuretic peptides constitute a family of three structurally related hormones and autacoids. All natriuretic peptides are synthesized as pre-prohormones encoded by the NPPA, NPPB, and NPPC genes. A- (atrial) and B-type natriuretic peptides (BNPs) are secreted from the atria and ventricles,35 ANP lowering blood pressure and attenuating cardiac hypertrophy, BNP reducing ventricular fibrosis [777]. Both 30 Low-density

lipoprotein is taken up by hepatocytes and vascular cells via the LDL receptor (LDLR). Homo- or heterozygous Ldlr gene defects raise LDL-emia. Whereas L1 LDL to L4 LDL are endocytosed via LDLR, L5 LDL is internalized by ScaRe1 in both ECs and EPCs. Expression of ScaRe1 is induced by L5 LDL (but not by L1 LDL). Via ScaRe1, L5 LDL disturbs the equilibrium between the pro-survival and proapoptotic members of the BCL2 family. Upon ScaRe1 stimulation, P38MAPK, which is countered by the PI3K–PKB–NOS3 pathway, is phosphorylated and activates caspase-3, thereby causing EC apoptosis [773]. 31 It disrupts FGF2 autoregulation via the FGF2–PI3K–PKB loop [771]. Exposure of L5 LDL with PAF acetylhydrolase to degrade PAF and PAF-like lipids removes its capacity to lower FGF2 signaling and induce EC apoptosis. 32 CuOx-LDL: copper-oxidized LDL. 33 Also known as NP , NPRa, and atrionatriuretic peptide receptor ANP . It is produced in the 1 A brain, heart, kidney, lung, adrenal gland, adipose tissue, and testis, in addition to vascular smooth myocytes [777]. 34 Increased content of intracellular cGMP activates its effectors, cGMP-dependent PKGs, phosphodiesterases (PDEs), and cyclic nucleotide-gated channels. In addition to intracellular cGMP accumulation, concentrations of cAMP, Ca2+ , and IP3 decline upon ANP exposure, the ANP– GC2a alleviating activity of adenylate cyclase (AC) and phospholipase-C in addition to Na+ influx and countering activation of PKC and MAPKs, thereby augmenting diuresis, provoking vasodilation, hindering cell proliferation, inflammation, and adverse hypertrophy [776]. 35 Low BNP concentrations are stored with ANP in atrial granules. However, BNP concentrations are greater in ventricles, where it is not stored in granules, but formed under the GATA4 control in response to cardiac stresses such as volume overload.

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Table 5.3 Tissular and cellular distribution of natriuretic peptide receptors (Source: [776]) Receptor GC2a (NP1 )

GC2b (NP2 )

GC2c (NP3 )

Organ Brain, heart, vasculature, kidney, adrenal glands, pituitary gland, lung, liver, ileum, thymus, ovary, placenta, testis Brain, heart, vasculature, adrenal gland, cartilage, lung, pituitary gland, thymus, ovary, placenta, testis Brain, heart, vasculature, kidney, liver, intestine

Cell EC, vSMC Renal epitheliocyte, mesangiocyte Fibroblast Granulosa cell Leydig cell VSMC Fibroblast Chondrocyte

EC, vSMC Fibroblast, mesangiocytes

ANP and BNP are also produced in the brain; ANP is additionally synthesized in the kidney and AT, whereas C-type natriuretic peptide (CNP), which is produced in the brain, heart, endothelia, and bone, primarily stimulates long bone growth [777]. CNP is not stored in granules. It is secreted upon exposure to growth factors and a stress field in cultured ECs. In humans, circulating BNP has a significantly longer half-life (∼20 mn) than ANP and CNP (∼2 mn). Membrane metalloendopeptidase (MME)36 degrades ANP and CNP, in addition to binding to the natriuretic peptide clearance receptor (GC2c), a pseudo-guanylate cyclase that constitutively mediates their internalization and elimination. On the other hand, BNP is initially cleaved by meprin-A in the kidney brush border and then further degraded by MME [777]. Both ANP and BNP activate the transmembrane guanylyl cyclase GC2a37 whereas CNP stimulates GC2b (Table 5.3).38 Transforming growth factor (TGF)β1, angiotensin-2, and endothelin-1 reduce GC2a synthesis [776]. A third receptor GC2c (NPR3)39 clears natriuretic peptides from the circulation via endocytosis for sequestration and degradation. The receptor GC2a lodges on adipocytes and can promote adiponectin production [777].

36 A.k.a.

neutral endopeptidase (NEP).

37 With a potency order ANP ≥ BNP  CNP [777].

Glycosylation of GC2a may influence receptor stability and ligand binding [776]. 38 Also abbreviated NP , NPRb, and ANP . It has the following selectivity preference CNP  2 B ANP ≥ BNP [777]. It is produced in the brain, heart, kidney, liver, lung, bone, and uterus, in addition to fibroblasts and vSMCs [777]. Activation of GC2b by CNP generates cGMP, but to a lesser extent than that of GC2a excited by ANP or BNP [776]. 39 Also dubbed NP , NPRc, and ANP . This most widely and abundantly expressed natriuretic 3 C peptide receptor has the following selectivity preference ANP > CNP ≥ BNP [777].

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Atrial natriuretic peptide acutely reduces plasmatic volume using three mechanisms: increased renal excretion of salt and water, vasodilation, and augmented vascular permeability via its endothelial receptor GC2a [778]. It is secreted by the heart upon atrial stretch and hence hypertension. ANP is also synthesized and metabolized in ECs. The natriuretic peptides, ANP, BNP, and CNP, are implicated in obesity and lipid mobilization [779]. The CNP subtype causes endothelium-dependent and independent vasodilation. A high-cholesterol diet can prevent efficacy of protein Tyr kinase inhibitors (PTKI), which are used to treat various cancer types, especially renal cell carcinoma (RCC), via suppression of cancerous cell proliferation or tumoral angiogenesis, rendering RCC refractory to PTKI treatment [780]. In cultured RCC and endothelial cells, exposure to LDLs activates the PI3K–PKB pathway, thereby promoting cell survival and proliferation and attenuating PTKI cytotoxicity.

5.1.3.3

Lipoprotein-A

Lipoprotein-A is an LDL-like particle linked to apolipoprotein-A , which is encoded by the APOA (LPA ) gene on chromosome 6. This ApoB100–ApoA complex is composed of apolipoprotein-B100 covalently attached to the very large hydrophilic glycosylated apolipoprotein-A . It thus resembles LDL by the presence of ApoB and a high content of cholesterol, but differs from LDL by its content in ApoA . It is enriched with oxidized phospholipids. Because ApoA and ApoB+ lipoproteins are secreted separately, the covalent linkage of ApoA to ApoB by a disulfide bridge occurs in the extracellular medium [781]. Lipoprotein-A shares structural features with plasminogen (kringle domains and a peptidase domain, which is catalytically inactive in LPa), the number of kringle-4 repeats determining the LPa-emia and hence the cardiovascular risk. Being similar in structure to that of plasminogen may explain its prothrombotic effect. Concentration of LPa obeys an autosomal dominant pattern of inheritance [727]. In the European population, circulating LPa concentration (LPa-emia) is lower in Northern (mean 4.9 mg/dl) than Central (mean 7.9 mg/dl) and Southern European cohorts (mean 10.9 mg/dl) [782]. Lipoprotein-A concentration is determined by ApoA synthesis in hepatocytes and catabolism, i.e., clearance. LPa-emia is elevated (>30–50 mg/dl; ≥75– 125 nmol/l) in CVD patients [783], the range of values measured by different techniques in various cohorts being similar and Blacks having a higher LPa-emia than Whites [784]. Standardization of LPa immunoassay is difficult, and LPa-emia threshold depends on the assay used. Sex (estrogens or testosterone) and thyroid hormones in addition to nicotinic acid and PCSK9 and CETP inhibitors lower LPa concentration, in addition to lipid apheresis [781].

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Concentration of circulating LPa in a population of individuals (48% women) mainly from North America (50%) and Western Europe (47%), with no previous history of CVD, correlates weakly with several conventional vascular risk factors. It represents a relatively modest risk factor for coronary heart disease and stroke [784]. Nevertheless, LP-a aggravates the risk for adverse cardiovascular events associated with other classical risk factors, such as LDLCS . It represents a risk for atherosclerosis, in particular CoAD, in addition to aortic valve calcification and stenosis and possibly venous thromboembolism [781]. Elevated LPa-emia is robustly associated with an increased CVD risk, in particular in diabetic individuals [782]. In vitro, LPa exerts inflammatory and thrombogenic actions. Lipoprotein-A enters the arterial intima and can favor inflammation, thrombosis, and foam cell formation [784]. Lipoprotein-A and lipoprotein-associated phospholipase-A2 (lpPLA2)40 act as causal and noncausal markers [784]. Lipoprotein-associated phospholipase-A2, which is encoded by the PLA2G7 gene (group-7 PLA2), circulates in the plasma mainly attached to LDLs. It may also be associated with atherosclerosis, according to some authors (but not all). The lipidic components of LPA and the protein ApoA undergo different intracellular fates in hepatocytes [783]. After uptake, the lipidic components leave the Rab5+ early endosomes for lysosomal degradation. On the other hand, ApoA dissociates from ApoB and moves to the trans-Golgi network and then possibly to Rab11+ recycling endosomes to be resecreted, about 30% of ApoA being recycled back to the extracellular space [785]. In the extracellular medium, ApoA reassociates with newly formed or circulating LDL, hence generating LPA , and is cleared via various receptors, enters the vessel wall or kidney, or is degraded. Endocytosis of ApoA and its recycling rely on the plasminogen receptor-KT (PlgRkt).41 Several hepatic receptors are involved in LPA catabolism. Its clearance and possible lysosomal degradation can rely on LDLR, VLDLR, LRP1, LRP2, ScaRb1, and syndecan-1 [783]. Once LPA is connected to LDLR and LRP1, clathrin-mediated endocytosis leads to lysosomal degradation of its lipid moiety and associated ApoA . The receptor ScaRb1 primes lysosomal degradation of the entire particle or the lipid content. 40 A.k.a.

platelet-activating factor acetylhydrolase (PAFAH). receptor PlgRkt is not only involved in LPA catabolism and ApoA internalization and recycling but also regulates plasminogen activation at the cell surface via urokinase- and tissuetype plasminogen activator. It also controls monocyte migration and matrix metallopeptidase (MMP) activation (e.g., MMP2 and MMP9) [108]. On the other hand, urokinase-type plasminogen activator receptor (uPAR or PlAUR) linked to the plasma membrane by a glycosyl phosphatidylinositol (PI) anchor, regulates plasmalemmal activation of plasminogen. Urokinase plasminogen activator stimulates migration of arterial smooth myocytes via uPAR and the pseudokinase protein Tyr kinase TYK2, which complexes with PI3, RhoA, and Rac1 (but not CDC42). Plasminogen can also carry oxidized phospholipids. In fact, ApoA may not be internalized by PlgRkt, but rather resorted to PlgRkt in the early endosome for slow recycling and prevention of rapid lysosomal degradation [783].

41 Plasminogen

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Plasmatic LPa concentration is primarily determined by variations in the LPA (APOA ) gene locus. In addition, APOA gene transcription is controlled by various agents, such as interleukin-6, which increases ApoA production in hepatocytes, and estrogens and bile acids, which decrease it [786].

5.1.3.4

Apolipoprotein-E

The glycoprotein ApoE, which is synthesized and secreted mainly by the liver, brain, and skin, in addition to macrophages, is a component of VLDLs, remnant lipoproteins, and HDLs. It facilitates their clearance via LDLR, LRP1, and syndecan-1. The three ApoE isoforms (ApoE 2 –ApoE 4 ) associated with three APOE alleles in humans differ in one or two amino acids (i.e., single amino acid substitutions) at two sites (positions 112 and 158): • ApoE 2 (ApoE2) possesses Cys112 and Cys158 • ApoE 3 (ApoE3) Cys112 and Arg158 • ApoE 4 (ApoE4) Arg112 and Arg158 These amino acid differences modify affinities for TG-rich lipoproteins and clearance receptors.42 Thus, they affect circulating concentrations of VLDLCS , IDLCS , and LDLCS , in addition to the remodeling of VLDL to LDL and receptor-mediated remnant clearance [786]. The ApoE4 protein prefers large TGRLs (VLDLs and chylomicrons), whereas ApoE3 and ApoE2 preferentially connect to small spherical HDLs [786]. Enrichment of ApoE4 on VLDL accelerates its clearance from blood circulation by the liver receptor (LDLR, LRP1, and syndecan-1) and outcompete LDL–LDLR binding, because of the 20-fold greater affinity of ApoE3 and ApoE4 for LDLR than that of ApoB100, elevating LDLCS concentration. The APOE genotype ( 2/ 2, 2/ 3, 2/ 4, 3/ 3, 3/ 4, and 4/ 4) strongly influences concentrations of ApoB-related lipoproteins including LPa and hence LPaCS .43 Difference in the affinity of ApoE subtypes for the lipoprotein clearance

42 Gliocytes

produce and secrete ApoE. ApoE can stimulate the MAP3K12–MAP2K7–ERK1/2 pathway in neurons, thereby phosphorylating Fos and activating production of amyloid-β precursor protein (APP) and amyloid-β secretion [787]. ApoE4, the most important genetic risk factor for Alzheimer’s disease, is more potent in stimulating APP transcription and amyloid-β secretion than ApoE3, which is more efficient than ApoE2. 43 In a cohort of more than 430,000 patients, mean LPa concentration rises from subjects with the 2/ 2 to those with 4/ 4 genotype ( 2/ 2 [ApoE2/2]: 23.4 ± 29.2; 2/ 3 [ApoE2/3]: 31.3 ± 38.0; 2/ 4 [ApoE2/4]: 32.8 ± 38.5; 3/ 3 [ApoE3/3]: 33.2 ± 39.1; 3/ 4 [ApoE3/4]: 35.5 ± 41.6; and 4/ 4 [ApoE4/4]: 38.5 ± 44.1 mg/dl) [786]. Individuals with the ApoE4/4 phenotype thus have a 65% higher LPa concentration than those with the ApoE2/2 phenotype.

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receptors, such as LDLR and LRP1, and hence competition between LPa and ApoE for a given receptor may thus affect LPa catabolism [786].44 Many ApoE receptors connect to other ligands. For example, VLDLR and LRP8 bind reelin; LRP5 and LRP6 are Wnt coreceptors; and LRP2 is an auxiliary receptor for sonic hedgehog [787]. In gliocytes, LDs store lipids for energy production. Mitochondrial dysfunction and redox stress due to elevated ROS formation induce neuronal lipid production and transport of lipids from neurons to gliocytes for storage in LDs that can contribute to the adjustment to redox stress. In neurons, lactate is converted to pyruvate and acetyl-CoA. In addition, lactate accumulation in neurons triggers the production of lipids that are subsequently transferred to gliocytes, in which they form LDs. Conversely, formation of glial LDs requires lactate transfer from gliocytes to neurons using monocarboxylate transporters in addition to fatty acid transporters, and apolipoproteins [788]. Glial lactate is then used for lipid synthesis within neurons. Monocarboxylate transporters enable gliocytes to secrete and neurons to absorb lactate. Lactate metabolites provide substrates for the synthesis of fatty acids, which are processed and transferred to gliocytes by FATP and apolipoproteins. In the presence of high ROS concentrations, impaired lactate transfer and low concentrations of FATP or apolipoproteins decrease glial LD formation. Apolipoproteins ApoD and ApoE participate in the transfer of lipids between neurons and gliocytes. Whereas ApoE2 and ApoE3 promote LD formation, ApoE4 leads only to the weak formation of LDs, even under conditions of overexpression and redox stress. Therefore, stress-primed LD formation, which relies on neuronal lactate, is not supported by ApoE4.

5.1.3.5

Lipoprotein Lipase (Lipase-D)

Lipoprotein lipase and its stimulator ApoA5 and inhibitors ApoC3 and AngptL4, which is secreted by AT during fasting, are implicated in the catabolism of TGRLs. Lipolysis of postprandial TGRLs by lipoprotein lipase augments levels of saturated and unsaturated FFAs in addition to those of hydroxylated linoleates hydroxyoctadecadienoic acids 9HODE and 13HODE, which mediate redox stress, thereby eliciting formation of TNFSF1, adhesion molecules, and ROS in ECs. Various LPL regulators include ApoA5, ApoC1 to ApoC3, and angiopoietin-like proteins AngptL3, AngptL4, and AngptL8 [789]. ApoC2 is the necessary cofactor for LPL, whereas ApoC1 and ApoC3 inhibit LPL [727].

44 The

ApoE 2 subtype, which is associated with recessive inheritance and low penetrance, has the weakest binding affinity to LDLR, LDLR binding for ApoE 3 and ApoE 4 being normal.

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5.1.4 Ceramides Ceramides are linked to several cardiovascular risk factors (inflammation, insulin resistance, and obesity) in addition to major adverse cardiovascular events in apparently healthy individuals. They serve as markers for assessing the cardiovascular risk. These molecules and related sphingolipids are involved in lipoprotein uptake and aggregation and hence cholesterol accumulation within macrophages in addition to regulation of NO synthesis and production of superoxide anions and cytokines [790]. Thus, they participate in atherogenesis. Inhibition of glycosphingolipid synthesis counters atherosclerosis in mice. Among four circulating ceramide species (Cer[d18:1/16:0], Cer[d18:1/18:0], Cer[d18:1/24:0], and Cer[d18:1/24:1]), the strongest association with major adverse cardiovascular events in apparently healthy individuals (primary prevention) is observed for Cer(d18:1/18:0) [790]. In patients with diagnosed disease (secondary prevention), among the culprit ceramides, Cer(d18:1/16:0) and Cer(d18:1/24:1) linkage with cardiovascular events is higher than that of Cer(d18:1/18:0). The role of Cer(d18:1/24:0) mimics that of LDLCS , which is typically associated with cardiovascular risk in primary prevention, whereas in CoAD patients, an inverse relationship or even no association can be observed due likely to hepatic LDLR regulation during inflammation. The Cer(d18:1/24:0) species, which links to lipoproteins, may be less active than other ceramide species. Adiponectin receptors (AdpnRs) encoded by the ADIPOR1 and ADIPOR2 genes differ according to the mammalian species and tissular distribution. They control glucose and lipid metabolism, at least partly, via ceramidase. Ceramide, sphingosine, and sphingosine 1-phosphate (S1P) are lipids that participate in regulating cell adhesion, differentiation, proliferation, migration, and apoptosis. Lysosomal and nonlysosomal ceramidases (acid, neutral, and alkaline N acylsphingosine amidohydrolases ASAH1–ASAH3)45 cleave ceramide to a FFAs and sphingosine, a precursor of the antiapoptotic factor sphingosine 1-phosphate.

45 Ceramidases

have a maximal activity in acidic, neutral, and alkaline environments, respectively. Alkaline ceramidases include three subtypes: ACer1 (ASAH3), Acer2 (ASAH3l), and ACer3. ASAH1 is a lysosomal ceramidase that works at an optimal pH value of 4.5 [791]. It targets ceramides with saturated medium acyl chains (C10–C14) or unsaturated long acyl chains (C18:1 or C18:2). It not only hydrolyzes ceramide into sphingosine but can also synthesize ceramide from sphingosine and FFAs [792]. This reverse enzymatic activity occurs at a distinct pH (backward reaction at pH 6.0 and forward reaction at pH 4.5). A multi-enzyme complex contains ASAH1, acid sphingomyelinase, and β-galactosidase (but not other lysosomal enzymes, such as α-iduronidase and α-galactosidase). ASAH2 localizes to the plasma membrane and can be secreted. It hydrolyzes various types of ceramides at an optimal pH value of about 7.0 [791]. ACER1 lodges in the ER. It processes ceramides with unsaturated long acyl chains (C18:1 and C20:1) and very long saturated (C24:0) or unsaturated (C24:1) acyl chains. ACER2 resides in the Golgi body and has an optimal pH value of about 9.0. It uses various ceramides. ACER3 is located in the ER and Golgi body. Its preferential substrates are ceramides carrying unsaturated long acyl chains (C18:1 and C20:1). It has an optimal pH of about 9.0.

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The zinc-binding GPCR subtype AdpnR2 possesses low basal ceramidase activity that is enhanced by adiponectin (Adpn) [793]. The AdpnR1 isoform also has weak intrinsic ceramidase activity. Prolonged glucocorticoid-based treatment counters inflammation, but develops insulin resistance. In addition, glucocorticoids induce production of AngptL4 and ceramides [794]. AngptL4 mediates glucocorticoid-induced lipolysis in WAT, induces expression of genes encoding enzymes of ceramide synthesis in the liver of glucocorticoid-treated mice: PP2a and PKCζ. Inhibition of AngptL4 (mainly), PP2a, or PKCζ lessens glucose intolerance observed in WT mice owing to chronic glucocorticoid administration.

5.2 Gluco- and Lipotoxicity Diabetes mellitus leads to diabetic cardiomyopathy. Mitochondrial dysfunction impairs oxidative metabolism despite the availability of substrates (amino acids, glucose, and fatty acids). Gluco- and lipotoxicity (hyperglycemia and dyslipidemia) provoke cardiomyocyte (CMC) dysfunction and death. Accumulation of metabolic intermediates within the myocardium perturbs gene transcription (e.g., transcription carried out by fatty acid-activated PPARs), mRNA translation (e.g., transcription controlled by nutrient-activated TOR), and protein post-translational modifications, in addition to the amount and action of signaling mediators [795]. In diabetic hearts, glucose uptake and oxidation are impaired, and hence fatty acids are almost exclusively used for ATP synthesis. Chronically elevated FA conversion to potentially toxic metabolites (e.g., ceramides, diacylglycerols, and ion channel-regulating acylcarnitines) and oxidation that increases ROS formation are responsible for lipotoxicity. Vascular endothelial growth factor is not only implicated in angiogenesis, especially under hypoxia, but also in neurogenesis, immunomodulation, wound healing, and metabolism, via its numerous VEGF and VEGFR isoforms and splice variants.46 The heart possesses all VEGF isoforms and receptors, VEGFb and VEGFR2 being the most highly abundant in normal conditions, a substantial and

Sphingomyelinases process sphingomyelin to ceramide and ceramidases ceramide to sphingosine, which is phosphorylated by sphingosine kinases (SphK1–SphK2). Sphingosine 1-phosphate is irreversibly cleaved by S1P lyase to ethanolamine phosphate and hexadecenal, which are incorporated into phosphatidylethanolamine (PE) and glycerolipids, respectively [791]. Sphingosine 1-phosphate can also be dephosphorylated to sphingosine by S1P-specific phosphatases (SPP1– SPP2) and broad-specificity lipid phosphate phosphohydrolase (LPP1–LPP3; a.k.a. phospholipid phosphatases, PLPP1–PLPP3, and phosphatidic acid phosphatases, PPAP2a–PPAP2c). Sphingosine 1-phosphate abounds in plasma; it mainly originates from red blood capsules. 46 For example, VEGFb 167 (>80% of total VEGFB transcripts) and VEGFb186 are VEGFb splice variants. VEGFb186 , but not VEGFb167 , possesses the HSPG-binding domain [795].

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releasable VEGFb pool lodging on the CMC surface [795].47 Vascular endothelial growth factor b modulates fatty acid uptake and oxidation, thereby protecting against the consequences of FA ingress and overprocessing. It is regulated by the PGC1α–ERRα (NR3b1) axis. Nutrients influence epigenetic background and hence transcription of the VEGFB gene; some species of dietary fatty acids affect the methylation status of the VEGFB promoter. In addition, VEGFb substantially increases transcription of genes implicated in cardiac contraction (e.g., SERCA and RyR) [799]. It also promotes cell (EC and CMC) survival. Hyperglycemia induced acute VEGFb release from CMCs in co-cultures with ECs linked to high glucose level-induced heparanase release from ECs [799]. Heparanase48 is secreted from ECs in response to elevated glucose concentration, releasing VEGFb from heparan sulfate proteoglycan, which then binds to EC VEGFR1, increasing fatty acid uptake, and CMC VEGFR1, attenuating apoptosis launched by the ERK–GSK3β pathway [799]. VEGFb activates the ERK–GSK3β axis in both CMCs and ECs, thereby attenuating H2 O2 -primed activation of caspase3 and polyADP ribose polymerase and hence the likelihood of cell death. Therefore, ECs sense glucose and then protect both CMCs and ECs via the auto- and paracrine action of VEGFb. In diabetic rats, cardiac heparanase and CMC VEGFb concentrations and ERK– GSK3β signaling decline, whereas CMC VEGFR1 expression rises, defining a state of VEGFb resistance [799]. In diabetes, circulating concentrations of glucose and fatty acids are chronically elevated; a depression of VEGF sensitivity may be an adaptation to prevent accumulation of lipotoxic species in the myocardium. Nevertheless, VEGFb resistance can also impair cardioprotection. In addition, VEGFb promotes transendothelial transport of circulating fatty acids that are subsequently used by cardiac and skeletal myocytes [795]. Resulting increased fatty acid oxidation raises mitochondrial acetyl-CoA concentration, which inhibits pyruvate dehydrogenase, the gatekeeper of pyruvate entry into the 47 Synthesis

of VEGFc and VEGFd is upregulated in heart failure, whereas VEGFb concentration declines [795]. 48 Heparanase is encoded by the HPSE gene under control of early growth response EGR1, among other transcription factors. It is overexpressed in diabetes. This endoglycosidase (endoglucuronidase) resides in the endosomal and lysosomal compartments for a relatively long period (half-life ∼30 h). Once it is secreted by degranulation, it degrades heparan sulfate. It thus releases various types of growth factors, cytokines, chemokines, and enzymes sequestered by heparan sulfate. Conversely, heparanase uptake is mediated by plasmalemmal heparan sulfate proteoglycans of the syndecan family, which limits its extracellular accumulation [796]. Under normal conditions, heparanase activity is restricted to some organs in addition to bloodborne cells (e.g., platelets, mastocytes, monocytes, neutrophils, and T lymphocytes). It is also synthesized in neovascular ECs and in some types of cancerous cells. It is implicated in inflammation, leukocyte migration, angiogenesis, and tumor growth and metastasis. It upregulates production of tumor progression agents (e.g., VEGF, HGF, TNFSF11, and MMP9), as it reduces the nuclear amount of syndecan-1, which prevents activity of histone acetyltransferases, thereby raising their activity and transcription of genes that favor an aggressive tumor phenotype [797]. It provokes VEGFc formation, thereby promoting tumoral lymphangiogenesis, which enables cancerous cell migration [798].

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tricarboxylic acid cycle, thereby preventing glycolysis (Randle cycle). Imbalance between fatty acid uptake and oxidation causes their accumulation, hence altering cardiac gene expression and signaling, especially the one driving insulin-mediated glucose uptake. Moreover, CMC-specific VEGFb overexpression provokes ceramide accumulation and mitochondrial dysfunction. Systemic excess lipids affect organismic metabolism and promote cardiometabolic disease with hypothalamic and pancreatic lipotoxicity and inflammation [717]. Local toxic and inflammatory effects of excess lipids in the hypothalamus impair its regulation of food intake, systemic energy expenditure, and peripheral metabolism. Ectopic fat depots and inflammation in the pancreas may alter insulin secretion. Endothelial dysfunction refers to a maladaptive endothelial phenotype characterized by reduced NO availability, NO being an important determinant of endothelial function, and hence abnormal vasoreactivity, in addition to augmented redox stress and expression of proinflammatory and pro-thrombotic factors. Nitric oxide is produced in vascular endothelia by the activated NOS3 subtype, which requires availability of its substrate, L arginine, and enzymatic cofactors (BH4 [tetrahydrobiopterin], FAD, FMN, and NADPH). Elevated endothelial cytosolic Ca2+ concentration, which can be initiated by GPCRs such as that of acetylcholine, promotes binding of calmodulin to and subsequent activation of NOS3. In addition, NOS3 phosphorylation (Ser1177) by AMPK, PKA, and PKB stimulates NO production independently of Ca2+ ion. Induced vasodilation results from reduced Ca2+ concentration inside adjacent vSMCs upon guanylate cyclase activation and cGMP formation. Major vasodilators, such as NO and PGi2 , are antiproliferative and antiinflammatory, whereas important vasoconstrictors, such as ET1 and Agt2, are mitogenic and proinflammatory. Insulin signals to participate in the vasomotor tone control using the vasodilatory NO-synthesizing PI3K and vasoconstrictory ET1-secreting MAPK branch, the latter also regulating adhesion molecule expression in vascular ECs (Table 5.4) [800]. Insulin tethers to its cognate receptor, which phosphorylates IRS1, which then connects to and activates PI3K. The latter produces PIP3 , which stimulates PDK1, which phosphorylates (activates) PKB. The latter phosphorylates NOS3 (Ser1177), which then produces NO. Insulin also stimulates the MAPK module and hence ET1 production in addition to PAI1, vcam1, and E-selectin in ECs [800]. On the other hand, the PI3K–PKB axis downregulates insulin-induced expression of PAI1 and adhesion molecules. Glucotoxicity, lipotoxicity, and inflammation that contribute to insulin resistance related to altered glucose transport also provoke endothelial dysfunction with increased vascular permeability [800, 801]. Glucotoxicity, lipotoxicity, and various types of cytokines inhibit the PI3K–PKB axis. A high intake of oils and fats enriched in ω6-fatty acids such as linoleic acid can cause endothelial dysfunction, redox stress, and inflammation [831]. On the other hand, ω3-fatty acids, such as eicosapentaenoic and docosahexaenoic acids, have antioxidant and anti-inflammatory effects.

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Table 5.4 Insulin regulates the vasomotor tone and other processes and the effect of gluco- and lipotoxicity (Source: [800]; cGMP cyclic guanosine monophosphate, ERK extracellular signalregulated kinase, ET endothelin, InsR insulin receptor, IRS insulin receptor substrate, JNK Jun N-terminal kinase, MAPK mitogen-activated protein kinase, NOS nitric oxide synthase, PDK phosphoinositide-dependent kinase, PI3K phosphatidylinositol 3-kinase, PLC phospholipase-C, S6K P70 ribosomal S6 kinase, sGC soluble guanylate cyclase) Process Vasodilation Vasoconstriction Cell adhesion Lipogenesis (hepatocyte) Glucotoxicity, lipotoxicity, inflammation

Pathway InsR–IRS1–PI3K–PDK1–PKB–NOS3–NO–sGC–cGMP InsR–SHC–GRB2/SOS–Ras–Raf–MAPK–ET1–ETR–PLC–Ca2+ InsR–MAPK (↑) InsR–IRS1–aPKC ERK −→ IRS1 JNK −→ InsR, IRS1 PKCα −→ IRS1 S6K −→ PI3K

Insulin binds to its receptor and then controls lipid uptake, lipolysis, and lipogenesis. Atypical protein kinase-C (aPKC) is required for insulin-stimulated glucose transport in myocytes and adipocytes and, in the liver, for activation of lipogenic enzymes

Endothelial lipotoxicity is caused by elevated concentrations of circulating nonesterified (free) fatty acids (neFAs), abbreviated in the present text neFA-emia. NeFAs are linked to obesity, insulin resistance, hypertension, and endothelial dysfunction [831]. In AT, neFA production increases in obese subjects; neFAs raise expression of IL6, but lower that of IL10 [802]. Augmented neFA-emia causes endothelial dysfunction, as FFAs: (1) disturb NOS3 activity, prevent prostacyclin production, and inhibit potassium channels, thereby impairing stimulation by insulin of endothelium-dependent vasodilation,49 (2) support α AR-mediated constriction, (3) provoke redox stress,50 and (4) trigger vascular cell proliferation and inflammation [831]. The FFA composition is more relevant to the vascular function than the total neFA amount [831]. An acute elevation of long- (lcFAs) but not medium-chain fatty acids (mcFAs) attenuates endothelium-dependent vasodilation. Endothelial function is inversely related to the proportion of saturated fatty acids (e.g., lauric and myristic acid) and positively related to the fraction of α-linolenic acid. The most abundant saturated fatty acid in human plasma, palmitate, can elicit formation of inflammatory cytokines in ECs.

49 In

ECs, insulin triggers the InsR–IRS1–PI3K–PDK1–PKB–NOS3 pathway. Once synthesized in ECs, NO is secreted and activates sGC in neighboring vSMCs. However, insulin also stimulates endothelin-1 synthesis in and secretion from human endotheliocytes. 50 In vSMCs, oleic and linoleic acids activate PKC, which stimulates NOx and hence ROS formation.

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Stearoyl-CoA desaturase SCD1, the rate-limiting enzyme of lipogenesis, converts saturated to monounsaturated fatty acids for incorporation into neutral lipids, hence lowering lipotoxicity [831]. Palmitic acid, a saturated fatty acid that abounds in HFD, increases SCD1 concentration. On the other hand, in myeloid cells, the toll-like receptor adaptor, toll–IL1R (TIR) domain-containing adaptor inducing interferon-β (TRIF), not only promotes inflammation to fight infection but also contributes to the hepatic metabolism. Activated TRIF represses SCD1, thereby reducing lipid accumulation in hepatocytes and preventing diet-induced hepatic steatosis [803]. Its effector, interferon regulatory factor IRF3, is a transcriptional suppressor that tethers to the Scd1 promoter. Small lipid-binding proteins (SLBPs) are receptors and transporters for hydrophobic ligands with distinct ligand selectivity, binding affinity, and action modes, in addition to binding partners,51 possessing both unique and overlapping functions according to the cell type. Whereas adipocyte FABP4 bound to linoleic acid translocates to the nucleus, once it is linked to oleate or stearate, it remains in the cytosol [831]. Lipidic autacoids, metabolites of arachidonic acids, short-lived endoperoxides, some prostaglandin types, and thromboxane-A2 , mediate endothelium-dependent vasoconstriction, which is exacerbated when NO production is altered, such as when FABP4 expression is triggered [831]. Lipocalin-2 also favors endothelial dysfunction related to aging and obesity. This lipid carrier elicits endotheliumdependent vasoconstriction and attenuates endothelium-dependent vasodilation. Furthermore, both FABP4 and Lnc2 are proinflammatory factors. Therefore, SLBPs, such as FABP4 and Lnc2, favor obesity-induced endothelial dysfunction, as they modulate various signaling cascades involved in vascular homeostasis maintenance.

5.3 Overweight and Obesity Imbalance between caloric intake (diet) and energetic expenditure (physical activity) is the major cause of obesity, an excess of body mass and adiposity. Carbohydrate and fatty acid surplus affects body metabolism and organ functioning. Saturated fatty acids (e.g., palmitate) favor ceramide accumulation and obesity-related insulin resistance more than unsaturated fatty acids (e.g., oleate). Sugar-sweetened beverages is the primary source of added sugars in the diet; sweeteners are high-fructose corn syrup and sucrose, both containing approximately equal amounts of fructose and glucose [804]. Excessive fructose consumption is

51 Among

SLBPs, adipocytic fatty acid-binding protein, FABP4, interacts with HSL (or lipaseE), whereas lipocalin-2 heterodimerizes with MMP9 [831]. The secretory protein FABP4 has an elevated circulating concentration in obese individuals [831].

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more detrimental than that of glucose, as it raises hepatic de novo lipogenesis and dyslipidemia, hence favoring insulin resistance and obesity (Sect. 6.5.3). Adiposopathy designates qualitative anomalies of the AT as a consequence of AT maladaptive remodeling. A lower degree of adiposopathy is associated with a better systemic metabolic profile and vascular function [805]. Macrophage infiltration and redox stress in adipose depots provoke secretion of numerous inflammatory agents and adipocytic hormones that impair functioning of the heart and vasculature. In addition, adipogenesis is associated with angiogenesis. The AT consists of multiple depots in the body and participates in the maintenance of physiological activities. However, excessive growth of adipose depots and ectopic accumulation of lipids, especially in the liver and skeletal muscles, alter the cardiovascular apparatus. Increased circulating lipidemia contributes to atherosclerosis, whereas augmented lipid deposition in the AT, liver, and skeletal muscle causes obesity and insulin resistance. Olfaction influences the anticipation of feeding. Inhibition of olfaction in lean and HFD-fed obese mice lowers nutrient intake and subsequently impedes further weight gain, reduces AT mass, and improves insulin resistance [806]. Olfactory sensory neurons also affect energy regulation. In mice with defective olfaction, fat consumption rises owing to elevated sympathetic nerve activity in AT, activated β-adrenoceptors on white and brown adipocytes promoting lipolysis [806]. In addition, thermogenesis increases in brown and inguinal fat depots. Conversely, ablation of the IGF1 receptor in olfactory sensory neurons enhances olfactory performance in mice, increases adiposity, and provokes insulin resistance. Bone morphogenetic protein BMP4 supports multipotent mesodermal stem cell differentiation into adipocytes, recruits and activates beige adipocytes in the subcutaneous WAT, increases the number of stromal vascular cells, and launches AT angiogenesis [807]. Pericytes are capable of adipogenic differentiation; these PDGFRβ+ progenitors within neovessels serve as adipocyte progenitors, PDGFRβ expression decaying during adipogenic differentiation. On the other hand, PDGFRα is a marker of adipogenic precursors that only lodge in the vAT. BMP4 represses PDGFRβ activity via lysosomal degradation, hence priming pericyte differentiation into adipocytes during beiging of the subcutaneous WAT, whereas angiogenesis engenders neovessels that maintain the pool of adipocyte progenitors. Blood concentration of erythritol, which is synthesized from glucose on the pentose phosphate pathway, is associated with increasing adiposity in young adults; it is higher than in individuals with stable adiposity [808]. Obesity can be associated with glomerular hypertrophy (glomerulomegaly) and focal segmental glomerulosclerosis (FSGS);52 it becomes a leading cause of chronic

52 Lesions

are often perihilar. FSGS is defined as a segmental sclerosis (perihilar, cellular, tip, collapsing, and not specified) of the glomerular tuft causing capillary obliteration [809]. Perihilar afferent arterioles and glomerular capillaries are dilated. Podocyte volume rises (podocyte

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kidney disease [809]. The glomerulus enlarges in response to obesity-induced elevation of renal plasma flow due to dilation mainly or solely of the afferent arteriole and constriction of the efferent arteriole by Agt2 and aldosterone that increases glomerular filtration rate and hence the amount of filtered and subsequently reabsorbed sodium and engenders a stable or slowly progressive proteinuria, which defines obesity-related glomerulopathy. Angiotensin-2 (obesity-mediated overactivation of the renin–angiotensin axis), insulin,53 the renal sympathetic nervous system,54 increased postglomerular oncotic pressure due to increased filtration fraction, and mechanosensors of tubular flow rate raise tubular sodium reabsorption. Insulin– PI3K–PKB and TOR signaling are implicated in podocyte hypertrophy [809].55 In the kidney, adipokines and altered metabolism of fatty acid and cholesterol and resulting lipid accumulation56 provoke insulin resistance in podocytes, tubular

hypertrophy), but at a lower rate than the increase in glomerular tuft volume, and the podocyte density declines. Because the glomerular volume expands, stress and strain exerted on these cells can detach them and cause a local denudation of the glomerular basement membrane. In addition, approximately 50% of patients with obesity-related glomerulopathy, a hyperfiltering nephropathy that does not manifest the typical signs of the nephrotic syndrome (hyperlipidemia, hypoalbuminemia, and edema) have mild diabetoid changes (focal or diffuse increase in the mesangial matrix and thickening of the glomerular basement membrane). Intracellular lipid vacuoles can accumulate in mesangiocytes, podocytes, and proximal tubular epitheliocytes. 53 Hyperinsulinemia secondary to insulin resistance increases the tubular reabsorption of sodium, as insulin stimulates epithelial sodium channel (ENaC) activity in the late distal tubule and, to a lesser extent, in the proximal tubule and loop of Henle. 54 Three factors associated with obesity activate the renal sympathetic nervous system: high leptin concentration, low adiponectin concentration, and obstructive sleep apnea [809]. 55 Podocytes possess insulin receptor and can adjust their morphology to postprandial changes in intracapillary pressure and glomerular filtration rate. Accumulation of non-esterified fatty acids in podocytes is linked to insulin resistance and podocyte apoptosis [809]. Insulin activates TORC1 via the PI3K–PKB axis and hence lipogenesis via PPARγ and SREBP1, angiogenesis via HIF1 and VEGF, and cell growth via S6K and 4eBP1. On the other hand, S6K inhibits TORC2 and IRS1, favoring insulin resistance. Nutrients and growth factors activate TORC2, which provokes actin remodeling via PKCα, Rho, and Rac and supports cell survival via FoxO1 and sodium reabsorption via the SGK1–ENaC axis [809]. Insulin also stimulates VEGF production in podocytes. Podocytes undergo hypertrophy via TOR to cover the enlarging glomerular tuft associated with excess weight. Once it is activated by TORC2, PKB2 favors podocyte survival. 56 Renal TG accumulation results from increased fatty acid synthesis mediated by SREBP1c, which promotes the formation of acetyl-CoA carboxylase, FAS, and stearoyl-CoA desaturase SCD1, in addition to CHREBP, which upregulates production of liver pyruvate kinase [809]. Moreover, SREBP1 upregulates expression of proinflammatory cytokines, such as TNFSF1, IL1β, and Ifnγ, which activate SREBP1, hence engendering a vicious cycle. Angiotensin-2 elicits SREBP1 activity, which also mediates profibrotic TGFβ signaling. On the other hand, FXR inhibits SREBP1c and ChREBP and stimulates PPARα, thereby impeding lipid accumulation. Renal TG accumulation can also occur by increased uptake via ScaRb3 and/or fatty acid transport protein (FATP) or by decreased fatty acid oxidation mediated by PPARα via peroxisomal acylCoA oxidase-1 and carnitine palmitoyltransferase in addition to sirtuin-3 via mitochondrial longand medium-chain acyl-CoA dehydrogenases [809]. On the other hand, G-protein-coupled bile acid receptor, GPBAR1, decreases inflammation and mitochondrial ROS generation and increases mitochondrial genesis and hence mitochondrial antioxidant generation and fatty acid oxidation.

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atrophy, chronic inflammation, redox stress, and interstitial fibrosis; mechanical forces arising from glomerular hyperfiltration cause a maladaptive adaptation.

5.3.1 Epidemiology Approximately one-third of the human population consists of overweight and obese people [810]. Obesity lowers life expectancy, as it augments the coupled metabolic and cardiovascular risks, that is, the likelihood of developing T2DM and atherosclerosis. Between 1978 and 2013, the proportion of overweight and obese adults (BMI ≥ 25 kg/m2 ) increased from 28.8 to 36.9% in men and from 29.8 to 38.0% in women [809]. The prevalence of obesity has increased to about 40% among adults and approximately 20% among adolescents [811]. In 2014, about 40% of adults were overweight, and 13% were obese worldwide [812]. In some countries in Oceania, North Africa, and the Middle East, the prevalence of obesity in 2013 exceeded 50% of the adult population. The prevalence of obesity is lower but still high in other countries, such as in North America (∼30%) and Western Europe (∼20%) [721]. Overweight and obesity increased at the end of the last century much faster in adults than in children and in women than in men. In children and adolescents (2–19 years), the prevalence rates of overweight and obesity defined as BMI greater than or equal to 85th or 95th percentile for a given age or sex, respectively, increased significantly among Hispanic females and Black males. Adult Black women had the highest rate of increase (annual average increase of 0.88%) compared with other ethnic groups [662]. Ethnic disparities exist in the prevalence of two related multifactorial diseases, obesity and diabetes. In general, in both adults and children, Blacks and Mexican Americans are at a higher risk than non-Hispanic Whites [662]. Among men, Blacks have a lower percentage of body fat than non-Hispanic Whites and Mexican Americans. Among women, Mexican Americans have a higher mean percentage body fat than non-Hispanic Whites and Blacks. Childhood adiposity is associated with adult left ventricular hypertrophy. In a population of 710 adults (aged 26–48 years), after age, sex, and race adjustments, association between childhood BMI and left ventricular mass index is explained more by adult BMI than systolic blood pressure, adiposity being a major predictor

Cholesterol accumulation results from augmented cholesterol synthesis by hydroxymethylglutaryl CoA reductase (HMGCR) stimulated by SREBP2 and uptake through LDLR, ScaRa, ScaRb3, and ScaRe1, and lowered cholesterol efflux through ABCa1 and ABCg1 via LXR and catabolism linked to bile acids and bile acid transporters. Activation of SREBP2 is caused by inflammatory cytokines, which interfere with the SCAP–SREBP2–LDLR and HMGCR axes, ER stress, which stimulates SREBP2 release from the ER, and advanced glycation end products (AGEs), which prime abnormal translocation of SCAP from the ER to the Golgi body [809].

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of left ventricular hypertrophy, especially the eccentric form, whereas hypertension causes concentric left ventricular hypertrophy [813]. Therefore, as longer exposure aggravates the risk for adverse remodeling, an independent predictor of cardiovascular events, obesity should be evaluated as excess adiposity multiplied by years of exposure (then expressed in kg–year).

5.3.2 Cardiovascular Effects of Obesity Obesity is strongly related to other cardiovascular risk factors, such as dyslipidemia, T2DM, and hypertension. Diverse fatty depots have a distinct impact on the cardiovascular apparatus. Obesity is linked to FAA-emia (plasmatic free fatty acid concentration), which provokes endothelial dysfunction and atherosclerosis (e.g., coronary arterial plaques). Systemic endothelial dysfunction and CVD is strongly associated with visceral adiposity, which does not represent predominant AT in healthy subjects. On the other hand, the scAT is a minor contributor or plays a neutral or even protective role [805]. Normally, subcutaneous and abdominal vAT represent about 80% and 5–20% of total body fat mass, respectively. According to the American Heart Association, cardiovascular health relies on a BMI lower than 25 kg/m2 and a fasting glycemia less than 100 mg/dl. Both the degree and duration of obesity influence CVD prognosis [721]. Metabolically benign obesity is defined by the presence of certain factors and conditions, such as low inflammatory marker concentrations in blood, high adiponectinemia, preserved insulin sensitivity, a low amount of vAT, and proper caloric intake and physical activity [721]. Cardiorespiratory fitness is improved by physical activity and exercise. The fatbut-fit concept relies on the following observations: (1) among obese men and women who are relatively fit using the sex- and age-specific quintiles from the Aerobics Center Longitudinal Study, the risk of CVD-induced mortality is reduced and (2) a mild to moderately obese fit man or woman can have a lower CVD-induced mortality risk than an unfit individual with a normal weight. Fit obese people have lower levels of most of the CVD risk factors.

5.3.2.1

Proatherogenic Fatty Acids

Oleic acid is the most abundant circulating proatherogenic ω9-monounsaturated fatty acid. It precludes vasodilation primed by acetylcholine and elicits proliferation of vSMCs. In smooth myocytes, oleic acid represses the formation of antiatherogenic sirtuin-1 and NR1c3 (PPARγ) [814]. Sirtuin-1 deacetylates NR1c3, thereby impeding inflammation, medial SMC proliferation and migration, and hence vascular wall remodeling, and protecting against redox stress. On the other hand, oleic acid activates NFκB and raises TGFβ1 release. Moreover, oleic acid

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augments NO production owing to NOS2 overexpression via NR1c3 inhibition and NFκB activation, thereby promoting the secretion of proinflammatory MMP1 and MMP3 [814].

5.3.2.2

Endothelial Dysfunction

Obesity is associated with hypertension secondary to arterial stiffness and vascular dysfunction with reduced endothelium-dependent dilation due to defective interplay between multiple endo- and paracrine messengers that provokes inflammation, redox stress, and subsequent structural modifications of the vessel wall. In young sedentary obese men and women, acetylcholine-stimulated blood flow declines because of impaired microvascular endothelial function with respect to lean and overweight subjects, whereas interstitial concentrations of hydrogen peroxide and superoxide rise in the vastus lateralis owing to augmented NOx activity [815]. After 8 weeks of exercise, H2 O2 concentration decreases in obese subjects and the microvascular endothelial function is restored, becoming similar to that in lean humans. In skeletal muscles, synthesis of the NAD(P)H oxidase subunits cytochrome-B245α, NOxO2, and NOxA2 increases in obese subjects, linking excessive NOx-derived ROS to microvascular endothelial dysfunction in obesity. Exercise reduces cytochrome-B245α and NOxA2 formation in the skeletal muscles of obese individuals. Endothelial dysfunction correlates with altered mitochondrial quality control in aging. Cellular senescence is associated in mitochondria with defective genesis and dynamics (fusion and fission) on a microscopic scale, and defective electron transport chain (ETC) and quality control, in addition to increased superoxide production and decayed activity of antioxidant Mn SOD2 on a nanoscopic scale [816].57 NOx4 is implicated in EC senescence. Although ECs have a low content of mitochondria (2–6% of the cell volume [∼28] and 32% in hepatocytes and CMCs, respectively, in rat ECs [816]),58 damaged mitochondrial dynamics participates in endothelial dysfunction. Myocardial arteriolar ECs contain mitochondria anchored to the cytoskeleton that release ROS in response to cell deformation caused by hemodynamic stress [816]. Under hypoxia, they form perinuclear clusters using microtubules and dynein. Hyperglycemia induces ROS overproduction, which provokes mitochondrial fragmentation [817]. In ECs, the ROS-sensitive channel TRPM2 is thus gated, eliciting Ca2+ influx, lysosomal permeabilization, and redistribution of lysosomal Zn2+ to mitochondria, where Zn2+ recruits DRP1, a mitochondrial fission factor (MFF). 57 Expression

of SOD2 is regulated by FoxO and SIRT1. content depends on the balance between mitochondrial genesis, which involves transcription of nuclear and mitochondrial genes, and mitophagy. Healthy mitochondria undergo fusion–fission cycles, whereas damaged ones depolarize and are cleared by mitophagy. Endotheliocytes create a large proportion of their energy from the anaerobic glycolysis.

58 Mitochondrial

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Overproduced ROS uncouple NOS3 and react with NO,59 thereby precluding the availability of NO, an antihypertensive, anti-thrombotic, and anti-inflammatory molecule. In ECs, the integral membrane protein, mitochondrial calcium uniporter regulator Mt CUR1, in addition to UCP2 and UCP3, support mitochondrial Ca2+ uptake by Mt CU [816]. Mitochondrial calcium at relatively low levels increases PGC1α expression and hence promotes mitochondrial genesis. Moreover, it stimulates mitochondrial NO production in addition to enzymes of the tricarboxylic acid cycle and oxidative phosphorylation. In addition, H2 O2 increases mitochondrial Ca2+ content due to at least partly decreased Ca2+ extrusion secondary to inhibited Na+ – Ca2+ exchanger.

5.3.2.3

Microvascular Dysfunction

Obesity affects large blood vessels in addition to the microvasculature, not only in vAT but also in the heart, brain, kidney, lung, and skeletal muscle, altering organ perfusion and contributing to impaired release and clearance of metabolites and neurohumoral factors, in particular adipokines, proinflammatory cytokines, and cardiomyokines [818]. A high circulating lipid load, which is linked to the amount and composition of the vAT, even before the onset of obesity, engenders global microvascular dysfunction in various organs [818]. Local low-grade inflammation and overall endothelial dysfunction influence vascular structure. Changes in arteriolar tone by vasoregulators released from perivascular nerves, irrigated organs, and the endothelium, in addition to circulating factors acutely regulates local blood flow resistance and hence supply of oxygen and nutrients. The endothelium produces and secretes vasodilators, such as NO, prostacyclin (PGi2 ), hydrogen peroxide (H2 O2 ), and endothelium-derived hyperpolarizing factor (EDHF), vasoconstrictors, such as endothelin-1 (ET1), vasoconstrictory prostanoids, and superoxide (O•− 2 ), in addition to vasoactive substances, the action of which depends on the vascular bed, such as uridine adenosine tetraphosphate (UP4 A). In obesity, the sympathetic nervous system is activated by leptin [818]. Endocrine messengers, such as the hormone insulin, target ECs. Within ECs, insulin can activate the PI3K–PKB–NOS3 axis, hence producing NO, or the MAPK module, ERK1 and ERK2, which stimulates ET1 formation and release. In the − (O•− 2 ) reacts with NO to form peroxynitrite (ONOO ). Endothelial NO synthesis depends on arginase-2 (Arg2) in mitochondria and L arginine carriers, in addition to NOS3. In ECs, mitochondrial arginase-2 is constitutively expressed, whereas the cytosolic arginase-1 (Arg1) is barely detectable [816]. Acetylcholine and serotonin activate NOS3 via cytosolic calcium ions and binding of calcium– calmodulin, which activates CamK2, which controls Nos3 gene transcription and the NOS3 phosphorylation state.

59 Superoxide

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healthy vasculature, insulin-induced activation of NOS3 and epoxyeicosatrienoic acids (EETs) predominate, whereas obesity favors insulin-mediated activation of ET1 and vasoconstrictory eicosanoids. Moreover, insulin-mediated capillary recruitment by adiponectin declines with adiponectinemia, whereas FFAs and inflammatory cytokines counter this process.

Visceral Adipose Tissue Microvasculature in Obesity Blood flow to the vAT rises after a meal in lean, but not obese subjects. Lipid accumulation in the visceral white adipocyte increases in size from 50 up to 200 μm, a length greater than the O2 diffusion distance and, with accompanying capillary rarefaction, diminishes AT oxygenation [818]. Impaired AT angiogenesis associated with capillary rarefaction, hypoxia, inflammation, and metabolic dysfunction results from overexpression of the antiangiogenic splice variant VEGFa165 b, which binds, but fails to efficiently activate VEGFRs [819]. Wnt5a is a proinflammatory secreted protein that is associated with metabolic dysfunction in obesity. Concentrations of Wnt5a and VEGFa165 b correlate in the scAT and vAT of obese individuals. In scAT, where angiogenic capacity is greater than in visceral depots, Wnt5a raises VEGFa165 b formation in vascular ECs. Moreover, Wnt5a can increase secretion of soluble VEGFR1 (VEGFR1S ), an inhibitor of angiogenesis. Secreted frizzled-related protein sFRP5, which acts as a Wnt5a decoy receptor, improves capillary sprout formation and reduced VEGFR1S production. Therefore, Wnt5a hampers AT angiogenesis via VEGFa165 b in obese humans. Acute hypoxia of hypertrophic adipocytes during short periods of high-fat feeding, launches angiogenesis via VEGF and proper matrix remodeling, thereby enabling further expansion [722]. On the other hand, chronic hypoxia occurs in expanded vAT with adipocyte hypertrophy and hyperplasia during prolonged high-fat feeding. Hypoxia lowers adiponectin and raises leptin release from adipocytes [818]. Moreover, chronic hypoxia induces sustained inflammation, inappropriate angiogenesis, and adverse matrix remodeling that becomes stiff and evolves to fibrosis.

Coronary Microcirculation in Obesity In obesity and hypercholesterolemia, coupling of the coronary blood flow with the myocardial metabolic demand is altered and coronary arterial resistance rises owing to disturbed regulation of the vasomotor tone and capillary rarefaction [818]. Obesity is also associated with the structural remodeling of the coronary microcirculation. Lower capillary density in obese patients is associated with arteriolar hypertrophic inward remodeling and stiffening [818].

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Cerebral Microcirculation in Obesity The brain microvasculature is only surrounded by neurons and gliocytes. Obesity can thus impact it only via modifications of interactions between vascular cells and their neighbors, hemodynamic changes, and circulating endocrine messengers. Obesity affects the structure of small cerebral arteries, arterioles, and capillaries associated with impaired metabolism, redox stress, and endothelial dysfunction and disrupts the neurovascular coupling [818]. In obese humans, cerebral blood flow declines with impaired vasodilation during hypercapnia due to capillary rarefaction, diminished NO contribution to basal cerebral microvascular tone control, altered release of vasodilatory prostanoids, and the effect of H+ on vascular smooth myocytic ion channels [818]. Inward remodeling and progressive arterial stiffening can be observed in obese rats with metabolic syndrome.

Renal Microcirculation in Obesity Obesity is linked to increased perirenal fat deposition, which causes low-grade inflammation, reduced antioxidant capacity, and renovascular endothelial dysfunction [818]. Obesity can be associated with nephrosclerosis and glomerulonephritis. Afferent arteriolar vasodilation increases renal blood flow and hence glomerular filtration. Arteriolar and capillary density in the outer renal cortex does indeed increase in obese animals. However, newly formed vessels are more tortuous and leaky, hence immature and dysfunctional. Structural alterations in the kidney of obese subjects result from impaired balance between pro- and antiangiogenic factors, concentrations of VEGF, VEGFR2, and AngptL2 increasing and that of the angiogenesis inhibitor TSp1 decreasing owing to redox stress [818].

Pulmonary Microcirculation in Obesity Obesity is associated with structural changes in the pulmonary microvasculature with increased medial thickness of pulmonary small arteries and veins and muscularization of pulmonary arterioles [818]. In general, vasoconstriction to serotonin and hypoxia is reduced in pulmonary resistance (but not conductance) arteries of obese rats.

Skeletal Muscle Microcirculation in Obesity At rest, skeletal muscle blood flow is moderately reduced, at least in mild obesity. Because exercise hyperemia involves many redundant regulatory mechanisms, it is relatively well maintained [818].

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Resting muscle sympathetic nerve activity is significantly higher in obese patients with metabolic syndrome and does not further increase during exercise [818]. Exercise-induced increase in skeletal muscle blood flow is reduced or at best preserved in obesity. Obesity is linked to a shift in the balance of neurogenic control of skeletal muscle blood flow, with increased α AR-mediated constriction at rest, which disappears during exercise, thereby compensating for a loss of β AR-primed vasodilation. In humans, endothelium-dependent skeletal muscle microvascular vasodilation in response to acetylcholine is preserved or reduced in obesity [818]. Contribution of both NO and vasodilatory prostaglandins to ACh-induced vasodilation is reduced, but compensated for by EDHF linked to EETs and/or H2 O2 . Superoxide dismutase, which converts superoxide to H2 O2 , is more active in obese individuals. In addition, reduced NO availability is counterbalanced by reduced PDE5 activity and ET1 sensitivity of skeletal muscle arterioles and local ET production. In addition, insulin resistance is associated with a shift from insulin-primed vasodilation to -constriction [818].

5.3.2.4

Obesity and Thrombosis

The healthy endothelium synthesizes and releases regulators of the coagulation cascade, platelet activation and aggregation, and fibrinolysis. This anti-coagulant barrier produces two transmembrane proteins, thrombomodulin and heparin sulfate proteoglycan, and release tissue factor pathway inhibitor (TFPI) [820]. Thrombomodulin tethers to thrombin to activate protein-C, thereby preventing FV I I I and FV activation and thrombin formation. HSPG is a cofactor of anti-thrombin-3. TFPI inhibits both FXa and TF–FV I I a complex. Endotheliocytes also produce tissue-type plasminogen activator, which activates the fibrinolytic cascade. The endothelium also precludes platelet activation via plasmalemmal ectonucleotide pyrophosphatase–phosphodiesterase, which degrades extracellular ATP and ADP, ENPP1, and ecto-5 -nucleotidase in addition to synthesis and release of prostacyclin and especially NO [820]. NO also hampers platelet aggregation. Krüppel-like factor, KLF2, an endothelial transcription factor, contributes to maintaining the anti-thrombotic endothelial surface, as it primes thrombomodulin and NOS3 expression [820]. Abdominal obesity is associated with an elevated risk for arterial and venous thrombosis in a context of inflammation and redox stress, which can alter antithrombotic endothelium. Obesity is linked to the risk for deep vein thrombosis and pulmonary embolism. Obese patients have chronic intra-abdominal hypertension, decreased blood flow velocity in the common femoral vein, and hence disturbed venous return [820].

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Obesity and Platelet Function The circulating platelet size, which is related to higher susceptibility of platelets to activation, increases in obese subjects [820]. Platelet density also rises in obese women. Platelets are anuclear fragments of megakaryocytes. Megakaryocyte maturation depends on inflammatory mediators (e.g., IL1, IL3, IL6, IL11, and IL18), NO, and thrombopoietin. In obesity, reduced NO production is compensated by augmented release of cytokines that influence megakaryopoiesis. Platelet guide inflammation and thrombosis are activated in these processes. Several platelet activation markers are elevated in obese patients, such as the mean platelet volume, circulating concentrations of platelet microparticles, thromboxaneB2 metabolites, soluble P-selectin, and platelet-derived TNFSF5 [821]. Platelet hyperactivity can be observed in obese women without cardiovascular risk factors [820]. Android obesity is linked to a four-fold higher rate of urinary excretion of 11-dehydroTxB2 with respect to lean women, a value comparable to that associated with cigarette smoking, hypercholesterolemia, and T2DM [820]. Abdominal obesity with inflammation and redox stress favors lipid peroxidation and hence increases 8-isoPGf2α concentration, a marker of redox stress that amplifies response of platelets to low stimulating agent concentrations. Platelet activation markers, such as TNFSF5 and P-selectin, have an upregulated expression in obesity. Upon platelet activation, TNFSF5, a trimer stored in α granules of resting platelets, is rapidly exposed on the platelet surface, where it is cleaved into a soluble fragment. TNFSF5S , which has auto-, para-, and endocrine effects, elicits platelet recruitment and vascular inflammation [820]. Selectin-P, which is also stored in α granules, rapidly translocates to the surface of activated platelets, where it facilitates monocyte recruitment to the vessel wall. Numerous adipokines contribute to endothelial dysfunction and can support platelet activation (Table 5.5). In addition, the sensitivity of platelets and their response to insulin, PGi2 , and NO decrease. Insulin receptors in the platelet plasma membrane, once they are liganded, autophosphorylate and then trigger insulin anti-aggregatory signaling and lower cytosolic Ca2+ concentration via NOS3 activation and PGi2 production, which launches the cGMP–PKG and cAMP–PKA pathways, respectively [820]. In addition, insulin can reduce platelet sensitivity to various platelet stimulants, such as ADP, adrenaline, collagen, and thrombin. Moreover, insulin regulates transcription factors that launch synthesis of tissue factor and plasminogen activator inhibitor PAI1 in obese individuals. Microvesicles released from activated platelets exert proinflammatory and procoagulant effects and trigger platelet aggregation. Circulating concentration of platelet-derived microvesicles is elevated in obese subjects compared with agematched lean subjects [820]. Activated platelets not only aggregate and release proinflammatory mediators but also shed LPC-rich microvesicles. These microvesicles serves as dockers that

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Table 5.5 Major adipokines contributing to endothelial dysfunction and platelet activation (Source: [820]; CCL/CXCL chemokines, CRP C-reactive protein, IL interleukin, PAI plasminogen activator inhibitor, PGi2 prostacyclin, SAa serum amyloid-A, TNFSF tumor-necrosis factor superfamily member, VEGF vascular endothelial growth factor) Process Endothelial dysfunction

Platelet activation

Adipokines With decreased concentration: adiponectin, apelin, clusterin, ghrelin, IGF1, IL10, lipocalin-2, omentin, PGi2 , SAa With elevated concentration: adipsin, angiotensinogen, calprotectin, cathepsin-S/L/K, CCL2/3/5/7/8/11, CRP, CXCL8, fetuin-A, IL1β/6, leptin, osteopontin, PAI1, resistin, tissue factor, TNFSF1, VEGF, visfatin With decreased concentration: apelin, PGi2 , SAa With elevated concentration: CRP, IL1β/6, PAI1, tissue factor, TNFSF1

dissociate pentameric C-reactive protein (or Ptx1; 5 CRP) into its proinflammatory and pro-thrombotic monomeric isoform (1 CRP) [822]. Adiponectin tethers to its receptors AdipoR1 and AdipoR2 on platelets. Although it impedes tissue factor formation in macrophages and activity, it exerts no effects on platelet activation and aggregation [820]. Leptin targets its receptors on ECs, macrophages, and platelets. At obesityrelevant leptinemia, leptin acts synergistically with ADP and thrombin for platelet aggregation [820]. Leptin may also activate platelets via the JaK2–PI3K–PKB– PDE3a–cAMP pathway in addition to PLCγ2, PKC, and PLA2. Hyperlipidemia enhances susceptibility to thrombosis. In addition, altered platelet lipidome may predispose to thrombosis. LDL and oxLDL60 prime platelet degranulation, α2B β3 -integrin activation, apoptosis, thrombin generation, and CXCL12 release [823]. The chemokine CXCL12 tethers to its receptors CXCR4 and CXCR7 and facilitates LDL and oxLDL uptake by platelets. Intraplatelet content of oxidized lipid metabolites, cholesteryl esters, sphingomyelin, ceramides, phospholipase metabolites (diacylglycerol), and acylcarnitines is elevated in atherosclerosis [823].

60 LDLs

taken up by platelets are oxidized by mitochondrial ROS, generating oxidized fatty acids (oxFAs), phospholipids (oxPLs), and lysophosphatidylcholine (oxLPC). Oxidized LDLs can activate platelets and promote uptake and generation of lipid metabolites. Lipids can be metabolized into ceramides, diacylglycerol, and sphingomyelin.

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Obesity and Coagulation Factors Obese subjects also have an elevated circulating concentration of von Willebrand factor, tissue factor, clotting factors FV I I and FV I I I , and fibrinogen but also of plasminogen activator inhibitor PAI1 and thrombin-activatable fibrinolysis inhibitor (TAFI), having an impact on fibrinolysis and contributing to a pro-thrombotic state [820]. Plasminogen activator inhibitor PAI1, or serpin-E1, is mainly secreted from the liver and AT. It represses plasminogen activators (tPa and uPA), thereby precluding dissolution of the fibrin clot. Obesity is linked to upregulated PAI1 formation in the vAT, hence hampering fibrin clearance and raising thrombosis risk. Thrombin not only contributes to clot formation but also stabilizes the clot via thrombin-activatable fibrinolysis inhibitor, which protects the fibrin clot against degradation. In obesity, the AT releases lower amounts of adiponectin, raising platelet susceptibility to aggregation and limiting fibrinolysis via PAI1 [820]. Obesity also impairs platelet sensitivity to insulin and promotes production of isoprostanes, which further render platelets reactive. In obese individuals, the AT favors immunocyte switch to an inflammatory phenotype, macrophage evolving to an M1 proinflammatory and TH2 cells to TH1 and TH17 polarization [820]. Adipose tissue M1 macrophages secrete tissue factor, which, combined with augmented hepatic synthesis of FV I I and FV I I I , heightens the coagulation risk. Tissue factor, a transmembrane glycoprotein, serves as a receptor for FV I I a and then activates the extrinsic coagulation cascade.61 Elevated tissue factor production in obesity is linked to its synthesis in macrophages and neutrophils in addition to adipocytes and stromal vascular cells. It can result from the action of insulin, TNFSF1, TGFβ, and leptin [820]. Production of ROS by NAD(P)H and xanthine oxidases, uncoupled NOS3, lipoxygenases, cyclo-oxygenases, microsomal P450 enzymes,62 and prooxidant CyP heme molecules augments. Furthermore, ROS production is favored by overactivation of the renin–angiotensin axis (RAA), as several RAA components are produced by dysfunctional adipocytes (Sect. 5.4.5.17). Concomitantly, endothelial dysfunction is accompanied by a decreased antioxidant defense, particularly NFE2L, in metabolic syndrome patients [820].63

61 Activation of the TF–FV I I a complex activates FX, thereby priming generation of thrombin and conversion of fibrinogen to fibrin. The generation of an insoluble fibrin clot is the final step of the coagulation cascade. 62 Microsomal P450 enzymes comprise CyP1a1/2, CyP2a6, CyP2b6, CyP2c8/9/19, CyP2d6, CyP2e1, CyP2j2, CyP3a4/5/7/43, and CyP4a11 [824]. 63 Redox stress causes dissociation of NFE2L from keap1, which initiates NFE2L proteasomal degradation. Hence, NFE2L translocates to the nucleus, where it binds to antioxidant response element and triggers transcription of antioxidant genes.

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Aldosterone and Mineralocorticoid Receptor

Obesity, hypertension, and hyperaldosteronism can be interrelated. The renin– angiotensin–aldosterone axis is activated in obesity and hypertension. In humans, obesity is associated with elevated aldosterone production in addition to metabolic syndrome and cardiac hypertrophy and fibrosis. In obese diabetic mice, concentrations of aldosterone and mineralocorticoid receptor (MR or NR3c2) rise [825]. Blockage of NR3c2 counters the pro-inflammatory cytokine profiles of AT in obese mice. In addition to the kidney, where aldosterone provokes sodium retention and volume expansion, NR3c2 lodges in the colon, brain, and AT, especially adipocytes in the perivascular adipose depots, and on leukocytes, particularly monocytes and macrophages, vascular smooth muscle and endothelial cells, and cardiac myocytes and fibroblasts. Mineralocorticoid receptor can favor arterial stiffness and insulin resistance [826]. In macrophages and dendrocytes, NR3c2 affect their phenotype and hence cytokine production [489]. In addition, the G-protein-coupled receptor, GPR30, may mediate some of the effects of aldosterone [825]. An early aldosterone-mediated PKCα activation promotes NR3c2 transactivation, rapid (within minutes) nongenomic aldosterone effect, enhancing its genomic action. Once it is liganded, NR3c2 translocates to the nucleus, where it connects to the steroid hormone (SHRE) and negative response element (nSRE) [827]. Among proteins, the synthesis of which is regulated by aldosterone, glucose 6-phosphate dehydrogenase may mediate the effect of NR3c2 on cellular function [825]. NR3c2 interacts with various modulating proteins of its activity, such as AT1 , EGFR, and probably striatin [825]. The small GTPase, Rac1, increases NR3c2 activity. In addition, the epigenetic regulator and demethylase KDM1a and membrane scaffold protein caveolin-1 modulate NR3c2 activity. Augmented NR3c2 activation in obesity can result from: (1) adipocyte production of aldosterone and aldosterone secretagogues, (2) increased NR3c2 synthesis, and (3) altered activity of NR3c2-interacting proteins [825]. Crosstalk among the AT, heart, and adrenal cortex on a macroscale is linked to interaction between aldosterone and adipokines on a nanoscale, especially between aldosterone and adiponectin [827]. Both AdipoR1 and AdipoR2 are identified in the normal human adrenal cortex. In healthy subjects, a high-salt diet (HSD) decreases concentrations of renin, Agt2, and aldosterone and increases adiponectinemia [827]. On the other hand, dietary sodium intake alters concentrations of almost all of the NR3c2 interactors and favors aldosterone-mediated vascular damage in animal models. Aldosterone-producing CyP11b2+64 cell clusters appear with aging; they are distinct from the zona glomerulosa of the young adrenal gland cortex, a continuous outer layer of aldosterone-producing cells, which becomes progressively discontinuous during aging [828]. 64 CyP11b2:

adrenal aldosterone synthase. Aldosterone synthesis requires the coordinated activity of several enzymes, in addition to CyP11b2, which converts deoxycorticosterone into aldosterone.

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Adrenal Gland Aldosterone is primarily synthesized in cells of the adrenal cortex. Classical aldosterone secretagogues include Agt2, ACTH, and K+ ion. However, adipocytes synthesize and secrete aldosterone in addition to mineralocorticoid-releasing factors that act directly on the adrenal gland and raise aldosterone secretion [826, 827]. Conversely, aldosterone promotes adipogenesis via the mineralocorticoid receptor.

Kidney The major action of aldosterone and its receptor is situated in the kidney, where they increase the amount of the ENaC on the apical membrane of epitheliocytes in the distal nephron and hence renal sodium reabsorption. Angiotensin-2 is the primary stimulus for aldosterone secretion in response to blood volume depletion.

Adipose Tissue Adipocytes produce aldosterone synthase and hence aldosterone, basally and upon exposure to angiotensin-2 [825]. Paracrine secretion of aldosterone by the pvAT favors vascular dysfunction in obese db/db mice, whereas NR3c2 blockage prevents endothelial dysfunction in diet-induced obesity (DIO). Overnutrition facilitates NR3c2 activation, which promotes adipogenesis via the TOR–S6K1–PPARγ and –C/EBPα pathways [826]. In fact, the TORC1 complex activates SREBPs via both TOR–S6K1-dependent and -independent signaling. The TORC2 complex controls PKB activity and thus participates in the early stage of adipogenesis. On the other hand, aldosterone hampers production and activity of UCP1 in brown adipocytes. Adipocytes can prime the adrenal secretion of aldosterone, as they release para- and endocrine aldosterone-stimulating factors, independently of plasmatic renin and K+ concentrations. The potent oxidized derivative of linoleic acid, (12,13)-epoxy 9-keto 10(trans)octadecenoic acid (EKODE), stimulates aldosterone genesis in rat adrenal glomerulosa cells. Concentration of EKODE correlates with aldosterone level in human subjects with an elevated BMI and in hypertensive African Americans [827]. Adipocytes also synthesize adiponectin, angiotensinogen, angiotensin, and mineralocorticoid receptor, in addition to aldosterone, in some conditions [827]. Angiotensin-2 produced by adipocytes, elicits expression of aldosterone synthase (CyP11b2) and promotes aldosterone secretion using the PP3–NFAT pathway.65 Leptin also promotes CyP11b2 formation and hence stimulates adipocytic

65 Both

aldosterone and cortisol can activate mineralocorticoid receptor.

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aldosterone synthesis using calcium ion. In addition, CETP inhibitors increase aldosterone synthesis in adipocytes via NOx [826]. Both NOx1 and NOx4 are involved in ROS-sensitive aldosterone production in cultured adipocytes. Conversely, aldosterone stimulates production of NOx2 and cytochrome-B245α subunit via NR3c2 and of NOx organizer NOxO2 via both AT1 and NR3c2, at least in rats [826]. NOx-derived ROS in WAT upregulates proinflammatory adipokines, engendering vascular insulin resistance and impaired vasodilation. Altered perivascular AT volume is linked to elevated aorta size and stiffness. Elevated aldosterone concentration and subsequent mineralocorticoid receptor activation linked to maladaptive AT expansion cause redox stress, inflammation via impaired adiposecretome (e.g., increased formation of TNFSF1, IL6, IL18, CCL2, CXCL8, TLR4, AngptL2, and Wnt5a and linked recruitment to AT of dendrocytes, B and T lymphocytes, macrophages, mastocytes, and neutrophils),66 and deregulated adipocyte autophagy,67 alters insulin metabolic signaling, and increases risk for hypertension [826]. Augmented activity of the aldosterone–NR3c2 couple is observed in obese patients with insulin resistance and hypertension. In adipocytes, NR3c2 activation not only favors dyslipidemia and insulin resistance but also alters arteriolar vSMC contractility via elevated vascular redoxsensitive PKG1 activity and lowered redox-sensitive rock activity in mice overexpressing adipocytic NR3c2 [826].

Heart In the heart, aldosterone causes inflammation, redox stress via NOxs, insulin resistance, left ventricular hypertrophy and cardiac fibrosis via genomic and nongenomic effects of the mineralocorticoid receptor [827]. Cardiomyocytes also possess AdipoR1, AdipoR2, and NR3c2, which is targeted by circulating aldosterone and cortisol. They can also synthesize aldosterone and adiponectin in pathological situations [827]. In mice, selective deletion of macrophage NR3c2 reduces cardiac fibrosis and blood pressure [825]. In CMCs, NR3c2 induces expression of ANP, a marker of cardiac hypertrophy. It interacts with KAT3b, a GATA4 transcriptional coactivator involved in cardiac hypertrophy [827]. The aldosterone–NR3c2 couple launches expression of connective tissue growth factor. In these cells, NR3c2 also rapidly raises concentration of Na+ –K+ –2Cl− cotransporter and concomitantly lowers that of Na+ –K+ pump

66 Low

doses of spironolactone, an MR antagonist, prevent macrophage infiltration and M1 macrophage polarization in aortic and cardiac walls [826]. On the other hand, the Western diet enriched in saturated lipids and refined carbohydrates increases αM -integrin, a macrophage marker in the cardiac wall. 67 In obesity, autophagosome maturation can be impaired. In addition, activity of AMPK, sirtuin, and TORC1 is altered [826]. However, in obese mice, defective autophagy may decrease WAT volume and enhance insulin sensitivity. Moreover, adipocytic MR activation by aldosterone promotes adipocyte autophagy.

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via PKC , elevating intracellular Na+ and Ca2+ concentration, hence favoring prohypertrophic ERK1/2 signaling and phosphorylation of Src, JNK, and NFκB [827]. Moreover, it boosts formation of the pro-hypertrophic cytokines cardiotrophin-1 and IL18, acting via P38MAPK on the one hand and the Rho–rock (Sect. 2.3.3.5) and PPAR–NFκB pathways on the other [827]. Aldosterone stimulates fibroblasts via the Ras–Raf–ERK axis. In these cells, aldosterone increases via NR3c2 expression of TGFβ, which favors the production of matrix proteins, but lessens NOS2 activity, thereby facilitating fibrosis. Aldosterone can also elicit proliferation of cardiofibroblasts via the kRas–ERK1/2 pathway [827].

Vasculature In the vasculature, aldosterone and NR3c2 favor macrophage infiltration, proinflammatory cytokine and superoxide production, but reduces endothelial progenitor cell migration and NO release, thereby leading to endothelial dysfunction and wall remodeling (Table 5.6) [825]. Endothelial and smooth myocytes possess 11β-hydroxysteroid dehydrogenase 11β HSD2 that allows aldosterone–NR3c2 interaction. Once it is activated, NR3c2 translocates to the nucleus and regulates transcription of genes that encode adipokines and SGK1 [826]. Mineralocorticoid receptor can be activated not only by aldosterone but also by glucocorticoids. Cortisol may participate in obesity-linked vasculopathy, especially when activity of 11β HSD1, which produces cortisol, is altered, or that of 11β HSD2, which converts cortisol to inactive, cortisone is impaired [825]. Aldosterone contributes to regulating not only insulin and redox signaling but also the vasomotor tone. However, whereas short-term exposure to aldosterone promotes vasodilation via NO, prolonged exposure causes vasoconstriction, as it attenuates NO availability, raises endothelin-1 expression and causes H2 O2 overexpression [826]. In aged mice with a vSMC-specific deletion of NR3c2, redox stress and vasoconstriction decline [825]. In vascular smooth myocytes, NR3c2 contributes to elevation in blood pressure due to aging [489]. In vSMCs, MR activation is involved Table 5.6 Effects of NR3c2 on the vasculature, that is, endothelial and smooth muscle cells and perivascular adipocytes (Source: [825]; ↑ increase, ↓ decrease, icam intercellular adhesion molecule, NO nitric oxide)

Cell type Endotheliocyte

Smooth myocyte Adipocyte

Effects Redox stress ↓ NO availability and vasodilation ↑ icam1 and leukocyte adhesion Redox stress ↑ vasoconstriction ↑ inflammatory factors ↓ insulin sensitizers Altered activity of vasoactive factors

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in vascular stiffness, as it raises vSMC proliferation, migration, calcification, in addition to CaV 1.2b channel activation and hence vasoconstriction [826]. Diet-induced obesity impairs endothelial regulation of the vasomotor tone; maximal acetylcholine-induced relaxation decreases down to 20% [829]. On the other hand, the MR antagonist eplerenone (200 mg/kg/day) of HFD-fed obese mice prevents this effect, as it downregulates the formation of membrane and cytosolic subunits of NAD(P)H oxidase P22PhOx and P40PhOx, but upregulates that of antioxidants (glutathione peroxidase-1 and superoxide dismutase SOD1 and SOD3). Eplerenone does not affect obesity-induced synthesis of prostaglandin synthases, PGhS1 and PtgIS. Therefore, obesity-induced endothelial dysfunction depends on the endothelial NR3c2 and redox stress. In mice, endothelium-specific ablation of the NR3C2 gene protects against DIOinduced endothelial dysfunction, but not obesity-linked insulin resistance and AT inflammation. Aldosterone infusion into lean mice achieving aldosteronemia similar to that in obese mice provokes endothelial dysfunction in WT mice due to redox stress, but not in EC NR3C2−/− mice [829]. In obese mice, Rac1 activation increases in ECs. In aortic ECs, the aldosterone–NR3c2 couple raises the plasmalemmal concentration of icam1 and subsequently leukocyte adhesion [825]. In ECs, NR3c2 participates in vascular stiffening, which accompanies obesity [827]. In ECs, NR3c2 activation is facilitated by adipocytic NR3c2 overactivation, increasing endothelial plasmalemmal Na+ channel (EdNaC) density, actin polymerization, and endothelial cortical stiffness, which decreases NOS3 activity [826]. Decayed NO amount provokes tissue transglutaminase TG2 release in the extracellular space, launching vascular and cardiac wall remodeling.68 The aldosterone–NR3c2 axis causes tissular remodeling. In resident vascular macrophages, NR3c2 activation mediates M1 polarization, chemotaxis, and inflammatory response [826]. In addition, AdC NR3c2 activation increases H2 O2 , affecting the vasomotor tone. Moreover, vascular NR3c2 overactivation stimulates pro-fibrotic TGFβ1, leading to fibrosis [826].

5.3.3 Obesity-Associated Chronic Inflammation and Insulin Resistance Cardiovascular disease is associated with adipocyte dysfunction and moderate chronic sterile inflammation. Inflamed AT is a local pathogenic environment. Impaired pvAT function affects associated blood vessel function in addition to causing systemic disorders via adverse delivery by blood of certain adipokine types.

68 TG2

is nitrosylated by NO and hence retained in the cytosol. Reduced NO amount causes TG2 secretion in the extracellular medium, where it elicits crosslinking of extracellular matrix proteins and fibrosis.

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Table 5.7 Adipokines and inflammation (Source: [802]; ↑ increase, ↓ decrease, APC antigenpresenting cell, DC dendrocyte, Eϕ eosinophil, EC EC, Mϕ macrophage, Mo monocyte, NK natural killer cell, T H helper T lymphocyte, CCL C–C-motif chemokine [ligand], CD cluster of differentiation, CmkLR chemokine-like receptor, CXCL C–X–C motif chemokine, CX 3 CL(R) CX3 C chemokine (receptor), IL interleukin, NFκB nuclear factor κ light chain enhancer of activated B cells, RBP retinol-binding protein, TGF transforming growth factor, TNFSF tumornecrosis factor superfamily member) Adipokine Adiponectin

Chemerin

Immunocyte recruitment ↓ Eϕ chemotaxis ↓ EC icam1 ↓ T-cell recruitment ↓ CXCL Chemotaxis via CmkLR1 especially on DC, NK, Mϕ

Leptin

Chemotaxis of Mo/Mϕ ↑ Mφ CCL3/4/5

RBP4

ND

Resistin

CD4+ T-cell chemotaxis ↑ Mφ CCL1/3/CXCL1 ↑ CX3CL1, CX3CR1 ↑ EC/SMC icam1/vcam1

Visfatin

Immunocyte activation ↓ IL17 in γ/δ T cell M1-like Mϕ phenotype CD4+ T-cell activation ↓ antitumoral DC immunity ↑ NKκB, TGFβ ↑ adiponectin ↓ TNFSF1, IL6 ↑ Mo/Mφ IL6/TNFSF1 T-cell activation, proliferation ↑ TH1/17 ↓ TH2 /TReg ↓ NK cytotoxicity Activation of APC, T cell Inhibited by TNFSF1 ↑ Mφ IL6/23/27 ↑ TH1/17 ↑ B-cell maturation ↑ leukocyte activation ↑ NFκB, TNFSF1, IL1β/6

Upon stressor exposure, adipocytes produce inflammatory cytokines supporting local infiltration and activation of immunocytes (Tables 5.7 and 5.8). Conversely, activated immune leukocytes secrete cytokines that alter adipocyte function and subsequently adipokine secretion pattern. Obesity-related AT inflammation impairs action of insulin in insulin-sensitive organs, such as the liver and skeletal muscle along with endothelium, in addition to AT. In obesity, many types of immunocytes accumulate in insulin-targeted organs and favor chronic inflammation and hence glucose intolerance and insulin resistance. Decreased uptake of excess fatty acids and an increased lipolysis rate in adipocytes increase fatty acid flux to the liver. Lipid accumulation in the liver causes NAFLD. Hepatic glucose egress and glycemia regulation are controlled by AT lipolysis rather than by insulin action on the liver. Availability of adiposederived fatty acids determines acetyl-CoA concentration in the liver, independently

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Table 5.8 Immunocytes infiltrating the AT (AT; Source: [802]; APC antigen-presenting cell, epiAT epididymal AT, ingAT inguinal AT, pvAT perivascular AT, scAT subcutaneous AT, vAT visceral AT) Cell type APC

B cell Eosinophil

Preferential location vAT>scAT

pvAT>vAT vAT>scAT vAT>scAT

Macrophage Neutrophil NK cell

vAT>pvAT vAT>scAT vAT>scAT epiAT>ingAT

NKT cell Cytotoxic CD8+ T cell γ/δ T cell TH1 TH2

epiAT>ingAT vAT>scAT

TH17

vAT>scAT epiAT>ingAT

TReg

vAT>scAT vAT>scAT vAT>scAT

Metabolic effects ↑ AdC ROS ↑ lactate production Differentiation of adipocytes via CSF2 Glucose intolerance via IgG Higher fasting insulinemia Insulin sensitivity Enhances beiging Insulin resistance Insulin resistance Insulin resistance Infγ production Promotes M1 macrophage phenotype Insulin resistance, hepatosteatosis Insulin resistance, steatohepatitis Regulates glucose tolerance via perforin Insulin resistance Insulin resistance Improves glucose tolerance via IL13–STAT3 and M2 macrophage phenotype induction Enhances beiging Insulin resistance, obesity Suppresses adipocyte differentiation Insulin sensitivity

of liver insulin sensitivity, but depends on increased gluconeogenesis via activation of pyruvate carboxylase by acetyl-CoA [717]. After food ingestion, nutrients are transported to and stored in the skeletal myocytes and adipocytes and subsequently used in periods of energy deprivation. Nutrient uptake relies on adequate organ perfusion. In skeletal muscles, insulin dilates arterioles and increases the capillary surface area, thereby facilitating nutrient and insulin delivery [720]. On the other hand, insulin resistance limits postprandial perfusion. Acute overfeeding reduces skeletal muscle perfusion, but not AT irrigation [830]. However, in ECs of the forearm circulation in healthy volunteers, insulin promotes ET1 synthesis and release [720]. In fact, in the skeletal muscle circulation, insulin can stimulate the release of both NO and ET1, thereby priming a neutral response. Insulin stimulates NO production, as it promotes NOS3 phosphorylation at Ser1177 and impedes that at Thr495 [831].

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In obesity, activated and inflamed endothelium initiates AT inflammation. In cultured ECs exposed to palmitic acid, which provokes mitochondrial damage and leakage of mitochondrial DNA into the cytosol, cytosolic DNA sensor cGMP– cAMP synthase activates stimulator of interferon genes (sting). Sting then tethers to interferon regulatory factor IRF3, causing its phosphorylation and nuclear translocation [832]. IRF3 connects to the ICAM1 promoter, icam1, eliciting monocyte adhesion on ECs (cGAS–STING–IRF3–icam1 pathway). Obesity is characterized by adipocyte hypertrophy and augmented infiltration of macrophages in adipose depots. Infiltrated macrophages in the AT generate ROS by NOx2, which causes lipid peroxidation. In adipocytes, excess nutrient (e.g., glucose and fatty acids such as palmitate) also provokes production by NOx4 of ROS in addition to a transient increase in activity of the pentose phosphate pathway, which is a major source of NADPH. This activates NOx and causes AT inflammation and insulin resistance [833]. The population of CX3 CR1+ sympathetic nerve-associated macrophages (SNAMϕs) in AT, which are distinct from adipose tissue macrophages (ATMϕs), increases in mouse models of obesity [834]. In humans, SNAMϕs also lodge in the paravertebral sympathetic ganglia. Fasting or cold exposure triggers noradrenaline (NAd) release from sympathetic neurons that innervate the AT. NAd stimulates adipocytic adrenoceptors that launch the Gs–cAMP–PKA axis, thereby promoting lipolysis in the WAT and hence fat mass reduction, in addition to heat production by brown adipose tissue (BAT). On the other hand, obesity and aging support SNAMϕ activity, decreasing response to cold and starvation. Fasting-induced lipolysis is also impaired in the elderly due to NAd degradation by ATMϕs. With aging, synthesis of NAd-degrading monoamine oxidase-A (MAOa) is indeed upregulated in ATMϕs via secreted GDF3, production of which is upregulated by the NLRP3 inflammasome [835]. Furthermore, in aged mice during fasting, adipocytes fail to synthesize hormone-sensitive lipase (HSL or lipase-E) and ATGL (Atgl or patatin-like phospholipase, PnPLA2). In addition, AT SNAMϕs limit NAd-primed lipolysis during aging and in obesity, clearance by SNAMϕs of extracellular NAd released from neurons contributing to obesity. In mice, diet-induced obesity raises the number of SNAMϕs [834]. Noradrenaline-scavenging AT SNAMϕs import and degrade NAd, as they possess sodium-dependent NAd transporter SLC6a2 for NAd import as well as MAOa for NAd degradation (clearance).69 Activated sympathetic nervous system elicits NAd uptake by SNAMϕs through SLC6a2, increasing NAd content, and shifts the SNAMϕ phenotype to a proinflammatory state. The specific deletion of SLC6A2 in SNAMϕs does indeed increase NAd concentration, thermogenesis, browning of white adipocytes and thus BAT mass, and weight loss in obese mice [834]. In the pvAT, recruitment of proinflammatory macrophages is linked to a depletion of the vasodilator hydrogen sulfide in the vessel [836]. In mesenteric arterioles,

69 MAOa

catalyzes oxidative deamination of neuro- and vasoactive amines (e.g., adrenaline, NAd, and serotonin) in the central nervous system (CNS) and peripheral organs.

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smooth myocytic and endothelial H2 S concentrations decline in vessels from obese mice with respect to those of lean mice. This effect depends on NOS2 activity of pvAT-resident proinflammatory macrophage. In skeletal muscles, insulin resistance is linked to a decreased signaling via insulin receptor substrate IRS1 and PI3K, whereas signaling via the MAPK module is maintained [687]. The kinase activity of InsR and, downstream from IRS1, of PI3K lowers in adipocytes of obese individuals, whereas IRS2-mediated PI3K activity remains normal. In the liver, the number and activity of insulin receptors and the ability of insulin to inhibit glycogenolysis do not change. Adaptive hyperinsulinemia is a compensatory state. All insulin signaling pathways are not homogeneously affected by insulin resistance that is linked to some pathways, whereas insulin sensitivity is maintained in others [687]. On the one hand, insulin resistance is observed in the brain, liver, skeletal muscle, and AT. On the other, hyperinsulinemia associated with insulin resistance can potentiate insulin action in insulin-sensitive cells. Hyperinsulinemia, which acts on the liver, kidney, and ovary, causes hypertriglyceridemia, increased sodium retention and hypertension, and hyperandrogenism, respectively. In mice, ablation of the insulin receptor in the vascular endothelium does not affect glucose metabolism, but lowers cardiac frequency and blood pressure in addition to protecting against adverse neovascularization in the retinal vasculature and blunting upregulated expression of VEGF, NOS3, and ET1 upon hypoxia [687]. In insulin resistance, elevated circulating insulin concentration favors production of vasoconstrictors and proinflammatory molecules, such as endothelin-1 and serpin-E1 (PAI1), thereby contributing to endothelial dysfunction [831]. However, endothelium-dependent hyperpolarization is maintained to elicit endotheliumdependent vasodilation in obese animals.

5.3.3.1

PI3K

The lipid and protein kinase PI3K is involved in the regulation of cell metabolism, growth, proliferation, migration, and survival. It produces the lipidic second messenger PIP3 to regulate cell fate and Ca2+ signaling, hence connecting cell contractility to metabolism control. It phosphorylates the lipidic substrates, PIs (PI4P and PIP2 ). Eight PI3K isoforms can be grouped into three categories according to the structure and preferred lipid substrate. Set-I PI3K isozymes form PIP3 from PI(4,5)P2 , set-I I PI3Ks generate PI(3,4)P2 and PI3P, and set-I I I PI3K primarily synthesizes PI3P. Among set-I PI3Ks, subset-I A heterodimeric PI3Ks (ubiquitous PI3Kc1α and PI3Kc1β and leukocytic PI3Kc1δ ) are activated by protein Tyr kinase receptors [837]. The single subset-I B member, PI3Kc1γ , which is confined to hematopoietic and cardiac and smooth myocytes, mainly signals from GPCRs. Nevertheless, PI3Kc1β can be activated by GPCRs and PI3Kc1γ by RTKs via the Ras–PI3Kr6 axis. PI3K signaling can be terminated by PTen, which dephosphorylates PIP3 .

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The latter serves as a docker for lipid-binding domain-containing proteins, such as PKB, which is activated by phosphorylation on Thr308 and Ser473 by PDK1 and TORC2, respectively. Activated PKB phosphorylates TSC2 and TORC1 involved in protein synthesis and cell growth, GSK3 implicated in glucose metabolism, and the FoxOS and P53 regulator of cell survival. In addition, both PI3Kc1α and PI3Kc1β are required for the maintenance of Ttubule network integrity via the correct location of junctophilin-2 and hence Ca2+ handling. Moreover, PI3Kc1α participates in controlling myofiber maturation and Z-disc alignment. PI3K mediates insulin action in the heart, especially insulin- and IGF1responsive PI3Kc1α . The latter controls CMC growth and contractility, as PKB phosphorylates (potentiates) CaV 1.2a action.70 In addition, PI3K can control Ca2+ signaling in response to catecholamines; it prevents positive inotropic action of β-adrenoceptors. β1-Adrenoceptor, the predominant subtype, and β2AR increase PI3K activity in adult rat CMCs [837]. Once β AR is activated by NAd and circulating adrenaline, it stimulates PKA that then phosphorylates Ca2+ -handling proteins. PI3K does not affect Ca2+ effectors directly, but operates via β ARK1, which phosphorylate liganded GPCRs, leading to their desensitization and internalization, thereby reducing the plasmalemmal density and β1AR-mediated positive inotropy. On the other hand, PI3Kc1γ is implicated in signaling triggered by β2AR using the cAMP–PKA axis confined to plasmalemmal nanodomains, hence controlling Ca2+ transient amplitude and decay kinetics, limiting the Ca2+ spike magnitude, but keeping a proper gating duration [837]. Calcium transient amplitude elevates and decay kinetics lowers in PIK3CG−/− adult ventriculomyocytes, which are more sensitive to stimulation by β2AR, which are compartmentalized in plasmalemmal compartments, via a kinaseunrelated mechanism. PI3Kc1γ serves as an anchoring protein that maintains within the same complexes PKA and PDE3a and PDE4a–PDE4b, thereby favoring PDE activation by PKA and subsequent cAMP degradation in restricted compartments. Whereas PI3Kγ-regulated PDE4 limits cAMP owing to subplasmalemmal domains close to CaV 1.2a, restraining Ca2+ influx and inotropic response, PI3Kγ-regulated PDE3 and PDE4 may control phospholamban activity and thus a cAMP pool at the ER for Ca2+ re-uptake during diastole, hence yielding proper lusitropy. In insulin resistance, the PI3K–PKB signaling activation is impaired, eventually causing contractile dysfunction and diabetic cardiomyopathy. It also prevents

70 In CMCs,

Ca2+ enters the cell mainly through CaV 1.2a in T tubules and triggers a massive Ca2+ release from the ER through RyR2 and binds to troponin-C, enabling myosin–actin interaction and CMC contraction. Calcium signaling is controlled by activating and inhibiting kinases and phosphatases, which target Ca2+ channels and their adaptors. In CMCs, PKA phosphorylates (activates) CaV v1.2a and RyR2 in addition to phospholamban, thereby having positive inotropic and lusitropic effects. Once it is activated by cytosolic Ca2+ and diacylglycerol, PKC phosphorylates Na+ –Ca2+ exchanger, transient receptor potential channels, and PLC [837].

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hypertension-induced fibrosis. On the other hand, angiotensin-2 has a rapid negative inotropic effect, likely via PKCα and PI3Kc1α inhibition [837]. In ECs, VEGF and FGF regulate angiogenesis via different PI3K subtypes (PI3Kc1α or hRas-activated PI3Kc1γ ), Ca2+ , and NO [837]. Both FGFR and VEGFR provoke NOS3 phosphorylation via the PI3K–PKB and PLCγ–Ca2+ – Cam–CamK2 pathways. In addition, Ca2+ -dependent cytosolic cysteine peptidases calpain-1 and -2 control cytoskeleton dynamics and hence cell migration, calpain-2 being involved in VEGF-induced angiogenesis. In vSMCs, the PI3K–PKB axis is required for correct CaV 1.2b functioning.71 PI3Kc1γ is the major isozyme, which is preferentially linked to Ca2+ signaling [837]. It is implicated in Agt2-mediated Ca2+ entry through CaV 1.2b and subsequent vasoconstriction. Set-I PI3Ks act both in the contractile and the synthetic phenotype. PI3Kc1γ participates in SMC migration induced by chemokine receptors such as CCR2 in addition to PDGFR [837]. In addition, PI3K raises TRPC6 translocation to the plasma membrane, especially in pulmonary arterial smooth myocytes in some cases of idiopathic pulmonary hypertension. Intimal hyperplasia is also related to interferon-γ released by T lymphocytes using PI3Kc1γ . PI3Kc1γ supports activation of macrophages in obesity, in addition to oxLDL internalization during atherogenesis. In a nonhematopoietic cell type, its action in HFD-induced inflammation and insulin resistance depends on its role in the control of adiposity [839]. In leukocytes, PI3Kγ recruits neutrophils to the AT, which is associated with macrophage activation in the AT and inflammation, hence triggering insulin resistance in the early stage of obesity.

5.3.3.2

PKB

Both acetylation and phosphorylation intervene in the initiation of insulin signaling. In the heart, insulin-stimulated glucose oxidation decreases in obesity. Impaired signaling by the Ins–PKB–GSK3β axis and hence phosphorylation of its substrates reduces cardiac glucose use [840]. Cardiac insulin resistance in obese mice is associated with increased PKB acetylation and decreased phosphorylation. Acetylation of PKB is increased by HFD, which decreases its phosphorylation state and activity. Deacetylation of PKB is a prerequisite for its activation, which enhances insulin signaling in obesity. 71 Smooth

myocyte contraction only partly relies on CaV v1.2b and RyR; it primarily depends on PLC–DAG/IP3 pathways that trigger Ca2+ influx through receptor-operated channels and Ca2+ release through IP3 R, respectively. Activated calmodulin stimulates MLCK that phosphorylates MLC20, hence launching contraction. Vasoconstriction is countered by vasodilation. In arterioles, Ca2+ , rather than IP3 crossing gap junctions of myoendothelial junctions, is the intercellular signal that provokes endotheliummediated feedback vasodilation [838]. Calcium ion enters vascular smooth myocytes through CaV 1.2b channels and subsequently ECs through gap junctions. However, the magnitude of Ca2+ signals in ECs depends on IP3 Rs, which amplify Ca2+ cues to propagating waves that activate IK channels to suppress vasoconstriction.

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389

Sirtuins

Sirtuins (SIRT1–SIRT7) are type-I I I histone and protein deacetylases and ADP ribosyltransferases that require NAD+ as a cosubstrate. The founding member of the SIRT family is the yeast silence information regulator-two (SIR2; i.e., SIRT), a factor that silences the mating-type locus, in addition to its participation in telomere regulation, maintenance of DNA integrity, and lifespan extension. Three sirtuin isozymes (SIRT1 and SIRT6–SIRT7) localize to the nucleus. In mammalian cells, instead of gene silencing, SIRT1 often promotes gene transcription by deacetylating specific transcription factors, corepressors, and coactivators (e.g., FoxOs, MyoD, NFκB, P53, and PGC1α). Sirtuins rapidly adjust the activity of histones, transcription factors, metabolic enzymes, and structural proteins to cellular needs. They operate in chromatin silencing, cell cycle regulation, cellular differentiation, and metabolism. They also enable organisms to cope with different stressor types and prevent aging. Lysine acetylation regulates the activity of enzymes involved in both fatty acid and glucose metabolism. Their specific functions differ, but different types of sirtuins cooperate to control cellular processes. In addition to their synergistic actions, sirtuins have also antagonistic effects. Members of the sirtuin family can set up cross-regulation. Obesity augments reliance of the heart on fatty acid oxidation, a major energy source in the adult heart. This phenomenon involves increased circulating fatty acid concentrations, altered transcriptional control of fatty acid oxidative enzymes in addition to post-translational control of fatty acid and glucose oxidation factors, and the development of cardiac insulin resistance, which decreases glucose oxidation [840].

Sirtuin-1 Sirtuin-1 autodeacetylates (self-activates; K230), similar to autocatalytic modifications of other types of enzymes (acetyltransferases, kinases, and phosphatases) [841]. On the other hand, the HDAC6 deacetylase is acetylated (inhibited) by KAT3b. Once it is autodeacetylated, sirtuin-1 efficiently deacetylates its substrates, such as P53 and histone lysines H1K26, H3K9, H3K56, and H4K16 [841]. The transcription factor P53 stimulates adipogenesis, as it activates the nuclear receptor NR1c3 (PPARγ) via KAT2b and C/EBPβ. Sirtuin-1 prevents adipogenesis, as it interacts with corepressors NCoR1 and NCoR2, thereby repressing NR1c3 (PPARγ) activity. The deacetylase sirtuin-1 protects against metabolic disorders engendered by overnutrition and aging in many cell types, such as adipocytes and macrophages [842]. Sirtuin-1 activity is reduced in adipocytes of obese and aged subjects. HFD primes sirtuin-1 cleavage by caspase-1 in the AT.

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Sirtuin-1 controls synthesis and secretion of several types of adipokines, cytokines, and chemokines (e.g., adiponectin, IL4, and CCL2). It deacetylates (inhibits) NFκB, thereby hampering its binding to its target gene promoters (e.g., Il2 and Ccl2). It also supports macrophage M2 phenotype, hence blocking infiltration of macrophages, limiting AT inflammation, and protecting against the onset and progression of obesity-related insulin resistance [842]. In mice, selective ablation of the Sirt1 gene in adipocytes accelerates glucose intolerance and hence HFD-induced InsRce during obesity onset with respect to specific Sirt1 depletion in myeloid cells such as macrophages [842]: • In adipocytes, SIRT1 targets NR1c3 (PPARγ). It plays a prominent role in glucose and lipid homeostasis during the early stage of obesity. • In macrophages, which interfere with adipocytes in obesity, SIRT1 counters the action of inflammatory mediators. Sirtuin-1 alleviates aggravation of metabolic disorders only at a later stage. Selective deletion of Sirt1 in adipocytes, but not in macrophages, exacerbates infiltration of macrophages in the AT at the early DIO stage, at least partly because of the increased formation of CCL2 and reduced production of adiponectin, macrophage migration being countered by CCL2 neutralization or adiponectin supplementation [842]. In AdC Sirt1−/− mice, the density of AT-resident macrophages rises and they evolve from the anti-inflammatory M2 to the proinflammatory M1 phenotype. Both CD4+ and CD8+ T lymphocytes control inflammation in visceral adipose depots. Infiltration of CD8+ T lymphocytes precedes accumulation of macrophages in WT and adipocyte- Sirt1-depleted mice [842]. Cytotoxic CD8+ T lymphocytes secrete TNFSF1, IL2, Ifnγ, and CCL5; CD4+ TH1 cells TNFSF1, IL12, and INFγ, these cytokines affecting adipocyte function and favoring the M1 macrophage phenotype. In the AT of obese subjects, activated natural killer T cells favor polarized M2 macrophages via the IL4–STAT6 axis [842]. Adipocytes release TH2 -type cytokines (e.g., IL4 and IL13) that are responsible for macrophage M2 polarization in the AT. However, IL4 is produced at a much higher level in eosinophils and other immunocytes than in adipocytes. Adiponectin primes change of peritoneal macrophages and Kupffer cells to the anti-inflammatory M2 phenotype also using the IL4–STAT6 pathway [842].

Sirtuin-1 and NFAT In adipocytes, sirtuin-1 deacetylates the transcription factor NFATc1 (or NFAT2), thereby controlling its binding to the Il4 gene promoter. The transcription factors of the NFAT family tether to the promoter and enhancer of the genes encoding IL2, IL4, and IL5, among other cytokines and plasmalemmal receptors.

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The NFAT factors share the Rel homology domain (RHD), a DNA-binding and dimerization sequence, with the transcription factors of the REL family, Rel72 and the NFκB heterodimer. All Rel proteins form homo- or heterodimers, except RelB, which only heterodimerizes. The PP3-dependent proteins NFATc1 to NFATc4, which are activated by cytosolic Ca2+ , bind to DNA as monomers, whereas tonicity-responsive NFAT5 tethers to DNA as a dimer and has a slower dissociation rate than the NFκB dimer.73 At rest, NFATcs are phosphorylated and sequestered in the cytosol. Upon stimulation, they are dephosphorylated by PP3 and translocate to the nucleus, where they regulate gene transcription alone or in cooperation with other transcription factors (e.g., AP1 and FoxP3).74 The NFAT factors are synthesized in T, B, and NK cells in addition to mastocytes. In particular, NFATc1 elicits Il4 gene transcription in T cells. In NFATC2-deficient CD4+ TH2 cells, chromatin accessibility is enhanced owing to permissive histone modification and DNA demethylation in the Il4 promoter region [843]. Deletion of NFATC1 and NFATC2 in T cells causes over- and underproduction of IL4, respectively, because of the differential regulation of these two factors [844]. In fact, both NFATc1 and NFATc2 are activators of Il4 gene transcription.75

Sirtuin-2 Among sirtuins, cytoplasmic sirtuin-2 is the most abundant in adipocytes [845]. Like sirtuin-1, sirtuin-2 hampers adipogenesis in addition to accumulation of lipids in adipocytes, as it deacetylates FoxO1, an inhibitor of adipogenesis that then localizes to the nucleus and represses activity of the nuclear receptor NR1c3 (PPARγ) [841]. The anti-adipogenic factors SIRT2 and asymmetric dimethylarginine (ADMA) countering the effects of pro-adipogenic factors that convert preadipocytes to mature adipocytes, such as adipokines and adipogenic transcription factors (e.g., NR1c3 a0nd C/EBPβ) [846]. During pre-adipocyte differentiation, SIRT2 synthesis is downregulated due to insulin-stimulated FoxO1 phosphorylation (inhibition) by the PI3K–PKB axis, FoxO1 translocating from the nucleus to cytosol,76 whereas the formation of NR1c3, C/EBPα, FABP4, GluT4, and FAS is upregulated [845]. 72 Rel:

reticulo-endotheliosis viral homolog proto-oncogene. affinity of NFAT5 for DNA is much lower than that of NFATc1 to NFATc4. The Nfat5 gene encodes multiple isoforms, among which NFAT5a and NFAT5c are the most well-known. The NFAT5c isoform has an additional N-terminal sequence of 76 amino acids with respect to NFAT5a. 74 VEGF signals via the PP3–NFATc1 axis in vascular ECs. It regulates endotheliocyte proliferation during valvuloseptal- and angiogenesis. 75 Demethylation at H3K4 by KDM1a engenders transcription of the NFATC1 gene, whereas an aging-associated increase in H3K9 methylation owing to reduced KDM3a activity represses NFATc1 synthesis. 76 The extent of deacetylation of FoxO1 by sirtuins affects its phosphorylation and DNA binding. FoxO1 is acetylated by KAT3a. 73 The

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Sirtuin-3 In mitochondria, the major NAD+ -dependent deacetylase sirtuin-3 targets numerous enzymes involved in fatty acid oxidation, such as long-chain acylCoA dehydrogenase (lcACDH) and β-hydroxyacyl-CoA dehydrogenase subunit (β HADH) [840]. Sirtuin-3 also deacetylates ETC complexes-I and -I I in addition to activating isocitrate dehydrogenase during caloric restriction. The cardiac fatty acid oxidation rate is higher (mean ∼14.2 nmol/g/s) in HFDfed mice (60% fat) than in mice (mean ∼9.2 nmol/g/s) fed with a low-fat diet (LFD; 4% fat) [840]. A high fatty acid oxidation rate can decrease heart beat efficiency. Activity of lcACDH and β HADH rises with their acetylation state. Hyperacetylation observed in HFD-fed mice is linked to a decreased sirtuin-3 synthesis, whereas KAT2b formation does not change. In addition, HFD and Sirt3 deletion diminish glucose oxidation, but acetylation of pyruvate dehydrogenase, the rate-limiting enzyme of glucose oxidation, is not altered. The inverse relation between fatty acid and glucose oxidation may be the main determinant for the low glucose oxidation in HFD-fed mouse hearts. Although sirtuin-1 expression does not change with the diet type in mice, that of sirtuin-6 is augmented in HFD-fed obese mice.

Sirtuins SIRT4 and SIRT5 Sirtuins cooperate to improve the mitochondrial metabolism. Sirtuin-1 stimulates mitochondrial gene transcription, whereas SIRT3, SIRT4, and SIRT5 modify various mitochondrial enzymes [841].

Sirtuin-6 The PGC1α factor, which regulates transcription of genes encoding enzymes of fatty acid oxidation, is acetylated (inhibited) by the nuclear acetyltransferase, KAT2a. On the other hand, the mitochondrial acetyltransferase KAT2b acetylates mitochondrial proteins. Sirtuin-6 deacetylates (activates) KAT2a, which acetylates PGC1α [840]. On the other hand, sirtuin-1 deacetylates (activates) PGC1α. In Sirt6−/− mice, the amount of AT is reduced, whereas SIRT6 overexpressing mice are protected against HFD-induced obesity [841].

Sirtuin-7 Sirtuin-7 specifically deacetylates histone lysine H3K18 and a small amount of other targets primarily involved in the activation of rDNA transcription. Whereas SIRT7 activates rDNA transcription, SIRT1 inhibits it [841].

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Sirtuin-7 tethers to SIRT1 and prevents its autodeacetylation, avoiding SIRT1 overactivity [841]. In Sirt7−/− mice, augmented SIRT1 activity represses activity of the nuclear receptor NR1c3 (PPARγ), thereby suppressing adipocyte differentiation and countering growth and maintenance of white adipose tissue and even diminishing WAT size; reduced SIRT1 activity restores adipogenesis.

5.3.3.4

Interferons

In obese individuals, NAFLD (Sect. 5.3.4) is linked to the development of insulin resistance. In NAFLD, both intrahepatic CD8+ and CD4+ T lymphocytes are activated. In humans, intrahepatic CD8+ T lymphocytes comprise conventional CD8+ T cells, which are usually activated by antigens, and CD8+ mucosal-associated invariant innate-like lymphocytes, which detect bacterial metabolites and constitute from 20 to 50% of the hepatic population of lymphocytes. In addition, innate-like lymphocytes, such as group-I innate lymphoid cells and natural killer (NK) and NK T cells reside in the liver; they rapidly respond to cytokines [812]. In mouse models of obesity (HFD-fed leptin-deficient [ob/ob] mice) and NAFLD patients, accumulation and activation of pathogenic CD8+ T lymphocytes in the liver by type-I interferon (Ifnα) is linked to a dysregulated glucose metabolism (augmented gluconeogenesis) and hepatic insulin resistance [847]. HFD-fed IFNAR1−/− mice are protected against NAFLD and hepatosteatosis. Hepatic CD8+ T lymphocytes augment their synthesis and release of the type-I I interferon Ifnγ and TNFSF1,77 provoking insulin desensitization, Ifnγ and TNFSF1 impairing insulin signaling in hepatocytes. In the liver of obese mice, production of interferon regulatory factors IRF3 and IRF7 is upregulated and transcription of interferon stimulatory genes increases. The gut flora participates in low-grade chronic hepatic inflammation of obese mice. Broad-spectrum anti-biotic treatment depletes commensal bacterial population and reduces the density of intrahepatic CD8+ T lymphocytes. Hepatic response to Ifnα may result from increased bacterial products entering the liver because of elevated intestinal permeability [847]. In addition, hepatic mitochondrial DNA, the concentration of which rises in NASH mice and patients, may act as TLR9 ligands to promote IfnI production. Intrahepatic immunocytes such as plasmacytoid dendrocytes, may be IfnI sources, CD8+ T lymphocytes forming the dominant IfnI -responsive cell types, the expanding population of which favors systemic insulin resistance in obesity.

77 TNFSF1

is involved in fibrosis in nonalcoholic steatohepatitis (NASH).

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5.3.3.5

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Adiponectin

The anti-inflammatory adiponectin (Sect. 5.4.5.1) increases the activity of AMPK78 and reduces that of the NAD(P)H oxidase subtype NOx4. Ablation of the NOX4 gene raises adiponectinemia. Adiponectin is secreted from AT in response to metabolic cues to sensitize the liver and muscle to insulin. Reduced adiponectinemia, which is usually observed in obesity, contributes to insulin resistance. Adiponectin prevents macrophage recruitment via its inhibition of several proinflammatory cytokines and chemokines (IL6, CCL2, and CXCL8). Adiponectin monomers multimerize, multimers being stabilized by covalent and noncovalent linkages. The largest high-molecular-weight (hMW) adiponectin resists peptidases. Sirtuin-1 supports formation of the FoxO1–C/EBPα complex, thereby upregulating the ADIPOQ gene transcription, but represses AdpnhMW secretion,79 as it inhibits the ER oxidoreductase paralog ERO1Lα, a catalyzer of disulfide bond formation with disulfide isomerase. Adiponectin secretion is indeed regulated by SIRT1, NR1c3 regulators, and ERO1Lα [848].80 Inhibition of SIRT1 and activation of NR1c3, especially during adipogenesis, enhances ERO1Lα synthesis and stimulates AdpnhMW secretion in mature adipocytes. Hence, SIRT1 precludes ERO1Lα action and hence AdpnhMW release. In adipocytes, ablation of Sirt1 raises ERO1Lα production without a marked change in synthesis of Adpn and C/EBPα and only a modest increase in the expression of FABP4 [848]. Methylation of DNA and expression of genes related to metabolism and inflammation in AT are altered in patients with T2DM. Low adiponectin concentrations predispose to T2DM, but high AdpnhMW concentrations predict increased mortality in T2DM subjects [849]. Plasmatic concentrations of FABP4 and AdpnhMW correlate positively with CVD-induced death [850]. FABP4, which is formed in adipocytes and macrophages and in foam cells, is an early predictor of adverse cardio- and cerebrovascular events, developing metabolic syndrome and T2DM, in addition to heart failure. In macrophages, FABP4 significantly increases content in triacylglycerol and cholesterol, as it downregulates transcription of genes involved in cholesterol egress and cholesterol ester hydrolysis. In addition, BNP and the N-terminal fragment of its prohormone contribute to AT metabolism and influence adiponectin secretion. 78 In

the AT, adenosine monophosphate (AMP)-activated protein kinase phosphorylates hormonesensitive lipase and acetyl-CoA carboxylase at inhibitory sites, repressing lipolysis and -genesis. 79 Circulating adiponectin consists of a set of multimers of the 30-kDa polypeptide via disulfide bonds (low- [AdpnlMW (trimers)], middle- [AdpnmMW (hexamers)], and high-molecular-weight complexes [AdpnhMW ]). Insulin sensitizers of the thiazolidinedione class increase the circulating AdpnhMW concentration [848]. The circulating AdpnhMW complex is the main mediator of insulinsensitizing activity of adiponectin. 80 In mammals, ERO1Lα and ERO1Lβ are encoded by the ERO1LA (ERO1L) and ERO1LB genes. The ERO1Lβ isoform is produced primarily in secretory cells. Its synthesis is induced by the unfolded protein response (UPR). The ERO1Lα isoform is formed in most cell types.

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395

Galectin

Galectin-3 (GaL3),81 is mainly secreted by macrophages and microgliocytes; it mediates interaction of macrophages with the extracellular matrix. In addition, GaL3 causes macrophage chemotaxis; the AT macrophage number falls in LGalS3−/− mice. Blood concentration of GaL3 rises in obese subjects, connecting inflammation with accumulation of AT macrophages, especially proinflammatory M1 CD11c+ (αX Itg+) macrophages, to obesity-induced insulin resistance [851]. Galectin-3 does indeed decrease insulin signaling and promotes AT inflammation. Galectin-3 impairs insulin action in adipocytes, hepatocytes, and myocytes. Administration of GaL3 reduces insulin-stimulated glucose uptake in these cell types in vitro. On the other hand, inhibition of GaL3 improves insulin sensitivity in obese mice. In obese mice, switching from an HFD to a standard diet (SD) lowers the density of AT αX Itg+ macrophages. Both heterozygous (LGalS3−/+ ) and homozygous (LGalS3−/− ) mice are protected from HFD-induced InsRce in the liver, skeletal muscle, and AT and from age-related insulin resistance, the diet being standard. The messenger GaL3 binds directly to insulin receptor and inhibits its signaling, the InsR–IRS1–PI3K–PDK1–PKB axis enabling transfer of the insulin-responsive glucose transporter GluT4 to the plasma membrane and repressing the transcription of lipolytic and gluconeogenic genes.

5.3.3.7

Hydroxyisobutyrate

Branched-chain amino acids (BCAAs) are implicated in the development of insulin resistance caused by excess lipid accumulation, once blood-borne lipids have crossed the blood vessel wall. Hydroxyisobutyrate (HIB), a catabolite of valine (a BCAA),82 is secreted from skeletal myocytes and serves as a paracrine regulator of transendothelial fatty acid transfer [852]. It thus stimulates fatty acid uptake by the skeletal myocyte and subsequent lipid accumulation. It works via PGC1α, which promotes endothelial uptake of fatty acids and regulates their consumption in addition to mitochondrial formation and angiogenesis. Increased BCAA catabolism can cause HIB secretion from skeletal muscles, raising transendothelial fatty acid transfer and subsequent muscular import, accu81 Galectin:

laminin-binding galactose-specific soluble lectin, which is encoded by the LGALS3 gene. It is also termed carbohydrate-binding protein CBP35 and galactoside-binding protein (GalBP). Galectin-3 is weakly expressed in mouse lymphocytes and neutrophils. 82 Hydroxyisobutyrate derives from hydroxyisobutyryl-CoA (HIBC) using HIBC hydrolase (HIBCH) and is subsequently processed by HIB dehydrogenase (HIBADH) [852]. The transcriptional coactivator PGC1α launches production of nearly every enzyme of BCAA catabolism. Valine is one of three BCAAs, which are essential dietary components.

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mulation of incompletely esterified lipotoxic intermediates such as diacylglycerol, and repressing insulin signaling. In diabetic subjects, the concentration of HIB in the skeletal muscle rises [852].

5.3.3.8

Optic Atrophy OpA1

Insulin resistance can be associated with mitochondrial dysfunction via direct effects, transcriptional repression of mitochondrial genes, and lipotoxicity. On the other hand, limited mitochondrial function in skeletal muscles can improve insulin sensitivity and protect against diet-induced obesity and insulin resistance via ROS, AMPK, and ER stress pathway, in addition to secretion of myokines (irisin, IL6, and FGF21) [853]. Mitochondrial dynamics, that is, repeated cycles of mitochondrial fusion and fission,83 leads to the exchange of mitochondrial genetic content, ions, metabolites, and proteins. Independently of its role in mitochondrial fusion, the mitochondrial fusion protein optic atrophy OpA1 maintains morphology of mitochondrial cristae and stabilizes ETC complexes [853]. Concentrations of Opa1 and Mfn2 decline in elderly insulin-resistant humans and T2DM patients [853]. In cardiac and skeletal myocytes exposed to hyperinsulinemia, OpA1 synthesis rises, promoting mitochondrial fusion and increasing mitochondrial oxygen consumption and ATP generation. On the other hand, OpA1 deficiency in muscles increases MuRF1 concentration,84 reduces ATP synthesis and the maintenance of mitochondrial membrane potential capacity, and causes glycolytic compensation, but also muscle atrophy, the severity of which depends on the degree of the progressive mitochondrial dysfunction. In mice, specific ablation in skeletal myocytes of the OPA1 gene not only provokes progressive mitochondrial dysfunction but also increases entire-body metabolic rate, improves global insulin sensitivity, and enables resistance to DIO via integrated stress response (Vol. 9, Chap. 2. “Hypoxia and Stress Response”), that is, activation of ER stress and secretion of FGF21 via activation of eIF2α and AMPK [853].85 Circulating FGF21 reduces weight gain and insulin sensitivity in response to HFD.

83 Fission is regulated by dynamin-related protein DRP1 and its partners fission protein FiS1, MFF,

and mitochondrial dynamics protein. Mitochondrial fusion is regulated by outer mitochondrial membrane (OMM) GTPases mitofusins Mfn1 and Mfn2 and inner mitochondrial membrane optic atrophy OpA1. 84 The ubiquitin ligase MuRF1 favors skeletal muscle atrophy. OpA1 represses MuRF1 expression in skeletal muscles. 85 In muscles, FGF21 is produced upon ATF4, PKB, and TORC1 activation in addition to ER and redox stress [853].

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397

Receptor for Advanced Glycation End Products

The receptor for advanced glycation end products (RAGEs), a member of the immunoglobulin superfamily of plasmalemmal molecules encoded by the AGER gene, interacts with a set of ligands that form in organs and circulate in blood. It especially binds to and transduces the action of end products (AGEs), which accumulate with aging, redox stress, hyperglycemia, inflammation, obesity, T2DM, and renal failure [854]. The RAGE is expressed on numerous cell types, that is, vascular endothelial and smooth muscle cells, and immunocytes, such as B and T lymphocytes, monocytes, macrophages, and neutrophils [854]. The RAGE can also tether to non-AGE ligands and then trigger generalized inflammation and redox stress, particularly via a sustained activation of the proinflammatory transcription factor NFκB and augmented production of microsomal prostaglandin-E synthase-1 (mPGeS1)86 and prostaglandin endoperoxide synthase PGhS187 in addition to MMPs [854]. Hyperglycemia accelerates RAGE ligand formation. The density of AT macrophages correlates with obesity and insulin resistance, at least in mice. The RAGE also transmits signaling from members of the S100 and calgranulin category,88 high-mobility group box-containing protein, HMGB1, which connects to toll-like receptors, macrophage differentiation antigen Mac1 (or integrin-αM β2 ), and lysophosphatidic acid [854]. The RAGE ligand SAa favors occurrence of an uremic environment in addition to ROS production in SMCs. The RAGE affects migration of monocytes and macrophages, cholesterol efflux, and pro- and anti-inflammatory cytokines. It activates diverse signaling cascades (Table 5.9). Its cytoplasmic domain interacts with the formin diaphanous-1, thereby influencing actin cytoskeleton dynamics, signaling particularly via GTPases of the RHO set (CDC42 and Rac1), and contributes to regulating serum response factor activity [854]. It also promotes PKB action and SMC migration. In vascular cells and macrophages, RAGE elicits hypoxia-stimulated expression of early growth response transcription factor EGR1 via Dia1 and hence proinflammatory and thrombotic gene expression. Binding of Dia1 to RAGE and hence signaling is suppressed upon phosphorylation by PKB, ERK1, and ERK2. In addition, RAGE downregulates glyoxalase-1 synthesis; GlO1 detoxifies the pre-AGE intermediate, methylglyoxal. In bone marrow-derived macrophages from diabetic mice, cholesterol efflux to ApoA1 and HDL is higher in mice devoid of the AGER gene than in WT mice.

86 Prostaglandin-E

synthase, which is encoded by the PTGES gene, is also dubbed mPGeS1. It catalyzes oxidoreduction of prostaglandin endoperoxide-H2 (PGh2 ) to prostaglandin-E2 (PGe2 ). 87 Prostaglandin-G/H synthase-1 encoded by the PTGS1 gene converts arachidonate to prostaglandin-H2 (PGh2 ). It is also labeled cyclo-oxygenase-1 and prostaglandin-H2 synthase-1. 88 Expression of RAGE and its ligand S100a12 in macrophages and apoptotic vSMCs is significantly greater in atherosclerotic lesions of diabetic patients [854].

398 Table 5.9 Effects of the receptor for advanced glycation end products (RAGE; Source: [854])

5 Hyperlipidemias and Obesity Process Inflammation Leukocyte migration and adhesion Cholesterol efflux SMC migration

Mediators ↑ ROS, NFκB ↑ Dia1, EGR1 ↑ icam1, vcam1 ↑ CCL2 ↓ PPARγ, ABCa1/g1 PKB

RAGE reduces reverse cholesterol transport from macrophages to plasma, liver, and feces owing to the decreased formation of cholesterol transporters ABCa1 and ABCg1 by PPARγ [854]. In obese individuals, insulin-related metabolism is defective. Expression of RAGE in the AT (i.e., on adipocytes, macrophages, and ECs) is significantly higher in obese than lean people and more in vAT than in scAT. Soluble RAGE (RAGES ), which is composed of the extracellular ligand-binding domains of RAGE that is cleaved by MMPs or adam10, diminishes inflammation and redox stress [854]. The RAGE splice variant also produces a soluble form (RAGEES ).

5.3.4 Non-Alcoholic Fatty Liver Disease Non-alcoholic fatty liver disease (NAFLD), also named hepatosteatosis and steatohepatitis, is related to insulin resistance. Insulin resistance in the AT causes excess supply of free fatty acids to the liver, which provokes lipotoxicity, redox stress, and apoptosis. It is now considered a hepatic manifestation of the metabolic syndrome. It results from cytoplasmic accumulation of lipids in hepatocytes. It is usually observed in overweight and obese individuals. NAFLD can affect up to 30% of the general adult population and up to 70% of diabetic and obese patients [855]. The main evolving stages range from simple steatosis, non-alcoholic steatohepatitis (NASH) with inflammation, to fibrosis and ultimately cirrhosis. Hence, NAFLD represents a disease spectrum. NASH is characterized by the co-existence of hepatic inflammation and fibrosis. Whereas hepatic steatosis has a benign clinical consequence, NASH has a much higher risk of evolving to cirrhosis and hepatocellular carcinoma. Simple steatosis is characterized by the ectopic accumulation of lipid droplets in the cytoplasm of hepatocytes without hepatocyte injury, inflammation, and fibrosis. Increased ingress in hepatocytes or the synthesis of triacylglycerol linked to augmented dietary intake, lipolysis in adipocytes, and de novo lipogenesis in hepatocytes in addition to decreased egress of TGs due to reduced mitochondrial β-oxidation and transport of TGs by VLDLs favor the progression of NAFLD [855].

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Evolution to NASH marked by hepatocyte injury, inflammation, and fibrosis results from redox stress, ER stress, and defective UPR, lipotoxicity, and gut dysbiosis [855]. A transition (pre-NASH stage) can be defined during the progression from hepatosteatosis to NASH [856]. The correlations between blood and liver concentrations of lipid species strongly decrease after this transition phase. Fluctuation of gene expression related to hepatic pathways of triacylglycerol synthesis and degradation precedes the transition from simple steatosis to NASH. In particular, diacylglycerol acyltransferase, lipoprotein lipase, and adipocytic triacylglycerol lipase play a crucial role in the transition. Liver ultrasound, computed tomography, and magnetic resonance imaging are used to diagnose hepatic steatosis with a relative high degree of accuracy. However, these techniques cannot distinguish NASH from simple steatosis. Liver biopsy is required for NAFLD diagnosis and staging. The term dynamical network markers (DNMs), based on nonlinear dynamic theory, refers to a group of molecules such as a set of metabolites with strong collective fluctuations [856]. A DNB criterion index measures the differential correlations and deviations of molecular expressions. Patatin-like phospholipase domain-containing PnPLA3 (or adiponutrin) is involved in lipid anabolism and catabolism. PnPLA3 is a lipase for TGs; it also acts as an acyl hydrolase (acylglycerol transacetylase). Transcription of the PNPLA3 gene on human chromosome 22 in adipocytes and hepatocytes is regulated by SREBP1c and hence the nutritional state, as SREBP1c is influenced by glucose and insulin. Protein PnPLA3 variant (I148M) is linked to NAFLD in women, not in men; the association varies among different ethnic groups [857]. The PnPLA3 I148M variant is more common in Hispanics. The PnPLA3 I148M variant may promote fibrosis via the hedgehog pathway, which primes activation and proliferation of hepatic stellate cells and excessive deposition of matrix constituents. According to genome-wide association studies, the TG hydrolase PnPLA3 is one of the major genetic modifiers influencing NAFLD progression [858]. Non-alcoholic fatty liver disease is independently associated with atherosclerosis in nondiabetic individuals. It is linked to a proinflammatory marker profile (low HDL-emia and adiponectinemia, but high triglyceridemia and CRP-emia [high plasmatic concentration of pentraxin-1]). In T2DM patients, hepatic lipidic content assessed by 1H magnetic resonance spectroscopy is higher in the absence of carotid plaques [859]. The type of cytokines implicated in the AT and liver differs. Obesity involves chronic low-intensity inflammation in the AT related to type-1 cytokines, that is, type-1 inflammation and a marked loss of eosinophils. However, obese mice prone to type-1 cytokine response exhibit more pronounced fibrosis. Hepatic fibrosis engendered by NAFLD is characterized by type-2 inflammation and recruitment of eosinophils [860]. Obese IL4- and IL10-deficient mice are resistant to NASH, protection being ensured by hepatic interferon-γ. In Ifnγ-deficient mice, hepatosteatosis progresses rapidly, fibrosis depending on both the TGFβ and IL13 signaling.

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Hypothyroidism is an independent risk factor for NAFLD. Hypothyroidism causes hypercholesterolemia, which favors NAFLD. However, cholesterol does not contribute to hypothyroidism-induced NAFLD [861]. Mild hypothyroidism, a metabolic disease characterized by low and high plasmatic concentrations of thyroid hormone89 and thyroid-stimulating hormone, respectively, is associated with NAFLD owing to the reduced insulin secretion in addition to insulin resistance in the AT [861]. The resulting lack of suppression of lipolysis raises the shuttle of fatty acids to the liver, where they are esterified and form TGs.90 Triglyceride accumulation in hepatocytes is associated with reduced fatty acid oxidation and VLDL assembly, provoking hepatic insulin resistance, TG accumulation synergizing reduced insulin secretion. Because glucose production is not suppressed after feeding, the resulting hyperglycemia stimulates de novo lipogenesis in the liver. On the other hand, in mice with severe hypothyroidism, the delivery of fatty acids to the liver decreases, protecting against NAFLD. Severely hypothyroidic SLC5A5−/− mice have hypercholesterolemia, but are protected against NAFLD.91 Drugs such as berberine,92 are aimed at improving mitochondrial function,93 sugar and lipid metabolism, AMPK signaling,94 increasing fatty acid β-oxidation

89 Thyroid

hormones (T3 and T4 ) bind to thyroid hormone receptors TRα and TRβ (NR1a1– NR1a2), which interact with other transcription factors to activate or inhibit the transcription of thyroid hormone-regulated genes. In the liver, NR1a1 and NR1a2 cooperate with NR1c1 (PPARα) to activate transcription of genes involved in the fatty acid β-oxidation [861]. 90 In NAFLD patients, de novo lipogenesis accounts for only about 25% of liver TGs, the main source (∼60%) of substrates for hepatic TG synthesis being fatty acids generated by AT lipolysis [861]. 91 The sodium–iodide symporter SLC5a5 (NIS) enables iodide ingress in the thyroid gland, the first step in thyroid hormone synthesis. In SLC5A5−/− mice, the marked reduction in plasmatic thyroid hormone concentrations suffices to lessen the response of AT to adrenergic signaling. Adrenoceptor-stimulated lipolysis and hence a circulating pool of glycerol and fatty acids in addition to activity of hormone-sensitive lipase (lipase-E) after overnight fasting decline [861]. Altered adrenergic stimulation of lipolysis in AT alleviates fatty acid delivery to the liver and subsequently hepatic TG synthesis. Hence, SLC5A5−/− mice are protected against NAFLD despite lowered fatty acid oxidation and thyroid hormone signaling in their livers. 92 Berberine is an isoquinoline quaternary alkaloid extracted from Coptis chinensis, Rhizoma coptidis, and Hydrastis canadensis, among other plants. 93 Berberine may increase sirtuin-3 expression, improving mitochondrial function and decreasing mitochondrial ROS generation [855]. 94 Berberine may inhibit mitochondrial ETC complex-I and activate AMPK, thereby diminishing lipogenesis, elevating energy consumption, and promoting FA oxidation [855]. It may elicit adiponectin multimerization and thus enhance insulin sensitivity via AMPK. It may also reduce lipid accumulation via AMPK phosphorylation (activation), thereby repressing transcription of genes targeted by SREBP1c (acetyl-CoA carboxylase ACC1, FAS, and stearoyl-CoA desaturase SCD1) and SREBP2 (hydroxymethylglutaryl-CoA synthetase [HMGCS] and reductase [HMGCR]) and hence TG and cholesterol synthesis.

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and insulin sensitivity,95 and lessening cholesterolemia and lipogenesis,96 in addition to inflammation97 and redox and ER stress.98

5.4 Adipose Tissue Adipose tissue includes mature adipocytes (>90% of the AT cell population), and pre-adipocytes, in addition to various other stromal cell types,99 such as fibroblasts, adipose tissue mesenchymal stem cells (ADMSCs; adipocyte stem and progenitor cells),100 vascular cells of microvessels (especially ECs), and resident (predominantly M2 macrophages101 and regulatory T cells [TR1 ] in as addition to protective eosinophils and, to a lesser extent, neutrophils [802])102 and infiltrating 95 Berberine

may trigger insulin secretion and synthesis of insulin receptor (via PKC) and insulin receptor substrate IRS2 in addition to supporting the IRS1–PKB axis and GluT4 translocation to the plasma membrane [855]. 96 Berberine may also upregulate LDLR expression owing to Ldlr mRNA stabilization and ERK activation in addition to downregulating that of C/EBPα, adiponectin, and leptin in metabolic syndrome [855]. In addition, it may reduce the production of PPARγ (NR1c3), PCSK9, and fatty acid translocase. 97 Berberine may alleviate the action of TNFSF1 and IL6 induced by palmitate, COx2 formation, and JNK1 phosphorylation [855]. 98 Redox stress induces expression of uncoupling protein UPC2 in NAFLD mice. Administration of berberine reduces UCP2 synthesis. Berberine may prime ER stress response partly via the ATF6–SREBP1c pathway [855]. 99 The stromal–vascular cell fraction corresponds to the non-adipocyte cell populations of AT. It encompasses AT stem cells, committed pre-adipocytes, fibroblasts, and vascular and immune cells. Adipose-derived stem cells contribute to cell renewal and AT repair [862]. They are ENPP1+ (CD39), epican+ (HSPG or CD44), Nt5E+ (CD73), Thy1+ (CD90), endoglin+ (CD105), pecam1− (CD31), and PTPRc− (CD45) cells. In fact, different subpopulations of adipose-derived stem cells can exist, expressing distinct surface markers such as the stem cell CD34 marker. They have the different ability to grow and differentiate according to the fat depot type. The adipogenic differentiation capacity of scAT-derived stem cells is higher than that of visceral AT-derived stem cells. Hence, the scAT and vAT can evolve to hyperplasia and hypertrophy in adverse conditions, respectively. Adipose-derived stem cells can differentiate into multiple cell lineages, such as CMCs, myoblasts, osteoblasts, chondrocytes, hepatocytes, pancreatocytes, ECs, and hematopoieticsupporting cells [862]. 100 Mesenchymal stem cells (MSCs) are multipotent stem cells that can engender adipocytes, osteoblasts, chondrocytes, and myocytes. They reside in almost all postnatal organs. AT stem cells can differentiate into cells of meso-, endo-, and ectodermal origin. They may or not be multipotent. 101 Macrophages are the most abundant immunocytes in vAT and scAT (>50% of all leukocytes) [802]. In lean humans, AT macrophages characterized by the surface markers macrosialin (CD68+) constitute less than 5% of all AT cells. Increased density of AT macrophages (up to 40% of all AT cells) in metabolic stress is associated with their phenotype changes (from M2 to M1). 102 In lean AT, both M2 macrophages and regulatory T cells release anti-inflammatory cytokines, such as interleukin-10 and TGF-β, which favor insulin sensitivity and prevents fat depot inflammation. In addition to regulatory T cells, insulin-sensitive fat depot-resident T lymphocytes encompass primarily TH2 cells.

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inflammatory cells (i.e., effector and memory T cells [e.g., TC1 , TH1 , and TH17 ],103 but also NK and NKT cells and granulocytes) [863]. All cell types contribute to the AT secretome, which comprises adipokines, gaseous messengers, various types of metabolites, and microRNAs. The nutritional status influences the adipocyte number. Overfeeding and hypercaloric diet with a high fat content cause AT expansion. Obesity from childhood is characterized by AT hyperplasia (elevated adipocyte number) due to the formation of new adipocytes from progenitor differentiation. The signaling effector PKB2 in adipocyte progenitors is activated by pro-adipogenic insulin and IGF1 in HFDinduced adipocyte proliferation [717]. A 5HT2A + neuronal cluster in the central nucleus of the amygdala, which is involved in feeding and reward, promotes food consumption, whereas the anorexigenic PKCδ+ neuronal cluster of the amygdala suppresses appetite [864]. Inhibitory 5HT2A + neurons innervate the parabrachial nucleus, which is implicated in appetite suppression. In AT, creatine metabolism is necessary for diet-induced thermogenesis, which limits weight gain, increased caloric intake being balanced by augmented heat production and, conversely, a long-term decreased food intake, reducing energy consumption. Creatine is synthesized by mitochondrial glycine amidinotransferase encoded by the GATM gene, in adipocytes and in other cell types. It promotes glucose tolerance and stimulates mitochondrial ATP turnover. Creatine supports cold- and β3-adrenoceptor-stimulated adaptive thermogenesis [865]. Adipocytespecific deletion of GATM depletes creatine and phosphocreatine. AdC GATM−/− mice are prone to DIO. Interleukin-10 modulates insulin signaling via the InsR–IRS1/2–PI3K–PKB–FoxO1 axis used in hepatic gluconeogenesis and lipid synthesis [802]. M2 macrophages control adipocytic lipolysis. In co-operation with eosinophils, M2 macrophages can manage generations of beige adipocytes [802]. Unlike resident macrophages and dendrocytes, neutrophils reside transiently in the AT [802]. They secrete IL4 and IL13. They contribute to the anti-inflammatory insulin-sensitive AT phenotype. In normal AT, regulatory T cells synthesize IL2 receptor subunit-α (CD25), tumor-necrosis factor superfamily members TNFSF4 and TNFSF18, cytotoxic T-lymphocyte antigen CTLA4, killer cell lectin-like receptor KLRg1 (CLec15a), in addition to FoxP3 [802]. 103 CD4+ and CD8+ T lymphocytes constitute clones defined by their functional and differentiated states. Interferon-γ and interleukin-4 represent type-1 and -2 cytokines, respectively. CD8+ memory T lymphocytes produce Ifnγ and TNFSF1. CD4+ effector T lymphocytes comprise helper TH0 , TH1 , and TH2 cells in particular. TH1 lymphocytes secrete interleukin-2, interferon-γ, and TNFSF2 implicated in adaptive immunity. TH2 lymphocytes release IL4 to IL6, IL10, and IL13 involved in humoral and allergic immunity. Action of cytolytic CD8+ effector T lymphocytes depends on CD4+ effector T lymphocytes. Cytotoxic CD8+ T lymphocytes are also classified according to cytokine type secretion. CD8+ T lymphocytes differentiate into subsets similar to CD4+ TH0 , TH1 , TH2 , and TH17 cells, which are referred to as TC0 , TC1 , TC2 , and TC17 cells, respectively, which are defined by expression of the same characteristic cytokines as their CD4+ counterparts. Although TC1 cells produce high amounts of interferon-γ, TC2 cells synthesize interleukins IL4, IL5, IL10, and IL13, but low levels of Ifnγ, TC0 cells forming IL4 in addition to Ifnγ and other cytokines and TC17 cells IL17.

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The diet-dependent AT hyperplasia is heterogeneous among fat depots. In humans, AT hyperplasia is observed in the femoral scAT, but not in the upper abdominal scAT [717]. Whereas expansion of intra-abdominal AT is associated with insulin resistance, a larger scAT improves insulin sensitivity. The number of adipocytes depends on the balance between cell death and the generation of new adipocytes from progenitor cells. Adipocyte progenitors are immature mesenchymal stromal cells associated with the AT vasculature. In adult humans, the turnover rate is estimated at 10% per year, a low value with respect to other organs [717]. Adipose-derived stem cells can secrete angiogenic growth factors and antiapoptotic cytokines (e.g., HGF, IGF, TGFβ1, and VEGF) [862]. Thus, they have immunomodulatory properties. On the other hand, inflammation can play an important role in adipose-derived stem cell differentiation. Some proinflammatory cytokines can suppress PPARγ expression, thereby impeding adipogenic differentiation, whereas an increase in proinflammatory cytokines usually induces adipocyte hypertrophy and hyperplasia (Table 5.10). Sphingosine kinase SphK1 participates in the regulation of the proinflammatory activity of adipose-derived stem cells [862]. Aging is associated with a loss of adipose-derived stem cell proliferation and differentiation potential. In older individuals, the differentiation rate is only high in the arm and thigh scAT [862]. The main local determinants of AT functionality, that is, the capacity of AT to store excess nutrients without adipocyte overloading, are the number and size

Table 5.10 Inflammation induced by metabolic disturbances affects adipose-derived stem cell behavior, inflammatory stimuli activating or attenuating their signaling (Source: [862]; ↑ increase, ↓ decrease) Process Self-renewal Proliferation Adipogenic differentiation Migration Senescence Angiogenesis Immunomodulation

Impact of inflammation on signaling mediators ↓ FGF, notch, Wnt ↑ TNFSF1, IL1β ↓ PPARγ (NR1c3) ↑ MMP2/9 ↑ TNFSF1 ↓ notch, Wnt ↓ FGF, notch, Wnt ↑ IL1β (NLRP3 inflammasome)

Altered self-renewal potential and augmented commitment to the adipogenic lineage relies on a coordinated inhibition of FGF, notch, and Wnt. In obese animals, these cells have a higher senescence rate and adipo- and osteogenic potential than those in lean animals. In obese patients, they have a higher migration and phagocytosis capacity and increased metabolic activity (increased glycolysis and succinate release and decreased succinate dehydrogenase activity), expression of proinflammatory cytokines and chemokines in addition to MMP2 and MMP9, and activation of the NLRP3 inflammasome compared with cells in lean subjects. Their angiogenic potential decreases in obesity

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of adipocytes. Metabolically healthy obese individuals have AT hyperplasia, the adipocyte number being higher than that of weight-matched insulin-resistant subjects [717]. Obesity is also associated with changes in the extracellular matrix. Adipocyte progenitor response is driven by M2-type macrophages and type-2 innate immunocytes during adaptive tissue remodeling [717]. Transient adaptive local inflammation triggers AT remodeling during AT hyperplasia in addition to WAT browning. On the other hand, chronic inflammation due to adipocyte overloading and lipotoxicity alters adipocyte metabolism. Extracellular matrix with its fibrillar and laminar structures contributes to the regulation of cell differentiation, metabolism, and inflammation in the AT. It consists of structural and adhesive proteins, collagen and elastin fibers, fibronectin, laminin, proteoglycans, among other species. Adipocytes, pre-adipocytes, and inflammatory leukocytes produce matrix metallopeptidases that degrade and remodel the matrix to regulate its rheology in addition to adipogenesis [717]. Chronic inflammation causes excess matrix, i.e., fibrosis. Collagen accumulation in the interstitial and pericellular spaces is up to four times higher in obese subjects than in lean people [717]. Hypoxia is one of the main causes of fibrosis in obesity. Activated HIF1α stimulates matrix constituent synthesis and crosslinking. It also launches inflammatory gene expression, leading to immunocyte recruitment. Adipocytes, macrophages, and mastocytes contribute to fibrosis in obesity. Fibrosis provokes AT stiffness and limits adipocyte proliferation. In addition to inflammation, lysophosphatidic acid, adiponectin, and leptin intervene in fibrosis. Adipokines released by adipocytes and stromal cells of the pvAT and eAT exert a paracrine action on blood vessels and myocardium, respectively, in addition to endocrine effects via the circulating adipokine pool. In particular, they participate in controlling blood coagulation. Conversely, the vascular wall and myocardium send paracrine or endocrine messengers to the AT. In addition, the sensory innervation of the pvAT influences adiposecretome. In particular, innervation of the AT affects responsiveness of the medial smooth muscle to NAd [866]. Adipose tissue dysfunction is characterized by a decreased release of homeostatic protective factors (e.g., adiponectin, NO, and some prostaglandin types) and increased secretion of stress adipokines (e.g., leptin, resistin, and visfatin) and development of low-grade chronic inflammation, which favors metabolic and vascular dysfunction [802].

5.4.1 Structural and Functional Types of Adipose Depots Functionally, the BAT, which is involved in the body temperature maintenance, oxidizes glucose and fatty acids to fulfill thermogenesis, whereas the white adipose tissue (WAT) stores TGs in LDs.

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White adipose tissue exists as vAT and scAT. These fat depots differ functionally and immunologically [802]. The former is metabolically more active than the latter, with higher levels of glucose uptake and fatty acid generation, the scAT taking up circulating FFAs and TGs. Moreover, the vAT has a greater sympathetic innervation. The vAT thus participates in the regulation of insulin sensitivity. It also contains more immunocytes, even under healthy conditions. However, excessive immunocyte infiltration and resulting chronic low-grade inflammation, especially in the retroperitoneal compartment, is linked to cardiovascular risk.

5.4.1.1

Brown Adipose Tissue

Energy uptake relies on nutrient absorption and energy expenditure is determined by basal metabolism, physical activity, and thermogenesis. In moderately cold temperatures, heat is generated by the BAT owing to increased uptake of nutrients, particularly carbohydrates (glucose), lipoprotein-derived TGs, cholesterol, and FFAs. Positron emission tomography coupled with X-ray computed tomography (PETCT) enables imaging of AT to assess both its volume from CT and metabolic activity using deoxy-(18F)fluoro-glucose uptake triggered by acute cold exposure. However, measurement of entire body BAT volume, distribution, and thermogenic activity is not obvious because brown adipocytes are structurally commingled with WAT, muscle, and blood vessels, forming narrow fascial layers adjacent to muscles, bones, and organs. The human BAT is indeed composed of stromal tissue, white adipocytes, and UCP1+ thermogenic brown and beige adipocytes, whereas, in rodents, BAT is homogeneous [867]. After 5-h tolerable cold exposure, BAT volume and activity measured by PETCT are lower on average in obese than in lean young men, although several obese young men have a substantial BAT volume [867]. Six activated BAT regions include cervical, supraclavicular, axillary, mediastinal, paraspinal, and abdominal (volume ∼1100 ml) with a potential for BAT genesis within anatomically inferior and posterior depots. However, less than one-half of these depots are stimulated by acute cold exposure [867]. Most young individuals, even obese ones, have a potential for BAT expansion, as they do not reach their maximal BAT content [867]. Source of BAT-stimulating NAd can vary. Whereas sympathetic stimulation preferentially activates superior depots, NAd released from pelvic tumors, such as pheochromocytoma and paraganglioma, in plasma targets nearby inferior retroperitoneal abdominal depots. The BAT increases the body’s energy expenditure not only upon cold exposure but also after overfeeding. The extent and activity of BAT are positively correlated, with energy expenditure during cold exposure, and negatively with age, BMI, and fasting glycemia [868]. In humans, BAT activation improves insulin sensitivity. The BAT, which exists not only in newborns and during childhood but also in adults, primarily localizes to the supraclavicular and paravertebral regions [805]. In newborn human infants, BAT is relatively well defined and resides in the inter-

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scapular, axillary, posterior cervical, supra-iliac, anterior mediastinal, and perirenal regions [869]. The BAT of human newborns is interspersed with WAT, the amount of BAT gradually declining in adulthood. In adult humans, in whom homeothermy does not rely on BAT thermogenesis, the BAT is less well defined. The main human depots lodge in the supraclavicular regions and the neck, with some additional paravertebral, mediastinal, para-aortic, and suprarenal sites [869].104 The BAT can be detected more or less easily according to age, gender, adiposity, and thermal environment [869]. It is observed most frequently in young lean women. It is much less well identified in response to cold and insulin in obese individuals. The BAT is highly vascularized to accommodate the greater demand for oxygen and nutrients. The dense vasculature enables a suitable amount of fatty acids and glucose to be delivered upon activation and warm blood irrigating it. The BAT is endowed not only with a rich blood and nerve supply but also a dense niche of perivascular mesenchymal cells, which serve as a source of preadipocytes. Brown adipocytes arise from specific adipogenic progenitors that are closer to skeletal muscle progenitors than to white adipocyte progenitors [869]. Brown adipocytes are characterized by multilocular LDs, whereas white adipocytes contain a unilocular large lipid vesicle. The gut microbiota can facilitate adaptive thermogenesis via a cold-induced switch in the cholesterol metabolism [870]. A greater proportion of absorbed dietary cholesterol is converted to bile acids using CyP7b1 involved in the alternative pathway of bile acid synthesis in the liver. Cold exposure also affects the bacterial composition of the gut flora. Mitochondria not only create ATP from oxidative phosphorylation but also orchestrate production of metabolites used for synthesis of nucleotides, lipids, and proteins, generate signaling ROS mediators, participate in regulating calcium concentration in addition to cellular proliferation, immune response, and cell death [816]. In vascular diseases, mitochondria change morphologically and functionally. Mitochondria abound in brown adipocytes. These cells have a high content of iron and cytochrome and a brownish color. Numerous mitochondria in brown adipocytes are large and possess laminar cristae, whereas they are small and elongated with randomly oriented cristae in white adipocytes [869].

Uncoupling Protein UCP1 In mitochondria of brown adipocytes, the ETC is uncoupled from ATP formation by uncoupling protein UCP1 in the inner mitochondrial membrane [868]. Fatty acid and glucose are processed as energy substrates, chemical energy being dissipated by acute adaptive thermogenesis. Energy of these nutrients is converted into a proton gradient, UCP1 catalyzing the inducible proton leak to release the energy of the 104 In

adult humans, BAT localizes to cervical, supraclavicular, mediastinal, paravertebral, suprarenal, and perirenal regions [818].

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proton gradient, that is, dissipating the mitochondrial proton motive force generated by the ETC as heat, instead of storing the energy via ATP generation [872]. In BAdC mitochondria, the large quantity of ETC complexes is associated with a weak amount of ATP synthase [871]. In addition, release of the proton motive force by the mitochondrial proton leak can alter the redox state of the ETC chain and reduce ROS production [871, 872]. The ETC substrate glycerol 3-phosphate (G3P) and resulting mitochondrial energization generate ROS via reverse electron transport or mitochondrial G3P dehydrogenase GPD2 [872]. Whereas ETC complex-I forms superoxide in the mitochondrial matrix via succinate, GPD2 synthesizes ROS in the mitochondrial intermembrane space. Cold-triggered sympathetic stimulation of brown adipocytes activates lipolysis, glucose uptake, and mitochondrial genesis. Acute adrenoceptor stimulation supports UCP1-dependent thermogenesis via ROS production by reverse electron transport through ETC complex-I . In mice depleted in the Ucp1 gene, not only is the ETC amount alleviated, but cold-activated metabolism also primes innate immunity via ROS and cell death in BAT [872]. Deficiency in UCP1 in BAdC mitochondria blunts their ROS-linked Ca2+ buffering capacity; BAdC mitochondria become highly sensitive to permeability transition induced by ROS and Ca2+ overload. The UCP1 protein, which is activated by FFAs and NAd (β3AR-mediated thermogenesis), allows protons to reenter the mitochondrion, bypass ATP synthesis, uncoupling the ETC, thereby, generating heat by nonshivering thermogenesis. Proton leak at the inner mitochondrial membrane via UCP1 is enhanced by longchain fatty acids and inhibited at rest by cytosolic purine nucleotides, mainly ATP [869]. Long-chain fatty acids are unable to dissociate from UCP1 until they are oxidized. UCP1 cotransports a fatty acid anion and H+ across the inner mitochondrial membrane. Proton is then released, whereas the fatty acid anion remains bound to UCP1, which can initiate another H+ transfer. Like other mitochondrial transporters, UCP1 is stabilized by its tethering to cardiolipin (CL). Fatty acids derived from TGs in LDs are metabolized by β-oxidation enzymes in the mitochondrial matrix and activate UCP1, which then generates heat using the electrons derived from β-oxidation. Replenishment of the intracellular TG stores depends on uptake from blood of FFAs bound to albumin or VLDLs and chylomicrons, and of insulin-dependent and -independent glucose followed by lipogenesis, along with glycolysis for ATP generation [869]. Human BAT takes up large amounts of glucose under thermoneutral conditions and rapidly adapts this uptake upon BAT stimulation. Insulin increases BAT glucose uptake five-fold via GluT4 (mainly) and GluT1. Uptake of circulating glucose by BAT after cold exposure is lower in insulin-resistant than in -sensitive individuals [869]. Activation of BAT by cold exposure (5–8 h) in healthy lean volunteers with large BAT depots (but not in those with small ones) increases insulin sensitivity. Prolonged cold exposure (10 d) markedly raises insulin sensitivity in diabetic subjects, but induces only a minor increase in BAT glucose uptake [869].

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Mitofusin Mfn2 The ubiquitous GTPase mitofusin Mfn2 is a major determinant of BAT thermogenesis. Mitofusin-2 controls OMM fusion, and mitochondrial fusion and fission, serving as quality control mechanisms to ensure mitochondrial function. In skeletal myocytes, defective mitofusin-2 formation and subsequent augmented mitochondrial fission are linked to insulin resistance. Liver-specific deletion of the MFN2 gene engenders glucose intolerance and enhances hepatic gluconeogenesis [873]. Deficiency of Mfn2 in hypothalamic POMC+105 neurons decreases energy expenditure and causes hyperphagia and hence obesity [874]. Ablation of the MFN2 gene in both white and brown adipocytes of SD- and LFD-fed mice provokes BAT dysfunction with lipid accumulation in the BAT, impaired ETC activity, and a blunted response to adrenoceptor stimulation [873]. Selective ablation of MFN2 in BAT improves glucose tolerance under HFD, but alters cold-stimulated thermogenesis. In HFD-fed obese mice with BAT MFN2 deletion, improved insulin sensitivity results from a gender-specific rewiring of the mitochondrial function. In females, mitochondria of brown adipocytes have a higher efficiency for ATP synthesis by FA oxidation, whereas in males, ETC complex-I activity declines and glycolysis to lactate rises linked to increased expression of the glycolytic enzyme isoform PKM2 in BAT [875]. Hence, selective excision of MFN2 in brown adipocytes improves insulin sensitivity in obese mice via increased glycolysis in male mouse BAT and coupled lipid import capacity and fatty acid oxidation in female mouse BAT, thereby preventing, whatever the gender, obesity-linked insulin resistance. On the other hand, Ucp1 deletion affects neither the ETC activity nor fatty acid oxidation capacity coupled with ATP synthesis. The presence or absence of adipose Mfn2 effects on liver parameters correlate with the existence or not of circulating FGF21 concentrations, respectively. Mitofusin-2 may differentially control FGF21 expression in white and brown adipocytes [874]. Mitofusin-2 is highly produced in BAT; its deficiency impairs ETC complex-I activity, destabilizing the connection between ETC complex-I and ETC complex-I I I , and disturbing the response to adrenoceptor stimulation, but protecting against HFD-induced insulin resistance [873]. In BAT, Mfn2 is required to sustain body temperature after cold exposure, Mfn2 expression induction being an element of the sympathetic response induced by thermal stress under obesity, but contributes to insulin resistance [875]. In response to cold exposure, thermogenesis in BAT is activated by adrenoceptor signaling, which raises lipolysis and fatty acid oxidation linked to proton leak through UCP1. Expression of Mfn2 and UCP1 is upregulated in BAT after acute cold exposure or treatment with β3 AR agonist in addition to feeding. On the other hand, NAd induces mitochondrial fission in brown adipocytes for proper activation of uncoupled ETC activity. Therefore, Mfn2 in BAT promotes cold tolerance via thermogenesis linked to proper lipid mobilization, fatty acid

105 POMC:

pro-opiomelanocortin.

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oxidation, and UCP1-dependent mitochondrial ETC activity, rather than favoring glucose uptake and glycolysis, processes improving glucose tolerance and insulin sensitivity. Mitofusin-2 (but not Mfn1) at the OMM interacts with the lipid dropletassociated protein perilipin-1, facilitating docking of mitochondria to LDs in BAT in response to adrenoceptor stimulation and hence heat production, TG lipolysis in LDs yielding fatty acids that enter the mitochondrion for β-oxidation to feed the ETC [873]. Mitofusin-2 also promotes between-mitochondrion and mitochondrion– ER contacts. As interaction of mitofusin-2 with perilipin-1 ensures proper lipolysis and subsequently fatty acid oxidation in response to adrenoceptor stimulation and food intake, it launches cold- and diet-induced thermogenesis in mice. Upon cold exposure, activated BAT reduces TGRL-emia (Plasmatic concentration of TGRLs) in lean mice and correct hyperlipidemia in ApoA5−/− mice, whereas denervation of mouse BAT causes hypertriglyceridemia [869]. Cold also normalizes the response to glucose and reduces insulin resistance in HFD-fed obese mice. Transplantation of BAT increases glucose tolerance. The BAT oxidative function declines with obesity and aging. Decreased thermogenic capacity, which fully depends on UCP1, favors obesity and can cause hyperphagia in rodents [869]. Conversely, augmented UCP1 activity ameliorates obesity, at least in mice. Activators of BAT (e.g., β3AR agonists and thyroid hormones) lowers lipidemia and glycemia. Acute BAT activation initially relies on the catabolism of intracellular triglycerides, whereas prolonged BAT activation increases TG clearance from plasma. The sympathetic nervous system stimulates BAT and contributes to its growth and hence activates thermogenesis. Three subtypes of β-adrenoceptors exist in brown adipocytes. However, their relative quantity varies according to the location and the mammalian species. In rodents, β3AR is predominant, whereas β1AR is generally preponderant in humans [869]. Although β1AR and β3AR regulate brown adipocyte proliferation and differentiation, respectively, β2AR alone may suffice for maintenance of BAT function [868]. Adrenergic stimulation of brown adipocytes enhances the lipolysis of TGs in LDs by ATGL and hormone-sensitive lipase (HSL or lipase-E) and releases fatty acids. The signaling cascade primed by β AR relies on cAMP, a target of hormonesensitive lipase.

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The BAT can also be stimulated by natriuretic peptides, thyroid hormone (e.g., triiodothyronine [T3 ]), retinoids, bone morphogenetic proteins BMP7 and BMP8b, fibroblast growth factor FGF21, VEGF, leptin, hypocretin (or orexin),106 and irisin [869]. In obese individuals, circulating natriuretic peptide concentrations decrease. These blood pressure modifiers also exert metabolic effects. Cardiac natriuretic peptides (ANP–BNP), which tether to GC2a and then activate the GC–cGMP–PKG pathway, can stimulate lipolysis in adipocytes with a potency similar to that of β AR agonist and promote WAT browning [876]. Natriuretic peptides can also increase the oxidative capacity of skeletal muscles. In HFD-fed mice, elevated natriuretic peptide signaling in muscles causes insulin resistance [876]. Overfed mice with a muscle-specific deficiency in the clearance receptor NPR3 (SkM Npr3−/− ) gain weight. However, in AT, enhanced NP signaling protects against DIO, raising energy expenditure, insulin sensitivity, and glucose uptake into BAT [876]. Mice with an AT-specific NPR3 deficiency (AT Npr3−/− ) are protected against detrimental effects of HFD-induced obesity (insulin resistance, inflammation, and hepatic steatosis). Oxygen consumption and carbon dioxide production increase without altering food intake and physical activity. In addition, expression of UCP1 and other mitochondrial proteins (and hence thermogenesis) along with that of the batokines FGF21, Nrg4, and BMP8b107 rise in the BAT of AT Nprc−/− mice. Overexpression of BNP or PKG also augments expression of genes involved in thermogenesis in proper chow- or HFD-fed mice [876]. Both FGF21 and BMP9, which are predominantly produced in the liver, activate BAT thermogenesis in addition to WAT browning [877]. FGF21, which is also synthesized in BAT and WAT, although to a lesser extent than in the liver, and in the skeletal muscle and pancreas, does not mediate cell proliferation, unlike other members of the FGF category. Under chronic cold exposure in mice, FGF21 expression decreases in the liver, but increases under PPARγ transcriptional control in BAT and WAT, where it markedly raises UCP1 production [877].

106 oρ ξις:

appetency, conation. growth factor-21 operates via adiponectin [876]. Neuregulin-4 abounds in the BAT, from which it is secreted; it decreases hepatic lipogenesis. Bone morphogenetic protein-8B increases thermogenesis via its central and peripheral actions.

107 Fibroblast

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Irisin is secreted by skeletal muscles during physical exercise, at least in rodents [877]. It is formed by cleavage of fibronectin type-I I I domain-containing protein FNDC5.108 It promotes the genesis of beige cells in white adipose depots, as it stimulates UCP1 production, at least in mice. It correlates with insulin desensitization. Thyroid hormones, which mediate overall energy expenditure, act as paraand endocrine messengers to regulate BAT [868]. Brown pre-adipocytes and adipocytes express a high level of iodothyronine deiodinase, DIo2, which converts thyroxine (T4 ) to the more active triiodothyronine (T3 ), which upregulates UCP1 production. In addition, thyroid hormone regulates energy balance in some types of hypothalamic neurons. Thyroid receptor isoforms specifically regulate UCP1 expression and thermogenesis. The TRα (NR1a1) isoform regulates adaptive thermogenesis, whereas TRβ (NR1a2) modulates UCP1 expression [877]. Among members of the BMP family of the TGF superfamily, BMP7 enhances thermogenesis in BAT [877]. It is produced in several hypothalamic nuclei and may regulate BAT via a central mechanism. BMP8b is also formed in BAT and in the hypothalamus, where it activates the sympathetic signaling to BAT, without changing the feeding behavior. BMP4 promotes differentiation of MSCs into white adipocytes. In primary human adipose stem cells, both BMP4 and BMP7 induce a white-to-brown adipocyte transdifferentiation. Phosphatase and tensin homolog deleted on chromosome 10, which counters PI3K action and hence reduces levels of phosphorylated PKB and FoxO1, supports the expression of UCP1 in BAT and WAT, and of its transcriptional regulator PGC1α [877]. In humans, PTEN haploinsufficiency increases the risk for obesity, but decreases that for T2DM, as it markedly improves insulin sensitivity. Brown adipose tissue produces more heat in female rats exposed to cold than in males [868]. Sex steroids, estrogens, and androgens have receptors on brown adipocytes. Testosterone and 17β-estradiol inhibits and stimulates mitochondrogenesis, UCP1 synthesis, and lipolysis, respectively. In humans, BAT is better developed in women than in men [868]. Excess weight gain during the early postnatal period, mainly due to overnutrition, can significantly alter body weight in adulthood. Excessive stimulation of energy expenditure at an early age of life can desensitize or reprogram thermogenesis regulation and subsequently impair energy expenditure in adulthood [878]. A set of 190 kinases enables brown adipocyte formation (i.e., UCP1 synthesis), pre-adipocyte differentiation, proliferation, and activation [879].109 Among these

108 In

muscles, PGC1α upregulates Fndc5 gene transcription. brown adipocytes develop from myogenic factor MyF5+ cells, whereas white and beige adipocytes originate from MyF5− precursors. However, brown pre-adipocytes can also derive from MyF5− precursors [879]. Transcriptional control of development and activation of brown adipocytes rely on early B-cell factor EBF2 and PR domain-containing protein PRDm16.

109 Classical

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kinases110 (e.g., PKA,111 ERK1, ERK2, and Src), AMPK112 regulates brown adipocyte formation and promotes UCP1 production. The BAT participates in regulating the vasculomotor tone and cardiovascular metabolism, in addition to thermogenesis [869]. In mice, it increases lipid clearance and improves glucose tolerance, thereby attenuating insulin resistance.

Batokines and MicroRNAs The BAT releases constitutive or inducible auto-, para-, and endocrine messengers, the so-called batokines, which are involved in between-organ crosstalk. Locally acting batokines operate on the BAT and its surrounding tissues. Endocrine batokines can improve metabolism in the heart and vasculature (Table 5.11). IGF1 secreted by BAT into the circulation ameliorates T1DM without changing insulin concentration [869]. Interleukin-6 and FGF21 reverse metabolic anomalies in HFD-fed and Ob/Ob insulin-resistant mice. However, secretion of T3 and FGF21 is not specific to BAT, rendering action of BAT uncertain with respect to these substances produced by several other organs; in particular, the liver and skeletal muscle also release FGF21 in the bloodstream.

110 Casein

kinase CsnK2 abounds in white adipocytes. Ablation of the CSNK2A1 and CSNK2B genes suppresses brown adipocyte formation. CK2 may affect maturation of white or brown adipocytes, but not differentiation [879]. Inhibition of CK2 elicits beige adipocyte development in response to β-adrenergic stimulation. Inhibition of JAK3 diminishes brown adipocyte formation; JAK3 may also play a distinct role in brown precursors and brown mature adipocytes [879]. The kinases PKG1, PI3K, and PKB are implicated in brown adipocyte formation and brown mature adipocyte activation [879]. 111 Activated PKA phosphorylates (activates) P38MAPK, which phosphorylates activating transcription factor ATF2, which interacts with PGC1α, coordinating the transcriptional program that activates brown adipocytes, and initiates transcription of the Ucp1 gene [879]. Its ubiquitous regulatory subunits PKAr1α and PKAr2α , which are encoded by the PRKAR1A and PRKAR2A genes, promote brown adipocyte formation. On the other hand, PKAr1β and PKAr2β , which are encoded by the PRKAR1B and PRKAR2B genes, play an opposite role. P38MAPK has a dual function in brown adipocyte formation and mature brown adipocyte function. 112 Among AMPKα catalytic subunits, AMPKα1 is more abundant than the AMPKα2 isoform. In mice, chronic cold exposure raises AMPKα1 concentration in BAT and epididymal WAT (epiWAT) [879]. β3-Adrenergic stimulation does not influence AMPKα1 activity in BAT, but only in epiWAT. Inhibition of AMPK reduces brown adipocyte survival and differentiation. In brown pre-adipocytes, AMPKα1 suffices for cell proliferation and differentiation, as elimination of AMPKα2 does not affect brown adipocyte formation, differentiation, and proliferation. The AMPKβ subunit influences the stability of the AMPK heterotrimer. Both AMPKβ1 and AMPKβ2 subunits are important for brown adipocyte formation [879]. The AMPKγ1 and AMPKγ2 subunits are widespread, the latter being the most abundant in the heart; AMPKγ3, which lodges in the skeletal muscle, is also present in intermediate amounts in brown pre-adipocytes.

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Table 5.11 Examples of batokines and their effects (Source: [869]) Batokine Fibroblast growth factor-21 Free fatty acids Interleukin-6

Nerve growth factor Neuregulin-4 Triiodothyronine

Effects Improved metabolism Cardioprotection, antihypertrophic action Improved metabolism Improved metabolism Cardioprotection (acutely), maladaptive remodeling (prolonged presence) Pro-survival in cardiac ischemia Improved cardiac function Decreased insulin resistance Cardioprotection Improved metabolism Cardioprotection

Neuregulin Nrg4 is formed in BAT activated by adrenoceptors under cold conditions. It protects against insulin resistance, hepatic steatosis, and myocardial ischemia [869]. Interleukin-6 improves insulin sensitivity, but contributes to adverse cardiac remodeling after sustained exposure [869]. Nerve growth factor may promote sympathetic innervation of BAT. It exerts a pro-survival activity in ischemic CMCs [869]. Certain types of adipocyte-specific branched fatty acids enhance the glucose effect and reduce AT inflammation in obesity [869]. MicroRNAs operate in AT formation and function. They are involved in the differentiation of white, beige, and brown adipocytes. They intervene in obesity, T2DM, and cardiovascular disease [880]. Certain microRNAs regulate brown adipocyte differentiation, such as miR26, miR34, miR133, miR155, miR378, and miR455 [880]. On the other hand, BAT activation can also regulate miR expression profiles. The CNS integrates afferent cues that regulate food intake and energy expenditure in a coordinated manner. In rodents, UCP1 expression in BAT is controlled by changes in metabolic state related to cold exposure or changes in food intake. These stimuli distinctly control sympathetic nervous system activity, which activates adrenoceptors and UCP1 expression in brown adipocytes, its signaling to BAT increasing in hypermetabolic conditions of lean animals [881]. In ADAM17−/− mice, elevated UCP1 concentration in BAT is linked to increased sympathetic outflow. Members of the MIR26 family, miR26a and miR26b, regulate human white and beige adipocyte differentiation, both types oxidizing nutrients at very high rates via nonshivering thermogenesis, their formation increasing in early adipogenesis. In adipose-derived stem cells, synthesis of mitochondrial UCP1 is upregulated, as miR26a and miR26b target ADAM17 [882].

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MiR34a is an anti-adipogenic agent with a dual role in both brown and beige adipocyte formation that has increased expression in obesity [880]. In HFD-fed obese mice, its inhibition reduces adiposity and improves oxidative function in AT. In addition, its reduced expression increases formation of the BeAT -specific marker TNFRSF9 and UCP1 in WAT (vAT) and promotes additional browning in BAT. Furthermore, inhibition of miR34 raises adipocytic SIRT1 level and hence deacetylation of PGC1α, which functions in WAT browning. MiR34a targets FGFR1 [880]. MiR133 targets PRDm16, thereby repressing brown adipogenesis in white adipocyte progenitors [880]. MiR155 operates both in brown adipocyte differentiation and WAT browning [880]. It targets C/EBPβ. It lowers UCP1 and PGC1α expression Synthesis of miR378 in BAT is induced by cold exposure; it promotes BAT (but not WAT) adipogenesis [880]. MiR378 targets PDE1B, the product of which yields cAMP and cGMP turnover. Formation of miR455 in BAT is induced by cold temperature exposure and BMP7, which is needed for the development of both BAT and recruitable BeAT [883]. It targets brown adipogenic inhibitors, such as Runx1T1 and necdin homolog, two adipogenic transcriptional repressors, in addition to HIF1α inhibitor (HIF1an), an hydroxylase that processes (inhibits) the AMPKα1 subunit. MiR455 suppresses necdin and Runx1T1 to initiate an adipogenic program and HIF1an to activate AMPKα1, which triggers a brown adipogenic program. Therefore, miR455 induces brown and beige adipogenesis via activation of the HIF1an–AMPK–PGC1α axis [883]. The WAT, and especially BAT, release microRNAs in exosomes that enter blood circulation, thereby supporting glucose tolerance and reducing hepatic synthesis and circulating level of FGF21 [884]. The AT is an important source of circulating exosomal miRs in both mice and humans. These exosomal miRs secreted by AT can act as both para- and endocrine messengers. These circulating miRs do indeed regulate body metabolism and mRNA translation in other organs such as the liver. In particular, exosomal BAT-derived miR99b controls FGF21 activity. Although the majority of BAT microRNAs in the bloodstream are contained in exosomes, the AT can also secrete miRs in microvesicles or are associated with Argonaute and HDLs [884]. The pvAT is endowed with a microvasculature. Insulin resistance is associated with the reduced formation of the anti-inflammatory microRNA, miR181b, in AT ECs [880]. MiR181b supports insulin-mediated PKB phosphorylation and hence reduces endothelial dysfunction. It targets Phlpp2 mRNA.113

113 PHLPP2:

pleckstrin homology (PH) domain and leucine-rich repeat-containing protein phosphatase. It dephosphorylates PKB (Ser473).

5.4 Adipose Tissue

5.4.1.2

415

Beige Adipose Tissue

Beige adipocytes (brown-like or “brite,” i.e., “brown in white” adipocytes) have some of the characteristics of brown adipocytes, such as abundant mitochondria and thermogenic activity They develop with WAT (browning) in response to various activators, such as NR1c3 (PPARγ) ligands, FGF21, irisin, cold, and β3-adrenoceptor agonists (Vol. 10, Chap. 1. Architecture and Structure of the Vasculature). Whereas β1AR recruits pre-adipocytes in WAT, β3AR provokes the emergence of beige adipocytes within WAT [868]. In humans, β1AR and β2AR may also regulate BAT activity. Upon removal of these stimuli, the beige adipocytes behave more like white adipocytes. Mitophagy determines the identity and function of adipocytes and its repression enables maintenance of the beige adipocyte phenotype, whereas enhanced mitophagy shifts the adipocyte phenotypes from beige to white. Therefore, autophagy can be beneficial or detrimental according to the context [885]. The WAT is distributed in visceral regions, whereas scAT is a mixture of white and beige adipocytes. Beige adipocytes may originate from multipotent pre-adipocytes located in various white adipose depots or from transdifferentiation of white adipocytes into beige adipocytes [869]. A set of markers defines brown, beige, and white adipocytes, • LHx8114 and ZiC1115 being related to brown. • TBx15116 to brown and beige. • HoxC9 (or Hox3b)117 and Shox2118 to beige.

114 LHx8:

LIM domain and homeobox-containing protein-8 (also called LHx7). This transcription factor is involved in the differentiation of certain neurons and mesenchymal cells [108]. The ZiC1 transcriptional activator is involved in neurogenesis. 115 ZiC1: zinc finger protein of cerebellum. The ZiC proteins ZiC1 and ZiC2 launch transcriptional activation of the APOE gene [194]. 116 TBx: T-box transcription factor. TBx15 may be a transcriptional regulator involved in the development of the skeleton, which controls the number of mesenchymal precursor cells and chondrocytes [108]. 117 Hox: homeobox gene product. HoxC9 specifies cell position on the anterior–posterior axis [108]. 118 Shox: short stature homeobox gene product. It may act as a growth regulator, in processing somatosensory information, and body structure formation [108].

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Table 5.12 Stimulators of BAT and BeAT mass (Source: [868]; BDNF brain-derived neurotrophic factor, BMP bone morphogenetic protein, DBC1 deleted in bladder cancer protein-1 [Fam5a], TBx T-box protein, VEGF vascular endothelial growth factor) Agent Main function Increases BAT mass and browns WAT FGF21 Regulation of fasting substrate metabolism Cardiac natriuretic peptides Regulation of sodium homeostasis Tbx15 Transcription Glucagon-like peptide Regulation of postprandial glucose metabolism PRDm16 Bidirectional switch between myogenesis and brown adipogenesis Increases BAT mass only BMP7 Pleiotropic actions Myostatin Inhibition of myogenesis Twist-1 Organogenesis, apoptosis Browns WAT only Irisin Myokine stimulated by exercise BDNF Neurotrophin (neuronal survival and axonogenesis Cyclooxygenase-2 (PGhS2) Rate-limiting enzyme in prostaglandin synthesis VEGFa Angiogenesis Sirtuin-1 Deacetylation DBC1 Sirtuin-1 inhibition Increases BAT activity only BMP8b Pleiotropic activity

• HoxC8 (or Hox3a), Inhbb,119 and Dpt120 for beige and white. • TcF21 (bHLHa23) to white adipocytes [868]. Many factors increase BAT and/or BeAT mass (Table 5.12) [868]. MicroRNAs regulate BeAT differentiation, that is, WAT browning [880]. Let7i5p causes differentiation of white adipocytes to a beige type, as it prevents UCP1 119 Inhβ b (or Inhβ2): inhibin-β b. The activins, which are dimers of β A or β B subunits encoded by the genes INHBA and INHBB, respectively, are TGFβ superfamily members implicated in reproduction and development. Inhibins and activins inhibit and activate the secretion of follitropin by the pituitary gland, respectively. They are also involved in regulating hypothalamic, pituitary, and gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, nerve cell survival, embryonic axial development, and bone growth, according to their subunit composition [108]. Activin ligands act as growth and differentiation factors in many cell types [194]. Activins are composed of β subunits only. Activin-β C can dimerize with activin-β A and -β B (activin-AC and-BC heterodimers), but not with inhibin-α. Inhibins are heterodimeric glycoproteins consisting of an α and β A or β B subunit. Inhibin- and activin-β B subunit is expressed in adrenal medullary cells. 120 DPt: dermatopontin. It is also called tyrosine-rich acidic matrix protein. It may mediate cell adhesion via integrin. It enhances TGFβ1 activity, hampers cell proliferation, accelerates collagen fibril formation, and stabilizes collagen fibrils.

5.4 Adipose Tissue

417

expression. Its overexpression in scWAT alters the formation and function of brite adipocytes via partial inhibition of Gs-coupled β3-adrenoceptor signaling of browning via the AC–cAMP–PKA–P38MAPK–PGC1α/ATF2–UCP1 pathway. MiR30b and miR30c control not only BAT function and hence energy homeostasis but also beige adipogenesis [880]. Their production rises in response to cold exposure in addition to stimulated β3AR signaling. They target the nuclear corepressor NRIP1.121 In scWAT and interscapular BAT, β3AR stimulation attenuates miR125b-5p expression. Overexpression of miR125-5p precludes beige adipogenesis in WAT.

5.4.1.3

White Adipose Tissue

The WAT is devoted to energy storage, fatty acids being exported for oxidation in other organs when energy is required during periods of food restriction or great exertion. The size of the WAT heightens when the energy balance is positive and declines when energy expenditure is in excess of intake. It receives a modest blood supply. Furthermore, WAT represents an endocrine tissue that secretes adipokines, which control eating behavior and insulin sensitivity, among other tasks. Brown adipokines can have more distinct effects than white adipokines [886]. The WAT is composed of white adipocytes (≤75%), fibroblasts, ECs, pericytes, macrophages, and pre-adipocytes, among other cell types. New white adipocytes are formed from a pool of precursors. White adipocytes store energy, whereas brown adipocytes dissipate energy, converting chemical energy into heat for thermoregulation. White adipocytes contain a single large LD (unilocular adipocytes) and few mitochondria. Certain hormones (e.g., adrenaline, NAd, glucagon, and adrenocorticotropin) bind to their receptors on adipocytes and trigger lipolysis by activated lipases, that is, the hydrolysis of triacylglycerol into fatty acids and glycerol. The pair of enzymes, hormone-sensitive (LipE) and monoacylglycerol (monoglyceride) lipase (MGL) convert diacylglycerol into fatty acids and glycerol [887]. Hormone-sensitive lipase is inhibited by insulin and activated by glucagon and adrenaline. Glycerol is exported from adipocytes via an aquaporin-type transporter and conveyed to the liver for oxidation or gluconeogenesis. Various regulators of lipid and lipoprotein metabolism are released from white adipocytes. Lipoprotein lipase secreted by adipocytes processes circulating triacylglycerols into chylomicrons and VLDLs to fatty acids. Other secreted proteins from WAT involved in lipid and lipoprotein metabolism include cholesteryl ester transfer protein and apolipoprotein-E [888]. The WAT also contributes to glucose homeostasis. Lipodystrophic mice, which possess only a small quantity of adipose depots, are diabetic; they exhibit both hyperglycemia and marked hyperinsulinemia [888].

121 NRIP:

nuclear receptor-interacting protein.

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The WAT participates in inflammation, some adipokines being inflammatory cytokines, whereas others are involved in the complement system [888]. Adipsin, a serine peptidase, is a constituent of the alternative complement pathway, which is also named complement factor-D; it is secreted from white adipocytes. Acylationstimulating protein (ASP or C3adesArg ; Sect. 5.4.5.21) is synthesized by WAT, which stimulates TG synthesis and glucose transport in adipocytes. In humans, its concentration rises in obesity, T2DM, and CVD, whereas exercise or weight loss lowers it. The WAT also stores cholesterol and is involved in the metabolism of steroid hormones. It does not synthesize steroid hormones, but it does express enzymes converting both glucocorticoids and sex hormones (Sects. 5.4.5.14 and 5.4.5.15) [888]. Adipokines can serve as auto-, para-, and endocrine messengers, which serve in metabolism and remote regulation (Sect. 5.4.5; Table 5.13)). vAT and scAT are characterized by a unique adipokine production pattern. The vAT synthesizes higher amounts of IL6 and PAI1, whereas the scAT produces larger quantities of adiponectin and leptin [887]. The leptin synthesis rate is indeed greater in scAT than in vAT. The amount of microRNAs synthesized by dicer in white adipose depots declines with aging and in humans with congenital generalized and HIV-associated lipodystrophy [884]. Lipodystrophies are acquired or genetic diseases with various degrees of AT deficiency and hence frequently altered adipokine and cytokine profiles (very low adiponectinemia and leptinemia), which causes ectopic TG accumulation, such as in the skeletal muscle and liver, and reduces insulin sensitivity [717]. Defective AT storage engenders elevated circulating concentrations of lipid metabolites, cholesterol, and TGs. They are often accompanied by dyslipidemia, insulin resistance, and CVD, especially in congenital generalized lipodystrophy (CGLD) patients. Type-1 to -4 CGLDs are autosomal recessive disorders caused by mutations in the AGPAT2 (CGLD1),122 Bscl2 (CGLD2),123 CAV1 (CGLD3),124 and CAVIN1

122 AGPAT2:

acylglycerol 3-phosphate acyltransferase-2 (or lysophosphatidic acid acyltransferaseβ). AGPAT2 affects adipocyte differentiation via PKB and PPARγ [717]. It catalyzes the second step of phospholipid synthesis. It operates in TG and glycerophospholipid formation from glycerol 3-phosphate. Mutations in the AGPAT2 gene cause defective TG storage and accumulation of intermediates such as lysophosphatidic acid. Increased LPA concentration hampers adipogenesis and AT expansion and increases AT fibrosis in obesity. 123 BSCL2: type-2 Berardinelli–Seip congenital lipodystrophy (LD genesis-associated seipin). Congenital generalized lipodystrophy is also dubbed Berardinelli–Seip congenital lipodystrophy. Mutations in the Bscl2 gene cause type-2 CGLD (CGLD2). 124 Cav: caveolin, a major component of caveolae involved in lipid regulation and endocytosis.

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Table 5.13 Adipokines secreted by the AT into the bloodstream and their effects (Sources: [826, 887]; ADRF adipocyte-derived relaxing factor, ASP acylation-stimulating protein, FFA free fatty acid, IGF insulin-like growth factor, PAI plasminogen activator inhibitor, TNFSF tumor-necrosis factor superfamily member [TNFSF1 being TNFα], VEGF vascular endothelial growth factor) Adipokine Adiponectin Adipsin ADRF Aldosterone

Angiotensinogen Angiotensin-2 Apelin ASP H2 O2 IGF1 Interleukin-6 Leptin

Omentin Osteopontin Resistin Serpin-E1 (PAI1) TNFSF1

VEGF FFA Glycerol

Effects Protection against inflammation, T2DM and CVD Vasodilation Complement pathway Vasodilation Vasodilation and vasoconstriction according to dose and exposure duration Inflammation, insulin resistance, hypertension Regulation of blood pressure and electrolyte homeostasis Vasoconstriction Vasodilation and vasoconstriction Influences the TG synthesis rate in AT Vasodilation and vasoconstriction Cell proliferation Inflammation, lipid and glucose metabolism, regulation of body weight Regulation of appetite and energy expenditure Inflammation Vasodilation and vasoconstriction Protection against inflammation, vasodilation Inflammation Insulin resistance, vasoconstriction Inhibition of plasminogen Affects insulin receptor signaling Vasoconstriction and -dilation Inflammation Angiogenesis Energy source Gluconeogenic precursor

(PTRF) genes (CGLD4),125 which encode lysophosphatidic acid acyltransferase-β, seipin, caveolin-1, and cavin-1.126 Partial lipodystrophies include familial partial lipodystrophy (FPLD). It engenders partial AT loss, mainly from the subcutaneous depots in the extremities

125 PTRF:

polymerase-1 and transcript release factor, also labeled cavin-1. Cavin stands for caveola-associated protein. 126 The three last-mentioned genes encode proteins involved in vesicular transfer and affect the formation or maturation of LDs.

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and upper trunk region.127 and acquired forms, most commonly occurring in patients with human immunodeficiency virus (HIV) receiving highly active antiretroviral therapy with protease inhibitors that reduce the levels of factors involved in adipocyte differentiation and function (e.g., ATF4, C/EBPα, DDIT3, PPARγ, and XBP1), and raises ROS concentration [717]. Congenital generalized lipodystrophy is characterized by a generalized loss of AT.128 HIV-associated lipodystrophy is associated with a decreased dicer concentration in AT. MiR16, miR201, miR221, and miR222 are highly expressed in fat depots. Defective miR processing in AT by dicer provokes whitening of BAT, InsRce, and altered circulating lipid levels.

5.4.1.4

Perivascular Adipose Tissue

The pvAT surrounds large arteries and veins, except in the cerebral circulation. It is accompanied by lymphatics and nerves. The pVAT has a unique embryonical origin from Tagln+ precursors of vSMCs.129 It is an intermediate between BAT and WAT, that is, beige AT (BeAT) in rodents, although it remains mainly a WAT in larger mammals (rabbits, pigs, and humans) [805]. It is structurally and functionally heterogeneous, not only among mammalian species but also according to its location, i.e., the vascular bed. It is separated from the vascular wall of large arteries, but is integrated into the wall of smaller vessels [889]. The pvAT surrounding large arteries contains its own vasculature, the vasa vasorum. It has beige or white AT-like characteristics in proximal and distal arteries, respectively. Its thermogenic function varies according to the vascular compartment. The pvAT can change its phenotype from WAT to BeAT and conversely, according to exposure to temperature and the nutritional status [805]. The pvAT interferes with the surrounding vascular wall via paracrine and endocrine effects, as pvAT-derived adipokines can reach the vascular lumen, hence the so-called vasocrine signaling. For example, the pvAT facilitates insulin effects and glucose uptake in lean (but not obese) mouse skeletal muscles [889]. The coronary vasa vasorum may convey harmful adipokines between pvAT and the vascular wall. The increased density of the coronary vasa vasorum precedes coronary endothelial dysfunction [890]. Adipokines, such as leptin, resistin,

127 In FPLD3, the PPARG gene, which encodes PPARγ, is mutated. mutations in the Pkb2 (AKT2) and PLIN1 genes impair adipocyte function and also cause FPLD. In FPLD2, mutations in the LMNA gene, which encodes lamin-A/C, weaken the nuclear envelope and cause cell death. 128 Despite the quasi-complete lack of metabolically active storage AT, mechanical fat in joints, orbits, palms, and soles is present [717]. 129 Tagln: transgelin. This actin crosslinker (gelling protein) is involved in calcium signaling. It is also called 22-kDa smooth muscle protein SM22α, whereas transgelin-2 is named SM22α homolog.

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Table 5.14 Perivascular and epicardial adiposecretome, obesity, and atherosclerosis (Sources: [890, 891] icam1S soluble intercellular adhesion molecule-1, IL interleukin, MIF macrophage migration inhibitory factor, NPR natriuretic peptide receptor, PAI plasminogen activator inhibitor, PGC peroxisome proliferator-activated receptorγ coactivator, sPLA2 secretory phospholipase-A2, TNFSF tumor-necrosis factor superfamily member) Augmented synthesis and secretion

Decreased synthesis

Chemerin, leptin, resistin, visfatin, adrenomedullin, angiotensinogen, TNFSF1, IL1β, IL1Rα, IL6, IL13, IL16, CCL2, CCL5, NPRc, sPLA2, PAI1, icam1S , PGC1α Adiponectin, adipsin, omentin, MIF

Table 5.15 Processes in which adipokines are involved (Source: [892]; ADRF adipocyte-derived relaxing factor, HGF hepatocyte growth factor, IL interleukin, PAI plasminogen activator inhibitor, ROS reactive oxygen species, TNFSF tumor-necrosis factor superfamily member, VEGF vascular endothelial growth factor) Process Glucose metabolism Lipid metabolism Angiogenesis Vasodilation Vasoconstriction Vasodilation or -constriction Immunity Inflammation Blood coagulation Pancreatic β-cell function Feeding behavior Maintenance of reproduction

Involved adipokines Adiponectin, resistin Retinol-binding protein, cholesteryl ester transfer protein Leptin, VEGF, HGF Adiponectin, ADRF, omentin, visfatin Resistin, angiotensin-2 Apelin, leptin, TNFSF1, IL6, ROS Adipsin TNFSF1, IL6 PAI1 Adiponectin, visfatin, IL6 Leptin Leptin, ghrelin

TNFSF1, and IL6, augment ROS formation directly or indirectly via production of endothelin-1 and angiotensin-2 (Table 5.14). The pvAT operates in various processes, such as angiogenesis, mesenchymal stem cell recruitment, vSMC proliferation, vEC function, vasomotor tone control, and CMC electrochemical activity (Table 5.15) [890].

Regulation of the Vasomotor Tone Endothelial regulators of the vasomotor tone include the vasodilating gases (e.g., NO), oxygen-derived free radicals (e.g., superoxide [O•− 2 ] and hydroxyl radical [OH• ]), and peptides (e.g., endothelins and angiotensins), contractant and relaxant

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eicosanoids comprising prostanoids (prostaglandins, prostacyclins, and thromboxanes), in addition to EDHF. Among peptides, whereas endothelin-1 and angiotensin2 cause vasoconstriction, endothelium-derived neuropeptide-Y (NPy)130 and ANP prime vasodilation. These regulators are also synthesized in vSMCs, inflammatory leukocytes, mesangiocytes, and adipocytes [779]. Vasoconstrictors include vasocontracting prostanoids131 and superoxide anion,132 in addition to angiotensin-2133 and endothelins (ET1–ET3).134 Obese patients have an elevated sympathetic signaling and vasomotor tone, that is, arteries present a stronger vasoconstriction than that observed in normal conditions. Sensory nerves comprising C and Aδ neural fibers surround vascular walls. They contain and release peptides, among which substance-P and calcitonin generelated peptide (CGRP) are the principal vasodilators [893]. Substance-P is an endothelial-dependent vasodilator, which also increases microvascular permeability, whereas CGRP operates as both endothelial-dependent and -independent vasodilator. However, sensory nerves are linked to the sympathetic and angiotensin signaling and hence substance-P and CGRP do not affect blood pressure at rest in normal conditions. Sensory nerves also localize to the pvAT, especially around small mesenteric resistance arteries, and cause vasodilation, despite their interaction with the sympathetic system [893, 894]. Stimulants of the pvAT sensory nerves (e.g., TRPV1 agonists) are thus vasodilators. Among TRPV1 agonists, capsaicin releases CGRP from sensory nerves [894]. Leptin can also be released to potentiate CGRP-mediated pvAT-derived sensory neurogenic vasodilation, leptin release being abolished by hypoxia [894]. Leptin receptors reside on sensory neurons. Deletion of leptin signaling in vagal afferent neurons causes hyperphagia and obesity. In WAT, leptin secreted from adipocytes increases the activity of innervating sensory nerves. However, other neurotransmitters can regulate the vasomotor tone. Angiotensin2 secreted by the pvAT augments sympathetic signal transmission in rat mesenteric arteries, which inhibits CGRP release from sensory nerves in this arterial bed [894].

130 Neuropeptide-Y

is involved in appetite regulation and stimulates adipogenesis [779]. It binds to its receptor on ECs, priming NO-dependent vasodilation, stimulating EC proliferation, and affecting endothelial permeability. It also strengthens thromboxane-mediated vasoconstriction. 131 Prostaglandin-H synthase PGhS1 (or cyclo-oxygenase COx1) is the sole enzyme forming vasocontracting prostanoids, at least in mice [779]. Aging and obesity are associated with PGhS activation. 132 This short-lived by-product of oxidative metabolism is mainly produced by vascular NAD(P)H oxidase and cytochrome-P450 epoxygenase, in addition to uncoupled NOS [779]. Superoxide inactivates NO via formation of peroxynitrite. 133 Obesity is also associated with activation of the RAA [779]. 134 Endothelin and the RAA interact, constituting a positive feedback loop, especially in obesity [779].

5.4 Adipose Tissue Table 5.16 Adipokines regulating the vasomotor tone (ADRF adipocyte-derived relaxing factor, Agt angiotensin, APPL adaptor containing phosphoTyr interaction, PH, and Leu zipper domain, H 2 S hydrogen sulfide, NO nitric oxide, PGi2 prostaglandin-I2 [prostacyclin])

423 Target cell EC SMC

Messengers (effectors) ADRF (via BK, IK, SK) ADRF (via KV , KATP , BK) H2 O2 (via sGC and Src–MAPK axis) Adiponectin NO (using the PI3K–PKB–NOS3 and APPL1–AMPK–NOS3 axes) H2 S Agt(1–7) (via KV ) PGi2

Superoxide ions can be generated by pvAT promote sensory neurogenic relaxation in the rat isolated mesenteric arterial bed. In lean individuals, pvAT is a paracrine regulator of the vasomotor tone and vasoprotector. It does indeed counter vasoconstriction, especially in the microvasculature.135 This anticontractile property is lost in obese subjects [896, 897]. The pvAT secretes adventitial vasodilators (adiponectin, adipocyte-derived relaxing factor [ADRF], angiotensin(1–7) , hydrogen peroxide, hydrogen sulfide, nitric oxide, palmitic methyl ester, and prostacyclin; Table 5.16). These pvAT vasodilators are counteracted by vasoconstrictors, such as angiotensin-2, superoxide anion, and TNFSF1. The pvAT operates via endothelium-dependent and -independent mechanisms. An endothelium-independent process relies on hydrogen peroxide and subsequent activation of soluble guanylate cyclase. At low concentrations (10–100 μmol/l) of H2 O2 , pre-constricted mesenteric vessels undergo further constriction, but higher concentrations (0.3–1 mmol/l) provoke a biphasic response, with an early constriction followed by dilation [891]. Hydrogen peroxide released from both adipocytes and macrophages can act via the Raf–ERK pathway in vascular smooth myocytes. Adipocyte-derived relaxing factor may hyperpolarize ECs. This hyperpolarization is then transmitted to adjacent smooth myocytes via myoendothelial junctions, thereby relaxing the arterial and arteriolar wall. Release of ADRF by pvAT relies on Ca2+ ion [866]. ADRF is linked to vascular potassium channels on vascular ECs and smooth myocytes (KV , possibly KV 7 [866],136 KATP ,137 BKV,Ca , IKCa , and SKCa ), the types of which depend on the vascular compartment and mammalian species [891]. In human internal mammary arteries, it acts via BK channels. Stimulated

135 Microvascular

changes are rare in obesity before the development of hyperglycemia and T2DM [895]. 136 The K 7 channel is targeted by vasopressin in aortic smooth myocytes [866]. V 137 The K ATP channel at most plays a minor role [866].

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β3-adrenoceptors also operate via SMC BK channels.138 The effect of ADRF may also be mediated by hydrogen sulfide, a gasotransmitter generated in the pvAT [866, 892]. In addition to ADRF, adipokines such as adiponectin, omentin, and visfatin are vasodilators. On the other hand, angiotensin-2 and resistin are vasoconstrictors released by adipocytes. Apelin, leptin, ROS, TNFSF1, and IL6 have both vasorelaxing and -contracting effects [892]. Plasmalemmal hyperpolarization linked to a product of arachidonic acid metabolism by phospholipase-A2 and cytochrome-P450 is the major contributor to endothelium-dependent vasodilation in human subcutaneous resistance arteries [898]. In these arteries, EDHF is thus a more important vasodilator than NO and PGi2 . In humans, small arteries and arterioles of the scAT are endowed with a myogenic tone, an increase in pressure triggering the PLC–DAG–PKC pathway [895]. In resistance arteries and arterioles of gluteal scAT from healthy volunteers, vasodilation caused by histamine, which targets myocytic H2 and endothelial H1 receptors, provokes endothelial NO release. In several vascular beds, nitric oxide- and prostacyclin-independent vasodilation is linked to activation of cytochrome-P450 epoxygenases in ECs, which generate vasodilatory EETs, but also oxygen-derived free radicals that lower NO availability [899]. CYP2c9 prevents endothelium-dependent NO-mediated enhancement in forearm blood flow in healthy volunteers and CoAD patients. Acetylcholine operates not only via NO but also in small arteries and arterioles of obese rodents via EDHF, that is, via KCa channels [895]. In healthy individuals, acetylcholine primes SMC hyperpolarization. In obesity, EDHF can compensate for impaired NO synthesis. Moreover, in scAT arteries of healthy subjects, insulin can reduce NAd-induced vasoconstriction [895]. Venous pvAT releases angiotensin(1–7) which activates KV channels and relaxes vSMCs using NO [891]. The pvAT counters vasoconstriction caused of thromboxane TxA2 and its stable metabolite TxB2 , which prime perioperative spasm, via ADRF and BK [891]. On the other hand, the pvAT enhances vasoconstriction induced by α1adrenoceptors stimulated by the perivascular sympathetic nerves, as they prime superoxide generation and activation of Src–MAPK pathway. In addition, angiotensin-2 derived from adipocytes potentiates vasoconstriction. The pvAT undergoes browning upon β3AR agonist exposure. In healthy organisms, pvBeAT regulates the vasomotor tone via autacoids. The final determinant of the vasomotor tone is the phosphorylation status of myosin light chain MLC20, which is regulated by myosin light chain kinase and phosphatase, MLCK being stimulated by elevated cytosolic Ca2+ concentration

138 Obesity

is characterized by an overactive sympathetic nervous system, which releases NAd at nerve terminals in pvAT. NAd can bind to β3-adrenoceptors, albeit with a lower affinity than to β1- and β2-adrenoceptors [891].

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and MLCP being inhibited by the PP1r12a subunit. Classically, PKG1 activated by cGMP decreases cytosolic Ca2+ content and phosphorylates BK channel and RhoA, thereby reducing rock activity and PP1r12a phosphorylation. The mesenteric pvAT around small resistance arteries is WAT; its anti-contractile effect can result from vascular potassium channels, such as KATP and KV channels (e.g., KV 7 [889]), in addition to messengers, such as H2 S, NO, PGi2 , and ATn(1–7) [897]. The aortic pvAT, a BeAT around a large highly distensible artery, also has anticontractile and -inflammatory properties. It secretes H2 O2 , which is predominantly synthesized by NOx4, the major isoform in adipocytes, without affecting NOS3 activity [897]. Whereas NOx4 produces mainly H2 O2 , the other Nox isoforms synthesize detrimental superoxide. Action of epididymal visceral (evWAT), interscapular (isBAT), inguinal subcutaneous (mixed WAT–BAT iscW/BAT), and pvAT (mixed WAT–BeAT pvW/BeAT) on arteries was investigated in WT and NOX4−/− mice [897]. In NOX4−/− mice, isBAT releases a reduced H2 O2 amount and does not exert an anti-contractile effect; BAT from WT mice mediates an anti-contractile action on vessels of NOX4−/− mice, the perivascular H2 O2 production being involved rather than that from local medial smooth myocytes. In BAT and BeAT (but not WAT), hydrogen peroxide activates PKA and mainly PKG1α via oxidant-induced dimerization in vSMCs, subsequently phosphorylating BK, which is implicated in the anti-contractile effect of BAT, and reduces phosphorylation of PP1r12a and MLC20 [897]. The pvAT, in particular, epicardial pericoronary adipose tissue, which is characterized by high rates of lipid deposition and lipolysis and high UCP1 expression, expands with obesity and diabetes [869]. A transition of pvAT happens from a protective form in lean healthy subjects to a damaging type in obese and diabetic individuals, pvAT containing more inflammatory cells (e.g., macrophages) and secreting fewer anti-inflammatory substances (e.g., adiponectin) and more proinflammatory adipokines (e.g., angiotensinogen, leptin, resistin, TNFSF1, IL6, chemokines CCL2 and CXCL8, and reactive oxygen and nitrogen species). In WT mice, 6-month high-sucrose, high-fat diet (HFHSD) raises body weight, fasting glycemia, postprandial insulinemia, and ornithinemia, and causes hypertension and arterial stiffening and fibrosis, but lowers argininemia [900]. Arginase-1 produced by vascular ECs is involved in obesity-induced vasculopathies via redox stress and declined availability in arginine and nitric oxide. In HFHSD-fed mice in addition to isolated vessels immersed in palmitate- and glucose-enriched media, vascular arginase activity rises and is coupled with redox stress. In mice, vascular dysfunction in DIO can be countered by arginase-1 inhibition and deletion of the ARG gene in vascular ECs (EC ARG1−/− ). Elevated arginase activity can cause depletion in arginine and subsequently in NO, hence impairing endothelial-dependent vasodilation. Arginase, a urea cycle enzyme, catalyzes hydrolysis of L arginine to urea and ornithine. It thus competes with nitric oxide synthase for their common substrate, L arginine. Ornithine is processed into polyamines by ornithine decarboxylase and proline by ornithine aminotransferase. Arginase has two isozymes; arginase-1 localizes to the cytoplasm

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Table 5.17 Regulators of vascular smooth myocyte (vSMC) proliferation (Source: [901]; Agt angiotensin, C3adesArg desarginated complement factor C3 fragment [or acylation-stimulating protein], CCL chemokine CC-motif ligand, FFA free fatty acid, H 2 S hydrogen sulfide, HBEGF heparin-binding EGF-like growth factor, IL interleukin, MIF macrophage migration-inhibitory factor, NO nitric oxide, PAI plasminogen activator inhibitor, ROS reactive oxygen species) Stimulators Inhibitors Dual regulators

C3adesArg , CCL2, CCL3, HBEGF, IL1β, IL6, leptin, MIF, PAI1, resistin, ROS, visfatin Adiponectin, Agt(1–7) , androgens, glucocorticoids, H2 S, IL10 Adrenomedullin, angiotensin-2, estrogens, FFAs, NO

and abounds in the liver; mitochondrial arginase-2 is the primary isoform in the kidney. Both isozymes lodge in vascular endothelial and smooth muscle cells; their synthesis can be upregulated by glucose and ROS [900]. Excessive arginase activity can also shift arginine metabolism to the production of ornithine, polyamines, and proline, polyamines promoting vascular smooth myocyte proliferation and proline collagen formation, thereby leading to perivascular fibrosis.

Vascular Smooth Myocyte Proliferation The pvAT releases tissue growth stimulators and inhibitors that operate on vSMCs (Table 5.17). Decreased concentration of adiponectin and increased concentration of TNFSF1 in PVAT favor intimal hyperplasia after endovascular injury, which relies on abnormal proliferation and migration of medial SMCs [901]. Visfatin secreted by the pvAT is a growth factor for vSMCs. It stimulates vSMC proliferation via ERK1, ERK2, and P38MAPK rather than JNK and the PI3K– PKB axis [901]. In addition, it can counter vSMC apoptosis induced by H2 O2 at nonphysiological concentrations.

Pathophysiological Role Obesity is associated with a reduced dilatory capacity of both conduit and resistance arteries. Hyperglycemia and hypertension as well as insulin resistance and obesity cause redox stress, which decreases NO availability and hence acetylcholine- and histamine-induced NO-mediated dilations of skeletal muscle arterioles [895]. In a pathological context, pvWAT is subjected to redox stress and inflammation. Factors derived from pvAT then provoke endothelial dysfunction and vascular remodeling with vSMC proliferation.

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Dysregulated synthesis and secretion of adipokines associated with immunocyte infiltration in the AT is linked to fat depot inflammation. Dysfunctional AT synthesizes harmful proinflammatory adipokines (e.g., leptin) rather than vasodilatory adipokines [892]. Dyslipidemia, insulin resistance, and hyperinsulinemia impair the vasomotor tone control in arteries [895]. Perfusion of the myocardium and skeletal muscles is altered in obese subjects, primarily because of the vasomotor dysfunction of resistance arteries. Hyperemia-induced forearm blood flow during exercise, local heating, or upon acetylcholine stimulation falls, even in obese children [895]. In pre-pubertal boys without insulin resistance, flow-mediated dilation in the brachial artery drops. In the mesenteric and skeletal muscle microcirculation, vasodilation mediated by the endothelium and induced by acetylcholine and sodium nitroprusside (SNP) via NO declines,139 increasing peripheral vascular resistance in obese people [895]. On the other hand, vasoconstriction by endothelin-1 in the forearm circulation of overweight and obese individuals is potentiated with respect to lean subjects [720]. Constriction of scAT arterioles engendered by NAd is not affected in obesity, but dilation induced by acetylcholine is impaired [895]. Arteriolar dilation mediated by EDHF, which is linked to BK, SK, and IK channels, may be less sensitive to redox stress than that induced by NO [895]. Insufficient AT perfusion increases formation of hypoxia inducible factor, HIF1α in adipocytes, which launches synthesis of several proinflammatory adipokines (TNFSF1, IL6, and CCL2) [895]. On the other hand, hypoxia promotes angiogenesis, as it triggers synthesis of angiogenic adipokines (e.g., VEGF, HGF, FGF2, leptin, PAI1, and IL6) [892]. Under hypoxia in obese subjects, the pvAT elicits formation of the vasa vasorum [889]. The vasa vasorum conveys macrophages from the systemic circulation to the pvAT. Inflammatory AT deregulates its effect on the vasomotor tone and recruits monocytes and induces local insulin resistance, vascular remodeling, and endothelial dysfunction [892]. The pvAT can not only affect endothelial function, decreasing NO availability and hence endothelium-dependent vasodilation and impairing the ability of the endothelium to respond to circulating messengers, but also the redox state, and can prime vascular wall inflammation. Conversely, endovascular injury engenders rapidly an overexpression of proinflammatory adipokines within the pvAT [890]. Several types of adipokines at pathophysiologically relevant concentrations alter endothelial function. Leptin, resistin, and TNFSF1 diminish endothelial NO production [890]. On the other hand, the pvAT exaggerates vasoconstriction in response to stimulation by the perivascular nerves via angiotensin-2 and increased superoxide production by NOx. In vSMCs of HFD-fed mice, overexpression and deletion of P22PhOx, a subunit of NOx1, NOx2, and NOx4, worsen the obesity state [889].

139 Sodium

nitroprusside, which is processed into NO, is a vasodilator of arterioles and venules.

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In metabolic syndrome, the pvAT exacerbates endothelial dysfunction via leptin and PKCβ in aortas, coronary, and mesenteric arteries [890]. Leptin mediates endothelial-dependent vasodilation via NO, but only at pharmacological concentrations (>160 ng/ml), whereas at relevant obese concentrations (50% of total phospholipids in cellular membranes [922]. Both PC and PE are synthesized by an amino-alcohol phosphotransferase reaction that uses diradylglycerol and either cytidine diphosphate (CDP)–choline or CDP– ethanolamine, respectively, in the last step of a synthesis route,the Kennedy pathway.154 The pathway that forms phosphatidylcholine and phosphatidyl etha154 Named

after E.P. Kennedy who, in collaboration with S.B. Weiss, elucidated this reaction established in 1956 [923]. A set of enzymes involved in the three steps of the synthesis of PC and PE:

(Step 1) choline kinases, ChKα and ChKβ and ethanolamine kinases, EtnK1 and EtnK2, which phosphorylate choline and ethanolamine, respectively. (Step 2) phosphocholine (PCyT1α–PCyT1β; or choline–phosphate cytidylyltransferases CCTα– CCTβ) and phosphoethanolamine cytidylyltransferase (PCyT2), which form cytidine diphosphate (CDP)–choline and CDP–ethanolamine from phosphocholine and -ethanolamine (or choline and ethanolamine phosphate), respectively, using cytidine triphosphate (CTP). (Step 3) choline and ethanolamine phosphotransferase CEPT1 and CDP–ethanolamine-specific ethanolamine phosphotransferase EPT1, which generate PC and PE, respectively.

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nolamine from choline and ethanolamine consists of three enzymatic steps and two branches, the CDP-choline and -ethanolamine subaxis. It relies on: (1) choline and ethanolamine kinases; (2) phosphocholine and -ethanolamine cytidylyltransferases, the rate-limiting enzymes; and (3) choline and ethanolamine phosphotransferases. The pathway that forms phosphatidylcholine and phosphatidylethanolamine from choline and ethanolamine consists of 3 enzymatic steps and 2 branches, the CDP–choline and –ethanolamine subaxis. It relies on: (1) choline and ethanolamine kinases; (2) phosphocholine and -ethanolamine cytidylyltransferases, the ratelimiting enzymes; and, (3) choline and ethanolamine phosphotransferases. Upon uptake of fatty acids, choline– and ethanolamine–phosphate cytidylyltransferases (but neither choline kinase nor choline phosphotransferase) localize to LDs [917]. In vitro, cytidylyltransferase is activated by diacylglycerol and phosphatidic acid. It binds to membranes depleted of PC. As a LD grows, a relative depletion of PC and enrichment in diacylglycerol and phosphatidic acid promote its binding and activation. 5.4.4.5

Neutral Lipid Synthesis Linked to Lipid Droplets

In adipocytes, LDs store neutral triacylglycerols. Triacylglycerol synthesis, i.e., lipogenesis, results from esterification of acyl-CoA derived from fatty acids and glycerol 3-phosphate. Fatty acids originate from three sources: blood circulation, lipolysis of intracellular TGs, and de novo fatty acid synthesis from glucose. Glycerol 3-phosphate arises from glycerol, glucose, and amino acids. In most cell types, neutral lipids are synthesized by enzymes permanently or transiently located in the ER. Phospholipids are produced for LD expansion and to avoid coalescence.

5.4.4.6

Lipid Droplet Proteins

Proteic constituents of LDs include components of COP1 coatomer and monomeric ARF GTPases. Both ARF1 and COP1 components act directly on the LD surfaces to remove phospholipids, as the Arf1–COP1 machinery increases the local surface tension and promotes budding of nano-LDs, promoting establishment of membrane bridges between LDs and the ER and allowing rapid displacement of ER-bound proteins, such as PnPLA2155 the rate-limiting enzyme in TG hydrolysis, which is involved in LD turnover, and GPAT4, to LD surface [924].

155 Intracellular

Ca2+ -independent patatin-like phospholipase domain-containing protein is also called desnutrin, calcium-independent phospholipase-A2, and ATGL.

5.4 Adipose Tissue

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The COP1 complex may stimulate lipolysis via inhibition of perilipin-2156 binding to LD surfaces and hence recruit PnPLA2 to lipid droplets [924]. At least five Rab isozymes are connected to LDs, especially Rab18, which mediates apposition of the LD phospholipid monolayer and ER membrane and may control lipolysis. In adipocytes, insulin-stimulated lipogenesis increases the residence of Rab18 into LDs [924].157 Rab18GTP interacts with the ER-tethering NRZ complex,158 which facilitates the fusion of vesicles from the Golgi body. Seipin is a ubiquitous oligomeric ER transmembrane protein implicated in LD genesis. Seipin is involved in nascent ER–LD contacts and the delivery of lipidic and proteic constituents of LDs from the ER [925]. Seipin is dispensable for the initial synthesis of neutral lipids from fatty acids and their aggregation inside the ER membrane, but mandatory for fatty acid incorporation into neutral lipids in preexisting LDs, hence intervening after the initial nucleation. Seipin helps to anchor newly formed LDs to the ER and stabilizes ER–LD tethering, thereby facilitating the incorporation of proteins and lipids through membrane bridges into growing (maturing) lipid droplets. During LD formation, seipin also assists the transfer of long-chain fatty acid–CoA ligase (synthetase) ACSL3 from endoplasmic reticulum to LDs. Numerous membrane transfer components, such as ArfGEF, coatomer COP1 components,159 and Rab GTPases (Rab1, Rab5, Rab7, Rab8a, Rab18, Rab32, and Rab40c) pertain to LDs and/or are involved in their creation [929].

156 A.k.a.

adipophilin and adipose differentiation-related protein (factor [ADRP (ADFP)]). small GTPase Rab18 lodges on membranes of the ER, Golgi body, endosomes, and peroxisomes [924]. 158 NRZ: NAG–RINT1–ZW10 (NAG: neuroblastoma-amplified gene product; RINT1: Rad50 interactor-1; ZW10: centromere- and kinetochore-associated protein zw10 homolog). Both ZW10 and RINT1 are cell cycle checkpoint proteins. 159 The Golgi body-derived coat protomer (coatomer) COP1 in the cytosol and cellular membrane operates along the early secretory pathway, whereas COP2+ vesicles export proteins from the ER and clathrin-coated vesicles transfer cargo along the late secretory and endocytic pathways [926]. COP1 contains ARF1, which controls CoP1 genesis, Arf1GEF (GBF1), and ArfGAPS such as ArfGAP1, with which most cargo interacts. The CoP1 complex carries cargo, such as the KDEL receptors, certain members of the P24 family of cargo proteins, and SNAREs [927]. Type-I transmembrane proteins of the P24 family (P24a–P24e) become major constituents [928]. 157 The

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In LDs, the transport protein particle complex TRAPPC2160 is a Rab1GEF and Rab18GEF [924].161 The small GTPase Rab18 linked to LDs may contribute to the connection and exchange between the ER and lipid droplets. It is also involved in the maintenance of the ER and the Golgi body [929]. The COP1–TRAPPC2–Rab18 signaling cascade puts LD and ER in close proximity to facilitate the lipogenic and/or lipolytic activities of Rab18 [924]. Lipolysis liberates fatty acids and glycerol from stored lipids. It is primed by hormones such as catecholamines that stimulate cAMP production and subsequent phosphorylation by PKA of LipE (HSL) and perilipins, which coat LDs, protecting them from LipE.

5.4.5 Adipose Tissue Secretome: Adipokines The AT serves not only as a triacylglycerol store and source of FFAs but also as an endocrine organ. It releases many endocrines in addition to auto-, juxta-, and paracrine messengers that intervene in metabolism and fat depot size and redistribution, and modulates diverse biological processes (Tables 5.19 and 5.20).

160 Several

types of TRAPP complexes (TRAPPC1–TRAPPC4) share the same core subunits, but contain distinct accessory subunits in Saccharomyces cerevisiae. In yeast, the different TRAPP complexes participate in distinct transfer processes: endoplasmic reticulum-to-Golgi body (ER–GB) transport (TRAPPC1), late transport steps in the GB (TRAPPC2), and autophagy (TRAPPC3 and TRAPPC4) [929]. Two TRAPP complexes exist in mammalian cells, TRAPPC2 and TRAPPC3, which share common core subunits (TraPP1–TraPP5), but differ in the peripheral subunits, TRAPPC2 contains TraPP9 and TraPP10, whereas TRAPPC3 possesses TraPP8 and TraPP11 to TraPP13 [929]. In mammals, TRAPP2 is implicated in intra-GB and/or GB-toplasma membrane (GB–PM) transport and in ciliogenesis in addition to the ER export of fibrillar procollagen; TRAPP3 controls ER–GB transport and autophagy. In mammals, both TRAPPC2 and TRAPPC3 act as a guanine nucleotide exchange factor (GEF) for Rab1 [929]. TRAPPC3 contributes to the control of autophagy via interaction between TraPP8 and TBC1D14, a RabGAP. TRAPPC3 also interacts with the COP2 vesicular coat [924]. TRAPPC2 is recruited to LDs upon lipid load via its interaction with COP1, the latter also mediating the establishment of tubular connections between the ER and the LD. Inactivation of TRAPPC2-specific subunits reduces lipolysis and provokes aberrantly large LDs [924]. 161 Recruitment of Rab18 onto the LD surface is impaired upon TRAPPC2 deletion, but the localization of Rab1 on the GB is not affected. The COP1–TRAPPC2 supercomplex recruits Rab18 onto the surface of small LDs. In addition to the Rab18GEF TRAPPC2, the Rab3GAP complex is a specific GEF for Rab18 [930]. Rab3GAP1 and Rab3GAP2, which elicit GTP hydrolysis by Rab3, work as a Rab18GEF complex. The Rab3GAP1–Rab3GAP2 complex is required for Rab18 linkage to the ER. Among the TBC1 domain-containing protein of the TBC1D set, TBC1D20 functions as a GTPase-activating protein for Rab1, Rab2, and Rab18 (Rab18GAP) [931, 932]. Warburg micro syndrome constitutes a spectrum of disorders characterized by severe eye, brain, and endocrine abnormalities. It is a rare autosomal recessive disorder caused by loss-of-function mutations in the RAB18, RAB3GAP1, RAB3GAP2, or Tbc1d20 genes. Enlarged GB and aberrant LD formation result from TBC1D20 LOF mutations [931]. In addition, fibroblasts with defective Rab18 and Rab3GAP1 exhibit aberrant LD genesis.

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Table 5.19 Adiposecretome and main adipokines (Part 1; Source: [889]) Adipocytokine Adiponectin

Major source Adipocyte

Apelin

Adipocyte

Leptin

Adipocyte (mainly subcutaneous)

Omentin

Adipocyte

Resistin

Monocytes, macrophages (adipocytes) Stromal cells

Visfatin

Receptor(s) Role AdipoR1/2 Antioxidant and -inflammatory Precludes NFκB and NOx activity NO synthesis via NOS3 phosphorylation by AMPK AplnR May reduce redox stress and inflammation May stimulate NOS3 (vasodilation) Positive inotropic effect LepR Regulates energy storage Induces NOx activity Proinflammatory Stimulates NOS expression Acts via JaK–STAT, SOCS, PI3K–PKB, and MAPK axes Increases insulin sensitivity Antioxidant, -inflammatory Reduces vSMC migration Raises NO availability Hampers intimal hyperplasia CAP1 Prooxidant, -inflammatory Decreases NO production Prooxidant, -inflammatory May increase NO production

Adipokines include protective anti-inflammatory (e.g., adiponectin, apelin, omentin, and interleukin-10) and detrimental and prooxidant and -inflammatory species (e.g., leptin, resistin, and visfatin) in metabolically healthy individuals and obese subjects, respectively (AMPK AMPactivated kinase, AT adipose tissue, CAP adenylate cyclase-associated protein, NF nuclear factor, NO nitric oxide, NOS nitric oxide synthase, NOx NAD(P)H oxidase, vSMC vascular smooth muscle cells)

The WAT is implicated not only in glucose metabolism (e.g., via adiponectin, resistin, and visfatin), lipid metabolism (e.g., via cholesteryl ester transfer protein, FABP4, lipoprotein lipase, retinol-binding protein, and sirtuins), and control of vasomotor tone (via adipokines and classical vasodilators such as NO and H2 S and vasoconstrictors such as endothelin-1 and angiotensinogen and its derivatives) along with medial SMC proliferation and migration (e.g., via omentin), but also in appetite regulation (e.g., via leptin), energy expenditure, angiogenesis (e.g.,

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Table 5.20 Adiposecretome and main adipokines (Part 2; Source: [889]; ET endothelin, IGF insulin-like growth factor, IL interleukin, PAME palmitic acid methyl ester, TNFSF TNF superfamily member [TNFSF1 is TNFα]) Adipocytokine ET1 IGF1 IL6 TNFSF1 NO, H2 S Agt(1–7) PGi2 PAME H2 O2 MicroRNAs

Major source All AT cell types All AT cell types Monocytes, macrophages Monocytes, macrophages

Receptor(s) Role ETA /B Prooxidant, -inflammatory IGF1R Regulates adipocyte and vSMC differentiation IL6R Prooxidant, -inflammatory TNFRSF1a Prooxidant, -inflammatory Vasodilation Antioxidant and -inflammatory Vasodilation Vasodilation Vasodilation Vasodilation MiR103-3b is linked to CCL13 MiR29a/143 regulate AT browning, inflammation MiR29a/143 are involved in adverse remodeling

via VEGF), blood coagulation and fibrinolysis (e.g., via plasminogen activator inhibitor-1 [serpin-E1]), immunity and hence inflammation (via cytokines and chemokines), and reproduction. Adipokines have a hemodynamic, metabolic, and immunological impact. They can influence vasomotor tone (adiponectin, leptin, omentin, resistin, visfatin, and TNFSF1), insulin sensitivity (adiponectin and resistin), and inflammation (leptin, chemerin, CCL2, and CXCL8) [890]. The AT also produces and secretes other types of auto-, para-, and endocrine regulators, such as VEGF and insulin-like growth factor IGF1, sex steroids, angiotensinogen, and ROS [866]. White and brown adipocytes synthesize and secrete AngptL4162 upon exposure to PPARα (or NR1c1). They also release the metal-binding and stress-response metallothioneins MT1 and MT2, which can play an antioxidant role [888].

162 A.k.a.

fasting-induced adipose factor (FIAF) and hepatic fibrinogen- and angiopoietin-related protein (HFARP).

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Table 5.21 Adipokines and metabolic syndrome (Source: [908]) Inhibitors Stimulators Associated anomalies Endothelial dysfunction

Insulin resistance

Adiponectin, nitric oxide Angiotensinogen, leptin, pentraxin-1, resistin, serpin-E1, TNFSF1 Context ↓ NO ↑ ATn2, ET1, icam1, vcam1, CCL2, TNFSF5, TNFRSF5, oxLDL Obesity, hypertension, glucose intolerance, low HDL concentration

Proinflammatory mediators favor endothelial dysfunction and insulin resistance, whereas amounts of protective adipocyte products decline with obesity (↑ increase, ↓ decrease)

Adipokines (or adipocytokines, i.e., adipocyte-derived cytokines)163 are sources of crosstalk between the AT and cardiovascular apparatus and explain the existence of intertwined metabolic and cardiovascular alterations [810]. Adipokines include hormones, growth factors, cytokines, chemokines, complement factors, enzymes, and constituents of the extracellular matrix. Adipocytes possess receptors for most of these substances. AT environment affect its secretome. Adipokines act on the hypothalamus and other organs, and they regulate metabolism in the liver, skeletal muscles, and heart. Energetic metabolism relies on metabolic circuits resulting from communications between various organs controlled by hormones (e.g., glucagon and insulin), growth factors (e.g., FGF21), and cytokines, among which adipokines (adipocytederived cytokines; e.g., adiponectin, adipsin, 15 C1q and TNF-related proteins [CTRP], leptin, omentin, resistin, retinol-binding protein RBP4, vaspin, and visfatin, in addition to the adipose tissue-derived cytokines IL1β and TNFSF1). Metabolism and immunity are linked by proteins of dual function. The AT produces and secretes proinflammatory adipokines, such as TNFSF1, interleukins IL1β and IL6, chemokine CCL2, leptin, serpin-E1 (Tables 5.21, 5.22 and 5.23).164 angiotensinogen, resistin, and pentraxin-1 (or CRP). Obesity and T2DM are characterized by high levels of proinflammatory cytokines. Obesity typically upregulates expression of proinflammatory adipokines (leptin, TNFSF1, IL6, and resistin) and downregulates that of anti-inflammatory adipokines (adiponectin, omentin, CTRP9, and secreted frizzled-related protein sFRP5) [805]. The cardiovascular risk associated with adipokine imbalance results from the paracrine action of adipokines released from epvATs and the endocrine effect of adipokines secreted by other fat depots.

163 Originally,

the term adipokine defines adipocyte-derived secreted immunomodulatory proteins. The broader meaning includes immunomodulatory proteins secreted by adipocytes and other cell types of AT. 164 A.k.a. plasminogen-activator inhibitor PAI1.

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Table 5.22 Effects of adipokines (Part 1; Source: [908]; CSF colony-stimulating factor, ET endothelin, EC EC, icam intracellular adhesion molecule, IL interleukin, LDL low-density lipoprotein, NF nuclear factor, NO nitric oxide, NOS NO synthase, PAI plasminogen-activator inhibitor, ROS reactive oxygen species, vSMC vascular smooth myocyte, TRAF tumor-necrosis factor receptor-associated factor, vcam vascular cell adhesion molecule) Adipokine Adiponectin

Leptin

Resistin

Effects ↓ icam1, vcam1, E-selectin ↓ NFκB ↓ vSMC proliferation and migration ↑ insulin sensitivity ↓ foam cell formation ↑ NOS3 production ↑ glucose transport ↑ ET1, ROS ↑ CSF1 release ↑ EC and vSMC proliferation and migration ↑ vSMC apoptosis ↑ angiogenesis ↑ sympathetic tone ↑ cholesterol accumulation under hyperglycemia ↑ ET1 release ↑ formation of adhesion molecules and chemokines ↓ glucose uptake and insulin action ↓ TRAF3

Several adipokines promote insulin sensitivity (e.g., adiponectin, leptin, and visfatin), whereas others induce insulin resistance (e.g., lipocalin-2, resistin, and RBP4, in addition to serpin-E1 (PAI1) along with TNFSF1 and IL6). However, adipokines, such as resistin and visfatin, play different roles in rodents and humans.

5.4.5.1

Adiponectin

Adiponectin165 is a member of the CTRP family,166 as it is structurally similar to complement factor C1q and tumor-necrosis factor. It is one of the most effective adipokines in correcting obesity-linked insulin resistance.

165 Adiponectin

(Adpn) is encoded by the ADIPOQ gene, which localizes to a susceptibility locus for T2DM and metabolic syndrome in chromosomal locus 3q27. It is also called AdipoQ (adiponectin C1q and collagen domain-containing protein), ACRP30 (30-kDa adipocyte complement-related protein), adipocyte C1q and collagen domain-containing protein (ACDC), adipose most abundant transcript product-1 (ApM1 and AdipQTL1, and gelatin-binding protein GBP28. 166 CTRP: C1q–TNF-related protein.

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Table 5.23 Effects of adipokines (Part 2; Source: [908]; AT 1 , angiotensin type-1 receptor, ATn angiotensin, FFA free fatty acid, TNFSF tumor-necrosis factor superfamily member) Adipokine Angiotensinogen

Interleukin-6

Pentraxin-1 (CRP)

Serpin-E1 (PAI1) TNFSF1 (TNFα)

Effects ↓ NO availability ↑ NFκB ↑ icam1, vcam1, CCL2, and CSF3 ↓ angiogenesis ↑ icam1, vcam1, E-selectin, CCL2 ↑ pre-adipocyte differentiation ↑ SMC proliferation and migration ↓ insulin receptor signaling ↑ hepatic CRP production ↓ NOS3 expression ↑ PAI1 expression in ECs ↑ release of ET1 and IL6 ↑ icam1, vcam1, selectins, CCL2 in ECs ↑ LDL uptake in ECs ↑ ROS ↑ AT1 on vSMCs ↑ SMC proliferation and migration ↓ angiogenesis ↑ EC apoptosis ↑ thrombogenesis ↑ TNFSF1, ATn2, FFAs ↑ restenosis ↓ NO availability ↓ adipocyte differentiation ↓ insulin signaling ↑ NFκB via ROS ↑ icam1, vcam1, E-selectin, CCL2 in EC and vSMC ↑ lipolysis and FFA level ↑ EC apoptosis

Adiponectin is produced in the brown and WAT. Adiponectin expression is launched by PGC1α, PPARγ (NR1c3), and BNP, GC2a residing on adipocytes [849]. In addition, NAD+ -SIRT and catecholamine–β-adrenoceptor axes may participate in determining adiponectin concentration. Transcription of the ADIPOQ gene in the human vAT is inhibited by glucocorticoids and TNFSF1, but stimulated by insulin and IGF1 [908]. Adiponectinemia ranges from 5 to 30 μg/ml in humans, representing up to 0.05% of total plasmatic proteins [933]. However, adiponectinemia depends on ethnicity. Adiponectin has an insulin-sensitizing effect [934]. Non-esterified fatty acids potentiate glucose-stimulated insulin secretion. However, adiposity often occurs

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Table 5.24 Adiponectin effects (Source: [934] ↑ increase, ↓ decrease) Glucose metabolism

Lipid catabolism

Miscellaneous

↑ glucose uptake ↓ gluconeogenesis ↑ insulin sensitivity ⊕ −→ glycogen synthase Fatty acid β-oxidation, generation of energy Triglyceride and LDL clearance −→ LDLR synthesis ⊕ −→ cholesterol efflux Weight loss Control of energy metabolism ↓ TNFSF1 concentration Brown adipocyte differentiation ⊕ −→ AMPK and PKA −→ PDGFRα and ERK1/2 signaling Detection of redox stress ↓ cell migration and proliferation −→ granulocyte differentiation −→ NFκB and inflammation −→ foam cell formation from macrophages

⊕ −→ stimulation, −→ inhibition

independently of plasmatic neFA concentration. Among adipokines that regulate β-cell function, concentration of adiponectin, a potentiator of glucose-stimulated insulin secretion, decreases with obesity and leptin, the concentration of which rises with adiposity, hampers insulin release [995]. Hypo-adiponectinemia contributes to the pathological conditions associated with overweight. Adiponectin represses endothelial inflammation and vSMC proliferation [934]. This vasodilator raises NO production via AdipoR1 and PI3K [720, 892]. In addition, adiponectin can decrease TNFSF1-induced production of ADMA, an inhibitor of NO synthesis, and improve the endothelial redox state, as it suppresses superoxide generation by NAD(P)H oxidase [720]. Adiponectin operates in the control of glucose and lipid metabolism and insulin sensitivity (Table 5.24). It has anti-inflammatory, antidiabetic, and antiatherogenic effects. Adiponectin exists in two forms, full length and a smaller globular fragment, which target the AdipoR2 and AdipoR1 receptors, respectively [891]. In human adipocytes, the expression of AdipoR1 is about 15-fold higher than that of AdipoR2. AdipoR1 supports the pro-angiogenic action of adiponectin in cultured ECs [939]. AdipoR2 can lead to insulin resistance, but helps revascularization. Both globular and full-length adiponectin induces vasodilation via NO in resistance arteries, at least in lean rats [933].

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Adiponectin monomers are assembled into multimers that can be categorized into three groups: high (AdpnhMW ), mid- (AdpnmMW ), and low-molecular-weight form (AdpnlMW ). The largest high-molecular-weight (HMW) adiponectin is resistant to peptidases. Adiponectin circulates in the bloodstream as oligomers (trimers, hexamers, and HMW forms that contain at least 18 monomers). Hexamer and HMW oligomers are assembled from the basic adiponectin trimer. Post-translational modifications (Lys hydroxylation and glycosylation) enable the formation of HMW oligomers. Adiponectin is also modified by sialic acids via O-linked glycosylation of threonine residues, which determines its clearance from the bloodstream [933]. In addition, Cys36 is succinylated, thereby impeding disulfide bond formation and Adpn oligomerization. In diabetes, adiponectin is succinylated. Its cognate receptors AdipoR1 and AdipoR2 belong to the 11-member PAQR family of transmembrane proteins (Table 5.25). In addition to AdipoR1, AdipoR2, and T-cadherin, the plasmalemmal calreticulin–CD91 complex may bind adiponectin on macrophages to facilitate the removal of apoptotic cells [939]. Both AdipoR1 and AdipoR2 activate AMPK, PPARα (NR1c1), and P38MAPK in the liver and skeletal muscle in addition to ECs [933]. However, AdipoR1 and AdipoR2 are the main effectors for AMPK and NR1c1 activation, respectively. The APPL1 adaptor is a direct interactor of both AdipoR1 and AdipoR2. It promotes the translocation of STK11 (LKB1) from the nucleus to the cytosol, where it activates AMPK [933]. In addition, adiponectin activates AMPK via the PLC– Ca2+ –Cam2Kβ pathway. The APPL1 adaptor also activates P38MAPK, triggers the STK11–AMPK– SIRT1 axis, and in cooperation with NR1c1, promotes glucose uptake, fatty acid oxidation, and mitochondrion genesis via PGC1α. APPL2 also interacts with both AdipoR1 and AdipoR2, but hinders adiponectin signaling in myocytes [933]. Other AdipoR1 signaling mediators include RACK1, CsnK2β, ERP46, and TNFSF3 [933]. In the liver, AMPK is activated by AdpnFL and, in the AT and skeletal muscle, by 3 Adpn [891]. Adiponectin thus exerts its metabolic effects (increased glucose uptake, glycogen deposition, and enhanced fatty acid oxidation in muscles following phosphorylation by AMPK of acetyl-CoA carboxylase, inhibition of triacylglycerol and fatty acid synthesis, and reduced glucose output in the liver) via AMPK. Whereas 6 Adpn and AdpnhMW activate NFκB in myocytes, 3 Adpn stimulates AMPK and enhances fatty acid oxidation in muscles [935]. AdpnhMW , but neither trimer nor hexamer, activates AMPK in the liver to reduce glucose production. Adiponectin localizes to the heart and vascular endothelium, where it interacts with T-cadherin (Cdh13), a glycosylphosphatidylinositol-anchored glycoprotein, which mediates the cardiovascular effects of adiponectin [805]. Cadherin-13 specifically bind 6 Adpn and AdpnhMW [933]. In pancreatic β cells and CMCs, adiponectin stimulates ceramidase, which processes ceramide into sphingosine, which is phosphorylated by sphingosine kinase to form sphingosine 1-phosphate [933]. Ceramidase also inhibits caspase-8

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Table 5.25 Members of the progestin and adipoQ receptor (PAQR) family with eleven genes and two pseudogenes Type PAQR1 PAQR2 PAQR3 PAQR4 PAQR5 PAQR6 PAQR7 PAQR8 PAQR9 PAQR10 PAQR11

Other aliases AdipoR1, ACDCR1 AdipoR2, ACDCR2 RKTG MPRγ, MPRα, Pγ LP MPRβ, LMPB1 MMD2 MMD1

Adiponectin binds to its receptors AdipoR1 (PAQR1) and AdipoR2 (PAQR2) in addition to cadherin-13 (or T-cadherin). Both PAQR1 and PAQR2 enhance ceramidase activity; adiponectin lowers ceramide concentration via these receptors. Accumulation of ceramide and glucosylceramide and hence hypo-adiponectinemia are involved in insulin resistance and atherosclerosis [936]. Both G-protein-coupled PAQR1 and PAQR2 tether to globular and full-length adiponectin; PAQR1 and PAQR2 have a high and intermediate affinity for globular adiponectin, respectively. The nuclear receptors NR1c1 and NR1c3 regulate PAQR1 and PAQR2 formation in adipocytes (but not in myocytes). PAQR1 and PAQR2 abound in the muscle and liver, respectively. PAQR1 and PAQR2 mainly target AMPK and NR1c1, respectively, and elicit fatty acid oxidation and glucose uptake upon adiponectin binding. Cadherin-13, a GPI-anchored cadherin, can compete with AdipoR1 and AdipoR2 for adiponectin binding or interfere with adiponectin signaling. It is a LDL receptor; LDL binding to Cdh13 activates ERK1, ERK2, and NFκB (ACDCR adipocyte C1q and collagen domain-containing protein receptor, LMPB lysosomal membrane protein in the brain, MMD monocyte-to-macrophage differentiation-associated factor or monocyteto-macrophage differentiation protein, MPR membrane progestin receptor, Pγ LP PPARγ-induced liver protein, RKTG Raf kinase trapping to the Golgi body)

and hence CMC apoptosis. This effect strengthens CMC apoptosis inhibition resulting from PKB and AMPK action. The SphK1 kinase activates PGHs2, thereby repressing inflammation primed by TNFSF1. In CMCs, where it is synthesized and secreted, adiponectin increases ScaRb3 translocation and fatty acid uptake via AMPK [933]. It enhances insulin-stimulated glucose transport via PKB. It stimulates lipoprotein lipase via actin remodeling boosted by the RhoA–rock axis. In addition, adiponectin promotes angiogenesis, as it elicits VEGF production via AMPK [933]. In the aorta of HFD-fed obese rats, adiponectin improves endothelial function, as it increases NO production via NOS3 phosphorylation by AMPK and PKB, once it connects AdipoR1 and AdipoR2, which cooperate [891, 933]. Moreover, adiponectin can repress basal and oxLDL-induced superoxide generation in addition to ROS production linked to hyperglycemia via the antioxidant cAMP–PKA pathway in ECs [891]. Furthermore, adiponectin inhibits NOS2, hence further reducing redox stress caused by excess NO radical.

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Adiponectin also impedes peroxynitrite-induced nitrosative stress. It can opsonize apoptotic cells and facilitate their clearance. Adiponectin promotes endothelial repair and angiogenesis after vascular injury via EPC mobilization from the bone marrow and spleen into the bloodstream and their recruitment to the injured vascular wall [933]. It stimulates survival, proliferation, and differentiation of bone marrow-derived EPCs and supports their migration via the PI3K–CDC42–Rac1 cascade, whereas activated AMPK is required for EPC recruitment by adiponectin to the vascular injury site. In diabetic patients, the circulating EPC density is altered partly because of redox stress [933]. Adiponectin suppresses proliferation and migration of human vSMCs induced by PDGFbb, FGF2, and HBEGF, as it tethers to these growth factors, in addition to those launched by IGF1, as AMPK inhibits ERK1 and ERK2 [933]. In macrophages, both globular and full-length adiponectin prevents production of proinflammatory cytokines induced by leptin and resistin [933]. It also suppresses NFκB signaling via the cAMP–PKA axis. It promotes the anti-inflammatory phenotype in macrophages and has an anti-inflammatory effect on vascular ECs. In human monocyte-derived macrophages, globular adiponectin upregulates the formation of the anti-inflammatory cytokine, IL10 [933]. Therefore, adiponectin exerts anti-oxidative, anti-inflammatory, and hence vasculoprotective actions. It impedes TNFSF1 production in adipocytes and macrophages. It can stimulate the synthesis of PGhS2 (or COx2) and thus prostaglandinE2 , a vasculoprotective autacoid [805]. Activation of AMPK by adiponectin in the myocardium protects mice against cardiac hypertrophy and ischemia–reperfusion injury. Its cognate receptors AdipoR1 and AdipoR2 mediate the antihypertrophic effect of adiponectin in CMCs. Hypoadiponectinemia is observed in obese subjects [805]. Adiponectin synthesis in adipocytes is hindered by ER and redox stresses. Adiponectin protects against the metabolic syndrome characterized by dyslipidemia (hypercholesterolemia, hypertriglyceridemia, and elevated LDL/HDL ratio), endothelial dysfunction, hyperglycemia (decreased glucose tolerance), hypertension, inflammation, insulin resistance, obesity, and redox stress. Insulin activates the PI3K–PKB pathway, thereby phosphorylating (inactivating) glycogen synthase kinase GSK3, a regulator of metabolism (and of cell fate and immunity), which stimulates adiponectin production. Indeed, GSK3 phosphorylates the transcription factor C/EBPα which is required for the synthesis of adiponectin, thereby impeding adiponectin formation [937]. Mice fed with an HFD and expressing PI3K-insensitive GSK3 are protected against the metabolic syndrome, adiponectin synthesis in the AT being significantly higher than in WT mice [937]. However, these mice, which exhibit hypocorticosteronemia and hypo-aldosteronemia, have a higher renal sodium excretion and hypertension.

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Adiponectin protects the ovarian function. Reduced adiponectin concentration and augmented adipocyte size are associated with insulin resistance in women with polycystic ovary syndrome (PCOS), who have decreased fertility and increased T2DM risk [938]. Altered AT function provokes metabolic dysfunction in women with PCOS.

5.4.5.2

C1q and Tumor-Necrosis Factor-Related Proteins

The C1q subclass of proteins includes immune complement C1q, C1qDC1 (or caprin-2), adiponectin, CTRPs, cerebellins, emilins, multimerins, otolin, and collagen-8 and -10. The C1q–TNF superfamily comprises more than 30 secreted multimeric proteins, which operate in the endocrine, immune, neuronal, reproductive, sensory, skeletal, and vascular systems. Members of the CTRP superfamily (i.e., adiponectin and C1q–TNF-related proteins, CTRP1–CTRP15), which are encoded by the genes of the C1QTNF group, are paralogs of adiponectin that contribute to the regulation of glucose and fatty acid metabolism (Tables 5.26 and 5.27) [939]. Adiponectin is synthesized in the pvAT, among other sites. Certain CTRPs are produced primarily in the stromal vascular cells of the AT. Table 5.26 Properties of the CTRP proteins (Part 1; Source: [939]; −→ inhibition) CTRP CTRP1

Tissue distribution Adipose tissue, placenta

Signaling AMPK, ERK1/2

Action sites Skeletal muscle

CTRP2

Adipose tissue, also lung, liver, testis, uterus Adipose tissue, kidney, bone, testis, uterus

AMPK

Skeletal muscle

PKB

Liver

MAPK

Endothelium

PKB

Heart Monocytes Skeletal muscle, eye

CTRP3

CTRP5

CTRP6

Adipose tissue, eye, brain, spleen, skeletal muscle, testis, uterus Placenta, also adipose tissue, spleen, lung, testis, prostate, uterus

AMPK

Synoviocytes Adipocytes

Function Glucose uptake Lipid oxidation Insulin sensitivity fatty acid oxidation glycogen deposition −→ gluconeogenesis and triglyceride synthesis EC migration and proliferation Cell survival Anti-inflammatory GluT4 translocation Lipid oxidation

Complement regulation Adipogenesis

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Table 5.27 Properties of the CTRPs (Part 2; Source: [939]) CTRP CTRP7 CTRP9

CTRP11

CTRP12

Tissue distribution Adipose tissue lung Adipose tissue

Adipose tissue, testis; also brain, kidney Adipose tissue

Signaling

Action sites

Function

AMPK

Lipid oxidation

AMPK–NOS3 AMPK

Skeletal muscle Endothelium Heart

MAPK, PKA

VSMC

MAPK

Pre-adipocyte

PKB

Adipose tissue Liver Pancreatic β cells Adipose tissue

CTRP13

CTRP15

Adipose tissue, brain, kidney

Skeletal muscle

AMPK

Adipocytes, hepatocytes Liver Hypothalamus Adipocytes, hepatocytes

Vasodilation Improved function after injury −→ migration and proliferation −→ adipogenesis

Glucose uptake −→ gluconeogenesis Insulin secretion −→ inflammation Glucose uptake −→ gluconeogenesis −→ food intake Lipid uptake

Cq1–TNF-related proteins circulate as monomers and heterotrimers [810]. They target the endocrine, immune, vascular, skeletal, and sensory systems. They can ensure cardioprotection [939]. Most members of the CTRP group are synthesized in the AT, which predominantly produces CTRP1 to CTRP3, CTRP5, CTRP7, CTRP9, CTRP12, and CTRP13 [939]. All CTRPs trimerize, hence forming their basic structural unit; some assemble into hexamer and high-molecular-weight oligomers that may have distinct biological and signaling properties; CTRP3, CTRP5, CTRP6, CTRP9, CTRP10, CTRP12, CTRP13, and CTRP15 multimerize. They also combine, thereby generating distinct functional ligands. In addition to homo-oligomers, the heterotrimers CTRP1–CTRP6, CTRP2–CTRP7, and adiponectin–CTRP2 are also secreted. Synthesis of CTRP3 and CTRP9 primarily in AT is downregulated in obesity [805]. Their circulating concentrations vary according to the metabolic state, sex, and genetic background [940].

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CTRP1 (C1qTNF1) The adipokine CTRP1 is produced in AT and in oxLDL- or IL1β-stimulated macrophages and ECs of atherosclerotic plaques [810]. Plasmatic CTRP1 concentration is elevated in hypertensive patients; CTRP1 mediates angiotensin-2-induced aldosterone production [941]. CTRP1 also stimulates P38MAPK and hence likely the P38MAPK–Arg pathway that mediates ATn2–primed endothelial dysfunction. In the plaque, CTRP1 activates ECs and macrophages that then produce adhesion molecules and inflammatory cytokines such as TNFSF1, thereby augmenting leukocyte infiltration and inflammation. Plasmatic CTRP1 originates from visceral AT in addition to monocytes and inflammatory sites; it is also secreted by vascular cells such as ECs [941]. In a small patient population, plasmatic CTRP1 concentration is linked to coronary atherosclerosis and impaired collateralization. In atherosclerotic lesions, CTRP1 co-localizes with macrophages and ECs of plaque microvessels. Exposure to CTRP1 and deletion of the Ctrp1 gene in ApoE-deficient mice facilitate and reduce the progression of atherosclerosis, respectively [941]. Plasmatic CTRP1 concentration rises significantly in coronary endarterectomy samples, atherosclerotic plaques, and in blood and peripheral blood mononuclear cells from patients with severe CoAD [941]. Therefore, CTRP1 can serve as a marker of atherosclerosis. As for adiponectin, CRTP1 synthesis is induced by the nuclear receptor NR1c3 (transcription factor PPARγ). In skeletal muscles, CTRP1 signals via AMPK; AMPKα is phosphorylated (activated) and hence phosphorylates (inactivates) acetyl-CoA carboxylase (ACC) [939]. In addition, CTRP1 activates PKB, ERK1, and ERK2 in differentiated mouse myotubes. As does adiponectin with its insulin-sensitizing effect, which also promotes NO synthesis and counters endothelin activity in vascular endothelia, CTRP1 induces glucose uptake and hence reduces glycemia and CTRP3, CTRP5, and CTRP9 (but not the other group members) support NOS3 activity via the AdipoR1–AMPK– PI3K–PKB–NOS3 axis [810].167 However, whereas adiponectin concentration declines in obese individuals, CTRP1 concentration rises in diabetic patients. Cq1–TNF-related protein 1 reduces damage after myocardial infarction in mice and platelet aggregation in primates [810]. However, CTRP1 concentration elevates in stable coronary atherosclerosis; it correlates positively with blood pressure and negatively with HDL concentration; it is not related to the BMI; it is also linked only to a low level of arteriogenesis. Endotheliocytes produce CTRP1 and respond to CTRP1, thereby activating the P38MAPK–NFκB pathway and increasing synthesis of adhesion molecules (selE, icam1, and vcam1) in ECs in addition to inflammatory cytokines and chemokines [941].

167 In

addition, CTRP3 and CTRP9 inhibit the proinflammatory LPS–TLR4 pathway [941].

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CTRP2 (C1qTNF2) The secreted protein CTRP2 enhances glycogen deposition and lipid oxidation in cultured myotubes. Fasting and high-fat feeding upregulate transcription of the Ctrp2 gene in WAT [942]. Cq1–TNF-related protein 2 activates AMPK in a dose-dependent manner. Both the full-length and truncated globular form enhance fatty acid oxidation.

CTRP3 (C1qTNF3) The adipokine CTRP3168 is synthesized in adipocytes, adipose stromal cells, and other cell types. In humans, circulating CTRP3 concentration correlates positively with adiponectin and negatively with waist circumference, blood pressure, and concentrations of fasting glucose, TGs, and cholesterol [939]. It lowers glycemia, as it suppresses the expression of gluconeogenic enzymes and hence hepatic glucose output. It also hampers hepatic TG synthesis, as it inhibits the formation of GPATs, AGPATs, and DGATs [943].169 In hepatocytes, it suppresses production of glucose 6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) involved in gluconeogenesis [940]. It also represses gluconeogenesis via activated PKB [939]. It protects against NAFLD and hepatic steatosis (or NASH) in mouse DIO models.170 Hepatic steatosis is caused by augmented FFA uptake and lipogenesis, especially hepatic TG synthesis, in addition to decreased β-oxidation171 and export of TGs and fatty acids [945]. Lipogenesis is regulated by nutritional and hormonal cues.172 Transcription of genes encoding lipogenic enzymes is primed by transcrip168 A.k.a.

cartducin and cartonectin. is synthesized and stored in AT and liver. Enzymes that catalyze steps in TG synthesis from glycerol 3-phosphate encompass members of the GPAT (glycerol phosphate acyltransferase [GPAT1–GPAT4]; mitochondrial glycerol 3-phosphate acyltransferase GPAM is also known as GPAT1 and GPAT3 as AGPAT8 and AGPAT9), AGPAT (acylglycerolphosphate acyltransferase [AGPAT1–AGPAT9]; AGPAT7 and AGPAT8 corresponding to LPC acyltransferase LPCAT4 and lysocardiolipin acyltransferase [LyCAT], respectively), lipin ([Lpin1–Lpin3]; LPIN) or phosphatidate phosphatase (PAP), and diacylglycerol acyltransferase (DGAT; [DGAT1– DGAT2]) families [944]. In the liver, TGs are synthesized via the glycerol phosphate pathway (glycerol 3-phosphate–LPA–PA–DAG–TG). Glycerol 3-phosphate, lysophosphatidic acid, phosphatidic acid, and diacylglycerol are sequentially processed. Their acylation is achieved by multiple isoforms of GPATs, AGPATs, and DGATs. 170 Hepatic steatosis, i.e., lipid accumulation in the liver, often develops in the metabolic syndrome. Steatohepatitis results from alcoholic liver disease and NAFLD. The latter can lead to NASH, cirrhosis, and hepatic carcinoma. Hepatosteatosis is linked to redox stress, inflammation of the liver and AT, and hepatocyte death. 171 The nuclear receptor NR1c1 (PPARα) and PGC1α regulate transcription of the gene encoding carnitine palmitoyltransferase, CPT1α, which is responsible for fatty acid oxidation. 172 The genes that encode lipogenic enzymes, such as FAS, are activated by insulin. The transcription factor USF1 stimulates FAS formation via the mediator subunit Med17 [945]. 169 Triacylglycerol

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tion factors, such as bHLHb11173 and members of the SREBP family.174 Production of the antiproliferative transcription cofactor B-cell translocation gene product BTG1 declines in obesity [946].175 In hepatocytes stimulated with insulin, the mediator complex subunit Med17, a transcriptional coactivator, is phosphorylated by CsnK2, enabling bHLHb11 to activate lipogenic enzyme-encoding genes [945].176 Hepatic synthesis of lipids is also precluded by the PAP lipin-1,177 which suppresses the activity of transcription factors of the SREBP family, thereby reducing production of TGs.178 Glutathione synthesis and NAD+ metabolism are altered in NAFLD; plasmatic and hepatic concentrations of glycine, betaine, and serine decline [948]. Supplementation of precursors of NAD+ and GSH significantly attenuate hepatosteatosis.

Mediator of RNA polymerase-2 transcription recruits RNA Pol2 and other elements of the general transcription machinery. 173 Also known as upstream stimulatory factor, USF1. This ubiquitous transcription factor belongs to the MYC family of DNA-binding proteins, which connects to DNA as a dimer (mainly as USF1– USF2a (bHLHb11–bHLHb12a) heterodimers but also as USF1–USF2b heterodimers in addition to USF1 and USF2a homodimers [194]. Food intake stimulates the release of insulin, which triggers anabolism, in particular, lipogenesis in the liver. In hepatocytes, insulin triggers phosphorylation of the transcriptional coactivator Med17, which activates USF1 for lipogenic factor synthesis. 174 SREBP (bHLHd1–bHLHd2). 175 The ubiquitous BTG1 protein was initially identified as a translocation gene in B-cell chronic lymphocytic leukemia. It is involved in cell proliferation, differentiation, and survival [946]. At least in adult mice, it is required to generate new neurons. It is a cofactor for various transcription factors. In macrophages, it represses the activity of NFκB. Hepatic overexpression of BTG1 reduces hepatic steatosis in db/db mice, a model of obesity. The BTG1 factor is inhibited by the ATF4 factor, which is implicated in glucose metabolism, autophagy, apoptosis, ER stress, and inflammation, especially in the liver and WAT. Conversely, overexpression of ATF4 precludes BTG1 action. It protects against diet-induced hepatic steatosis, as it regulates hepatic lipid metabolism and prevents activity of ATF4 and SCD1, the rate-limiting enzyme, in the synthesis of monounsaturated fatty acids. This FoxO3a target suppresses transcription of the gene encoding stearoyl-CoA desaturase SCD1, which is involved in fatty acid synthesis [946]. A high-carbohydrate diet favors BTG1 production via the TOR–S6K1–CREB pathway. 176 This phosphorylation (at Ser53) occurs in the liver of fed mice and insulin-stimulated hepatocytes, only if Med17 is not previously phosphorylated by P38MAPK, which is activated during fasting [945]. 177 Lipin-1 is also a transcriptional coregulator that enhances fatty acid oxidation via PPARα and PGC1α [947]. This PAP catalyzes the synthesis of diacylglycerol. 178 On the other hand, upon phosphorylation of lipin-1 by TORC1 and casein kinase CsnK1, the RING-type ubiquitin ligase complex SCFβTRCP (β TRCP: β-transducin repeat-containing protein) polyubiquitinates lipin-1 for degradation [947]. This ligase also operates in the cell cycle, apoptosis, and metabolism. The SCF complex comprises four core subunits: the RING box protein RBx1, scaffold cullin-1, adaptor S-phase kinase-associated protein Skp1, and substrate receptor Fbox protein. F-box proteins were first identified as components of SCF ubiquitin ligase complexes, in which they bind substrates for ubiquitin-mediated proteolysis. In humans, numerous F-box proteins exist that also function in other types of proteic complexes. The F-box protein β TRCP has two distinct paralogs: β TRCP1 (or F-box/WD repeat-containing protein FBxW1) and β TRCP2 (or FBxW11).

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In addition, CTRP3 stimulates chondrogenic precursor cell proliferation [940]. In monocytes, adipocytes, and colonic fibroblasts, it can reduce the secretion of proinflammatory cytokines IL6 and TNFSF1 in response to lipopolysaccharide stimulation. In the vasculature, CTRP3 promotes angiogenesis and thus increases capillary density [940]. It promotes VEGFa formation via the PKB–HIF1α pathway. In vitro, it induces EC proliferation and migration, as it activates ERK1, ERK2, and P38MAPK [939]. In the heart, CTRP3 activates PKB (but not AMPK), attenuating CMC apoptosis in ischemic cardiac regions in addition to increasing revascularization and reducing fibrosis in mice after myocardial infarction [805]. In vitro, CTRP3 inhibits TGFβinduced profibrotic gene expression in cardiofibroblasts.

CTRP4 (C1qTNF4) The CTRP4 protein is synthesized and secreted in the CNS by neurons, but not astrocytes, in addition to other organs [949]. It oligomerizes and its circulating concentration increases in leptin-deficient obese mice. Refeeding after overnight fasting triggers CTRP4 formation in the hypothalamus. Suppression of food intake by CTRP4 correlates with a decreased transcription of the genes encoding orexigenic neuropeptides NPy and AgRP in the hypothalamus.

CTRP5 (C1qTNF5) The protein CTRP5 is widespread, with highest levels detected in the eye and AT [939]. Saturated fatty acids upregulate CTRP5 expression in adipocytes, where it operates in an autocrine fashion to reduce adiponectin and resistin secretion. In muscles, this autocrine regulator acts in response to reduced mitochondrial content. It stimulates AMPK, hence inducing GluT4 translocation. It enhances lipid oxidation using via the AMPK–ACC axis.

CTRP6 (C1qTNF6) The adipose-tissue factor CTRP6 is involved in adipogenesis via the marker lipogenic genes and ERK1/2 pathway [950]. The complement system is implicated in the host defense against infection and in inflammatory diseases (Vol. 12, Chap. 2. Chronic Inflammation). CTRP6 suppresses the alternative pathway of the complement system, as it competes with complement factor-B [951].

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CTRP7 (C1qTNF7) Like CTRP9, CTRP7 expression is induced in the human failing heart. Whereas CTRP9 is produced in adult human CMCs, CTRP7 mainly originates from cardiofibroblasts. Unlike CTRP9 in adult rat CMCs, CTRP7 does not have an anti-oxidative action via AMPK, adiponectin receptors, and calreticulin.

CTRP8 (C1qTNF8) The CTRP8 messenger is expressed predominantly in the lung and testis. It homotrimerizes and heteromerizes with the complement component-1-like agent C1qL1,179 a secreted multimeric protein that also heteromerizes with CTRP1, CTRP9, and CTRP10 [952]. It connects to leucine-rich G-protein-coupled relaxin and insulin-like family peptide receptor RXFP1, at least in human glioblastoma cells. The CTRP8–RXFP1 complex launches cell migration via PI3K, PKC, and increased production and secretion of the lysosomal peptidase cathepsin-B [953].

CTRP9 (C1qTNF9) Among all CTRP paralogs, CTRP9, a secreted glycoprotein that experiences multiple post-translational modifications, has the highest degree of amino acid identity to adiponectin. It forms predominantly trimers; adiponectin and CTRP9 form heterotrimers [935]. In humans, CTRRP9a and CTRP9b are encoded by distinct genes. CTRP9a is secreted as a multimer. CTRP9b interacts with CTRP9a or adiponectin to be released [952]. The action of CTRP9 is initiated from both AdipoR1 and AdipoR2 in addition to calreticulin. CTRP9 activates AMPK, PKB, ERK1, and ERK2. In the skeletal muscle, activated AMPK increases mitochondrial content and upregulates the transcription of genes that enable lipid oxidation. CTRP9 also improves insulin function and affects glucose metabolism and insulin activity. The CTRP9 protein also controls the functioning of cardiac and endothelial cells. It increases NO production using the AMPK–NOS3 pathway, thereby priming vasodilation [939]. In a model of femoral artery injury, CTRP9 overexpression reduces intimal hyperplasia, as it represses vSMC proliferation and migration using the cAMP–PKA–ERK pathway. C1q–TNF-related protein 9 activates synthesis TRdx1 and SOD2 via AMPK and SIRT3, hence attenuating cell death in response to H2 O2 . It attenuates apoptosis and fibrosis via the AdipoR1–AMPK axis in addition to redox stress in diabetic mice after ischemia–reperfusion injury [805].

179 A.k.a.

C1q-related factor (CRF) and C1qTNF14.

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On the other hand, in response to hypertension, myocardial capillary ECs can favor adverse hypertrophy of CMCs. CTRP9 secreted by ECs primes the activation of the pro-hypertrophic kinase ERK5, which phosphorylates (activates) the prohypertrophic transcription factor GATA4 [954]. Formation of the pro-survival cardiokine, CTRP9, is markedly downregulated after myocardial infarction. It promotes implanted stem cell survival and ensures cardioprotection [955]. In vitro, CTRP9 supports proliferation and migration of adipose tissue-derived mesenchymal stem cells (ADMSCs) and protects them against death induced by hydrogen peroxide. C1q–TNF-related protein 9 promotes superoxide dismutase SOD3 synthesis and secretion from ADMSCs, protecting CMCs against redox stress. At physiological doses, C1q–TNF-related protein 9 prolongs transplanted ADMSC retention and survival. In mice subjected to myocardial infarction, implantation of ADMSCs within the myocardium does not exert significant cardioprotection. On the other hand, once their transplantation is combined with CTRP9, cardioprotection yielded by CTRP9 alone is potentiated. C1q–TNF-related protein 9 promotes ADMSC proliferation and migration via the ERK1/2–MMP9 axis and cell survival via the ERK1/2–NFE2L2 axis and antioxidant production. N-Cadherin is a CTRP9 receptor that mediates ADMSC signaling [955].

CTRP10 (C1qTNF10–C1qL2) C1q–TNF-related protein 10 corresponds to complement component C1q subcomponent-like protein C1qL2. Whereas C1QTNF1 to C1QTNF3, C1QTNF5, and C1QTNF7 transcripts are predominantly formed in the AT, the eye and placenta produce the highest levels of C1QTNF10 and C1QTNF6 transcripts, respectively [956]. CTRP10 trimers further assemble into oligomers via disulfide bond in their N-terminal cysteine residues.

CTRP11 (C1qTNF11) The CTRP11 protein is predominantly formed in WAT and BAT and in the former primarily in stromal vascular cells [939]. Overnight fasting and refeeding upregulates CTRP11 production. It can impede adipogenesis in a paracrine manner between adipocytes and stromal vascular cells, as it precludes expression of the two major transcriptional regulators of adipogenesis NR1c3 and C/EBPα.

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CTRP12 (C1qTNF12) Expression of the antidiabetic adipokine CTRP12180 is downregulated in obesity [957]. It is synthesized more in subcutaneous than in visceral adipose depots and more in adipocytes than in the stromal vascular cells. In humans, AT predominantly produces CTRP12, whereas its expression is more widespread in mice [939]. Krüppel-like factors, KLF3 and KLF15, down- and upregulate CTRP12 production, respectively. In the scAT, insulin elicits CTRP12 synthesis and secretion [958]. Conversely, CTRP12, which acts in an insulin-independent manner, improves insulin signaling in the liver and AT and hence insulin sensitivity [957]. It activates the PI3K–PKB pathway to suppress gluconeogenesis and promote glucose uptake. The CTRP12 adipokine has at least two isoforms, a full-length and a cleaved globular isoform, which build distinct oligomers and have different functions. Furin (or PCSK3) cleaves CTRP12 (Lys91) [939]. The full-length isoform forms trimers and larger complexes [957]. The cleaved globular isoform mainly dimerizes. Whereas CTRP12L activates PKB in hepatocytes and adipocytes, CTRP12S stimulates ERK1, ERK2, and P38MAPK. Only full-length CTRP12 support insulin-induced glucose uptake in adipocytes. In addition, CTRP12 suppresses gluconeogenesis in cultured hepatocytes. A modest increase in circulating CTRP12 concentration suffices to lower glycemia and improve insulin sensitivity in mice, as CTRP12 enhances insulin signaling in the liver and AT, but not in skeletal muscle [939].

CTRP13 (C1qTNF13) In humans, CTRP13 is preferentially expressed in AT and, in mice, in the brain and AT (mainly by stromal vascular cells) [939]. In cultured adipocytes, hepatocytes, and myotubes, CTRP13 activates AMPK and hence promotes glucose uptake. In vitro, it activates AMPK and inhibits G6Pase and PEPCK, thereby lowering gluconeogenesis in hepatocytes. It also partly reverses lipid-induced insulin resistance in hepatocytes, as it suppresses the JNK signal. In the brain, CTRP13 serves as an anorexigenic factor. A reciprocal regulation exists in the hypothalamus between CTRP13 and the orexigenic neuropeptide agouti-related protein (AgRP) homolog, CTRP13 repressing AGRP gene transcription and AgRP upregulating Ctrp13 gene transcription (food intake modulatory

180 A.k.a.

adipolin.

5.4 Adipose Tissue

459

hypothalamic feedback loop) [939]. Food restriction in mice lowers the CTRP13 level and raises those of orexigenic neuropeptides NPy and AgRP in the hypothalamus. However, when food restriction is coupled with physical activity, hypothalamic expression of both CTRP13 and AgRP augments [939]. This neural circuit may be disrupted in anorexia.

CTRP14 (C1qTNF14–C1qL1) Whereas CTRP10 corresponds to C1qL2, the C1QTNF14 gene product is the complement component C1q subcomponent-like protein C1qL1, which is also termed C1q-related factor (C1qRF or simply CRF), the transcripts of which are most abundant in regions of the nervous system involved in motor function (e.g., cerebellar Purkinje cells in addition to the accessory olivary nucleus, pons, and red nucleus [959].

CTRP15 (C1qTNF15) The CTRP15 protein181 is predominantly produced in skeletal muscles, more in oxidative slow-twitch fibers than in glycolytic fast-twitch fibers [939]. Its circulating concentration rises upon refeeding after fasting. It may serve as a postprandial hormone produced and secreted by skeletal muscles in response to nutrient flux. It diminishes FFA-emia, as it upregulates the expression of ScaRb3, FABPs, and FATPs, thereby promoting FFA uptake in hepatocytes.

5.4.5.3

Adipsin (Complement Factor-D)

Type-2 diabetes mellitus linked to a pancreatic β-cell failure, which engenders insulinopenia and hyperglycemia, is associated with adipsin deficiency. Its production declines in many animal models of obesity and diabetes. The adipokine adipsin, or complement factor-D, secreted mainly or exclusively by the AT, maintains β-cell functioning [960]. It catalyzes the rate-limiting step of the alternative pathway of complement activation and hence supports the formation of the C5–C9 membrane attack complex. It also elicits the generation of numerous signaling molecules, such as the anaphylatoxins C3a and C5a [960]. The CFd–C3b complex cleaves complement factor-B and catalyzes the formation of the C3 convertase (C3bBb), which can act on C3 to liberate C3a. The C3a peptide generated by adipsin is a potent insulin secretagogue via the C3a receptor C3aR1 only when coupled to hyperglycemia [960]. C3a augments concentrations

181 A.k.a.

myonectin.

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of the messenger and energy provider ATP as well as the signaling mediator Ca2+ , thereby supporting mitochondrial ETC. The receptor C3aR1 primes Ca2+ influx and activates ERK, Rho, and NFκB.

5.4.5.4

Apelin

The ubiquitous peptide apelin and its Gi/Gq-protein-coupled receptor APJ are formed in the CNS, particularly in the hypothalamus. It is considered an adipokine because it is synthesized and secreted by adipocytes. The PI3K–PKB pathway is involved in apelin synthesis and secretion in adipocytes. Insulin stimulates apelin production and activity via the PI3K–PKB axis and MAPK module [961]. Proinflammatory cytokines launch apelin production via PI3K and JNK in adipocytes. The apelin gene encodes a 77-amino acid preproprotein and apelin propeptide. Several active apelin forms exist (e.g., apelin36 [apelin(42–77) ], apelin19 [apelin(59–77) ], apelin17 [apelin(61–77) ], and apelin13 [apelin(65–77) ]), and the pyroglutamated form of apelin13 (pGlu apelin13 ), which is protected from peptidase degradation [962]. Apelin is involved in the regulation of food intake and energy metabolism in addition to fluid homeostasis, cell proliferation, and angiogenesis. Apelin receptor, which is encoded by the APLNR gene,182 predominantly localizes on ECs in organs with a high metabolism rate, such as the skeletal muscle, myocardium, and AT. It inactivates FoxO1 and hence FABP4, thereby avoiding excessive fatty acid accumulation and improving fatty acid uptake, glucose utilization, and insulin sensitivity [963]. It is a potent vasodilator via NO. It also represses vasoconstriction mediated by angiotensin-2 [961]. Conversely, ATn2 downregulates the formation of apelin and APJ, as it hampers ERK1/2 and P38MAPK signaling.183 Apelin decreases glycemia mainly because of increased glucose uptake in the myocardium, skeletal muscles, and AT via AMPK and NOS3 (Table 5.28) [962]. In addition, apelin increases the insulin-stimulated glucose transport in insulinresistant adipocytes. Moreover, ingested glucose rapidly induces the paracrine secretion of apelin in the mouse gut; the amount of glucose transporter SLC5a1184 declines in enterocytes, whereas that of GluT2 rises upon AMPK activation, thereby priming glucose absorption. A transient increase in glycemia in the portal vein may rapidly induce insulin secretion. Apelin may improve insulin sensitivity. Apelin also launches the secretion of glucagon-like peptide, GLP1 [962]. In adipocytes, apelin inhibits lipolysis induced by β-adrenoceptor (but not basal lipolysis) via the Gq/Gi–AMPK pathway [962]. Apelin injection for 2 weeks decreases the size of fat depots in SD- and HFD-fed mice. Chronic apelin 182 A.k.a.

angiotensin receptor like-protein AgtRL1. APJ receptor resides on ECs, vSMCs, and CMCs, among other cell types. 184 The solute carrier family member SLC5a1 is also named sodium–glucose cotransporter SGLT1. 183 The

5.4 Adipose Tissue Table 5.28 Apelin signaling and effect (Source: [962])

461 Pathway AMPK

AMPK–NOS3 PDE3b PDE3b–PI3K–PKB

Effects Intestinal glucose absorption Lipolysis, fatty acid oxidation Mitochondrial genesis Glucose uptake Insulin secretion Glucose uptake

It plays a beneficial role in energy metabolism and insulin sensitivity

administration in obese and insulin-resistant mice increases fatty acid oxidation in muscles via AMPK in addition to mitochondrial genesis in skeletal and cardiac myocytes via PGC1α [962]. Apelin may also prevent the development of obesity via the maintenance of proper vascular permeability. Apelinemia rises in obesity and T2DM, but does not correlate with BMI, compensating for defective insulin signaling [964]. Apelin is implicated in obesityrelated hypertension owing to a crosstalk between ATn2–AT1 and apelin–APJ signaling via ERK1, ERK2, and P38MAPK [961]. In a rat model of obesityrelated hypertension, apelinemia and transcription of apelin and APJ in perirenal AT decrease because of ATn2 inhibition.

5.4.5.5

Asprosin

Asprosin185 is a fasting-induced glucogenic protein secreted by WAT, which circulates in the bloodstream at nanomolar levels. During fasting, it binds to the surface of hepatocytes, activates PKA, and elicits a rapid release of glucose from the liver, especially for cerebral functioning. Glucose may serve as a suppressor of asprosin release via a negative feedback loop. Asprosin also primes hepatic glucose production using the second messenger cAMP [965]. It is the C-terminal cleavage product of profibrillin encoded by the FBN1 gene.186 Humans with insulin resistance have elevated asprosinemia, the FBN1 mRNA sources including WAT, BAT, and the skeletal muscles. Inappropriately elevated glucose production by the insulin-resistant liver is a major factor of the metabolic syndrome.

185 ασπρoν:

white. is a structural component of microfibrils of the load-bearing extracellular matrix (diameter 10–12 nm), which provides structural support against sustained mechanical forces in organs, such as the lung and arteries, where these microfibrils form the periphery of elastic fibers. These microfibrils also form elastin-independent networks in tendon and renal glomerulus, among others, in which they yield tensile strength and play an anchoring role. Fibrillin-1 also interacts with growth factors, such as BMPs, GDFs, and latent TGF-β-binding proteins, in addition to integrins and proteoglycans and other proteic constituents of the extracellular matrix [108].

186 Fibrillin-1

462

5.4.5.6

5 Hyperlipidemias and Obesity

Chemerin

Chemerin is involved in immunity as a chemokine that targets macrophages and dendrocytes. This adipokine is also implicated in lipid and glucose metabolism in the liver and skeletal muscles, yielding a link between chronic inflammation and obesity [966]. Hyperchemerinemia is indeed associated with obesity, insulin resistance, and systemic inflammation in obese adults and children [967].187 In children, chemerinemia is associated with the amount of soluble adhesion molecules icam1 and E-selectin (but not soluble vcam1 and P-selectin) in the circulation. In human subjects, levels of TGs, total cholesterol, CRP protein, and leptin, in addition to BMI and waist circumference, correlate positively with chemerin, whereas systolic and diastolic blood pressure, LDLCS , and HDLCS do not correlate significantly and adiponectin correlates negatively [968]. Chemerin is synthesized at high levels in the WAT and liver. It is secreted as inactive prochemerin. It is cleaved (activated) by serine peptidases of the inflammatory and coagulation cascade. Chemerin acts via its cognate chemokine-like receptor CmkLR1 and regulates adipogenesis via NR1c3 in addition to inflammation and glucose metabolism [969]. ECs possess CmkRL1 that activate icam1 and E-selectin expression. The auto- and paracrine messenger chemerin operates in adipocyte differentiation. In adipocytes, it also stimulates lipolysis and modulates the expression of genes involved in glucose and lipid metabolism (glucose transporter GluT4, adiponectin, and leptin) [968].

5.4.5.7

Leptin

Leptin188 is almost exclusively secreted by white and brown adipocytes. Leptin formation is upregulated by glucocorticoids and proinflammatory cytokines and downregulated by cold exposure, adrenoceptor activation, stimulation by growth and thyroid hormone, and melatonin, in addition to smoking [887]. Obesity is associated with elevated leptinemia and hypothalamic leptin resistance. This endocrine agent participates in the regulation of energetic balance, as it suppresses appetite and promotes energy expenditure. Its secretion increases or decreases under conditions of a positive or negative energy balance, respectively. Leptin serves as an adipostat that informs on the status of energy storage in the AT

187 Chemerinemia

correlates negatively with age, decreasing from the pre-pubertal, pubertal, to post-pubertal period in healthy lean children (only in boys, not in girls) [967]. In aged adults, it correlates positively with age. 188 λ πτoτης: thinness, leanness; λ πτυνω: make thin (λ πτoν: mite, tiny coin; λ πτoς: peeled [thin covering removed]). Blood leptin concentration rises with feeding and declines with fasting. In rodents, its absence is a strong feeding inducer, but its presence is not a potent feeding inhibitor. Excessive feeding upregulates leptin synthesis, whereas fasting downregulates it. Various stimuli (e.g., cytokines, glucocorticoids, and insulin) acutely adjust leptin production.

5.4 Adipose Tissue

463

to adapt appetite and metabolism via the leptin receptor [970]. In humans, leptin concentration increases with AT mass and decreases during weight loss. This hormone targets the brain to establish a negative feedback loop for weight control. However, obese humans exhibit hyperleptinemia without hypophagia (leptin resistance). Leptin impairs several metabolic actions of insulin (i.e., stimulation of glucose transport, glycogen synthase and PKA, lipogenesis, and protein synthesis, in addition to inhibition of lipolysis) [971]. Conversely, insulin potentiates leptin-induced NO release. Whereas leptin impedes lipogenesis, angiotensin and acylationstimulating protein stimulate it, glucose being a substrate for lipogenesis [887]. Leptin exerts a paracrine effect on adipocytes. Its synthesis and secretion by adipocytes is induced by IL6 and inhibited by TNFSF1 [908]. Leptin stimulates proliferation and migration of ECs and vSMCs. In ECs, leptin increases production of NO and ROS in addition to CCL2 [908]. It stimulates angiogenesis. Leptin also increases the peripheral sympathetic tone. Therefore, leptin regulates blood pressure by two opposing mechanisms [866, 892]. On the one hand, vasodilation via NO release and stimulation of EDHF, which involves neither KATP nor BK channels, and attenuation of angiotensin-2 action; on the other, leptin provokes vasoconstriction via central excitation of the sympathetic nervous system. Leptin represses intimal hyperplasia. It targets its endothelial receptor, which activates STAT3 and prevents synthesis via PPARγ (NR1c3) and release of endothelin1 and subsequent vSMC proliferation [972]. On the other hand, obesity is linked to endothelial leptin resistance. In vSMCs, leptin elicits MMP2 formation [805]. Leptin favors platelet aggregation and arterial thrombosis via its receptor [908]. Leptin is a proinflammatory adipokine for monocytes, macrophages, neutrophils, NK cells, and T lymphocytes [805]. On macrophages, leptin elicits release of monocyte colony-stimulating factor (CSF1) [908]. Leptin facilitates cholesterol accumulation in macrophages, especially in hyperglycemia. Hyperleptinemia in obese individuals supports atherosclerosis, but protects against ischemia-induced cardiac damage [805].

5.4.5.8

Lipocalin

Lipocalin-2189 is highly expressed in adipocytes [973]. It is widespread and exists as monomers and homo- and heterodimers with gelatinase [831]. It can undergo N-linked glycosylation (Asn65). Lipocalin-2 belongs to the lipocalin superfamily, which comprises RBP4, FABP4, ApoD, and prostaglandin-D synthase (PGdS). Lipocalins generally bind small hydrophobic ligands, but can also tether to soluble extracellular

189 A.k.a.

growth factor-stimulated superinducible protein-24, neutrophil gelatinase-associated lipocalin, and siderocalin.

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5 Hyperlipidemias and Obesity

macromolecules and specific plasmalemmal receptors. Lipocalin-2 binds to the receptors LRP2 (megalin) for its cellular uptake and brain-type organic cation transporter SLC22a17 [831]. It is implicated in cell survival and apoptosis. The formation of lipocalin-2 is induced by TNFSF1, an insulin resistanceinducing factor, in cultured adipocytes. Its production elevates upon exposure to insulin resistance-mediating agents in obese subjects via CCAAT/enhancer-binding protein. The proinflammatory transcription factor NFκB upregulates lipocalin-2 synthesis. In mice, WAT is a dominant site of Lcn2 formation, especially in obesity. Ligands of lipocalins include retinoids, arachidonic acid, leukotrieneB4 , platelet-activating factor, steroids, odorants, pheromones, and, bacterial siderophores [973].190 The 25-kDa secretory glycoprotein lipocalin-2 can bind weakly to some common ligands of lipocalins (e.g., leukotriene-B4 and plateletactivating factor) [831]. Lipocalin-2 deficiency attenuates the metabolism of arachidonic acid, the concentration of which elevates with aging and obesity, at least in mice [831]. Inflammatory lipid species processed by arachidonate lipoxygenase are significantly reduced in WAT of LCN2−/− mice. Lipocalin-2 elicits the production of inflammatory lipid species and adipokines in the AT and magnifies systemic insulin resistance [831]. However, its depletion impairs adaptive thermogenesis and hence cold tolerance, uncoupling protein UCP1 level decreasing in the BAT in LCN2−/− mice. Lipocalin-2 causes aging- and obesity-associated insulin resistance, hyperglycemia, and hyperinsulinemia, as it upregulates formation and activity of arachidonate 12-lipoxygenase and stimulates TNFSF1 production in the AT [974].

5.4.5.9

Omentin

Omentin-1, or intelectin-1, is abundantly produced in human vAT. Omentinemia decreases in obese subjects and in patients with T2DM [805]. Omentin has two isoforms, omentin-1 being the major isoform in human blood [892]. Omentin-1 ensures protection of the cardiovascular apparatus. It causes vasodilation via NO [892] and suppresses TNFSF1-induced inflammation of vascular endothelia via the AMPK–NOS3 pathway [805].

190 Lcn2 is used by cells of the innate immunity to sequester siderophores and thus deprive bacteria

of iron.

5.4 Adipose Tissue

5.4.5.10

465

Resistin

In rodents, resistin191 is mainly synthesized in adipocytes, but, in humans, the main sources are monocytes and macrophages. Resistin production in monocytes and macrophages rises in response to proinflammatory stimuli such as IL6 [805]. Adipocytes also produce resistin-like molecule RetnLα [888]. Release of resistin is stimulated by hyperglycemia and growth and gonadal hormones in addition to inflammation [887]. Circulating concentration increases in diet-induced and genetic forms of obesity in rodents [908]. Resistinemia also rises in obese humans. Adipose tissue-specific resistin affects glucose metabolism and insulin sensitivity. It causes insulin resistance in the liver and skeletal muscle [908]. Neutralization of resistin by specific antibodies decreases glycemia and improves insulin sensitivity. Resistin impairs endothelial function and thus reduces expression of NOS3 and hence NO concentration [892].

Resistin and Inflammation Resistin has a proinflammatory effect on smooth myocytes. It induces human aortic smooth myocyte proliferation [908]. Resistin elicits ET1 release and activates ECs. It upregulates production and secretion by ECs of chemokines (e.g., CCL2) and adhesion molecules (e.g., icam1 and vcam1). It thus promotes monocyte–EC interaction in addition to activation of macrophages [805]. Furthermore, it primes synthesis in ECs of pentraxin-3, an inflammatory mediator that contributes to the regulation of innate immunity and is involved in atherosclerosis. Resistin induces C/EBPβ- and NFκB-dependent production of cytokines (e.g., TNFSF1, IL1α, and IL1β) and chemokines (e.g., CCL2–CCL3, CCL3L1, CCL4–CCL5, CCL8, and CXCL1–CXCL3), at least in human articular chondrocytes [975].

191 Resistin:

resistance to insulin. It suppresses the ability of insulin to stimulate glucose uptake into adipocytes. It is encoded by the RETN gene, which is transcribed during adipocyte differentiation. It homodimerizes or multimerizes, circulating in blood in low- and high-molecularweight isoforms (7–22 ng/ml) [975]. Patients with acute coronary syndrome have significantly higher resistinemia (1.18 ± 48 μ g/l) than patients with stable angina pectoris (0.66 ± 0.40 μ g/l). It is also termed C/EBP -regulated myeloid-specific secreted cysteine-rich protein XCP1, CysH-rich secreted protein-A12α-like protein-2, protein found in inflammatory zone 3 (FIZZ3), and adipocyte-secreted factor (AdSF). It belongs to the RELM family of resistin-like molecules that contains ReLMα and ReLMβ secreted by the AT and the gastrointestinal tract, respectively.

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Resistin competes with lipopolysaccharide for binding to TLR4 in human myeloid and epithelial cells and launches the NFκB and MAPK pathways, thereby initiating the expression of TNFSF1, IL1β, and IL6 [975]. Chronic inflammatory atherosclerosis is primed by migration of circulating monocytes into the subendothelial space, where they differentiate into macrophages. Macrophages take up modified lipoproteins (VLDL, IDL, and LDL) and transform into foam cells. Macrophage class-A (ScaRa1–ScaRa3) and -B scavenger receptors (ScaRb1–ScaRb3) internalize oxLDLs. Macrophages infiltrating atherosclerotic lesions secrete resistin, which affects EC function and vSMC migration, especially during plaque rupture [975]. Resistin causes lipid accumulation, as it promotes oxLDL uptake via ScaRa and ScaRb3 by macrophages (and hence foam cell formation) and lowers concentration of ABCa1 used for cholesterol efflux. In addition, resistin affects fatty acid metabolism, as it raises cholesterol esterification and the availability of non-esterified (free) fatty acids in macrophages.

Resistin and Endothelial and Smooth Myocytic Function Resistin is involved in angiogenesis, possibly via TNFSF12, along with endothelial and smooth myocytic dysfunction, thrombosis, and inflammation [975]. Resistin activates ECs and triggers endothelin-1 synthesis and release, in addition to adhesion molecules and chemokines [908]. On the other hand, it downregulates the formation of TRAF3, an inhibitor of TNFSF5-mediated EC activation [908, 975]. At high concentrations, resistin from eAT of ACS patients increases endothelial permeability [975]. In addition, at clinically relevant concentrations, resistin significantly lowers NOS3 synthesis. On the other hand, it raises ROS production. It operates via the P38MAPK and JNK kinases. Resistin can provoke vSMC proliferation via ERK1, ERK2, and PKB in addition to migration via integrin activation [975].

5.4.5.11

Retinol-Binding Protein-4

Retinol-binding protein RBP4 belongs to the lipocalin family of proteic carriers of small hydrophobic molecules. It transports retinol (vitamin A) in the blood circulation from the liver to other organs. It binds to transthyretin, a carrier of thyroid hormones. Its circulating concentration is influenced by iron and ferritin. The liver has the highest RBP4 level; AT has the second highest RBP4 production rate [976]. Its synthesis is controlled by many agents (Table 5.29). The adipokine and plasmatic retinol carrier RBP4 tethers to plasmalemmal receptors in addition to retinoic acid receptors NR1b1 to NR1b3 and NR2b1 to NR2b3. It is involved in glucose metabolism and insulin sensitivity. Circulating concentration of RBP4 correlates with insulin resistance and metabolic syndrome

5.4 Adipose Tissue Table 5.29 Regulation of RBP4 production in human adipocytes (Source: [976]; ↑ increase, ↓ decrease)

467 Regulator Estradiol Iron donor Iron buffer Leptin NR1c3 (PPARγ) TNFSF1 (TNFα)

RBP4 production and secretion ↑ ↑ ↓ ↑ ↑ ↓

and serves as an independent predictor of adverse cardiovascular events. It links obesity to the metabolic syndrome. Omental AT is an important RBP4 source in severely obese patients. Increased rbp4emia (plasmatic RBP4 concentration), which is linked to increased vAT mass (i.e., overweight and obese subjects) in addition to T2DM, provokes synthesis of the gluconeogenic enzyme PEPCK in the liver and impairs insulin signaling in the skeletal muscle [977]. However, transcription of the Rbp4 gene is downregulated in scAT of obese post-menopausal women and Rbp4-emia is similar in normal, overweight, and obese women [978]. A weight loss of 5% engenders only a small decline in adipose RBP4 production without marked change in Rbp4-emia. In the AT, synthesis of RBP4 and GluT4 are reciprocally related. In mice, they are inversely correlated. In adipocytes, GluT4 concentration declines in overweight individuals and more so in obese subjects; it correlates positively with that of RBP4 [978]. Formation of RBP4 thus appears to be distinctly regulated in mice and humans. In skeletal muscles, RBP4 decreases insulin sensitivity, at least partly via a decreased phosphorylation of IRS1 [978]. Concentration of RBP4 is elevated in atherosclerotic lesions, especially in regions enriched with macrophage-derived foam cells [979]. RBP4 favors macrophage-derived foam cell formation owing to upregulation of ScaRb3 synthesis via TLR4 and the Src–JNK–STAT1 axis and cholesterol uptake.

5.4.5.12

Vaspin (Serpin-A12)

Visceral AT-derived serpin (vaspin or serpin-A12) targets various peptidases such as kallikrein-7. It is predominantly secreted from vAT [980], and originates mainly in non-adipocytes. Vaspin is also expressed in the hypothalamus, pancreatic islets, stomach, and skin [981].

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5 Hyperlipidemias and Obesity

Vaspin synthesis depends on both age and gender [981]. Insulin sensitizers enhance vaspin synthesis. Glucose significantly increases vaspin formation in omental fat depots. Vaspinemia evolves with a daily profile, with a peak during the early morning fasting period and a significant postprandial drop 2 h after breakfast. Vaspin plays a local and endocrine role. In the liver, it binds to the HSPa5192 (GRP78)–DnaJc1 receptor complex.193 In ECs, it targets a plasmalemmal complex made up of HSPa5 and voltage-dependent anion channel and exerts protective and proliferative effects. It inhibits NFκB in this cell type. Vaspinemia correlates with the degree of obesity and insulin resistance. It can compensate for falling insulin sensitivity. It normalizes glycemia and modifies the transcription of genes involved in the genesis of insulin resistance, such as those encoding adiponectin, leptin, resistin, TNFSF1, and GluT4 [981]. This insulin sensitizer has an anti-inflammatory action. In humans, obesity, insulin resistance, and T2DM are associated with increased vaspin transcription in the AT. In obese subjects and T2DM patients, vaspin concentration is 0.36 to 0.52 ng/ml higher than in healthy controls [983]. Higher vaspinemia is observed after physical activity in untrained individuals [981]. In obese mice, vaspin administration reduces food intake and improves glucose tolerance and insulin sensitivity [980].

192 The

molecular chaperone 70-kDa heat shock protein HSPa5 is also termed 78-kDa glucoseregulated protein (GRP78), a typical marker of ER stress. The ER is sensitive to stressors that reduce its protein folding capacity and can cause accumulation and aggregation of unfolded proteins, in particular, redox stress. An inflammatory response induces ER stress and UPR to recover a proper ER function or activate apoptosis. Proinflammatory mediators, such as TNFSF1, IL1, and IL6, provoke ER stress via ROS production. HSPa5 triggers UPR. Transcription of the genes encoding the ER chaperones HSPa5 and protein disulfide isomerase PDIa3 (a.k.a. endoplasmic reticulum protein ERP57; ERP60, and ERP61, in addition to glucose-regulated protein GRP57 and GRP58, is upregulated. Several factors are required for optimal protein folding, such as ATP, Ca2+ , and an oxidizing environment to allow disulfide bond formation. The protein chaperone PDIa3 catalyzes isomerization, reduction, and oxidation of disulfides. It interacts with the lectin chaperones calreticulin and calnexin to modulate folding of newly synthesized glycoproteins. HSPa5 is involved in the translocation of newly synthesized polypeptides across the ER membrane and their subsequent folding, maturation, transport, or retrotranslocation. It binds transiently to newly synthesized proteins in the ER and with higher affinity to misfolded, underglycosylated, or unassembled proteins. HSPa5 also localizes to the plasma membrane with MHC class-I molecules. It is a receptor for the uptake of certain viruses, and is also the receptor of α2-macroglobulin, in addition to LRP1 [982]. 193 DnaJc1: DnaJ (Hsp40) homolog, subfamily-C, member-1, which is also named murine tumoral cell DnaJ-like protein MTJ1. DnaJ-like proteins collaborate with members of the HSP70 family in protein conformation and oligomerization. The transmembrane protein DnaJc1 is a co-chaperone for HSPa5 ATPase activity.

5.4 Adipose Tissue

5.4.5.13

469

Visfatin (NAmPT)

Visfatin194 is a nicotinamide phosphoribosyltransferase195 (NAmPT) and adipokine mainly produced in and secreted from vAT rather than scAT [901].196 It is also released from pvAT. In the rat thoracic aorta, visfatin expression in pvAT is about four-fold and two-fold higher than that in scAT and vAT, respectively [901]. Intracellular visfatin works as a NAmPT and is implicated in the differentiation and survival of vSMCs [984]. Extracellular visfatin may operate as a paracrine messenger. Visfatin provokes vasodilation via NO production. It also stimulates angiogenesis via VEGF and MMPs [892]. Visfatin, which acts as a NAmPT, synthesizing NMN from nicotinamide, stimulates vSMC proliferation via NMN-mediated activation of ERK1, ERK2, and P38MAPK [984]. Visfatin mimics insulin in cultured adipocytes, myocytes, and hepatocytes. In mice, it activates the insulin receptor and lowers glycemia [984]. Visfatin is implicated in obesity. Visfatinemia rises in overweight and obese individuals in addition to T2DM patients [985]. In Asian Indians, visfatinemia correlates with obesity, even after adjusting for age, sex, and diabetes [986]. Visfatinemia is associated with vAT, but not with subcutaneous fat depot. Among Iranian children and adolescents (aged 7–16 years), obese subjects have significantly higher insulinemia and leptinemia and lower adiponectinemia and visfatinemia [987].

5.4.5.14

Glucocorticoids

Adipocytes synthesize 11β-hydroxysteroid dehydrogenase 11β HSDH1, which converts inactive glucocorticoid precursors (cortisone) to active glucocorticoids (cortisol) [901]. Glucocorticoids hinder vSMC proliferation and migration and hence restenosis after angioplasty. Concentration of the HSD11B1 mRNA increases in eAT near the proximal tract of the right coronary artery in CoAD patients.

5.4.5.15

Sex Hormones

Initiation and progression of CVDs differ according to the gender owing to distinct protective and harmful effects of sex chromosomes and gonadal hormones, especially estrogens and androgens [988].

194 A.k.a.

pre-B-cell colony-enhancing factor PBEF (PBEF1). catalyzes the condensation of nicotinamide with 5-phosphoribosyl 1-pyrophosphate to synthesize nicotinamide mononucleotide (NMN), an intermediate in the synthesis of nicotinamide adenine dinucleotide. 196 The name visfatin refers to its major synthesis site, visceral fat. 195 It

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Adipose tissue produces sex hormones from precursors. Estrogens suppress or support vSMC proliferation according to the vSMC phenotype [901]. Androgens reduce intimal hyperplasia in rabbits and exert an antiproliferative, proapoptotic, and anti-migrative action on cultured vSMCs. However, testosterone concentration declines with aging (i.e., mano- or andropause [a drop of 1–2% per year]), hypogonadism being defined by a testosterone concentration lower than 8–11 nmol/l ( DCA = LCA > CA [1003]. Polyunsaturated fatty acids, such as arachidonic, linolenic, or docosahexaenoic acid, in addition to intermediates of bile acid synthesis, are also FXR ligands [1002]). In cholestasis or inborn metabolic disorders, bile acid intermediates, which exist in large amounts, can be FXR agonists. On the one hand, FXR can launch gene transcription as a monomer (e.g., the UGT2B4 gene) or a heterodimer with RXR (e.g., the Pltp gene). On the other, FXR can repress gene transcription indirectly via NR0b2 (e.g., the CYP7A1 gene), or directly as a monomer (e.g., the APOA1 gene) or heterodimer (e.g., the APOC3 gene) [1002]). PGC1α is a FXR cofactor. Four isoforms (FXRα1–FXRα4) do not play the same role in gene transactivation. The FXR regulates bile acid synthesis, transport, and detoxification (Table 5.34). Its target genes include NR0B2,217 Fabp6,218 ABCB11, Pltp219 and APOC2. Activated FXR reduces triglyceridemia, inhibits bile acid synthesis, and raises transport of bile acids from the intestinal lumen into the enterocytes and back to the liver. Activated FXR induces expression of NR0b2,220 which interacts with two other nuclear receptors that transactivate the CYP7A1 gene, NR2a1,221 and NR5a2.222 NR0b2 represses CYP7A1 gene transcription, as it causes dissociation of coactivators linked to NR2a1 and NR5a2 in addition to histone deacetylation of the promoter [1002]).

216 The

isoprenoid farnesol derives from the hydrolysis of farnesyl diphosphate. It participates in the control of HMGCR stability. However, it does not directly interact with FXR [1000]. 217 A.k.a. small heterodimer partner (SHP). 218 Fatty acid-binding protein-6 is involved in enterohepatic bile acid metabolism. It is required for efficient apical to basolateral transport of conjugated bile acids in ileal enterocytes with the affinity order of potency taurine-conjugated > glycine-conjugated > unconjugated bile acids. It binds to bile acids with the order of potency DCA > CA > CDCA [108]. It stimulates gastric acid and pepsinogen secretion. It functions as the cytosolic receptor for bile acids that have undergone sodium-dependent active transport using SLC10a2 (or iBAT) [194]. 219 PLTP: phospholipid transfer protein. 220 A.k.a. SHP. 221 A.k.a. hepatic nuclear factor HNF4. 222 A.k.a. liver receptor homolog LRH1.

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483

Table 5.34 Effects of FXR on bile acid (BA) processing (Source: [1002]; ⊕ −→ stimulation, −→ inhibition, BAAT bile acid–CoA:amino acid N acetyltransferase, BACS bile acid coenzyme A (CoA) synthetase, SLCO solute carrier organic anion transporter, SulT2a1 bile salt sulfotransferase, UGT UDP-glucuronosyl transferase) Hepatocyte BA synthesis

BA conjugaton BA export BA import BA detoxification Enterocyte BA uptake BA endocytosis BA egress

−→ synthesis of CyP7a1 FXR⊕ −→ NR0b2; NR0b2–NR5a2 −→ CyP7a1 NR0b2–NR2a1 −→ CyP8b1 FXR⊕ −→ FGF19; FGF19–FGFR4⊕ −→ JNK; JNK −→ CyP7a1, CyP8b1 ⊕ −→ synthesis BAAT, BACS ⊕ −→ synthesis ABCb11, ABCc2 −→ synthesis SLC10a1, SLCO1b1 ⊕ −→ synthesis CyP3a4, SulT2a1, UGT2b4 ⊕ −→ synthesis SLC10a2 ⊕ −→ synthesis FABP6 ⊕ −→ synthesis OSTα/β

Table 5.35 Impact of the FXR on lipid metabolism (Source: [1002]; ⊕ −→ stimulation, −→ inhibition, ↑ increase, ↓ decrease, CETP [exchanges cholesterol esters from HDLs with TGs in TGRLs (e.g., VLDLs and chylomicrons)], PLTP phospholipid transfer protein, SREBP sterol regulatory element-binding protein, TG triglyceride) TG metabolism

HDL metabolism

⊕ −→ synthesis NR1c1 (in humans) ⊕ −→ synthesis SREBP1c (in mice) ⊕ −→ synthesis ApoC2, VLDLR, Sdc1⊕ −→ LPL −→ synthesis ApoC3 −→ LPL ↑ TG clearance −→ synthesis ApoA1 (↓ [HDL]) ⊕ −→ synthesis PLTP (HDL remodeling) ↑ CETP activity

Lipoprotein lipase (LPL) is involved in the degradation of TG-rich lipoproteins. ApolipoproteinC3 (ApoC3) inhibits LPL, whereas ApoC2 and ApoA5 activate LPL. The VLDLR receptor enhances TG hydrolysis by LPL. Syndecan-1 (Sdc1) binds remnant particles before their transfer to receptors

The FXR has an impact on lipid metabolism (Table 5.35). Activated FXR decreases plasmatic concentrations of ApoA1 and HDLCS .223 In cultured hepatocytes, glucose induces Fxr gene expression, probably via metabolites of the pentose phosphate pathway, hence reducing cholesterolemia, whereas insulin counters this effect [1002]). Glucose also represses APOC3 gene expression. On the other hand, bile acids modulate gluconeogenesis, as they prevent 223 HDL

carries cholesterol from cells to hepatocytes, where it can be excreted into the bile as either free cholesterol or after conversion into bile acids.

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formation of PEPCK, glucose 6-phosphatase, and fructose (1,6)-bisphophatase via NR0b2 and NR2a1. The FXR participates in the lipoprotein metabolism. It targets the genes encoding phospholipid transfer protein, which permits the transfer of phospholipids and cholesterol from TGRLs to HDLs, in addition to ApoC2 and ApoE [1000]. In hepatocytes, the FXR successively undergoes phosphorylation by casein kinase CsnK2 (Ser327), sumoylation by UbC9 and pias1 (Lys325), and ubiquitination by RNF4, these post-translational modifications define a CsnK2–RNF4 activation– degradation axis [1004]. Sumoylation enhances ligand-primed FXR activation and transcriptional coactivation. Subsequent ubiquitination enables a maximal FXR activation for optimal up- or downregulation of responsive genes involved in bile acid homeostasis and liver regeneration, but leads to FXR proteasomal degradation.

5.5.2 LXRs The sterol-responsive nuclear receptors, LXRs, are major determinants of cellular cholesterol homeostasis. Whereas NR1h3 (LXRα) is expressed in a cell-specific manner, NR1h2 (LXRβ) is ubiquitous. The LXR ligands comprise oxysterols ([24S,25]-epoxycholesterol and 20(S)-, 22(R)-, 24(S)-, and 27-hydroxycholesterol) and intermediates of cholesterol synthesis, particularly desmosterol [1000]. They regulate transcription of genes involved in lipid absorption, metabolism, and excretion. They are activated by high cellular sterol content; they then induce formation of the cholesterol efflux transporters ABCa1 and ABCg1 in addition to ApoE to export excess cellular cholesterol. They also limit the uptake of lipoproteinderived cholesterol, as they induce formation of the ubiquitin ligase MyLIP,224 which ubiquitinates LDLR for degradation [1005]. In the intestine, LXR activates transcription of the genes encoding FABP6 and three ATP-binding cassette transporters (ABCa1, ABCg5, and ABCg8) [1000]. Both ABCg5 and ABCg8, which may form a functional heterodimer, limit intestinal absorption of sterols. On the other hand, ABCa1, which facilitates export of phospholipids and cholesterol from various cell types, may promote efflux of cholesterol from the enterocyte back into the lumen. In addition, ABCa1, ABCg5, and ABCg8 may remove phospholipids and/or sterols out of hepatocytes and macrophages, in addition to enterocytes. In the liver, LXR activates transcription of the genes encoding controllers of bile acid synthesis and metabolism (e.g., CyP7a1), cholesterol esterification (ACAT) and flux, fatty acid synthesis and esterification (LXR activates SREBP1c (bHLHd1c), which raises production of acetyl-CoA synthase and carboxylase, FAS, stearoylCoA desaturase, glycerol phosphate acyltransferase, and CTP:phosphocholine

224 MyLIP:

of LDLR.

myosin regulatory light chain-interacting protein. It is also named inducible degrader

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485

cytidylyltransferase), and lipoprotein metabolism [1000]. Other hepatic LXR target genes include ABCA1, ABCG5, and ABCG8. Hepatic ABCa1, ABCg5, and ABCg8 can enable efflux of cholesterol and phospholipids into blood or bile. Oxysterols repress SREBP2 (bHLHd2) activity and hence formation of its targets, such as LDLR and HMGCR, whereas hepatic SREBP1c-dependent genes are transcribed. Macrophages express LXRα and LXRβ, but neither FXR nor PXR [1000]. Agonists of LXR increase production of ABCa1, ABCg1, ApoE, and LXRα. ABCa1 enables efflux of phospholipids and cholesterol to acceptors, such as ApoA1 and ApoE. ABCg1 may be involved in controlling the efflux of cellular cholesterol to HDL and/or secretion of ApoE. In macrophages, upon endocytosis of modified lipoproteins or efferocytosis (uptake of dying and dead cells [e.g., apoptotic and necrotic cells]), that is, when cellular pools of oxysterol inflate, LXRs promote cholesterol efflux via ABCa1 and ABCg1, thereby suppressing TLR-mediated inflammation. Moreover, LXRs provoke transcription of genes that encode proteins that are involved in elongation and unsaturation of fatty acids and hence synthesis of long-chain polyunsaturated fatty acids (lcpuFAs; e.g., ω3-fatty acids), in addition to pro-resolving lipid mediators [1006]. LcpuFAs counter transcription primed by NFκB via altered histone acetylation in enhancer and/or promoter regions.225 In addition, LXRs increase production of the receptor kinase MerTK, which supports efferocytosis by macrophages, hence suppressing TLR4-mediated inflammation in addition to further upregulating ABCA1 and ABCG1 gene transcription [1006]. In macrophages, LXR targets the gene encoding endo- and exonuclease and phosphatase family domain-containing protein, EEPD1,226 which localizes to the plasma membrane and promotes cholesterol egress, as it controls ABCa1 amount and activity [1007]. The LXR participates in the lipoprotein metabolism. Synthesis of CETP and LPL is primed by oxysterols and the LXR [1000]. CETP is implicated in the transfer of cholesteryl esters between plasma lipoproteins. Lipoprotein lipase catalyzes hydrolysis of lipoprotein TGs. FXR induces production of ApoC2, the LPL cofactor.

225 Activation

of LXRs by desmosterol and oxysterols causes sumoylation of LXRs, leading to LXR binding (without RXRs) to NFκB and AP1 response elements. 226 Members of the EEPD family cleave phosphodiester bonds in nucleic acids and phospholipids [1007]. Moreover, several members operate as lipid phosphatase, targeting mainly inositol phosphates, which regulate intracellular molecular transfer. The DNA 5 -endonuclease EEPD1 promotes 5 -end resection at DNA double-strand breaks at stalled replication forks [194]. It is required for DNA repair by homologous recombination, but prevents DNA damage repair by nonhomologous end joining. EEPD1 may prevent phosphorylation of ABCa1, which primes degradation by calpain of ABCa1 and attenuates ApoA1-dependent cholesterol efflux, thereby stabilizing ABCa1 and favoring cholesterol export [1007].

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5.5.3 PXR The PXR is also activated by bile acids with the rank order of potency 3ketoLCA > LCA > DCA = CA [1003]. Other ligands comprise pregnenolone, progesterone, androstanol, and hyperforin, in addition to the drug dexamethasone and various xenobiotics (e.g., rifampicin and phenobarbital) [1000]. Activated PXR increases the formation of numerous cytochrome-P450 (CyP3a11, CyP3a4, and various CyP2b group members) involved in the metabolism of many xenobiotics and endogenous substrates before their excretion into the bile. On the other hand, activated PXR represses CyP7a1 production. In the liver, PXR ligands (e.g., LCA and 3-keto LCA) elicit formation of SLC01b1, which imports organic anions and sulfated and glucuronidated bile acids from blood into the hepatocyte, in addition to CyP2b and CyP3a [1000]. Many of these compounds are excreted into the bile through ABCb1 on the canalicular membrane.

5.5.4 RXRs Retinoid X receptors (RXRα–RXRγ [NR2b1–NR2b3]) are activated by retinoids (vitamin A derivatives). They regulate transcription of genes involved in cell proliferation and differentiation and glucose, TG, cholesterol, and bile acid metabolism. They heterodimerize with several other types of nuclear receptors (PPARs, LXRs, and FXRs) to control gene expression. In addition, they sensitize cells to insulin. They also protect against atherosclerosis. Platelets possess intracellular retinoid X receptors. Stimulation of platelets by PPAR ligands slightly raise cytosolic cAMP concentration, thereby activating PKA [1008]. Ligands of RXRs prevent platelet response (i.e., platelet aggregation, granule secretion, integrin activation, and calcium mobilization) and hence thrombosis upon exposure to ADP and thromboxane-A2 via inhibition of Gq in addition to thrombin and docosahexaenoic acid, a ligand for the glycoprotein receptor for collagen GP6 [1008]. The RXR ligands activate PKA via cAMP and activated NFκB, the inactive NFκB–Iκ Bα complex tethering to and inactivating the PKA catalytic subunit and this inhibition being relieved after platelet activation by collagen or thrombin. Activated PKA inhibits the RhoA and Rac1 GTPases

5.6 Control of Body Weight and Energy Homeostasis The control mechanism of body weight involves numerous regulators of food intake and metabolism, especially peptidic hormones (e.g., ghrelin and leptin) and neuropeptides (e.g., NPy and agouti gene-related peptide [AgRP]), which target their cognate receptors in the cerebral feeding centers, particularly in the hypothalamus (Table 5.36).

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Table 5.36 Orexigenic (i.e., appetite stimulators) and anorexigenic messengers (i.e., satietogenic mediators and appetite suppressors) Agent Orexigenic messengers Agouti gene-related peptide Ghrelin Hypocretin-1/2 (orexin-A/B) Melanin-concentrating hormone Neuropeptide-Y Neuropeptide LENSSPQAPARRLLPP Neuropeptide AVDQDLGPEVPPENVLGALLRV Uridine Anorexigenic messengers Adiponectin Amylin Apolipoprotein-A4 C1q and tumor-necrosis factor-related protein-3 Cholecystokinin Cocaine- and amphetamine-regulated transcript-derived peptide Enterostatin Gastrin-releasing peptide Glucagon-like peptide-1 Growth and differentiation factor-15 Insulin Leptin Melanocyte-stimulating hormone-α/β/γ Neuromedin-B Oxyntomodulin Peptide Tyr–Tyr Pro-opiomelanocortin Resistin Thyrotropin-releasing hormone

Alias AgRP Ghrl Hcrt1/2 MCH NPy LEN PEN U Adpn Amy ApoA4 CTRP3 Cck CART Est GRP GLP1 GDF15 Ins Lep MSH NMb Oxm PYY POMC Retn TRH

The satiety informant enterostatin is formed in the stomach and intestine by cleavage of secreted pancreatic procolipase (proClps), the remaining Clps part serving as a cofactor for pancreatic lipase during lipid processing. High-fat diet increases procolipase production and subsequently enterostatin release into the gastrointestinal lumen

Nutrients provide energy and building blocks for organismal growth. Cell growth is coordinated with nutrient availability by a central controller TOR, in particular in response to amino acids (Vols. 2, Chap. 2. Cell Growth and Proliferation and 9, Chap. 2. Hypoxia and Stress Response). In response to nutrients, TOR stimulates anabolism (protein, lipid, and nucleotide synthesis) and represses catabolism,

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especially autophagy.227 The TOR kinase forms two structurally and functionally different complexes, TORC1 and TORC2. Nutrients, growth factors, and cellular energy status coregulate TORC1 activity. The active RAG heterodimer binds to raptor and then recruits TORC1 to the lysosome, where it is activated by RHEB, itself stimulated by the PI3K–PDK1–PKB pathway, which dissociates TSC2 from the RhebGAP TSC complex (Tables 5.37 and 5.38). Upon growth factor and/or amino acid deprivation or stressor exposure, TSC2 moves to the lysosomal surface, where it inhibits RHEB and hence TORC1 [1009]. The intergenic 2p25.3 region of chromosome 2 close to the TMEM18 gene possesses genetic variants that are strongly associated with obesity in children and adults. The transmembrane protein-18 localizes to the nuclear envelope, interacts with components of the nuclear pore complex, nucleoporin NDC1, and the nucleocytoplasmic transport component aladin,228 and may thus be involved in the transport of molecules across the nuclear envelope [1010]. Moreover, it participates in the control of appetite. Its expression in the murine hypothalamic paraventricular nucleus (PVN), which is implicated in feeding behavior and energy expenditure, influences the nutritional state. Its ablation, both globally and within the hypothalamus in mice, increases food intake and hence body weight with gender-specific changes, whereas its selective overexpression in the PVN of WT mice raises energy expenditure and reduces food intake and adipose depot mass [1010]. The FTO gene229 that encodes α-ketoglutarate–dependent dioxygenase and its neighboring genes IRX3230 and RPGRIP1L231 also contributes to controlling energy balance [1010].

5.6.1 Signal Integration by the Brain Energy homeostasis requires the transmission to the brain of information on energy fluxes in and from organs, especially nutrient levels in blood and stores, and energy needs of organs to regulate food intake and maintain energy store at appropriate levels in a given condition. The autonomic nervous system responds via control of activity of organs that play an important role in energy homeostasis in addition to secretion of metabolic

227 In the budding yeast, Saccharomyces cerevisiae, Tor gene mutations confer resistance to growth

inhibition provoked by rapamycin. alacrima, achalasia, adrenal insufficiency neurologic disorder. 229 FtO: fat mass and obesity-associated protein. 230 IRX3: iroquois homeobox-containing gene-3. 231 RPGRIP1L: RPGR-interacting protein-1-like protein, also called nephrocystin-8 (NphP8) and fantom (Ftm), which is involved in the organization of apical junctions in addition to regulation of proteasomal activity in the primary cilium. 228 Aladin:

5.6 Control of Body Weight and Energy Homeostasis

489

Table 5.37 Nutrients, growth factors, TOR kinase, and TORC1 (Source: [1009]) Agent Flcn, FnIP1/2

Features GAPs for RagC/D Stimulated by AA ARF1 Stimulated by Gln RHEB Stimulated by the GF–PI3K–PDK1–PKB axis Ragulator–RAgA/B–RagC/D–SLC38a9–vATPase at lysosomal surface Ragulator GEF Gator-2 MIOS–Sec13–Seh1L–WDR24–WDR59 pentamer) Inhibits gator-1 SLC1a5 Sodium-dependent AA (Gln) transporter SLC3a2–SLC7a5 Dimeric antiporter: imports Leu, exports Gln Activate TORC1 Arginine Activates TORC1 Binds to castor1–castor1 and castor1–castor2, disrupting (castor)2 –gator2 SLC38A9 Lysosomal AA transporter Arg sensor Binds to ragulator and Rags Glutamine Stimulates TORC1 lysosomal translocation via ARF1 Glutaminase De-amination of glutamine to glutamate Glutamate dehydrogenase Leu cofactor Converts glutamate to α KG which activates TORC1 via PHD SLC15a4 Lysosomal H+ -coupled histidine transporter His export SLC36a1 Proton and AA symporter, mainly in endosomes Interacts with RagC/D SLC36a4 Proton and AA symporter, mainly in the Golgi body Interacts with TOR, raptor, and Rab1a Rab1a Stimulates TORC1 interaction with GB RHEB TasR1/3 AA-induced TORC1 lysosomal translocation (Part 1) Stimulators involved when nutrients are available and upon growth factor stimulation (AA amino acid, ARF ADP ribosylation factor, Flcn folliculin, FnIP folliculin-interacting protein, gator GAP activity toward Rag, KG ketoglutarate, Mios WD repeat-containing protein Mio, PHD prolyl hydroxylase, ragulator Rag and TORC1 regulator complex, RHEB Ras homolog enriched in brain, Tas1R1[3] taste GPCR1 member 1[3], WDR WD repeat-containing protein)

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Table 5.38 Nutrients, growth factors, TOR kinase, and TORC1 (Source: [1009]) Agent TSC2

AMPK

Axin

Castor-1 Sestrin-2

Gator-1 eIF2α K4

Features Inhibits RHEB Activated upon phosphorylation by AMPK Repressed upon phosphorylation by PKB Stimulated by glucose deprivation Activated by sestrins Activated by Axin–STK11 at the lysosome Inhibits RHEB via TSC2 Phosphorylates raptor to inhibit TORC1 Axin–STK11 phosphorylates (activates) AMPK at the lysosome via vATPase–ragulator Inhibits ragulator (RagA/B-GEF) and then TORC1 Stimulated by AA depletion GDI for RagA/B Binds to and inhibits gator-2 Cytosolic Leu sensor that links to Leu DEPDC5—-NPRL2–NPRL3 trimer GAP for RagA/B phosphorylates (inhibits) eIF2, repressing protein synthesis, but enabling selective production of ATF4, which elicits synthesis of AA transporters, AA metabolic enzymes, and autophagic factors

(Part 2) Inhibitors involved upon nutrient deprivation and growth factor depletion (AMPK AMP-activated protein kinase, Axin axis inhibition protein-1, castor cellular arginine sensor for TORC1, DEPDC DEP domain-containing protein, gator GAP activity toward Rag, NPRL nitrogen permease regulator-like protein, PKB protein kinase-B, STK protein Ser/Thr kinase, TSC tuberous sclerosis complex)

hormones. Control of chemical messengers that regulate hunger and food intake enables handling of metabolic disorders and obesity. Messengers, such as leptin and ghrelin, act on certain brain regions, using NPy, agouti-related peptide, melanocortins, hypocretins, and melanin-concentrating hormone (MCH), among other mediators [1011]. Feeding behavior is regulated by the CNS, especially some regions of the forebrain232 and brainstem233 participate in regulating feeding [1012]. 232 The

forebrain, also called the prosencephalon, is the anterior part of the brain. It comprises the cerebral hemispheres, thalamus, and hypothalamus. 233 The brainstem is the trunk connecting the spinal cord to the brain. It consists of the medulla oblongata, pons, and midbrain.

5.6 Control of Body Weight and Energy Homeostasis

491

In addition, the hypothalamus is a major hub controlling energy homeostasis that integrates nutritional, metabolic, endocrine, and thermal signals. Numerous neuropeptides are released by the hypothalamus.

5.6.1.1

Hypothalamus

Two major opposing hypothalamic clusters encompass the lateral hypothalamus, including the perifornical region, which drives feeding and the ventromedial hypothalamic (VMH) nucleus that inhibits it, provoking satiety. In fact, many interconnected hypothalamic clusters regulate food intake and energy homeostasis, such as the arcuate (ArcN),234 paraventricular,235 dorsomedial hypothalamus (DMH), and VMH nuclei, and the lateral hypothalamic area (LHA).236 In the hypothalamus, hormonal signals that control appetite and meal size and frequency in addition to status of primary storage organs (e.g., lipids in AT and glycogen in the liver) regulate the energy balance.

Arcuate Nucleus Energy homeostasis is regulated by neuronal populations of the ArcN. In particular, insulin and leptin liberated in blood proportionally to nutrient levels interact with their cognate neuronal receptors, principally in the hypothalamus. Both basal and meal-stimulated insulin secretion depend on available lipids [1014].

234 Neurons

of the ArcN are sensitive to concentrations of ghrelin, Cck, GLP1, NMb, and ApoA4, most of these peptides being made in the brain. However, their activity relies on ghrelinemia, insulinemia, and leptinemia. In addition, these neurons are sensitive to local concentrations of glucose, some long-chain fatty acids, and some amino acids (e.g., leucine) [1014]. 235 Neurons of the PVN possess MC , MC , and various types of NPy receptors. NPy receptors are 3 4 targeted not only by NPy but also by peptide-YY and pancreatic polypeptide. They are involved in the control of appetite and circadian rhythm, among other behavioral processes. Neurons of the PVN synthesize and secrete neuropeptides that have a catabolic action, such as corticotropinreleasing hormone (CRH) and oxytocin, which reduces food intake. 236 Neurons of the lateral hypothalamus synthesize and secrete anabolic peptides, such as MCH,an orexigenic peptide, and hypocretins (Hcrt1–Hcrt2; or orexins [OxA–OxB]; from Greek oρ ξις: appetency), which both favor food intake. Hypocretin+ neurons of the LHA belong to the dopamine+ mesolimbic circuit.

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In the ArcN, medial NPy+ AgRP+ neurons stimulate feeding,237 whereas lateral POMC+238 and CART+239 neurons cause hypophagia (Table 5.39) [1012]. These neurons project to the PVN that controls feeding and yields preganglionic autonomic output to the brainstem. POMC+ CART+ neurons also innervate MCH+ and Hcrt+ neurons in the LHA and sympathetic preganglionic neurons in the spinal cord [1011]. The NPy+ AgRP+ neurons innervate many of the POMC+ CART+ neuron targets. Feeding regulators, such as ghrelin, glucocorticoids, leptin, and melanocortins operate at least partly via the NPYergic circuit. Neuropeptide-Y, one of the most powerful orexigenic agents, provokes food intake via its receptors (Y1 –Y5 [in mice, also Y6 ]). The combined action of hypothalamic Y1 and Y5 receptors in the PVN, DMH, and VMH mediates hyperphagia [1018].240

237 Fasting

increases the hypothalamic formation of orexigenic NPy and agouti-related peptide and decreases that of anorexigenic pro-opiomelanocortin (POMC) and cocaine- and amphetaminerelated transcript-derived peptide. 238 POMC+ neurons regulate orexinergic signaling by hormones, such as cholecystokinin, ghrelin, insulin, and leptin. POMC is a precursor of three melanocortins, α-, β-, and γ-melanocytestimulating hormone (α MSH–γ MSH). Pre-POMC is sequentially cleaved in the hypothalamus; prohormone convertase PC1 (or PCSK1) produces ACTH that is processed into α MSH to γ MSH by PC2 (or PCSK2). All three melanocortins activate the arcuate, ventromedial, paraventricular, periventricular, and supra-optic nuclei, in addition to the pre-optic area [1015]. In humans, α MSH is the predominant POMC-derived neuropeptide in the central regulation of body weight. α MSH and β MSH activate melanocortin receptors in the arcuate (MC3 ) and paraventricular nuclei (MC4 ) [1016]. Activation of these two GPCRs is antagonized by agouti-related peptide. α MSH and β MSH bind to MC4 with high affinity, ACTH to MC4 with a lower affinity, and γ MSH exclusively to MC3 [1017]. α MSH and γ2 MSH are anorexigenic, the latter subtype with a slower time course of action, but not β MSH [1015]. On the other hand, α MSH and β MSH activate the dorsomedial nucleus, but γ2 MSH is weakly active in this cluster. Therefore, in addition to common neural circuits, distinct hypothalamic circuits are activated by different subtypes of POMC products. Many neuroendocrine cells activated by MSH in the arcuate, paraventricular, periventricular, and supra-optic nuclei (but neither DMH nor VMH) project outside the blood–brain barrier [1015]. The POMC+ neurons of the ArcN project to other hypothalamic nuclei, in particular PVN, where stimulation of the postsynaptic melanocortin receptor MC4 decreases body weight [1017]. In humans, heterozygous loss-of-function mutations in the Pomc or MC4R gene cause overweight and moderate obesity. Deficiency of POMC also reduces glucocorticoid levels and provokes redhair pigmentation, as pituitary POMC is the precursor of ACTH, which stimulates glucocorticoid secretion via MC2 in the adrenal cortex, in addition to MSH, which, via MC1 , controls hair pigmentation. 239 CART: cocaine- and amphetamine-regulated transcript-derived peptide. 240 The genes encoding the Y and Y receptors, which are predominant Y receptors in the PVN, 1 5 are coexpressed in the same neuron types and regulated in a coordinated manner by the same promoter [1018]. Double germline and adult-onset hypothalamus-specific deletions of the genes encoding the Y1 and Y5 receptors lower spontaneous hypophagia in male mice and fasting-induced food intake in both male and female mice, hypophagia being stronger in germline than in adult-onset deletions. The receptors Y1 and Y5 play a redundant role in feeding regulation. Single germline Y1 deficiency in male and female mice attenuates fasting-induced intake, but not spontaneous food intake. Single

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Table 5.39 Neuronal populations of the arcuate nucleus and their regulators Neuron type NPy+ AgRP+ neurons

POMC+ CART+ neurons

Stimulators (orexigens) Ghrelin

Inhibitors (anorexigens) Insulin, PYY, adiponectin, leptin, resistin, TNFSF1, IL1β (anorexigens) (orexigens) Insulin PYY Adiponectin, leptin, resistin IL6/10 TNFSF1 (exercise)

Neuropeptide-Y (NPy) reduces energy expenditure and stimulates food intake via the Y1 and Y5 receptors in the PVN. Pro-opiomelanocortin and its products such as α-melanocyte-stimulating hormone (α MSH) preclude feeding. Agouti-related protein homolog antagonizes α MSH, thereby increasing feeding and body weight. Cocaine- and amphetamine-regulated transcript-derived peptide inhibits NPy-induced feeding. Other hypothalamic orexigenic peptides, such as MCH and hypocretins, are produced in neurons of the lateral hypothalamus. In the brainstem connected to the hypothalamus, neurons of the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMNV) receive and integrate inputs from the vagus nerve upon sensing nutrient accumulation in the stomach and duodenum. The brainstem regulates responses to fasting via ascending projections to the hypothalamus and short-term satiety via descending projections from the hypothalamus

In the hypothalamus, insulin and leptin decrease anabolic (pro-feeding) and increase catabolic neuropeptides, whereas ghrelin has opposite effects [1019]. These hormones target hypothalamic neuropeptides to regulate food intake and modulate metabolism via the efferent autonomic nervous system. Adiponectin and leptin synergistically activate POMC+ neurons in the ArcN [1020]. Conversely, NPy+ AgRP+ neurons are inhibited by adiponectin via PI3K, independently of AMPK, PI3K being a substrate for both adiponectin and leptin in the regulation of energy balance and glucose metabolism via melanocortin activity. Adiponectin stimulates POMC neurons at various glucose concentrations. Adiponectin (AdpnR1–(AdpnR2) and leptin receptors (LepR) localize to POMC+ and NPy+ AgRP+ neurons of the ArcN involved in glucose and fatty acid

germline Y5 deficiency augments spontaneous and fasting-induced feeding in both genders. The remaining receptor compensates to protect against hypophagia. Resulting hypophagia is associated with decreased NPy synthesis in the ArcN, whereas that of POMC remains unchanged. In male (but not female) Y1−/− Y5−/− mice, body weight elevates; adiposity rises in both genders, but in females, lean tissue mass decreases. Adult-onset PVN-specific Y1−/− Y5−/− chow diet-fed mice have a trend toward reduced fasting-induced food intake, but no significant differences in spontaneous food intake. On the other hand, in HFD-fed mice, spontaneous and fasting-induced food intake diminishes significantly. Despite hypophagia, these mice become obese. Obesity is more obvious in germline HT Y1 - and Y5 -deficient mice than in adult-onset PVN Y1 - and Y5 -deficient mice, the latter model exhibiting obesity only with HFD and not normal chow. Other neural circuits can compensate for double ablation, whether germline or adult-onset, of the genes encoding the Y1 and Y5 receptors.

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metabolism regulation. Whereas AdpnR1 is widespread, at least in the rat brain, AdpnR2 lodges in specific brain regions, such as the cortex, hypothalamus, and hippocampus [1021]. In the arcuate and paraventricular nuclei, Adipor1 and Adipor2 reside on neurons and astrocytes. AdpnRs and LepRs initiate several overlapping signaling cascades that involve the JaK2–STAT3, insulin receptor substrates IRS1 and IRS2, FoxO1, AMPK, and PI3K [1020]. Adiponectin potentiates leptin effects on melanocortin-dependent thermogenesis and AT mass. In the rodent brain, resistin lowers food intake and controls the formation and the enzymatic activity of various hypothalamic molecules implicated in feeding, likely via auto- and paracrine loops [1022]. It reduces hypothalamic production of NPy and POMC (but not AgRP) and Adpn. On the other hand, it increases the activity of metabolic enzymes (e.g., AMPKα). Both resistin and melanocortin agonists may influence AT 11β HSD1, which converts inactive into active glucocorticoids, thereby favoring obesity and insulin resistance. Resistin stimulates hepatic glucose production and inhibits muscle and AT glucose utilization. The activity of the hypothalamus–pituitary–adrenal (HTA) axis and the plasmatic concentration of cortisol increase in obese humans [1019]. In obese rats, negative feedback control of the HPA axis is impaired, owing to a lower concentration of mineralocorticoid receptors in the hippocampus, but an unchanged number of glucocorticoid receptors. In obese humans, forward drive to the HPA axis increases and sensitivity to the HPA axis feedback falls. Glucocorticoids participate in the regulation of metabolic homeostasis and can provoke hyperphagia, hyperinsulinemia, and insulin resistance via augmented NPy content in the arcuate nucleus [1019]. Peptide-Y3–36 , a cleavage product of PYY released after a meal by endocrine cells of the gut (L cells of the distal gastrointestinal tract), is the most common circulating PYY form that targets the Y2 receptor, which serves as inhibitory receptors on both orexinergic NPy+ neurons and anorexinergic POMC+ neurons. Peptide-Y3–36 potently and reversibly inhibits POMC+ neurons via postsynaptic Y2 receptors [1023]. In fact, PYY3–36 inhibits both POMC+ and NPy+ neurons [1024]. It increases G-protein-gated inwardly rectifying K+ channel flux, decreasing inward calcium current, hyperpolarizing the membrane potential, and inhibits the presynaptic release of excitatory glutamate. It reduces feeding in obese humans; its basal concentration drops in obese subjects. In the hypothalamus, overeating is associated with insulin and leptin resistance via aberrant production of proinflammatory molecules, such as TLR4 and IKK, activation of the IKKβ–NFκB axis, dysfunction of insulin and leptin signaling via IKKβ, and endoplasmic reticulum stress. On the other hand, physical exercise suppresses hyperphagia and associated hypothalamic activation of the IKKβ–NFκB axis via anti-inflammatory interleukins IL6 and IL10 [1025]. The cytokine IL6 has both pro- and anti-inflammatory effects; it inhibits acute phase inflammation, as it causes the formation of IL10, IL1R antagonist, and soluble TNFSF1 receptors. During exercise, IL6, which is released by contracting skeletal muscles, provokes metabolic changes in the liver, AT, and hypothalamus.

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Activated microgliocytes synthesize and release cytokines that consequently launch inflammation. According to the circumstances, microglial TNFSF1 and can promote neural survival or cause neuronal dysfunction. TNFSF1 operates via widespread TNFR1 and TNFR2 expressed mainly in immunocytes. High-carbohydrate and -fat diet stimulates microglial reactivity in the mediobasal hypothalamus (MBH). Chronic overfeeding and DIO decreases the number of POMC+ neurons [1026]. Microgliocytes in the MBH hypersecrete TNFSF1, which stimulates ATP production in POMC+ neurons, favoring mitochondrial fusion, increasing the firing rate and excitability, triggering mitochondrial stress, and contributing to obesity. In the hypothalamus of rats, TNFSF1 launches proinflammatory signaling, triggering production of cytokines and cytokine-responsive protein, such as IL1β, IL6, IL10, and SOCS3 [1027]. TNFSF1 activates gene transcription via activator protein-1 in the hypothalamus. In addition, TNFSF1 induces expression of neurotransmitters involved in the control of feeding and thermogenesis. It provokes expression of orexigenic NPy and MCH (up to 1.3-fold) in addition to anorexigenic POMC (up to 8.0-fold) and CRH (up to 1.8-fold) [1027]. Both insulin and TNFSF1 reduces feeding (45 and 25%, respectively) during the same time window, but TNFSF1 does not modulate the anorexigenic effect of insulin in the hypothalamus.

Lateral Hypothalamic Nucleus Mingled neurons of the lateral hypothalamus release melanin-concentrating hormone or hypocretins (which are not coexpressed). Hcrt+ and MCH+ neurons have similar projection patterns. Major targets comprise brainstem motor centers such as nuclei of cranial motor neurons in the trigeminal, hypoglossal, and facial nerves, implicated in chewing, licking, and swallowing, in addition to the sympathetic and parasympathetic preganglionic nuclei in the medulla and spinal cord, involved in salivation, esophageal and gastric motility, gastric acid secretion, and the regulation of pancreatic hormone secretion of pancreatic hormones, and the ascending arousal system, which includes noradrenergic locus ceruleus,241 serotoninergic dorsal and median raphe nuclei,242 and histaminergic tuberomammillary nucleus of the posterior hypothalamus [1011]. In addition, Hcrt+ and MCH+ neurons are connected to the cerebral cortex. MCH+ neurons also project and stimulate to the nucleus accumbens, which receives sensory and feeding behavioral cues from the cerebral cortex. Moreover, the NAcc is involved in the GABAergic circuit via the GABAergic ventral pallidum (VP), thereby disinhibiting feeding [1011]. Food restriction elicits synthesis of MCH and hypocretins. Leptin secreted by the WAT during times of plenty is a necessary (but not sufficient) satiety stimulus.

241 From

Latin cæruleus: azure, blue, dark blue. seam.

242 ραϕη:

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Hedonic Stimuli

Feeding, which provides substrates for metabolism, relies not only on homeostatic signals by sensors of energetic balance transmitted to the brain but also circadian cues and rewarding behavior (Tables 5.40 and 5.41) [1011]. Feeding is not only a necessary element of existence, but it relies on hedonic mechanisms; humans tend to eat large amounts of palatable food (beyond homeostatic need), taste and smell representing major palatability or food aversion signals. Gustatory and olfactory information modulates feeding behavior.

Table 5.40 Major neural circuits linked to circadian, homeostatic, and sensory afferent feeding inputs to the brain and neurotransmitters (Source: [1011] AgRP agouti-related protein, AIC agranular insular cortex, ArcN hypothalamic arcuate nucleus, CART cocaine- and amphetamine-regulated transcript-derived peptide, CGRP calcitonin-gene related peptide, DMH dorsomedial hypothalamic nucleus, GIC granular insular cortex, ILC infralimbic cortex, LHA lateral hypothalamic area, NAcc nucleus accumbens, NPy neuropeptide-Y, NTS nucleus of the solitary tract, PBN pontine parabrachial nucleus, POMC pro-opiomelanocortin, PVN hypothalamic paraventricular nucleus, SCN suprachiasmatic nucleus, SPVZ subparaventricular zone, VMH ventromedial hypothalamic nucleus, VPMpcTN ventral posteromedial parvicellular thalamic nucleus [subparafascicular nucleus]) Input Circadian Leptin Vagal, taste Connectivity

Neural circuits SCN–SPVZ–DMH/PVH ArcN–LHA (NPy/AgRP, POMC/CART) NTS–PBN PBN–LHA/DMH/VMH PBN–PVN PBN–VPMpcTN–(CGRP)–GIC GIC–AIC/ILC/NAcc

Table 5.41 Major output circuits regulating feeding (Source: [1011])

Output Autonomic Endocrine Motor Cortex

Source Neural circuit (neurotransmitter) ArcN–(POMC)–NAcc LHA–(MCH)–NAcc–(GABA)–VP–(GABA)–LHA PVH (ADH, CRH, TRH) LHA (Hcrt) VP–MDTN–AIC/ILC

The hypothalamic paraventricular (PVH) and arcuate nuclei (ArcN) and the lateral area (LHA) yield the main autonomic, endocrine, and motor responses contributing to feeding, using melaninconcentrating hormone (MCH) antidiuretic hormone (ADH; or vasopressin), corticotropin- (CRH) and thyrotropin-releasing hormone (TRH), and hypocretin (Hcrt: or orexin), respectively. Projections to the prefrontal cortex originate from the LHA and the nucleus accumbens (NAcc) via the ventral pallidum (VP) and mediodorsal thalamic nucleus (MDTN) to the agranular insular (AIC) and infralimbic (ILC) cortical regions (CART cocaine- and amphetamine-regulated transcriptderived peptide, GABA γ-aminobutyric acid, POMC pro-opiomelanocortin)

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Food and drink flavor provides a major gustatory hedonic value or taste aversion. Flavors are discriminated by taste receptors, which recognize the four classic tastes (bitter, salty, sour, and sweet). Most of the taste cells in the tongue respond to more than one of these four tastes. Taste information is relayed by the NTS and the parabrachial nucleus [1011]. The latter projects to a gustatory nucleus in the thalamus, the lateral frontal cerebral cortex, central nucleus of the amygdala, and several hypothalamic clusters such as the lateral hypothalamus. Odorants are detected by GPCRs of olfactory receptors, including the vomeronasal organ, the peripheral sensory organ of the accessory olfactory system at the base of the nasal septum. Olfactory signals are transmitted by bipolar olfactory receptor neurons in the olfactory neuroepithelium, which also contains microvillar, sustentacular, and globose and horizontal basal cells.

5.6.2 Brain–Gut Axis The autonomic regulation of digestion involves the central, parasympathetic, sympathetic, and enteric nervous systems (ENS) in addition to neuroendocrine messengers originating from the cerebral cortex and adrenal medulla. The brain–gut or gut–brain axis refers to bidirectional communication between the gut and brain via vagal and spinal afferent neurons and humoral factors, such as intestinal hormones, gut flora-derived messengers, and cytokines, which transmit information from the gut to the brain and from the brain to the gut via autonomic neurons and neuroendocrine factors [1028]. The gut–brain axis thus uses four major communication supports: nervous influx carried by vagal and spinal afferent neurons, immune messages by cytokines, endocrine signals by gut hormones (>20 hormone types), and intestinal floraderived cues, as lipopolysaccharide and peptidoglycan components can act on the CNS [1028].

5.6.2.1

Vagus Nerve and Nucleus of the Solitary Tract

The brain receives various signals from the gastrointestinal tract via sensory afferents and blood circulation. Afferent signals transmitted by the vagus nerve inform of gastric stretch in addition to hepatic concentrations of glucose and lipids [1011]. Sensory terminals terminate in the medial and dorsomedial parts of the NTS, which is protected from hormones by a blood–brain barrier, and directly in the gastromotor vagal neurons, whereas others relay to the dorsal motor vagal nucleus, which innervates the entire gastrointestinal tract. Direct projections from the NTS and relayed fibers via the parabrachial nucleus innervate the paraventricular, dorsomedial, lateral hypothalamic and arcuate nuclei of the hypothalamus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and visceral sensory thalamus [1011].

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Area Postrema

The area postrema is a circumventricular organ close to the NTS in the dorsomedial medulla oblongata outside the blood–brain barrier. In humans, this bilateral structure resides immediately dorsal to the subpostremal nucleus, which separates the NTS from the area postrema. The area postrema receives visceral afferent inputs from the glossopharyngeal and vagus nerves. Neurons in the area postrema can respond to circulating gastrointestinal hormones, which also serve as neuropeptides, such as amylin, cholecystokinin, and glucagon-like peptide GLP1, and transmit them to the medullary NTS and pontine parabrachial nucleus [1011].

5.6.2.3

Secretin Set

The secretin (SCT) set of the brain–gut axis243 consists of structurally and functionally related peptidic hormones, such as secretin (Sct), glucagon (Gcg), glucagon-like peptides GLP1 and GLP2, gastric inhibitory polypeptide (GIP; or glucose-dependent insulinotropic polypeptide), adenylate cyclase-activating polypeptide AdCyAP1 (a.k.a. pituitary adenylate cyclase-activating peptide [PACAP]), PACAP27, PACAP38, and helodermin-like peptide (Table 5.42) [1012]. Other SCT set members, vasoactive intestinal peptide (VIP), peptide histidine– isoleucine (PHI) and its human analog peptide histidine–methionine (PHM), and peptide histidine–valine (PHV) are co-synthesized from the same precursor.244 These peptidic hormones are widespread, being detected in the CNS and in the gastrointestinal, respiratory, and reproductive tracts. Peptides of the SCT category and their receptors are detected in various brain regions, especially the hypothalamus. They operate via common and distinct class-I I , subclass-B1 GPCRs.

Class-I I G-Protein-Coupled Receptors G-protein-coupled receptors are key players in cellular communication. Class-A rhodopsin-like receptors represent the majority; class B includes secretin-like and adhesion GPCRs, F frizzled, and C (22 members), the main neurotransmitters, glutamate (mGlu1–mGlu8) and GABA (GABAA –GABAB ), and sweet and umami taste (T1R1–T1R3) and calcium-sensing receptors (CaS) [1029]. Class-C GPCRs 243 The first hormone discovered in humans (in 1902), secretin, a 27-amino acid-containing peptide

encoded by the SCT gene, provides the name of this hormone category. This hormone category is also named the SCT–VIP set. 244 Peptide histidine–isoleucine was originally isolated from the porcine upper intestine. It activates AC in rats and inhibits VIP binding to its receptors. PHI exists in the body in two forms: shorter (27 amino acids) and longer peptide (42-amino acid), which is named peptide histidine–valine.

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Table 5.42 Peptides of the secretin (Sct) family and appetite regulation (Source: [1012]; ND not determined, AdCyAP1 adenylate cyclase-activating polypeptide-1 [or pituitary adenylate cyclase-activating polypeptide (PACAP)], GLP glucagon-like peptide, GHRH growth hormonereleasing hormone, PHI peptide histidine–isoleucine, VIP vasoactive intestinal peptide, MC4 melanocortin-4 receptor, POMC pro-opiomelanocortin, AP area postrema, ArcN arcuate nucleus, CeA central amygdala, DMH dorsomedial hypothalamus, DMNV dorsal motor nucleus of the vagus, DVC dorsal vagal complex, lPBN lateral parabrachial nucleus, LHA lateral hypothalamic area, MlPON medial pre-optic nucleus, NAcc nucleus accumbens, NTS nucleus tractus solitarius, PVN paraventricular nucleus, SCN suprachiasmatic nucleus, vHpc ventral hippocampus, VMH ventromedial hypothalamus, VTA ventral tegmental area) Feeding behavior Anorexigenic

Peptide AdCyAP1

Primary function Hypothalamic formation of POMC, MSH, MC4

Glucagon

Glucose release

Anorexigenic

GLP1

Incretin

Anorexigenic

GLP2

Anorexigenic

GHRH

Intestinal mucosal growth GH release

PHI

Prolactin regulation

Anorexigenic

Secretin

Bicarbonate and water release from pancreas

Anorexigenic

VIP

Vasodilation

ND

Orexigenic

Brain targets Hypothalamus (ArcN, PVN, VMH) Forebrain [striatum] (Nacc) Hypothalamus (ArcN, LHA) Brainstem (DVC) Hypothalamus (ArcN, DMH, LHA, PVN, VMH) Brainstem (NTS, lPBN, AP, DMV) Mesolimbic reward system (VTA, NAcc) Amygdala (CeA) Hippocampus (vHpc) Hypothalamus (ArcN) Hypothalamus (ArcN, DMH, SCN/MlPON) Hypothalamus (PVN) Amygdala (CeA) Hypothalamus (ArcN, PVN) Brainstem (NTS) Amygdala (CeA) Hypothalamus (PVN, SCN)

are homo- or heterodimers, dimerization being mandatory for signaling and binding to both protomers required for full activity (e.g., GABAB1 –GABAB2 , T1R3–T1R1, and T1R3–T1R2). Class-I I GPCRs couple mainly to the Gs–AC–cAMP pathway but also with Gq and Gi, thereby activating PLC. They are stimulated by neuropeptides, such as STN, GLP1 and GLP2, growth hormone-releasing hormone (GHRH),245 AdCyAP1, CRH, VIP, parathyroid hormone (PTH), and calcitonin-related peptides [1030].

245 A.k.a.

growth hormone-releasing factor, somatocrinin, somatoliberin, and somatorelin.

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They promote neuronal survival, especially during stroke, as they support cell response to oxidative, metabolic, and excitotoxic stress. Corticotropin-Releasing Hormone and Related Neuropeptides Corticotropin-releasing hormone is a potent mediator of endocrine, autonomic, and immune responses to stress [1030]. It also modulates behavior (motor function, arousal, feeding, and anxiety-related behavior). It is released from hypothalamic parvocellular neurons of the paraventricular nucleus into portal vessels to activate the HTA via ACTH secreted from the anterior pituitary gland. ACTH stimulates glucocorticoid secretion from the adrenal gland. Other regions with high concentrations of CRH+ neurons include the bed nucleus of the stria terminalis, with projections to brainstem areas involved in autonomic function, and interneurons of prefrontal, cingulate, and insular cortical areas. The effects of CRH are mediated by two receptors CRHR1 and CRHR2. The former abounds in cerebellar, neocortical, hippocampal, and sensory relay structures of the rat brain. The latter can also modulate stress response upon HPA activation. Urocortin-1 and -2 are members of the CRH family of neuroprotective neuropeptides. They are produced in multiple regions of the brain, with high levels in certain neuronal populations of the midbrain and brainstem [1030]. Parathyroid Hormone and Related Neuropeptides Parathyroid hormone (PTH) is a glycoprotein that primarily functions in controlling calcemia, its secretion from the parathyroid glands being regulated by feedback via calcemia. Parathyroid hormone and two related peptides, parathyroid hormonerelated peptide (PTHRP) and 39-amino acid tuberoinfundibular peptide TIP39, target three receptors (PTH1 –PTH3 ). PTH and PTHRP primarily interact with PTH1 and TIP39 PTH2 . The receptors PTH1 and PTH2 in addition to their cognate hormones are widely expressed in the CNS (PTH2 : limbic, hypothalamic, and sensory areas, especially PVN, nerve terminals in the median eminence, superficial layers of the spinal cord dorsal horn, and caudal part of the sensory trigeminal nucleus) [1030]. The calcitonin family of peptidic neuroprotectors comprises amylin (or islet amyloid polypeptide [IAPP]), calcitonin, α- and β-CGRPs by alternate splicing of a single transcript type, and adrenomedullin (Adm). These members are widespread in peripheral organs and in the peripheral and CNS. Adrenomedullin and CGRP cause vasodilation, calcitonin decrease bone resorption, amylin reduces nutrient intake (Sect. 5.6.7.1). The CGRP and adrenomedullin-2 receptor requires the single transmembrane domain-containing protein RAMP1 coupled with calcitonin receptor and adrenomedullin-1 RAMP2 or RAMP3, and amylin RAMP1, RAMP2, or RAMP3 [1030]. The calcitonin receptor stimulates AC and phospholipase-C in addition to phospholipase-D and inhibition of AC.

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Secretin Secretin is a gastrointestinal hormone released by S cells of the duodenum and proximal jejunum in response to acid milieu to stimulate bicarbonate secretion from the pancreas to neutralize gastric chyme acidity [1012]. It thus acts on epitheliocytes of the pancreatic and biliary ducts, launching secretion of alkaline bicarbonate-rich fluid. It also slows gastric emptying [1030]. Secretin is also a neuropeptide synthesized in multiple brain regions (hypothalamic ArcN, supra-optic nucleus [SON], and PVN, NTS of brainstem, cerebellum, area postrema, central amygdala, hippocampus, and cerebral cortex), being an element of the brain–gut axis that controls digestion and feeding behavior [1012, 1030]. The Sct receptor (SctR) localizes to gastric and intestinal epitheliocytes, pancreatic acinar cells, Brunner’s glands, gastric and intestinal smooth myocytes [1030]. It is also identified in the hypothalamic ArcN, PVN, and SON [1012], and in the cerebellum, cerebral cortex, thalamus, striatum, hippocampus, and brainstem [1030]. Secretin stimulates lipolysis and fatty acid uptake in adipocytes [1031]. Secretin is also a neurohypophysial factor detected throughout the hypothalamoneurohypophysial axis and released from the posterior pituitary gland (neurohypophysis) upon exposure to plasmatic hyperosmolality. In the hypothalamus, it stimulates vasopressin production and release, thereby regulating body water content, as vasopressin elicits water reabsorption in the renal collecting duct via the formation and translocation of aquaporin-2 [1032]. In addition, it has a direct effect on renal water reabsorption and modulates water and electrolyte transfer in pancreatic ductal cells, cholangiocytes, and epididymal epitheliocytes. Secretin stimulates the synthesis of aquaporin-2 under hyperosmotic conditions and its translocation from intracellular vesicles to the plasma membrane [1033]. Peripheral and central secretin exert an anorexigenic effect via SctR on intestinal vagal afferents and the central melanocortin system [1012]. Secretin stimulates the dorsal division of the parvocellular neurons of the PVN, which are involved in the control of central autonomic outflow.246 In addition, central and peripheral secretin upregulates synthesis of the melanocortin receptor MC4 in the PVN, which supports secretin action. In the ArcN, POMC+ neurons are endowed with SctR, hence contributing to the secretin effect.

246 The

PVN is composed of magno- (from Latin magnus: large) and parvocellular neurons (from Latin parvus: small). Vasopressin (AVP; or antidiuretic hormone [ADH]) and oxytocin (Oxt) are synthesized from the precursors, propressophysin (i.e., ADH–neurophysin-2) and pro-oxyphysin (i.e., vasopressin–neurophysin-2–copeptin), in separate magnocellular neurons of the supra-optic and paraventricular nuclei of the hypothalamus, these neurosecretory cells forming the supraoptico- and paraventriculohypophyseal tracts, and secreted by the neurohypophysis (posterior pituitary gland). Parvocellular neurons consist of neurosecretory cells projecting to the median eminence in addition to caudally projecting pre-autonomic cells (neurons projecting to either the medullary [nucleus tractus solitarius and rostral ventrolateral medulla] or the spinal autonomic control centers [intermediolateral cell column]) [1034].

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The neurotransmitter secretin, a regulator of feeding behavior and neuroendocrine signaling in the hypothalamus, is released upon membrane depolarization of hypothalamic cells via voltage-gated sodium and calcium channels [1012].

Adenylate Cyclase-Activating Polypeptide AdCyAP1 Adenylate cyclase-activating polypeptide, AdCyAP1, or PACAP, is widespread. Pituitary adenylate cyclase-activating peptide was first identified as a 38-amino acid peptide (PACAP38) from ovine hypothalamus in rat anterior pituitary cells, a Cterminally truncated form, PACAP27, being subsequently identified [1030]. Adenylate cyclase-activating polypeptide is detected in various regions of the brain (central thalamic nuclei, amygdala, olfactory bulb, frontal cortex, basal ganglia, nucleus accumbens, dentate gyrus, superior colliculus, substantia nigra, pituitary gland, locus ceruleus, pontine and raphe nuclei, and hippocampus), its highest expression occurring in the hypothalamic ArcN, PVN, VMH, and SON, adrenal glands, and gonads [1012, 1030]. It can localize to POMC+ neurons of the ArcN, but not POMC+ neurons within the brainstem. Receptors of AdCyAP1 include AC-activating polypeptide receptor AdCyAP1R1 (PAC1 or PACAPR1) and the VIP receptors VIP1 (VPAC1 or PACAPR2) and VIP2 (VPAC2 or PACAPR3). The AdCyAP1 receptor has a high affinity for AdCyAP and a much lower affinity for VIP [1030]. On the other hand, VIP1 has a similar affinity for VIP and PACAP27 and a lower affinity for PACAP38 and VIP2 has a similar affinity for AdCyAP1 and VIP. AdCyAP1R1, VIP1 , and VIP2 activate AC and the IP3 –Ca2+ axis. Adenylate cyclase-activating polypeptide abounds in the brain, especially in the magnocellular region of the hypothalamic PVN and SON, and pituitary and adrenal glands, whereas VIP1 and VIP2 reside primarily in the lung, liver, and testis [1030]. These receptors are identified in approximately half of POMC+ neurons and a significant proportion of NPy+ neurons [1012]. AdCyAP1 can stimulate release of both oxytocin and vasopressin and modulate the activity of other hypothalamic neuronal populations, augmenting production of GnRH, somatostatin, and CRH [1030]. This anorexigenic mediator promotes hypothalamic production of POMC and α MSH, in addition to MC4 , and α MSH secretion [1012]. Hypophagia is caused by AdCyAP1 primarily via VIP1 in the PVN and then the melanocortin system. In the VMH, the AdCyAP1–VIP1 couple may chiefly stimulate energy expenditure; hypophagia exerted by intra-VMH AdCyAP1 results from activation of ionotropic NMDA-type glutamate receptors. Binge (i.e., excessive indulgence in eating and drinking alcohol during a given period) transiently separates homeostatic feeding from hedonic feeding behavior. Within the NAcc, AdCyAP1 mimics GABA, reducing the intake of palatable food without altering homeostatic feeding [1012]. On the other hand, within the VMH, AdCyAP1 mimics AMPA, diminishing homeostatic feeding without altering hedonic feeding. Fasting and HFD decreases and increases AdCyAP1 synthesis in VMH, respectively.

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Adenylate cyclase-activating polypeptide can have both an- and orexigenic effects. VMH AdCyAP1+ neurons interact with anorexinergic Arc POMC+ neurons [1012]. Orexinergic Arc AgRP+ neurons communicate with excitatory afferent PVN AdCyAP1+ neurons. Autonomic PVN neurons projecting to preganglionic sympathetic neurons in the spinal cord are involved in hepatic glucose production and subsequent elevation in glycemia [1012]. In the brain, exposure to PACAP38 also significantly increases glycemia. In addition, the AdCyAP1–VIP1 couple is involved in light-regulated feeding behavior. Adenylate cyclase-activating polypeptide interacts with other peptides to modulate feeding. Central administration of AdCyAP1 increases the concentrations of hypothalamic hypophysiotropic neurohormones, such as vasopressin (ADH), gonadotropin-releasing hormone (GnRH1–GnRH2), somatostatin (SSt), and CRH [1012]. AdCyAP1, which counters ghrelin action, but favors activity of GLP1 and leptin, supports leptin-stimulated hypophagia and hypothermia.

Vasoactive Intestinal Peptide Vasoactive intestinal peptide is distributed throughout the gastrointestinal tract and CNS (e.g., cerebral cortex, thalamus, and hypothalamic suprachiasmatic nucleus [SCN] and PVN) [1012]. It operates as a neuroendocrine hormone and a neurocotransmitter. Its precursor, pre-proVIP, engenders PHI, PHM, the human equivalent of PHI, and PHV, a C-terminally extended form of PHM [1030]. This hormone causes vasodilation via the VIP1 and VIP2 receptors. The former is produced in the cerebral cortex and hippocampus and the latter in the thalamus, midbrain, and in the magnocellular regions of the PVN and SON in addition to SCN of the hypothalamus [1012]. Neuroprotection by VIP relies on activity-dependent neurotrophic factor (ADNF), a 14-kDa gliocyte-derived neuroprotective protein homolog to the chaperone heat shock protein HSP60, and its truncated products, ADNF14 (14amino acid [VLGGGALLRSIPA]) and ADNF9 (9-amino acid [SALLRSIPA]), neuroprotective protein (ADNP or ADNP1)247 and its derived protein, novel ADNF9-like active peptide (NAP, which contains 8-amino acids [NAPVSIPQ]), the most potent neuroprotector, in addition to IGF1 [1030, 1035]. Neuroprotection ensured by VIP is thus indirect; it requires astrocytes, which, once they are stimulated by VIP, secrete the neuroprotectors ADNF and ADNP. In fact, the structurally related peptides expressed in the central and peripheral nervous 247 Activity-dependent

neuroprotective protein, or activity-dependent homeobox gene-derived neuroprotector, ADNP1 is a VIP-responsive protein and homeodomain- and zinc finger-containing transcription factor encoded by the ADNP gene in astrocytes, which interacts with components of the BAF complex. The ADNP protein is also involved in erythropoiesis [1036]. The homologous protein ADNP2, which is encoded by the Adnp2 gene, is also dubbed homeodomain- and zinc finger-containing protein ZNF508. Both ADPN1 and ADPN2 regulate globin synthesis.

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system, AdCyAP1, PHM, and VIP,248 act as neuroprotectors via the Gs–AC– cAMP–PKA–MAP2K–ERK pathway and operate via a similar mechanism, priming ADNP1 synthesis [1037]. Moreover, VIP triggers the release of a set of astroglial neuroprotective factors (e.g., interleukins IL1 and IL6, neurotrophin-3 peptidase nexin-1 [serpin-E2], chemokines CCL3 and CCL5, ADNF, and ADNP). In astrocytes, ADNP1 production is modified by VIP via VIP2 [1038]. Plasmatic VIP concentration increases after a carbohydrate-rich meal or water loading [1012]. VIP impedes food intake and contributes to circadian feeding behavior and control of release of anorexigenic hormones, such as GLP1, insulin, leptin, and PYY in both the fasting and postprandial periods. Moreover, it can stimulate α MSH release. Intracranial injection of VIP suppresses insulin secretion and elicits secretion of adrenaline and glucagon in addition to sympathetic nerve activity [1030]. Other VIP effects include electrolyte secretion, smooth myocyte relaxation, and protection against redox stress.

Glucagon Proglucagon is synthesized in pancreatic α cells and processed into glucagon (Gcg) (Sect. 5.6.7.10). Proglucagon also contains glucagon-like peptides, GLP1 and GLP2. Glucagon is a hyperglycemic factor. Peripheral Gcg induces satiety via the NTS, area postrema, and central nucleus of the amygdala (CeNA), influencing meal size rather than between-meal interval via hepatic vagal afferents [1012]. It counters hypoglycemia and insulin action, as it stimulates hepatic glucose synthesis and secretion. On the other hand, its hypothalamic action prevents hepatic glucose production [1012]. Diet-induced obesity abolishes the anorexigenic effects of glucagon. The glucagon receptor (GcgR) triggers the Gs–AC–cAMP and PLC–IP3 –Ca2+ pathways [1030]. It is expressed mainly in the liver and kidney and, to a lesser extent, in the heart, AT, adrenal glands, pancreas, spleen, thymus, and throughout the gastrointestinal tract of rats [1030]. The glucagon receptor is also detected in the cerebral cortex, olfactory tubercle and bulb, amygdala, hippocampus, hypothalamus, thalamus, medulla, lateral septum, subfornical organ, and anterior pituitary gland of rats [1030]. It is observed

248 AdCyAP1,

PHM, and VIP are widespread, with a similar distribution (cerebral cortex, pituitary and adrenal glands, nerve endings of the respiratory system, gastrointestinal tract, and reproductive system). PACAP also lodges in the amygdala, septum, thalamus, brainstem, and spinal cord; PHI in the temporal lobes, striatum, and medulla; VIP and PACAP in immocytes; and VIP and PHI in the suprachiasmatic nuclei and hippocampus [1037]. They regulate the pituitary and adrenal glands and pancreas, relax smooth myocytes in blood vessels in addition to the respiratory, gastrointestinal, and reproductive tracts, and influence immunity. In the CNS, AdCyAP1, PHM, and VIP function as neurotransmitters, neuromodulators, and neurotrophic factors.

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in AgRP+ neurons of the ArcN and in the dorsal vagal complex (DVC) of the brainstem [1012], that is, a collection of three neighboring medullary nuclei: (1) the viscerosensory NTS, (2) the area postrema, and (3) the DMNV. Elevated circulating Gcg concentration linked to high-protein diet results from the GcgR– PKA–ERK1/2–KATP cascade in the DVC [1012]. In addition, circulating Gcg suppresses glucose sensing via hypothalamic neurons (LHA, DMH, and VMH). Central Gcg reduces concentrations of Cam2Kβ and its affector AMPK in the ArcN, where central Gcg exerts an acute anorexigenic action via the PKA–Cam2Kβ– AMPK pathway [1012].

Glucagon-Like Peptide-1 Also processed from the pre-proglucagon, GLP1, is a hormone secreted from the intestinal L cells in response to nutrient intake (Sect. 5.6.7.8). It acts as an incretin with GIP, stimulating glucose-dependent insulinotropic action [1012]. It can induce closure of the KATP channel, yielding glucose sensitivity in β cells. In addition, GLP1 provokes calcium influx via Rab3, Gi, and Gq [1030]. Moreover, GLP1 is one of the leptin effectors; leptin may enhance the central GLP1 activity [1012]. Glucagon-like peptin-1 operates via the GLP1 receptor (GLP1R). The coordinated action of GLP1R, GIPR, and GcgR reduces obesity in rodents [1012]. The GLP1 receptor, a prototypical member of the family-B GPCRs, is a major target for T2DM treatment. It can launch cAMP production by AC via the Gs subunit, Ca2+ mobilization, and G-protein-independent ERK1/2 signaling. Glucagon-like peptide-1 and oxyntomodulin initiate ligand-directed signaling. Ligand-determined signaling, i.e., ligand-induced differential signaling, refers to functional selectivity, i.e., the ability of different ligands (full and partial agonists, inverse agonists, antagonists, and allosteric modulators) of a given receptor to elicit distinct cellular responses due to ligand- and effector-binding specificity of the receptor. In other words, receptors trigger biased signaling, a given agonist preferentially activating a given signaling cascade over others (biased agonism). Biased GPCR agonists distinctly activate signaling pathways with different potencies, whereas unbiased agonists activate these pathways with equal efficiency. In addition, biased ligands stabilize receptor conformations that differ from those primed by unbiased ligands. Distinct biased ligands of the same receptor can stabilize different receptor conformations, which engage different intracellular effectors. Multiple subtypes of G proteins can be engaged with distinct efficacies and kinetics and different types of GPCR ligands and allosteric modulators bias the G-protein coupling, which then depends not only on messenger type but also on the relative amounts of the GPCR subunits and intracellular regulators. Gprotein-coupling profiles of GPCRs are related to the variety of α (16 subtypes with distinct properties), β, and γ subunits of G proteins, a given receptor being coupled with multiple types of G protein subunits with varying potency and kinetics [1039]. Furthermore, GPCRs trigger canonical and alternative signaling cascades. They signal not only via heterotrimeric GTP-binding proteins but also

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via other effectors such as β-arrestins. Biased ligands selectively change the propensity of GPCR coupling to either G proteins or β-arrestin. On the one hand, liganded GPCRs are phosphorylated by kinases and connect to arrestin, which triggers GPCR desensitization and endocytosis, thereby hindering G-protein coupling with the receptor. Arrestin attracts adaptors and clathrin for GPCR internalization and desensitization. Nevertheless, inhibition of endocytosis does not reduce the initial cAMP production, but the later cAMP accumulation, agonistbound GPCR signaling, such as that primed by Gs-coupled β2-adrenoceptor, PTH, TSH, sphingosine 1-phosphate, and dopamine, continuing once it is internalized after a short delay (arrestin-based endocytic signaling) [1040]. On the other hand, G-protein-independent signaling uses arrestin, which recruits and activates the ERK cascade. β-Arrestin thus generates its own signaling via the cRaf–MAP2K–ERK axis when the GPCR–Arr complex resides on endosomes (arrestin-based cortical signaling). Arrestin tethers to GPCRs according to two modes [1041]. Stable binding of arrestin to phosphorylated amino acids in the intracellular loop of the M1 muscarinic acetylcholine receptor, which persists for minutes after agonist removal, upregulates ERK, whereas transient binding of arrestin to the unphosphorylated receptor lessens ERK signaling, as it attracts a protein phosphatase. Therefore, the ERK signaling bias is determined by binding modes of arrestin to the receptor. Polar transmembrane residues of family-B GPCRs function not only for protein folding, but they also control activation transition, ligand-biased signaling, clusters of residues within the receptor stabilizing receptor conformation [1042].249 In the rat brain, GLP1R is identified in the lateral septum, subfornical organ, thalamus, hypothalamus, interpeduncular nucleus, posterodorsal tegmental nucleus, area postrema, inferior olive, and NTS [1030]. In rodents, peripheral GLP1 activates the sympathetic nervous system, increasing tyrosine hydroxylase synthesis, sympathetic outflow, and hence cardiac frequency and blood pressure [1030]. Upon nutrient entry into the gastrointestinal tract, peripherally secreted GLP1 connects to GLP1R on adjacent celiac and gastric neurons, which form branches of the vagal afferents [1012]. Peripheral GLP1 can also cross the blood–brain barrier and activate central GLP1R in the NTS. GLP1 sends satiation cues and triggers insulin secretion via the vagovagal reflex. The GLP1 receptor alters feeding behavior using various neural relays, such as hypothalamus (ArcN, PVN, and LHA), hindbrain nuclei (parabrachial nucleus, area postrema, medial NTS), ventral hippocampus (vHpc), and nuclei embedded within the mesolimbic reward circuitry (VTA and NAcc) [1012]. It delays gastric emptying

249 Polar

transmembrane residues avoid the core of the hydrophobic lipidic bilayer; they are buried within the interior of the protein, often lining internal water-filled cavities and forming hydrogen bonds with buried water molecules and other polar residues [1042]. The fine control of GLP1R signaling is linked to changes in interactions formed by these buried polar residues. Family-B GPCRs (e.g., GPCRs of GLP1, AdCyAP1 and VIP (VIP1 –VIP2 ), secretin, and PTH) do not share the conserved polar residues that are essential for the functioning of family-A GPCRs (e.g., β2AR); they possess their own unique set of conserved intramembranous polar residues that play a role analogous to those in family-A GPCRs.

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and has a hypophagic effect. Central GLP1 modulates utilization of circulating glucose via the PKA–AMPK axis, ERK1, and ERK2 [1012]. Central GLP1 is produced in the caudal nucleus of the NTS and ventrolateral medulla. Its receptor GLP1R lodges in the hypothalamus (ArcN, PVN, and DMH) in addition to the medullary dorsal vagal complex (area postrema, viscerosensory NTS, and DMNV) and pontine parabrachial nucleus in the brainstem. GLP1 activates CRH+ Nucb2+ (nesfatin-1+) neurons and, to a lesser extent, Oxt+ neurons in the PVN [1012]. In the PVN, GLP1 stimulates the hypothalamic– pituitary–adrenal axis and catecholamine release. The calcium-binding glucose-responsive insulinotropic and anorexigenic hormone and neuropeptide nesfatin-1250 operates in hypothalamic circuits regulating feeding and energy homeostasis. It may also directly modulate peripheral arterial resistance. It increases preproinsulin formation and insulin secretion and hampers feeding, at least in rodents. Glucose stimulates prepronesfatin synthesis and nesfatin-1 release. In DIO and T2DM, prepronesfatin production is upregulated. Nucb2+ neurons in the PVN regulate feeding via the hypothalamus and brainstem [1043]. Axon collateral branches target multiple neurons that can be located in different nuclei, for example, a single Nucb2+ neuron in the PVN projects to both the DVC and the ArcN, which are involved in feeding regulation. In the hindbrain, GLP1+ neurons project to the GLP1R+ nucleus accumbens and ventral tegmentum of the mesolimbic reward system [1012]. In the NAcc, activated GLP1R represses feeding via AMPA- (GluRs) and kainate-type glutamate (GluKs) receptors, and subsequent stimulation of GABAergic medium spiny neurons. In addition, central GLP1 increases dopamine signaling in amygdala. Activated GLP1R in the CNS increases activity of tyrosine hydroxylase, the rate-limiting enzyme of dopamine synthesis in the ventral tegmental area. VTA Dopaminergic neurons also project to the amygdala. Both GLP1 and GLP2 can prevent apoptosis and thus promote gut mucosa enlargement [1030].

Glucagon-Like Peptide-2 Also derived from preproglucagon, GLP2 is an anorexigen (Sect. 5.6.7.9). GLP2 stimulates PKA and then reduces glutamate-induced excitotoxic injury in hippocampal cells [1030]. Central, but not peripheral, administration of GLP2 stimulates hypothalamic nuclei (ArcN, PVN, DMH, VMH, and LH) via GLP2R in cooperation with the melanocortin system (MC4 ) [1012].

250 A.k.a.

DNA-binding neuropeptide NEFA and nucleobindin-2. It is encoded by the NUCB2 gene. Prepronesfatin is cleaved into nesfatin-1.

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Gastric Inhibitory Peptide Gastric inhibitory peptide promotes energy expenditure, but does not affect food intake.

Growth Hormone-Releasing Hormone Growth hormone-releasing hormone (GHRH; or GHR factor [GHRF]) derives from a precursor. It is released from neurosecretory cells in the hypothalamic ArcN. The predominant site of its receptor GHRHR is the pituitary gland, the hypothalamic peptide GHRH stimulating secretion of the pituitary growth hormone (GH). Hypothalamic hormones control the activity of target cells in the anterior pituitary gland. Hypothalamic GHRH in cooperation with somatostatin regulates via GHRHR the production of GH by pituitary somatotroph cells (or somatotropes) and secretion via the AC–cAMP–PKA axis and calcium influx [1030]. Growth hormone contributes to regulating cellular metabolism, proliferation, and differentiation, many of its effects being mediated by insulin-like growth factor, IGF1 [1030]. The IGF1 receptor is observed in the hypothalamus and pituitary gland. In the hypothalamus, IGF1 can regulate GH secretion, as it stimulates somatostatin release. In the pituitary gland, IGF1 reduces GHRHR synthesis and hence GH secretion. The GHRH+ neurons localize primarily to the ArcN and also DMH and VMH [1012]. Arc GHRH+ neurons project to the perifornical region, the lateral preoptic area, and hypothalamic suprachiasmatic and medial pre-optic nuclei (MlPON). The latter two areas are major central sites of GHRH orexigenic action. This action depends on the circadian rhythm, as it favors and impedes feeding during the light and dark phases respectively, in rats, selectively reducing protein intake from dark onset feeding, without affecting carbohydrate intake [1012]. Hence, GHRH participates in regulating the circadian feeding rhythm. Somatostatin may inhibit GHRH+ neurons.

5.6.2.4

Melanocortin Pathway

Melanocortins are a group of small anorexigenic peptidic hormones derived by the cleavage of POMC synthesized in the pituitary gland and hypothalamic neurons. Melanocortins include melanocyte-stimulating hormone (α MSH, β MSH, and γ MSH) and, in the anterior pituitary gland, ACTH. After feeding and energy excess, POMC+ neurons are activated by circulating hormones, such as leptin and insulin, and neurotransmitters, and then inhibit feeding and stimulate metabolism. Signaling by POMC+ neurons is controlled by neighboring neurons that synthesize GABA, NPy, and AgRP. Pro-opiomelanocortin is cleaved not only to ACTH and MSH but also to endorphins [1044].

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In the brain, POMC processing engenders essentially to α MSH and γ MSH. POMC neurons localize in particular to the ArcN of the hypothalamus, and project to multiple brain regions. Loss of α MSH production by POMC-deficient neurons favors obesity. In hypothalamic neurons that produce the polypeptide hormone precursor POMC, cleavage of POMC by PCSK1 creates ACTH1–39 from the middle part of the POMC precursor; ACTH1–39 is further processed by PCSK2 to ACTH1–17 [1044]. ACTH1–17 generates mature α MSH1–13 , which, once it is released into the synaptic cleft, stimulates the postsynaptic melanocortin Gs-coupled receptor MC4 in the hypothalamus and suppresses appetite. ACTH1–17 is processed during transit of POMC along the secretory pathway within hypothalamic neurons and α MSH1–13 is stored in vesicles. ACTH1–17 is sequentially cleaved by carboxypeptidaseE (CPe), peptidyl glycine α-amidating mono-oxygenase (PAM) to desacetyl α MSH1–13 , the newly exposed C-terminal glycine being converted to an amide, and N acetyltransferase, which acetylates the N-terminal serine residue. Shortlived α MSH1–13 is inactivated to α MSH1–12 by proline carboxypeptidase (prCP) in the hypothalamus.251 Proline carboxypeptidase concentration is linked to inflammation, hypertension, and metabolic disorders (obesity and diabetes). Melanocortins and their receptors are implicated in feeding, lipolysis, thermogenesis, hyperalgesia, pigmentation, memory, sexual behavior, and central regulation of cardiovascular activity and inflammation. They stimulate melanogenesis in melanocytes and steroidogenesis in adrenal cortical cells. Five melanocortin receptors (MC1 –MC5 ) are characterized by their cell-specific expression pattern and binding affinity, MC2 being the ACTH receptor. Agoutirelated protein, which is involved in controlling feeding behavior via the central melanocortin axis, and agouti signaling peptide antagonize melanocortin receptors; they prevent cAMP production mediated by stimulated melanocortin receptors especially in the hypothalamus and adrenal glands. Agouti-related protein suppresses the activity of MC3 and MC4 , as it promotes their endocytosis using arrestin; ASiP targets MC1 and hinders α MSH signaling. The orexigenic hormone ghrelin produced by the stomach and released into the blood circulation during fasting, involves melanocortin signaling. Ghrelin stimulates the liberation of NPy and AGRP, inhibiting anorexigenic POMC+ neurons and increasing food intake. Furthermore, it upregulates hypothalamic prCP formation [1045]. The feeding-inhibitory melanocortin pathway based on leptin-responsive POMC+ CART+ neurons in the ArcN operates via α MSH on pre- and postsynaptic melanocortin receptors MC3 and MC4 , AgRP antagonizing α MSH at these receptors [1011]. The MC4 receptor is synthesized in several hypothalamic nuclei (PVN, LHA, and ArcN) in addition to other brain regions, such as parasympathetic and sympathetic preganglionic neurons in the medulla and spinal cord.

251 The

serine peptidase prolyl carboxypeptidase (prCP) operates in the renin–angiotensin and kallikrein–kinin axes. In hypothalamic POMC+ neurons, it processes α MSH1–13 [1045].

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Leptin activates POMC+ neurons that innervate sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord, hence contributing to increasing arterial pressure and cardiac frequency as well as regulating energy expenditure and insulin secretion and sensitivity [1011]. Elevated leptin concentration in DIO causes hypertension via LepR of neuronal circuits in the DMH [1046]. The MC4 receptor is detected in several other brain regions involved in feeding regulation such as the nucleus accumbens, which possesses GABAergic neurons projecting to the LHA [1011]. Therefore, the melanocortin system contributes not only to the homeostatic control of feeding, but also to its hedonic aspects.

5.6.2.5

Neuropeptide-Y Family

Members of the NPY family, NPy, peptide Tyr–Tyr (PYY), and pancreatic polypeptide (PPy), are produced by cells at distinct levels of the gut–brain axis [1028].

Neuropeptide-Y Synthesis Sites Neuropeptide-Y is produced at all levels of the brain–gut axis, that is, enteric neurons (enteric neural plexus, interneurons, descending inhibitory motoneurons of the myenteric plexus [where it co-localizes with vasoactive intestinal polypeptide and nitric oxide synthase], and noncholinergic secretomotor neurons [SMNs] of the submucosal plexus), primary afferent neurons originating in the dorsal root ganglia (particularly injured mid-sized and large sensory neurons), several neuronal circuits of the brain, and postganglionic sympathetic neurons (where it co-localizes with NAd and ATP) [1028]. It preferentially resides in sympathetic neurons supplying the vasculature, but is absent from sympathetic neurons innervating the gastrointestinal mucosa. In the spinal cord, an abundant NPy+ neuropil is made of at least three different neuronal populations: (1) NPy+ GABA+ inhibitory interneurons, (2) descending noradrenergic neurons originating in the locus ceruleus, and (3) primary afferent nerve endings. In the spinal cord of rats and mice, the NPy receptor Y1 lodges on seven distinct neuron types of the dorsal horn and Y2 exclusively on the central terminals of primary afferent neurons in the superficial laminae [1028]. In the brain, NPy is the most abundant neuropeptide, especially in the NTS, ventrolateral medulla, periaqueductal grey, locus ceruleus, thalamus, hypothalamus (e.g., the arcuate and paraventricular nuclei), septum, hippocampus, amygdala, basal ganglia, nucleus accumbens, and cerebral cortex [1028]. This neurotransmitter is used by noradrenergic neurons originating in the locus ceruleus and issuing both ascending and descending projections in the CNS, NPy+ AgRP+ neurons of the ArcN, and various circuits of the limbic system.

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Production of Peptide-YY and Pancreatic Polypeptide On the other hand, PYY and PPy are exclusively formed by endocrine cells of the digestive tract. Peptide-YY is produced by enteric neurons of the stomach, pancreatic endocrine cells, and endocrine L cells that abound in the lower gastrointestinal tract in proportion to nutrient intake [1028]. It is postprandially released from intestinal L cells. Gastric acid secretion, cholecystokinin, bile acids, and C12-length and short-chain FFAs (butyrate) stimulate PYY secretion. PYY+ L cells also produce glicentin and glucagon-like peptides GLP1 and GLP2. These cells possess bitter and sweet taste receptors in addition to taste-related gustducin and thus serve as chemosensors. In the rodent brain, PYY+ neurons localize to the hypothalamus, pons, medulla and spinal cord [1028]. Pancreatic polypeptide is synthesized by pancreatic endocrine F cells in addition to endocrine cells in the small and large intestines, distinct from PYY+ cells [1028]. It is also postprandially secreted. Its release and actions on feeding and digestion require the parasympathetic vagus nerve.

Cleavage of Neuropeptide-Y and Peptide-YY Once released, the N-terminus of NPy and PYY is truncated by dipeptidyl peptidase DPP4 and other enzymes (e.g., aminopeptidase-P and membrane metalloendopeptidase [MME]),252 yielding NPy3–36 and PYY3–36 fragments [1028]. PYY3–36 is a preferential agonist of the Y2 receptor.

Neuropeptide-Y Receptors The NPy receptors (Y1 –Y2 and Y4 –Y5 in humans), which are involved in circadian rhythm control, are also activated by PYY and pancreatic polypeptide, with given affinities for the different members of the NPY family and their fragments. Whereas NPy and PYY do not markedly differ in their affinities for Y1 , Y2 , and Y5 , PYY3–36 , the most common circulating form, is a preferred agonist of Y2 and PPy binds preferentially to the Y4 receptor [1028]. Whereas full-length PYY1–36 activates Y1 , Y2 , and Y5 , PYY3–36 preferentially operates via Y2 and accessorily Y5 . The major NPy receptor subtypes in the brain are the widespread Y1 and Y2 receptors, whereas Y4 and Y5 are restricted to some brain regions, such as the NTS, area postrema, nucleus ambiguus, hypothalamus, thalamus, and amygdala. In the brain, NPy binds to its Gi/o-coupled receptors, which trigger AC inhibition. It increases feeding and lipid (energy) storage facilitates learning and memory, as it modulates hippocampal activity, impedes anxiety, and regulates mood

252 A.k.a.

atriopeptidase, enkephalinase, neprilysin, and neutral endopeptidase-24.11.

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and stress resilience. Both PPy and PYY signal to the brain, thereby attenuating food intake and depression-related behavior [1028]. Neuropeptide-Y stimulates vSMC contraction, modulates release of pituitary hormones, and hinders insulin release. Its signaling mediators encompass Ca2+ channels in neurons and K+ channels in CMCs and vSMCs.

Neuropeptide-Y and Nociception In the gut–brain axis, the neurotransmitter NPy, an important regulator of emotion processing, inhibits nociceptive transmission in the spinal cord and brainstem arising from intestinal obstruction, inflammation, and damage [1028]. Its Y1 , Y2 , and Y5 receptors are widely expressed in cerebral areas involved in anxiety, mood, cognition, and stress resilience regulation. NPy acts as a stress mediator in the central and peripheral nervous systems and in the cardiovascular apparatus and gastrointestinal tract (e.g., stress-induced defecation and feeding), in addition to metabolism, immunity, and stress adaptation. The sensory innervation of the gastrointestinal tract (mesentery, serosa, muscularis, and mucosa) relies on two major neuronal populations: (1) spinal afferent neurons originating from the dorsal root ganglia and (2) vagal afferent neurons originating primarily from the nodose ganglion. Spinal afferent neurons, which contain low NPy amounts, terminate in the spinal cord, where interneurons and descending noradrenergic neurons have a higher NPy content. NPy tethers to Y1 and Y2 in the spinal cord and counters thermal, chemical, and mechanical hyperalgesia. NPy can control pain transmission in the spinal cord primarily via Y2 and subsequent inhibition of transmitter release from the terminals of primary afferent neurons and via Y1 and subsequent inhibition of postsynaptic neurons in the dorsal horn [1028]. Vagal afferent neurons also contribute to visceral nociception, particularly visceral chemonociception. In the gut–brain axis, NPy may function as both a transmitter and modulator of the communication between vagal afferents and their projection neurons in the NTS. NPy operates via Y2 (most likely presynaptic) and Y4 in the NTS, which control the chemonociceptive input from the stomach to the brainstem [1028]. Neuropeptide Y can limit the impact of psychological stress on the gut– brain axis. The amygdala, a major brain region coordinating behavioral stress responses, contains high concentrations of NPy, anxiolytic excitatory postsynaptic Y1 , emotion-supporting presynaptic Y2 and Y4 , and Y5 [1028]. Intestinal hormones, such as ghrelin, PYY, PPy, GLP1, and GLP2 also influence emotion, anxiety, and mood. Ghrelin released from the upper gastrointestinal tract upon hunger functions as an anxiolytic and antidepressant [1028]. PYY3–36 promotes hedonic sensation via direct access to the brain and activation of vagal afferents to the brainstem. The effect of PPy preferentially mediated by Y4 diminishes anxiety-like and depression-related behaviors via peripheral action and

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the area postrema [1028]. On the other hand, GLP1 favors anxiety-related behavior, whereas GLP2 attenuates a depression-like state [1028].

Neuropeptide-Y and Peptide-YY Effects on Feeding and Digestion The gastrointestinal tract produces and releases multiple para- and endocrine messengers that regulate feeding and satiety to ensure energy homeostasis. In gut– brain signaling, the satiety factors PPy and PYY slow the gastrointestinal transit of chyme and prevent further food intake. Pancreatic polypeptide release is under vagal cholinergic control. It activates Y4 on neurons in the brainstem, hypothalamus, and amygdala [1028]. It can enter the brain preferentially in circumventricular organs, which are structures in the brain characterized by their extensive vasculature and lack of a normal blood–brain barrier, such as the area postrema and subfornical organ. Pancreatic polypeptide decreases food intake and increases energy expenditure via the vagus nerve. Peptide-YY is a relatively selective Y2 receptor agonist. Both PYY1–36 and PYY3–36 hamper gastric acid secretion, gastrointestinal transit, and food intake in both lean and obese subjects via stimulation of Y2 on vagal afferent neurons and in the hypothalamus [1028]. In the brain, PYY3–36 reduces feeding primarily via presynaptic Y2 on orexinergic NPy+ AgRP+ neurons in the ArcN, inhibiting their action and hence disinhibiting POMC+ neurons, as orexinergic and anorexinergic neurons exert a mutual inhibition. In the brain, Npy mainly targets Y1 (also Y5 ) in the hypothalamus, brainstem, nucleus accumbens, and corticolimbic system implicated in the regulation of appetite. In the digestive tract, NPy and PYY preclude gastrointestinal motility and water and electrolyte (e.g., Cl− ) secretion [1028]. Both PYY and NPy exert a tonic gastrointestinal anti-secretory activity via epithelial Y1 and neural Y2 receptors. Antagonism of Y1 and Y2 explains colonic transit acceleration by Y1 and inhibition by Y2 . Although Y2 plays a major role in the anti-secretory and pro-absorptive action of NPy and PYY, Y1 on SMNs and epitheliocytes and Y4 on enterocytes also contribute to these effects. The enteric and cerebral neuropeptide NPy and the gut hormones PYY and PPy play specific roles at various levels of the brain–gut axis. They inhibit gastrointestinal motility and secretion, thereby affecting signals sent to the brain via a neural route or the bloodstream [1028]. Polypeptide Y acts preferentially via Y4 and Y5 . It hampers gastric emptying via the vagus nerve and acid secretion via Y1 and Y2 and its action in the brainstem and stomach, hence reducing appetite, in addition to intestinal electrolyte and water secretion and intestinal peristalsis by neural and non-neural mechanisms [1028]. Inhibition of PYY1–36 and PYY3–36 on gastrointestinal motility and secretion is mediated by Y1 on enterocytes, myenteric, and submucosal neurons and ECs, Y2 on myenteric and submucosal neurons and extrinsic primary afferent nerve fibers, and Y4 on enterocytes [1028]. PYY may act as an ileal and colonic brake set

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into operation when lipids reach the lower gut that slows gastric emptying and intestinal transit, which, in coordination with its anti-secretory action, facilitates nutrient absorption. The receptor of ethanolamide oleoylethanolamide (OEA) and LPC, glucosedependent insulinotropic receptor GPR119 (or GPCR2) respond to luminal nutrients and release PYY and GLP1 from L cells [1028]. The NPy–PYY system has an impact on the composition and function of the gut flora. The appetite stimulator, NPy, has an anti-bacterial effect against Escherichia coli, Enterococcus faecalis, and Lactobacillus acidophilus [1028]. In addition, NPy released from sympathetic nerves in lymphoid tissues in contact with immunocytes (e.g., dendrocytes, granulocytes, B, T, and NK lymphocytes, monocytes, and macrophages), acts as a neuro-immune transmitter that activates or represses immunity according to its concentration, activated Y receptor subtype, and immunocyte types [1028]. It modulates immunocyte transfer, helper Tcell differentiation, NK-cell activity, phagocytosis, cytokine secretion, and ROS production. It activates antigen-presenting cell function. It stimulates Y1 and exerts a gastrointestinal proinflammatory action. NPy promotes NOS2 expression and subsequent redox stress and inflammation. In the mouse gastrointestinal tract, NPy+ nerves contact immunocytes such as IgA-producing lymphocytes in the ileum lamina propria. In humans, it stimulates proliferation of lymphocytes in the colonic lamina propria. Nonetheless, the proinflammatory NPy effect may be countered by its induced vasoconstriction, the sympathetic action on splanchnic resistance arteries being co-mediated by the sympathetic triad, that is, ATP, NAd, and NPy. NPy potentiates NAd- and ATP-primed mesenteric vasoconstriction via postjunctional Y1 receptor [1028]. On the other hand, colonic PYY concentration declines in patients with inflammatory bowel disease. Conversely, the gut microbiota influences the gut–brain axis via PYY and NPy. For example, dysbiosis of the intestinal flora upregulates the formation of brain-derived neurotrophic factor (BDNF) in the hippocampus [1028]. Longterm treatment of mice with the probiotic Lactobacillus rhamnosus reduces anxiety and improves stress coping via the vagus nerve. Neuropeptide Y participates in the regulation of emotion and affective behavior. NPy+ neurons in the arcuate and paraventricular nuclei protect against behavioral disturbances in response to infection and immunity stimulation. Proinflammatory cytokines reach the brain via the bloodstream in addition to exciting vagal afferent neurons; they then elicit cytokine formation by microgliocytes and astrocytes. Although the human gut has a limited repertoire of glycoside hydrolases, the gut microbiota synthesizes these enzymes, which process dietary carbohydrates to acetate, propionate, and butyrate [1028]. ScFAs can interact with FFAR3+ PYY+ enteroendocrine cells in the intestinal epithelium.

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5.6.3 Enteric Nervous System The digestive tract from the esophagus, stomach, small intestine, to the colon is innervated by the CNS and its walls are endowed with the ENS, which regulates locally the behavior of digestion organs independently of the CNS, both the ENS and the CNS sharing the structure and neurochemistry [1047]. The ENS, a quasi-autonomous component of the nervous system that participates in digestion control (food degradation, nutrient absorption, and waste removal, in addition to mechanical mixing and rhythmic propulsive muscular contractions), cooperates with the sympathetic and parasympathetic branches of the autonomic nervous system (remote control). It communicates with CNS reflex and command centers and sympathetic ganglia-associated neural circuits via a bidirectional information flux between the ENS and CNS through the vagus and pelvic and sympathetic nerves in addition to between the ENS and sympathetic prevertebral ganglia [1048]. The ENS (neuron number O[108 ]) consists of a huge number (O[103 ]) of small ganglia, the great majority of which form the myenteric and submucosal plexuses [1048]: 1. The myenteric plexus is a continuous circuit from the esophagus to the internal anal sphincter. It resides between the outer longitudinal and inner circular smooth muscle. 2. The submucosal plexus exist only in the small and large intestines. These plexuses also contain gliocytes, which nourish neurons, mastocytes, which ensure protective inflammation, and the blood–ENS barrier, which prevents entry of harmful substances. They contain sensors of chemical species of the alimentary bolus during the oro-aboral transit and physicochemical properties of the digestive tract environment. These sensors monitor the state of food processing and propulsion within the digestive tract. The nervous system controls not only food transit time and mucus secretion but also bacterial species of the digestive ecosystem. Conversely, the intestinal biota influences neurotransmitter levels. Neurons also project from the ENS to the prevertebral ganglia in addition to organs implicated in food intake and processing, the trachea, gallbladder, and pancreas. The digestive tract smooth muscle is organized in layers of smooth myocytes that operate as mechanical units, as they are interconnected via gap junctions, under the control of pacemaker cells, interstitial cells of Cajal. The relative roles of the ENS and CNS differ considerably along the digestive tract [1048]: • The CNS determines the movements of the esophagus via neural pattern generators and plays a major role in monitoring the state of the stomach and in controlling its contractile activity and acid secretion via the vagovagal reflexes. Voluntary control of defecation is exerted via the CNS, with its defecation centers in the lumbosacral spinal cord and pelvic connections.

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• The ENS in the small intestine and colon involved in the sensory-motor control contains reflex circuits with their sensory afferent neurons, which are sensitive to chemical and mechanical stimuli, interneurons, and motor neurons that regulate muscular activity, transmucosal fluxes, local blood flow, and endocrine and immune functions. Major neurotransmitters of the ENS include dopamine, glutamate, NAd, NO, and serotonin, which triggers peristalsis. The gastrointestinal tract is also a source of neuropeptides, enkephalins, and benzodiazepines. Networks of enteric ganglia are connected by interganglionic bundles. Most enteric motor neurons, which act on effector cells (epitheliocytes, smooth myocytes of the digestive tract walls, pacemaker, neuroendocrine, mucosal gland and vascular cells, and immunocytes) localize to the myenteric plexus [1049]. Hence, the myenteric plexus comprises intestinofugal (IFNs) and enteric primary afferent neurons (EPANs), ascending (AINs) and descending interneurons (DINs), excitatory (ECMMNs) and inhibitory circular (ICMMNs) and longitudinal muscle motoneurons (LMMNs), SMNs and vasomotor neurons (VMNs), in addition to projections of EPANs of the submucous plexus. Enteric primary afferent neurons of myenteric and submucous plexuses respond to luminal chemical stimuli and wall stretch [1049]. These neurons receive a slow synaptic input, probably mediated by tachykinins from other EPANs, and are connected to AINs, DINs, LMMNs, ECMMNs, and ICMMNs: • Excitatory circular muscle motoneurons employ acetylcholine and tachykinins [1049]. • Inhibitory circular muscle motoneurons contain NO, ATP, VIP, and AdCyAP1 (or PACAP). • Ascending interneurons possess synthases for acetylcholine, tachykinins, and opioids. • Descending interneurons include cholinergic, somatostatin and ChAT+ (choline acetyltransferase), serotonin and ChAT+, NOS, VIP, and ChAT+, in addition to NOS and VIP+ DINs. • Secretomotor neurons in the myenteric ganglia comprise two groups, cholinergic and VIP+ SMNs. • Intestinofugal cholinergic neurons project from the myenteric ganglia to the prevertebral ganglia. They form short intestino-intestinal inhibitory reflex pathways, activation of which reduces both motor and secretomotor activity. In addition to the reflex motor response of the longitudinal muscle, many polarized enteric motor pathways control the motor activity of the intestinal circular smooth muscle [1049]. Small rings of circular smooth muscle can contract independently with a spatiotemporal coordination due to interconnected neural modules with repeated and overlapping circumferential, ascending, and descending circuits, peristalsis corresponding to the sequential contraction of the circular smooth muscle initiated by local stretch due to the intraluminal content that propels the alimentary bolus.

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The circumferential circuit, which is activated by mechanical stimulation of EPANs, synapses with local inhibitory and excitatory motoneurons. The ascending excitatory circuit involves EPANs, AINs, and ECMMNs. The descending inhibitory circuit relies on a different EPAN type with long anal projections connected to ICMMNs. The descending excitatory circuit includes mechanosensitive EPANs in addition to ECMMNs. The secreto- and vasomotor neural pathways comprise EPANs of both the submucosal and myenteric plexuses and cholinergic and noncholinergic SMNs and VMNs in the submucosal ganglia. In mice, the ENS neuroglial network is composed of overlapping clonal units. Neurons and gliocytes, which regulate gut function, derive from neural crest progenitors [1050]. The ENS regulates the gut flora, in addition to food flux and processing. Peristalsis influences gut microbiota composition, as it suppresses the growth of the proinflammatory bacterial community, at least in zebrafish [1051]. For example, an expanded population of Vibrio combined with a loss of Faecalibacterium prausnitzii or Escherichia can cause neutrophil recruitment. Conversely, the introduction of Escherichia or Shewanella ameliorates the intestinal state. The intestinal microbiota is linked to the body metabolism and circadian clock via the circadian transcription factor, nuclear factor interleukin-3-regulated protein NFIL3 [1052]. The epitheliocyte circadian transcription factor NFIL3 activates transcription from the IL3 promoter, but represses the transcriptional activity of Per1 and Per2. Its activity oscillates diurnally in intestinal epitheliocytes. It controls a circadian program of lipid metabolism, as it is involved in regulating lipid uptake in and export from intestinal epitheliocytes. Flagellin and lipopolysaccharide produced by certain intestinal bacteria control the amplitude of the circadian oscillation of NFIL3 via group-3 innate lymphoid cells (ILC3), STAT3, and the epithelial clock [1052].

5.6.4 Peptidic Messengers: Maturation by Cleavage Neuropeptides constitute the largest class of messengers (>100 neuropeptides). Most peptidic hormones and neurotransmitters are synthesized as precursors that undergo proteolysis. A carboxypeptidase cleaves a peptide bond at the carboxy (C)-terminus of a peptide or protein to liberate a single amino acid residue, whereas an aminopeptidase hydrolyzes peptide bonds at the opposite amino [N]-terminus of the protein. The pancreatic carboxypeptidases CPa1, CPa2, and CPb are involved in food digestion. However, most carboxypeptidases operate in protein maturation, especially the synthesis of neuroendocrine peptides, in addition to growth factor production, blood coagulation, and wound healing, among other processes.

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Carboxypeptidases are usually classified into three major categories: 1. Metallocarboxypeptidases utilize a metal in their active site. 2. Cysteine (thiol) carboxypeptidases use cysteine residues as active sites. 3. Serine carboxypeptidases employ serine residues as active sites. For example, the zymogens253 and apoenzymes,254 carboxypeptidases, CPa and CPb, which are formed from precursors (pre-procarboxypeptidases) and activated 2+ by trypsin, in addition to CPd are metallocarboxypeptidases (CPxZn , x: a, b, d). 255 Another type of classification refers to their substrate preference : • Carboxypeptidase-A (A: aromatic or aliphatic) targets amino acids containing aromatic or branched hydrocarbon chains. • Carboxypeptidase-B (B: basic) cleaves positively charged amino acids (Arg or Lys). • Carboxypeptidase-C has a preference for hydrophobic residues. • Carboxypeptidase-D processes basic amino acids in addition to peptides with hydrophobic residues. • Carboxypeptidase-E (CPe), or carboxypeptidase-H (CPh), also called enkephalin convertase, is a prohormone processor. The membrane-bound metallocarboxypeptidase CPd of the trans-Golgi network removes the C-terminal Arg and Lys residues from peptides with an optimal pH ranging from 5 to 7. It participates in the synthesis of neuropeptides and peptide hormones following the action of furin, an endopeptidase of the trans-Golgi network. Carboxypeptidase-E, or CPh, is responsible for C-terminal trimming of peptidic hormones and neurotransmitters [1053]. Hence, many substrates of CPe are neuropeptides. Pro-protein convertases cut the precursor at specific sites to generate intermediates containing a C-terminal basic residue (lysine or arginine). These intermediates are then cleaved by CPe to remove the basic residues. This sequential processing suffices to produce the active peptide for many peptides, but, for some peptides, additional processing steps, such as C-terminal amidation, are subsequently required. Conversion of C-terminally Gly-extended peptides into C-terminally α-amidated peptides is achieved by a sequential two-step process catalyzed by PAM.256

253 A

zymogen is an inactive molecule that is converted into an enzyme when activated by another enzyme. 254 An apoenzyme is a protein that forms an active enzyme upon combination with an additional small molecule, the cofactor, either an inorganic molecule such as metal (e.g., Fe2+ , Mg2+ , Mn2+ , or Zn2+ ) or a small organic molecule, a coenzyme (e.g., biotin [vitamin-B7 (or -H) or coenzyme-R] or coenzyme-A). 255 For example, cathepsin-A is a lysosomal carboxypeptidase in addition to β-galactosidase and neuraminidase. 256 A.k.a. PHM and peptidyl α-hydroxyglycine α-amidating lyase (PAL), as it has two enzymatically active domains. These catalytic domains work sequentially to mature neuroendocrine

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Table 5.43 Peptidases of the PCSK family (LPC lymphoma pro-protein convertase, MBTPS1 membrane-bound transcription factor peptidase site-1, NARC neural apoptosis-regulated convertase, NEC neuroendocrine convertase, PACE paired basic amino acid cleaving enzyme, PC prohormone (pro-protein) convertase, SKI subtilisin/kexin isozyme, SPC subtilisin-like pro-protein convertase) Type PCSK1 PCSK2 PCSK3 PCSK4 PCSK5 PCSK6 PCSK7 PCSK8 PCSK9

Other aliases PC1, PC3, SPC3, NEC1 PC2, SPC2, NEC2 Furin, SPC1, PACE PC4, SPC5 PC5, PC6, SPC6 SPC4, PACE4 PC7, PC8, SPC7, LPC MBTPS1, S1P, SKI1 PC9, NARC1

The isozyme PCSK3 corresponds to furin and the subtype PCSK8 to site-1 peptidase S1P (i.e., membrane-bound transcription factor peptidase, site 1 [MBTPS1]). The serine peptidase PCSK8 catalyzes the first activation step of the membrane-bound transcription factors SREBPs and hence controls the lipid composition of cells. In response to cholesterol deprivation, PCSK8 cleaves SREBPs between two membrane-spanning domains, releasing them from membranes of the ER and GB in co-operation with SCAP, sterols blocking the ER–GB transport and cleavage using the sterol-sensing escort SCAP. Other MBTPS1 substrates include BDNF, N acetylglucosamine 1-phosphotransferase α–β subunits (GNPTαβ), and activating transcription factor ATF6, another membrane-bound transcription factor that activates genes in the ER stress response

The precursors contain multibasic amino acid cleavage sites with the consensus Lys(Arg)–Xn –Arg (X: any amino acid, except Cys; n = 0, 2, 4, 6). These multibasic sites are often cleaved by peptidases of the PCSK family (PCSK1– PCSK9),257 PCSK1 and PCSK2 (Table 5.43).258 The production of active PCSK2 requires the chaperone secretogranin-5, which also inhibits this enzyme [1053]. The major peptidases that cleave basic sites include the trans-Golgi network enzyme PCSK3 (or furin), and two secretory vesicle enzymes, PCSK1 and PCSK2. Other furin-like peptidases of the trans-Golgi network include PCSK7259 and

peptides. This bifunctional enzyme is engendered from a pre-proprotein. Alternative splicing leads to soluble and integral membrane bifunctional PAMs in addition to a soluble monofunctional mono-oxygenase [1054]. Inclusion of exon A between the PHM and PAL domains (PAM1) in addition to monofunctional PAM4 is associated with multiple sulfated O glycans. The transmembrane domain is eliminated with Tyr965 sulfation (soluble bifunctional PAM3). 257 PCSK: pro-protein convertase subtilisin/kexin. Members of the PCSK family are related to the yeast peptidase kexin and bacterial subtilisin. 258 PCSK2 abounds in the intermediate lobe of the pituitary gland (hypophysis), where it may process β-lipotropin to β-endorphin and cleave ACTH into α-melanocyte-stimulating hormone precursor. 259 A.k.a. pro-protein convertase PC7 or PC8.

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PCSK5.260 These peptidases produce intermediates that contain C-terminal Lys and/or Arg residues. These C-terminal basic residues are removed by CPd in the trans-Golgi network or by CPe in secretory vesicles. Pro-protein convertase subtilisin/kexin 1 cleaves prodynorphin at a monobasic site, but cleaves the monobasic processing site of prosomatostatin inefficiently [1055]. Dynorphin convertase, a thiol peptidase present at high concentrations in the brain, pituitary gland, and ileum, may be involved in the monobasic processing of precursors other than prodynorphin. PCSK6 cleaves prosomatostatin at its monobasic site in the secretory pathway [1055]. Among four PCSK6 isoforms produced by mRNA alternative splicing, PCSK6c is formed in β cells, but not in α cells of the islets of Langerhans. Both PCSK1 and PCSK2 process pro-insulin to insulin in the pancreatic β cells in addition to POMC to ACTH and other products in the anterior and intermediate lobes of the hypophysis (pituitary gland) [1055].261 Several peptidic regulators of food intake derive from a single precursor, proPCSK1n,262 or proSAAS,263 PCSK1n being a potent inhibitor of PCSK1 [1056]. Proteolytic cleavage of proPCSK1n at a K–X–X–R site engenders the fragments proPCSK1n(34–41) and proPCSK1n(42–61) [1053]. The C-terminal peptide corresponds to the proPCSK1n(221–260) fragment. PCSK1n is initially cleaved in the Golgi body or trans-Golgi network by furin and/or furin-like enzymes and carboxypeptidase-D. Resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides. The small forms are generated by secretory granule prohormone convertases and CPe.. Pro-protein convertase subtilisin/kexin 1n has similar characteristics to chromoand secretogranins, which form a category of neuroendocrine proteins,264 which are cleaved in neuroendocrine tissues to generate neuroendocrine hormones and neurotransmitters. ProPCSK1n is cleaved in the brain and pituitary gland. A different post-translational processing of proglucagon (proGcg) generates distinct sets of peptides with opposing activities, the corresponding sequences

260 A.k.a.

proprotein convertase PC5 or PC6. cells overexpressing the PCSK1 inhibitor PCSK1n, processing of POMC (241 amino acids), itself synthesized from the 285-amino acid pre-pro-opiomelanocortin (pre-POMC), into either ACTH, or β-lipotropin, or β-endorphin is greatly reduced. In the ArcN, POMC is cleaved to α α MSH, a neurotransmitter, which acts via melanocortin receptors MC3 and MC4 on neurons of other hypothalamic regions to cause hypophagia. POMC+ neurons have a catabolic effect; they raise energy expenditure. 262 PCSK1n: pro-protein convertase subtilisin/kexin type-1 inhibitor. 263 A protein precursor that contains the amino acid sequence Ser–Ala–Ala–Ser. 264 Granins are regulated neuroendocrine secretory proteins lodging in dense-core secretory vesicles of endocrine and neuroendocrine cells storing amine and peptide hormones and neurotransmitters. They include chromogranin-A, -B, also called secretogranin-1, and -C, or secretogranin-2, and secretogranin-3, and -5, some of which can be precursors of auto-, para-, and endocrine messengers. Chromogranins can also be involved with the sorting of proteins into the regulated secretory pathway. 261 In

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in proglucagon being linked by pairs of basic amino acids (Lys–Arg or Arg– Arg) [1055, 1057]. Proglucagon is initially cleaved at the Lys79–Arg80 site and engenders glicentin and major proglucagon fragment (MPGF). The amino acid sequence of glicentin contains that of glucagon; hence, in the pancreas, glicentin serves as an intermediate in the formation of glucagon. • In the pancreas, proglucagon engenders glicentin-related pancreatic polypeptide (GRPP; proGcg(1–30) ), glucagon (proGcg(33–61) ), intervening peptide IP1 (proGcg(64–69) ), and MPGF (proGcg(72–158) ). Glicentin (proGcg(1–69) ) is cleaved at the Lys31–Arg32 and Lys62–Arg63 sites in GRPP, glucagon, and IP1, whereas MPGF accumulates and is slowly and partly processed to GLP1 after cleavage at the Arg109–Arg110 site. In pancreatic islet α cells, proglucagon is cleaved by PCSK1 to glucagon-like peptide-1 (GLP1 [proGcg(72–107/108) ]) and by PCSK2 to glucagon. These cells possess a high concentration of PCSK2, which processes proglucagon to glucagon, but a very low PCSK1 concentration.265 • In the gut and brain, proglucagon generates glicentin (enteroglucagon [proGcg(1–69) ]), truncated GLP1 (t GLP1 [proGcg(78–107) ]), intervening peptide IP2 (proGcg(111–122/123) ), and glucagon-like peptide-2 (GLP2 [proGcg(126–158) ]). Hence, in intestinal endocrine L cells, proglucagon cleaved by PCSK1, which is both necessary and sufficient for complete proglucagon processing, engenders GLP1, a potent incretin hormone, and GLP2, a regulator of gut mucosal growth and integrity. An additional cleavage of GLP1 at Arg109 yields short active forms of GLP1, collectively termed truncated GLP1, GLP1(7–37) (proGcg(78–108) ) and 2 its desglycyl C-terminally amidated counterpart, GLP1(1–36) –amide (GLP1NH (1–36) [proGcg(78–107) ]). Production of active t GLP1 involves the cleavage of the proglucagon at the monobasic Arg109 processing site. The amidated and Glyextended forms have similar activities and overall metabolism [1057]. In humans, almost all of GLP1 secreted from the gut is amidated. Glicentin engenders GRPP and oxyntomodulin (enteroglucagon [proGcg(33–69]) ]) using PCSK2. The pancreatic β cell secretes glucagon that counterbalances the hypoglycemic action of insulin, whereas the intestinal L cell releases a potent insulinotropic hormone, t GLP1(7–36) , the active truncated GLP1 form produced from GLP1(1–36) cleavage by PCSK1 or PCSK3. The proglucagon gene engenders various related peptides that target distinct class-I I GPCRs for specific effects (Table 5.44).

265 PCSK1

can cleave the Lys31–Arg32, Lys79–Arg80, and Arg109–Arg110 processing sites of proglucagon [1055].

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Table 5.44 Class-I I peptide hormone receptors (Source: [1030] AdCyAP adenylate cyclaseactivating polypeptide, CalcRL calcitonin receptor-like receptor, CRH corticotropin-releasing hormone, CGRP calcitonin gene-related peptide, GHRH growth hormone-releasing hormone, GIP gastric inhibitory peptide, PTH parathyroid hormone, PTHRP PTH-related peptide, VIP vasoactive intestinal peptide) Receptor SctR GcgR

Ligand Secretin Glucagon

GLP1R GLP2R GHRHR AdCyAP1R1

GLP1 GLP2 GHRH AdCyAP1

VIP1 (VIPR1) VIP2 (VIPR2) CRH1 (CRHR1) CRH2 (CRHR2) PTH1 (PTHR1) PTH2 (PTHR2) CalcR

VIP, AdCyAP1 VIP, AdCyAP1 CRH, urocortin Urocortin-1/2/3

CalcRL GIPR

PTH1, PTHRP PTH2 Calcitonin, amylin, CGRP CGRP, adrenomedullin GIP

Primary function Pancreatic secretion Glycogenolysis and gluconeogenesis, insulin secretion Secretion of insulin and glucagon Proliferation of intestinal epitheliocytes Growth hormone secretion Glucose homeostasis, nociception, learning, memory, circadian rhythm Neuromodulation, T-cell differentiation Circadian rhythm Secretion of adrenocorticotropin hormone (ACTH) Stress-related behavior, neuroendocrine function Ca2+ homeostasis (bone and kidney), tissue differentiation Renal vasodilation Ca2+ homeostasis (calcitonin) Glucagon secretion (amylin) Microvascular tone Secretion of insulin

The proglucagon gene engenders various related peptides, glicentin, glicentin-related pancreatic polypeptide (GRPP), intervening peptide IP1, major proglucagon fragment (MPGF), glucagon, and the glucagon-like peptides, GLP1 and GLP2, which target distinct receptors for specific effects

5.6.5 Gastrointestinal Epithelial Barrier The intestinal mucosa is covered by a single layer of epitheliocytes fastened by tight junctions that limits intestinal permeability. The intestinal epithelium is structurally organized in crypts and villi. Intestinal cells of crypts and villi include absorptive enterocytes and secretory cells, such as Paneth, goblet, tuft, and enteroendocrine cells in the small intestine (Table 5.45). Thus, the intestinal epithelium consists of [1058]:

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Table 5.45 Intestinal epitheliocytes (Source: [1059]) Intestinal epitheliocyte type Intestinal epithelial stem cell Enterocyte

Paneth cell

Goblet cell Enteroendocrine cell

Tuft cell Microfold cell (M cell)

Role Renewal of the epithelium Nutrient absorption Transcytosis of IgA from the lamina propria to the lumen side, secretion of antimicrobial proteins Construction of stem cell niche Secretion of mucus, antimicrobial agents (α-defensins, cathelicidins, lysozyme, Reg3γ) Secretion of mucus, RELMβ, TFF3 Transport of luminal antigens across epithelia Secretion of regulators of appetite and digestion Cck (I cells), GLP1, GLP2 (L cells), 5HT (enterochromaffin cells) Induction of type-2 immune response (secretion of IL25) Transport of luminal antigens to dendrocytes across epithelia

Enterocytes, which are implicated in nutrient absorption, represent the vast majority of villous cells in the small intestine. Goblet cells, which are scattered throughout the epithelium and form a protective mucus layer. Enteroendocrine cells (∼1% of epitheliocytes), which produce hormones and hence regulate various functions of the intestinal epithelium. Paneth cells (lifespan ∼2 months), which are clustered in the crypt bottom, where they represent the single type of differentiated cells; they produce antimicrobial peptides, which control the gut microbiota, in addition to growth factors for the maintenance of the neighboring stem cells. Microfold or membranous (M) cells, which cover the surface of the gut-associated lymphoid follicles. Cup cells (up to 6% of ileal epitheliocytes). Tuft cells (0.4% of intestinal epitheliocytes). In the large intestine, secretory cells, mainly goblet cells, are more abundant than absorptive enterocytes. This epithelial barrier constitutes the first line of defense against intruding microorganisms via secretion of mucins and antimicrobial proteins and transport of secretory IgA.

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The intestine is one of the organs with the highest self-renewal capacity. The entire epithelium is renewed in 4–7 days [1058]. Multipotent LGR5+266 bHLHa45+, and OlfM4+267 intestinal stem cells (ISCs) localize to the base of intestinal crypts [1060, 1061]. They continuously proliferate to maintain their pool and to engender differentiated intestinal cell types. Regeneration of the intestinal epithelium is initiated by ISCs, which engender transit-amplifying precursor cells. These can generate all intestinal cell types, which move toward the lumen until they are eventually shed [1062]. Wnt, BMP, notch, and hedgehog regulate the ISC fate. The differentiation status of a cell is determined by its position along the villus axis, which is tightly regulated by opposing gradients of morphogens such as Wnt and BMP, Wnt–β Ctnn signaling being the highest at the crypt base, where it promotes stem cell expansion and transit-amplifying cells proliferate, whereas BMPs abound near the lumen axis and inhibit proliferation [1062]. During intestinal regeneration, opposing gradients of Wnt and BMP signaling ensure proper differentiation along the villus axis. Intestinal subepithelial myofibroblasts (ISEMFs), which are adjacent to ISCs near the crypt base, secrete BMP antagonists, such as gremlins Grem1 and Grem2 and noggin, thereby promoting ISC proliferation at the crypt base. ISEMFs influence cell fate in the regenerating intestine, as they secrete AngptL2, which primes an autocrine positive feedback loop via integrin-α5 β1 and NFκB, which downregulates formation of BMP2 and BMP7 [1063]. Angiopoietin-like protein AngptL2 maintains the ISC niche and enables regeneration of intestinal epithelium after injury, as it prevents BMP synthesis. It does not operate via the PI3K–PKB–β Ctnn and PI3K–NFκB axes [1063].268 Upon damage, DLL1+ committed cycling or quiescent progenitors, mainly from the secretory lineage lodging at the border of the stem cell niche dedifferentiate and repopulate the stem cell niche, regenerating LGR5+ cells [1061]. Stem cells rapidly divide to engender highly proliferative progenitor cells, transit-amplifying cells, which are transient indispensable integrators of stem cell niche components. Transit-amplifying cells have a finite lifespan [1066]. They arise from stem cells, proliferate, and then gradually differentiate, hence being related to progenitor and precursor cells. Stem, transit-amplifying, progenitor, and precursor cells are all characterized by a gene expression pattern linked to an epigenetic

266 LGR5:

leucine-rich repeat-containing GPCR-5, a R-spondin receptor. olfactomedin-4. It supports cell adhesion, probably via lectins and cadherins [108]. Overexpression of OlfM4 can induce cell differentiation and apoptosis [194]. 268 The Wnt–β Ctnn and PTen–PI3K–PKB pathways interact to drive long-term hematopoietic stem cell proliferation and simultaneously inhibit differentiation and apoptosis via upregulation of inhibitor of differentiation ID2 (bHLHb26) [1064]. Maintenance of self-renewal and induction of differentiation in embryonic stem cells in response to Wnt signaling relies on a regulatory circuit involving β-catenin, cadherin-1, the PI3K–PKB axis, and Snai2 in a time-dependent manner. Short-term upregulation of β-catenin enhances ESC self-renewal via the PI3K–PKB cascade [1065]. Long-term Wnt activation promotes ESC differentiation via the β Ctnn-Snai2 axis. 267 OlfM4:

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signature. Intestinal transit-amplifying cells migrate toward the tip of the villus axis, where they undergo apoptosis and are shed into the lumen of the healthy intestine. Whereas primed stem cells generate transit-amplifying cells, quiescent stem cells only proliferate when transit-amplifying cells are formed and begin to synthesize sonic hedgehog [1067]. Generation of transit-amplifying cells is thus independent of autocrine SHh signaling, which instead promotes proliferation of quiescent stem cells. The intestinal lineage fate is mainly regulated by two transcription factors, bHLHb39269 and bHLHa14,270 which are controlled by notch signaling. The bHLHa14 factor commits cells to the secretory lineage; it controls transcription factors linked to secretory cell types.271 On the other hand, notch represses bHLHa14 via HES1 and HES5, thereby directing cells to the absorptive lineage. Transcriptional repression by chromatin modifiers is a mechanism that establishes and maintains cell identity. The chromatin polycomb repressive complex, PRC1 monoubiquitinates histone H2a (Lys119) owing to its Ub ligase RNF51, the activity of which is enhanced by Ring1b. The complex PRC2 methylates H3 (Lys27) because of its methyltransferase subunits KTM6a and KTM6b; it prevents CDKN2A gene transcription. Loss of PRC1 impairs ISC function and causes intestinal stem cell exhaustion. Deletion of PRC2 specifically affects the proliferation of cryptic transit-amplifying cells, provoking the accumulation of intestinal cells of the secretory lineage, especially goblet and enteroendocrine cells. This accumulation does not result from impaired proliferation of progenitor cells upon CDKN2A activation, but from regulation of the transcription factors responsible for secretory lineage commitment [1061]. The complex PRC2 does indeed control the equilibrium between secretory and absorptive lineage differentiation programs, that is, the cell fate balance between secretory cells and enterocytes. It controls proliferation of cells within the crypt and represses the transcription factor bHLHa14, thereby favoring the generation of enterocytes rather than secretory cell types in the adult intestine [1061]. Therefore, PRC2 exerts a dual role in the intestinal epithelium, as it represses CDKN2A expression, hence preserving progenitor cell proliferation, and restricts secretory commitment by targeting master regulators of cell differentiation, thus coordinating cell differentiation, avoiding erroneous activation of secretory lineage differentiation. The ligand-activated transcription factor aryl hydrocarbon receptor (AHR or bHLHe76) recognizes xenobiotics, such as environmental toxins, dietary com-

269 BHLHb39:

class-B basic helix–loop–helix protein-39. It is also called hairy and enhancer of split protein HES1. 270 Also known as atonal homolog AtoH1 or AtH1. It is required for secretory commitment downstream from the notch signal. 271 Indeed, it does activate specific transcription factors that induce terminal differentiation into endocrine, Paneth, or goblet cells. Growth factor independent-1 transcriptional repressor GFI1 and SAM pointed domain-containing ETS factor SPDEF specify the goblet cells’ fate.

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pounds,272 and microbial virulence factors,273 in addition to natural compounds, such as tryptophan metabolites (e.g., kynurenines) and microbiota-derived factors. Activated bHLHe76 primes CYP1 gene transcription and hence production of inducible CyP1a1, CyP1a2, and CyP1b1,which metabolize xenobiotics, in addition to UGT1a6. Cytochrome-P450-1, which are detoxifying mono-oxygenases, oxygenate bHLHe76 ligands for clearance, the resulting metabolites having decreased activity and increased water solubility, thereby controlling bHLHe76 ligand availability and terminating bHLHe76 activation [1070]. On the other hand, inhibition of CyP1a1 increases the presence of bHLHe76 ligands and prolongs bHLHe76 activation. Sustained bHLHe76 activation by ligands resisting clearance or by constitutively active bHLHe76 has deleterious effects. Constitutive expression of CyP1a1 in intestinal epitheliocytes causes a loss of type-3 innate lymphoid cells (ILC3) and TH17 cells, which depend on bHLHe76 for survival, increasing susceptibility to enteric infection. In addition, environmental factors, such as redox stress and chemical pollutants, modulate CyP1 activity. Constitutive CyP1a1 activity may be counter-balanced by increased intake of dietary bHLHe76 ligands [1070]. Host–microbia interactions rely on simple metabolites from lipids, sugars, and peptides that act as signaling mediators (e.g., glycans, lipidic messengers, and neurotransmitters). Commensal bacteria produce GPCR ligands, such as N acyl amides (e.g., endocannabinoids and long-chain N acyl amide commendamide), eicosanoids (prostaglandins and leukotrienes), and sphingolipids, that act as messengers. N acyl amide synthase genes abound in the gastrointestinal bacteria and the resulting lipids interact with GPCRs to regulate gastrointestinal tract function [1072]. A set of GPCRs exist for fatty acids (FFARs). They differ according to their specificity for FAs of various chain lengths and degrees of saturation and for FA derivatives such as OEA and oxidized FAs (Table 5.46) [1071]. The FFARs form a subset of nutrient sensors in the gastrointestinal tract, pancreas, AT, and regions of the CNS such as the hippocampus, in addition to leukocytes. The FFAR1 receptor (GPR40) is activated by medium- and long-chain saturated and unsaturated FAs [1071]. The FFAR2 receptor (GPR43) reacts to shortchain FAs produced in the gut by microbial fermentation of carbohydrates. The FFAR3 receptor (GPR41) is also activated by short-chain FAs also formed from the fermentation of complex carbohydrates in the colon. The GPR84 receptor for medium-chain FFAs is activated particularly by capric (C10), undecanoic (C11), and lauric (C12) acids. Glucose-dependent insulinotropic receptor GPR119 tethers to ethanolamide OEA, an FA derivative rather than an FFA, and LPC. The GPR120

272 For

example, indole 3-carbinol produced by the breakdown of the glucosinolate glucobrassicin existing at relatively high levels in cruciferous vegetables (e.g., broccoli, cabbage, cauliflower, brussels sprouts, collard greens, and kale) [1068]. 273 For example, phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis [1069].

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Table 5.46 Fatty acid receptors (Source: [1071]; HODE hydroxyoctadecadienoic acid, LPC lysophosphatidylcholine) Type FFAR1

Ligand C16–C22

Gα subunit Gq/11

FFAR2

C2–C4

Gq/11, Gi/o

FFAR3 GPR84 GPR119

C3>C4>C2 C9–C14 LPC

Gi/o Gi/o Gs

GPR120 GPR132

C14–C22 9HODE

Gq/11 Gq/11, Gi/o, G12/13

Cellular expression Pancreatic α/β cell, K cell of the small intestine, L cell of the large intestine, splenocytes, dendrocytes, monocytes, B/T lymphocytes, natural killer cells Adipocyte, enteroendocrine cells (L cell of the ileum and colon), immunocytes Adipocyte Monocytes, macrophages Pancreatic β cell, enteroendocrine cells Enteroendocrine cells Macrophages

Free fatty acids (FFAs) stimulate insulin secretion acutely, but chronic hyper-FFA-emia causes lipotoxicity and hence decreased β-cell function and insulin resistance

receptor is optimally activated by saturated and unsaturated FAs with chains containing 14–18 C and 16–22 C, respectively. The GPR132 receptor is targeted by oxidized FFAs derived from linoleic and arachidonic acids and thus may be a sensor of lipid overload and redox stress. Lipidic ligand-activated GPCRs, such as S1P4 , PGi2 R, PGe2 R4, GPR119, and GPR132 intervene in pathogenesis correlated with changes in the gut flora, in particular obesity, diabetes, and atherosclerosis [1072]. The orphan G-protein-coupled receptor, GPR15, or brother of bonzo, a chemoattractant receptor that assists the homing of T cells to the colon, lodges on lymphocytes. It mediates their transfer to the lamina propria of the colon in addition to the skin and recruitment of effector T lymphocytes to the inflamed intestinal region [1073]. Secreted protein and GPR15 ligand GPR15L, which is encoded by the C10ORF99 gene in epitheliocytes exposed to the environment, especially after immunological challenge, is a potential cytokine, which is also a ligand of the transmembrane and secreted sushi domain-containing protein SusD2.274

274 SusD2

interacts with the secreted protein galectin-1, which is synthesized by cancerous cells and promotes immunity evasion, angiogenesis, and metastasis [1074].

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5.6.6 Endocrine Cells of the Gastrointestinal Tract Mucosae and the Pancreas Electrochemically excitable enteroendocrine cells constitute the enteric endocrine system. They produce gastrointestinal hormones or peptides in response to various types of stimuli [1075]. A majority of enteroendocrine cells synthesize and secrete multiple types of peptidic hormones, coexpressing for example, GIP, neurotensin, peptide-YY, proglucagon, and secretin. However, in humans, ghrelin and somatostatin are generally not co-produced with other peptidic hormones in the small intestine. As autacoids, they target the ENS; as endocrine messengers, they are liberated into the bloodstream. The reflex circuitry of the ENS comprises sensory transducers in the mucosa (entero-endocrine cells), afferent neurons, interneurons, and motor neurons. The ENS is involved in nutrient absorption, as it controls gastrointestinal peristalsis,275 segmentation,276 secretion, and blood flow. It also participates in the response to injury. It adapts the neuronal morphology and function and pattern of released neurotransmitters in response to inflammation. Luminal nutrients also excite enteroendocrine cells. Satiety is modulated by sensory signals arising from the inner surface of the digestive tract [1076]. Enteroendocrine cells possess a neuropod (i.e., a cytoplasmic process elongating toward the neuron) that serves as an enteroendocrine cell–neuron contact. The resulting neuro-epithelial circuit contains elements for neurotransmission, that is, pre-, post-, and transsynaptic proteins. Enteroendocrine cells relay information to local sensory neurons via synthesis and release of neurotransmitters that target their cognate receptors on nearby sensory neurons. These cells form DOPA decarboxylase and Tyr hydroxylase for dopamine synthesis. Therefore, in addition to paracrine transmission, innervation of enteroendocrine cells enables a fast transmission of sensory signals from the digestive tract lumen with precise topographical representation of sensory signals and feedback onto enteroendocrine cells. Gastric enteroendocrine cells (G cells), which release cholecystokinin, α and γ-endorphin, gastrin, somatostatin, substance-P, and vasoactive intestinal peptide. Enterochromaffin-like cells secrete histamine. Intestinal enteroendocrine cells, which are spread throughout the intestine, although their population in the large bowel is generally less diverse than in the small intestine, include [1075]:

275 The

smooth muscle of the gastrointestinal tract undergoes contraction–relaxation cycles, thereby generating peristaltic waves that propel a food ball (i.e., food bolus in the esophagus, once food is chewed and swallowed, and liquid chyme, once it is processed and digested in the stomach). 276 Segmentation occurs during feeding, hence mixing food without propelling nutrients, and between meals. In the large intestine, several times per day, mass movements push the chyme toward the rectum.

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• D cells, which synthesize somatostatin, the major inhibitory hormone of the digestive tract277 • I cells, which secrete cholecystokinin • K cells, which free gastric inhibitory peptide278 • L cells, which release proglucagon-derived peptides (glicentin, GLP1, GLP2, and oxyntomodulin) and peptide-YY279 • M cells, which produce motilin • N cells, which liberate neurotensin • S cells, which secrete secretin • Enterochromaffin cells, which release serotonin280 They contain enolase-2+ secretory vesicles, which encompass chromogranin-A+ large dense-core vesicles and synaptophysin+ small synaptic-like microvesicles. Pancreatic enteroendocrine cells in the islets of Langerhans produce insulin and glucagon, the parasympathetic nervous system stimulating insulin secretion and inhibiting glucagon secretion and the sympathetic nervous system having the opposite effect. They also synthesize amylin, ghrelin, pancreatic polypeptide,281 and somatostatin.

277 Somatostatin

also localizes to neurons of the myenteric plexus. differences in production of peptidic hormones, K cells are highly similar to L cells. Secretion of GIP is stimulated by glucose, cAMP, and linoleic acid. 279 The L cell is an open-type endocrine cell with a long cytoplasmic process reaching the gut lumen equipped with microvilli that protrude into the lumen. These microvilli can sense nutrients in the lumen and then trigger secretion. L cells are sensitive to glucose. The L cells are most dense in the ileum. They contain multiple types of ion channels, AC isozymes, and PDE subtypes [1077], in addition to glucose transporters and glucokinase, a pancreatic, but not intestinal glucose sensor [1078]. Glucokinase activation potentiates GLP1 secretion from murine enteroendocrine GLUTag cells (derived from a murine colonic tumor), but not from primary murine L cells. Secretion of GLP1 depends on a transmembrane difference in electrical potential at the cell surface, linked to activity of NaV , CaV 1, and CaV 2.1 channels [1079]. Elevation of cAMP concentration is an effective stimulus for GLP1 secretion from L cells; the cAMP level depends on the activity of cAMP-producing (ACs) and cAMP-hydrolyzing enzymes (PDEs). All membrane-bound ACs are stimulated by Gs; AC1, AC3, and AC8 are activated and AC5 and AC6 are inhibited by Ca2+ – calmodulin, and AC2, AC4, and AC7 are activated by Gβγ in addition to phosphorylation by PKC. Among the ACs, only AC2 abounds in enteroendocrine cells. PDE2a and PDE4d are the predominant isoforms in L cells, with moderate levels of PDE1c, PDE3a, PDE8a, PDE8b, and PDE11a; GLP1 secretion depending on PDE2a, PDE3a, PDE3b, and PDE4d. Inhibition of PDE2 to PDE4 stimulates GLP1 secretion. Inhibition of PDE3, but not PDE2, prevents GLP1 secretion in response to guanylin [1077]. 280 The secretory stimulation pathway is launched from β-adrenergic and PACAP receptors. The secretory inhibition pathway is elicited by ionotropic GABAA and cholinergic receptors. 281 In humans, pancreatic polypeptide (PP) is formed from a precursor protein PPy encoded by the PPY gene. The precursor engenders carboxyamidated PP and an icosapeptide. is secreted by pancreatic PP cells of Langerhans islets. The vagus nerve, gastrin, secretin, and cholecystokinin provoke its secretion. It regulates pancreatic endo- and exocrine secretion in addition to gastrointestinal secretion and hepatic glycogen concentration. 278 Despite

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Incretins are metabolic hormones that have an incretin effect, that is, they stimulate insulin release from pancreatic β cells of the islets of Langerhans after eating in response to ingested glucose, and inhibit glucagon secretion from α cells. The two major incretins are glucagon-like peptide GLP1 and gastric inhibitory peptide.282 GIP-secreting K cells and GLP1-secreting L cells reside throughout the small intestine. In addition, in humans, enteroendocrine cells can produce both GIP and GLP1 [1057]. The sodium–glucose cotransporter SGlT1 (SLC5a1) may be involved in glucose-induced GLP1 secretion. Both insulinotropic GLP1 and GIP are rapidly inactivated by dipeptidyl peptidase DPP4. Upon lipid ingestion, enteroendocrine cells of the small intestine release neurotensin (NTs). Neurotensin and its receptors (NTs1 –NTs3 ) favor uptake of dietary fatty acids. They inhibit AMPK, which stimulates lipolysis, reduces fatty acid absorption, and impedes lipid storage, in response to oleate [1081]. Increased fasting plasmatic concentration of proNTs, a stable neurotensin precursor fragment produced in equimolar amounts to neurotensin, are associated with increased T2DM and CVD risk. Serotonin regulates gastrointestinal motility. Various enteric luminal stimuli act on excitable serotonin-secreting enterochromaffin cells via voltage-gated Na+ and Ca2+ channels, thereby participating in the gut–brain signaling axis. Serotoninergic enterochromaffin cells in the gut epithelium are chemosensors for catecholamines, irritants, and metabolites [1082]. Catecholamines, such as adrenaline and NAd, target α2a-adrenoceptor of the epithelial basolateral surface, activate Ca2+ -permeable channel TRPC4 via Gαi , and prime Ca2+ signaling in enterochromaffin cells. The chemical irritant allyl isothiocyanate, a compound of wasabi, activates the Ca2+ -permeable channel TRPA1. The microbial product isovalerate stimulates the olfactory receptor OlfR558 in Mus musculus. Once enterochromaffin cells are activated, voltage-gated Ca2+ channels trigger release of serotonin, which excites serotonin-sensitive primary afferent sensory nerves via serotonin receptors at synaptic connections. The response of the ENS to dietary and microbial metabolites then influences gastrointestinal activity.

5.6.7 Satiation Signals Hunger and appetite are major triggers of eating decisions; satiation and satiety are main stop signals [1014]:

282 A.k.a. glucose-dependent insulinotropic polypeptide. GIP+ K cells lodge in the duodenal and jejunal epithelia. Its secretion, which depends on sodium–glucose cotransporter SGlT1 (SLC5a1) and is modulated by the KATP channel, is triggered by the ingestion of carbohydrates or lipids. It has an antiapoptotic effect on pancreatic β cells, which form at high levels KIR 6.2, ABCc8, SLC5a1, GPR40 (or FFAR1), GPR119, and GPR120 [1080].

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531

Table 5.47 Gastrointestinal hormones that affect satiation (Source: [1014]) Peptide Amylin Apolipoprotein-A4 Cholecystokinin Enterostatin Gastrin-releasing peptide Ghrelin Glucagon-like peptide-1 Neuromedin-B Oxyntomodulin Peptide-YY

Effect on food intake Decrease Decrease Decrease Decrease Decrease Increase Decrease Decrease Decrease Decrease

Satiation factors respond to specific nutrient stimuli (e.g., cholecystokinin to proteins and lipids, glucagon-like peptide, GLP1, to carbohydrates and lipids, and peptide-YY primarily to lipids), mixed meals of different contents eliciting release of diverse cocktails of gastrointestinal hormones. Most of these peptides (Cck, GLP1, GLP2, oxyntomodulin, ApoA4, GRP, NMB, PYY, and ghrelin) are also synthesized in the brain

• Satiation is related to the feeling of fullness that initiates the decision to stop eating. • Satiety ensures fasting during a prolonged duration until hunger primes the eating decision. A diet enriched in fat or simple carbohydrates does not afford a strong signal of satiety. Upon repletion, elevated cerebral signals launched by insulin and leptin increase sensitivity to satiation signals. Endogenous satiation factors secreted in response to food ingestion activates specific receptors that cause cessation of eating (Tables 5.47 and 5.48). Some satiation signals are released from enteroendocrine cells in the mucosa of the gastrointestinal tract in response to food intake. Local sensory nerves expressing receptors for these gut peptides signal to the brain.

5.6.7.1

Amylin

Amylin, which is secreted by pancreatic β cells with insulin, inhibits gastric acid secretion and lowers glucagon concentration and food intake [1014]. Amylin signals via the calcitonin receptor modified by receptor activitymodifying proteins. It may act as a hormone and directly stimulate neurons in the area postrema in the hindbrain rather than visceral afferent nerves. Its anorexigenic action is potentiated by insulin action and strengthens the effects of cholecystokinin and gastrin-releasing peptide [1014].

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Table 5.48 Hormones of entero-endocrine cells of the large bowel (Source: [1075]; ↑ increase, ↓ decrease) Peptide Glicentin

Glucagon-like peptide-1

Glucagon-like peptide-2

Oxyntomodulin Peptide-YY

Serotonin Somatostatin

5.6.7.2

Effects Stimulation of enterocyte proliferation Inhibition of gastric acid secretion Stimulation of insulin secretion Regulation of gut mobility Incretin effect (↑ insulin and ↓ glucagon secretion) Gastrointestinal motility (delays gastric emptying) Postprandial satiety Stimulation of enterocyte proliferation Inhibition of enterocyte and crypt cell apoptosis Increased nutrient uptake through transporters Reduced intestinal trans- and paracellular permeability; anti-inflammatory effect Inhibition of gastric acid secretion Stimulation of intestinal blood flow via NOS3 Relaxation of murine gastric smooth myocytes Inhibition of gastric emptying Inhibits gastric emptying, intestinal motility, gastric acid secretion, pancreatic exocrine function Suppresses appetite Stimulates enterocyte proliferation Stimulates intestinal motility and secretion Inhibits digestive endo- and exocrine function Stimulates colonic peristalsis

Apolipoprotein-A4

Apolipoprotein-A4 is synthesized by intestinal mucosal cells during the packaging of digested lipids into chylomicrons. It is also produced in the ArcN[1014]. The ArcN is endowed with a relatively leaky blood–brain barrier. It integrates diverse hormonal and neural signals and controls energy homeostasis. Two main categories of neurons include POMC+ and AgRP+ NPy+ neurons283 Apolipoprotein-A4 attenuates food intake in rats. It may interact with cholecystokinin. Both intestinal and hypothalamic ApoA4 are regulated by absorption of lipids, but not carbohydrates.

283 AgRP:

agouti-related peptide, an antagonist of the MC3 and MC4 receptors. Neuropeptide-Y stimulates food intake. AgRP+ NPy+ neurons have an anabolic effect.

5.6 Control of Body Weight and Energy Homeostasis

5.6.7.3

533

Bombesin Analogs

Members of the BN (bombesin) family of peptides, which includes the amphibian peptide bombesin and its mammalian analogs, gastrin-releasing peptide (GRP) and neuromedin-B, reduce food intake in humans [1014].

5.6.7.4

Cholecystokinin

The prototypical satiation signal is the duodenal peptide cholecystokinin (Cck) secreted by duodenal I cells in response to dietary lipids or proteins. It influences gut motility and primes gallbladder contraction, and secretion of pancreatic enzymes and gastric acid. Moreover, it activates its receptors on duodenal sensory nerves [1014]. The Cck1 receptor on nearby branches of vagal sensory nerves decreases the size of the meal once it has begun, reducing hunger and increasing the fullness sensation. Neurons of various subnuclei of the NTS284 receive vagal information from the cardiovascular and respiratory apparatuses and from the gastrointestinal tract. Cholecystokinin activates NTS PPG+ neurons285 via adrenergic and glutamatergic neurons [1083]. NTS PPG+ neurons integrate signals related to long-term energy balance and short-term nutritional status to produce an output to feeding and autonomic circuits. During meals, cholecystokinin, glucagon-like peptide GLP1, and distension of the stomach and intestine trigger nerve impulses in sensory nerves afferent to the hindbrain in communication with neurons in the NTS.

284

This visceral sensory relay station receives inputs from the cardiovascular, respiratory, and gastrointestinal apparatuses. Its neighboring structures include the wall of the fourth ventricle, area postrema, subnucleus commisuralis, fasciculi of nuclei gracilis and cuneatus, spinal nucleus of the trigeminal nerve, DMNV nerve, hypoglossal nucleus parasolitary nucleus, vestibular and cochlear nuclei, and parvocellular reticular formation. The solitary tract consists of fibers from the inferior ganglion of the vagus nerve, glossopharyngeal nerve, and geniculate ganglion of the facial nerve. Sensory afferent fibers from chemo- and mechanoreceptors of the gastrointestinal tract, which use GABA and glutamate as inhibitory and excitatory neurotransmitters, relay signals of satiety to the NTS during feeding. The NTS is involved in generating and synchronizing peristalsis in the upper gastrointestinal tract during swallowing via the nucleus ambiguus in the medulla oblongata, which supplies motor fibers to the vagus, glossopharyngeal, and accessory nerves innervating the pharynx, larynx, and esophagus in addition to parasympathetic fibers to the vagus. 285 PPG: pre-proglucagon. Neuronal circuits in the hypothalamus and hindbrain are implicated in the control of food intake and energy expenditure, and hence AT mass. Glucagon-like peptide GLP1 is produced in the CNS by PPG+ neurons of the NTS in the hindbrain that project to various regions of the brain such as the hypothalamus.

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5.6.7.5

5 Hyperlipidemias and Obesity

Enterostatin

The pentapeptide enterostatin is linked to the intestinal processing of lipids. It is cleaved in the intestinal lumen by colipase secreted by the exocrine pancreas. It then enters the blood circulation. It reduces food intake in rats fed with lipids (but not with carbohydrates or proteins) [1014].

5.6.7.6

Ghrelin

Ghrelin nutritionally produced in endocrine cells of the gastric fundus and duodenum is the most potent circulating orexigen. Its concentration increases during food deprivation and peaks before meals. It stimulates eagerness for ingesting palatable food and drink. Ghrelin differs from all other peptide hormones by its octanoylation, which enables its binding to its receptor [1085]. Ghrelin targets growth hormone secretagogue receptor,286 which possesses two subtypes (GHSR1a–GHSR1b) and localizes to vagal sensory nerves in addition to the hypothalamus, especially the lateral hypothalamus, which produces the orexigenic hypocretin, and ventromedial and arcuate nuclei. Ghrelin thus acts on the vagus nerve. Furthermore, it directly stimulates neurons in the ArcN [1014]. In the ArcN, NPy+ AgRP+ neurons possess ghrelin receptor; ghrelin induces immediate early gene expression [1011]. Ghrelin also targets GHRS in the NTS in the hindbrain,287 ventral tegmental area (VTA),288 nucleus accumbens, and ventral hippocampus [1084]. Ghrelin acts in the LHA in a sex-specific manner. In male and female rats, ghrelin delivered to the LHA favors food intake and appetite for sucrose [1084]. However, it motivates food search and raises body weight only in females, which have a slightly higher GHSR concentration in the LHA than males. Acute GHSR blockade in the LHA reduces food intake, body weight, and sucrose-motivated behavior in female, but not male rats. In Ghsr−/− female rats, the reward-driven behavior is abolished, but does not affect basal food intake [1084]. Ghrelin regulates GH release, appetite, metabolism, neurotransmission, behavior, stress response, and immune function via the Gq/11-coupled GHSR1a receptor. When its constitutive activity is unusually high, this ghrelin receptor stimulates CamK4, PLC, and PKC, hence leading to phosphorylation of cAMP-responsive element-binding protein (CREB), activation of Ca2+ channels, and inhibition of 286 A.k.a.

ghrelin receptor and growth hormone-releasing peptide receptor (GHRP). Growth hormone secretagogues (GHSs), or GH-releasing peptides (GHRPs), elicit GH secretion, not via GH-releasing hormone receptor, but via GHS receptor (GHSR), which tethers to ghrelin. 287 The hindbrain, also called the rhombencephalon, is the lower part of the brainstem. It comprises the cerebellum, pons, and medulla oblongata. 288 The ventral tegmental area in the midbrain sends dopaminergic neural projections to the limbic and cortical areas.

5.6 Control of Body Weight and Energy Homeostasis

535

K+ channels [1085]. On the other hand, Gi/o-coupled GHSR1a activated by ghrelin launches the β Arr–ERK1/2 and Src–PKB pathways. The constitutive internalization of GHSR1a relies on the sequential activation of Rab5 and Rab11 linked to early endosomes and endosomal recycling compartments, respectively. The agonist-independent activity of GHSR1a may modulate food intake and body weight. GHSR1a signals in a ligand-independent manner basally or when it is heterodimerized with other GPCRs. Liganded GHSR1a heterodimerizes with receptors of serotonin 5HT2C (control of food intake), dopamine D1 (enhanced dopamine signaling), somatostatin Sst5 (regulation of insulin release), and melanocortin MC3 (body weight regulation). It also heterodimerizes with its truncated form (GHSR1b; without the distal domain of its C-terminus, which is used for receptor internalization, β-arrestin recruitment, and signal termination), which has a dominant negative effect on ghrelin receptor function. GHSR1b is primarily located in the endoplasmic reticulum, where it tethers to GHSR1a, hence lowering plasmalemmal GHSR1a amount, as the GHSR1a–GHSR1b heterodimers accumulate at the ER [1086]. Ghrelin is associated with the anticipation of meal ingestion, as its concentration peaks shortly before scheduled meals. This orexigenic peptide may dampen the effects of GLP1 and PYY(3–36) on gastric emptying and food intake. In rats, cells with a truncated GHSR1a form respond more intensively to ghrelin than cells with the full-length receptor, thereby potentiating the fattening effect of ghrelin, without affecting appetite [1087]. This GHSR1a mutant form (GhsrQ343X ) preserves plasmalemmal GHSR concentration. Rats have a more stable body weight under caloric restriction, whereas under standard conditions, body weight and adiposity increase and glucose tolerance lowers. Ghrelin restores the NO–ET1 balance, hence improving endothelial function [720]. Ghrelin stimulates NO production in ECs using the PI3K–PKB–NOS3 pathway. Its concentration decays in obese subjects.

5.6.7.7

Glicentin

Glicentin inhibits gastric acid secretion, but, at least in rats, does not affect food intake [1014]. Glicentin and oxyntomodulin are also detected in the CNS, principally in the hypothalamus and brainstem.

5.6.7.8

Glucagon-Like Peptide-1

The circulating hormone glucagon-like peptide, GLP1, is produced by intestinal L cells, especially in the ileum and colon, and α cells in the pancreas from cleavage by PCSK1 or PSCK3 of proglucagon [1088]. The transcription factor Pax6 in enteroendocrine cells activates proglucagon synthesis [1057]. β-Catenin also stimulates proglucagon expression in the intestine, but not in the pancreas, via the

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transcription factor bHLHb19 (TCF4).289 Increased pancreatic GLP1 production may result from impaired β-cell function linked to a defective inhibition by insulin of GLP1 production. The neuropeptide GLP1 is also synthesized by PPGcg+ (pre-proglucagon) neurons primarily in the brainstem by the NTS, which projects to the appetite centers in the hypothalamus (ArcN and PVN) activated by leptin and the vagus nerve (Sect. 5.6.2.3). This anorexigen can activate the area postrema and neural circuits that mediate vagal satiety signaling. High glucose concentrations upregulate the formation of PCSK1 (but not that of PCSK2). Its secretion is elicited by nutrients. Lipids cause GLP1 secretion. GLP1 diffuses across the basement membrane and lamina propria to be taken up by a capillary. In rats, glucose-dependent insulinotropic peptide (GIP) stimulates GLP1 secretion via the vagus nerve, but not in pigs and humans [1057]. A local paracrine control is exerted by neighboring somatostatin+ D cells. Gastrin-releasing peptide in addition to its C-terminal decapeptide neuromedin-C are powerful stimulants of GLP1 secretion [1057]. A neural pathway stimulates GLP1 secretion. Both muscarinic receptors M1 and M2 may be involved in the control of GLP1 release. Catecholamines may also be implicated via β-adrenoceptors. In fact, the sympathetic innervation (NAd transmitter) to the gut inhibits GLP1 secretion, whereas the extrinsic vagal innervation (cholinergic activity) is not implicated or may play only a minor role. Nevertheless, vagal stimulation provokes GRP release. Glucagon-like peptide-1 is rapidly catabolized (inactivated) in the blood circulation by dipeptidyl peptidase DPP4 on the luminal surface of ECs (half-life 5000 mg/d) requires sodium restriction [1181]. A low sodium intake is more efficient at lowering the risk for CVD than a moderate sodium intake. A low sodium intake affects the sympathetic nervous system and renin–angiotensin–aldosterone cascade.

6.5.1 Dietary Sodium Intake Vasopressin, an antidiuretic (ADH) and vasoconstrictor hormone, participates in the maintenance of the water balance and plasmatic sodium concentration. Hypernatremia (hyperosmolality) triggers vasopressin release to reduce water clearance by the kidney [1182]. Dietary salt raises blood pressure in most people with hypertension and in about one-third with normotension [30]. The osmotic effect of sodium salt expands the extracellular volume. Dietary salt aggravates age-related hypertension. Moreover, hypertensive subjects have a limited ability for renal sodium excretion and cutaneous retention of osmotically inactive sodium. In the skin, macrophages, which release VEGF that primes lymphangiogenesis, are major players in the response to interstitial hypertonicity caused by high extracellular Na+ concentrations. A modest reduction in salt intake lowers blood pressure in normotensive and hypertensive people. A concentration of the extracellular cation Na+ increasing from the low to the upper end of the normal range (135–145 mmol/l) is linked not only to an osmotic water efflux and systolic and diastolic blood pressure but also to concentrations of total cholesterol, LDLCS , and ApoB [1183]. Augmentations in lipid concentrations and blood pressure linked to a 10-mmol/l increase in sodium concentration are similar to those associated with 7- to 10-year aging. Furthermore, a concentration of the extracellular cation Na+ , which increases from the low to the upper end of the normal range, raises lipid content (∼30%) in cultured adipocytes. Adipocytes and cutaneous macrophages respond to intracellular hypertonicity by NFAT5 formation.

6.5.2 Dietary Lipid Intake Elevated consumption of lipids engenders a nutritional state associated with redox stress and insulin resistance caused by mitochondrial H2 O2 [1184].

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Catalase, which lodges primarily in peroxisomes and, to a lesser extent, in the mitochondrial matrix, has a lower binding affinity for H2 O2 (in the low millimolar range) than that of glutathione peroxidase and peroxiredoxin (in the low micromolar range), which both reside throughout the cell and require the cofactors glutathione and thioredoxin, respectively. However, catalase contributes markedly to mitochondrial H2 O2 clearance. Catalase thus cooperates with GPOx and PRdx in the control of mitochondrial H2 O2 concentration. Moreover, catalase is the single player in H2 O2 removal in defective mitochondria, owing to a diminished contribution from the glutathione peroxidase–glutathione reductase and peroxiredoxin–thioredoxin–thioredoxin reductase axes [1184]. The catalase content in mouse cardiac mitochondria rises (∼0.5-fold) in response to high-fat diet, more than other antioxidant proteins, but this augmentation does not prevent H2 O2 induced reduction in cardiac insulin signaling, whereas a 50-fold overexpression of catalase prevents diet-induced loss in insulin signaling [1184]. Therefore, catalase may prevent toxic peaks in H2 O2 concentration, whereas GPOx and PRdx may regulate mitochondrial H2 O2 concentration. High dietary intake of saturated and trans-fatty acids27 (TFAs) and cholesterol and low intake of fruits, vegetables, and fish raise the cardiovascular risk. Dietary features for cardiometabolic health include more minimally processed foods (fruits, nonstarchy vegetables, nuts, seeds, whole grains, fish, seafood, yogurt, and vegetable oils) and fewer red meats, sodium-preserved meats, and foods rich in refined grains, starches, and added sugars [1186]. However, consumption of high-fat dairy food (e.g., milk, cheese, yogurt, and butter, in addition to hidden dairy fat in numerous mixed foods) with its branchchain fatty acids and medium-chain saturated lipids, can have beneficial or at least neutral effects on diabetes, whereas dairy fat intake correlates with improved insulin sensitivity [1187]. In CoAD patients, analysis of TFAs in RBC membranes shows that low TFA concentrations are associated with low HDLCS concentrations, whereas, in general, high TFA concentrations are linked to high LDLCS and low HDLCS concentrations [1185].28 Low TFA consumption owing to avoidance of TFA-containing foods (e.g., whole-fat dairy and processed food containing partly hydrogenated oils)29 does not improve the cardiometabolic status. Ruminant-derived TFAs are particularly associated with a reduced risk of sudden cardiac death. Furthermore, high TFA concentrations (yet lower than in other populations) are related to lower concentrations of TG and fasting glucose in addition to lower blood pressure [1185]. 27 TFAs

are unsaturated fatty acids containing double bonds in trans configuration. They are found in trace amounts in dairy products (e.g., trans-palmitoleic acid) and generated by the food industry, especially during hardening of unsaturated fats. Major TFA sources comprise cakes, cookies, pies, and pastries [1185]. In animals and plants, fatty acids usually occur in cis configuration. However, in milk, dairy products, and meat, some trans-isomers occur naturally in small quantities. 28 The natural TFAs vaccenic and trans-palmitoleic acids have beneficial effects. However, transisomers may be hazardous. 29 Oils are only partly hydrogenated, a portion of cis-isomers being converted into trans-isomers.

6.5 Unhealthy Diet

577

6.5.3 Dietary Carbohydrate Intake The American diet can contain large amounts of sugar-sweetened beverages, especially fructose sweeteners, that contribute to insulin resistance and obesity. Excessive fructose consumption is more detrimental than excessive glucose intake, as fructose raises hepatic de novo lipogenesis [804]. Women are more susceptible than men to type-2 diabetes upon fructose consumption. In male and female rats, ingestion of fructose for 2 weeks reduces fatty acid oxidation and engenders hypertriglyceridemia and hepatic steatosis [1188]. Hyperinsulinemia is observed in fructose-fed female, but not male, rats. In fructosefed female rats, glucose tolerance is altered and activity in the liver of insulin receptor substrate IRS2 declines. Hepatic synthesis of fructokinase, which controls fructose metabolism, is markedly induced by fructose ingestion in female, but not in male, rats, increasing the AMP/ATP ratio. Therefore, only in fructose-fed female rats does AMPK activity rise and hence the formation of hepatic nuclear factor HNF4 and SREBP1 lowers [1188]. Glucose suppresses food intake via by the hypothalamic AMPK–malonylCoA axis, whereas centrally administered fructose increases food intake [1189]. In the hypothalamus, initial fast steps of fructose metabolism30 deplete hypothalamic ATP concentration, whereas the slower regulated glucose metabolism elevates hypothalamic ATP concentration. Therefore, fructose activates hypothalamic AMPK, which phosphorylates (inactivates) ACC. Biotin (vitamin-B7)-dependent ACC catalyzes the irreversible carboxylation of acetyl-CoA to malonyl-CoA via its biotin carboxylase and carboxyltransferase activities. Fructose thus reduces hypothalamic malonyl-CoA content, which increases food intake. On the other hand, glucose diminishes AMPK activity, raises hypothalamic malonyl-CoA, decreases, and increases expression of orexigenic and anorexigenic neuropeptides, respectively, thereby suppressing food intake. Although total caloric intake in glucose-supplemented rats is significantly higher than that in fructose-supplemented rats, only fructose supplementation provokes hypertriglyceridemia, increases body weight, and alters aortic function [1190]. In female rats supplemented with fructose (at a much higher amount than fructose consumed daily by obese humans), hepatic production of the lipogenic enzymes stearoyl-CoA desaturase-1, FAS, and sterol regulatory element-binding protein1 in addition to that of microsomal triglyceride transfer protein, an essential protein for assembly and secretion of very low-density lipoproteins (VLDL)TG , increase, whereas the synthesis of carnitine palmitoyl transferase-1A, the rate-limiting enzyme controlling mitochondrial fatty acid uptake and subsequent oxidation, decreases. In addition, only glucose supplementation increases plasmatic concentration of adiponectin and production of NR1c1, thereby increasing NO and ceramidase activ30 Fructose

ysis.

processing bypasses the rate-limiting step catalyzed by phosphofructokinase in glycol-

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ity. Insulin signaling in the liver and aorta is impaired in both glucose- and fructosesupplemented rats, the effect being more pronounced in fructose-supplemented rats. Moreover, fructose alters endothelial function. Fructose has slight effects on endothelium-dependent vasodilation (i.e., NO-dependent action of acetylcholine and bradykinin) but impedes NO donor (sodium nitroprusside [SNP])-mediated vasodilation [1190]. On the other hand, glucose augments SNP-mediated vasodilation and lowers vasoconstriction induced by phenylephrine likely because of increased NOS3 phosphorylation and subsequently, elevated basal NO concentration. The actin-associated vasodilator-stimulated phosphoprotein (vasp) is similarly phosphorylated at Ser239 by PKG whatever the sweetener sugar type, but its phosphorylation at Ser157 by PKA is impaired by fructose consumption, owing to an elevated synthesis of PDE4 (∼50%), which specifically hydrolyzes cAMP [1190]. In addition, vasp production is markedly reduced upon fructose supplementation.

6.5.4 Dietary Protein Intake The dietary amino acid (AA) requirements are influenced by (1) dietary factors (e.g., AA content and proportions and amounts of other substances), (2) physiological characteristics of subjects (e.g., age, genetic background, and physical activity), (3) environmental factors (e.g., temperature, dietary habits, and air pollution), and (4) eventual pathological conditions [1191]. Amino acids provide nitrogen and sulfur, which are not made in the body. These building blocks form peptides, proteins, and low-molecular-weight substances (e.g., creatine, dopamine, glutathione, NO, purines, pyrimidines, and serotonin) [1191].31 Amino acids participate in regulating the oxidation of fatty acids and glucose in a cell-specific manner via metabolites and signaling messengers. Metabolic enzymes are proteins synthesized from AAs. Carnitine synthesized from lysine, methionine, and serine permits transfer of LCFAs from the cytoplasm into the mitochondrion for β-oxidation. Arginine, leucine, glycine, tryptophan, and glutamine activate the TOR pathway to stimulate protein synthesis. Creatine produced from arginine, glycine, and methionine, enables energy storage as phosphocreatine. Glutathione formed from cysteine, glycine, and glutamate, taurine (a sulfur-containing metabolite of methionine), glycine, proline, and hydroxyproline protect cells from redox stress. The product of arginine catabolism, NO supports oxidation of fatty acids and glucose to CO2 and H2 O. The vasodilator NO increases renal blood flow and hence glomerular filtration rate, the kidney being involved in AA reabsorption and in excretion of ammonia, urea, and sulfate. Melatonin and serotonin, which are metabolites of tryptophan, preclude the production of inflammatory cytokines.

31 Glutamine

yields about 50 and 35% of ATP in lymphocytes and macrophages, respectively.

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The amounts of energy released from the oxidation of dietary fat, protein, and starch to water and CO2 are 9, 4, and 4 kcal/g, respectively, whereas the resting metabolic rate (RMR) of healthy humans decreases with age, ranging from 55 in 5-year-old children to 25 kcal/kg of BW/day in 45-year-old adults, minimal physical activity requiring 30 kcal/kg/day (1.2 × RMR) [1191]. Exercise engenders a transient AA catabolism; the rate of AA oxidation increases. Its effect depends on the type of exercise. Exhaustive endurance exercise reduces the rate of protein synthesis without affecting protein breakdown, whereas prolonged resistance exercise increases the rate of protein degradation. Dietary protein intake contributes to energy metabolism, appetite,32 and lifespan control. Only about 8% of dietary protein is used for gluconeogenesis and the remainder for protein turnover and AA oxidation. On the other hand, elevated argininemia enhances insulin sensitivity, stimulates oxidation of fatty acids and glucose in skeletal muscles, promotes energy expenditure, and reduces white adipose tissue (WAT) mass in obese humans. In addition, adequate consumption of dietary proteins can control satiety, inhibiting release of appetite-promoting ghrelin and stimulating secretion of appetite-suppressing peptide-YY and glucagon-like peptide GLP1 [1191]. Safety of increased protein intake for weight control and muscle synthesis is governed by the protein absorption rate in the gastrointestinal tract and hepatic capacity to deaminate proteins and produce urea for excretion of excess nitrogen [1193]. Proteins are processed by peptidases to generate amino acids and di- and tripeptides in the lumen of the digestive tract. These digestion products are used by gut bacteria or absorbed into enterocytes. Intestinal bacteria can produce beneficial (e.g., SCFAs) and deleterious (e.g., cresol, skatole, and sulfide) metabolites from AA. Dietary AA and protein oxidation produce metabolites, such as ammonia, NO, homocysteine, and sulfate. Ammonia is converted into urea, primarily in the liver and, to a lesser extent, in the small intestine. The kidney removes H+ by combining it with glutamine-derived NH3 to generate NH+ 4 , which is excreted in the urine. When vitamin-B6, -B9, and -B12 are adequately supplied, homocysteine, an oxidant, is rapidly recycled into methionine in the liver. Consumption of protein above safe upper limits should be avoided. Excessive protein intake (>35% of total energy intake) causes hyperaminoacidemia, hyperammonemia, and hyperinsulinemia [1193]. Chronically elevated protein intake (>2 g/kg/day) can provoke digestive, renal, and vascular anomalies [1191]. Hyperhomocysteinemia hampers NO synthesis in ECs.

32 In

the vinegar fly Drosophila melanogaster, yeast, which contains amino acids, carbohydrates, vitamins, and sterols, may cover nutritional requirement. Preference for yeast and AA is governed by restriction of dietary essential AAs. Gut bacteria influence feeding decision. Commensal bacterial species Acetobacter pomorum and Lactobacilli suppress protein appetite [1192].

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6.5.5 Healthy Diet Dietary regimens and specific nutrients (e.g., magnesium, selenium, fructose, ω3-fatty acids, glutamine, histidine, isoleucine, valine, capsaicin, vanillic acid, quercetin, rutin, naringin, epigallocatechin gallate (a component of green tea), Cudrania tricuspidata fruits, aloe vera, red ginseng, honey, barley, and pre- and probiotics) have been studied [1194]. Vegetables, fruits, whole grains, legumes, nuts, and dairy are major sources of minerals (e.g., calcium, magnesium, and especially potassium), which attenuates the hypertensive effect of sodium. Diet should contain high amounts of fiber, vitamins (especially antioxidant vitamins C and E), antioxidants, minerals, phenolics, and unsaturated fats (e.g., ω3-fatty acids) and avoid food rich in refined grains, starches, sugars, salt, and trans-mono- and -polyunsaturated fatty acids. Active polyphenols that include flavonols (in onions, broccoli, tea, and various fruits), flavones (in parsley, celery, chamomile tea), flavanones (in citrus fruits), flavanols (e.g., catechins and procyanidins [in cocoa, apples, grapes, red wine, tea]), anthocyanidins (in berries), and isoflavones (in soy) are beneficial. Polyphenols increase NO availability, exert antioxidant and anti-inflammatory effects, and hinder improper platelet reactivity. Cocoa polyphenols enhance endothelial function, as they increase NOS activity. In addition, cocoa flavanols exert antioxidant effects in vitro. In humans, cocoa lowers plasmatic concentration of F2 -isoprostanes and thus counteracts lipid peroxidation. Daily chocolate consumption (Vol. 7, Chap. 2. “Context of Cardiac Diseases”) during a 4-week period improves in a sustained manner the endothelial function of patients with moderate class-I I congestive heart failure.33 In these patients, flavonol-rich 70% cocoa-containing chocolate provokes a significant short(2 h) and long-term (4 weeks) vasodilation of the brachial arterial circuit without affecting neurohumoral activation, in addition to fasting glycemia and insulin sensitivity in comparison with control subjects [1195]. The effect of endothelialdependent vasodilators (NO) is favored over vasoconstrictors (e.g., endothelin-1 and angiotensin-2). Epicatechin, which mimics the endothelial effects of flavanolrich cocoa, not only augments NO availability but also reduces endothelinemia. Moreover, blood pressure in patients with hypertension (not in subjects with low blood pressure) and platelet adhesion decreases, but amelioration in platelet function is not sustained. Intake of olive oil, one of the main components of the Mediterranean diet (Vol. 7, Chap. 2. “Context of Cardiac Diseases”), can reduce blood pressure owing to the assumed role of its minor components, such as α-tocopherol, polyphenols, and other phenolic compounds, which are not present in other oil types. An antioxidantrich dietary pattern such as a traditional Mediterranean diet, especially when 33 Congestive

reactivity.

heart failure is characterized by impaired endothelial function and increased platelet

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enriched with virgin olive oil, decreases cholesteryl ester transfer protein activity and increases the ability of HDLs to esterify cholesterol, paraoxonase-1 arylesterase activity, and HDL vasodilatory capacity [1196]. However, the hypotensive effect of olive oil results from its high oleic acid content [1197]. On the other hand, soybean oil, which has a low oleic acid content, and the oleic acid structural analogs, elaidic and stearic acids, do not induce hypotension. Increased oleic acid concentration in cell membranes and the resulting changes in cell membrane lipidic composition and structure influence activity of adrenoceptors and attenuate blood pressure. Oleic acid, but neither elaidic nor stearic acid, regulates the activity of β2a- and α2d-adrenoreceptors and subsequently the signaling cascade involving G protein, adenylate cyclase, and the vasodilatory cAMP–PKA axis [1197]. In addition, oleic acid reduces concentrations of the inhibitory Gi2 and Gi3 subunits in addition to the vasoconstrictory effectors, subunits of the Gq/11 class and PLCβ, and hence of the second messengers inositol trisphosphate, diacylglycerol, and calcium. Dietary supplementation with polyunsaturated fatty acids is used in individuals at risk for CVD. The ω6-polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), ensures cardioprotection. It is processed by 12-lipoxygenase to its reduced oxidized oxylipin, 12(S)-hydroxy (8Z,10E,14Z)-eicosatrienoic acid (12[S]HETrE), which impedes platelet activation and aggregation, Rap1 activation, and hence injuryinduced thrombosis, via a Gs-coupled receptor signaling in platelets [1198]. Metabolic stress is often associated with insulin resistance, ectopic lipid accumulation, and lipogenesis,34 especially in the liver. Palmitoleate, an ω7monounsaturated fatty acid which is found in foods (animal and vegetable oils) and is produced in small amounts by the human body, ameliorates the adverse evolution of hyperglycemia and hypertriglyceridemia [1199].35 Palmitoleic acid downregulates the expression of proinflammatory adipocytokines (TNFSF1 and resistin) formed in the WAT and of lipogenic genes (sterol regulatory elementbinding protein SREBP1, FAS, and stearoyl-CoA desaturase SCD1) in the liver. Palmitoleate also reduces metabolic stress [1200]. It counteracts endoplasmic reticulum (ER) stress and represses lipid-induced inflammasome activation in

34 I.e.,

fatty acid and triglyceride synthesis from glucose and other substrates predominantly in the liver. 35 Dietary intake of palmitoleate is relatively weak. However, it abounds in plant and marine sources. Palmitoleate is the second most abundant mono-unsaturated fatty acid (C16:1) in most blood lipid pools. It abounds in AT. It is synthesized from palmitic acid by stearoyl-CoA desaturase, which has two isoforms (SCD1 and SCD5) in humans. It prevents β-cell apoptosis induced by glucose or saturated fatty acids [1199]. It promotes insulin sensitivity, as it suppresses inflammation. In the skeletal muscle, this AT-derived lipidic regulator stimulates insulin action, as it raises activity of the glucose transporters GluT1 and GluT4, thereby improving glucose utilization. In the liver, palmitoleate produced in the AT regulates the lipid metabolism and suppresses hepatosteatosis in mice deficient in fatty acid-binding proteins.

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human macrophages, thereby slowing down atherosclerosis progression. Chronic palmitoleate supplementation in hyperlipidemic mice lowers blood concentrations of interleukins IL1β and IL18. Aspalathin, a glucosyl dihydrochalcone36 from rooibos (Aspalathus linearis),37 can improve glucose metabolism and hence insulin resistance, which can be associated with saturated FFAs such as palmitate, in adipocytes [1201]. Aspalathin represses the activity of NFκB and AMPK, activates PKB, and upregulates the formation of the nuclear receptors NR1c1 and NR1c3 (PPARα and PPARγ), which regulate carbohydrate and lipid metabolism. Two major antioxidant and anti-thrombotic dihydrochalcones of green rooibos tea, aspalathin and nothofagin, inhibit thrombin (FI I a) and activated clotting factor FXa. They also prevent thrombin-catalyzed fibrin polymerization and platelet aggregation [1207]. In addition, they reduce the ratio of plasminogen activator inhibitor PAI1 to tissue-type plasminogen activator (PAI1/tPA). Cinnamon, a traditional antidiabetic remedy from the aromatic bark of Cinnamomum cassia, has anti-hyperglycemic and antihyperlipidemic effects. It upregulates expression of NR1c1 and NR1c3, which activate their target genes (Aco,38 Fas, GLUT4, Lpl, and SCARB3), thereby improving insulin sensitivity, reducing fasting glycemia and amounts of FFAs and LDLCS , in addition to the blood level of aspartate aminotransferase, an indicator of hepatocyte damage, in high-caloric dietinduced obesity (DIO) and db/db mice [1203]. Catechins, which are compounds in plants, fruits, and their derivatives used as foods and drinks (e.g., pears, apples, tea, cocoa, and wine) can reduce blood pressure via vasodilation and attenuate atherosclerotic lesions [1204]. The flavonoid cyanidin in red berries and other fruits blocks the binding of interleukin-17A to the IL17Rα subunit to alleviate inflammation caused by IL17aproducing TH17 lymphocytes [1205]. Sulforaphane,39 which derives from a glucosinolate of broccoli, and quercetin, an aglycone derived from flavonol glycosides, can prevent redox stress, as they upregulate expression of NFE2L2, a regulator of cellular resistance to oxidants. Sulforaphane inhibits glucose production in cultured hepatocytes via nuclear translocation of NFE2L2 and subsequent decreased production of gluconeogenesis enzymes [1206]. It improves glucose tolerance in rodents fed a high-fat or -fructose

36 This

flavonoid is also alternatively called dihydrochalcone C glucoside, the dihydrochalcone being linked by a carbon atom to glucose. 37 Rooibos (red bush) is a member of the Fabaceae (a.k.a. Leguminosae) and Papilionaceae tribes, from the Crotalarieae family of plants (commonly known as the legume, pea, or bean family) growing in South Africa’s fynbos, that is, a shrubland (i.e., a vegetation dominated by shrubs). 38 ACO: acyl-CoA oxidase. 39 Sulforaphane is a compound of the isothiocyanate group of organosulfur compounds, which originates from its glucosinolate precursor, glucoraphanin, upon processing by myrosinase. It is found in cruciferous vegetables (e.g., broccoli, cauliflower, Brussels sprouts, and cabbages). Analysis of gene expression patterns associated with T2DM in the liver and gene signatures linked to substances that counteract the effects of diabetes detected sulforaphane [1206].

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diet. Moreover, concentrated broccoli sprout extracts reduce blood concentrations of fasting glucose and glycated hemoglobin (HbA1c) in obese T2DM patients. Resveratrol is a stilbene-type phytoalexin of legumes and fruits (e.g., grapes and berries) that acts via AMPK, sirtuin-1, and NFE2L2 to protect against pancreatic βcell dysfunction, improves insulin sensitivity, and hinders inflammation in addition to poly-ADP ribose polymerase and phosphodiesterase activities [1207]. The transcription factor NFE2L2 permits adaptation and survival under stress conditions. It controls the basal and induced formation of cytoprotective proteins, such as antioxidant, detoxifying, and anti-inflammatory enzymes, in addition to proteins that assist in the repair or removal of damaged molecules [1208]. It thus maintains cellular redox balance, as it controls synthesis, utilization, and regeneration of glutathione, thioredoxin, and NADPH in addition to the production of ROS by mitochondria and NADPH oxidase, hence regulating outcome upon oxidant exposure. Upon stimulation by growth factors or stressors, it counteracts increased ROS production in mitochondria via the transcriptional upregulation of the uncoupling protein, UCP3; maintains nuclear respiratory factor, NRF1, and PGC1α and hence mitochondrial genesis; and promotes purine nucleotide synthesis. It impedes oxidant-mediated opening of the mitochondrial permeability transition pore and subsequent mitochondrial swelling. The factor NFE2L2 raises production of antioxidants and detoxifying agents (e.g., drug processors), such as glutathione peroxidases (e.g., GPOx2), glutathione S transferases, superoxide dismutase, catalase, glutamate–cysteine ligase, heme oxygenase HOx1, and NAD(P)H dehydrogenase quinone (or NADPH:quinone oxidoreductase) NQO1 [1209]. NFE2L2 also influences ATP synthesis, as it acts on the mitochondrial membrane potential, mitochondrial fatty acid oxidation, availability of substrates (NADH, FADH2 , and succinate), and supports the structural and functional integrity of mitochondria [1208]. This short-lived protein is ubiquitinated for proteasomal degradation by three ubiquitin ligase complexes, keap1–Cul3–Rbx1, Cul1-based SCFβTRCP upon phosphorylation by GSK3 [1210],40 and Syvn1–Sel1L41 during ER stress linked to misfolded glycoproteins and nonglycosylated proteins [1211]. In cultured CMCs and ECs, NFE2L2 counters production of ROS and redox stress-induced insulin resistance [1212]. Aspalathin can ameliorate a hyperglycemic-induced shift in substrate preference and protect the myocardium against cell apoptosis.

40 GSK3

launches ubiquitination by SCFβTRCP and proteasomal degradation of various transcription factors and other proteins (Snai, β-catenin, Gli2–Gli3, the phosphatases CDC25a and PHLPP1, Rho/RacGEFs FGD1 and FGD3, BCL2L3, securin, and prolactin receptor [1210]. 41 Syvn1: synoviolin; Sel1L: suppressor of Lin12-like protein-1.

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6.5.6 Diet and Gut Flora The human gastrointestinal tract constitutes a bacterial ecosystem. This microbiome possesses the highest microbial density in the colon. Gut microbes are codependent on one another and depend on their host, as they require a metabolic support from members of the community for survival and receive a favorable environment from the host. The intestine affords an adequate environment for bacteria via its quasiconstant temperature and periodic supply of nutrients and water. Reciprocally, resident microbes process indigestible food and assist nutrient digestion via saccharolytic or proteolytic pathways. The saccharolytic pathway provides the majority of SCFAs. The proteolytic pathway, which is related to protein fermentation, also produces SCFAs, but leads to other co-metabolites, such as ammonia, various amine types, thiols, phenols, and indoles, some of which are toxic. Microbial metabolites, such as SCFAs produced by bacterial fermentation of indigestible dietary polysaccharides, regulate the intestinal epithelial barrier and immunity [1059].42 Resident bacteria also restrict the colonization of aggressive microorganisms, as they compete for nutrients. Conversely, the immune system influences microbiome composition. Loss of the NLRP6 inflammasome increases colitis-associated bacteria.43 NLRP12 contributes to the maintenance of the intestinal microbiome [1213]. In NLRP12 deficiency, gut populations of commensal protective bacteria of the Lachnospiraceae family decays and colitogenic bacteria of the Erysipelotrichaceae family predominate; the resulting dysbiosis correlates with bowel inflammation. Intestinal inflammation is associated with the abnormal activation of inflammatory signaling pathways, such as components of the NFκB, MAPK, and STAT sets, and cytokines. In addition to administration of beneficial commensal Lachnospiraceae isolates, neutralizing inflammatory cytokines IL6 and TNFSF1 also restores a protective microbiome. Most of the gut microbial community is composed of five phyla (Actinobacteria, Bacteroidetes, Verrucomicrobia, Firmicutes, and Proteobacteria). Food constituents determine gut commensal microbiota composition in the gastrointestinal tract.44 Composition of the diet influences not only the content, but also the metabolism, of the human intestinal flora. Some diets promote the growth of beneficial microorganisms, whereas others support harmful microfloral activity [1214].

42 Lactate

produced by Lactobacillus promotes proliferation of intestinal epitheliocytes. Butyrate strengthens the epithelial barrier via hypoxia and HIF1β [1059]. 43 The NLRP6 inflammasome impedes canonical and alternative NFκB axes. 44 Bifidobacterium is a dominant genus of the intestinal microbiota during the first year after birth [1059]. The microbial diversity gradually rises during the next 3 years, the microbiota composition varying according to genetic and dietary factors. Furthermore, the gut microbiota exhibits a circadian rhythm, with daily oscillations in the flora composition and gene transcription linked to the metabolism regulation.

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Sulfur compounds, sulfate and sulfite, increase the growth of potentially pathogenic microorganisms or the production of potentially harmful bacterial products in the gastrointestinal tract. The colon hosts a specialized set of gram− anaerobic sulfate-reducing bacteria (SRB; i.e., species of the genera Desulfotomaculum, Desulfovibrio [64–81%], Desulfobulbus, Desulfobacter, and Desulfomonas) [1214]. They use dissimilatory sulfate reduction to reduce sulfite and sulfate to sulfide (e.g., H2 S). Sulfate-reducing bacteria compete with methanogenic bacteria (MB) for nutrients, methanogenesis and sulfate reduction being mutually exclusive in the colon. Sources of dietary sulfate include preservatives, dried fruits treated with sulfur dioxide, dehydrated vegetables, fresh and frozen shellfish, packaged fruit juices, baked goods, white bread, and the majority of alcoholic beverages [1214]. The main amounts of sulfur-containing amino acids derive from cow’s milk, cheese, eggs, meat, and cruciferous vegetables. Consumption of a high-protein diet can increase the production of potentially harmful bacterial metabolites [1214]. When dietary proteins escape digestion in the proximal part of the digestive tract and reach the colon, in addition to host-derived proteins (pancreatic and intestinal enzymes, mucins, glycoproteins, and sloughed epitheliocytes), undigested proteins are fermented by the colonic microflora with endproducts of small- and branched-chain fatty acids (e.g., isovalerate, isobutyrate, and methyl butyrate) and produced potentially harmful metabolites (e.g., ammonia, amines, phenols, sulfide, and indoles). Moreover, ingestion of large amounts of animal proteins increases the activity of certain bacterial enzymes (e.g., βglucuronidase, azo- and nitroreductase, and 7α-hydroxysteroid dehydroxylase). High simple sugar diets slow bowel transit time and heighten fermentative bacterial activity, thereby augmenting the toxic residence time. Refined carbohydrates are processed quickly in the ascending colon, whereas high-fiber foods are metabolized more slowly, releasing fermentation endproducts (e.g., hydrogen gas and SCFAs) more gradually [1214]. Furthermore, high sugar intake increases the flux of bile acids that feed some species of intestinal bacteria. Conversely, metabolism of the gut microbiota can contribute to genesis of CVDs via the trimethylamine (TMA) and trimethylamine N oxide (TMAO), SCFA, and primary and secondary bile acid pathway [1215]. Microbial metabolites can enter the blood circulation and then exert their action directly or after being further processed by host enzymes. In addition, bacterial constituents (e.g., lipopolysaccharide and peptidoglycans) can be conveyed in the bloodstream and affect remote organ functioning. Some bacterial products interact with host messengers (e.g., ghrelin, glucagon-like peptide GLP1, leptin, and peptide-YY). Others stimulate the parasympathetic nervous system.

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Immunoregulatory CD4+ CD8αα+,45 CD4, and CD8 double-positive intraepithelial lymphocytes (dpIELϕs), which originate from intestinal CD4+ T cells, their differentiation depending on bacterial factors, participate in intestinal immunity.46 Among species of probiotic bacteria, Lactobacillus reuteri support the development of these cells, as they create specific indole derivatives of dietary tryptophan that activate aryl hydrocarbon receptor (AHR, or bHLHe76) in CD4+ T cells, downregulating the expression of zinc finger and BTB domain-containing protein ZBTB7b and eliciting their differentiation from precursors [1218].

6.6 Effects of the Gut Microbiota Gut flora contains predominantly four bacterial phyla: Gram− Bacteroidetes and Proteobacteria and Gram+ Actinobacteria and Firmicutes. The bowel microbiota supports the maturation and functioning of mucosae and immunity, owing to crosstalk between the intestinal commensal bacteria and components of the immune system, and controls the regeneration of the intestinal epithelium [1219]. Immature mucosal defense contributes to increased susceptibility of newborn infants to pathogens. For example, exposure of neonatal mice to commensal bacteria immediately after birth, which recruits IL22+ group-3 innate lymphoid cells (ILC3) into lungs of newborn mice via intestinal dendrocytes, is required for a robust host defense against bacterial pneumonia [1220].47

T-cell surface glycoprotein CD8 exists as CD8αα or CD8αβ dimer, the CD8 α- and β-chain being encoded by the CD8A and CD8B genes. The CD8αα homodimer lodges on subpopulations of developing thymocytes, gut intraepithelial lymphocytes, and a subset of NK cells and dendrocytes [1216]. The CD8αβ heterodimer, which is expressed predominantly on thymocytes and peripheral cytotoxic T cells and their progenitors, is an efficient coreceptor of the αβ-T-cell receptor. The CD8αα dimer is a weaker coreceptor than CD8αβ for TCR activation, although both bind to major histocompatibility complex (MHC) class-I molecules with similar affinity, but the homodimer tethers to thymic leukemia antigen, an alternative MHC class-I molecule in the mouse, with much higher affinity than the heterodimer. The homodimer is involved in virus-specific memory T-cell generation, upregulation of its expression correlating with the increased formation of BCL2 and IL7Rα [1216]. 46 The intestine is implicated in lymphopoiesis; precursors that can differentiate into T cells reside in the epithelium and lamina propria. Moreover, the thymus exports immature cells to the intestine. Intraepithelial lymphocytes contain several subsets, including TCRαβ+ CD8αα+ IELs, which originate from a subset of CD8αα+ double-positive thymic cells [1217]. 47 Symbiotic and commensal bacteria of the digestive tract lumen are detected by intestinal mucosal dendrocytes. Commensal colonization begins during birth and evolves rapidly during the first month of life. Cesarean deliveries and antibiotics during early life alter intestinal commensal colonization in the newborn and increase the risk for infection. Commensal bacteria raise the number of IL22+ ILC3 in the small intestine. Whereas T lymphocytes and NK cells are primary mediators of mucosal defense against respiratory pathogens in adults, the mouse lung in the postnatal period is populated by only a few IL22+ T and NK cells, but a significant density of IL22+ ILC3 [1220]. Whereas migration of ILC3 in the small intestine relies on the chemokine 45 The

6.6 Effects of the Gut Microbiota

587

Commensal microorganisms produce various metabolites (SCFAs, organic acids, vitamins, cofactors, amino acid derivatives, amines, and indoles). Microbial metabolites (e.g., SCFAs, deoxycholate, and aminobenzoate) stimulate the vagus nerve, which signals to the central nervous system, and enteroendocrine cells, which produce gastrointestinal hormones, thereby defining a gut flora–brain axis. In addition, Lactobacillus and Bifidobacterium produce the neurotransmitter GABA and Clostridium tryptamine [1059]. Commensal gastrointestinal bacteria in the human gut communicate with host cells, N acyl synthases catalyzing synthesis of GPCR ligands, the bacterial metabolites N acyl amides [1072]. Agonists of glucose-dependent insulinotropic receptor GPR119 (GPCR2) produced by the human gut flora regulate metabolic hormones and glucose homeostasis as efficiently as usual ligands. The human microbiota-derived and GPCR agonist commendamide is a longchain N acyl amide that shares a broad structural similarity with lipid-like ligands, such as eicosanoids, endocannabinoids, and sphingolipids. Lipid-like-tethering GPCRs, such as those interacting with N acyl amides, are implicated in diabetes and obesity (GPR119), atherosclerosis (GPR132 and PtgIR), colitis (S1P4 , PtgIR, and PTGER4), autoimmunity (GPR132), and cancer, among other diseases [1072]. Gut microbes produce TMAO from dietary nutrients, such as choline, lecithin (or phosphatidylcholine), and L carnitine, which is linked to atherogenesis [1221]. TMAO enhances platelet responsiveness to multiple agonist types via Ca2+ signaling, thereby heightening thrombotic risk [1222]. Acute dietary deficiency in micronutrients, such as vitamins (e.g., vitamin A and folate) and minerals (e.g., iron and zinc), affects the structure of the bacterial community [1223]. Bacteroides vulgatus shows the largest response to vitamin A deficiency, which increases its density. In the bowel of neonatal and adult mice, anaerobic, spore-forming bacteria, Clostridiales, protect against pathogen invasion [1224]. Protection is enhanced by adding succinate to drinking water, which favors colonization of the neonatal gut by clusters IV and XIV a Clostridia and concomitantly excludes Salmonella typhimurium. Administration of Clostridiales, but not Bacteroidales, protects neonatal mice from infection.

receptors CCR9 and α4 β7 Itg on ILC3, their movement in the lung depends on CCR4, which is activated by CCL17 expressed by the pulmonary epithelium and CCL20. Intestinal ItgαE + ItgαM + dendrocytes exposed to commensal bacterial flora promote CCR4 expression of the lung-homing IL22+ ILC3.

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Dysbacteriosis, or dysbiosis,48 refers to an altered bowel flora linked to qualitative and quantitative change in bacterial types of the gut microbiota and in its activities. The abnormal composition of the intestinal flora is characterized by a decreased diversity in beneficial bacteria and increased presence of opportunistic pathogens. Dysbacteriosis contributes to many types of chronic and degenerative diseases [1214]. Lipopolysaccharide from intestinal bacteria and Gram− bacteria activates TLR4 in ECs, which cooperates with its coreceptor CD14, thereby engendering cerebral cavernous malformations that can cause stroke [1226]. These abnormal vascular structures arise from the loss of an adaptor complex that inhibits MAP3K3–KLF2/4 signaling in cerebral ECs.

6.6.1 Gut Flora and Obesity Leptin-deficient obese mice are characterized by a largely reduced amount of Bacteroidetes and a proportional increase in Firmicutes with respect to wildtype siblings and their mothers, all fed the same diet [1219]. High-fat and -polysaccharide diets in wild-type rodents cause similar changes in microbial flora. The ratio of Firmicutes/Bacteroidetes in the distal gut microbiota can also increase in obese humans [1219]. However, the larger quantity of Firmicutes associated with diet-induced obesity is related to the growth of the Mollicutes class in the Firmicutes phylum [1227]. In humans, the gut microbial flora is also altered with obesity. The relative abundance of Bacteroidetes increases as some individuals lose weight when undergoing a fat- or carbohydrate-restricted low-calorie diet [1227]. Individuals with obesity who have a high bacterial gene count (i.e., process ingested food more efficiently) possess a greater proportion of bacterial species associated with an antiinflammatory status (e.g., Faecalibacterium prausnitzii) and a smaller proportion of species linked to a proinflammatory status (e.g., Bacteroides species). Obesity, i.e., an excess of AT, results from an imbalance between nutrient intake and expenditure. It is a risk factor for insulin resistance, metabolic syndrome, and diabetes. It is often associated with hyperglycemia, hypertriglyceridemia, dyslipidemia, and hypertension. The metabolic syndrome is characterized by impaired lipid accumulation, loss of insulin sensitivity, and chronic low-grade inflammation. Excessive nutrient intake activates immunocytes (e.g., dendrocytes, macrophages,

48 The

word “dysbiosis” was coined by I.I. Metchnikoff (1845–1916), the opposite of symbiosis, a state of co-existence in mutual harmony [1225]. A dysbiotic microbiota is an altered intestinal microbial community, consisting of bacteria, yeast, viruses, and parasites, and characterized by quantitative and qualitative changes in its composition, metabolic activities, and local distribution of its members. Dysbiosis is currently linked to inflammatory bowel disease, obesity, diabetes, allergy, asthma, and cancer, most of these afflictions involving inflammation, a phenomenon often caused by an impaired immune response to intestinal bacteria.

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and mastocytes) and hence cytokine production, especially in metabolic organs and adipose depots. Insulin triggers glucose uptake in cells and hence glycolysis for cytosolic and mitochondrial ATP synthesis, in addition to glycogenesis and lipogenesis for storage and protein synthesis. Loss of insulin sensitivity primes fasting hyperglycemia and increases hepatic lipid synthesis, dyslipidemia, and fat accumulation in ATs. Insulin resistance causes permanent hyperglycemia. Inflammation and altered insulin activity are linked via [1227] (1) activation of the Iκ B kinase complex, ERK1, ERK2, and JNKs that decreases Tyr phosphorylation of IRSs and hence insulin signaling; (2) production of cytokines in the visceral AT, which affects synthesis of NR1c1, IRS1, and GluT4; and (3) interactions between gut microorganisms and host metabolism, modification in gut microbial composition altering the symbiotic relationship between intestinal bacteria and the host. Altered bowel microbiota composition is linked to the development of obesity, insulin resistance, and diabetes in the host via several mechanisms: (1) increased energy harvest from the diet and host lipid storage,49 (2) impaired fatty acid metabolism and composition in the AT and liver, (3) modulation of intestinal secretion of peptide-YY and glucagon-like peptides GLP1, and GLP2 (4) activation of the LPS–TLR4 axis (5) regulation of intestinal barrier integrity by GLP2, an intestinal growth factor with anti-inflammatory effects and intestinal barrier stabilizer [1228]. The Western diet favors an obesogenic gut flora. Gut microbiota participates in regulating the transcription of host genes that control the metabolic processes. The intestinal flora stimulates hepatic and AT lipogenesis via carbohydrate response element-binding protein (CHREBP)50 and sterol responsive element-binding protein, SREBP1,51 eventually promoting lipid accumulation in the liver and AT [1228]. Moreover, with respect to germ-free mice, the density of small

49 Germ-free

mice have less adipose depot than conventional mice, although they ingest more food than their conventional littermates. Transplantation of fecal microbiota of conventional mice to germ-free mice increases the amounts of fat and hepatic triglyceride and causes insulin resistance without adding to the amount of food [1219]. Mice colonized with the fecal microbiota of co-twins with obesity had a greater increase in body weight and adipose depots than mice colonized with the fecal microbiota of lean co-twins [1227]. 50 CHRE- binding protein is a transcription factor activated in response to high glucose concentrations in the liver independently of insulin. Simple sugars derived from the digestion of dietary carbohydrates are taken up by the liver and converted to fatty acids for long-term storage when they exceed the amount needed to meet the body’s short-term energy requirements. This process is carried out by many enzymes in the liver: pyruvate kinase, which regulates the final step of glycolysis, acetyl-CoA carboxylase, which catalyzes the first step of fatty acid synthesis, and FAS, all regulated by CHREBP [1229]. 51 Sterol regulatory element-binding protein, SREBP1c, is an insulin-activated isoform of the SREBP family of transcription factors. It is the main insulin-responsive regulator of the transcription of genes encoding hepatic lipogenic enzymes in rodents fed high-carbohydrate diets [1229].

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intestinal villi capillaries and monosaccharide uptake from the gut into the portal blood increase. The gut microbiota thus favors the development of the digestive tract epithelium and influences intestinal function and motility, thereby promoting nutrient extraction from the alimentary bolus. Microbiota of the digestive tract supports the storage of circulating triglycerides into adipocytes, as it suppresses intestinal secretion of an inhibitor of AT lipoprotein lipase, AngPtL4 [1228].52 Lactobacillus paracasei upregulates AngPtL4 synthesis in colonic epitheliocytes [1219]. Hypothalamic AngPtL4 expression is regulated by appetite regulators and mediates their anorexigenic effects via inhibition of hypothalamic AMPK activity. This first regulatory route is complemented by others. In lean germ-free mice, activation of hepatic and muscular fatty acid oxidation is greater. This reaction is mediated by two complementary mechanisms [1228]: (1) Elevated activity of AMPK, which controls cellular energy status and stimulates enzymes of mitochondrial fatty acid oxidation, such as acetyl-CoA carboxylase and carnitine palmitoyltransferase-1, decreased glycogen storage, and increased hepatic insulin sensitivity. (2) An elevated PGC1α synthesis induced by AngPtL4, a coactivator of nuclear receptors and enzymes involved in fatty acid oxidation. The gut microbiota suppresses the release of AMPK, which is primarily expressed in the liver, skeletal muscle, and brain, in response to metabolic stress (e.g., hypoxia, glucose deprivation, exercise), thereby lowering mitochondrial fatty acid oxidation, ketogenesis, glucose uptake, and insulin secretion but raising cholesterol and triglyceride synthesis [1227]. Intestinal microbiota synthesizes a large amount of glycoside hydrolases that break down plant polysaccharides to monosaccharides and, as end products of bacterial fermentation, SCFAs. SCFAs enter the blood circulation and act in the gut as signaling mediators. They tether to free fatty acid receptors, FFAR2 (GPR43), FFAR3 (GPR41), and GPR109a on intestinal enteroendocrine cells, thereby stimulating secretion of peptide-YY, which slows intestinal transit and raises nutrient absorption [1228]. Mice deficient in FFAR3 can be lean compared with their wild-type littermates [1227]. Conventional and germ-free Ffar3−/− mice colonized by only Bacteroides thetaiotaomicron and Methanobrevibacter smithii are markedly leaner than their wild-type littermates despite similar levels of chow consumption, whereas wild-type or GFfar3−/− germ-free mice do not differ [1219]. Activated FFAR2 can suppress insulin sensitivity in ATs and promote insulin sensitivity in the liver and skeletal muscle [1227]. Butyrate serves as an energy substrate for enterocytes. Type-2 diabetes is associated with a reduced amount in butyrate-producing bacteria, decreasing insulin sensitivity [1215]. Short-chain fatty acids such as butyrate activate FFA2 and FFA3 , which affect inflammation and enteroendocrine regulation. The SCFA–FFA3 couple induces expression of peptide-YY in gut epithelial L cells. Short-chain fatty acids via both FFA2 and FFA3 also trigger GLP1 secretion by intestinal L cells. In WAT

52 Angiopoietin-like

protein-4 is also called hepatic fibrinogen- and angiopoietin-related protein and fasting-induced adipose factor.

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591

of the mouse, SCFAs via FFA2 suppress insulin-mediated lipid accumulation and stimulate energy expenditure in the liver and muscle. The obese gut microbiome is characterized by a depletion in proteins involved in motility (chemotaxins and flagellar assembly mediators) and an abundance of glycoside hydrolases, which process indigestible alimentary polysaccharides; phosphotransferases, which are implicated in the import of simple sugars (e.g., glucose, fructose, and acetyl galactosamine); β-fructosidase, which degrades fructosecontaining carbohydrates (e.g., sucrose) to lactate, butyrate, or acetate; and other transport proteins and fermentation enzymes [1228]. Choline is an essential nutrient for the synthesis of phosphatidylcholine, which is a major component of cell membranes. Phosphatidylcholine is a major component of VLDLs, which carry triglycerides. Defective export of triglycerides by VLDLs causes hepatosteatosis. Gut flora is able to convert choline to TMA, thereby controlling the availability of choline and indirectly affecting triglyceride storage in the liver [1227]. Bowel microbiota subjected to a high-fat diet converts dietary choline into hepatotoxic methylamines, hence reducing the availability of choline, which is necessary for the assembly and secretion of VLDLs, and eventually leading to insulin resistance, hepatic steatosis, and lipoperoxidation [1228]. Gut flora can modulate host lipid metabolism via modified bile acid conjugative pattern, affecting emulsification and absorption properties of bile acids and indirectly impacting on hepatic lipidic storage and lipoperoxidation [1228]. Gut microbiota may contribute to the high-fat-diet-induced obesity via farnesoid X receptor (FXR; NR1h4/5), the bile acid receptor responsible for the regulation of bile acid synthesis and hepatic triglyceride accumulation [1227]. The gut microbiota influences the metabolism and signaling of bile acids, as they produce agonists of G protein-coupled bile acid receptor-1 (TGR5) and process antagonists of FXR such as tauroβ-muricholic acid, a primary bile acid, thereby relieving FXR inhibition [1215]. Conversely, bile acids affect the cecal microbiota composition of rats. Gut bacteria experiencing high-fat and -sugar diets can initiate a chronic inflammatory state of obesity and insulin resistance via lipopolysaccharide (endotoxin), which derives from the outer cell membrane of Gram− bacteria. The resulting low-grade endotoxinemia reduces the amount of epithelial tight junction proteins (occludin and ZO1), thereby increasing intestinal permeability [1228]. Lipopolysaccharide can cross the gastrointestinal mucosa through leaky intestinal tight junctions and infiltrate chylomicrons and then the liver or adipose depots, triggering an innate immune response via TLR4, ERK1, ERK2, JNKs, P38MAPKs, and IKKβ [1227]. Prebiotic carbohydrates and antibiotics lower endotoxinemia and inflammatory cytokine formation in the liver and enhance intestinal GLP2 production [1219]. Endocannabinoids mediate the influence of intestinal microbiota on gut permeability in addition to endotoxinemia and adipogenesis. Endocannabinoids increase the synthesis of occludin-1 but decrease that of claudin-1 [1219].

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In murine models of metabolic syndrome, the amount of intestinal barrierprotective bacterial species (Bifidobacterium species) diminishes and that of endotoxin-producing species (Desulfovibrionaceae) rises [1219]. Microbial metabolites produced from aromatic amino acids (tyrosine, tryptophan, and phenylalanine) may affect host immunity. Indole, one of the major tryptophan-derived microbial metabolites, produced by tryptophanase in Bacteroides thetaiotaomicron, Proteus vulgaris, and Escherichia coli, among other species, can be sulfated in the liver to create 3-indoxylsulfate or undergo further bacterial metabolism, producing indole 3-pyruvate, 3-lactate, and 3-acetate [1227]. Indoxylsulfate activates the aryl hydrocarbon receptor (bHLHe76), thereby regulating the transcription of IL6 and several enzymes of the CYP450 superfamily (e.g., CyP1a1, CyP1a2, and CyP2S1). Indole 3-propionate is a pregnane X receptor (NR1i2) agonist, which has a beneficial action on the gut barrier, as it upregulates expression of junctional proteins and downregulates TNFSF1 production in enterocytes [1227]. The pattern recognition receptor, TLR5, affects microbial composition and intervenes in the genesis of metabolic syndrome. Tlr5−/− mice exhibit hyperphagia and develop a metabolic syndrome, food restriction improving metabolic anomalies, but lean Tlr5−/− mice display insulin resistance, although in other animal colonies with TLR5 deficiency, intestinal inflammation and metabolic dysfunction are not observed [1219]. Gut flora fermentation of prebiotics (e.g., nondigestible prebiotic carbohydrates), which serve as food for beneficial probiotics (i.e., symbiotic and commensal intestinal bacteria), promotes L-cell differentiation in the proximal colon of rats and increased glucagon-like peptide GLP1 response to a meal in healthy humans. Prebiotics preserve the intestinal barrier and increase secretion of GLP1 and GLP2 [1228]. An elevated Firmicutes/Bacteroidetes ratio is related to obesity-associated dysbiosis. Germ-free mice have a defective epithelial barrier and production of IgA and antimicrobial peptides but are resistant to obesity induced by high-fat and highsugar diets [1059]. Prebiotics (i.e., fermentable food constituents, such as pectin, inulin, and resistant starch) determine the gut flora and can protect against excessive inflammation.

6.6.2 Gut Flora and Cancer Metabolites released by invading pathogens, such as TMA and 4-ethylphenylsulfate, are pathological agents. In the mouse small intestine, increased densities of Helicobacteraceae, Lactobacillaceae, Enterobacteriaceae, Clostridiaceae, and Peptostreptococcaceae and decreased densities of Bifidobacteriaceae, Porphyromonadaceae, and Alcaligenaceae favor local carcinogenesis [1059].

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6.6.3 Gut Flora and Hypertension In animal models of hypertension (spontaneously hypertensive rats [SHRs] and rats receiving a chronic infusion of angiotensin-2), the expression of several tight junction proteins declines in the gut [1230]. In addition, increased intestinal permeability results from elevated sympathetic nerve activity before the development of hypertension. A decreased mesenteric blood flow is also linked to intestinal hyperpermeability [1231]. Alcohol abuse also raises bowel permeability. Phenolic compounds from tyrosine breakdown by opportunistic bacteria disrupt the epithelial barrier of the digestive tract. Cigarette smoking and psychological stress have an impact on the sympathetic nervous system and hence gut permeability. In animal models of hypertension, gut–brain communication involving the paraventricular nucleus of the hypothalamus increases with a dysfunctional sympathetic command to the gastrointestinal tract [1230]. Dysbacteriosis, which happens after the onset of gut hyperpermeability, is associated with an increased density of inflammatory cells within the intestinal wall, the densities of T lymphocytes, monocytes, and macrophages increasing in the intestinal epithelium [1230]. Among substances released by the gut flora, especially SCFAs, polyamines, and activators of the aryl hydrocarbon receptor, within the framework of hypertension, SCFAs, which can inhibit histone deacetylases, may modulate the immune environment of the bowel [1231].53 Short-chain fatty acids stimulate host Gprotein-coupled receptors, which have an impact on renin secretion. The renal and vascular OlfR78 elevates blood pressure, whereas FFA3 (GPR41) lowers blood pressure [1215].

6.6.4 Gut Flora and Atherosclerosis The gut microbiota not only supports weight gain and impairs glucose metabolism in addition to atherosclerosis via metabolism of substrates such as choline, phosphatidylcholine, and carnitine to TMA, and subsequent conversion to TMAO by flavin mono-oxygenase-3, and via B2 lymphocytes [1232]. The processing of dietary choline to TMA by intestinal microbiota and to TMAO by flavin mono-oxygenase in the liver suppresses reverse cholesterol transport [1233]. The absence of intestinal microbiota reduces fecal bile acid excretion and heightens macrophage-to-feces reverse cholesterol transport [1234]. In chow-fed

53 The

gut flora is involved in the stimulation of immunity, the synthesis of vitamin-K and of the B group, intestinal motility, digestion and nutrient absorption, resistance to pathogen colonization, the metabolism of plant compounds and drugs, and the production of SCFAs and polyamines [1214].

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germ-free mice, plasmatic cholesterol concentration does not change, but hepatic cholesterol content, along with biliary secretion of cholesterol and bile acids, increase. However, these results may depend on diet, genetic background, and microbiota composition, in addition to differences between mammalian species in cholesterol and bile acid metabolism [1233]. Metabolites from proteins and amino acids in the diet, such as cresyl sulfate and indoxylsulfate, may contribute to pathogenesis [1235]. Increased dietary intake of precursors of toxic metabolic products of the intestinal microbiome, such as TMAO from phosphatidylcholine, has an impact on health.54 Circulating TMAO concentration correlates positively with atherosclerotic plaque size [1215]. In hyperlipidemia, signals sent by the intestinal microbiota activate B2 lymphocytes in the spleen, which then augment their production of MHC class-II molecules and IgGs. Furthermore, commensal intestinal microbes orchestrate recruitment and ectopic activation of B2 lymphocytes55 in perivascular AT, which, in cooperation with increased concentrations of circulating IgGs, participates in atherosclerosis via toll-like receptors, but independently of lipid metabolism [1235]. In rodents, administration of Lactobacillus plantarum significantly reduces infarct size and improves left ventricular function after myocardial infarction [1215]. Administration of the Lactobacillus rhamnosus GR1 attenuates left ventricular hypertrophy and heart failure after myocardial infarction.

54 TMAO

is the hepatic oxidation product engendered by flavin mono-oxygenase from the microbial metabolite TMA formed by TMA lyases. 55 B2 lymphocytes cells represent almost all B lymphocytes, including follicular and marginal zone B lymphocytes. The spleen is the major B2-cell reservoir (∼80% follicular B lymphocytes and ∼10% marginal zone B lymphocytes) [1235]. Follicular B lymphocytes participate in T-celldependent antibody response to antigen. Marginal zone B lymphocytes are situated at the interface with the blood circulation and represent the first line of defense against antigens; they engender a rapid T-cell-independent antibody response to antigen.

Chapter 7

Genetic Risk Factors

Precision medicine relies on subject-specific data used as input variables in computer-aided medical tools, especially genetic information linked to pathophysiological mechanisms. Adequate gene expression and transcriptional programming is mandatory for correct functioning of the cardiovascular apparatus. Genetic data are aimed at determining individuals at risk for surveillance, early diagnosis, and adequate treatment, in addition to family screening. Complex traits are influenced by both the environment and a combination of variants in several genes. In diseases, some genes can correlate with a given trait because they are directly involved in the development of these affections (i.e., causal mechanism) or the pathophysiological process secondarily alters the expression of the genes (i.e., reactive mechanism). The long DNA molecule (∼2 m) is packaged into chromatin to lodge in the small nucleus. Chromatin, that is, the DNA–histone complex, is organized into repeating units, nucleosomes (i.e., 147-bp double-stranded DNA around a histone octamer). Chromatin compacts and organizes DNA and must be remodeled for gene accessibility to the transcriptional machinery. Chromatin fibers (diameter 5–100 nm) comprise DNA wrapped around nucleosomes. Heterochromatin is the condensed transcriptionally silent form, whereas euchromatin contains transcribed genes. Histone variants demarcate boundaries between hetero- and euchromatin. Interaction between distal parts of the chromatin fiber engenders higher-order folding and compaction [1236]. In vitro, DNA compaction starts with it wrapping around core histone octamers to form nucleosomes. The current hierarchical model states that primary 11-nm DNA–nucleosome polymers assemble into 30-nm fibers that further fold into 120-nm chromonema, 300- to 700-nm chromatids (fibers), and, ultimately, mitotic chromosomes; heterochromatin is generally depicted as 30 and 120-nm fibers [1237]. However, chromatin has a higher propensity to aggregate in a dilute medium than in the crowded nucleoplasm [1236]. Yet, higher-order chromatin structures can exist in vivo. © Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0_7

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Almost all chromatin in the nucleus is predominantly organized as a disordered flexible polymer (diameter 5–24 nm), whatever the chromatin state, decondensed euchromatin, which often contains active DNA regions, and generally condensed and inactive heterochromatin. These chromatin chains can achieve different compaction levels and high-packing densities [1237]. In mitotic chromosomes (nuclear subregion with chromatin volume concentration >40%), polymers bend back on themselves to pack at high density, whereas during interphase (nuclear subregion with chromatin volume concentration ranging from 12 to 52%), the chromatin chains extend further [1237]. The genome is thus generally packaged in the nucleus as disordered polymer chains. Higher-order chromatin states, such as euchromatin, heterochromatin, and mitotic chromosomes, may be generated by differential packaging density of the same type of fiber rather than by a distinct organization. The ring-shaped cohesin complex1 regulates the separation of sister chromatids during cell division2 and mediates cohesion between replicated sister chromatids [1238]. Sister chromatids are thus tethered together by the cohesin complex from their birth to their separation at anaphase owing to acetylation of its SMC3 subunit3 by cohesin acetyltransferases esco1 and esco2 [1239].4 Whereas esco1 is active throughout the cell cycle, esco2 modifies cohesin only during the S phase; it is a substrate of the ubiquitin ligase APC/C, which is activated at mitotic exit. In fact, Esco1 regulates almost exclusively the activities of cohesin involved in DNA repair, transcriptional control, chromosome loop formation and/or stabilization, and the maintenance of chromosomal structure. Cohesin also contributes to damaged DNA repair5 and gene expression regulation in both proliferating and postmitotic cells. Transcription by RNA polymerase pushes the cohesin complex off genes, which slides along DNA, the displaced complex continuing to hold sister chromatids together [1240]. Transcriptional activity thus redistributes the cohesin complex to the 3 -ends of convergently oriented gene pairs.

1 The

cohesin complex consists of four core subunits (structural maintenance of chromosome ATPases SMC1 and SMC3, double-strand break repair protein Rad21 [a.k.a. nuclear matrix protein NXP1 and sister chromatid cohesion protein SCC1 homolog], and stromal antigen Stag1 or Stag2) [1238]. Other cohesin subunits comprise meiotic cohesin subunit Stag3 and meiotic recombination protein Rec8. Binding partners include sororin (or cell division cycle-associated protein CDCA5) and the interactors wings apart-like homolog, Wapal, and accessory regulators of cohesion maintenance, PDS5a and PDS5b (or SCC112a–SCC112b), which can be associated with Stag1 and Stag2. The cohesin complex encircles DNA at specific chromosomal sites. 2 Cohesin binds densely in centromeric regions of chromosomes where it helps to mount sister chromatids onto spindle microtubules from opposing poles. 3 SMC3: structural maintenance of chromosome protein-3. It is also termed basement membraneassociated chondroitin proteoglycan, i.e., bamacan, and chondroitin sulfate proteoglycan CSPG6. 4 Esco1(2): establishment of cohesion-1 homolog-1(2). 5 Newly replicated sister chromatids are held together by cohesin to facilitate not only chromosome segregation but also DNA repair. Cohesin connects to large regions surrounding double-strand breaks.

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597

Genome expression is regulated not only during DNA transcription but also during transport, splicing, and translation, and via turnover of generated transcripts. The gene is a hereditary unit situated in a given DNA segment on a given chromosome.6 It determines a particular trait. A Mendelian trait is controlled by a single locus and obeys to a simple Mendelian inheritance pattern. The genotype of any individual refers to the complete heritable genetic identity, that is, the entire cellular database of protein-coding genes. The genotype is translated to a phenotype at the organism level and within each cell type. Certain genes are not transcribed in some cell types owing to the epigenetic control of DNA accessibility (i.e., at the level of chromatin structure and organization; Chap. 7 and Vol. 11, Chap. 1. Gene Transcription and Epigenetic Control). Traditional genetic studies in humans or animal models establish the relation between genotype and phenotype. An allele refers to an alternative form of a gene. Every individual possesses a pair of alleles for any trait received from each parent. Two alleles of a given gene are either identical or differ, i.e., the organism is homozygote or heterozygote with respect to this gene. If, in a given subject, a gene exists in two alleles (A and a), three combinations of alleles (genotype) are possible (AA, Aa, and aa). Homozygotes AA and aa exhibit different forms of the trait (phenotype). According to Mendel’s principles of heredity, dominance results from the relation between two alleles of a gene, in which one allele (A) masks the expression (phenotype) of the other allele (a). In other words, heterozygous Aa individuals display the same phenotype as AA individuals, the allele A being dominant and allele a recessive. The terms amorph, hypomorph, and hypermorph are applied to alleles and transcripts that do not have activity or have a lower or higher activity than wild-type alleles or transcripts, respectively. The term antimorph corresponds to dominant phenotypes in which the mutant protein produced interferes with the action of the wild-type one. Neomorphic mutations refer to a dominant phenotype, a heterozygote expressing the mutated allele. The genetic background explains the heritability of certain vasculopathies and determines the susceptibility or resistance to cardiovascular risk factors. Genes and their products are related to a complex regulation that maintains the organismal homeostasis when environmental conditions change. Genetic variants can disturb elements of this regulatory network and its ability to restore and preserve homeostasis in a varying environment. Dysregulated processes lead to cardiovascular diseases (CVDs). The nucleotide sequences of DNA are subjected to mutations (i.e., permanent alterations), such as those occurring upon defective DNA damage repair and errors

6 χρωματιζω: color; σωμα): body. The human diploid chromosome number formed by two complete sets of chromosomes originating from each parent equals 46. The basic elements of a chromosome include a centromere, short and long chromosomal arms, telomeres, and replication origins.

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during replication (Vol. 9, Chap. 1. Ciliopathies). Two main categories include somatic mutations that can affect cell division and germline mutations that happen in gametes or their precursors and hence are transmitted to the progeny. According to the length of the nucleotide sequence, mutations are classified into short genic and long chromosomal mutations (i.e., comprising multiple genes). Monogenic disease, or single gene disorder, arises from a single mutated gene. For example, dysfunctional signaling from bone morphogenetic protein (BMP) is observed in primary or idiopathic pulmonary arterial hypertension (Vol. 13, Chap. 2. Pulmonary Hypertension). Mutations in the Bmpr2 gene that encodes the receptor BMPR2 are responsible for about 80% of heritable pulmonary arterial hypertension. An autosomal dominant disorder results from a single mutated copy of the gene of an autosome (i.e., any chromosome other than a sex chromosome), whereas two copies of the gene are mutated in an autosomal recessive disorder. Many types of small-scale mutations exist. Base substitutions mutations:

change a given codon. They include three subcategories of

Missense mutation substitutes one DNA base and hence one amino acid type during translation of the encoded protein. Silent mutation encodes a different amino acid without modifying the function of the protein produced. Nonsense mutation introduces a premature stop signal and hence engenders a truncated protein. Insertion adds extra base pairs, that is, a DNA sequence, hence generating an altered protein. Deletion removes a DNA sequence. Frameshift mutation results from loss (deletion) or addition (insertion) of DNA bases in a reading frame (set of codons). Repeat expansion increases the number of nucleotide repeats. In gain-of-function mutations (GOF [activating]), the effect of the encoded protein is stronger or supplanted by a new function. In loss-of-function mutations (LOF [inactivating]), the protein is partly or fully inactivated. The mutation type can promote • Haploinsufficiency, i.e., insufficient protein production and function, the presence of a single functioning copy of the gene being insufficient to produce a normal effect • A dominant negative effect, that is, a deleterious protein action, an overexpressed mutant polypeptide disrupting the activity of the wild-type gene product

7 Genetic Risk Factors

599

The simplest dominant negative mutation is linked to proteins that oligomerize, the mutant protein reducing the activity of the complex, hence impeding the process in which they are involved.7 The development of expression arrays allows the quantification of all transcript levels in a given cell type, thereby monitoring molecular phenotypes in a given disease. Congenital venous malformations, which are composed of dilated and distorted veins (Sect. 1.4.2), are linked to activating mutations in the PIK3CA gene, which encodes PI3Kc1α (P110α PI3K), and related genes of the PI3K–PKB pathway in about 30% of cases that lack TEK alterations [1242]. Mutations in the TEK gene, which encodes the protein Tyr kinase receptor TIE2, are found in approximately 50% of sporadic nonfamilial venous malformations [1243]. Endothelial expression of PIK3CAH1047R (constitutively active P110α PI3K mutant) engenders endotheliocyte hyperproliferation and reduces pericyte coverage of blood vessels and expression of arteriovenous specification markers in a mouse model of human venous malformations. These alterations regress upon PI3K inhibition by rapamycin. Genes that carry causal variants can interact. Therefore, analysis of a disease comprises elucidation of gene interactions. In activated endotheliocytes, the highly connected genes, Atf4,8 INSIG1,9 and Xbp1,10 constitute hubs [1244]. Gene modules are clusters of genes with a higher degree of connectivity with other members of the same module than with genes in different modules. For example, several of the most highly connected genes implicated in the unfold protein response are needed in the reaction of endotheliocytes to oxidized lipids. The ABCC6 gene, a cis-acting quantitative trait gene, is the major causal gene in cardiac calcification.11 Common forms of CVDs involve the interplay of numerous gene products and environmental factors (e.g., overnutrition, sedentary lifestyle, and smoking). Gene products interact with each other and the environment; these interactions can change in disease. Differentiated cells can evolve to a new identity upon ectopic expression of the pluripotency transcription factors KLF4, MyC, Oct4, and Sox2 forming the

7A

first example is given by signal transduction. Plasmalemmal protein Tyr kinase receptors (RTK) often dimerize once their extracellular domain is liganded and one monomer phosphorylates the cytoplasmic region of the other monomer. This cross-phosphorylation triggers the signaling cascade. In heterozygous individuals who express a normal (RTKN ) and mutant (RTKM ) allele encoding a RTK lacking the cytoplasmic portion, only the dimer RTKN RTKN works and the dimers RTKN RTKM and RTKM RTKM are inactive. A second example is given by the mutant factor P53, which prevents the normal P53 transcriptional regulator from binding to the P53-responsive element in the promoter of its target genes [1241]. 8 ATF: activating transcription factor. 9 InsIG: insulin-induced gene product. 10 XBP: X-box-binding protein. 11 ATP-binding cassette protein ABCc6 isoform-1 transports glutathione conjugates as leukotrieneC4 and N ethylmaleimide S glutathione. Isoform-2 inhibits TNFSF1-mediated apoptosis [108].

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KMOS set that trigger genomic reprogramming and dedifferentiation into induced pluripotent stem cells and eventually to the formation of teratomas. This reprogramming is, in general, inefficient, owing to cellular intrinsic barriers mediated by P53, CKI2aP16 , and CKI2aP19 . Reprogramming also inflicts extensive damage of nearby cells and subsequently senescence, thereby activating NFκB, secreting cytokines (especially interleukin-6), forming a cytokine-rich environment associated with senescent cells, and favoring KMOS-driven reprogramming of neighboring cells [1245]. Among gene insertion and deletion and single nucleotide variants, about 4% cause a loss of gene function, especially genes encoding drug targets for lipid lowering or conferring risk for highly penetrant genetic diseases [1246]. Prevention of CVDs partly relies on searching for candidate genes for susceptibility to these maladies, although genetic variants can lodge outside protein-coding genes (i.e., intergenic regions). In addition to genetic mutations that affect blood vessels (e.g., Marfan syndrome), genome-wide association studies (GWAS) have identified hundreds of risk loci for metabolic and CVDs associated with impaired lipid and glucose metabolism in addition to abnormal arterial pressure and inflammation. A genomic risk score based on a large number of single nucleotide polymorphisms (SNPs) can improve risk prediction based on traditional factors used in clinical scores. SNPs perturb the amount and activity of proteins. Multiple chromosomal sites can contribute synergistically to the disease risk. The number of detected genes often represents only a fraction of those expected to be involved in a given disease. In coronary artery disease (CoAD), the heritability explained by the top SNPs is approximately 10%, whereas estimates of heritability from family studies range between 30 and 50% [1247]. Therefore, most often, the genetic susceptibility to chronic disease of any individual cannot be ascribed to a few genetic variants. Some highly polygenic traits need incorporation of a large number of SNPs, in addition to the high density of independent causal SNPs within each genetic locus. Genetic risk scores (GRS), which complement clinical risk scores, can be implemented after removal of the most highly correlated SNPs, each GRS corresponding to a variable that summarizes the degree of the presence of many high-risk variants [1248]. Numerous genetic loci are identified in association with hypertension, atherosclerosis, and thrombosis. For example, a CoAD GRS (GRS for CoAD) involving more than 49,000 SNPs validated on five cohorts improves risk prediction and hence early identification of individuals with increased risk for at least preventive lifestyle modifications [1249]. Familial hypercholesterolemia (FHCS) remains underdiagnosed despite widespread cholesterol screening, whereas genomic screening can prompt the diagnosis, thereby preventing cardiovascular morbidity and mortality [1250]. Pathogenic variants in three genes, APOB, Ldlr, and Pcsk9, account for the majority of FHCS cases. Carriers of FHCS variants have about 70 mg/dl greater maximal LDLCS level than noncarriers.

7.1 Messenger RNA Synthesis and Splicing

601

In addition to a genetic predisposition, epigenetic context can influence gene expression in disease. Gene transcription is epigenetically regulated by DNA methylation and histone modifications in addition to microRNAs and other a priori nonprotein-coding RNAs. Hence, the epigenetic framework is established not only by DNA modifications and reversible covalent post-translational modifications of histones but also by interactions with nonprotein-coding RNAs. Genetic studies can also be used to guide development of therapies to evaluate their efficacy and safety. For example, agonists of the GLP1 receptor (GLP1R) are used to treat type-2 diabetes (which may be linked to diminished incretin activity), owing to their low risk for hypoglycemia, in addition to weight loss, which is greater in obese individuals than in nonobese patients and a blood pressure-lowering effect in hypertensive individuals, but not in normotensive subjects. Glucagon-like peptide GLP1 reduces glycemia in type-2 diabetic patients, as it stimulates insulin secretion and inhibits glucagon release. The long-acting mimetics of the GLP1 hormone, GLP1R agonists, trigger insulin secretion after oral intake of glucose, but not after intravenous injection of glucose [1251].12 However, certain genes encoding targets of various drugs used to treat type2 diabetes and obesity have variants linked to metabolic traits. In particular, a rare missense variant, the gain-of-function Ala316Thr SNP (minor GLP1R allele rs10305492) in the Glp1r gene, which encodes glucagon-like peptide-1 receptor, is associated with lower fasting glycemia [1251]. It protects against atherosclerosis; hence, GLP1R agonists do not increase the cardiovascular risk.

7.1 Messenger RNA Synthesis and Splicing Genes encoding proteins are transcribed as precursor messenger RNAs, which contain nonprotein-coding sequences that must be removed before protein synthesis, i.e., mRNA translation (Vol. 1, Chap. 5. Protein Synthesis and 11, Chap. 1. Gene Transcription and Epigenetic Control). Gene transcription requires initiation, elongation, and termination. Enhancers and promoters initiate transcription. In addition, enhancers modulate transcriptional elongation. The RNA polymerase-2-associated factor PAF1 lodges on some enhancers and prevents their hyperactivation [1254]. In humans, more than 2000 transcription factors regulate the activation of approximately 23,000 genes. Among them, P53 controls the expression of numerous genes. It favors cell apoptosis and senescence in addition to DNA repair in a cell

12 Among

GLP1R agonists, liraglutide and exenatide administered once and twice daily, respectively, are commonly prescribed. Liraglutide may achieve a tighter glycemic control with slightly reduced side effects than exenatide [1252]. In addition, once-daily liraglutide reduces to a greater extent glycemia than once-weekly exenatide [1253].

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type-specific manner, even in a given organ [1255].13 It provokes cell cycle arrest upon acute exposure to stressors, especially in response to DNA damage. In the heart, P53 is a master regulator of the transcriptome [1256]. In adult cardiomyocytes, this transcriptional hub, which cooperates with other transcription factors (e.g., MEF2a, myocardin, and PGC1α), targets more than 20 gene sets (>1000 genes) related to cardiac architecture and function (mitochondrial genesis, CMC metabolism [oxidative phosphorylation and glycolysis], and excitation– contraction coupling). On the other hand, cardiac P53 deficiency confers resistance to acute mechanical stress. However, deletion of P53 in older mice triggers adverse cardiac hypertrophy. Other P53-related factors, P63 and P73, contribute to regulating the transcriptional network [1256]. In the post-transcriptional phase, the spatiotemporal control of protein synthesis is ensured by RNA-binding proteins and microRNAs, which regulate mRNA stability and translation. The spatiotemporal specificity of alternative splicing14 is governed by combinations of cis-regulatory elements and trans-acting factors that support or prevent spliceosome assembly [1257]. Other regulators of alternative splicing include transcription and chromatin regulators that recruit splicing components and affect the kinetics of exposure of competing splice sites in addition to post-translational and signaling pathways that influence function and/or localization of splicing regulators. Hundreds of regulators positively or negatively control alternative splicing, among which multitask transcription factors (∼ one-third of these regulators) and DNA-binding proteins play dual direct and indirect roles in splicing regulation. The zinc finger and CCCH-type motif-containing proteins (e.g., GTF3a,15 ZnF385a,16 CTCF,17 YY1,18 and WT1)19 play a role in RNA binding and regulation. For

13 Signaling

dynamics varies among cell types. Certain molecules affect P53 dynamics and determine cell-specific P53 signaling dynamics in DNA repair and activity of the ataxia-telangiectasia mutated kinase [1255]. 14 Alternative splicing selects splice sites in pre-mRNAs to generate structurally and functionally distinct mature mRNAs and hence protein variants. Alternative splicing of pre-mRNAs thus expands the functional capacity of the genome. 15 General transcription factor GTF3a (TF I I I a) is involved in RNA polymerase-3-dependent transcription and ribosomal large subunit genesis [108]. 16 ZnF385a affects location and translation of a mRNA subset [108]. 17 Chromatin-binding transcriptional repressor CCCTC-binding factor (CTCF) tethers to chromatin insulators and prevents interaction between the gene promoter and nearby enhancers and silencers. In particular, it regulates the APOA1/C3/A4/A5 gene cluster [108]. 18 Transcription factor yin and yang-1 connects to sites overlapping the transcriptional start site and exerts positive and negative control on numerous genes [108]. 19 Wilms tumor protein WT1 regulates transcription of numerous genes. Isoforms lacking the KTS motif may act as transcription factors, whereas isoforms containing the KTS motif may bind mRNA and act in mRNA splicing [108].

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example, BTB domain-containing transcription factor NAcc1,20 controls expression of specific splicing regulators. However, defective mRNA splicing engenders up to 15% of all inherited diseases, whereas mutations affecting splicing agents comprise 50–60% of all disease-causing mutations [1258]. Adaptation during perinatal cardiac growth requires switching of titin isoform from the fetal to the adult type. Titin is a very long filamentous protein spanning each hemisarcomere from the Z disc to the M line of the sarcomere. The A-band region of titin is inextensible and contains repeats of immunoglobulin (Ig) and type3 fibronectin (FN3) modules. Upon sarcomere extension, titin is stretched in its extensible region residing in the I band that comprises tandem arranged Ig-like domains, a PEVK segment (i.e., a segment rich in proline [P], glutamate [E], valine [V], and lysine [K]) and an N2 region. Titin is encoded by a single gene, TTN, which contains 363 exons in humans. Alternative splicing of TTN premRNA in the extensible region engenders different titin isoforms of varying sizes: a small cardiac stiff (N2B) and large compliant titin isoform (N2BA), the small isoform containing the N2B element only and the large isoform having both the N2B and N2A elements. The N2B element contains three Ig-like domains and a 572-aa sequence, whereas the N2A element consists of 4 Ig-like domains and a 106-aa sequence. In addition, the N2A subtype is associated with a larger PEVK segment than the N2B subtype. The N2BA titin contains a much longer PEVK segment than do N2B titins, along with an additional tandem Ig segment. In addition to the expression level of titin isoforms, the phosphorylation state of titin affects the resting tension of cardiomyocytes. Phosphorylation of the myocardial N2B spring element of titin by PKA or PKG reduces cardiomyocytic stiffness [1259]. Loss-offunction mutations in RNA-binding motif-containing protein RBM20, a splicing regulator, cause a dilated cardiomyopathy (DCM)-like phenotype owing to the persistent production in the adult heart of the giant fetal titin isoform N2BAG, which is characterized by additional spring elements, both in the tandem Ig-like and PEVK regions. Non-ischemic cardiomyopathies, diseases of the myocardium associated with mechanical and/or electric dysfunction, can be classified according to structural and functional changes into dilated (congestive DCM), hypertrophic (HCM), and constrictive (restrictive) cardiomyopathy (RCM), in addition to arrhythmogenic cardiomyopathy (or arrhythmogenic right ventricular cardiomyopathy [ARVC] or dysplasia [ARVD]). The last-mentioned cardiomyopathy type is an inherited form characterized by fibrofatty myocardial replacement (Tables 7.1 and 7.2). Each of these categories is further categorized by its etiology (infection, systemic inflammation,21 toxicosis, or inherited disorder) and idiopathic cardiomyopathies in the absence of an identifiable cause. However, several cardiomyopathy types remain 20 Nucleus

accumbens-associated protein-1 recruits HDAC3 and HDAC4 and operates as a transcriptional corepressor in neurons. 21 Inflammatory cardiomyopathies are engendered by myocarditis that causes chronic remodeling and involves innate immunity cells (dendrocytes, macrophages, neutrophils, and natural killer

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Table 7.1 Gene mutations causing arrhythmogenic cardiomyopathy (Part 1; Source: [1260– 1262] PTPLa protein Tyr phosphatase-like protein A) Gene Arvd3 Arvd4 CDH2 CTNNA3 DES

DSC2 DSG2 DSP

Protein ARVD3 ARVD4 Cadherin-2 (neuronal [N]-cadherin) Catenin-α3 (ARVC13, ARVD13) Desmin (ARVC7, ARVD7)

Desmocollin-2 (ARVC11, ARVD11) Desmoglein-2 (ARVC10, ARVD10, CdhF5) Desmoplakin (ARVC8, ARVD8)

Subcellular location ND ND Area composita Area composita Intermediate filament (links the nuclear membranes to the sarcolemma and Z-disc) (DES mutations cause MFM1 [myofibrillar myopathy-1]) Desmosome Desmosome

Desmosome (DSP mutations cause Carvajal syndrome) FRMD4A FERM domain-containing protein-4A Adherens and tight junctions (?) HACD1 Hydroxyacyl-CoA dehydratase-1 (ARVC6, ARVD6, PTPLa) Question mark (?) denotes uncertainty. Familial arrhythmogenic right ventricular cardiomyopathy (ARVC) or dysplasia (ARVD) has an autosomal dominant pattern of transmission. It is characterized by fibrofatty replacement of the right ventricular myocardium and ventricular arrhythmias. In cardiomyocytes, the area composita of intercalated discs is a mixed type between-cell junction that contains desmosomal and tight and adherens junction constituents (desmocollin Dsc2, desmoglein Dsg2, desmoplakin, plakoglobin, plakophilin-2 and -4, desmosomal cadherins, in addition to afadin; armadillo repeat-containing protein deleted in velocardiofacial syndrome [ARCVF]; cadherins Cdh2 and Cdh11; catenin-α, -β, and -δ1; and vinculin, in addition to tight junction zona occludens protein ZO1)

unclassified, such as left ventricular noncompaction cardiomyopathy, although it is still frequently associated with monogenic neuromuscular disorders, and endocardial fibroelastosis.

cells). Viral, bacterial, fungal, protozoal, and parasitic infections can provoke inflammatory cardiomyopathies. Non-infectious myocardidites are usually associated with autoimmune disorders, (e.g., sarcoidosis, scleroderma, and systemic lupus erythematosus), toxins (e.g., arsenic and cocaine), and hypersensitivity (e.g., some antibiotics species). In developed countries, the most common cause of inflammatory cardiomyopathies is lymphocytic viral myocarditis [1263]. In Latin America, it is often caused by Chagas disease.

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Table 7.2 Gene mutations causing arrhythmogenic cardiomyopathy (Part 2; Source: [1260– 1262]) Gene ITGA8 (?)

Protein Integrin-α8

JUP

Junction plakoglobin (catenin-γ) (desmoplakin-3) (ARVC12, ARVD12) ribosomal protein SA (LAMR1) pseudogene-6 Lamin-A/C Plakophilin-2 (ARVC9, ARVD9) Phospholamban

RPSAP6 (?) LMNA PKP2 PLN RYR2 SCN5A

Ryanodine receptor-2, (ARVC2, ARVD2) NaV 1.5

Subcellular location Focal adhesion (receptor for fibronectin and cytostatin) Desmosome (JUP mutations cause Naxos disease)

Nucleus (retroposon LAMR1P6) Nuclear envelope Desmosome Mitochondrion (SERCA inhibition) Mitochondrion

Plasma membrane (SCN5A mutations cause Brugada syndrome and familial DCM) TGFB3 Transforming growth factor-β3 Intra- and extracellular media (ARVC1, ARVD1) (targets endoglin–Tβ R1–Tβ R2) (TGFB3 mutations cause aortic aneurysms) TMEM43 Transmembrane protein-43 Nuclear envelope (ARVC5, ARVD5, luma) TTN Titin Sarcomere Question mark (?) denotes uncertainty. Integrins cooperate with syndecan-4, a plasmalemmal heparan sulfate proteoglycan to transduce signals for focal adhesion assembly and stress fibers on fibronectin

Primary cardiomyopathies result from lesions only or predominantly of the myocardium. Secondary cardiomyopathies involve a multisystem disorder (e.g., amyloidosis, Anderson–Fabry disease, sarcoidosis, among others), in addition to other causes (e.g., ischemia and toxins). Primary cardiomyopathies comprise genetic, acquired, or mixed forms. In the MOGES classification, M refers to the phenotype, O to organ involvement (i.e., with or without extracardiac tissue involvement), G to genetic transmission (e.g., autosomal dominant or recessive), E to pathogenesis (e.g., mutation type), and S to disease stage. The RCM and DCM are linked predominantly to diastolic and systolic dysfunction, respectively [1264]. Dilated cardiomyopathy is characterized by an enlarged and weakly contractile left ventricle. Most DCMs engendered by gene mutations are inherited in an auto-

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somal dominant pattern [1265]. It also results from hypertension, valvulopathies, inflammatory and infectious diseases, and toxin exposure. Restrictive inherited or acquired cardiomyopathies are characterized by nondilated left and right ventricles with increased myocardial stiffness that leads to impaired ventricular filling and hence diastolic dysfunction. They can be caused by myocarditis, infection (e.g., Chagas disease), amyloidosis, hemochromatosis, sarcoidosis, endomyocardial fibrosis [1266]. They can be classified as infiltrative (e.g., amyloidosis and sarcoidosis), storage disease (e.g., hereditary hemochromatosis and mucopolysaccharidosis), non-infiltrative (e.g., diabetic cardiomyopathy and scleroderma), and endomyocardial types (carcinoid heart disease and endomyocardial fibrosis) [1267]. Inherited cardiomyopathies also include DCM, HCM, and RCM. They are associated with altered cytoskeleton (force transmission disease—cytoskeletal cardiomyopathy) and sarcomere (force generation disease—sarcomeric cardiomyopathy), arrhythmogenic cardiomyopathy being linked to defective desmosome (cell junction cardiomyopathy) and channelopathy-linked cardiomyopathies (short and long QT [SQTS and LQTS] and Brugada syndromes and catecholaminergic polymorphic ventricular tachycardia [CPVT]; CMC electrical dysfunction), which provokes defective ion transfer across the plasma membrane and electromechanical coupling. Inherited cardiomyopathies most often result from mutations in genes encoding sarcomeric and other types of CMC proteins. However, most genes implicated in one of the cardiomyopathy types are also involved in other cardiomyopathy forms. Three major types are DCM, HCM, and RCM [1268, 1269]. Adaptive and maladaptive cardiac secondary remodeling masks specific effects of the gene mutation. Inherited non-ischemic dilated cardiomyopathy is often caused by mutations in the genes encoding plasmalemmal (e.g., SCN5A mutations), cytoskeletal (e.g., mutations in the genes encoding dystrophin, sarcoglycan, and filamin-C), sarcomeric (e.g., TTN [titin] truncated variants in addition to mutations of the MYH7, TNNT2, and TPM1 genes), mitochondrial (e.g., mutations in the PLN gene [phospholamban]), desmosomal, nuclear membrane (e.g., LMNA missense and truncating mutations), and RNA-binding proteins (e.g., RBm20 SNPs, RBM20 regulating mRNA splicing) [1265]. Occasionally, a di- and oligogenic origin is observed. Inherited restrictive cardiomyopathy is linked to mutations in sarcomeric proteins (troponins TnnI [TNNI3] and TnnT [TNNT2], α-actin [ACTC], myosin-binding protein-C [MYBPC3], and β-myosin heavy chain [MyH7]). Inherited restrictive cardiomyopathy are also complications of primary hyperoxaluria,22 and storage

22 Primary

hyperoxaluria (PH) results from mutations in the AGXT, GRHPR, or HOGA1 genes for PH type-I to -I I I , which encodes alanine–glyoxylate aminotransferase, glyoxylate and hydroxypyruvate reductase, and hydroxy oxoglutarate aldolase-1, respectively.

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diseases, such as Fabry disease,23 Gaucher disease,24 hereditary hemochromatosis25 glycogen storage disease,26 mucopolysaccharidosis,27 and Niemann–Pick disease [1267].28 It is characterized by increased stiffness of the left ventricular wall without augmented wall thickness and impaired diastolic function. It is the least common type among the three most prevalent forms. A SNP in the gene (E143K; i.e., Glu143 changed to Lys) encoding myosin essential light chain (MELC)29 is associated with RCM in humans, who only exhibit RCM symptoms when carrying a homozygous mutation (Table 7.3). Mutations in genes encoding MLCs account for 1–5% of all cardiomyopathies of genetic

23 Fabry

disease is caused by mutations in the GLA gene that encodes galactosidase-α. disease is engendered by mutations in the GBA gene that encodes acid β-glucosidase. 25 Hereditary hemochromatosis is provoked by mutations in the Hamp, Hfe1, Hfe2, HJV, PNPLA3, SLC40A1, and TfR2 genes, which encode hepcidin antimicrobial peptide, hereditary hemochromatosis protein HFE1, HFE2, and juvenile HFE2, patatin-like phospholipase domain-containing protein (a.k.a. adiponutrin and calcium-independent PLA2 ), ferroportin-1 (a.k.a. HFE4 and ironregulated transporter IREG1), and transferrin receptor-2 (or HFE3). 26 Glycogen storage disease (GSD) is linked to deficiency in glucose 6-phosphatase (GSD1A), translocase T1 (GSD1B) and T2 (GSD1C); α-glucosidase (GSD2); amylo-α-(1,6)-glucosidase 4α-glucanotransferase (or glycogen debrancher; GSD3); amylo-(1,4-to-1,6)-transglucosidase (or glycogen brancher GBE1; GSD4); glycogen phosphorylase (GSD5); hepatic phosphorylase (GSD6); and phosphofructokinase (GSD7). 27 Mucopolysaccharidosis type-I (or Hurler syndrome), and -I I (or Hunter syndrome) are due to mutations in the IDUA and IDS genes, which encode αL iduronidase (MPS1) and iduronate 2sulfatase (MPS2), respectively. 28 Type-A and -B Niemann–Pick diseases are caused by mutations in the SMPD1 gene, which encodes acid sphingomyelinase. Type-C results from mutations in the NPC1 or NPC2 gene. 29 Sarcomeric hexameric class-I I myosins consist of two myosin heavy chains (MHCs) and two MELCs and two myosin regulatory light chains (MRLCs). Myosin consists of the head (S1), neck (S2), and tail. The myosin head possesses a motor domain containing a catalytic (ATP-binding pocket) and actin-binding region. The myosin neck has a lever arm composed of attachment sites for MELC and MRLC. The myosin tail formed by the rod parts of MHCs corresponds to the thick filament of sarcomeres. In human hearts, MHC6 predominates in atria and MHC7 is the quasi-exclusive isoform in ventricles. Myosin light chain governs the interaction between actin and myosin. In the fast skeletal muscle, MELC comprises MLC1 and MLC3, both encoded by the MYL1 gene. The atrio- and ventriculomyocyte is endowed with MELCA (or MLC4) and MELCV (or MLC3) encoded by the MYL4 and MYL3 gene, respectively, and with MRLCA (or MLC7) and MELCV (or MLC2) encoded by the MYL7 and MYL2 gene, respectively. In the adult heart, MLC2V is highly produced in the epicardium and weakly in the endocardium [1271]. Both MELC and MRLC can be phosphorylated. Defective MLC phosphorylation causes severe cardiac dysfunction and cardiomyopathy. Myosin essential light chain is implicated in the structural stability of the myosin; it serves as a structural component of the actomyosin cross-bridge. Two MELC isoforms are expressed in fast skeletal muscle (long and short), whereas only the long MELC exists in the heart. The MELCL subtype enters into direct contact with actin. Myosin containing MELCL has a slower cross-bridge kinetics than myosin comprising MELCS [1270]. The A57G SNP in the cardiac MELC causes familial hypertrophic cardiomyopathy. The MELC N-terminus acts in prepositioning the cross-bridge for optimal force production [1272]. 24 Gaucher

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Table 7.3 Predominant myosin heavy (MHC) and essential (MELC) and regulatory (MRLC) light chains in the adult human heart cavities Chamber Atrium Ventricle

MHC MHC6 MHC7

MELC MLC4 MLC3

MRLC MLC7 MLC2

origin; they engender DCM, HCM, and RCM. Although only 55% of mutant protein is expressed, mice develop RCM [1269]. In a transgenic mouse model of RCM overexpressing mutant E143K, hypercontractility of ventricular MELC and elevated active and passive tension measured in skinned papillary muscle results from a higher affinity of myosin to actin, augmented myosin ATPase activation by actin, a slower rate of ATP-dependent dissociation of the actomyosin complex, and lowered interfilament lattice spacing, in addition to fibrosis, MRLC hypophosphorylation, which may contribute to cardiac dysfunction owing to morphological and functional alterations (e.g., change in force, myofilament calcium sensitivity, ATPase activity, cross-bridge kinetics) and, above all, adverse remodeling [1269]. However, increased calcium sensitivity caused by troponin-I hypophosphorylation in response to impaired β-adrenoceptor signaling, which is frequently observed in cardiomyopathy models [1268], is not observed, phosphorylation level of troponin-I remaining unchanged [1269]. Hypertrophic cardiomyopathy characterized by left ventricle thickening is the most common inherited form. Variants depend on the mutated sarcomeric proteinencoding gene, 70% of mutations involving the MYBPC330 and MYH7 genes.31 Mutations of the TNNT2, TNNI3, TPM1, ACTC1, MYL2, MYL3, and CSRP3, which encode cardiac troponin-I, α-tropomyosin, cardiac α-actin, myosin light chain, and cysteine- and glycine-rich protein-3, respectively, are uncommon HCM causes [1273]. Hypertrophic cardiomyopathy is a typical monogenic disorder with an autosomal dominant pattern. In HCM, some mutant sarcomeric proteins increase Ca2+ sensitivity and contractility of CMCs (e.g., troponin-T32 or α-tropomyosin), whereas others have the opposite effect; several myocardial anomalies are thus implicated [1274]. Mutations in nonsarcomeric genes such as the PRKAG2 gene33 are also implicated in HCM and in glycogen accumulation, ATP supply deficiency, and ventricular pre-excitation) [1275]. In cardiomyocytes and fibroblasts, the heterozygous K475E SNP lessens AMPK basal activity and sensitivity to AMP, and hence increases phosphorylation of S6K and 4eBP1 linked to TORC1 activation, as does

30 MyBPc3:

cardiac myosin-binding protein-C. MYH7 gene encodes MHC7. 32 The human heart expresses four TnT isoforms (cTnT1–cTnT4) by alternative splicing, the fetal subtypes including cTnT1, cTnT2, and cTnT4. In the adult human heart, cTnT1 and cTnT2 are barely detectable and cTnT4 is a minor isoform. 33 The PRKAG2 gene encodes the noncatalytic AMPK-γ2 subunit. 31 The

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the AMPKN488I mutation. On the other hand, the AMPKR384T and AMPKR531Q SNPs cause infantile congenital HCM with an increase in AMPK Thr172 phosphorylation and basal activity and reduced AMP and ATP binding [1274].

7.1.1 Gene Accessibility by Chromatin Relaxation The chromatin unit, at least in vitro, is the nucleosome, a DNA segment wrapped around a histone octamer ([H2a]2 –[H2b]2 , [H3]2 –[H4]2 ), the histone tails (i.e., Ntermini of four histone types and C-termini of H2a and H2b) protruding from the nucleosome. Chromatin is also organized into segregated contact domains, or topologically associating domains (TADs), which participate in gene transcription regulation. During interphase, TADs are organized into two polarized compartments, active and inactive X chromosomes adopting different folding and compartmentation configurations, inactive X chromosome being partitioned into two contiguous compartments separated by the DXZ4 element, and active X chromosome according to the p and q arms [1276]. A stable heritable epigenetic state results from (1) a trigger, epigenator; (2) its responding element, epigenetic initiator, which determines the location of the epigenetic chromatin environment; and (3) an epigenetic maintainer, which sustains the chromatin environment that propagates in the next generations [1277].

7.1.1.1

DNA Methylation

The main epigenetic modification of DNA is the covalent attachment of a methyl group to the cytosine in cytosine–guanosine (CpG) dinucleotides.34 Most cytosines are methylated when the DNA bases cytosine and guanine are apposed, although DNA sequences containing a high number of the CpG motifs, CpG islands (CpGIs), which are mainly promoter-associated genomic regions, are generally unmethylated.35 Methylation of CpG dinucleotides in the gene promoter and

34 The

letter p indicates that a cytosine nucleotide apposed to a guanine nucleotide in the linear sequence of bases is connected to guanine by a phosphodiester bond. When the DNA bases cytosine and guanine are apposed, a methyl group can be added on position 5 of the base cytosine, generating a CMe pG dinucleotide. This modification not only alters DNA structure but also affects transcription factor binding. Maintenance of a particular cellular state after cell division depends on the faithful transmission of methylated CpGs in addition to inheritance of the repertoire of transcription factors from the mother cell to the daughter cells. 35 However, insertion of CpG-free DNA into a CpGI provokes methylation of the entire CpGI in human pluripotent stem cells [1278].

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body represses and elicits gene transcription, respectively. Conversely, cytosine methylation can be added or removed by proteins that associate with transcription factors. Approximately 1,600 human transcription factors recognize and access their specific DNA-binding sites and then control gene transcription. Their binding to DNA is prevented or promoted by the methylated CMe pG dinucleotide [1279]. 1. Connection to DNA of most major classes of transcription factors (e.g., basic helix–loop–helix [bHLH], basic leucine zipper [bZip], and E26 proteins [ETS]) is inhibited by CMe pG. 2. On the other hand, transcription factors, such as homeodomain-containing proteins (Hox), nuclear factors of activated T cells (NFAT), and POU proteins, that is, transcription factors belonging to sets that are enriched in regulators of embryo- and fetogenesis, prefer to tether to methylated DNA.

7.1.1.2

Histone Modifications

Packaging of the genome in chromatin, the DNA–histone complex, locally changes to enable regulated gene transcription. Chromatin remodeling is the first step of transcriptional regulation. Specialized proteins read, write, or erase histone marks, that is, post-translational modifications (mainly acetylation, methylation, phosphorylation, sumoylation, and ubiquitination; Vol. 11, Chap. 1. Gene Transcription and Epigenetic Control). Numerous types of chromatin post-translational modifications are achieved by specialized enzymes and modifying complexes. These modifications influence the affinity for adjacent DNA and histones, the basic positively charged proteic scaffold around DNA. Post-translational modifications of the five histone types include acetylation (ac), ADP-ribosylation (ar), which comprises MARylation (MAR; i.e., adding monoADP ribose) and PARylation (PAR; i.e., polyADP ribose), biotinylation (bi), butyrylation (bu), citrullination (ci), crotonylation (cr), formylation (fo), glycosylation (gl; i.e., O-GlcNAcylation), malonylation (ma), methylation (me), myristoylation (my), palmitoylation (pa), phosphorylation (ph), proline isomerization, propionylation (pr), succinylation (sc), sumoylation (su),36 and ubiquitination (ub). According to convention and for the sake of homogeneity with the notation commonly used in this book series for post-translational modification site AAj (AA: target amino acid; j : position of amino acid substrate in the polypeptidic chain), a post-translational modification of a given histone site (HiAAj ; i: histone species) engenders a histone mark that is denoted HiAAj ptm (ptm: ac, ar, bi, gl, me, ph, su, ub, etc.). The superscript 1 to 3 attached to methylation refers to mono-,

36 Sumo:

small ubiquitin-like modifier.

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di-, or trimethylated forms. A modified residue is written as AAPtm (e.g., KAc , KCr , and KMe stand for acetylated, crotonylated, and methylated lysine). Various enzymes change the histone structure, such as histone acetyltransferases, deacetylases, and kinases along with ATP-dependent nucleosome remodeling factors. Accessibility of genes is controlled by: 1. ATP-dependent chromatin (nucleosome)-remodeling complexes that not only influence nucleosome positioning and composition and hence restructure nucleosomal DNA, enable nucleosome sliding and repositioning, in addition to exchanging and evicting histones and entire nucleosomes from chromatin, using the energy of ATP hydrolysis, but also coordinates transcriptional repression or activation along with elongation, and re-establishes a proper chromatin structure after transcription 2. Histone chaperones that control the supply of free histones and cooperate with chromatin remodelers 3. Histone variants 4. Post-translational modifications of histones Most genes have a nucleosome-depleted region (NDR) at their promoters and an array of regularly spaced nucleosomes phased with respect to the transcription start site, usually located inside the first nucleosome. The ATP-dependent histone remodelers operate as complexes, ranging from heterodimers to complexes possessing more than 12 subunits. They include an ATPase of the SMARCA2/4,37 of the SMARCa1/5,38 or of the chromodomain (CHD) group.39 ATP-dependent chromatin-remodeling enzymes pertain to [1282]: 1. the SWI/SNF,40

37 Swi2

or SNF2 ATPase in Saccharomyces cerevisiae. in Drosophila melanogaster. 39 At least in yeasts, the ISWI and CHD remodelers abound in NDR flanking transcription units, where they bind to segments of linker DNA flanking transcription factor-binding sites (TFBSs). As remodeler binding correlates with nucleosome turnover and transcriptional elongation rate, the ISWI and CHD remodelers may first associate with naked DNA within NDRs and subsequently relocate to gene bodies following nucleosome disruption by RNA polymerase-2 transit, linker DNA promoting their remodeling activity [1280]. Among the nucleosome-spacing enzymes in yeasts, CHD1 forms nucleosome arrays with short spacing at genes and ISW1 with long spacing [1281]. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Whereas CHD1 causes short spacing with H1 eviction and chromatin unfolding, ISW1 provokes longer spacing, H1 binding, and chromatin condensation. 40 SWI/SNF: mating type switching/sucrose nonfermenting complex (or category). The SWI/SNF family members include SNF2 and Sth1 in yeasts, brahma (Brm) in Drosophila melanogaster, and mammalian Swi/SNF-related, matrix-associated, actin-dependent regulators of chromatin SMARCa2 (Brm homolog) and SMARCa4 (brahma-related gene product BRG1), two homologous ATPases [1282]. SMARCa2 and SMARCa4 associate with different promoters during cellular proliferation and differentiation. SMARCa2 interacts with components of the notch pathway; SMARCa4 binds to zinc finger-containing proteins [1283]. The chromatin-remodeling complexes SWI/SNF and RSC (RSC: remodeler of the structure of chromatin) are related; they share 38 ISwi

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2. ISWI,41 3. INO80,42 4. and CHD set.43 Numerous types of histone post-translational modifications that modulate chromatin structure include [1291]: • Acetylation, which is regulated by the opposing action of histone acetyltransferases (type-A and predominantly cytoplasmic type-B HATs) and deacetylases (Class I to Class IV HDACs)

two identical subunits and at least four other homologous components [1284]. Both types can mobilize, eject, and remodel nucleosomes, but they do not have spacing activity. Whereas nucleosome spacing and phasing relative to the transcription start site require the ISW1 and CHD1 chromatin remodeling enzymes, the RSC complex is involved in setting the size of the nucleosome-depleted regions [1284]. The SWI/SNF complex and the related human SMARCa2and SMARCa4-based complexes in addition to human ACF (from ACF1: ATP-utilizing chromatin assembly and remodeling factor), chrac (chromatin accessibility complex), and RSF (from RSF1: remodeling and spacing factor; a.k.a. hepatitis B virus X-associated protein [HbxAP, or XAP8)] complexes can function as nucleosome-spacing factors [1285]. 41 ISWI: imitation switch complex (or category). Three different chromatin remodeling complexes isolated from Drosophila embryos contain the ISWI ATPase: NURF (from NuRF: nucleosome remodeling factor, also called ISwi), ACF (from ACF1: ATP-utilizing chromatin assembly and remodeling factor, also called ChrAC and ChrAC175), and chrac (from ChrAC: chromatin accessibility complex, also called ISwi) [1286, 1287]. The nucleosome remodeling factors NURF and CHRAC mediate the sliding of nucleosomes along DNA without major trans displacement of histone. The ISWI set comprises the homologs Isw1 and Isw2 in yeast and SMARCa5 (SNF2h) and SMARCa1 (SNF2l) in mammals [1282]. The human ISWI homolog remodeler SMARCa5 functions as a dimeric sensor of linker length, with one ATPase molecule contacting extranucleosomal DNA on each side of a nucleosome [1280]. The heterodimer RSF (from RSF1: remodeling and spacing factor), which contains SMARCa5, facilitates regular spacing between nucleosomes and permits transcription initiation from a nucleosomal promoter. Bromodomain adjacent to zinc finger domain-containing protein BAZ1b, also called Williams–Beuren syndrome (WBS) chromosomal region 9 protein WBSCR9 and Williams syndrome transcription factor WSTF, transiently associates with the ATPase subunit SNF2h in the WSTF–ISWI chromatinremodeling complex (WICH) [1288]. BAZ1b is the kinase that phosphorylates H2a.x. SMARCa5 and its binding partners BAZ1a (or ATP-utilizing chromatin assembly and remodeling factor ACF1) and BAZ1b are rapidly recruited to UVc-induced DNA damage to promote transcription recovery [1289]. The human chrac complex contains BAZ1a and histone-fold proteins (ChrAC15 and ChrAC17) [1286, 1287]. The yeast chromatin remodeling enzymatic complex ISW1, which serves as a quality control factor and nuclear export surveillance factor, binds to the premature messenger ribonucleoparticles (mRNA–RNP), retaining export-incompetent transcripts near their transcription sites [1290]. 42 Ino: inositol-requiring protein. The protein Ino80 is a DNA helicase and component of the chromatin-remodeling INO80 complex, which is involved in transcriptional regulation and DNA replication and repair. 43 CHD: chromodomain (chromo: chromatin organization modifier) helicase DNA-binding protein. In humans, the set includes CHD1, CHD1L, and CHD2 to CHD9. The human CHD7 remodeler requires extranucleosomal DNA for its remodeling activity, as does SMARCa5 [1280].

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Table 7.4 Enzymes that antagonize or reverse histone methylation (Source: [1292]) Enzyme family JMJC

LSD PADI

Enzyme or enzyme group JHDM1 (KDM2) JHDM1f (PHF8) JHDM2 (JmjD1, KDM3) JHDM3 (JmjD2, KDM4) JARID (KDM5) UTX/Y (KDM6) JmjC only LSD1 (KDM1a) LSD2 (KDM1b) PADI4

Substrate specificity H3K36 me1 , H3K36 me2 ND H3K9 me1 , H3K9 me2 H3K9 me2 , H3K9 me3 , H3K36 me2 , H3K36 me3 H3K4 me2 , H3K4 me3 ND Asn hydroxylation H3K4 me1 , H3K4 me2 ND H3R2, H3R8, H3R17, H3R26, H4R3

Histone methylation is regulated by demethylimination and demethylation (ND not determined, JHDM JmjC-domain-containing histone demethylase, JmjC jumonji-C, LSD lysine-specific histone demethylase, PADI peptidylarginine deiminase, PHF PHD finger-containing protein)

• Methylation, which results from the balance between histone Lys methyltransferases (HKMTs), Arg methyltransferases (type-I and -I I PRMTs), and demethylases (Table 7.4) • Phosphorylation, which is governed by histone kinases (e.g., MAPK1 and JaK2) and phosphatases (e.g., PP1) • Other modifications, such as ADP-ribosylation, deimination, glycosylation, Proisomerization, sumoylation, ubiquitination, and histone tail clipping Numerous chromatin-associated factors interact with modified histones, histone marks acting as platforms for recruitment, assembly, and retention of chromatinassociated factors. Among epigenetic enzymes, some histone methyltransferases determine the transcriptional program in cardiomyocytes. The KMT1c–MEF2C complex maintains heterochromatin needed for the silencing of developmental genes in the adult heart [1293]. However, KMT1c favors cardiac hypertrophy, as it represses antihypertrophic genes. Altered energy sensing and supply and loss of metabolic flexibility, capacity (reserve), and efficiency compromise cardiac contractility upon stressor exposure. Maladaptive metabolic remodeling initiates and maintains contractile dysfunction in heart failure [1294]. The nuclear receptor and transcription factor NR1c1 (PPARα) plays a major role in cardiac metabolism regulation. Its transcriptional activity depends on various partners that determine the metabolic phenotype in the diseased heart. Metabolic reprogramming can result from epigenetic modifications induced by NR1c1 [1295]. Members of the melastatin-related transient receptor potential category TRPM6 and TRPM7 are chanzymes (both ion channels and protein Ser/Thr kinases). The

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C-terminal kinase domain of TRPM6 is cleaved from the channel domain in a cell type-specific fashion, when the channel conductance is functional. The cleaved kinase that remains active translocates to the nucleus, where it phosphorylates specific histone residues. TRPM6-cleaved kinases (TRPM6CKs) tether to subunits of protein Arg methyltransferase PRMT5 [1296]. The PRMT5 complex may direct TRPM6CK to a specific histone location. Histone phosphorylation by TRPM6CK hinders methylation of Arg adjacent to amino acids phosphorylated by TRPM6CK, thereby regulating gene transcription. Epigenetic inheritance of histone modifications over successive cell generations during DNA replication enables epigenetic maintenance of gene silencing during chromosome duplication and hence of daughter cell specificity determined by the subset of genes that are transcribed.44 Moreover, short and long nonprotein-coding RNAs can regulate the positioning of histone modifications, as they interact with enzymes that determine histone marks. Small RNAs (content 20–30 nucleotides; i.e., miRs, piRNAs, and siRNAs) regulate gene expression and genome stability, as they target nascent chromatinbound nonprotein-coding RNAs that serve as assembly platforms, hence recruiting chromatin-modifying complexes to specific chromosomal regions [1300]. Short RNAs (content 50–200 nucleotides) transcribed from repressed polycomb target genes can form stem–loop structures and interact with the polycomb repressive complex, PRC2, repressing gene transcription in cis [1301]. Long intergenic nonprotein-coding RNAs also regulate chromatin states and epigenetic inheritance. For example, hotair, a lncRNA from the HOXC locus, represses transcription of the HOXD locus; it interacts with PRC2 and labels remotely (in trans) gene silencing [1302]. In fact, this scaffold lincRNA assembles two histone-modifying enzymes, as it not only tethers to PRC2 but

44 In Drosophila, parental trimethylation of Lys27 of histone H3 by the methyltransferase polycomb

repressive complex PRC2 is inherited in daughter cells after DNA replication [1297]. Early in embryogenesis, in the cells of bodily segments, some HOX genes are heritably activated, whereas others are repressed. They are related to epigenetic memory, transient signals establishing a permanent stimulation or inhibition of gene expression [1298]. Nucleosomes carrying the repressive histone mark H3K27 me3 are involved in the epigenetic memory, that is, in the maintenance of the cellular memory of gene expression states. Once established at a repressed HOX gene, inheritance of repressive H3K27 me3 chromatin depends on the transmission of parental trimethylated H3K27 nucleosomes associated with this gene to daughter DNA in the following cell generations. Long-term memory relies on efficient copying of the mark after each replication cycle that requires PRC2 [1298]. Hence, propagation of the chromatin mark H3K27 me3 requires trimethylation of newly incorporated nucleosomes by PRC2 attached to the polycomb response element [1297, 1298]. In fission yeast, Lys9 of histone H3 (H3K9) is methylated in heterochromatin, epigenetic inheritance requiring binding sites for sequence-dependent ATF– CREB family transcription factors and chromodomain sequence-independent mechanisms that propagate methylated H3K9 [1299].

7.1 Messenger RNA Synthesis and Splicing

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also to HCLR repressor complex,45 enabling RNA-mediated assembly of PRC2 and KDM1a and coordinating their connection to chromatin for coupled H3K27 methylation and K4 demethylation [1303]. Assembly of histones on gene promoters interferes with the initiation of transcription by RNA polymerase-2 and general transcription factors. ATP-dependent chromatin remodelers, histone-modifying enzymes, FACT, and GTF2 relieve inhibition by histones of gene transcription, as they trigger chromatin decondensation and remove nucleosomes, thereby exposing DNA for transcription. Nucleosome positioning represents another type of transcriptional regulator. Gene activation can be achieved by movement of the +1 nucleosome, exposing the TATA box motif of the promoter. However, interaction of transcriptional regulators with nucleosomes can stimulate gene transcription. Histone tail acetylation and the activating histone mark H3K4 me3 recruit transcription factors. TAF14, a subunit of GTF2f, binds to acetylated H3K9, mediator to histone H4 tail, Swi/SNF and RSC/SMARC complex to acetylated nucleosomes. A nucleosome occluding the TATA box and transcription start sites can enhance transcription with respect to naked DNA, rather than impeding it [1304]. Both histone acetylation and trimethylation of H3K4 support transcription of isolated repressive chromatin from the chromosomal locus of the yeast PHO5 gene by RNA polymerase-2, GTF2s, PHO4 gene activator, and the SAGA, SWI/SNF, and mediator complexes. The mediator complex, a general transcription factor, is supposed to connect to transcriptional activators bound to enhancers and RNA polymerase-2 linked to promoters to initiate transcription. During transcriptional activation, the mediator undergoes a compositional change in which its kinase module dissociates from the remainder of the complex upon interaction with RNA Pol2 [1305]. However, depletion of mediator complex subunits only causes a modest decrease in transcription [1305]. Transcription is abrogated only upon simultaneous depletion of all mediator complex modules (head, middle, and tail modules). Furthermore, different mediator modules support RNA polymerase-2, but in a distinct manner. The mediator complex is thus essential for RNA Pol2-mediated transcription but does not participate in the pre-initiation complex (PIC) formation, although it stimulates PIC creation [1305].

45 Complex

formed by histone deacetylase (HDAC [H]), corepressor element-1 (CoRE1) silencing transcription factor (CoREST [C]), Lys-specific demethylase LSD1 (L; a.k.a. KDM1a), and RE1silencing transcription factor (REST).

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7.1.2 Gene Transcription Gene transcription begins when RNA polymerase-2 (RNA Pol2) tethers to DNA and starts to catalyze the synthesis of a complementary mRNA. • Transcription is initiated when RNA Pol2 binds to the gene promoter. Enhancer, which interacts with the promoter, controls the gene transcription rate via activator proteins that facilitate DNA looping, which brings the enhancer closer to the promoter, whereas repressors prevent this looping. In addition, RNA Pol2 is assisted by general transcription factors. • The DNA double helix then unwinds and RNA Pol2 reads the template strand and adds nucleotides to the growing RNA. • Transcription stops at a terminator and mRNA 3 -end is polyadenylated. Alternative polyadenylation generates distinct 3 -ends in transcripts made by such as mRNAs, leading to the production of two mRNA isoforms with different 3 -untranslated regions (3 UTR APA) [1306]. RNA Pol2

• The constitutive UTR (cUTR), that is, the 3 UTR region upstream from the proximal polyadenylation site (PAS), is found in both short and long isoforms. • The alternative UTR (aUTR), downstream from the proximal PAS, is present only in the long isoform. RNA-binding proteins, microRNAs, and lncRNAs can interact with aUTRs. Protein-coding genes include signal-regulated genes and those with a relatively constant transcription rate (housekeeping genes) that are associated with alternative modes of the PIC assembly [1307]. Gene promoters contain cis-regulatory elements that bind transcriptional activators and core promoter motifs such as the TATA box. Genes without a well-defined TATA box, but with a CpGI promoter, generally have a cell maintenance function [1308]. However, many cell-specific genes are also linked to CpGI promoters. In addition, promoters with a TATA-like element rather than a consensus TATA box are also generally associated with a managing function. Housekeeping genes can be endowed with a +1 nucleosome covering the transcription start site. The TATA box-binding protein (TBP), a general transcription factor required at an early step for PIC assembly, is recruited by both the GTF2d (TFI I D) and the SAGA complex.46 Core promoters of highly regulated genes typically contain a TATA element, which is targeted by TBP, connection of which to DNA is assisted by the histone acetyltransferase and deubiquitinase SAGA complex.

46 SAGA:

Spt–Ada–Gcn5 acetyltransferase. This complex contains KAT2a (or GCN5), the transcription initiation protein suppressors of Ty3 homolog SupT3H (Spt3) and SupT7L, Spt20, transcriptional adaptors (Ada) TAda1L, TAda2b, and TAda3, factors TAF5L, TAF6L, TAF9, TAF10, and TAF12, and ataxins Atxn7 and Atxn7L3, among other subunits [1309].

7.1 Messenger RNA Synthesis and Splicing

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In housekeeping genes, RNA Pol2 is positioned at the promoter by a combination of TBP-associated factors (TAF) to form the transcription PIC (GTF2a–GTF2b– GTF2d–GTF2e–GTF2f–GTF2h–Pol2). Once it is phosphorylated by GTF2h, RNA Pol2 recruits elongation and mRNA processing factors. The heat shock transcription factor, HSF1, regulates expression of 21 genes, among which the numbers of GTF2d- and SAGA-linked core promoters are nearly equal. The SAGA-regulated genes have a greater change in transcriptional rate upon HSF1 binding, hence being more strongly regulated than GTF2d-regulated genes [1308]. Therefore, SAGA-regulated TATA-box promoters of genes with constitutive expression are more responsive to exposure of a given common activator (HSF1) than GT2d-controlled TATA-like promoters of genes, the transcription of which is regulated by environmental and developmental signals or cell-typedependent cues. SAGA-linked TATA-box promoters are more dynamic because the ATP-dependent helicase bTAF147 preferentially removes TBP from SAGAregulated promoters (elevated TBP turnover) and thus increases the response to activator in addition to nucleosome repositions, as nucleosome occupancy and positioning can also explain the difference in responsiveness. Before mRNA is exported to the cytoplasm and translated in the ribosome, it matures.

7.1.3 Formation Modes of Messenger RNA Subtypes Four basic mechanisms and their combinations can engender many mRNAs from a single gene:

47 The

RNA polymerase-2- and bTFI I d transcription factor-associated protein bTAF1 is also called 170-kDa bTFI I d transcription factor-associated subunit, TATA-box-binding protein (TBP)associated factor TAFI I 170 and TAF172, and Mot1 homolog. BTAF1 has a DNA-dependent ATPase activity that dissociates TBP from DNA, freeing TBP, which can then associate with other TATA boxes or TATA-less promoters. BTAF1, DR1, TBP, RNA Pol2, TAF1, and the SAGA subunit SPT20 co-localize to a large number of promotors. Initiation of transcription by RNA Pol2 requires the assistance of TBP and TBP-associated factors (TAFs), which form two complexes, TFI I d (GTF2d) and its orthologous complex, bTFI I d (bTAF1–TBP), which can replace TFI I d [194]. The GTF2d complex is composed of TBP and more than eight TAFs. However, most TBP is present in the bGTF2d complex, which has DNA-dependent ATPase activity. BTAF1 belongs to a separate group within the SWI2/SNF2 category of ATPases, which includes the Rad54 ATPase involved in DNA repair and its orthologs, Rad54L1 and Rad54L2 [1310]. BTAF1 represses SAGA-dependent TATA box-containing gene and activates GTF2d-dependent TATA-less genes. It cooperates with the downregulator of transcription DR1 (a.k.a. TATA-binding protein-associated phosphoprotein and negative cofactor NC2), which inhibits PIC formation, as it competes with GTF2a and GFT2b for TBP binding [1310]. The combined action of bTAF1 and DR1 mobilizes TBP from preferred TATA box-containing promoters, allowing TBP redistribution to disfavored TATA box-less promoters.

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1. Alternative promoter usage, which generates several primary transcripts according to their initiation site 2. Differential termination or 3 -post-transcriptional processing 3. Alternative splicing of a given primary transcript 4. Recombination of genomic sequences, such as for immunoglobulin genes Alternative splicing is defined by a combinatorial rearrangement of exons or a fraction of exons at splice sites (i.e., at junctions between exons and introns). Pre-messenger RNA splicing consists in removing introns and ligating exons to engender mature mRNAs ready for translation into proteins. Parts of introns can also be concatenated into mature RNAs. Transcription is a source of genetic instability that can form genotoxic DNA– RNA hybrids, or R-loops, between the nascent mRNA and its template. Introns counteract transcription-associated genetic instability, hence protecting the genome [1311]. They prevent R-loop accumulation and subsequent DNA damage during transcription, as they promote the assembly of ribonucleoproteic complexes. Recruitment of the spliceosome and subsequent mRNP assembly, but not splicing per se, prevents R-loop formation. Introns are spliced out via two trans-esterifications, which involve the upstream and downstream intronic splice sites (SS; i.e., 5 SS and 3 SS) and the branch point sequence (BPS). Nonprotein-coding sequences are eliminated in two sequential reactions [1312]: 1. In the first reaction, step-I splicing factors cut one end of the noncoding intron, freeing the 5 -exon and looping the intron back on itself to form an intron lariat. 2. In the second reaction accomplished by an activated spliceosome (C complex) and step-I I splicing factors, the intron lariat is excised, and the exon ends are ligated. More than 95% of transcripts from human protein-coding genes are alternatively spliced. Hence, a given gene encodes numerous protein isoforms that have different or even opposing functions [1313]. Specific inhibitors and activators of the splicing control can affect pathophysiological processes. The spliceosome is a ribonucleoproteic RNA-directed metalloribozyme, the small nuclear U-rich RNA U6 (snRNA-U6 or simply U6) coordinating the catalytic Mg2+ ions. The spliceosome is composed of five small nuclear ribonucleoproteins (snRNPs) and numerous proteic splicing factors. Each snRNP contains an snRNA (among U1–U2 and U4–U6) and a unique set of proteins. A cycle of assembly and disassembly occurs between snRNPs and pre-mRNAs. In a first stage, U1 recognizes the 5 SS in the intron (at the junction between the target intron and upstream exon). U1 interacts with the 5 SS, U2 with the BPS, U5 with the free 5 exon generated after the first splicing step, and U6 with the 5 SS after U1 linkage.

7.1 Messenger RNA Synthesis and Splicing

619

Four main modes of alternative splicing exist [1314]. Splicing mode I Exon (E) skipping: a cassette exon is spliced out of the transcript together with its flanking introns (I; the sequence E1–I1–E2–I2–E3 engendering E1–E3). Hence, a cassette exon is alternatively included or excluded in the final transcript. Splicing modes I I and I I I Alternative 3 SS and 5 SS selection (the sequence E1–I1–E2–I2–E3 engendering e1–E2–E3 and E1–e2–E3: e1: truncated E1 at alternative 3 SS, upstream from the terminal splice region, allowing full-length exon incorporation e2: truncated E2 at alternative 5 SS; downstream from the start splice region enabling full-length exon integration) Splicing mode I V intron retention: an intron remains in the mature mRNA transcript. Splicing mode V mutually exclusive exons: two exons are alternatively included or skipped. These rearrangements may occur either in the coding or the noncoding region of the mRNA. When occurring in the 5 - or 3 -untranslated regions, splicing does not affect the protein sequence, but can regulate its expression [1313]. Cis-acting elements, such as exon splicing enhancers and exon splicing silencers and intron splicing silencers and intron splicing enhancers, are bound by transacting elements (splicing factors) that facilitate or repress splicing [1313]. A region rich in pyrimidine nucleotides (cytosine [C] and thymine [T]) helps the spliceosome assembly via various splice factors. At the 3 -end of the intron, a AG splice site (i.e., linked to purines adenine [A] and guanine [G]) is preceded by a polypyrimidine (pY) tract and a branch point that requires a consensus splice site (YNYURAY; R: purine, U: uridine) [1313]. Two major classes of splice factors (SF) include Ser–Arg-rich motif-containing (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). Other RNA-binding proteins are considered as SFs outside of these major classes, many regulating cell-specific splicing events, such as epithelial splicing regulatory proteins ESRP1 and ESRP2, cytidine–uridine–guanidine (CUG) triplet repeat RNAbinding protein CUGBP1 and CUGBP2 (CELF1 and CELF2), RNA-binding Fox homolog RBFox1 and RBFox2, and muscleblind-like protein (MBnl1) [1313]. The ribonucleoproteic remodeling ATPases and helicases and splicing factors play an essential role in the two-step splicing reaction, as they recruit the proper spliceosomal complexes. Serine–arginine-rich motif-containing splicing factors (SRSF) stimulate premRNA splicing via their interaction with exon splicing enhancers and activation of 5 - or/and 3 -splice sites. The activity of SRSFs is regulated by phosphorylation by three main sets of splicing kinases: CDC2-like kinases (CLK1–CLK4), dualspecificity Tyr-regulated kinases (DYRK1a–DYRK1b and DYRK2–DYRK4), and SR-rich splicing factor protein kinases (SRPK1–SRPK3). The kinases PIM1,

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7 Genetic Risk Factors

PIM2,48 and pre-mRNA processing factor (splicing kinase) PRPF4 also participate in the control of the phosphorylation of splicing factors. The spliceosome is in a catalytically inactive state and is remodeled into a catalytically active form by RNA helicase [1315]. Diverse spliceosome complexes intervene at different steps of pre-mRNA splicing. The pre-spliceosomal A complex made up of U1 and U2 associates with the tri-snRNP formed by U4, U5, and U6, generating the pre-catalytic B complex. Dissociation of U1 and U4 and recruitment of the 19 complex (NTC; or CDC5L complex in humans)49 and NTCrelated proteins (NTRs) trigger structural re-arrangement and formation of the activated B complex. The Bact complex is converted to the catalytically activated B complex, in which an invariant adenine nucleotide of the BPS initiates the first transesterification and generates a free 5 -exon and an intron–exon lariat. The catalytic step-I spliceosomal C complex catalyzes the second transesterification, which ligates two exons. The ligated exons are released from the postcatalytic P complex, but the intron lariat remains bound to the intron lariat spliceosomal complex (ILS complex). Finally, the postsplicing complex is disassembled, the intron lariat is released, and snRNPs and NTC proteins are recycled. Genetic intronic and exonic mutations can alter RNA splicing; transcript variants can contribute to diseases [1317]. Introns of mRNAs are removed during splicing, which generates uninterrupted open reading frames (ORF), i.e., coding segments with a start and stop codon, the sequence of consecutive, non-overlapping codons (triplets of nucleotides), which are translated into proteins. Intronic mutations, even those that affect a region more than 30 nucleotides from a splice site, can impair splicing, whereas the likelihood with which exonic mutations alter splicing is lower. At least 10,689 exons are targets of alternative splicing. The percentage of spliced transcripts for each exon among 16 explored human tissues was estimated [1317]. Among 658,420 SNPs mapped to exonic and intronic sequences containing the regulatory code for about 120,000 exons in approximately 16,000 genes, 54,3525 are common SNPs (minor allele frequency >1%) and 114,895 are most often rare SNPs (minor allele frequency Co2+ > Mn2+ > Sr2+ > Ba2+ > Cu2+ > Fe2+ . It is highly expressed in the brain, lung, and distal convoluted tubule of the nephron [194]. cytosolic purine 5 -nucleotidase-2. Indeed, it preferentially hydrolyzes inosine monophosphate and other purine nucleotides [108]. It is allosterically activated by various compounds such as ATPn [194]. 86 ATRAP: angiotensin-2 receptor AT -associated protein. It potentiates AT internalization 1 1 upon angiotensin-2 stimulation and favors receptor desensitization via phosphorylation, impeding signaling [194]. 87 ClCn6: neuronal voltage-gated chloride channel. 88 CSK: ubiquitous cytoplasmic Src tyrosine kinase. It participates in the regulation of cell growth, differentiation, migration, and immune response. It phosphorylates (inactivates) Tyr residues in the C-terminal tails of SRC family kinases (Fyn, HCK, LCK, Lyn, Src, and Yes), hence suppressing signaling from various types of plasmalemmal receptors (e.g., BCR and TCR) [108, 194]. 89 ATXN: ataxin. Ataxin-2 is involved in EGFR internalization (inhibition) at the plasma membrane [108]. 90 CRIP1: gene encoding cysteine-rich protein-1. 91 RAB11FIP1: gene encoding Rab11 family-interacting protein-1. 85 Nt5c2:

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8. The fifth DBP-linked SNP, rs16948048, localizes to the 17q21 locus, upstream from the ZNF652 and PHB genes.92 • Four loci are significantly related to systolic arterial pressure (ATP2B1,93 CYP17A1, PLEKHA7,94 and SH2B3). • 6 to DBP (ATP2B1, CACNB2,95 CSK–ULK3,96 SH2B3, TBX3–TBX5,97 ULK4). • 1 to hypertension (ATP2B1) [1339]. Systolic BP is linked to 13 SNPs, the strongest connection being the SNP rs2681492 in the ATP2B1 gene. A signal was identified on the 12q24 locus for SH2B3 (rs3184504) and nearby ATXN2 (rs653178). The PLEKHA7 gene (11p15.1 locus; rs381815) and the 2q31–2q33 sequence adjacent to the Pms198 and MSTN99 (rs7571613) suggest an association. The block on chromosome 12q24 linked to DBP is linked to mutations in the SH2B3 (rs3184504), ATXN2 (rs653178), and Trafd1 genes (rs17630235). It includes the Acadh10, ALDH2, ATXN2, BRAP, C12ORF30, C12ORF51, CUX2, Erp29, FAM109A, Mapkapk5, PTPN11, RPL6, SH2B3, TMEM116, and TRAFD1

92 Phb:

prohibitin. This intracellular antiproliferative protein hampers DNA synthesis and hence angiogenesis. 93 ATP2b1: plasma membrane calcium ATPase-1 (or PMCA1). This magnesium-dependent enzyme couples ATP hydrolysis with export of bivalent calcium ions from the cytosol [108]. It constitutes a family of plasmalemmal pumps with ATP2b2 (or PMCA2), ATP2b3 (or PMCA3), and ATP2b4 (or PMCA4), the member diversity being augmented by alternative splicing of transcripts. In mammals, PMCA1 is formed in Ca2+ -transporting epitheliocytes and bone mesenchymal cells. Four PMCA1 splice variants are described (PMCA1a–PMCA1d); PMCA1a and PMCA1c are detected in the brain, spinal cord, heart, and skeletal muscle; PMCA1b is ubiquitous; PMCA1d is observed in the heart and skeletal muscle [194]. 94 PlekHa7: pleckstrin homology domain-containing protein-A7. It is implicated in the formation and maintenance of zonula adherens with nezha (or calmodulin-regulated spectrin-associated protein CamSAP3) and KIFc3. Nezha is a microtubule minus-end-binding protein that regulates noncentrosomal microtubule organization. PlekHa7 operates via nezha, which anchors microtubules at their minus-ends to zonula adherens, leading to the recruitment of KIFc3 [108]. It redistributes the cadherin-1–catenin complex to the zonula adherens [194]. It also interacts with the adherens junction protein catenin-δ1. Hence, PlekHa7, nezha, and KIFc3 are involved in maintaining the integrity of the zona adherens. 95 Ca β2: voltage-gated calcium channel β2 subunit. It increases peak calcium flux, shifts the V voltage dependencies of activation and inactivation, modulates G-protein inhibition, and controls the α1-subunit membrane targeting [108]. It forms a high voltage-activated, long-lasting calcium channel with the α1d subunit encoded by the CACNA1D gene [194]. Channel activity is enhanced by coupling with the α2 subunit encoded by the CACNA2D1 gene. 96 ULK3: uncoordinated-51-like kinase-3. 97 TBx: T-box transcription factor. 98 PMS1: postmeiotic segregation increased protein-1. It is a DNA mismatch repair protein. 99 Mstn: myostatin (or GDF8). It is synthesized as a preprotein that matures by two cleavages. It contributes to the control and maintenance of skeletal muscle mass, as it impedes skeletal muscle growth.

7.3 Blood Pressure and Hypertension

631

genes.100 In addition, ATP2B1,101 TBX3–TBX5,102 and PLEKHA7103 are associated with DBP. Five genes, ANXA1, Far2, GZMB, MYADM, and TSPAN2, which encode annexin-A1, fatty acyl-CoA reductase FAR2, granzyme-B,104 myeloid-associated differentiation marker,105 and tetraspanin Tspan2, are correlated with SBP and DBP [1340].

100 TRAFD1:

TRAF-type zinc finger domain-containing protein-1. It controls excessive innate immune responses via a negative feedback [108]. ACADH10: acyl-CoA dehydrogenase family member-10. It is implicated in mitochondrial fatty acid oxidation [194]. AlDH2: mitochondrial aldehyde dehydrogenase-2. It is involved in alcohol metabolism. BrAP: BrCa1-associated protein. It is also named impedes mitogenic signal propagation, and RING finger protein RNF52. It precludes MAPK activation, as it represses formation of the Raf–MAP2K complexes via inactivation of the KSR1 adaptor ([pseudo]kinase suppressor of Ras), which scaffolds signaling MAPK complex. It also acts as a Ras-responsive ubiquitin ligase that, upon Ras activation, autopolyubiquitinates, thereby relieving inhibition of the Raf–MAPK signaling [108]. This cytoplasmic protein recognizes and interacts with the nuclear localization signals of target proteins for nuclear import [194]. CUx2: cut-like homeobox gene product-2 -(or CUTL2). This transcription factor may be involved in neural specification [108]. ERP29: endoplasmic reticulum protein-29. This 29-kD reticuloplasmin participates in the processing of secretory proteins within the endoplasmic reticulum (ER). Fam109a: family with sequence similarity member-109A. It is also called 27-kDa inositol polyphosphate phosphatase-interacting protein IPIP27a, and sesquipedalian-1 (Ses1). It acts in endocytosis. It is involved in receptor recycling from endosomes to the trans-Golgi network and plasma membrane [108]. It links phosphatidylinositol (4,5)-bisphosphate 5-phosphatase OCRL (InPP5f) to the recycling of receptors at sorting and recycling endosomes [194]. MAPKAPK5: mitogen-activated protein kinase-activated protein kinase-5. It is regulated by P38MAPK [194]. It is activated by cellular stressors and proinflammatory cytokines. It phosphorylates HSP27 (HSPb1), provoking actin meshwork rearrangement. It also hinders TORC1 signaling. It phosphorylates FoxO3, P53, ERK3, ERK4, and RHEB [108]. PTPn11: nonreceptor protein Tyr phosphatase-11 (or SH2 domain-containing protein Tyr phosphatase SHP2). It acts downstream from various receptor and cytoplasmic protein Tyr kinases. It dephosphorylates rock2, stimulating its RhoA binding, in addition to CDC73 [108]. RPl6: ribosomal protein-L6. It is also called Tax-responsive enhancer element-binding protein TaxREB107 (or TxREB1). TMem116: transmembrane protein-116. 101 I.e., 12q21 locus (rs2681472). 102 I.e., 12q24 locus (rs2384550). 103 I.e., 11p15 locus (rs11024074). 104 A.k.a. granzyme-2, cathepsin-G-like protein CGL11, cytotoxic T-lymphocyte-associated serine esterase CTLA1. 105 Myeloid-associated differentiation marker contributes to membrane raft organization and establishment of the endothelial barrier [108]. It impedes PKC signaling, actin filament polymerization, heterotypic intercellular adhesion, cell migration, but promotes protein targeting to plasma membrane and cell spreading.

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7 Genetic Risk Factors

In Amish subjects, mutations in STK39,106 the product of which interacts with WNK kinases107 and cation–chloride cotransporters in the distal nephron (Vol. 6, Chap. 3. “Cardiovascular Physiology”), alter renal Na+ excretion and cause monogenic forms of BP dysregulation [1341]. Other genes that encode BP regulators include Wnk1, Bmpr2,108 GPX1,109 TAF1,110 GYS1,111 CAST,112 IKBKAP,113 MEF2A,114 and PPARA [1340].115 Among BP- and hypertension-related genetic variants (i.e., significantly associated with increased BP), the SNP rs3184504 (a missense SNP leading to the R262W permutation in the SH2b3 protein) localizes to the third exon of the SH2B3 gene. It is linked to the expression of four genes in the genetically inferred causal BP gene sets (three genes in cisSNP, SH2B3, ALDH2, and NAA25,116 and two genes in transSNP, Il8117 and Tagap) [1340].118 The genetic subnetwork 106 STK:

protein Ser/Thr kinase, also called SPAK (Ste20/SPS1-related proline- and alanine-rich protein kinase). It can mediate stress-activated signaling. It inhibits SLC4a4, SLC26a6, in addition to CFTR (a.k.a. ABC35 and ABCc7) [108]. 107 WNK: lysine-deficient (with-no-K [Lys]) kinase. 108 BMPR2: bone morphogenetic protein receptor-2. 109 GPOx1: glutathione peroxidase-1. 110 TAF1: TATA box-binding protein (TBP)-associated factor-1. It is the largest component and core scaffold of the TFI I D basal transcription initiation factor complex (or GTF2d). It can autophosphorylate or transphosphorylate other transcription factors, such as P53, GTF2a1, and GTF2f1 [108]. It can also serve as histone acetyltransferase for histones H3 and H4. 111 GyS1: glycogen synthase-1. 112 Cast: calpastatin (calpain inhibitor). 113 Iκ BKAP: inhibitor of NFκB kinase (IKK)-associated protein. 114 MEF2a: myocyte enhancer factor-2A. This transcriptional activator binds specifically to the MEF2 element of numerous muscle-specific genes. It also activates many growth factor- and stress-induced genes [108]. It is implicated not only in skeletal and cardiac muscle development but also in neuronal differentiation and survival. 115 PPARα: peroxisome proliferator-activated receptor-α (nuclear receptor NR1c1). Once it is liganded, it regulates lipid metabolism and peroxisomal oxidation of fatty acids. It is activated by palmitoyl oleoyl glycerol phosphocholine in addition to oleoylethanolamide, a natural lipid regulating satiety [108]. It stimulates transcription of the ACOX1 and P450 genes that encode peroxisomal acyl-CoA oxidase, ACOx1, and cytochrome-P450 enzymes. Transactivation requires heterodimerization with NR2b1 [108]. It is antagonized by NR2c2. 116 NAA25: Nα-acetyltransferase-25 auxiliary subunit of the NatB complex, which catalyzes acetylation of the N-terminal methionine residues of peptides beginning with Met–Asp–Glu [108]. 117 IL8: interleukin-8, in fact the chemokine CXCL8 attracts neutrophils, basophils, and T cells, but not monocytes. CXCL8(6–77) has a five- to tenfold higher activity on neutrophil activation, CXCL8(5–77) a greater activity on neutrophil activation, and CXCL8(7–77) a larger affinity for CXCR1 and CXCR2 than CXCL8(1–77) , respectively [108]. 118 TAGAP: T-cell activation Rho GTPase-activating protein. T-cell activation requires stimulation of the receptor TCR–CD3 complex followed by the recruitment of intracellular signaling effectors (e.g., PLCγ1, GRB2, and PI3K), the interaction of which is mediated by adaptor proteins, such as linker for activation of T cells (LAT), T-cell receptor (TCR)-interacting molecule (TRIM), and T-cell signal-transduction and lymphocyte-specific linker (Lnk or SH2b3). The Lnk adaptor protein is also called SH2b3 adaptor protein. It can prevent T-cell activation. It is involved

7.3 Blood Pressure and Hypertension

633

related to the key driver gene SH2B3 comprises another major key driver gene ALDH2 (cis), Tagap (trans), and Cxcl8 (trans), which are in the genetically inferred causal BP coexpression network modules in addition to ARHGEF40 (trans),119 TAGAP (trans), MYADM (trans), FOS (trans), PPP1R15A (trans),120 and S100A10 (trans),121 which are in the top BP signature gene set [1340]. The SH2B3derived between-protein interaction subnetwork is enriched with genes involved in intracellular signaling in addition to T-cell activation and differentiation, i.e., STAT1,122 KCNJ2,123 and PTPRO.124 This SH2B3-derived subnetwork is thus

in signaling primed by growth factor and cytokine receptors. It is phosphorylated by LCK and connects to the TCR ζ-chain. Mutations in the SH2B3 gene are linked to susceptibility to autoimmune malabsorptive type-13 celiac disease (based on the immunophenotype of enteric intraepithelial lymphocytes), also called susceptibility to gluten-sensitive type-13 enteropathy, in addition to susceptibility to insulin-dependent (type-1) diabetes mellitus. The celiac disease is a multifactorial disorder of the small intestine influenced by environmental and genetic factors. It is characterized by malabsorption upon inflammation of the intestinal mucosa after ingestion of wheat gluten and/or related rye and barley proteins. It is strongly associated with specific HLA class-I I genes HLA-DQ2 and HLA-DQ8 located on chromosome 6p21. The immune system in the gut is abnormally sensitive to gliadin, a fragment of gluten (a protein found in wheat, rye, and barley), owing to certain variants of the HLA-DQA1 and HLA-DQB1 genes of the MHC class-I I antigen-presenting receptor (the proteins produced from the HLA-DQA1 and HLA-DQB1 genes form an antigen-binding DQαβ heterodimer that attaches to protein fragments). The SNP rs653178 in the ATXN2–SH2B3 locus is also linked to a susceptibility to peripheral arterial disease [1342]. Autoimmune thrombotic anti-phospholipid syndrome, which is defined by a thrombophilia caused by anti-phospholipid antibodies interacting with hemostatic and inflammatory mediators and hence generating a procoagulant (prothrombotic) state, is linked to a strong haplotypic association with the ATXN2–SH2B3 locus [1343]. 119 ARH: autosomal recessive hypercholesterolemia. A.k.a. RhoGEF40 (Rho guanine nucleotideexchange factor-40) and solo. 120 PP1 r15a : protein phosphatase-1 regulatory subunit-15A. It dephosphorylates the translation initiation factor eIF2a (or eIF2S1), thereby restarting protein synthesis after stress exposure [108]. It hampers the TGFβ1 signaling dephosphorylation. 121 S100a10 induces the dimerization of annexin-A2 [108]. 122 STAT: signal transducer and activator of transduction. It mediates cellular response to interferons and SCF among other growth factors. Upon Ifnγ exposure, STAT1 is phosphorylated and then homodimerizes and translocates into the nucleus, where it binds to the IFNγ-activated sequence (GAS) to launch transcription of target genes [108]. 123 The KCNJ2 gene encodes inward rectifier potassium channel K 2.1, which has a greater tenIR dency to allow K+ influx than efflux. Their voltage dependence is regulated by the concentration of extracellular K+ ions; when [K+ ]e rises, the channel opening shifts to a more positive voltage range [108]. The inward rectification is mainly due to the blockage of outward current by internal magnesium. 124 Receptor protein Tyr phosphatase-O regulates the relation between the glomerular pressure and filtration rate [108].

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7 Genetic Risk Factors

enriched with BP-related genes. It also includes the PLCE1,125 PRRC2A,126 ADRB2,127 RHOA, and SOCS1 genes.128 In summary, the SH2B3 genetic subnetwork linked to rs3184504 includes 19 genes in a cis or trans manner [1340]. Deletion of the SH2B3 gene exacerbates angiotensin-2-induced hypertension via inflammation and T-cell activation. The FOS gene is linked to hypertension and is considered a marker of neuronal activity in various regions such as those implicated in BP control (e.g., spinal sympathetic neurons). In essential or primary hypertension, a genetic predisposition favors the cumulative effect of lifestyle factors. Hypertension existing as a polygenic trait can follow a Mendelian mode of inheritance linked to mutations in some genes [1336]. Gene mutations cause monogenic forms of secondary hypertension (e.g., primary aldosteronism among >25 gene mutations), in particular related to the regulatory function of the kidney and adrenal gland (i.e., water and sodium homeostasis; Table 7.6). Twelve gene mutations cause hypertension (Table 7.7) [1344]. Among them, five gene mutations are activating (GOF mutations), hence directly increasing BP; the remainder are LOF mutations with an elevated BP that results from a feedback loop. Hypertension of genetic origin results from disturbances of only two categories of processes: 1. Renal sodium handling (Table 7.8) 2. Steroid hormone metabolism and mineralocorticoid receptor activity (Table 7.9) Multiple single-nucleotide polymorphisms are associated with hypertension (Table 7.10) [1336]. • The variant rs13333226, which localizes to the promoter of UMOD gene (encoding uromodulin) expressed in the kidney affects sodium homeostasis, as uromodulin is related to the salt-retaining sodium–potassium–chloride cotransporter NKCC2. • Another SNP exists in the promoter of the Nos3 gene involved in the vasomotor tone. • The SNP rs5068 affects the NPPA–NPPB genetic locus.

125 PLC 1:

phospholipase-C 1. proline-rich coiled–coil-containing protein-2A. It may participate in the regulation of pre-mRNA splicing [108]. 127 ARβ2: β2-adrenoceptor. 128 SOCS: suppressor of cytokine signaling protein. SOCS1 precludes cytokine signaling via the JaK–STAT3 pathway in addition to IGF1R signaling [108]. It is also a substrate recognition component of the ECS (elongin-BC–Cul2/5–SOCS) ubiquitin ligase complex. 126 PrRC2a:

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Table 7.6 Examples of gene mutations (monogenic forms of hypertension; (Source: [1336]; ACTH adrenocorticotropic hormone, CYP11B1 11β-hydroxylase [confers ACTH responsiveness], CYP11B2 aldosterone synthase, SCNN1B, SCNN1G genes encoding β and γ subunits of epithelial Na+ channel [ENaC], TAL thick ascending limb of the loop of Henle) Locus 1p36.13

Genes CLCNKB

3p21.3

CACNA1D

4q31.2

NR3C2

8q24.3

CYP11B1/B2

11q24.3

KCNJ1

11q24.3

KCNJ5

12p12.3

WNK1

15q21.1

SLC12A1

16p12.2

SCNN1B/1G

16q13

SLC12A3

17q21.2

WNK4

Disease Autosomal recessive type-3 Bartter syndrome Impaired Cl− re-absorption in TAL, altered Na+ re-absorption Hypokalemia Hyperreninemia and -aldosteronemia Sporadic aldosterone-producing adenoma Autosomal dominant type-1 pseudohypoaldosteronism Autosomal dominant hyperaldosteronism-1 (↑ aldosterone secretion by ACTH due to chimeric gene [5 -regulatory sequences of CYP11B1 fused with the distal coding sequences of CYP11B2]) Autosomal recessive type-2 Bartter syndrome Autosomal dominant type-3 hyperaldosteronism Autosomal dominant type-2C pseudohypoaldosteronism Autosomal recessive type-1 Bartter syndrome Autosomal dominant Liddle syndrome Autosomal recessive Gitelman syndrome Autosomal dominant type-2B pseudohypoaldosteronism

Hypertension and mutations in genes encoding signaling mediators, such as those of the Ras–Raf–MAPK axis, can cause cardiac hypertrophy with cell enlargement and proliferation. Activation of receptor protein Tyr kinases by growth factors and the effectors, small GTPase Ras and kinase Raf, contribute to cardiac hypertrophy.

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7 Genetic Risk Factors

In RASopathies, i.e., impaired Ras–Raf–MAPK module signaling, such as the congenital Noonan syndrome,129 activating mutations in the RAF1 gene (e.g., cRaf L613V SNP) cause cardiac hypertrophy.

Table 7.7 Gene mutations causing hypertension (Source: [1344]) Gene CUL3 CYP11B1 CYP11B2 CYP17A HSD11B2 KCNJ5 KLHL3 NR3C SCNN1B/G Wnk1/4

Product Cullin-3 11β-Hydroxylase Aldosterone synthase Steroid 17-hydroxylase 11β-Hydroxysteroid dehydrogenase-2 Inward K+ rectifier Kelch-like-3 Mineralocorticoid receptor ENaC channel subunit With no Lys kinase

Disease Gordon’s hypertension syndrome Type-4 congenital adrenal hyperplasia Type-1 hyperaldosteronism Type-5 congenital adrenal hyperplasia Cortisol 11β-ketoreductase deficiency Type-3 familial hyperaldosteronism Familial hyperkalemic hypertension Early-onset autosomal dominant hypertension Pseudoaldosteronism Type-2 pseudohypoaldosteronism

The final steps in the synthesis of cortisol and aldosterone from 11-deoxycortisol and deoxycortisone, respectively, are catalyzed by cytochrome P450 isozymes, CyP11b1 (steroid 11βhydroxylase) and CyP11b2 (aldosterone synthase). The former is synthesized in the adrenal zona fasciculata under ACTH control; the latter is produced in the zona glomerulosa and is regulated by ACTH, angiotensin-2, and potassium Table 7.8 Mono- and polygenic hypertension syndromes and renal sodium handling (ClCK kidney-specific chloride channel, SLC9a3 solute carrier family-9 member 3, or Na+ –H+ exchanger NHE3) Nephron site Thick ascending limb of the loop of Henle

Ion carrier Apical SLC12a1 Apical KIR 1.1 Apical SLC9a3 Basal ClCKA and ClCKB

Effects Regulators Na+ , K+ , Cl− reabsorption NO K+ reabsorption; NO Na+ import; NO Cl− export (continued)

129 The

Noonan syndrome is a genetic disorder characterized by short stature, webbed neck, chest deformities, facial anomalies, undescended testes, and frequently by diverse cardiac anomalies such as congenital heart defects. It can be accompanied by hypertrophic cardiomyopathy with asymmetric or symmetric septal hypertrophy, in addition to eventual cardiac malformations (pulmonary and mitral valve dysplasia, atrial and ventricular septal defect, coarctation, and tetralogy of Fallot).

7.3 Blood Pressure and Hypertension

637

Table 7.8 (continued) Basal Na+ –K+ ATPase (antiporter) Distal convoluted tubule Collecting duct

Apical SLC12a3 Basal Na+ –K+ ATPase Basal ClCKB Apical ENaC Basal KIR 1.1 Basal Na+ –K+ ATPase

K+ influx, Na+ outflux Sodium electrochemical gradient for SLC12a1; NO Na+ and Cl− reabsorption WNK1/4, Cul3–KlhL3 NO Cl− reabsorption Na+ re-absorption NO, WNK1/4 K+ secretion NO, WNK1/4 NO

The apical membrane of epitheliocytes of the nephron forms the interface between the tubular lumen and cytosol (wetted surface); the basal membrane contacts the basement membrane connected to the interstitial fluid and then the peritubular capillary Table 7.9 Steroidogenesis (Source: [1345]) Mineralocorticoids CyP21a2 3β HSD Cs CyP11a1 → pregnenolone → progesterone → CyP11b2 11-deoxycorticosterone CyP11b1 → corticosterone → aldosterone Glucocorticoids CyP17 3β HSD Cs CyP11a1 → pregnenolone → 17-hydroxypregnenolone →

17-hydroxyprogesterone CyP21a2 11-deoxycortisol CyP11b1 → → cortisol Sex hormones CyP17 CyP17 Cs CyP11a1 → pregnenolone → 17-hydroxypregnenolone → HSD HSD dehydroepiandrosterone 3β → androstenedione 17β→ testosterone CyP17 CyP17 Cs CyP11a1 → pregnenolone → 17-hydroxypregnenolone → HSD 17β HSD dehydroepiandrosterone 3β → androstenedione Cyp19 estradiol → estrone →

Steroid hormones are derivatives of cholesterol (Cs). Cholesterol mobilized from cytosolic lipid droplets or lysosomes is transported to the inner mitochondrial membrane, where it is converted to pregnenolone in a sequence of two reactions, both catalyzed by CyP11a1. Pregnenolone reenters the cytosol. Steroid hormones include glucocorticoids (e.g., cortisol), mineralocorticoids (e.g., aldosterone), androgens (e.g., testosterone), estrogens (estradiol and estrone), and progestogens, or progestins (e.g., progesterone). The basic structure is composed of the cyclopentanoperhydrophenanthrene ring. Their synthesis requires a set of oxidative enzymes located in mitochondria and ER (HSD hydroxysteroid dehydrogenase, CyP cytochrome-P450, CyP11a1 cholesterol desmolase, or cholesterol side-chain cleavage enzyme [mitochondria; adrenal cortex, ovary, and testis], CyP11b1 11β-hydroxylase [mitochondria; zona fasciculata (middle) and reticularis (inner) of the adrenal cortex], CyP11b2 aldosterone synthase [mitochondria; zona glomerulosa (outer) of the adrenal cortex], CyP17 17α-hydroxylase, or (17,20)-lyase [ER; adrenal cortex, ovary, and testis], CyP19 aromatase [ER; adipocytes; ovary and testis; CyP21a2: 21hydroxylase [ER; adrenal cortex], a.k.a. CyP21b)

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7 Genetic Risk Factors

Table 7.10 Examples of single-nucleotide polymorphisms (SNPs) associated with hypertension (Source: [1336]; ADM adrenomedullin, AGT angiotensinogen, CASZ1 castor homolog 1, zinc finger, CLCN6 chloride transport protein-6 [anion–proton antiporter], GUCY guanylate cyclase, MTHFR methylene tetrahydrofolate reductase, NPPA(B) atrial (brain) natriuretic peptide, NPR3 natriuretic peptide clearance receptor, MCD medullary collecting duct, TAL thick ascending limb of the loop of Henle) Locus 1p36.22

1q42.2 2p13.1 3p25.3

Genes CASZ1, MTHFR, CLCN6, NPPA–NPPB SLC16A1, CAPZA1, ST7L, MOV10 AGT SLC4A5 HRH1

3p24.1

SLC4A7

4q12 4q21.2 4q24 4q32.1 5p13.3 7q36.1 10p12.3

PDGFRA Fgf5 SLC39A8 GUCY1A3/1B3 NPR3 ABP1, KCNH2, NOS3, ACCN3 CACNB2

10q25.3 11p15.4 16p12.3

ADRB1 ADM UMOD

1p13.2

SNP rs88031534, rs506835, rs1736750421 rs1703061320, rs293253821 rs200477623 rs7571842, rs101778337 rs34759125 (histamine receptor H1 of smooth myocytes and neurons [nucleus tractus solitarius]) rs1308271121 (TAL and MCD) rs87160626 rs1699807318 rs1310732521 rs1313957121 rs117377120 rs391822629 rs1101416619, rs437381421, rs181335321 rs180125336 (Arg389Gly) rs712922021 rs1333322622

The transcriptional coactivator YAP in the Hippo (STK4–STK3) pathway, which regulates organ size, increases TEF1 activity and is involved in Raf-induced cardiac hypertrophy and hyperplasia [1346]. Genome-wide association studies are aimed at identifying DNA loci (i.e., individual genes and coexpressed modules as genes interact in regulatory networks)130 that correlate with hypertension. In association with blood mRNA expression profiles, the objective is searching molecular mechanisms underlying BP regulation [1340]. Blood-pressure-linked genetic coexpression network modules enriched with expression-associated SNPs include:

130 Gene

coexpression networks consist of modules of genes with a high level of coregulation. Genetic variants can disturb subnetworks, the overall dysregulation of which alters homeostasis.

7.3 Blood Pressure and Hypertension

639

Module 1, which contains genes involved in chromatin modification, intracellular transport, and regulation of gene expression (with the key driver genes ATXN2, BHLHB2 (or BHLHE40),131 Hipk1,132 MAPKAPK5, Nmt1,133 Nsf,134 PRRC2A, RAB11FIP1,135 SH2B3, and VIM).136 Module 2, with genes implicated in hemostasis, platelet activation, and wound healing. Mmodule 3 with genes contributing to immunocyte-mediated cytotoxicity, cellular defense response, and inflammatory response (with the key genes ADGRG1,137 GZMB, GZMH,138 KLRD1,139 PRF1,140 and TGFBR3).141 Genes involved in multiple biological processes are tightly coregulated in relation to BP control.

131 This

transcriptional repressor is also called differentially expressed in chondrocytes protein DEC1. It is involved in the regulation of the circadian rhythm, as it prevents activity of the clock and clock-controlled genes [108]. It operates in the autoregulatory feedback loop independently of the Per–Cry loop. It also inhibits its own production in addition to that of DBP (D element-binding protein [D site of albumin promoter (albumin D-box)-binding protein], a.k.a. Tax-responsive enhancer element-binding protein, TaxREB302) and bHLHe41 (a.k.a. bHLHb3 and DEC2). It also acts as a corepressor of RXR and the RXR–LXR heterodimers. The LXRs (LXRα–LXRβ or NR1h3–NR1h2) target ABCa1, ABDg5, and ABCg8, which are involved in sterol transport across the plasma membrane in enterocytes; ABCa1, which enables the initial step of HDL formation, lipidation of lipid-free ApoA1I, cholesterol ester transfer protein (CETP), SREBP1c, and CyP7a1 in hepatocytes; and LPL, ABCa1, the ABCg1 isoforms ABCg1a and ABCgx (full-length ABCg1 having 678 amino acids [ABCg1(678) ], and the splice variant 666 [ABCg1(666) ] [1347]), ApoE, and CETP in blood [1000]. 132 Homeodomain-interacting protein kinase-1 is the protein Ser/Thr kinase involved in transcriptional regulation and TNF-mediated apoptosis [108]. It phosphorylates death-domain-associated protein, DAP6 (or DAxx), in response to cellular stress, in addition to the transcription factor MyB (inactivation). 133 NMT1: N myristoyltransferase-1. 134 NSF: N ethylmaleimide-sensitive factor. 135 Rab11 family-interacting protein-1 is involved in endosomal recycling and in controlling membrane trafficking in phagocytosis [108]. 136 Vimentin belongs to the class-I I I intermediate filaments constituents. Vimentin stabilizes typeI collagens [108]. 137 Adhesion G-protein-coupled receptor-G1 (AdGRg1; or GPR56) is involved in cell adhesion, especially for developing neurons and hematopoietic stem cells [108]. It tethers to collagen-3 encoded by the COL3A1 gene, thereby preventing neuronal migration and activating the RhoA pathway. It controls cancer progression, as it precludes VEGFa production and hence angiogenesis. On the other hand, AdGRg1 N-terminal fragment activates VEGFa production. 138 GzmH: granzyme-H. 139 KLRd1: killer cell lectin-like receptor-D1. 140 Prf1: perforin-1. It acts in secretory granule-dependent cell death and in defense against virusinfected and cancerous cells. Once it is bound to calcium, it inserts into the membrane of target cells, oligomerizes, and forms large pores, which enables cytolysis by the uptake of cytotoxic granzymes [108]. 141 Transforming growth factor-β receptor-3 captures TGFβ for presentation to receptors [108].

640

7 Genetic Risk Factors

7.4 Genetic Determinants of Dyslipidemias Genome-wide association studies have identified numerous (∼100) lipid-associated loci, which are related to dyslipidemia (elevated fasting and postprandial plasmatic concentrations of triglyceride-rich lipoproteins [TGRL; i.e., very low-density lipoproteins, VLDLs, and intermediate-density lipoproteins, IDLs] and LDLCS , but low HDLCS concentration). SNPs linked to atherosclerosis risk factors and related traits were explored in various populations (a total of 188,578 individuals) of European, East and South Asian, and African ancestry (Tables 7.11, 7.12, 7.13, 7.14, and 7.15) [1348]. The genome was screened for common variants associated with serum lipids in a large population (>100,000 individuals) of Europeans in addition to individuals of East and South Asians and African Americans [1349]. Among 95 significantly associated loci, SNPs include loci near lipid regulators (e.g., CYP7A1,142 Table 7.11 Loci primarily associated with LDL–cholesterol and total cholesterol (TC; Part 1; Source: [1348])

142 Cytochrome-P450

Locus ABCB11 ACADH11 ANXA9-CERS2 APOH-PRXCA ASAP3 BRCA2 CMTM6 CSNK1G3 DLG4 EHBP1 FAM117B FN1 GPR146 HBS1L INSIG2 KCNK17 LOC84931

Associated traits TC LDL LDL LDL TC LDL LDL, TC LDL LDL, TC LDL TC LDL TC TC LDL, TC TC LDL, TC

CyP7a, or cholesterol 7α-mono-oxygenase (hydroxylase), catalyzes a ratelimiting step in cholesterol catabolism and bile acid synthesis.

7.4 Genetic Determinants of Dyslipidemias Table 7.12 Loci primarily associated with LDL–cholesterol and TC (Part 2; Source: [1348])

Table 7.13 Loci primarily associated with triglycerides (Source: [1348])

641 Locus MIR148A MTMR3 PHC1–A2ML1 PHLDB1 PPARA PXK SNX5 SOX17 SPTLC3 TOM1 UGT1A1 VLDLR VIM–CUBN

Associated traits LDL, TC, TG LDL TC TC LDL, TC TC LDL LDL, TC LDL TC LDL, TC LDL, TC TC

Locus AKR1C4 INSR LRPAP1 MET MPP3 PDXDC1 PEPD VEGFA

Associated traits TG TG LDL,TC, TG TG TG TG HDL, TG HDL, TG

NPC1L1,143 and SCARB1)144 in addition to loci not previously implicated in lipoprotein metabolism, such as GALNT2,145 PPP1R3B,146 and TTC39B.147 Small LDLs are more predictive of coronary atherosclerosis than large LDLs, as they have a reduced LDLR-binding affinity and hence a longer blood residence time, a greater proteoglycan-binding affinity in arteries, and greater oxidative susceptibility in addition to association with other risk markers (reduced HDL level,

143 Niemann–Pick-C1-like

protein, NPc1L1, enables uptake of cholesterol across the plasma membrane of enterocytes and may operate in the transport of multiple lipids. 144 Scavenger receptor ScaRb1 is a receptor for phospholipids, cholesterol ester, lipoproteins, and phosphatidylserine along with apoptotic cells. 145N Acetylgalactosaminyltransferase, GalNT2, catalyzes the initial reaction in O linked oligosaccharide synthesis, the transfer of an N acetyl D galactosamine residue to a serine or threonine residue on the protein substrate, such as Muc1a, Muc1b, and Muc5ac [108]. 146 Protein phosphatase-1 regulatory subunit PP1 r3b controls glycogen metabolism as a glycogentargeting PP1 subunit, as it enhances the rate of glycogen synthesis. 147 Tetratricopeptide repeat protein TTC39b reduces plasmatic HDLCS concentration [194].

642 Table 7.14 Loci primarily associated with HDL–cholesterol (TC; Source: [1348])

Table 7.15 Major lipid-associated SNPs linked to coronary atherosclerosis (Source: [1348])

7 Genetic Risk Factors Locus ADH5 ANGPTL1 ATG7 CPS1 DAGLB FAM13A FTO GSK3B HAS1 HDGF–PMVK IKZF1 KAT5 MARCH8–ALOX5 MOGAT2–DGAT2 OR4C46 PIGV–NR0B2 RBM5 RSPO3 SETD2 SNX13 STAB1 TMEM176A ZBTB42–AKT1 Trait HDLCS LDLCS TG

Associated traits HDL HDL HDL HDL HDL HDL HDL, TG HDL HDL HDL HDL HDL HDL, TC HDL HDL HDL, LDL, TG HDL HDL, TG HDL HDL HDL HDL HDL

Genomic sites Cetp APOB/E, Ldlr, SORT1 APOA1/A5, Lpl, TRIB1

The SORT1 SNP is preferentially associated with an increased concentration of tiny LDLs (subgroup-I V )

increased IDL level, insulin resistance, and content in oxidized lipid, ApoC3, and glycated ApoB; Table 7.16) [1350, 1351]. Primary (familial) dyslipoproteinemias are caused by single or multiple gene mutations. According to the Fredrickson classification (Table 7.17), they are categorized into five main types [1352, 1353]. Dyslipoproteinemias are associated with the overproduction or defective clearance of TGs and low-density lipoproteins (LDLs) or the underproduction or excessive clearance of high-density lipoproteins (HDLs). Type-1 hyperlipoproteinemia (hyperchylomicronemia) includes dyslipidemias with normal or only slightly increased concentration of VLDL:

7.4 Genetic Determinants of Dyslipidemias Table 7.16 Content (%) of apolipoprotein-B-containing lipoprotein subspecies (Source: [1350]; CE cholesteryl ester, PL phospholipids, TG triglycerides, UC unesterified cholesterol [total not always equal to 100%])

Type VLDLI VLDLI I IDLI IDLI I LDLI LDLI I LDLI I I LDLI V

643 Protein 11 18 17 17 18 19–21 22–24 26–29

CE 8 24 35 37 43 45 44–46 40–42

UC 6 9 10 11 9 9–10 7–8 7

TG 58 29 16 3 7 3–4 3 5–6

PL 17 22 21 21 22 22–23 21 18–19

The roman numeral is related to the lipoprotein size in decreasing order (from large, medium, small, to tiny). Whereas CETP mediates TG enrichment of lipoproteins, that is, transfer of TGs into LDLI I , LDLI I I , and LDLI V , hepatic lipase (or lipase-C) causes lipolysis. In blood, lipolysis by lipoprotein lipase (LPL; lipase-D) of VLDLI yields IDLs and then LDLI I I and, from larger VLDLI , LDLI V . Lipolysis of VLDLI I engenders IDLI and LDLI I . Hepatocytes also release IDLI I , which is processed by LPL into LDLI

Type-1A, due to LPL deficiency or defective apolipoprotein-C2 (ApoC2)148 Type-1B, or familial ApoC2 deficiency Type-1C, due to a circulating inhibitor of LPL Type-2 hyperlipoproteinemia (hyperbetalipoproteinemia), the most common form, is classified according to whether VLDL (TG) concentration remains normal (type-2A) or increases (type-2B): Type-2A, sporadic (owing to dietary factors), polygenic, or truly familial, owing to mutations either in the Ldlr or APOB gene149 Type-2B, resulting from oversynthesis of substrates (TGs and acetyl-CoA) increase in ApoB100 synthesis, or decreased LDL clearance, including: • Familial combined hyperlipoproteinemia (FCH) • Lysosomal acid lipase (cholesteryl ester hydrolase) deficiency (cholesteryl ester storage disease)

148 Apolipoprotein-C2

is a component of chylomicrons, VLDL, LDL, and HDL. Both proApoC2 and ApoC2 activate LPL. In normolipidemia, it is mainly linked to HDL; in hypertriglyceridemia, it is predominantly found in VLDL and LDL [108]. 149 Apolipoprotein-B is a constituent of chylomicrons (ApoB48), LDL (ApoB100), and VLDL (ApoB100). It enables internalization of LDLs by the ApoB and ApoE receptor. The LDLR receptor belongs to a family of membrane receptors that include VLDLR, LRP1a, LRP1b, LRP8, LRP2 to LRP6, LRP8, LRP9, and LRP12. The primary ligands of LDLR are cholesterol-rich LDL and abnormal ApoB+ ApoE+ β VLDL [1355]. Interaction of LDL with LDLR is mediated by a single ApoB molecule.

644

7 Genetic Risk Factors

Table 7.17 Fredrickson classification of familial dyslipoproteinemias (Sources: [1352, 1354]; ↑ increase, ↓ decrease, Apo apolipoprotein, IDL intermediate-density lipoprotein, LDL low-density lipoprotein, LPL lipoprotein lipase, VLDL very-low-density lipoprotein) Type 1 1A 1B 1C 2A 2B

3 4

5

Defect Disease Familial chylomicronemia or lipase-D deficiency LPL deficiency (familial hyperchylomicronemia) ApoC2 deficiency (C2 anapolipoproteinemia) LPL inhibition (familial chylomicronemia) LDLR deficiency (familial hypercholesterolemia) ↓ LDLR, ↑ ApoB (familial combined hyperlipidemia) Defective ApoE2 synthesis (familial dysbetalipoproteinemia) ↑ VLDL production ↓ VLDL clearance (familial hypertriglyceridemia) ↑ VLDL production ↓ lipase H (familial mixed hyperlipidemia)

Lipoprotein type (elevated level)

Lipid type (elevated level)

Chylomicron

Triglycerides

Chylomicron

Triglycerides

Chylomicron

Triglycerides

LDL (β LP)

Cholesterol

LDL, VLDL (preβ LP)

Cholesterol, triglycerides

IDL VLDL

Triglycerides, cholesterol Triglycerides

VLDL, chylomicron

Triglycerides, cholesterol

Apolipoprotein-C2 is a lipase activator. Lipases remove chylomicrons and triglycerides from blood. Apolipoprotein-E, a component of VLDLs and chylomicrons, clears them from the blood and hence removes both cholesterol and triglycerides

• Secondary combined hyperlipoproteinemia, usually in the metabolic syndrome Type-3 hyperlipoproteinemia (a.k.a. floating and broad β disease, in addition to dysbetalipoproteinemia), is typified by augmented concentration of both chylomicrons and IDLs, i.e., TGs and cholesterol. Type-4 hyperlipoproteinemia (hyperprebetalipoproteinemia or familial hypertriglyceridemia) is marked by an elevated VLDL concentration and normal LDL concentration. Type-5 hyperlipoproteinemia (a.k.a. hyperprebetalipoproteinemia and chylomicronemia, mixed familial hyperlipoproteinemia, and mixed hyperlipidemia) is characterized by an elevated concentration of VLDL and chylomicrons in addition to glucose intolerance and hyperuricemia. Unclassified familial extremely rare forms include hyperalphalipoproteinemia and polygenic hypercholesterolemia.

7.4 Genetic Determinants of Dyslipidemias

645

Familial hypercholesterolemia is a genetic disorder characterized by a high plasmatic concentration of LDL–cholesterol (LDLCS ). Homozygotes, but not heterozygotes, develop early atherosclerosis. This autosomal codominant monogenic condition is caused by mutations in the genes encoding the LDL receptor (Ldlr [e.g., W23X, W66G, and W556S mutations]), apolipoprotein-B100 (APOB [e.g., R3500Q mutation]), or Pcsk9, leading to a diminished LDLCS clearance. Mutations in several genes affect HDL metabolism, such as those that encode the ABCa1 carrier,150 lecithin–cholesterol acyltransferase (LCAT), ApoA1,151 and lipoprotein lipase (Vols. 4, Chap. 2. Signaling Lipids and 11, Chaps. 4. Lipids and 5. Lipoproteins). The glycoprotein LCAT performs two types of activity. Using its phospholipaseA2 activity, LCAT can hydrolyze fatty acid from phosphatidylcholine. With its acyltransferase activity, it can transfer the fatty acid to free cholesterol and hence form cholesterol ester. It rapidly converts discoidal HDLs into spherical HDLs. Discoidal HDLs consist of 100 to 200 lipids wrapped by two molecules of apolipoprotein-A1. Lipoprotein lipase, or lipase-D, hydrolyzes TGs in circulating lipoproteins (e.g., chylomicrons, VLDLs, and LDLs) into two free fatty acids and a monoacylglycerol molecule. It processes not only triacylglycerols but also diacylglycerols. It is attached to the luminal surface of capillary endothelia by heparan sulfate proteoglycans, especially in the adipose tissue (AT), heart, and skeletal muscles. Apolipoprotein-C2 acts as a coactivator of LPL on the luminal surface of vascular endothelia.

150 ATP-binding

cassette superclass member-A1 (a.k.a. cholesterol efflux regulatory protein [CERP]) is a cAMP-dependent and sulfonylurea-sensitive anion transporter. This traffic ATPase binds ATP in the presence of Mg2+ ions. This cholesterol efflux pump is responsible for cellular ApoA1-mediated cholesterol export. Oxysterol receptors LXRs (NR1h2–NR1h3) and the bile acid receptors FXRs (NR1h4–NR1h5) heterodimerize with their RXR partners (NR2b1–NR2b3). The LXR–RXR dimer stimulate formation of the reverse cholesterol shuttles ABCa1 and ApoE [1356]. The FXR–RXR dimer represses synthesis of the rate-limiting enzyme of bile acid synthesis CyP7a1 (cholesterol 7α-hydroxylase). In fact, LXRs, which enhance efflux through cholesterol acceptors, promote the synthesis of ABCa1, ABCg1, ABCg4, ABCg5, and ABCg8. Whereas ABCg5 and ABCg8 limit the intestinal absorption of sterols and support the excretion of sterols from the liver into bile, ABCa1 facilitates the efflux of phospholipids and cholesterol from cells and represses cholesterol absorption in the intestine. Apolipoproteins ApoE, ApoC1, ApoC2, ApoC4, and ApoD serve as acceptors in ABCa1-mediated cholesterol efflux [1356]. In addition, LXRs elicit production of the lipoprotein remodelers, LPL, CETP, and phospholipid transfer protein (PLTP). MicroRNAs miR33a and miR33b embedded within introns of SREBP2 and SREBP1, respectively, repress ABCa1 synthesis [194]. 151 Apolipoprotein-A1 participates in the reverse transport of cholesterol from organs to the liver for excretion, as it promotes cholesterol efflux from cells and acts as a cofactor for the LCAT [108].

646

7 Genetic Risk Factors

Severe hypertriglyceridemia can result from rare cases of homozygous mutations in the APOA5, APOC2, Lpl, Lmf1,152 Gpihbp1,153 and Gpd1 genes [738].154 On the other hand, heterozygous loss-of-function APOC3155 mutations reduce feeding levels of TGs. Genes responsible for inherited lipid disorders comprise DNA variants with usually deleterious loss-of-function and occasionally gain-of-function mutations that produce aberrant plasmatic lipid concentrations [1379]. Common DNA variants with small or moderate effects also modulate plasmatic lipid concentrations in a polygenic fashion. Investigations into genes associated with lipid levels discovered 175 genetic loci linked to LDLCS , HDLCS (a noncausal atherosclerosis marker), TGs, or total cholesterol. Genetic determinants of lipoproteinemias involve contributions from multiple DNA sequence variants [1354]. Many genes with common variants also experience rare mutations causing dyslipidemias (Sect. 5.1).

152 LMF1:

lipase maturation factor-1. It is involved in the maturation of specific proteins in the ER and transfer, especially lipoprotein lipase through the secretory pathway. Each LMF1 molecule chaperones at least 50 LPL molecules [108]. 153 GPIHBP1: GPI-anchored HDL-binding protein-1. 154 GPD1: soluble cytosolic glycerol 3-phosphate dehydrogenase. It catalyzes the reversible redox reaction of dihydroxyacetone phosphate and reduces nicotine adenine dinucleotide to glycerol 3phosphate and NAD+ [194]. It is involved in the transport of reducing equivalents from the cytosol to mitochondria in cooperation with the mitochondrial GPD2 isoform. It is also important for TG synthesis. 155 Apolipoprotein-C3 is a component of TG-rich VLDLs and HDLs. Inside the cell, it promotes hepatic VLDL1 assembly and secretion [108]. In the extracellular space, it attenuates hydrolysis and clearance of TGRLs, as it inhibits LPL and hepatic uptake of TGRLs.

7.4 Genetic Determinants of Dyslipidemias

647

Genomic sequence variants can be mapped to predisposition to a disease or a trait of interest.156 Blood concentrations of lipoproteins and lipids are heritable risk factors for CVDs. Susceptibility alleles at 18 loci are reproducibly associated with concentrations of LDLCS , HDLCS , and TGs [1358]. Among chromosomal regions linked to these agents, two are associated with LDLCS (1p13 near Celsr2,157 PSRC1,158 and SORT1159 and 19p13 near Cilp2160 and PBX4);161 one with HDLCS (1q42 in GALNT2);162 and five with TGs (7q11 near TBL2163 and Mlxipl,164 8q24 near TRIB1,165 1q42 in GALNT2, 19p13 near CILP2 and PBX4, and 1p31 near ANGPTL3).166 Plasma lipid concentration with a high probability of artery disease is strongly associated with variants in various genetic loci of lipid metabolism localized to:

156 Like individual-level data,

aggregate SNP data do not conceal the identity of individuals, whose SNP profiles constitute the genotype data pool and must then be efficiently protected, but keeping accessibility [1357]. 157 CELSR2: Cadherin EGF LAG seven-pass G-type receptor-2. 158 Proline- and serine-rich coiled-coil protein-1 is implicated in the cell division cycle. In particular, it recruits KIF2a and ankyrin repeat domain-containing protein AnkRD53 to the mitotic spindle [108]. 159 SORT1: sortilin-1. It is a sorting receptor in the Golgi body (GB) and a clearance receptor on the cell surface. It is involved in protein transfer from the GB to lysosomes and endosomes [108]. It can promote mineralization of the extracellular matrix during osteogenic differentiation, as it scavenges extracellular LPL. It may be required in adipocytes for the formation of SLC2a4+ (GluT4+) storage vesicles. 160 CILP2: cartilage intermediate layer protein-2. 161 PBx4: preB-cell leukemia homeobox gene product-4. 162 GalNT2: N acetylgalactosaminyltransferase-2. It catalyzes the initial reaction in O-linked oligosaccharide synthesis and transfer of an N acetyl D galactosamine residue to a serine or threonine residue of the target protein [108]. 163 Tβ L2: transducin-β-like protein-2. 164 MLX-interacting protein-like protein (MLXIPL), also called bHLHd14 and carbohydrateresponsive element-binding protein (CHREBP), is a transcriptional repressor. 165 Tribbles homolog Trib1 interacts with MAP2K, hence regulating activation of MAPKs. 166 Angiopoietin-related protein-3 acts partly as a hepatokine involved in lipid and glucose metabolism regulation [108]. It suppresses plasmatic TG clearance, as it inhibits LPL via PCSK3 and PCSK6. It can also inhibit endothelial lipase (LipG), raising the plasmatic concentration of HDLCS and phospholipids. In adipocytes, it can activate lipolysis, releasing free fatty acids (FFAs) and glycerol. It is involved in angiogenesis. In endotheliocytes, it activates FAK, MAPK, and PKB, enabling cell migration.

648

7 Genetic Risk Factors

1. Genes, such as ABCA1, APOB, Celsr2, Cetp,167 Dock7,168 GALNT2, GCKR,169 Hmgcr,170 Ldlr, LIPC,171 LIPG,172 Lpl, Mlxipl, NCAN,173 Pcsk9, and TRIB1) 2. Gene clusters (e.g., MVK–MMAB,174 APOA5–APOA4–APOC3–APOA1, in addition to APOE–APOC1–APOC4–APOC2 clusters)175 3. Genetic sites near: (a) The MVK–MMAB cluster and GALNT2 gene for variants attached to HDLCS , in addition to the above-mentioned genes and gene clusters (b) The SORT1 gene for variants ascribed to LDLCS (c) The TRIB1, Mlxipl, and ANGPTL3 genes for variants related to TGs (d) The NCAN gene for variants linked to both TGs and LDLCS [1359]

167 Inhibitors of cholesteryl ester transfer protein (CETP) increase plasmatic HDLCS

concentration, but do not reduce the risk for atherosclerosis [1379]. Single nucleotide polymorphisms in or near the Cetp gene also modify plasmatic LDLCS concentration in the inverse direction to that of [HDLCS ]. 168 DOCK7: dedicator of cytokinesis-7. 169 GcKR: glucokinase regulator. 170 HMGCR: hydroxy methylglutaryl–coenzyme-A reductase. 171 LipC: hepatic lipase-C. 172 The SNP Asn396Ser in the LIPG gene that encodes endothelial lipase, which processes HDLs, increases plasmatic HDLCS concentration. 173 Ncan: neurocan. 174 MvK: mevalonate kinase (i.e., ATP–mevalonate 5-phosphotransferase); MMAb: methylmalonic aciduria type-B protein (i.e., cob(I )alamin adenosyltransferase). MvK represents a regulatory node in cholesterol synthesis. MMAb catalyzes the final step in the synthesis of the cofactor adenosylcobalamin, a vitamin B12-containing coenzyme for methylmalonyl-CoA mutase [194]. 175 Other genes, such as APOE, Hmgcr, and APO , influence LDLCS concentration. Single A nucleotide polymorphisms in at least six genes that control plasmatic TG levels (APOA4, APOA5, APOC3, ANGPTL4, LPL, and TRIB1), which encode LPL or its regulators, are associated with atherosclerosis.

7.4 Genetic Determinants of Dyslipidemias

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Among 22 loci associated with dyslipidemia in different European populations, 6 additional identified loci include ABCG5,176 TMEM57,177 Ctcf–Prmt8,178 DNAH11,179 Fads2–Fads3,180 and MADD–FOLH1 [1360].181 • With LDLCS near the ABCG8,182 MAFB,183 Hnf1a,184 in addition to Timd4 genes185

176 ATP-binding

cassette transporter, ABCg5, operates in the import and export of dietary cholesterol in and from enterocytes and sterol excretion into bile [108]. 177 Transmembrane protein-57 is also termed macoilin. This brain-specific protein is primarily linked to postmigratory neurons [194]. Its expression progressively decays during brain maturation. 178 The transcriptional repressor CCCTC-binding zinc finger factor (CTCF) binds to chromatin insulators and prevents interaction between promoter and nearby enhancers and silencers. In particular, it connects to the promoters of the BAG1, MYC, PIM1, and Plk gene [108]. It regulates the APOA1–APOC3–APOA4–APOA5 gene cluster. It also controls MHC class-I I gene expression. Membrane-associated protein Arg N methyltransferase PRMT8 can both catalyze the formation of ωN monomethylarginine and asymmetrical dimethylarginine. It can also mono- and dimethylate Ewing sarcoma gene product (EWS) [108]. 179 Axonemal dynein heavy chain-11 produces force toward the minus-ends of microtubules in respiratory cilia [108]. 180 Fatty acid desaturases FADS2 and FADS3 are implicated in lipid metabolism. They introduce double bonds between given carbons of the fatty acyl chain [194]. They catalyze synthesis of highly unsaturated fatty acids from precursors such as the polyunsaturated fatty acids linoleic (LA) and α-linolenic acids (α LA), desaturating LA and α LA into γ-linoleic acid (γ LA) and stearidonic acid, respectively [108]. 181 MAPK-activating death domain-containing protein (MADD) participates in regulating cell proliferation, survival, and death via alternative mRNA splicing [108]. It is also called Rab3 GDP/GTP-exchange factor (Rab3GEP). It activates Rab3a, Rab3c, and Rab3d, and it links to TNFRSF1a. Folate hydrolase FolH1, also termed glutamate carboxypeptidase-2, has a type-I V dipeptidyl peptidase activity. In the intestine, it enables folate uptake [108]. In the brain, it modulates excitatory neurotransmission, as it processes the neuropeptide N acetylaspartylglutamate (NAAG) to release glutamate. 182 The transporter ABCg8 carries dietary cholesterol in and out of enterocytes and permits sterol excretion into bile [108]. 183 The transcription factor MAFb (v-maf musculoaponeurotic fibrosarcoma proto-oncogene homolog-B; the oncogene vMAF being identified in the avian transforming retrovirus isolated from chicken musculoaponeurotic fibrosarcoma) is a transcriptional activator and repressor. It represses ETS1-mediated transcription of erythroid-specific genes in myeloid cells. It is required for differentiation of monocytes, macrophages, osteoclasts, podocytes, and pancreatic β cells [108]. It activates promoters of the genes encoding insulin and glucagon. 184 Hepatocyte nuclear factor-1α, or transcription factor TcF1, activates tissue-specific expression of multiple genes, especially in pancreatic islets and the liver [108]. 185 TIMD4: T-cell immunoglobulin and mucin domain-containing protein. This phosphatidylserine receptor enhances engulfment of apoptotic cells [108]. It is also involved in regulating T-cell proliferation and lymphotoxin (TNFRSF1b and TNFRSF3) signaling.

650

7 Genetic Risk Factors

• With HDLCS near the ANGPTL4,186 HNF4A,187 lecithin–cholesterol acyltransferase, PLTP,188 and TTC39B genes189 and the FADS1–FADS2–FADS3 cluster • With TGs near the SLC35G5190 and PLTP genes and the FADS1–FADS2– FADS3 cluster [1361] Mono- and polygenic dyslipoproteinemias (Tables 7.18, 7.19, 7.20, and 7.21) can be associated with: 1. Altered endocytosis and abnormal degradation caused by mutations in the gene encoding the low-density lipoprotein receptor (LDLR), ATP-binding cassette transporters, Niemann–Pick-like proteins, and the convertase PCSK9, among others 2. Defective lipid transport, attachment, and detachment to lipoproteins due to abnormal apolipoproteins, lecithin–cholesterol acyltransferase, CETP, lipases, and so on Candidate genes are tested by genotyping SNPs in large groups of patients to detect susceptibility loci with a significant association with the investigated disease. Homozygous gene deletion of the APOE191 or Ldlr gene causes severe hypercholesterolemia and atherosclerosis. The risk of inherited premature atherosclerosis is linked either to a classical Mendelian pattern or complicated context, with

186 Angiopoietin-related

protein-4 is produced upon hypoxia in endotheliocytes [108]. Under hypoxic conditions, its unprocessed form accumulates in the subendothelial matrix, where it limits the formation of actin stress fibers and focal contacts in endotheliocytes. It participates in the regulation of angiogenesis and tumorigenesis. It prevents proliferation, migration, and tubulogenesis of endotheliocytes and reduces vascular leakage. It is also involved in regulating glucose and lipid metabolism in addition to insulin sensitivity. 187 Hepatocyte nuclear factor-4α (a.k.a. NR2a1 and TcF14) controls synthesis of serpin-A1 (α1antitrypsin), ApoC3, and transthyretin, in addition to HNF1α (or TcF1) [108]. 188 Phospholipid transfer protein carries diacylglycerol, phosphatidic acid, sphingomyelin, phosphatidylcholine, phosphatidylglycerol, cerebroside, and phosphatidyl ethanolamine [108]. It enables transfer of excess surface lipids from TGRLs to HDLs, hence facilitating the formation of smaller lipoprotein remnants, contributing to LDL formation, and assisting in the HDL maturation. It also intervenes in the uptake of cholesterol from cells for its transport to the liver for degradation and excretion. 189 Tetratricopeptide repeat-containing protein TTC39b reduces plasmatic HDLCS concentration [194]. 190 Solute carrier family member SLC35g5 is also named acyl–malonyl-condensing enzyme 1-like protein-2 (AMAC1L2). 191 Apolipoprotein-E mediates internalization and catabolism of lipoproteins. It connects to the LDLR (ApoB and ApoE receptor) and specific ApoE receptor for chylomicron remnants in the liver [108]. Three major isoforms of human apolipoprotein-E exist (ApoE2–ApoE4). A single locus with three common alleles (APOE2–APOE4 or 2– 4) is responsible for the ApoE polymorphism [194]. Class-β phenotypes (β2–β4) represent homozygosity for one of the alleles and class-α phenotypes represent heterozygosity for two different alleles. ApoE subclass-β4, which results from homozygosity for the 4 allele, is linked to type-I I I hyperlipoproteinemia [194].

7.4 Genetic Determinants of Dyslipidemias

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Table 7.18 Genetic (primary) dyslipidemias Disorder Familial hypercholesterolemia (autosomal dominant disorder) type-2 hyperlipoproteinemia Familial apolipoprotein-B100 deficiency hypobetalipoproteinemia GOF Pcsk9 mutations Autosomal recessive hypercholesterolemia Type-2 familial HDL deficiency (familial hypoalphalipoproteinemia) Tangier disease (severe form) Sitosterolemia (autosomal recessive pattern) Autosomal dominant coronary artery disease

Mechanism Effect Mutations in the Ldlr gene Decreased LDL clearance ↑ blood [LDLCS ] Mutations in the APOB gene ↓ LDL–LDLR connection ↓ LDL catabolism (clearance) Increased LDLR degradation Mutations in the Ldlrap1 gene Mutations in the ABCA1 or APOA1 gene Prevention of export of cholesterol from cells Mutations in the ABCA1 gene Mutations in the ABCG5 or ABCG8 gene Mutations in the LRP6 gene

(Part 1) Hypercholesterolemias (Sources: [194, 1362]; ↑ increase, ↓ decrease, ABC ATP-binding cassette transporter, Apo apolipoprotein, CS cholesterol, LDL low-density lipoprotein, LDLR LDL receptor, LDLRAP LDLR adaptor protein, LRP LDLR-related protein, PCSK proprotein convertase subtilisin/kexin). The autosomal dominant pattern means that a parent with familial hypercholesterolemia has a 50% likelihood of passing a gene mutation to each child, regardless of sex. Two main types of familial hypercholesterolemia are the homozygous and heterozygous forms

Table 7.19 Genetic (primary) dyslipidemias Disorder Familial hypertriglyceridemia

Familial combined hyperlipidemia Hepatic lipase deficiency

Mechanism Effect Multiple defects (APOA5/C2, lipase-D, Lmf1, Gpihbp1, Gpd1) ↑ VLDL synthesis, ↓ VLDL clearance Multiple defects ↑ [ApoB], ↓ [LDLR] LIPC gene mutations ↑ [CS], [TG], [HDL], ↓ [LDL], large LDLs

(Part 2) Pure and mixed hypertriglyceridemias (Sources: [194, 1362]; ↑ increase, ↓ decrease). Hepatic lipase synthesized from the LIPC gene in hepatocytes and released into blood convert VLDLs and IDLs to LDLs and assists in transporting HDLs to the liver

652

7 Genetic Risk Factors

Table 7.20 Genetic (primary) dyslipidemias Disorder Type-5 hyperlipoproteinemia (late-onset mixed hyperlipemia) Endothelial LPL deficiency

Familial ApoC2 deficiency Familial chylomicronemia

Mechanism Effect Mutations in the APOA5 gene LPL impairment Inactivated LPL (lipase-D) Decreased chylomicron clearance ↑ [CM], [VLDL] ↓ [HDL], [LDL] Impaired LPL activity Missense mutations in the Gpihbp1 gene GPIHBP1 oligo- and multimerization

(Part 3) Hyperchylomicronemias (Sources: [194, 1362]; ↑ increase, ↓ decrease). Primary hyperchylomicronemia is linked to a marked hypertriglyceridemia. It includes familial LPL (lipase-D) deficiency, familial apolipoprotein ApoC2 deficiency, primary type-V hyperlipoproteinemia, idiopathic hyperchylomicronemia, which is caused by an LPL inhibitor or LPL auto-antibody, and mutations in the gene encoding glycosylphosphatidylinositol-anchored high-density lipoproteinbinding protein, GPIHBP1, or lipase maturation factor LMF1 Table 7.21 Genetic (primary) dyslipidemias Disorder Primary hypoalphalipoproteinemia Familial dysbetalipoproteinemia (autosomal dominant or recessive) type-3 dyslipoproteinemia Familial ApoA/ApoC3 deficiency Familial LCAT deficiency (Norum disease)

Mechanism Effect Possibly related to ApoA1/A4/C3 Defective ApoE2 synthesis ↑ [CS] and [TG] ↓ CM and VLDL clearance ↓ TG, ↑ HDL LCAT gene mutations Inhibition of LCAT synthesis or impaired LCAT activity (i.e., Cholesterol–lipoprotein attachment

(Part 4) Dysalpha- and dysbetalipoproteinemias (Sources: [194, 1362]; ↑ increase, ↓ decrease, CM chylomicron)

inheritance linked to multiple causal genes possibly coupled with a dependence on environmental factors (e.g., tobacco). Familial hypercholesterolemia, a classical Mendelian autosomal dominant, incompletely dominant, or codominant disorder with hyper-LDL-emia and premature atherosclerosis, is caused by mutations in the Ldlr gene. Five types of Ldlr gene mutations are identified: In type-1 Ldlr gene mutations (elimination of the Ldlr gene promoter and frameshift, nonsense, and splicing mutations), LDLR synthesis fails. In type-2 mutations, the most common, intracellular LDLR transport is defective; LDLR does not move at a normal rate between the ER and GB.

7.4 Genetic Determinants of Dyslipidemias

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In type-3 mutations, LDLR is delivered to the plasma membrane but cannot bind LDL. In type-4 mutations, the rarest ones, the LDLR–LDL complex cannot be internalized. In type-5 mutations, the LDLR–LDL complex is endocytosed, but LDL cannot be released from endosomes, and LDLR cannot recycle back to the plasma membrane. Homozygotes are more severely affected than heterozygotes. Moreover, among homozygotes those with receptor-defective alleles and residual receptor activity are less severely affected than patients with receptor-inactivating mutations. Phenotypical variations exist not only among homozygotes, but also among heterozygotes. Additional forms of hypercholesterolemia exist with autosomal dominant or recessive patterns. Two defective alleles for the gene that encodes the LDLR adaptor protein, LDLRAP1,192 are needed to acquire autosomal recessive hypercholesterolemia [1363]. Autosomal dominant premature CoAD is linked to a mutation in the Lrp6 gene.193 A form of autosomal dominant hypercholesterolemia is associated with gainof-function mutations in the Pcsk9 gene. Proprotein convertase subtilisin/kexin PCSK9 catalyzes its own cleavage. Afterward, the LDLR antagonist PCSK9 is secreted and binds to LDLRs, which are then degraded. Loss-of-function mutations in the Pcsk9 gene raises plasmalemmal LDLR and reduces plasmatic LDLCS concentrations. The agents PCSK9 in addition to NPC1L1 and HMGCR regulate LDLs, but not other liproteins and lipids [1364]. Mutations in the Pcsk9 and APOB genes are identified in familial hypercholesterolemia patients in whom mutations of the Ldlr gene were ruled out. Tangier disease is characterized by mutations in the ABCA1 gene. The dimeric ATP-binding cassette carrier ABCa1 promotes cholesterol efflux from cells to nascent HDLs, i.e., reverse cholesterol transport. Rare variants in this gene are linked to familial hypoα lipoproteinemia and Tangier disease. Generation of discoidal HDLs is mediated by ABCa1, which transfers cellular cholesterol and phospholipids to apolipoprotein-A1 and then dissociates into monomers [1365].

192 A.k.a.

autosomal recessive hypercholesterolemia protein (ARH, ARH1, and ARH2). This clathrin-associated sorting protein (CLASP) allows efficient LDLR endocytosis in polarized cells such as hepatocytes and lymphocytes, but not in nonpolarized cells such as fibroblasts [108]. 193 The plasmalemmal coreceptor LDLR-related protein LRP6 is a component of Wnt signaling that enables the formation of signalsomes.

654

7 Genetic Risk Factors

Monogenic codominant hypercholesterolemias represent a genetically heterogeneous group of disorders that include: 1. Familial hypercholesterolemias due to various types of mutations in the Ldlr gene 2. Familial ApoB100 deficiency caused by mutations in the APOB gene impairing LDLApoB100 binding to LDLR 3. Type-3 autosomal dominant hypercholesterolemia engendered by mutations in the Pcsk9 gene Monogenic codominant hypercholesterolemias are linked to an increased predisposition to premature CoAD, despite an interindividual variability among subjects carrying the same mutation of one of the candidate genes due to environmental, metabolic, and other genetic factors. Combined codominant monogenic hypercholesterolemias and hypoalphalipoproteinemias can result from mutations in the Ldlr and Lcat genes. The Ldlr mutation is associated with high concentrations of LDLCS and ApoB and the Lcat mutation with low concentrations of HDLCS and ApoA1 [1366]. In carriers of Lcat mutations, LCAT activity and hence the cholesterol esterification rate lower. The LCAT mutation is most often not detected in patients with primary hypoalphalipoproteinemia. Mutations in the Pcsk9 gene cause a type of autosomal dominantly inherited hypercholesterolemia (HCHOLA3) [1367]. The PCSK9 enzyme of the proteinase-K category (secretory subtilase family) regulates the plasmatic cholesterol concentration. It tethers to LDLR and causes its degradation. It thus alters metabolism of LDLCS , which is removed from the blood via LDLR on the surface of hepatocytes, as it reduces hepatic LDL uptake. It also interacts with other receptors, such as the VLDLR, LRP1 (ApoER1), and LRP8 (ApoER2) proteins [1368]. In humans, the gain-of-function substitution of Asp374 by Tyr (D374Y) raises (≥10-fold) the affinity of PCSK9 for LDLR, partly simulating the absence of LDLR (see LDLR−/− mice) [1368]. In mice, transfer of the PCSK9DY gene to the liver provokes long-term hyperlipidemia with hyper-LDL-emia. Estrogen deficiency is associated with increased LDLCS . The gene encoding Gprotein-coupled estrogen receptor (GPER) has a common hypofunctional missense variant P16L that is linked to a significant increase in LDLCS [1369]. The activated GPER receptor upregulates LDLR expression, at least partly via PCSK9. Glycosylphosphatidylinositol (GPI)-anchored HDL-binding protein GPIHBP1 on capillary endotheliocytes binds to LPL, a TG regulator, in the subendothelial space and shuttles it to the capillary lumen. Lipoprotein lipase hydrolyzes TGs in chylomicrons and VLDLs into two FFAs and a monoacylglycerol molecule, producing chylomicron remnants and IDLs, respectively. It is attached to the luminal surface of the capillary endothelium by heparan sulfate proteoglycans and/or glycosyl phosphatidylinositol, especially in the heart, skeletal muscles, and AT, in addition to the lung, brain, kidney, and lactating mammary gland, along with macrophages. It regulates the supply of fatty acids to cells for storage or oxidation. Indeed, it interacts with and anchors lipoproteins to the vascular wall and tethers to lipoprotein receptors,

7.4 Genetic Determinants of Dyslipidemias

655

thereby facilitating lipoprotein uptake [1370]. It promotes lipid exchange between lipoproteins. It can also mediate the selective uptake of lipoprotein-associated lipids and lipophilic vitamins without the concomitant uptake of lipoproteins. Only GPIHBP1 monomers can bind to LPL. Missense mutations in the Gpihbp1 gene cause di- and multimerization of GPIHBP1 via disulfide bridges and thus familial chylomicronemia [1371]. Among GPIHBP1 mutants, GPIHBP1W109S , which cannot link to LPL, has a reduced tendency toward di- and multimerizing. In the rare autosomal recessive familial hyperchylomicronemia, inactivating mutations in the Lpl gene or, less frequently, loss-of-function mutations in genes encoding LPL regulators are characterized by severe hypertriglyceridemia (10– 100 ×normal value) [1372]. Lipoprotein lipase requires a specific cofactor, ApoC2, to be fully active [1370]. Apolipoprotein-A5194 activates intravascular TG hydrolysis by proteoglycanbound LPL. A truncation mutation in the APOA5 gene causes familial hyperchylomicronemia. The genetic LPL deficiency causes type-1 familial hyperlipoproteinemia. On the one hand, the LPL variant engendered by the gain-of-function S447X SNP confers an antiatherogenic lipid profile characterized by a low TG concentration [1373]. On the other, several LPL variants resulting from loss-of-function mutations are associated with hypertriglyceridemia and hence atherosclerotic risk. In the absence of accumulation of TGRLs, an LPL-independent rescue pathway must exist. Apolipoprotein-C3, a pleiotropic regulator of the TGRL metabolism synthesized mainly in the liver and, to a lesser extent, in the intestine, links to ApoB+ lipoproteins (chylomicrons, VLDLs, and HDLs). It regulates the plasmatic TG concentration, as it inhibits lipoprotein lipase via ApoC2 and limits clearance of TGs from plasma, hence increasing the concentration of plasma TGs via an LPLindependent mechanism [1372]. In addition, ApoC3 inhibits hepatic lipase (LipC), promotes intrahepatic VLDL assembly and secretion, and precludes hepatic clearance of TGRL remnants. It can also raise endotheliocyte apoptosis and promote inflammation. It can hamper uptake of TGRLs via lipoprotein receptors. Blockage of ApoC3 synthesis in patients with LPL deficiency lowers concentrations of TGs, chylomicrons, and VLDLs [1372]. In particular, it reduces the concentration of ApoB48 in addition to of ApoE in plasma and chylomicrons. Loss-of-function mutations in the APOC3 gene are associated with hypotriglyceridemia and a reduced CoAD risk, but not hyperhdlemia.

194 Apolipoprotein-A5

is a minor apolipoprotein mainly linked to HDL and, to a lesser extent, VLDL and chylomicrons. It is a potent stimulator of LPL and a weak activator of lecithin– cholesterol acyltransferase [108]. It inhibits hepatic VLDLTG production (but not VLDLApoB formation).

656

7 Genetic Risk Factors

In endotheliocytes, the most common form of apolipoprotein-E, ApoE3, activates ApoER2 (or LRP8) and subsequently NOS3 and cell migration. It also attenuates monocyte–endotheliocyte adhesion, favors endothelial repair, and prevents intimal hyperplasia and thrombosis. The LRP8 receptor abounds in membrane rafts and caveolae. On the other hand, ApoE4 stimulates neither NOS3 nor cell migration. Genetic variants of the circulating ApoE or its receptor LRP8 confer a greater risk for premature atherosclerosis and myocardial infarction [1374]. The Lrp8 variant (LOF SNP R952Q) is not functional. The ApoE4 variant precludes ApoE3–LRP8 action. Among genetic loci associated with lipoprotein levels and atherosclerosis, a nonprotein-coding SNP at the chromosomal 1p13 locus (SORT1; rs12740374) creates a binding site for the C/EBPα transcription factor and alters the hepatic expression of the SORT1 gene that encodes sortilin-1 [1375]. Sortilin acts as a multiligand receptor. In particular, it can bind ApoA5, ApoB100, LDL, and LPL (lipase-D), at least in hepatocytes [1376]. In human hepatocytes, sortilin participates in the degradation of nascent VLDLs [1375]. Usually, membrane sortilin is cleaved in the GB by adamlysin-10 and its cytosolic domain acts as a chaperone for the secretion of ApoB and VLDLs [1376]. When it is overproduced, the processing capacity of the sheddase adam10 is exceeded and Sort1 endowed with its cytosolic tail conveys ApoB and VLDL bound to it from the GB to lysosomes, where these cargos are degraded. Moreover, sortilin allows LDL uptake not only in hepatocytes, leading to lysosomal degradation, but also in macrophages in the vessel wall, causing cholesterol accumulation and foam cell formation [1377]. Sortilin can thus lower the secretion of ApoB and VLDLs and augment LDL uptake in hepatocytes and macrophages, independently of LDLR and in addition to other mediators of modified LDL uptake by these cells (e.g., scavenger receptors ScaRa and ScaRb3) [1376]. In macrophages, LDL uptake decreases the LDLR expression, but increases the Sort1 level.

7.5 Atherosclerosis Genetic approaches are aimed at identifying genes in addition to molecular interactions linked to atherosclerosis, an example of multifactorial cardiovascular maladies with a prominent vascular inflammation that involves numerous genetic loci [1378]. Multiple monogenic disorders are responsible for inherited forms of premature atherosclerosis, among which many primarily affect plasmatic concentrations of lipids and lipoproteins (Tables 7.22, 7.23, and 7.24). A dose-dependent log-linear correlation exists between the magnitude of LDLCS concentration (>0.5–1.0 mmol/l [20–40 mg/dl]) to which the vasculature is exposed and the atherosclerosis risk; this effect augments with increasing duration of LDLCS exposure [225]. Rare gene mutations that reduce LDLR function elevate LDLCS concentration and atherosclerosis risk, whereas rare variants that lower LDLCS concentration diminish the risk for atherosclerosis.

7.5 Atherosclerosis

657

Table 7.22 Inherited premature atherosclerosis (Part 1) Metabolism of lipids and lipoproteins (Sect. 7.4; Source: [1363]; CoAD coronary artery disease) Disorder Autosomal dominant hypercholesterolemia Autosomal recessive hypercholesterolemia Dysbetalipoproteinemia Sitosterolemia Autosomal dominant CoAD

Mechanism Effect Mutations in the APOB, Ldlr, and Pcsk9 genes Mutations in the Ldlrap1 gene Mutations in the APOE gene Mutations in the ABCG5 or ABCG8 gene Mutations in the Lrp6 gene

In sitosterolemia, phytosterols from vegetable oils and plant-based foods accumulate in blood and organs. Sitosterolemia patients usually have normal to moderately elevated plasmatic concentrations of total sterol, but very high concentrations of plant sterols (sitosterol, campesterol, stigmasterol, avenasterol) and 5α-saturated stanols Table 7.23 Inherited premature atherosclerosis (Part 2; Sources: [194, 1362, 1363]; ND not determined, ACTA2 smooth muscle actin-α-2, CBS cystathionine β-synthase, MMADHC mitochondrial methylmalonic aciduria and homocystinuria type-D protein, MTHFR methylenetetrahydrofolate reductase, MTR 5-methyltetrahydrofolate–homocysteine methyltransferase, MTRR 5-methyltetrahydrofolate–homocysteine methyltransferase reductase, RECQL2 [RECQ3] RecQlike protein-2 [a.k.a. RecQ protein DNA helicase-3 and Werner syndrome ATP-dependent exonuclease (WRN)], TGFBR TGFβ receptor) Disorder Familial thoracic aortic aneurysm and dissection (TAAD) Homocystinuria Familial anti-phospholipid syndrome Hutchinson–Gilford progeria syndrome Werner syndrome Williams–Beuren syndrome, supravalvular aortic stenosis syndrome, infantile hypercalcemia

Mechanism Effect Mutations in the ACTA2 or TGFBR2 gene Annulo-aortic ectasia Mutations in the CBS, MMADHC, Mthfr, MTR, and MTRR genes ND Thromboses, hemolysis Mutations in the LMNA gene (lamin-A) Unstable nuclear envelope, rapid aging, arteriosclerosis Mutations in the RECQL2 (WRN) gene Rapid aging, arteriosclerosis Chromosomal 7q11.23 region deletion (26–28 genes) Arterial stenoses, hypertension, hypercalcemia

Familial hypercholesterolemia (FHCS) causes premature atherosclerosis. It is characterized by marked elevation in plasmatic LDLCS concentration (4.5– 12 mmol/l). This autosomal codominant disorder is usually caused by LOF mutations in the Ldlr gene and, less commonly, from LOF mutations in the

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7 Genetic Risk Factors

Table 7.24 Inherited premature atherosclerosis (Part 3; Source: [194, 1362, 1363]; ND not determined) Disorder Type-1 familial partial lipodystrophy (FPLD1) Type-2 familial partial lipodystrophy (FPLD2) Fibromuscular dysplasia (FMD) Pseudoxanthoma elasticum (PXE) Familial xanthomatosis acid lipase deficiency Wolman disease cholesteryl ester storage disease (autosomal recessive pattern) Cerebrotendinous xanthomatosis

Mechanism Effect ND Hypertension, diabetes Altered 1q21–1q22 locus Mutations in the LMNA gene Insulin resistance ND Focal arteriopathy (commonly renal and carotid arteries) Mutations in the ABCC6 gene Vascular calcifications, arterial stenoses Mutations in the LIPA gene Lysosomal lipase deficiency Lipid accumulation in the spleen, liver, bone marrow, lymph nodes, intestine, and adrenal glands Mutations in the CYP27A1 gene Plasmalemmal accumulation of cholestanol

The LIPA gene encodes lysosomal acid lipase, which processes cholesteryl esters and TGs. The CYP27A1 gene encodes hepatic mitochondrial sterol 27-hydroxylase, which catabolizes cholesterol to form bile acids). Its mutations impede cholesterol processing to chenodeoxycholic acid and another derivative, cholestanol, formed by hydroxysteroid dehydrogenase HSD3β2 accumulates in blood and cells

APOB gene, which lower binding of ApoB+ lipoproteins to LDLR, or GOF mutations in the Pcsk9 gene. Heterozygous FHCS (heFHCS) affects 1:200 to 1:300 people worldwide [225]. Homozygous FHCS (hoFHCS) is a much rarer anomaly (plasmatic LDLCS concentration >13 mmol/l). Some gene variants associated with high concentrations of HDLCS are linked to high risk for CVD [729]. Mutations in the Cetp gene leading to low activity of cholesteryl ester transfer protein can elevate HDLCS concentration. A LOF mutation in the SCARB1 gene, ScaRb1 being a major HDL receptor, engenders high HDLCS concentrations associated with a high CoAD risk, as do the ABCA1 and LIPC gene variants. Variants in or near the APOA gene and hence plasmatic LPA concentration are associated with atherosclerotic risk [1379]. Lipoprotein-A can serve as an independent and likely causal risk factor for cardiovascular disease. In classical homocystinuria mainly linked to homozygous or compound heterozygous, the patient inheriting two independent loss-of-function mutations in the same gene, mutations in the CBS gene that encodes cystathionine β-synthase

7.5 Atherosclerosis

659

inherited in an autosomal recessive manner or recessive inheritance of mutations in the Mthfr gene yield a defective sulfur metabolism and favor the appearance of thrombosis and, hence, myocardial infarction. Vascular complications of WBS syndrome195 typically comprise stenoses in large (e.g., characteristic supravalvular aortic stenosis [SVAS]) and mid-sized arteries [1363]. It is caused by heterozygous deletions of about 1.6 Mb of the chromosomal sub-band 7q11.23 between the WBS region and the Magi2 gene.196 Deletions arise by inter- or intrachromosomal crossover. The WBS deletion region contains the following genes [1380]: BAZ1B,197 BCL7B,198 CLDN3 and CLDN4,199 CLIP2,200 EIF4H,201 ELN,202 FKBP6,203 FZD9,204 GTF2I RD1,205 Limk1,206 Mlxipl,207 Rfc2,208 STX1A,209 and TBL2.210 Disruption of the ELN gene, which encodes elastin by translocation or partial deletion, is often associated with SVAS. Pseudoxanthoma elasticum is a disorder characterized by connective tissue calcification that affects the arterial media, among other organs. Gene expression changes in blood can reflect the interaction of genetic predisposition and disease progression in addition to the state of metabolism and immunity and environmental modifiers implicated in disease mechanisms, along with the eventual thrombosis.

195 A.k.a.

Williams syndrome, supravalvular aortic stenosis syndrome, infantile hypercalcemia, and chromosome 7q11.23 deletion syndrome. 196 Membrane-associated guanylate kinase-related protein with inverted domain organization, WW, and PDZ domain-containing protein MAGI2 may act as a scaffold at synapses, as it enables assembling neurotransmitter receptors and cell adhesion proteins [108]. It may also regulate activin-mediated signaling in neurons. It potentiates PTen-mediated PKB1 inactivation. It also plays a role in NGF-induced recruitment of RapGEF2 to late endosomes and neurite outgrowth. 197 BAZ1b: bromodomain adjacent to zinc finger domain protein-1B, also called WBS chromosomal region protein WBSCR9 or WBSCR10. 198 BCL7b: B-cell lymphoma protein-7B. 199 Cldn: claudin. 200 CLiP2: CAP–GLY domain-containing linker protein-2, also called WBS chromosomal region protein WBSCR3 or WBSCR4. 201 EIF4h: eukaryotic initiation factor-4H, or WBSCR1. 202 Eln: elastin. 203 FKBP6: FK506-binding protein-6. 204 FZD9: frizzled-9. 205 GTF2I RD1: GTF2I repeat domain-containing protein-1 or WBSCR11. 206 LIMK1: LIM domain-containing kinase-1. 207 MLXIPL: MLX-interacting protein-like protein, a.k.a. bHLHd14, WBSCR14, and WSbHLH. 208 RFC2: replication factor complex activatory subunit-2. 209 Stx1a: syntaxin-1A. 210 Tβ L2: transducin-β-like protein-2, or WBSCR13.

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7 Genetic Risk Factors

The relation to CVD of a given variant of a susceptibility gene can be weak, moderate, or strong [1381], such as Ace (angiotensin convertase); AGT,211 AGTR1,212 APOB, APOC3, and APOE, CBS,213 CDKN2A and CDKN2B,214 Cetp,215 CYBA,216 CYP11B2,217 F2 and F5,218 GNB3,219 GPI X,220 IL1B and Il6,221 ITGB3,222 Lpl, Mthfr, MTR, MTRR,223 Nos3,224 Pai1,225 PON1,226

211 AGT:

gene encoding angiotensinogen (ATng), or serpin-A8. gene encoding AT1 (type-1 angiotensin-2 receptor). 213 Cβ S: cystathionine β-synthase. 214 CDKN2A(B): cyclin-dependent kinase inhibitor-2A (B). 215 CETP: cholesterol ester transfer protein. 216 CyBα: cytochrome-B245 α-polypeptide (NOx subunit). 217 CyP11B2: cytochrome-P450–11B2. 218 FI I : coagulation factor-I I (thrombin); FV : coagulation factor-V (proaccelerin). 219 GNβ3: guanine nucleotide-binding protein (G protein) β3 subunit. 220 GP9: platelet glycoprotein-9 (-I X). 221 IL1β(6): interleukin-1β (-6). 222 Itgβ3: platelet integrin-β3. 223 MTHFR: methylene tetrahydrofolate reductase; MTr: methyl tetrahydrofolate–homocysteine methyltransferase (methionine synthase); MTrR: methyl tetrahydrofolate–homocysteine methyltransferase reductase (methionine synthase reductase). These enzymes are involved in the folate and methionine (Met)–homocysteine (HCys) metabolism in addition to in methylation reactions. Folate polyglutamates are converted to methylene tetrahydrofolate, which is a methyl donor in the synthesis of deoxythymidine monophosphate, which is used for DNA synthesis, from deoxyuridine monophosphate. Methylene tetrahydrofolate is converted by MTHFR into active methyl tetrahydrofolate, which yields a methyl in the remethylation of homocysteine to methionine. Methionine is then catabolized to the universal methyl donor S adenosylmethionine, which is processed into S adenosylhomocysteine by methionine synthase reductase. Methyl tetrahydrofolate and homocysteine are transformed by methionine synthase into tetrahydrofolate and methionine, respectively, methionine synthase requiring vitamin-B12 (cobalamin) as a coenzyme. Homocysteine is also processed into cysteine using vitamin B6 and subsequently to glutathione. The MTr cofactor cobalamin(I ) is oxidized to cobalamin(I I ) (also denoted cob(I I )alamin, the central cobalt atom having an oxidation state of +2), thereby inactivating MTr; MTrR processed cobalamin(I I ) to methyl cobalamin(I I I ) (CH3 cob(I I I )alamin) to restore cobalamin(I ) and thus maintain the MTr activity [1382]. Therefore, methionine synthase reductase reactivates methionine synthase. The Mthfr variant C677T leads to an enzyme with reduced activity, hence altering the folate metabolism and causing DNA hypomethylation [1383]. In addition, reduced MTHFR activity raises homocysteinemia, which favors inflammation and redox stress. Homocysteine can also affect DNA hypo- and hypermethylation. The SNP on MTR A2756G (rs1805087) is associated with a higher concentration of total cholesterol and LDLCS [1382]. The SNP on MTRR A66G (rs1801394) is a marker of congenital heart defects. The gene mutations, MTHTR C677T, MTHFR A1298C, MTR A2756G, and MTRR A66G, can synergistically raise the incidence of dyslipidemia in Chinese hypertensive subjects. 224 NOS3: nitric oxide synthase-3 (endothelial). 225 PAI1: plasminogen activator inhibitor-1, or serpin-E1. 226 POn1: paraoxonase-1. 212 AGTR1:

7.5 Atherosclerosis

661

Table 7.25 Genes particularly linked to coronary atherosclerosis and key driver genes (key regulatory genes for sets of functionally relevant genes; Source: [1247]; (GWAS genome-wide association study, ALG8 asparagine-linked glycosylation homolog-8, CEBPD CCAAT/enhancerbinding protein-δ, CES carboxylesterase, DECR2 peroxisomal dienoyl coenzyme-A reductase-2, DCI dodecenoyl coenzyme-A δ-isomerase, DNAJC7 DnaJ (Hsp40) homolog, subfamily C, member 7, EHHADH enoyl co-enzyme-A hydratase/3-hydroxyacyl coenzyme-A dehydrogenase, ETFDH electron-transferring flavoprotein dehydrogenase, FCER1G high affinity Fc receptor of IgE 1γ polypeptide, FCGR1A high affinity Fc receptor of IgG 1α polypeptide, FYB Fyn-binding protein, GC group-specific component [vitamin-D–binding protein [VDBP]), GFER growth factor augmenter of liver regeneration, GLO1 glyoxalase-1, KCNA5 voltage-gated K+ channel, shaker-related subfamily, member 5, MAP3K6 mitogen-activated protein kinase kinase kinase-6, NCKAP1L NCK-associated protein 1-like, PLG plasminogen, PPIL1 peptidylprolyl isomerase-like protein-1, PRMT1 protein Arg methyltransferase-1, PTPRC protein Tyr phosphatase receptorC, PZP pregnancy-zone protein, SGK1 serum/glucocorticoid-regulated kinase-1, SLC10A6 solute carrier family-10A6 (sodium/bile acid cotransporter), SQLE squalene epoxidase, UBE2S ubiquitin conjugase-E2S, VPS52 vacuolar protein sorting homolog-52, ZC3H7B zinc finger CCCH-typecontaining protein-7B) Superset Lipid metabolism and transport Immunity Antigen Unassigned

GWAS signal genes CYP4A11, LDLR, LPL, LRRC19, MAT1A, ME1, NAT2, SREBF1, TMEM27, TMEM116 CTSS, HLAB, HLADRB1, HLADQB1, OAS1 AS3MT, CD2AP, FLOT1, HCG4, TAF11 ALS2CR13, ARL3, CEACAM3, LMO4, NTSC2, SURF6, TIE1

Key driver genes CES3, Dci, Ehhadh, Etfdh, GC, HGR, PLG, PZP, SLC22A5, SQLE FCER1G, FCGR1A, FYB, Nckap1L, PTPRC Decr2, Gfer, GLO1, PP1L1, VPS52 ALG8, CEBPD, DNAJC7, KCNA5, Map3k6, Prmt1, Sgk1, SLC10A6, UBE2S, ZC3H7B

SELE,227 Sod2,228 and Sod3,229 Tnf.230 Multiple genetic factors with modest effects are necessary for the development of atherosclerosis. Gene variants raising the concentrations of plasmatic ApoB+ lipoproteins increase the atherosclerotic risk [1379]. Gene networks linked to atherosclerosis involve those implicated in metabolism and transport of lipids, blood coagulation, and immunity, along with additional ones with no obvious functional assignation (Table 7.25) [1247]. For example, a gene

227 SelE:

selectin-E. mitochondrial superoxide dismutase. 229 SOD3: extracellular superoxide dismutase-3. 230 TNF: tumor-necrosis factor (TNFα or TNFSF1). 228 SOD2:

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7 Genetic Risk Factors

network involved in antigen processing is strongly associated with CoAD. The key driver genes of this network include GLO1231 and Ppil1 [1247].232 Genome-wide association studies have detected 27 loci connected to CoAD. However, large-scale GWASs that involve tens of thousands of cases and controls may explain at most 15 to 20% of the heritability of cardiovascular pathological conditions. The top 20 key drivers derived from the causal module include BACH2,233 BANK1, Cd72, CD79B, Cd200,234 Celsr1, COBLL1, Ctgf, Ebf1, FAM3C,235

231 Glo1:

glyoxalase-1. It catalyzes the conversion of hemimercaptal formed from methylglyoxal and glutathione to S lactoylglutathione [108]. It is involved in the regulation of TNFSF1-induced transcriptional activity of NFκB. 232 PPIL1: peptidylprolyl isomerase-like protein-1. It accelerates protein folding. It catalyzes the cis–trans isomerization of proline imidic peptide bonds in oligopeptides [108]. It may be involved in pre-mRNA splicing. 233 bach2: BTB and CNC homology-2, basic leucine zipper transcription factor-2. The transcription factors of the MAF family are members of the basic leucine zipper (bZip) superfamily with AP1 (i.e., heterodimers made up of Fos, Jun, ATFs, and JDPs [Jun dimerization proteins JDP1 (a.k.a. BaTF3 and ATF-like-3) and JDP2]), members of the ATF–CREB family (ATF1–ATF7, CREB, CREM [cAMP-responsive element modulator]), antioxidant and prolongevity members of the CNC family (Cap ‘n’ Collar [apostrophes replacing for shortening purpose both the “a” and the “d” in “and”]; i.e., NFE2, NFE2L1–NFE2L3, and bach1–bach2), C/EBP, and members of the PAR bZip family (PAR: proline and acidic amino acid-rich basic leucine zipper; i.e., albumin Dsite-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor). Bach2 inhibits HOx1 and causes apoptosis in response to redox stress. This transcriptional regulator acts as a repressor or activator. It binds to MAF recognition elements and coordinates transcriptional regulation mediated by MAFk [108]. Members of the MAF family can be divided into two groups, small and large MAF proteins. The large MAFs include MAF, MAFa, MAFb, and NRL (neural retina leucine zipper protein). The small MAFs comprise MAFf, MAFg, and MAFk. The small MAFs heterodimerize with the members of the CNC family of transcription factors in the stress response and detoxification pathways [1384]. 234 BAnk1 and cluster of differentiation proteins CD72, CD79b, and CD200 correspond to Bcell scaffold with ankyrin repeats protein-1, B-cell differentiation antigen; B-cell antigen receptor complex-associated protein β chain; and type-I membrane glycoprotein OX2 with complement receptor function synthesized by macrophages. 235 CELSR: cadherin, EGF-like, LAG-like, and 7-pass receptor. It is involved in the transmission of directional cues to align individual cells within an epithelial sheet or multicellular clusters [194]. It recruits to cell contacts frizzled-6. CoBlL1, CTGF, and EBF stand for cordon-bleu–like protein-1, connective tissue growth factor, and early B-cell factor, a transcriptional activator, respectively. The Fam3c protein (family with sequence similarity protein-1) is also called interleukin-like EMT inducer. It promotes epithelial-to-mesenchymal transition [108].

7.6 Chromosomal 9p21 Risk Locus

663

LARGE,236 PKIG, PPAPDC1B, RALGPS2, RASGRP3, SAV1, SPIB, TNFRSF13C and Tnfrsf17, and TSPAN13 [1378].237 Tissue-specific SNPs identify the abdominal AT as an important gene regulatory organ for blood lipids and the Pcsk9 gene, a CoAD risk locus [1385]. PCSK9 is secreted from abdominal AT into the portal vein and then affects hepatic LDLR degradation. In abdominal AT, SNPs in ABCA8238 and ABCA5239 (rs4148008) in addition to STARD3240 (rs11869286) are associated with HDL, in EVI5241 (rs7515577) with total cholesterol, and in TMEM258242 (rs174546) with LDL.

7.6 Chromosomal 9p21 Risk Locus The chromosomal 9p21.3 CoAD risk locus containing multiple highly correlated SNPs is close to the CDKN2A and CDKN2B genes, which encode cyclindependent kinase inhibitors CKI2a and CKI2b, which interact with CDK4 and CDK6 (Vol. 9, Chap. 1. “Ciliopathies”). This nonprotein-coding region produces antisense long nonprotein-coding RNA in the CDKN2B gene locus (Vol. 11, Chap. 2. “Nonprotein-Coding Transcripts and Regulation of Protein-Coding Gene Transcription”). The 9p21 locus is associated with the extent and severity of atherosclerosis. A higher risk allele frequency is observed in subjects with multivessel disease. In addition to myocardial infarction, SNPs in the chromosomal site 9p21 are related to stroke, fusiform abdominal aortic and saccular intracranial aneurysms, and

236 Large

is a glycosyltransferase-like protein. The bifunctional xylosyl- and glucuronyltransferase large-1 (or acetylglucosaminyltransferase-like protein-1A) is involved in the synthesis of the phosphorylated O mannosyl trisaccharide, a carbohydrate of α-dystroglycan, which is required for binding laminin-G-like domain-containing extracellular proteins [108]. 237 PKIγ: cAMP-dependent protein kinase inhibitor-γ; PAP2D1b: phosphatidic acid phosphatase type-2 domain-containing protein-1B; RalGPS2: RalGEF with PH domain and SH3-binding motif protein-2; RasGRP3: Ca2+ - and DAG-regulated Ras guanyl-releasing protein-3; Sav1: salvador homolog-1; SPIb: Spi1- (or PU.1)-related transcription factor; TNFRSF13c(17): tumor-necrosis factor receptor superfamily member-13C (-17); and TSpan13: tetraspanin-13. Tetraspanins are components of membrane complexes with integrins, among others, and function in cell adhesion, development, activation, growth, and migration. 238 ATP-binding cassette superclass member-A8 is an ATP-dependent lipophilic drug transporter. It carries substrates for ABCc2 (e.g., estradiol β-glucuronide taurocholate, and LTc4 ) and for the organic anion transporter OAT3 (SLC22a8) [194]. 239 ATP-binding cassette superclass member-A5 may play a role in autolysosome processing. 240 Steroidogenic acute regulatory protein (StAR)-related lipid transfer protein carries cholesterol. 241 Ecotropic viral integration site-5 protein homolog contributes to the regulation of cell cycle progression. 242 Transmembrane protein-258 is a component of the oligosaccharyltransferase complex involved in protein glycosylation [1386]. It also controls the ER stress response.

664

7 Genetic Risk Factors

peripheral artery disease [1387]. In addition, ablation of the CDKN2B gene alters the vascular response to mechanical injury [1387]. Approximately 25% of Europeans carry two copies of the risk allele and have a 40% increased CoAD risk in general and a twofold premature CoAD risk, in addition to carotid atherosclerosis, peripheral arterial disease, and abdominal aortic and intracranial aneurysms [1364]. The risk conferred by this locus is independent of other known risk factors (plasmatic lipid levels, BP, obesity, diabetes mellitus, markers of inflammation, age, and sex). Fifty-nine SNPs have been identified at the 9p21.3 locus, which can disrupt or create TFBSs [1388]. The CoAD risk alleles linked to SNPs rs10811656 and rs10757278 localize to one of 33 detected enhancers in the 9p21 locus and disrupt a binding site for the transcription factor STAT1, which inhibits CDKN2Bas expression, hence leading to the absence of STAT1 binding in the case of homozygous CoAD risk haplotype. The SNPs rs10811656 and rs4977757 disrupt binding sites for the TEAD transcription factor.243 In human vascular endotheliocytes, activated interferon-γ affects the structure of the chromatin and transcriptional regulation in the 9p21 locus, that is, STAT1 binding, long-range enhancer interactions, and altered expression of neighboring genes. In human aortic smooth myocytes (hAoSMCs) homozygous for the nonrisk allele, overexpression of TEAD3 and TEAD4 prime CKI2a production, but not for the risk allele, which is linked to reduced CKI2a and CKI2b concentrations and hence increased proliferation [1391]. TGFβ which interacts with TEAD factors and then elicits CKI2a synthesis, does not hamper proliferation of hAoSMCs homozygous for the risk allele. Carriers of the 9p21 risk allele have an increased microvessel density in coronary atherosclerotic plaques, which is linked to elevated intraplaque inflammation and risk of bleeding and rupture [1387]. However, intraplaque endotheliocytes are partly covered by perivascular cells, i.e., smooth myocytes or pericytes, which both produce actin-α2. These microvessels are thus immature, as the lack of a complete perivascular cell layer impedes endothelium stabilization. Actin-α2 coverage of new blood vessels is also reduced in hindlimb ischemia in CDKN2B−/− mice. Endotheliocytes depleted in CDKN2B and exposed to hypoxia have a greater angiogenic capacity, but impaired neovessel maturation, as migration and proliferation of CDKN2B−/− vSMCs is only slightly disturbed and hence SMC investment of new vessels is weak.

243 TEAD:

transcriptional enhancer activator domain-containing factor. The transcription factors of the TEAD family (TEAD1–TEAD4) are required for cardiogenesis and myogenesis, among other tasks[1389]. They need coactivators to activate gene transcription used in cell differentiation and proliferation in addition to stem cell maintenance. Several TEAD-interacting transcriptional coactivators can be classified into three groups: (1) YAP and Taz, (2) vestigial-like proteins (VgL1– VgL4), and (3) nuclear receptor coactivators of the SRC family (SRC1–SRC3) [1389, 1390]. The TEADs also act as cancer activators or suppressors.

7.7 Aortopathies

665

In hypoxic endotheliocytes and smooth myocytes, loss of CDKN2B expression is associated with an augmented production of TGFβ1, which has a cell- and contextspecific activity, and a decline in the formation of inhibitory SMAD7 [1387]. Subsequently, SMAD3 activation rises, ultimately upregulating the expression of TGFβ1induced protein-1, which has antagonistic effects on EC and SMC [1387]. • In endotheliocytes, TGFβ1 has pro- or antiangiogenic effects according to the signaling pathway, either promoting EC migration and tube formation via ALK1 and SMAD1–SMAD5 or inhibiting angiogenesis via ALK5 and SMAD2– SMAD3. • In vSMCs, TGFβ1 promotes either the contractile phenotype via myocardin and serum response factor or the synthetic phenotype and stimulates proliferation via PDGFaa. The focal adhesion molecule TGFβ1I1 regulates nuclear receptor signaling, extracellular matrix sensing, and cellular senescence, in addition to TGFβ auto-induction. It precludes EC spreading and tubulogenesis in vitro, but favors SMC proliferation and migration. It either promotes TGFβ signaling via SMAD3 or hinders it via SMAD7 [1387]. Several risk regions, such as those that contain the ABO244 and SH2B3 gene245 are linked to multiple coronary lesions (Tables 7.26, 7.27, 7.28, 7.29, 7.30 and 7.31) [1364].

7.7 Aortopathies Genetic diseases with autosomal dominant transmission can affect the aorta. They are subdivided into two categories: inherited syndromic and nonsyndromic maladies. Turner syndrome, which is related to the X chromosome (monosomy X [i.e., a single copy of the X chromosome in the cell), engenders congenital cardiac defects, bicuspid aortic valve (30%), aortic anomalies such as co-arctation (12%), and generalized dilation of major arteries [1393]. Marfan syndrome, the most frequent heritable connective tissue disorder, is caused by mutations in the FBN1 gene246 (Table 7.32). It is most frequently associated with dilation of the aortic root and ascending aorta, thoracic aortic

244 The ABO blood group corresponds to transferase-A, or α(1,3)N acetylgalactosaminyltransferase

and transferase-B, or α(1,3)-galactosyltransferase. adaptor SH2b3 is involved in signaling from growth factor and cytokine receptors. 246 Fbn1: fibrillin-1. Fibrillin-1 is a structural component of microfibrils (diameter 10–12 nm) of the extracellular matrix, which act as a scaffold for elastin deposition and contribute to load bearing. These microfibrils can also form elastin-independent meshworks (e.g., cornea and glomerulus), where they also provide tensile strength, in addition to their anchoring role. Fibrillin1 interacts with growth factors (e.g., BMPs and GDFs) and latent TGFβ-binding proteins (LTBPs), 245 The

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7 Genetic Risk Factors

Table 7.26 Significant loci for coronary artery disease (Part 1; old and new SNPs; Sources: [1364, 1392])

Chromosome 1

Closest gene(s) IL6R MIA3 PCSK9 PPAP2B SORT1

2

ABCG5/G8 AK097927 APOB LINC00954 VAMP5/8–GGCX WDR12 ZEB2

3

MRAS

SNP rs4845625 rs6689306 rs17465637 rs67180937 rs11206510 rs17114036 rs9970807 rs599839 rs7528419 rs6544713 chr2:44074126:D rs16986953 rs515135 chr2:21378433:D rs16986953 rs1561198 rs7568458 rs6725887 chr2:203828796:I rs2252641 rs17678683 rs9818870 chr3:138099161:I

Putative relevant function Immunity Collagen secretion LDLR recycling Intercellular interactions ApoB secretion, LDL turnover, ↓ VLDL secretion rate Cholesterol uptake, secretion ND Lipid transfer ND Vesicular transfer ND Gene transcription Cell growth and differentiation

The intestinal microbiome contributes to the variation in blood lipids. Multiple SNPs localize to the intergenic region. The causal gene can encode a microRNA or long nonprotein-coding RNA rather than a protein. Four microRNAs (miR128–1, miR130b, miR148a, and miR301b) are associated with LDLCS uptake and cholesterol efflux. SNPs can affect lncRNA–microRNA interactions. Some loci can coordinately regulate the transcription of several genes rather than a single gene. Chromatin looping can place gene enhancers in proximity to gene promoters. A SNP situated in the regulatory element alters transcription factor-binding affinity and hence gene transcription. A minor allele linked to increased or decreased expression of the causal gene in the relevant cell type can thus protect against atherosclerosis

aneurysms, and aortic dissection. Concentration of adamts1 in the heart of Marfan syndrome patients is lower than that of organ transplant donors, whereas NOS2

in addition to integrins and other matrix protein and proteoglycans [108]. It participates in cell adhesion via its binding to integrins.

7.7 Aortopathies

667

Table 7.27 Significant loci for coronary artery disease (Part 2; old and new SNPs; Sources: [1364, 1392])

Chromosome 4

Closest gene(s) EDNRA GUCY1A3 REST–NOA1

5 6

SLC22A4/A5 ADTRP-C6ORF105 ANKS1A KCNK5 PHACTR1 PLG TCF21 (BHLHA23) SLC22A3–LPAL2–LPA

SNP rs1878406 rs4593108 rs7692387 rs72689147 rs17087335 rs273909 rs6903956 rs17609940 rs10947789 rs56336142 rs12526453 rs9349379 rs4252120 rs4252185 rs12190287 rs12202017 rs2048327 rs55730499 rs3798220

Putative relevant function Vasoconstriction Nitric oxide signaling Maintenance of vSMC quiescence Organic cation transport ND Potassium ion flux PP1 activity regulation Calcification Fibrinolysis Gene transcription LPA activity

production rises [1394]. Pharmacological inhibition of NOS2 rapidly reverses aortic dilation and medial degeneration in young adamts1-deficient mice and young or old mice with Marfan syndrome. The vascular type-4 Ehlers–Danlos syndrome provoked by mutations in the COL3A1 gene affects the entire vasculature and heart, among other organs. Loeys–Dietz syndrome is characterized by arterial tortuosity and aneurysms throughout the arterial tree. It results from mutations in the TGFBR1 or TGFBR2 gene. Arterial tortuosity syndrome is associated with arterial tortuosity, elongation, stenoses, and aneurysms of the large and mid-sized arteries. It is engendered by mutations in the SLC2A10 gene.247

247 Solute

GluT10.

carrier superclass member SLC2a10 facilitates glucose transport, hence its other alias,

668

7 Genetic Risk Factors

Table 7.28 Significant loci for coronary artery disease (Part 3; old and new SNPs; Sources: [1364, 1392])

Chromosome 7

8

9

Closest gene(s) HDAC9 NOS3 ZC3HC1 7q22 (BCAP29) LPL TRIB1 ABO CDKN2BAS KIAA1462 9p21

SNP rs2023938 rs2107595 rs3918226 rs11556924 rs10953541 rs264 rs2954069 rs579459 rs2519093 rs10757274 rs2505083 rs2487928 rs3217992 rs2891168 rs4977574

Putative relevant function Repression of MEF2 Nitric oxide production Cell proliferation regulation B-cell receptor signaling Lipolysis of TG-rich LPs SMC proliferation, plasma lipid metabolism IL6, E-selectin, LDL levels Cell proliferation, platelet function

CDKN2B, CDKN2BAS1

Aneurysm-osteoarthritis syndrome is responsible for approximately 2% of familial thoracic aortic aneurysms and dissection. In fact, it causes tortuosity, aneurysms, and dissections throughout the arterial tree. It results from a mutation in the SMAD3 gene. On the other hand, nonsyndromic familial thoracic aortic aneurysms and dissections (nsTAAD) show cystic medial necrosis. They are generated by mutations in the ACTA2, MYH11, MYLK, PRKG11, or the TGFB2 gene.

7.8 Aneurysms In the European and Japanese populations, susceptibility loci associated with intracranial aneurysms have been detected on chromosomal regions 2q, 8q, and 9p [1395]. Susceptibility loci on chromosomal loci 8q and 9p are likely linked to SOX17, which is necessary for endotheliocyte formation and maintenance and CDKN2A, respectively.

7.8 Aneurysms

669

Table 7.29 Significant loci for coronary artery disease (Part 4; old and new SNPs; Sources: [1364, 1392])

Chromosome 10

Closest gene(s) CXCL12

KIAA1462 LIPA

CYP17A1-CNNM2-NT5C2 11

PDGFD SWAP70

12

ZNF259—APOA1– –APOA5–APOC3 ATP2B1 KSR2 SH2B3

SNP rs501120 rs2047009 rs1870634 rs2505083 rs1412444 rs11203042 rs1412444 rs12413409 rs11191416 rs974819 rs2128739 rs10840293

Putative relevant function Endothelial regeneration, neutrophil migration ND Hydrolysis of cholesteryl esters and triglycerides Steroidogenic pathway SMC proliferation

rs964184

WBC and vSMC migration TG-rich LP metabolism

rs7136259 rs2681472 rs11830157 rs3184504

Calcium homeostasis Blood pressure Cell proliferation Cytokine signaling

The APOA1–APOC3–APOA4–APOA5 locus is associated with LDLCS and triglyceride levels

Both thoracic aortic aneurysms and abdominal aortic aneurysms (AAAs) can be associated with genetic disorders of connective tissue, such as Marfan, Ehlers– Danlos, and Grönblad–Strandberg syndrome (pseudoxanthoma elasticum), a disorder characterized by mineralization of elastic fibers caused by mutations in the ABCC6 gene. Transforming growth factor-β receptor Tβ R2 is involved in mechanotransduction in vSMCs. Mutations of the TGFBR2 gene cause the Loeys–Dietz syndrome characterized by arterial aneurysms. The 9p21.3 locus is associated not only with CoAD, but also with AAAs. On the other hand, the Il6r gene248 variant (Asp358Ala) is characterized by a lower risk of AAA and, in lymphoblastoid cell lines, a reduced expression of the Stat3, MYC, and ICAM1 genes in response to IL6 stimulation [1396].

248 Activated interleukin-6 receptor can participate in the regulation of the immune response, acute-

phase reactions, and hematopoiesis [108].

670

7 Genetic Risk Factors

Table 7.30 Significant loci for coronary artery disease (Part 5; old and new SNPs; Sources: [1364, 1392])

Chromosome 13

Closest gene(s) COL4A1/A2

14

VEGFR1 HHIPL1

15

ADAMTS7–MORF4L1 FURIN MFGE8–ABHD2 SMAD3

SNP rs4773144 rs9515203 rs11838776 rs9319428 rs2895811 rs10139550 rs7173743 rs4468572 rs17514846 rs8042271 rs56062135

Putative relevant function Basement membrane

Angiogenesis ND Vascular repair Proteolysis Angiogenesis TGFβ signaling

The APOA1–APOC3–APOA4–APOA5 locus is associated with LDLCS and TG levels

Table 7.31 Significant loci for coronary artery disease (Part 6; old and new SNPs; Sources: [1364, 1392]; PC phosphatidylcholine, PE phosphatidylethanolamine)

18

Closest gene(s) BCAS3 RAI1–PEMT–RASD1 (RASD1–SMCR3–PEMT) SMG6–SRR UBE2Z UBE2Z–GIP–ATP5G1–SNF8 PMAIP1–MC4R

19

APOE–APOC1

Chromosome 17

LDLR ZNF507 21

KCNE2 SLC5A3–MRPS6–KCNE2

22

POM121L9P-ADORA2A

SNP rs7212798 rs12936587 rs216172 rs46522 rs663129 rs56289821 rs2075650 rs4420638 rs1122608 rs12976411 rs4420638 rs9982601 rs9982601 rs28451064 rs180803

Putative relevant function Angiogenesis Conversion of PE into PC RNA decay Ubiquination Apoptosis; obesity LDL and VLDL clearance LDL clearance ND Cardiac ion flux

Adenosine signaling

7.8 Aneurysms

671

Table 7.32 Genetic diseases with autosomal dominant transmission that affect the aorta (GluT glucose transporter, PKG cGMP-dependent protein kinase-G) Malady Aneurysm-osteoarthritis syndrome

Arterial tortuosity syndrome Ehlers–Danlos syndrome Loeys–Dietz syndrome Marfan syndrome

Involved gene mutation ACTA2 (smooth myocyte α-actin), MYH11 (smooth myocyte myosin heavy chain), MYLK (myosin light chain kinase), PRKG1 (PKG1), TGFB2 (TGFβ2) SLC2A10 (GluT10) COL3A1 (type-3 procollagen) TGFBR1 or TGFBR2 (Tβ R1 or Tβ R2) FBN1 (fibrillin-1)

Fibrillin-1 is a large glycoproteic structural component of calcium-binding microfibrils in elastic connective tissue of the media. Transforming growth factor TGFβ2 is a cytokine that favors the occurrence of fibrosis. It operates via its receptors Tβ R1 and Tβ R2 and SMAD homolog effectors

Abdominal aortic aneurysms are linked to variants in the DAB2IP249 and LRP1 genes,250 which encode the scaffold disabled homolog-2-interacting protein and the endocytic receptor LDLR-related protein-1, respectively [1396].

249 Disabled

homolog Dab2-interacting protein is involved in numerous signaling pathways linked to innate immunity, inflammation, and cell apoptosis, proliferation, migration, and maturation, and hence angiogenesis [108]. In response to VEGFa, it precludes the VEGFR2–PI3K angiogenic signaling and consequently endotheliocyte migration and tubulogenesis. It regulates the unfolded protein response. It prevents the Ifnγ-mediated JaK–STAT cascade and thus smooth myocyte proliferation and adverse intimal expansion. It operates as a GTPase-activating protein for ARF6 and Ras. 250 Low-density lipoprotein receptor (LDLR)-related protein LRP1 is also called α2-macroglobulin receptor (α2MR). It is involved in endocytosis and hence cellular lipid homeostasis along with phagocytosis of apoptotic cells. It permits plasmatic clearance of chylomicron remnants and activated LRP-associated protein LRPAP1 (α2MRAP) in addition to local metabolism of complexes between plasminogen activators and their endogenous inhibitors [108].

672

7 Genetic Risk Factors

Analysis of genetic variations related to AAA shows that the LDLR,251 LRP1, IL6R, SORT1,252 and DAB2IP genes in addition to the chromosomal 9p21 locus explain partly the heritability of AAA (as well as CoAD) [232]. Genomic loci representing a risk for AAA are identified in chromosome 1 (SORT1 and IL6R in addition to 1q32.3 [Smyd2]),253 9 (DAB2IP and cdkn2bas1 [or anril]), 12 (LRP1), 13 (locus 13q12.11 [LINC00540]), 19 (LDLR), 20 (locus 20q13.12 near PCIF1–MMP9–ZNF335),254 and 21 (21q22.2 [ERG]),255 ERG, IL6R, and LDLR being modifiers of Mmp9 [1399]. MMP9 secreted into the intima by atherosclerotic inflammatory leukocytes damages the extracellular matrix. Inflammation, matrix degradation, and LDL metabolism are important processes in AAA genesis. In the aortic wall, the genetic variant at 20q13.12 is associated with PLTP, the expression of which is significantly higher in aneurysmal aortic walls; the variant rs181914932 is upstream from PCIF1 [1399]. The variant at the chromosomal site 13q12.11 that encodes LINC00540 may regulate the FGF9 gene in the 12q12.11 locus. The locus near the SORT1 gene may control not only SORT1 transcription but also the distant BCAR3 and NOTCH2 genes.

7.9 Stroke Stroke can be of hemorrhagic and ischemic origin. Hemorrhagic strokes can be primarily intraparenchymal or subarachnoid. Ischemic strokes, i.e., most strokes, can be categorized according to their etiology: • Atherosclerosis of extra- and intracranial arteries • Cardiogenic embolism due to: 251 Low-density lipoprotein receptor tethers to the major plasmatic cholesterol-carrying lipoprotein,

LDL, for endocytosis upon clustering into clathrin-coated pits [108]. is a transmembrane protein of the trans-Golgi network. It is a sorting receptor in the GB and a clearance receptor on the cell surfaces [108]. It serves as a receptor for neurotensin. It is implicated in protein transfer from the GB to lysosomes and binds with high affinity to ApoB. Hepatic sortilin-1 lowers ApoB100 secretion from hepatocytes [1397]. On the other hand, it promotes LDL uptake and lysosomal catabolism [1397] but reduces LDLR-mediated LDL uptake owing to augmented secretion of PCSK9, circulating PCSK9 priming LDLR degradation in lysosomes [1398]. It can internalize extracellular lipoprotein lipase. In adipocytes, it may contribute to the formation of storage vesicles containing the glucose transporter GluT4 (SLC2a4) that enables responsiveness to insulin [108]. 253 The Smyd2 gene encodes SMYD2 (SET and MYND domain-containing protein), that is, KMT3c. It controls HSP90 methylation [1399]. It also plays a role in the differentiation of embryonic stem cells. 254 PCIF: phosphorylated CTD-interacting factor. 255 Erg: Ets erythroblastosis virus E26 proto-oncogene product homolog, a transcriptional regulator in hematopoietic and endothelial cells [1399]. It operates in angiogenesis. In AAA, both the media and adventitia are more vascularized than in normal walls. 252 Sortilin-1

7.9 Stroke

673

1. Cardiac wall anomalies (cardiomyopathies, post-infarction hypo- and akinetic ventricular regions, atrial and ventricular aneurysms, atrial myxomas, papillary fibroelastomas, and other tumors, septal defects, and patent foramen ovale) 2. Cardiac valvulopathies 3. Cardiac arrhythmias, atrial fibrillation being the most frequent cardiac arrhythmia and a major cause of stroke • Occlusion of small penetrating arteries (lacunar stroke), dissection, vasculitis, genetic disorders, among others Excessive cytotoxic Ca2+ influx through ionotropic N methyl D aspartate (NMDA) receptor and inappropriately activated Ca2+ signaling causes neuronal cell death. Release of excess glutamate during and after an ischemic insult leads to glutamate receptor hyperactivity, triggering calcium overload and ROS generation [1400]. Nitric oxide generates peroxynitrite and subsequently releases Zn2+ from intracellular stores in the neurons of the cerebral cortex. Free Zn2+ ions open mitochondrial permeability transition pores, thereby releasing cytochrome-C, generating ROS, activating P38MAPK, and causing K+ efflux. Transient cerebral ischemia causes a heterogeneous pattern of cell death in the brain [1401]. In the hippocampus, response from NMDA GluR is augmented in more susceptible pyramidal cells of the CA1 region and is associated with delayed neuronal death, whereas that in the resistant CA3 pyramidal cells is transiently depressed. Nutrient deprivation differentially shifts the intracellular equilibrium between protein Tyr kinase and phosphatase activities that modulate NMDA GluR activity in CA1 and CA3 pyramidal cells, PTKs and PTPs promoting NMDA GluR activity in CA1 and CA3 neurons, respectively. The NMDA GluR channel is composed of a GluN1 and one or more GluN2 subunits, in some cases linked to a GluN3 subunit. On the one hand, GluN2a+ NMDA GluR favors ischemic tolerance and attenuates ischemia-primed neuronal death in rats. On the other, GluN2b+ NMDA GluR abolishes ischemic tolerance and triggers neuronal death [1402]. Ischemia damages not only neurons but also gliocytes, in addition to the myelin sheath produced by oligodendrocytes around axons, abolishing action potential propagation. Oligodendrocytes subjected to hypoxia and augmented extracellular K+ concentration experience an influx of H+ , Ca2+ , and Mg2+ ions and a decreased K+ conductance, the low pH, and change in K+ favoring Ca2+ influx from the extracellular medium through the H+ -gated divalent cation-permeable TRPA1 channel [1403]. Pharmacological TRPA1 inhibition reduces ischemia-induced myelin damage, but not axon damage. A very small proportion of strokes are attributable to monogenic disorders,256 almost all being multifactorial, i.e., linked to many genetic and environmental

256 Monogenic

(Mendelian) disorders are responsible for probably less than 1%, this proportion being larger among younger patients with stroke.

674

7 Genetic Risk Factors

risk factors [1404]. Genome-wide association studies have detected risk loci associated with ischemic stroke that are implicated in atrial fibrillation (PITX2257 and ZFHX3),258 coronary (chromosomal region 9p21, ABO,259 ALDH2,260 and Hdac9)261 and carotid atherogenesis (MMP12), hypertension (ALDH2,262 and HDAC9)263 pericyte and smooth myocyte differentiation (FOXF2),264 coagulation (HABP2),265 and neuroinflammation (TSPAN2) [1404]. Two loci (chromosomal region 1q22, which contains PMF1266 and SLC25A44)267 and APOE)268 are related to hemorrhagic stroke (Table 7.33). Hereditary cerebral amyloid angiopathy (HCAA) results from amyloid deposits in blood vessel walls of the central nervous system. It causes stroke, epilepsy, progressive dementia, and other neurological defects starting in mid-adulthood. Mutations in the APP gene are the most common cause of HCAA, i.e., it engenders the Dutch (the most common form), Flemish, Italian, Piedmont, Arctic, and Iowa 257 Pitx2:

pituitary paired-like homeodomain transcription factor-2. zinc finger homeobox gene product-3 (homeodomain transcription factor). 259 The blood group ABO refers to ABO transferases encoded by the ABO gene (a.k.a. A3GALNT and A3GALT1), i.e., transferase-A, or α(1–3)N acetylgalactosaminyltransferase (α3GalNT) and transferase-B, or α(1–3)-galactosyltransferase (α3GalT1). The A and B alleles encode glycosyltransferase, which adds N acetylgalactosamine and D galactose of the H antigen (group-O determinant, i.e., the phenotypic marker of the O blood group), converting it into A and B antigen, respectively. The ABO antigens are expressed not only on red blood capsule membranes but also on the surface of other cells, such as epitheliocytes, vascular endotheliocytes, and platelets. 260 AlDH2: mitochondrial aldehyde dehydrogenase-2. It protects myocardium against ischemia– reperfusion injury, as it detoxifies reactive aldehydes such as 4-hydroxynonenal (4HNE) formed during cardiac ischemia–reperfusion injury, promotes autophagy during ischemia, activating AMPK and inhibiting TOR, and conversely prevents autophagy during reperfusion, stimulating PKB and TOR [1405]. 261 HDAC9: histone deacetylase-9. An SNP rs2107595 in the Hdac9 gene is associated with large artery stroke in Caucasians, in addition to CoAD risk in a Chinese Han population [1406]. Patients with CoAD have higher plasmatic HDAC9 concentrations than controls. 262 Aldehyde dehydrogenase-2 deficiency exacerbates adverse cardiac remodeling due to hypertension, as it suppresses autophagy [1407]. Maladaptive cardiac remodeling is often accompanied by activation of both anabolism and catabolism and hence dysfunctional organelles, which should be degraded and recycled by autophagy triggered by the AMPK–TOR, JNK–FoxO3a, or beclin1 pathway. The AlDH2 deficiency precludes expression of beclin-1 and promotes interaction between BCL2 and beclin-1. 263 Histone deacetylases contribute to the regulation of cardiac remodeling via autophagy. Class-I isozymes HDAC1 and HDAC2 are required in autophagy [1408]. On the other hand, class-I I A HDACs, such as HDAC5 and HDAC9, function as signal-responsive repressors of adverse cardiac hypertrophy, as they inactivates MEF2 independently of deacetylase activity. 264 Loss of forkhead box-containing transcription factor FoxF2 reduces coverage of endothelia by smooth myocytes and pericytes. 265 HaBP2: hyaluronan-binding protein-2, which is also called Factor-V I I -activating peptidase. This extracellular serine peptidase is involved in coagulation, fibrinolysis, and inflammation. 266 PMF1: polyamine-modulated factor-1; both its long and short isoforms interact with NFE2L2. 267 SLC25a44: solute carrier superclass class-25 mitochondrial carrier member-44. 268 ApoE: apolipoprotein-E. 258 ZFHx3:

7.9 Stroke

675

Table 7.33 Genetic loci associated with ischemic stroke (Sources: [230, 1404] CAA familial cerebral amyloid angiopathy, cadasil cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, carasil cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, CSVD cerebral small vessel disease, EDS4 type-4 vascular Ehlers–Danlos syndrome (vEDS), melas mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes, PXE pseudoxanthoma elasticum, RVCL retinal vasculopathy and cerebral leukodystrophy) Disease Implicated gene (protein) Monogenic disorders causing small artery occlusion (lacunar) ischemic stroke Cadasil NOTCH3 Carasil HTRA1 (serine peptidase PesS11 [HtrA1]) CAA APP (amyloid-β precursor protein) CST3 (cystatin-3) ITM2B (integral membrane protein-2B) Familial autosomal dominant COL4A1 (procollagen-4α1 chain) (COL4A1 syndrome) porencephaly RVCL TREX1 (3 repair exonuclease-1) Monogenic diseases causing large and small artery occlusion ischemic stroke genetic disorders with stroke as manifestation EDS4 (vEDS) COL3A1 (procollagen-3α1 chain) Fabry disease GLA (galactosidase-α) Homocystinuria CBS (cystathionine β-synthase) Marfan syndrome FBN1 (fibrillin-1) Melas MTTL1 (mitochondrially encoded tRNA leucine-1) Moyamoya disease ACTA2 (smooth muscle α-actin) Rnf213 (RING finger-containing protein-213) PXE ABCC6 Sickle cell disease HBB (hemoglobin-β subunit) Common genetic variants Atherosclerosis HDAC9, Cdc5l, MMP12, TSPAN2 Atrial fibrillation ZFHX3 Cardioembolism PITX2, ZFHX3 CSVD FOXC1, FOXF2, PITX2 Thromboembolism ABO (rs505922)

676

7 Genetic Risk Factors

cerebral amyloid angiopathy (CAA) types [1362]. Mutations in the CST3 gene, which encodes cystatin-3 (or cystatin-C), a potent inhibitor of lysosomal peptidases, cause the Icelandic CAA type. Mutations in the ITM2B gene, which encodes integral membrane protein-2B,269 are responsible for familial British and Danish CAA types. Moyamoya disease270 is a rare chronic cerebrovasculopathy characterized by a progressive stenosis of the terminal segment of the internal carotid artery and its main branches, occlusion resulting from hyperplasia of vSMCs and luminal thrombosis. This cerebrovasculopathy primes a compensatory collateralization of small arteries in the basal ganglia. Familial Moyamoya disease is inherited in an autosomal dominant manner. The prevalence and incidence of Moyamoya disease are the highest in East Asia, particularly in Japan. Mutations in the ACTA2 gene, which encodes the vSMC-specific isoform of α-actin, in families with thoracic aortic aneurysms and dissections and Moyamoya disease, are observed in patients of Northern European descent, such as heterozygous R179H in ACTA2 exon 6 [1409]. Another Moyamoya disease-associated gene encodes the AAA+ ATPase module and ubiquitin ligase domain-containing mysterin (or RNF213),271 a ubiquitous soluble cytosolic protein [1410]. Moyamoya syndrome is related to similar angiographic features in children with sickle cell disease and Down syndrome.

protein ItM2b plays a regulatory role in the processing of β-amyloid-A4 precursor protein (APP); it prevents β-amyloid peptide aggregation and fibril deposition. This peptidase inhibitor precludes the access of secretases to APP cleavage sites and hence secretion of Aβ(1–40) and Aβ(1–42) peptides, which form protofilaments and fibrils that are toxic oligomers [108]. Like several transmembrane proteins, a two-step ItM2b proteolytic cleavage provokes ectodomain shedding by adam10 into the extracellular space and then intracellular domain release by intramembrane peptidase IMP4 (a.k.a. presenilin-like protein PSL1 and signal peptide peptidaselike protein SPPL2b), which may function as a signaling molecule [194]. 270 In Japanese, moyamoya means puff of smoke. The compensatory arteriogenesis appears as a “puff of smoke” on cerebral angiographies. 271 Mysterin: moyamoya steno-occlusive disease-associated AAA+ and RING finger-containing protein. 269 The

Conclusion

Post hoc, ergo melius hoc [That which comes after is better than that preceding] (A. A. Cournot [1801–1877])

Pathophysiology is aimed at describing structural and functional manifestations and at determining conditions and mechanisms involved in a given disease. Explorations are carried out on various length scales associated with molecules, cells, tissues, organs, and physiological apparatuses, i.e., from molecular pathophysiology to anatomic pathology. Once information is collected, a reductionist approach is most often used to assess underlying biological processes. However, any biological mechanism has a complex behavior. Biological complexity results from connections between regulatory signaling circuits on the nanoscopic scale, crosstalk between cell populations on the micro- and mesoscopic scales, and interferences between physiological apparatuses on the macroscopic scale. The concept of homeostasis1 is related to self-regulating physiological systems in a living organism that exchange matter and energy with the environment. Homeostasis is defined by a relatively stable equilibrium between interdependent elements aimed at maintaining internal conditions, adjusting, and optimizing them for survival. Complex interactions maintain functioning of the organism within a normal range, balancing matter exchange and stabilizing bodily temperature and fluid composition. Therefore, the homeostasis concept underlines the stability of the internal milieu subjected to perturbations. Upon exposure to a disturbance, a transient response enables the return to the previous state. Homeostasis operates at the level of the cell, tissue, organ, and organism. The spatiotemporal organization of living systems characterized by feedback and feedforward is associated with self-organization and nonlinear dynamics, which was

1 oμoιαζω:

to be like; oμoιoς: like in mind, distressing; στασις: placing, setting.

© Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0

677

678

Conclusion

introduced by H. Poincaré, distinct types of initial conditions leading to different behaviors. Mutually influencing variables can evolve toward a restricted region of the phase space, the so-called basin of attraction endowed with its single attractor. At bifurcation points, attractors shift and the behavior can change to chaotic motions. Chaotic dynamics enables quick adjustments, which are mandatory in physiology. Bottom–up mechanisms emerge from autonomous dynamics of biological entities that integrate local working conditions [1411]. For example, second messengers can trigger signaling waves that amplify and spread signals. On the other hand, various biological processes undergo cycles of oscillatory activity that are synchronized both in space and time and enable anticipation. They follow a top– bottom process. Circadian clocks function simultaneously and coordinately under the control of the master clock. Biomathematical modeling of biological processes in normal and pathological conditions are developed to determine respective effects of involved phenomena, to assess the role of influence parameters on the entire process, and predict outcomes on various scales. Biomathematical modeling of biological mechanisms on the nano- and microscopic scales can generate phenomenological models on the mesoand macroscopic scales that serve not only to exhibit major pathophysiological features but also to derive proper inverse problems to be solved to estimate biological quantities that cannot be directly measured. Many biological processes implicate the displacement and deformation of soft tissues and hence fields of mechanical stress and strain. Biomechanics is aimed at contributing to the improvement of diagnosis methods, the elaboration of proper therapeutic strategies, and the conception of new surgical procedures and medical implantable devices, in addition to the development of measurement techniques. In particular, assessment of local blood flow features in patient-specific simulations carried out in the altered cardiac pump or a diseased segment of the vasculature enables the clinical decision to be improved. In this context, computational models are aimed at: 1. 2. 3. 4.

Investigating the origin, development, and progression of cardiovascular diseases Implementing new diagnostic modalities Predicting the outcome of therapies Optimizing drug administration modes, implantable device design, and surgical reconstruction techniques

Cellular and tissular engineering, which lead to regenerative and reparative medicine, are aimed at restoring the form and function of damaged organs of the human body or replacing them (e.g., vascular grafts). Cells and tissues are conditioned in vitro mimicking the physiological environment and then implanted. Bioreactors incorporate various sources of cell signaling, i.e., not only major involved chemical messengers but also physical and mechanical agents. Hence, the short- and long-term evolving mechanical stress field must be deduced from functional imaging and adequate modeling.

Conclusion

679

Materials of regenerative medicine are optimized for proper delivery of nutrients, growth factors, and morphogens. Controlled cell placement enables adequate growth of the interactive cell populations involved and engenders the required structure–function relation and rheology of the organ. In addition, pre-implantation seeding or post-implantation taxis of specialized cells create a suitable vascularization and innervation in addition to immunity. Biomechanics also participates in conceiving, designing, implementing, and optimizing health-related nanotechnology.2 Optimal design of nanoparticles is linked to their size, shape, surface charge, and rheology. Nanotechnology is used in genome analysis, cellular and tissular engineering, and in prevention, early detection, imaging, monitoring, and therapeutics. Nanomaterials endowed with adequate ligands can be used for drug delivery owing to their ability to cross biological barriers and target specific cell populations such as cancerous cells.3 Controlled pharmacokinetics and -dynamics enable the proper release of drug amounts at optimal periods during the circadian cycle using physical and chemical signals. Cybermedicine, or precision medicine, includes computer-aided procedures for optimized diagnosis, treatment, image-guided therapy, prognosis, and follow-up. The new generation of medical tools is based upon experience in sensor fusion, computer vision, robotics, and virtual reality. Navigation and positioning tools efficiently assist medical and surgical procedures. They optimize gestures in the patient-specific anatomy to minimize per-operative complications and guide the operator to achieve the desired material placement, especially in the beating heart. Telemedicine is based on systems of electronically communicating data from one site to a distant site with data fusion by superimposing patient-specific data. Telepresence operation procedures have two major components, a remote site with a 3D camera system and responsive manipulators with sensory input and an operating workstation with a 3D monitor and dexterous handles with force feedback. A remotely controlled robot is capable of executing the procedure at the site of the operation, in which, nonetheless, there are specialists ready to execute the tasks. Hence, vascular diseases provide a unique opportunity for interdisciplinary research aimed at developing computer-aided medicine and surgery.

2 Nanomaterials

usually correspond to objects with dimensions in the range of 1–100 nm. In the medical field, they may include objects up to 1 μm in size. 3 Nanoparticles can be used to concentrate the energy of ultrasound beams for the thermal ablation of cancers. Tumoral cells can also be destroyed by a magnetic field; nanoparticles coated with aminosilane are taken up faster by cancerous cells than by normal cells. Moreover, the treatment can be repeated, as nanoparticles form stable deposits within tumors.

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CP, Sakalihasan N, Courtois A, Ferrell RE, Eriksson P, Folkersen L, Franco-Cereceda A, Eicher JD, Johnson AD, Betsholtz C, Ruusalepp A, Franzén O, Schadt E, Björkegren JL, Lipovich L, Drolet AM, Verhoeven E, Zeebregts CJ, Geelkerken RH, van Sambeek MR, van Sterkenburg SM, de Vries JP, Stefansson K, Thompson JR, de Bakker PI, Deloukas P, Sayers RD, Harrison S, van Rij AM, Samani NJ, Bown MJ (2017) Meta-analysis of genome-wide association studies for abdominal aortic aneurysm identifies four new disease-specific risk loci. Circ Res 120:341–353 1400. Bossy-Wetzel E, Talantova MV, Lee WD, Schölzke MN, Harrop A, Mathews E, Götz T, Han J, Ellisman MH, Perkins GA, Lipton SA (2004) Crosstalk between nitric oxide and zinc pathways to neuronal cell death involving mitochondrial dysfunction and p38-activated K+ channels. Neuron 41:351–365 1401. Gee CE, Benquet P, Raineteau O, Rietschin L, Kirbach SW, Gerber U (2006) NMDA receptors and the differential ischemic vulnerability of hippocampal neurons. Eur J Neurosci 23:2595–2603 1402. Chen M, Lu TJ, Chen XJ, Zhou Y, Chen Q, Feng XY, Xu L, Duan WH, Xiong ZQ (2008) Differential roles of NMDA receptor subtypes in ischemic neuronal cell death and ischemic tolerance. Stroke 39:3042–3048 1403. Hamilton NB, Kolodziejczyk K, Kougioumtzidou E, Attwell D (2016) Proton-gated Ca2+ permeable TRP channels damage myelin in conditions mimicking ischaemia. Nature 529:523–527 1404. Chauhan G, Debette S (2016) Genetic risk factors for ischemic and hemorrhagic stroke. Curr Cardiol Rep 18:124 1405. Ma H, Guo R, Yu L, Zhang Y, Ren J (2011) Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde. Eur Heart J 32:1025–1038 1406. Wang XB, Han YD, Sabina S, Cui NH, Zhang S, Liu ZJ, Li C, Zheng F (2016) HDAC9 variant rs2107595 modifies susceptibility to coronary artery disease and the severity of coronary atherosclerosis in a Chinese Han population. PLoS One 11:e0160449 1407. Shen C, Wang C, Fan F, Yang Z, Cao Q, Liu X, Sun X, Zhao X, Wang P, Ma X, Zhu H, Dong Z, Zou Y, Hu K, Sun A, Ge J (2015) Acetaldehyde dehydrogenase 2 (ALDH2) deficiency exacerbates pressure overload-induced cardiac dysfunction by inhibiting Beclin-1 dependent autophagy pathway. Biochim Biophys Acta Mol Basis Dis 1852:310–318 1408. Cao DJ, Wang ZV, Battiprolu PK, Jiang N, Morales CR, Kong Y, Rothermel BA, Gillette TG, Hill JA (2011) Histone deacetylase (HDAC) inhibitors attenuate cardiac hypertrophy by suppressing autophagy. Proc Natl Acad Sci USA 108:4123–4128 1409. Roder C, Peters V, Kasuya H, Nishizawa T, Wakita S, Berg D, Schulte C, Khan N, Tatagiba M, Krischek B (2011) Analysis of ACTA2 in European Moyamoya disease patients. Eur J Paediatr Neurol 15:117–122 1410. Morito D, Nishikawa K, Hoseki J, Kitamura A, Kotani Y, Kiso K, Kinjo M, Fujiyoshi Y, Nagata K (2014) Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state. Sci Report 4:4442

Conclusion 1411. Lloyd D, Aon MA, Cortassa S (2001) Why homeodynamics, not homeostasis? Sci World J 1:133–145

Notation Rules: Abbreviations, Aliases, and Symbols

Pronounceable abbreviations formed by omitting letters from the end of a word (e.g., chap., cont., ed., fig., and vol. for chapter, continued, editor, figure, and volume, respectively) or sequential word groups, often from Latin origin, such as e.g., etc., and i.e., which, in Springer books, are not italicized, represent and replace long names or word sequences, thereby lightening the text. They take period, as the final letter is not included, except physical units of measurements associated with prefixes of the metric system (e.g., from exa- (E [1018 ]) to atto (a [10−18 ]). It is usually acceptable to start or end a sentence with an abbreviation that begins or ends with a capital letter. This precept was avoided as much as possible—but nevertheless adopted for avoiding repetitions. Abbreviations include: • Letter acronyms, which are based on initials of nouns and are pronounceable as words, such as NSAIDs (nonsteroidal anti-inflammatory drugs), and SARS (severe acute respiratory syndrome), or strings of letters, such as ppm (part[s] per million), or on mixtures of initial and noninitial letters, pronounced as words or not, such as AIDS (acquired immunodeficiency syndrome) and PMNL (polymorphonuclear leukocytes; also abbreviated PML and PMN and also called granulocytes, i.e., basophils, eosinophils, neutrophils, and mastocytes), syllabic acronyms, which are based on syllables, such as modem (modulator– demodulator), and hybrid acronyms, which contain initials, inner letters, and syllables, such as SQUID (superconducting quantum interference device, not the cephalopod, used to measure weak signals, quantum-based magnetometers serving for example in magnetoencephalography) • Abridged aliases, or alternative shortened names • Contractions, which are shortened versions of the written form of a word created by omission of all internal letters • Initialisms, also dubbed alphabetisms, which are formed from a string of initials of several nouns read letter-by-letter and pronounced separately, such as ASCII © Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0

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(for American Standard Code for Information Interchange) and CPU (central processing unit) • Portmanteau words, which combine parts of at least two related words and give rise to a regular word, such as aldol formed from aldehyde and alcool, which acts as both a hydrogen donor and acceptor, and amphetamine (αmethylphenethylamine), an addictive, mood-altering drug The usual name of the translation initiation factor is eukaryotic translation initiation factor, which is abbreviated eIF. However, the word “translation” that is indispensable to comprehend the molecule role is omitted and the adjective “eukaryotic” is useless in this book series dealing with mammalian species, essentially humans, as is the adjective “mechanistic”1 (National Cancer Institute [NCI] Dictionary of Cancer Terms2 and IUPHAR/BPS Guide to Pharmacology),3 or “mammalian”, used to designate the protein Ser/Thr kinase target of rapamycin. Therefore, the shortened alias that is retained in this textbook is TIF. Similarly, the shortened aliases for translation elongation, release, and termination factors are TEF, TRF, TTF, instead of eEF, eRF, and eTF.

Common Latin Abbreviations In addition to common abbreviations such as “a.k.a.” (also known as) and “w.r.t.” (with respect to), Latin-derived shortened expressions are used to lighten sentences: • “a.p.” [ante prandium]: before a meal • “c.” [circa]: around (in the sense of approximately during a period) • “cf.” [confero]: to bring together, collect, gather, unite, join; meaning “see also” and “compare with” • “e.g.,” [exempli gratia]: for example • “et al.” [et alii]: and others • “etc.” [et cetera]: and so forth • “i.e.,” [id est]: in other words, that is • “o.d.” [omni die]: every day

1 “Mechanistic”

is an adjective related to the fact that the protein action is determined by rapamycin (sirolimus). This drug was initially discovered as an antifungal substance produced by Streptomyces hygroscopicus from a soil sample of Easter Island in Chilean Polynesia, its native name being Rapa Nui. This macrolide (bacteriostatic antibiotic) also have immunosuppressive and antiproliferative effects. The macrolides, tacro-, pimecro-, and sirolimus are immunosuppressants or immunomodulators coating drug-eluting stents. Rapamycin complexes with the 12-kDa FK506binding protein (FKBP12), FK506 being another immunosuppressive macrolide, and operates as an inhibitor of the target of rapamycin complex TORC1. 2 www.cancer.gov/publications/dictionaries/cancer-terms. 3 www.guidetopharmacology.org.

Notation Rules: Abbreviations, Aliases, and Symbols

• • • • • • •

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“N.B.” [nota bene]: pay attention “p.p.” [post prandium]: after a meal “q.v.” [quod vide]: in what to see (cross-reference) “v.i.” [vide infra]: see below “v.s.” [vide supra]: see above “viz.” [videlicet]: obviously; meaning “more specifically” “vs.” [versus]: toward, facing; mistakenly meaning “against”4

Chemical Notations In chemistry, notations are used to denote chemical elements, represent the chemical binding types and structures by means of a linear series of symbols, and describe a compound. A chemical formula expresses information on the proportion of constituent atoms of relatively simple compounds using chemical element symbols with numbers (number of a given type of groups, atoms, submolecular components, or subunits, in addition to neutron numbers for isotopes) and, when necessary, other symbols, such as parentheses for multiple identical chemical groups, brackets, dashes for linkages, and plus (+) and minus (−) superscripts for ions. Molecular formulas indicate the numbers of each type of atom in a molecule. They encompass structural formulas that inform on the bonding of atoms in a molecule and include various types of skeletal formulas (a standard notation for cyclic and complex molecules, commonly adding information on interatomic bounds), which are graphical representations of the spatial relation between constituent atoms, and condensed formulas (e.g., glucose: C6 H12 O6 ). The punctuation mark “slash” (/), which commonly denotes division, is also usually interpreted as “and” or “or”. However, this special character is quite equivocal in biochemistry, as it can not only mean “and” when referring to two isoforms (e.g., ERK1/2) or two targets (e.g., protein Ser/Thr kinase) and “or” when it is related to two molecule noun synonyms (e.g., PPARα/NR1c1, the nuclear receptor NR1c1 being peroxisome proliferator-activated receptor-α), but also can designate a intermolecular linkage (e.g., the AHR/ARNT heterodimer composed of the class-7 group-E basic helix–loop–helix transcription factors aryl hydrocarbon receptor [bHLHe76] and AHR nuclear translocator [bHLHe2]). In the present textbook, aliases of a given substance are given using a pair of round (parentheses) or square (crotchets or brackets) brackets, the latter being employed inside the

4 Origin

may be rather from Latin adversus, which has a first meaning: turned toward, fronting, facing, before, in front, in addition to a second sense: opposite, in opposition.

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former,5 and slash (e.g., proapoptotic BCL2 family members BCL2L4/BAX and BCL2L7/BAK1), whereas en-dashes (without spacing before and after) are used for molecular complexes. For the sake of homogeneity, short aliases of enzymes have similar spelling. • The short alias suffix DH designates dehydrogenase, such as ACADH (acylCoA dehydrogenase), G6PDH (glucose 6-phosphate dehydrogenase), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase), which are encoded by the ACAD, G6PD, and GAPDH genes, respectively. • The short suffix Ox is used for oxidases, such as CcOx1 and CcOx4I1 (cytochrome-C oxidase subunit-1 and -4 isoform-1),6 NOx1 (nicotinamide adenine dinucleotide phosphate [NADPH] oxidase-1), and XOx (xanthine oxidase), which are encoded by the COX1, COX4I1, NOX1, and XDH (XO) genes, respectively. • The short suffix POx is related to peroxidases, such as GPOx1 (glutathione peroxidase-1) and MPOx (myeloperoxidase), which are encoded by the GPX1 and MPO genes, respectively. • The short suffix Rdx stands for redoxin, such as PRdx1 (peroxiredoxin-1), which are encoded by the PRDX1 gene. • The short suffix Rd denotes reductase, such as GRd (glutathione reductase) and TRdxRd1 (thioredoxin reductase-1), which are encoded by the GSR and TXNRD1 genes, respectively.

Numerical Terms in Chemical Names Numerical terms are used in chemical names to indicate a number of identical structural units in a chemical compound. Multiplying prefixes and infixes are used to denote principal characteristic groups, saturated unbranched acyclic hydrocarbons, sites of unsaturation, and number of ionic centers. According to the IUPAC (International Union of Pure and Applied Chemistry) chemical nomenclature, multiplying affix, which indicates how many particular atoms or functional groups are attached at a particular point in a molecule, derive from both Greek and Latin languages. The etymology of prefixes is only loosely 5 Parentheses

are also standard notation marks for arguments of a function, coordinates of a point; brackets for intervals; curly brackets (braces) for elements of a set; and angle brackets (chevrons) for an average over time, inner product of 2 functions or vectors, and binary operation. 6 Cytochrome-C oxidase subunit-1 to -3 (CcOx1 [usually abbreviated as MtCO1 (i.e., Mt CO1)] to CcOx3 [MtCO3 (i.e., Mt CO3)]) are components of the electron transport chain complex-I V used for mitochondrial oxidative phosphorylation that are encoded by the genes MTCO1 to MTCO3 in the mitochondrial DNA (Mt DNA). It other subunits (CcOx4–CcOx8) with their isoforms (CcOx4I1–CcOx4I2, CcOx5a–CcOx5b and CcOx5bL1–CcOx5bL7, CcOx6a1–CcOx6a2, CcOx5b1–CcOx6ab2, and CcOx6c, CcOx7a1–CcOx7a2, CcOx7b1–CcOx7b2, and CcOx7c, and CcOx8a–CcOx8c) are encoded by genes in the nuclear DNA (Nu DNA).

Notation Rules: Abbreviations, Aliases, and Symbols

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based on the corresponding Greek words. In some cases, a Latin root is preferred, or mixtures of Greek and Latin roots (e.g., nona- for 9 [rather than ennea-, which is also used in geometry] and undeca- for 11 [rather than hendeca- also used in geometry]). The affix for a number greater than 12 is constructed in the opposite order to that of the constituent Arabic numerals (i.e., units, tens, hundreds, etc.). Names for multiples of tens beyond twenty are formed by adding the infix -conta- to the name of the corresponding units, with insertion of an a only for 30 to 39. For simple substituents, i.e., which are not themselves substituted, the multiplying affixes di-, tri-, and tetra-, are generally used; for substituted substituents, the multiplying prefixes bis-, tris-, and tetrakis- are used. Designation of fatty acid derivatives hence employs Greek roots for numbers in their affixes (e.g., mono- or hen-: 1; di-, dis-, or do-: 2; tri- or tris-: 3; tetra-: 4; penta-: 5; hexa-: 6; hepta-: 7; octa-: 8; ennea-: 9; deca-: 10; dodeca-: 12; eicosa-: 20; henicosa-: 21 [docosa-, . . ., nonacosa-: 22, . . ., 29]; triaconta-: 30 [hentriaconta-, . . ., nonatriaconta-: 31, . . ., 39]; etc.).7

Phosphoinositides Phosphoinositides include phosphatidylinositol (PI) and its phosphorylated derivatives, phosphatidylinositol phosphates (PIP), each category of molecules yielding numerous secondary messengers. Different phosphatidylinositol phosphates result from phosphorylation of single or multiple sites of the inositol ring of phosphatidylinositol, the phosphorylation sites being in parenthesis: 1. Phosphatidylinositol monophosphates PI(3)P, PI(4)P, and PI(5)P 2. Phosphatidylinositol bisphosphates PI(3,4)P2 , PI(3,5)P2 , and PI(4,5)P2 (often written in a simplified but ambiguous form PIP2 ) 3. Phosphatidylinositol trisphosphate PI(3,4,5)P3 (or PIP3 ) Multiple inositide kinases and phosphatases interconvert phosphoinositides. Phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) kinases are denoted according the target phosphorylation site and incorporating any present phosphorylation sites (in parenthesis), such as PIiK (i: 3, 4, or 5) and PI(i)Pj K (phosphatidylinositol i-phosphate j -kinase; i = j ). The aliases PI, PIP, and PIPP refer to phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol polyphosphate phosphatases. Phosphoinositides are synthesized or degraded using regulated enzymes stimulated by activated plasmalemmal receptors, such as phosphatidylinositol 3-kinases

7 When

alone, the number 1 is represented by “mono-” and 2 by “di-”. In association with other numerical terms, the number 1 is represented by “hen-” and 2 by “do-”.

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(PI3K), which generates the second messenger phosphatidylinositol trisphosphate (PIP3 ), and phospholipase-C, which hydrolyzes the messenger phosphatidylinositol (4,5)-bisphosphate (PIP2 ) into inositol trisphosphate (IP3 ), a Ca2+ mobilizer, and diacylglycerol, an activator of protein kinase-C and -B. Cardiac ion channel activity depends on PIP2 density. Membrane phospholipids are also substrates of phospholipase-A2, which generates arachidonic acid, the stem molecule for eicosanoids.

Fatty Acid Derivatives Natural fatty acids comprise hydroxy, epoxy (furanoid), methoxy, and oxo substituents (Table 1).8 Most natural fatty acids have 16–22 carbon atoms. • Among Epoxy and furanoid fatty acids, leukotoxins are monoepoxy fatty acids formed in the lung and other organs from linoleate. Furanoid fatty acids are present mainly in phospholipids; urofuranic acids are short-chain dibasic furanoid fatty acids in human plasma. • Hydroxy means containing the hydroxyl group. 2-Hydroxy fatty acids (length C16–C26) are constituents of sphingolipids. 3-Hydroxy fatty acids (length C6– C16) are formed during β-oxidation of fatty acids. Fatty acid–hydroxy–fatty acid (FAHFA) is a fatty acid with a centrally located hydroxyl group to which a further fatty acid is linked as an estolide. The palmitoyl ester of 9-hydroxy-stearic acid in the adipose tissue of mice has antidiabetic and anti-inflammatory effects. • Methoxy refers to the alkoxy group O–CH3 , a functional group consisting of a methyl group bound to oxygen. It is usually describe an ether (R–O–CH3 ). Methoxy fatty acids exhibit antibacterial, antifungal, antiviral, and anticancerous activities. • Oxo refers to the ketone functional group, in addition to other prefixes such as keto. Ketone (alkanone) is an organic compound with the structure R–C=O–R , where R and R are carbon-containing substituents. Ketones and aldehydes are compounds that contain a carbonyl group (a carbon–oxygen double bond). Fatty acid derivatives are lipidic mediators that include dihydroxyeicosatrienoic acids, docosanoids (anti-inflammatory resolvins and protectins), eoxins (proinflammatory substances produced in eosinophils and mastocytes), epoxyeicosatrienoic acids, epoxytrienoic acids, hepoxilins (monohydroxyepoxyeicosanoids), hydroxyeicosatetraenoic acids, hydroperoxyeicosatetraenoic acids, isoprostanes, leukotrienes, lipoxins (trihydroxyeicosatetraenoic acids [20:4]), and prostanoids (prostaglandins, prostacyclins, and thromboxanes). Aliases are formed from letters associated with their chemical structure, the Greek-derived numerical infix originating from the fatty acid (FA) number (i:j ),

8 Christie

WW (2016) LipidHome. www.lipidhome.co.uk.

Notation Rules: Abbreviations, Aliases, and Symbols

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Table 1 Functional groups Functional group

Alcoholate Alcohol Aldehyde Amides Amines Carboxylic acid Carboxylate salt Esters of carboxylic acid Ethers Imines Hydroperoxides Peroxides Peroxols Ketones Sulfides Thiols

Formula

Affix Prefix Group name OxidoHydroxyFormylCarbamoylAminoCarboxy-

-olate -ol -al -carboxamide -amine -oic acid

R-oxycarbonyl-

-carboxylate

–OR C=NR

R-oxyR-imino-

-imine

–O–OR –OOH =O –SR –SH

R-peroxyHydroperoxyOxoR-sulfanylSulfanylSulfhydryl-

–O− –OH –CH=O –CONH2 –NH2 –COOH –CO− 2 –COOR

Suffix

-peroxol -one -thiol

Oxygenated fatty acids exist in the form of esters via their carboxyl groups. Hydroxy fatty acids also exist in ester linkage to other fatty acids via their hydroxyl groups Table 2 Notation of fatty acid derivatives using letters (L) associated with their chemical structure L1 H: hydroxy HP: hydroperoxy E, Ep: epoxy DH, DiH: dihydroxy TH: trihydroxy

L2 D, Do: docosa E: epoxy O: octadeca P: peroxy

L3 D: di Do: docosa E: eicosa M: mono P: penta T: tetra Tr: tri

L4 E: enoic (A: acid) D: di

L5

E: enoic

which refers to the numbers of carbon atoms [i C] and of double bonds (Tables 2, 3 and 4). Eicosanoids (20-carbon [C20] fatty acids containing four double bonds and their metabolites), or icosanoids (preferred IUPAC name), are lipidic mediators. They derive from ω3- or ω6-fatty acids (Table 5), in particular those generated from arachidonic acid (alias AA), an ω6-fatty acid. The subgroups of eicosanoids include leukotrienes, lipoxins, and prostanoids (prostaglandins [alias PG], prostacyclins,

770

Notation Rules: Abbreviations, Aliases, and Symbols Table 3 Aliases of fatty acid derivatives (Part 1) Type Dihydroxydocosapentaenoic acid Dihydroxyeicosaenoic acid Dihydroxyeicosatetraenoic acid Dihydroxyeicosatrienoic acid Dihydroxyoctadecamonoenic acid Dihydroxyoctadecadienoic acid Dihydroxyoctadecenoic acid Docosahexaenoic acid Docosapentaenoic acid Docosatetraenoic acid Eicosapentaenoic acid Eicosatrienoic acid Epoxyeicosadienoic acid Epoxyeicosaenoic acid Epoxydocosapentaenoic acid Epoxyeicosatetraenoic acid Epoxyeicosatrienoic acid Epoxyhydroxyeicosatetraenoic acid Epoxyoctadecadienoic acid Epoxyoctadecamonoenic acid

Alias DHDPE (DiHDPA) DHEE (DiHEE) DHETE (DiHETE, DEA) DHET (DiHETrE) DHOME (DiHOME) DHODE (DiHODE) DHOE DHA DPE DTE EPA (EPE) ETE EED EEE EDPE (EpDPE, EDP) EETE (EpETE, EEQ) EETrE (EpETrE, EET) EHETE EODE EOME (EpOME)

FA number] 22:5 20:1 20:4 20:3 18:1 18:2 18:1 22:6 22:5 22:4 20:5 20:3 20:2 20:1 22:5 20:4 20:3 20:4 18:2 18:1

The numerical infix originates from the fatty acid (FA) number (i:j ), which refers to the numbers of carbon atoms [i C] and double bonds. Arachidonic (AA), eicosapentaenoic (EPA), and docosahexaenoic acids (DHA) are precursors of eicosanoids

which correspond to prostaglandin-I (PGi2 deriving from the ω6-arachidonic acid and PGi3 from the ω3-eicosapentaenoic acid (EPA)], and thromboxanes [thromboxane-A2 and -B2 ]). Numeral subscripts associated with prostaglandins refers to the number of double bonds in the side chains (e.g., PGe1 and PGe2 for prostaglandin-E1 and -E2 ). Many prostaglandin species exist, a letter being assigned to each type (type-D, -E, -F, H, and -I [aliases PGd, PGe, PGf, PGh, and PGi]). This notation is thus distinct from that used for molecule isoforms (e.g., ACi: adenylate cyclase isoform-i and Ci: complement component-i [i: natural number]; F: coagulation [or clotting] factor- [: Latin (Roman) numeral]). Prostaglandin-H2 synthase (alias PGh2 S) and prostaglandin endoperoxide-H synthases PGhS1 and PGhS2 (or cyclooxygenases COx1 and COx2) produce the precursor prostaglandin-H2 of 2 prostanoid families of eicosanoids: prostaglandins and thromboxanes. Leukotrienes are other eicosanoid types that act as inflammatory mediators produced in leukocytes by the oxidation of arachidonic acid by arachidonate 5lipoxygenase (alias ALOx5). These lipid messengers serves in autocrine and paracrine signaling to regulate immunity. Their structure is characterized by

Notation Rules: Abbreviations, Aliases, and Symbols

771

Table 4 Aliases of fatty acid derivatives (Part 2) Type Heptyloxytridecenoic acid Hexyloxytetradecenoic acid Hydroperoxydocosahexaenoic acid Hydroperoxyeicosadienoic acid Hydroperoxyeicosapentaenoic acid Hydroperoxyeicosatetraenoic acid HPETE ethanolamide Hydroperoxyoctadecadienoic acid Hydroperoxyoctadecatrienoic acid Hydroxydocosahexaenoic acid Hydroxyeicosapentaenoic acid Hydroxyeicosatetraenoic acid HETE ethanolamide Hydroxyepoxyeicosatrienoic acid Hydroxyoctadecadienoic acid Hydroxyoctadecatrienoic acid Pentyloxypentadecenoic acid Trihydroxyeicosadienoic acid Trihydroxyeicosatrienoic acid Trihydroxyoctadecamonoenic acid

Alias HTrDE (HTDA) HxTDE (HTDA) HPDHE (HPDoHE) HPEDE HPEPE HPETE HPETEE HPODE HPOTrE (HPOTE) HDHE (HDoHE) HEPE HETE HETEE HEET HODE HOTrE PPDE (PPDA) THED THET THOME

FA number] 13:1 14:1 22:6 20:2 20:5 22:4 22:4 18:2 18:3 22:6 20:5 20:4 20:4 20:3 18:2 18:3 15:1 20:2 20:3 18:1

four double bonds, hence the notation LTa4 , LTb4 , LTc4 , LTd4 , and LTe4 for leukotriene-A4 , -B4 , -C4 , -D4 , and -E4 .

Enzyme Sets Synthases and synthetases catalyze condensation reactions without and with nucleoside triphosphate as a source of energy, respectively (e.g., citrate synthase and succinylCoA synthetase). In this book series, this dictinction is omitted. Enzymes are classified into many sets according to their activity. 1. Hydrolases break single bonds using water. 2. Isomerases provoke structural changes within a molecule (change in shape). They encompass epimerases, isomerases, and racemases. 3. Ligases join two substrates using energy (ATP or another energy source), DNA ligases closing breaks in DNA molecules using energy supplied by ATP or NAD+ . 4. Lyases cleave a specific form of bond and rearrange electronic organization to generate a double bond, breaking one bond or adding one bond, the reactant

772

Notation Rules: Abbreviations, Aliases, and Symbols Table 5 Examples of polyunsaturated fatty acids (PUFA) of the ω3 and ω6 sets Type Alias ω3-Polyunsaturated fatty acids Hexadecatrienoic acid HTA α-Linolenic acid ALA Stearidonic acid SDA Eicosatrienoic acid ETE Eicosatetraenoic acid ETA Eicosapentaenoic acid EPA Heneicosapentaenoic acid HPA Docosapentaenoic acid DPA Docosahexaenoic acid DHA ω6-Polyunsaturated fatty acids Linoleic acid LA γ-Linolenic acid GLA Dihomo-γ-linolenic acid DGLA Arachidonic acid AA

Lipid number 16:3 18:3 (octadecatrienoic acid) 18:4 (octadecatetraenoic acid) 20:3 20:4 20:5 21:5 22:5 22:6 18:2 (octadecadienoic acid) 18:3 (octadecatrienoic acid) 20:3 (eicosatrienoic acid 20:4 ([5,8,11,14]-eicosatetraenoic acid

PUFAs possess more than a single carbon–carbon double bond, whereas monounsaturated fatty acids (MUFA) have one double bond in the fatty acid chain and all other single-bonded carbon atoms. ω3-Fatty acids have a double bond 3 carbons away from the methyl (–CH3 ) end, or ω end, i.e., in the –3 position; ω-6 fatty acids have a double bond 6 carbons away from the ω end, i.e., in the –6 position

being broken without adding water and creating a double bond. They comprise aldolases, carboxylases, and dehydratases. 5. Oxidoreductases catalyze oxidation or reduction reactions, in which electrons are transferred from one molecule (the reductant) to another one (the oxidant). They include dehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, and reductases. 6. Transferases transfer a functional group from one molecule to another. In particular, kinases (phosphotransferases) transfer a phosphoryl group (phosphorylation) from a donor (usually ATP) to an acceptor molecule, a sugar (e.g., glucokinase), protein (e.g., protein Tyr or Ser/Thr kinases), lipid (e.g., phosphatidylinositol 3-kinases), another nucleotide (e.g., hexameric nucleoside diphosphate kinase [NDK/NDPK], which exists as NDKa/NDPKa and NDKb/ NDPKb isoforms encoded by the NEM1 and NME2 gene, respectively, among

Notation Rules: Abbreviations, Aliases, and Symbols

773

other subtypes),9 or a metabolic intermediate (e.g., cytosolic phosphoenolpyruvate [PEP] carboxykinase PEPCK1 or simply PCK1, which is involved in gluconeogenesis, as it forms phosphoenolpyruvate from oxaloacetate using GTP and releasing GDP and CO2 , the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the tricarboxylic acid cycle; the PCK2 subtype is mitochondrial). Conversely, phosphatases dephosphorylate their substrates, whereas phosphorylases cause a displacement reaction in which phosphate from an inorganic phosphate becomes covalently attached to an acceptor.

Protein Posttranslational Modifications Numerous types of protein posttranslational modifications are achieved by specialized enzymes and modifying complexes. The notation commonly used in this book series for any posttranslational modification target site is AAj (AA: amino acid; j : position of amino acid subtrate in the polypeptidic chain). In the nucleus, chromatin modifications influence affinity for adjacent DNA and histones, the basic positively charged proteic scaffold around DNA. Posttranslational modifications of the five histone types include acetylation (ac), adpribosylation (ar), which comprises marylation (MAR: monoADP ribose) and parylation (PAR: polyADP ribose), biotinylation (bi), butyrylation (bu), citrullination (ci), crotonylation (cr), formylation (fo), glycosylation (gl; i.e., oglcnacylation), malonylation (ma), methylation (me), myristoylation (my), palmitoylation (pa), phosphorylation (ph), proline isomerization, propionylation (pr), succinylation (sc), sumoylation (su),10 and ubiquitination (ub). The notation for posttranslational modifications of histone sites (HiAAj ; i: histone species) differs from that used for histone marks (HiAAj ptm; ptm: ac, ar, bi, gl, me, ph, su, ub, etc.). The superscript 1–3 attached to methylation refers to mono-, di-, or trimethylated forms. A modified residue is written as AAPtm (e.g., KAc , KCr , and KMe stand for acetylated, crotonylated, and methylated lysine).

9 Both

NDPKa and NDPKb are involved in the synthesis of nucleoside triphosphates other than ATP. NDPKa acts also as a nucleoside diphosphate kinase, protein Ser/Thr kinase, geranyl and farnesyl pyrophosphate kinase, and 3 –5 -exonuclease. The NDPKc isoform is encoded by the NME3 gene. The NDPKd subtype encoded by the NME4 gene lodges in the mitochondrion. NDPK5 to NDPK7 are encoded by the NME5 to NME7 gene, NDPK5 being likely a pseudokinase. 10 Sumo: small ubiquitin-like modifier.

774

Notation Rules: Abbreviations, Aliases, and Symbols

Molecular Aliases Molecules are also labeled by aliases, especially those that have very long chemical names.11 For example, ABC stands for ATP-binding cassette transporter and SLC for solute carrier superclass member. Aliases that designate different types of molecules and those without an obvious meaning should be eliminated for a rapid understanding in multidisciplinary frameworks by investigators of other scientific fields; they are thus not used in the present textbook. In biology, a given molecule usually possesses many aliases. Conversely, a given alias commonly refers to various types of molecules.12 For example, P35 is an alias for annexin-A1, brain syntaxin-1A, ficolin-2, interleukin-12A, the cyclin-H assembly factor ménage à trois homolog-1, regulatory subunit-1 of cyclindependent kinase CDK5, and uroplakin-3B, among others. It is substituted by AnxA1, Stx1a, Fcn2, IL12a, MAT1, CDK5r1 , and UPk3b aliases, respectively. Protein P39 corresponds to the subunit D1 of the lysosomal V-type H+ ATPase (ATP6v0d1), the transcription factor Jun, a component of activator protein AP1, and regulatory subunit-2 of cyclin-dependent kinase CDK5 (CDK5r2 ). Extracellular signal-regulated protein kinases ERK1 and ERK2, members of the mitogen-activated protein kinase (MAPK) module (last tier), are also abbreviated P44 and P42 (also P40 and P41). However, both P42 and P44 correspond to the 26S protease regulatory AAA ATPase subunit (PSMC6). The alias P42 is also utilized for cyclin-dependent kinase CDK20, cyclin-dependent kinase-like protein CDKL1, and 43-kDa NuP43 nucleoporin. The alias P44 can also refer to interferon-induced protein IFI44 (or microtubule-associated protein MTAP44) and androgen receptor cofactor P44 (a.k.a. methylosome protein MeP50 and WD repeat-containing protein WDR77). The numbering of mitogen-activated protein kinase (MAPK) isoforms, which are categorized into three families (ERK, JNK, and P38MAPK), is neither straight-

11 Latin

alias: at another time, some other time, at other times.

12 See

1. Information Hyperlinked over Proteins (iHOP; www.ihop-net.org) and the corresponding reference: Hoffmann R, Valencia A (2004) A gene network for navigating the literature. Nature Genetics 36:664. 2. Online Mendelian Inheritance in Man (OMIM; www.omim.org), An Online Catalog of Human Genes and Genetic Disorders (1966–2016) McKusick–Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine. 3. Universal Protein Resource (UniProt) Consortium (2002–2016; www.uniprot.org) European Bioinformatics Institute, Swiss Institute of Bioinformatics and Protein Information Resource. 4. BioGRID: General Repository for Interaction Datasets (www.thebiogrid.org), a database of physical and genetic interactions for model organisms. 5. GeneCards human gene database (www.genecards.org). Crown Human Genome Center, Department of Molecular Genetics, the Weizmann Institute of Science.

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forward nor founded on unicity (ERK2 is also called MAPK1 and MAPK2 and MAPK15 refers to both ERK7 and ERK8). In the present text, stress-activated members of the P38 family (P38α–P38δ)13 are designated as P38MAPKs to avoid confusion with other molecules, the alias of which is also P38. Indeed, the alias P38 stands for: (1) extracellular signal-regulated kinase ERK3 and ERK6, (2) adaptor CRK (chicken tumor virus regulator of kinase, or v-crk sarcoma virus CT10 oncogene homolog), (3) growth factor receptor-binding protein GRB2-related adaptor protein GRAP2 (a.k.a. GRID, GADS, GRB2L, GRF40, GRPL, and Mona), (4) ubiquitin ligase RING finger protein RNF19a, or dorfin, (5) 38-kDa DNA polymerase-δ-interacting protein PolδIP2 (a.k.a. polymerase [DNA-directed] PDIP38 and PolD4), (6) activator of 90-kDa heat shock protein ATPase homolog AHSA1, and (7) aminoacyl tRNA synthase complex-interacting multifunctional protein AIMP2, or tRNA synthase complex component JTV1.

Types of Aliases Aliases include all written variants, i.e., any abbreviation such as acronyms that are spoken as a word.14 Aliases are either entirely written with uppercase letters, or the first letter is in uppercase and the following letters in lowercase (e.g., AIDS or Aids). An abbreviation formed from a string of initials and spoken as individual letters is usually entirely capitalized (e.g., DNA, which stands for deoxyribonucleic acid; HIV, for human immunodeficiency virus; and HTML, for HyperText Markup Language). Abbreviations can be strings of constituent letters of sequence of words, such as pixel and voxel for two-dimensional [2D] picture and three-dimensional [3D] volume elements.15 These mixtures of initial and noninitial letters are generally spoken as words and written in lowercase. Aliases can be partly written with uppercase letters, mixed-case variants being words deriving from an acronym by affixing, so the root acronym remains obvious (e.g., DNase, an alias for deoxyribonuclease). Derived acronyms are also expressed in mixed case (e.g., mRNA, rRNA, snRNA, and tRNA for messenger, ribosomal, small nuclear, and transfer RNA, respectively).

13 Protein

P38α is also called MAPK14, cytokine suppressive anti-inflammatory drug (CSAID)binding protein CSBP, CSBP1, or CSBP2, and stress-activated protein kinase SAPK2a; P38β as MAPK11 and SAPK2b; P38γ as MAPK12, ERK6, and SAPK3; and P38δ as MAPK13 and SAPK4. 14 ακρo-: end, tip (ακρoκωλιoν: extremities of the body; ακρoπoυς: extremity of the leg [πoυς: foot; κωλην: leg; κωλoν: limb]; ακρoρρινιoν: tip of the nose); oνυμα: name. 15 In computed tomography, the voxel size depends on the matrix size, selected field of view, and CT section thickness.

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Notation Rules: Abbreviations, Aliases, and Symbols

In general, abbreviations exclude the initials of short function words, such as “and,” “or,” “of,” or “to.” However, they are sometimes included to make them pronounceable (e.g., radar [originally RADAR] for radio detection and ranging. Acronyms are generally written with all letters in uppercase (all-uppercase acronyms). Yet, some acronyms, anacronyms, are treated as regular words, spoken as words, and written in lowercase (e.g., laser [originally LASER], an acronym for light amplification by stimulated emission of radiation, which consists only of initial letters, and sonar [originally SONAR] for sound navigation and ranging, which includes noninitial letters in addition to initials of short function words). A substance’s noun can be its abbreviation that derives from its chemical name (e.g., amphetamine: α-methylphenethylamine, which also contains noninitial letters and is pronounced as a word). Acronyms can give rise to molecule names by adding a scientific suffix such as “-in,” a common ending of molecule nouns (e.g., sirtuin, a portmanteau that comes from the alias SIRT, which stands for silent information regulator-2 [two]). However, both cardinal (size, molecular weight, etc.) and ordinal (isoform discovery order) numbers in names are most often represented by digits rather than initial letters. Other scientific prefixes and suffixes can be frequently detected throughout the present text. Many prefixes are used to specify position, configuration, behavior, quantity, direction, motion, structure, timing, frequency, and speed. Many molecules are portmanteau words, or blends, parts of multiple words and their meanings being combined into a new word (e.g., calmodulin stands for calcium modulated protein; caspase for cysteine-dependent aspartatespecific protease; chanzyme for ion channel and enzyme; chemokine for chemoattractant cytokine16 ; emilin for elastin microfibril interfacer; endorphins and endomorphins for endogenous morphines; ephrin for erythropoietinproducing hepatocyte (EPH) receptor kinase interactor17 ; granzyme for granule enzyme; moesin for membrane-organizing extension spike protein; porin for pore-forming protein; prompt for promoter upstream transcript18 ; restin for Reed–Steinberg cell-expressed intermediate filament-associated protein, an alias for cytoplasmic linker protein CLiP1 (or CLiP170); serpin for serine protease inhibitor; siglec for sialic acid-binding Ig-like lectin; and transceptor for transporter-related receptor.

16 Cytokines

are peptidic, proteic, or glycoproteic regulators that are secreted by cells of the immune system. These immunomodulating agents serve as auto- or paracrine signals. 17 Ephrin is a macronym (i.e., a nested or multilayered acronym) the first letters corresponding to the acronym EPH, which means erythropoietin-producing hepatocyte receptor kinase. Ephrin stands for EPH receptor interactors. In this acronym, the abbreviated word receptor is redundantly included. 18 The uppercase initial P in Prompt was used in the first volumes of this book series to avoid confusion with the command-line interpreter prompt or prompt book to direct precise timing of actions on the theater stage. However, the context eliminates ambiguity; the word is entirely written in lowercase letters.

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Certain types of aliases that contain noninitial letters are pronounced as a string of letters, such as PMNL (polymorphonuclear leukocytes, also called granulocytes, i.e., immunocytes possessing granules storing enzymes that are released during inflammation) and HSPG (heparan sulfate proteoglycan, i.e., glycoproteins containing at least one covalently attached heparan sulfate chains, a type of glycosaminoglycan, which comprise secretory vesicle proteoglycan [serglycin], membrane-bound elements [e.g., syndecans and glypicans], and secreted matrix constituents [e.g., agrin, perlecan, and collagen-18]). Some abbreviations are new words or new meanings of known words (e.g., rock: Rho-associated coiled-coil-containing protein kinase, also called Rho kinase, which, in the context, cannot be confused with the geological term). In biochemistry, abbreviations also represent relatively long (e.g., Cam stands for calmodulin, which is itself a portmanteau, and Trx for thioredoxin) or short molecular nouns (e.g., Plxn for plexin and Ttn for titin). In addition, single-letter symbols of amino acids are often used to define a molecule alias (e.g., tyrosine can be abbreviated as Tyr or Y, hence SYK stands for spleen tyrosine kinase). Aliases use, in general, capital letters and can include hyphens and dots. Yet, as a given protein can represent a proto-oncogene19 encoded by a gene that can give rise to an oncogene (tumor promoter) after gain- or loss-of-function mutations,20 the same acronym represents three different entities.21

19 In

1911, P. Rous isolated a virus that was capable of generating tumors of connective tissue (sarcomas) in chickens. Proteins were afterward identified, the activity of which, when uncontrolled, can provoke cancer, hence the name oncogene given to genes that encode these proteins. Most of these proteins are enzymes, more precisely kinases. The first oncogene was isolated from the avian Rous virus by D. Stéhelin and called Src (from sarcoma). This investigator demonstrated that the abnormal functioning of the Src protein resulted from mutation of a normal gene, or proto-oncogene, which is involved in cell division. 20 Loss-of-function mutations cause complete or partial loss of the function of gene products that operate as tumor suppressors, whereas gain-of-function mutations generate gene products with new or abnormal function that can then act as oncogenes. Typical tumor-inducing agents are enzymes, mostly regulatory kinases and small guanosine triphosphatases, that favor the proliferation of cells that normally need to be activated to exert their activities. Once their genes are mutated, these enzymes become constitutively active. Other oncogenes include growth factors (a.k.a. mitogens) and transcription factors. Mutations can also disturb signaling axis regulation, thereby raising protein expression. Last, but not least, chromosomal translocation can also provoke the expression of a constitutively active hybrid protein. 21 Like Latin-derived shortened expressions and foreign words, which are currently written in italics, genes can be italicized. However, this usage is not required in scientific textbooks published by Springer. Italic characters are then used to highlight words within a text to target them easily. Proteins are currently romanized (ordinary print), but with a capital initial, as in most (if not all) scientific articles. Nevertheless, long names (not aliases) of chemical species are entirely in lowercase, as the context suffices for disambiguation, confusion with a usual word (e.g., hedgehog: animal versus protein [but Hedgehog gene]; notch: indentation or incision vs. morphogen [but Notch gene]) being easily avoided.

778

Notation Rules: Abbreviations, Aliases, and Symbols

In addition, a given abbreviation can designate distinct molecules without necessarily erroneous consequence in a given context (e.g., PAR: polyADP ribose or protease-activated receptor and GCK: germinal center kinases or glucokinase; in the latter case, the glucokinase abbreviation should be written as GcK or, better, GK).

Molecule Aliases and Adopted Notation Rules Numerous aliases that designate a single molecule can result from the fact that molecules have been discovered independently several times with possibly updated functions. Some biochemists render in upper case the name of a given molecule, whereas others partly use lower case (e.g., cell division cycle guanosine triphosphatase of the RHO family CDC42 or Cdc42, adaptor growth factor receptor-bound protein GRB2 or Grb2, chicken tumor virus regulator of kinase CRK or Crk, guanine nucleotideexchange factor Son of sevenless SOS or Sos, etc.). Acronyms are then not always entirely capitalized. The printing style of aliases should not only avoid confusion, but also help the reader to remember the meaning of the alias. In the present textbook, the choice of lower- and uppercase letters in molecule aliases is dictated by the following criteria. (1) An uppercase letter is used for initials of words that constitute molecule nouns (e.g., receptor tyrosine kinase RTK). An alias of any compound takes into account added atoms or molecules (e.g., PI: phosphoinositide and PIP: phosphoinositide phosphate) in addition to their number (e.g., PIP2 : phosphatidylinositol bisphosphate, DAG: diacylglycerol, and PDE: [cyclic nucleotide] phosphodiesterases). (2) A lowercase letter is used when a single letter denotes a subfamily or an isoform when it is preceded by a capital letter (e.g., PTPRe: protein tyrosine phosphatase receptor-like type-E). Nevertheless, an uppercase letter is used in an alias after a single or several lowercase letters to distinguish the isoform type (e.g., RhoA isoform and DNA-repair protein RecA for recombination protein-A), but OSM stands for oncostatin-M, not Osm used to designate the unit osmole22 to optimize molecule identification. These criteria enable the use of differently written aliases with the same sequence of letters for distinct molecules (e.g., CLIP for corticotropin-like intermediate peptide, CLiP: cytoplasmic CAP-Gly domain-containing linker protein, and iCliP: intramembrane-cleaving peptidase). As the exception proves the rule, current aliases, such as PKA and PLA that designate protein kinase-A and phospholipase-A, respectively, have been kept. Preceded by only two uppercase letters, a lowercase letter that should be 22 Osmole:

the amount of osmotically active particles that exert an osmotic pressure of 1 atm when dissolved in 22.4 l of solvent at 0 ◦ C.

Notation Rules: Abbreviations, Aliases, and Symbols

779

used to specify an isoform can bring confusion with acronyms of other protein types (e.g., phospholamban alias PLb). Nouns (e.g., urokinase-type plasminogen activator [uPA]) or adjectives (e.g., intracellular FGF isoform [iFGF] in addition to endocardial endotheliocyte [eEC] and vascular smooth myocyte [vSMC]) that categorize a subtype of a given molecule or cell correspond to a lowercase letter to emphasize the molecule or cell species. Hence, an uppercase letter with a commonly used hyphen (e.g., I[R]SMAD that stands for inhibitory [receptor-regulated] SMAD; V-ATPase for vesicular adenosine triphosphatase23 ; MT1-MMP for membrane type-1 matrix metalloproteinase; and T[V]-SNARE for target [vesicle-associated] soluble N ethylmaleimide-sensitive factor-attachment protein receptor) is then replaced by a lowercase letter (e.g., i[r]SMAD, vATPase, mt1MMP, and t[v]SNARE), as is usual for RNA subtypes. Similarly, membrane-bound and secreted forms of receptors and coreceptors that can derive from alternative mRNA splicing are defined by a lowercase letter (e.g., sFGFR for secreted extracellular FGFR form and sFRP for soluble frizzled-related protein, which are now written FGFRS and S FRP, respectively). Localization of molecules is highlighted using left subscripts and superscripts (e.g., m IJK and S IJK for membrane-bound and secreted forms of the molecule IJK; see Lists of Notations - Subscripts and Superscripts). Left superscripts denote molecules linked to a given cell and organelle type (e.g., EC IJK and SMC IJK designate an endotheliocytic and smooth myocytic origin of the molecule IJK; PM IJK and Ve IJK a plasmalemmal and vesicular situation) enable to easily differentiate a given type of protein involved in intercellular interactions, between-organelle crosstalk, and material transfer between subcellular compartments. (3) Although l, r, and t can stand for molecule-like, -related, and -type, respectively, when a chemical is related to another one, in general, uppercase letters are used for the sake of homogenity and to clearly distinguish between the letter L and numeral 1 (e.g., KLF: Krüppel-like factor, CTK: C-terminal Src kinase (CSK)-type kinase, and SLA: Src-like adaptor). (4) An uppercase letter is most often used for initials of adjectives contained in the molecule name (e.g., AIP: actin-interacting protein; BAX: BCL2-associated X protein; HIF: hypoxia-inducible factor; KHC: kinesin heavy chain; LAB: linker of activated B lymphocytes; MAPK: mitogen-activated protein kinase; and SNAP: soluble N ethylmaleimide-sensitive factor-attachment protein). In the last-mentioned example, the left superscript N corresponds to the attachment of a molecule (e.g., glycan in N-linked glycosylation) to a nitrogen atom of a protein. Similarly, the left superscript O defines the attachment of a molecule to an oxygen atom in an amino acid residue of a peptide (e.g., O-

23 In

yeasts, the vacuole represents the lysosome in mammalian cells.

780

Notation Rules: Abbreviations, Aliases, and Symbols

linked glycosylation, in which a carbohydrate group is covalently attached to a peptide). (5) Lowercase letters are used when alias letters do not correspond to initials (e.g., Fox [forkhead box]), except for portmanteau words that are entirely written in minuscules (e.g., gadkin: γ1-adaptin and kinesin interactor and beclin: type2 B-cell lymphoma (leukemia) protein (BCL2) interactor).24 This rule applies whether alias letters correspond to successive noun letters (e.g., Par: partitioning defective protein; Pax: paxillin; BrK: breast tumor kinase; and ChK: checkpoint kinase, whereas CHK denotes C-terminal Src kinase [CSK]-homologous kinase) or not (e.g., Fz: frizzled and HhIP: hedgehog-interacting protein),25 except for composite chemical species (e.g., DAG: diacylglycerol). However, some current usages have been kept for short aliases of chemical species name (e.g., Rho for Ras homolog rather than RHo). (6) Most molecule aliases containing at least four alphabetical characters (e.g., argonaute, drosha, smoothened, and sprouty) are treated as nouns; they contain only lowercase letters, particularly those that are also common nouns and adjectives (e.g., aubergine, dicer, fringe, fritz, frizzled, hairy, hedgehog, itch, jagged, kringle, masterming, notch, numb, patched, roundabout, serrated, shutdown, slingshot, slit, spire, split, etc.), as the context enables disambiguation. Other aliases keep their uppercase letters (e.g., UVRAG [ultraviolet wave resistance-associated gene product], UV being the usual abbreviation for ultraviolet), especially those that represents members of molecular sets (e.g., TNFSF1 that interacts with its receptor TNFRSF1a).26 In the book series “Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems”, tumor-necrosis factor-α (TNFα) is considered as the first member of TNF ligand superfamily (TNFSF1) and TNFβ, also called lymphotoxin-α, represents the second member (TNFSF2), whereas in the iHOP repository created by R. Hoffmann and in the GeneCards database of the Weizmann Institute of Science TNFSF1 and TNFSF2 correspond to TNFβ and TNFα, respectively. Aliases of mediators of the TOR pathway (TOR: target of rapamycin) utilize only lowercase letters (e.g., deptor: DEP domain-containing TORbinding protein; gator: GTPase-activating protein toward Rag; lamtor: late endosomal and lysosomal adaptor, MAPK and TOR activator; protor: protein observed with rictor; ragulator: Rag and TORC1 regulator complex; raptor: 24 The

BECN gene encodes beclin (alias Becn), a homolog of the autophagy-related protein Atg6. It encodes a protein involved in the determination of segmental polarity and intercellular signaling during morphogenesis. Homologous gene and protein exist in various vertebrate species. The name of the mammal hedgehog comes from hecg and hegge (dense row of shrubs or low trees), as it resides in hedgerows, and hogg and hogge, owing to its pig-like, long projecting nose (snout). The word Hedgehog hence is considered as a seamless whole. 26 Or TNFR1. 25 The Hedgehog gene was originally identified in the fruit fly Drosophila melanogaster.

Notation Rules: Abbreviations, Aliases, and Symbols

781

regulatory associated protein of TOR [the molecule cannot be mistaken for raptor, a bird of prey (e.g., eagle, peregrine falcon, goshawk, harrier, hawk, or owl)]; reptor: repressed by TOR; and rictor: rapamycin-insensitive companion of TOR). Therefore, this rule is used for any alias when it is straightforwardly pronounceable to avoid ugly mixtures of upper- and lowercase letters and to enable capitalization of the alias first letter at the beginning of a sentence as any noun, although uppercase letter-written aliases help searching in the text (a nowadays obvious task reading pdf files). According to the above-mentioned rules, these aliases should contain only uppercase letters, particularly those of some types of intercellular adhesion molecules, such as icam1 (and not ICAM1 [intercellular adhesion molecule-1]); pecam1 (and not PECAM1 [platelet–endotheliocyte adhesion molecule-1]); and vcam1 (and not VCAM1 [vascular cell adhesion molecule-1]), among others (e.g., ambra rather than AMBRA [activating molecule in beclin-1-regulated autophagy protein]), or uppercase letters mixed with lowercase letters, as are other types of cell adhesion molecules, such as bcam rather than bCAM (basal cell adhesion molecule); glycam rather than GlyCAM (glycosylation-dependent cell adhesion molecule); madcam rather than MAdCAM (mucosal vascular addressin cell adhesion molecule) and other mediators such as barkor (beclin1-associated autophagy-related key regulator). Nevertheless, this rule can bring confusion. The regulators of the circadian rhythm, periods, are clearly distinguished from a time interval, as they are associated in the sentence with cryptochromes (Cry1–Cry2), their function is mentioned, or their isoform aliases (Per1–Per3) are used. (7) Proteic complexes are abbreviated in uppercase letters, such as the FACT complex, which designates the heterodimeric histone chaperone that facilitates chromatin transcription (instead of FaCT); MAML complex, which stands for mastermind-like coactivator complex constituted of MamLi proteins (MamL1–MamL3); NURD complex for nucleosome remodeling and deacetylation complex; TRAPPC complex for transport protein particle complex made up of TraPPi components (ii: integer); and the polyadenylation TRAMP complex of the nuclear exosome involved in RNA quality control and degradation. This rule enables two similar aliases to be distinguished (e.g., NExT: notch extracellular truncation domain and NEXT: nuclear exosome-targeting complex [instead of NExT]). Proteic complexes can be defined by the initial letters of their components. For example, the helicase CMG complex corresponds to the CDC45L–MCM– GINS trimer, the ribosomal RNA-processing LNXD complex to the LAS1L– Nol9–XRN2–DXO tetramer, the nuclease MRN complex implicated in DNA double-strand break detection to the MRe112 Rad502 NBS12 hexamer, the other ribosomal RNA-processing PBW complex to the Pes1–BOP1–WDR12 trimer, the chaperone R3P complex to the RuvBL1, RuvBL2, RPAP3, and PIH1D1 tetramer, and the translation termination SRTRF complex to SMG1– ReNT1–TRF1–TRF3 tetramer.

782

(8)

(9)

(10)

(11)

Notation Rules: Abbreviations, Aliases, and Symbols

Proteic complexes can also be abbreviated using alias of their subunits that pertain the same family of proteins. For example, the GINS complex, that is, the GINS1–GINS2–GINS3–GINS4 tetramer was created from the first letters of the Japanese numbers 5, 1, 2, and 3 (go, ichi, ni, and san) in reference to the four proteic subunits of the complex (Sld5, Psf1, Psf2, and Psf3) described in the yeast Saccharomyces cerevisiae. In general, aliases of genes and their corresponding transcripts are written with majuscules when the corresponding protein aliases contain only lowercase letters (e.g., the ADAM10 gene encodes a disintegrin and metallopeptidase domain-containing protein-10 [adam10], also called adamlysin-10 and the AMBRA1, CALCOCO1 and 2, and NOTCH1 to 4 genes encodes ambra-1, calcium-binding and coiled-coil domain-containing proteins calcoco-1 and -2, and notch-1 to -4, respectively) or at least a single minuscule (e.g., the PINK1 and PTEN gene encodes PTen-induced kinase PInK1 and phosphatase and tensin homolog deleted on chromosome 10 [PTen], respectively). Conversely, gene and transcript aliases are written with a first capital letter followed by minuscules when the corresponding protein aliases contain only uppercase letters (e.g., the PIN4 rotamase of the mitochondrial matrix is encoded by the Pin4 gene). Aliases designating a molecule category, group, or set (i.e., class, subclass, hyperfamily, superfamily, family, subfamily) and their members are written in capital letters, such as IGSF (IGSFi: immunoglobulin superfamily member i), KIF (KIFi: kinesin family member i), SLC (SLCi solute carrier superclass member i), TNFSF (TNFSFi; tumor-necrosis factor superfamily member-i), and TNFRSF (TNFRSFi; tumor-necrosis factor receptor superfamily memberi). Whereas Src (sarcoma-associated [Schmidt–Ruppin A2 viral oncogene homolog) is a kinase, the alias SRC stands for the SRC family, which encompasses many kinases, the SRC family kinases, and similarly for the NUMB family of clathrin-associated sorting proteins and monomeric (small) GTPases of the RHO category, among others. To highlight its function, substrate aliases (e.g., ARF GTPases) contained in a molecule alias are partly written with lowercase letters (e.g., ArfRP, ArfGEF, ArfGAP stand for ARF-related protein, ARF guanine nucleotide-exchange factor, and ARF GTPase-activating protein, respectively). Abbreviation of chemical species, the name of which share a suffix related to a function, such as -globin (e.g., cytoglobin, hemoglobin, myoglobin, neuroglobin, and secretoglobin)27 ; -granin (e.g., chromogranin and secre-

27 From

Latin globus: round body, ball, globe, sphere.

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togranin)28 ; -kinin (e.g., bradykinin and cholecystokinin)29 ; -medin (e.g., neuromedin, olfactomedin, and somatomedin)30 -nectin (e.g., adiponectin, fibronectin, osteonectin, and vitronectin)31 ; -poietin (e.g., angiopoietin, ery-

28 From

Latin granum: grain, seed, small kernel and granulum: a small grain. Chromoand secretogranin constitute the granin family of ubiquitous secretory proteins detected in secretory vesicles containing amine and peptide hormone and neurotransmitter of endocrine and neuroendocrine cells. These prohormones engender peptidic messengers with auto-, para-, and endocrine effects. Chromogranins include ChgA/CgA, or parathyroid secretory protein PSP1, and ChgB/CgB, or secretogranin-1 (Scg1/Sg1), which are encoded by the CHGA and CHGB genes. Secretogranins comprise Scg2/Sg2 (chromogranin-C), which engenders secretoneurin, a protein involved in chemotaxis of monocytes and endotheliocytes and regulation of endotheliocyte proliferation, Scg3/Sg3, and Scg5/Sg5 (secretory granule neuroendocrine protein SGNE1), which are encoded by the SCG2, SGC3, and SCG5 genes. 29 βραδυ: slow; κιν ω: set in motion; χoλη: gall, bile; κυστις: bladder. Bradykinin is an inflammatory mediator that causes vasodilation. It also inhibits angiotensin-2 formation. Cholecystokinin boosts gallbladder contraction as well as pancreatic and gastric acid secretion to facilitate digestion within the small intestine. 30 From Latin mediator, medianus (in the middle), and mediastinus (a common servant). Somatomedins constitute a group of proteins that promote cell growth and division. Somatomedin-A, -B, and -C correspond to IGF2, IGF1, and vitronectin, respectively. Neuromedins serve in interneuronal communications. Neuromedin-B stimulates smooth muscle contraction. Neuromedin-C is a cleavage product of the precursor that also engenders gastrin-releasing peptide (GRP). Neuromedin-K (also called tachykinin-3 and neurokinin-B) and -L (also neurokinin-A and substance-K) belong to the kanassin subset of the tachykinin group. Precursors of tachykinins, α-, β-, and γ-preprotachykinins, are generated by the TAC1 gene due to alternative splicing of the TAC1 transcript. Protachykinin-1 encoded by the TAC1 gene is cleaved into NMl (NKa), neuropeptide-K (NPk) and- γ, substance-P, and C-terminal–flanking peptide. In airways, tachykinins couple with NK1 to NK3 receptors to cause bronchoconstriction, plasma protein extravasation, and mucus secretion, and to attract and activate immunocytes. In addition, NMl stimulate intestinal contraction. Neuromedin-N, which shares the same precursor as neurotensin (pro[NT/NMn), supports chemotaxis of lymphocytes. Neuromedin-U is a potent constrictor on the uterus. It abounds in the gastrointestinal tract. It stimulates contractions of the gastrointestinal walls and urinary bladder. Neuromedin-S is formed in the suprachiasmatic nucleus. Neuromedin NMu and NMs are anorexigenic neuropeptides. Olfactomedins (OlfMs/Olfms) are characterized by a domain discovered in glycoproteins of the olfactory neuroepithelium. In humans, they constitute a family of 13 members. They mediate signaling in early neurogenesis and hematopoiesis. 31 Nectins and nectin-like molecules are transmembrane proteins primarily involved in cell adhesion (from Latin necto: to bind, connect, fasten together, join, tie). Nectar (ν κταρ) is a sugary fluid secreted by plants to attract insects for pollination. Adiponectin has antiapoptotic, anti-inflammatory, and antioxidative effects. It favors fatty acid oxidation via AMPK and improves insulin sensitivity. Fibronectin is an abundant soluble glycoprotein of body fluids and insoluble component of the extracellular matrix. It tethers to plasmalemmal integrins as well as matrix constituents, such as collagen, fibrin, and heparan sulfate proteoglycans. It operates in cell adhesion, differentiation, migration, and proliferation. Vitronectin is another glycoprotein that abounds in blood and the extracellular matrix. It connects to proteoglycans and certain types of integrins.

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thropoietin, and thrombopoietin)32 ; -regulin (e.g., amphiregulin, epiregulin, neuregulin)33 ; -statin (e.g., cystatin, enterostatin, follistatin, myostatin, and somatostatin)34 ; -tensin (e.g., angiotensin and neurotensin),35 and -trophin

Osteonectin, which is encoded by the SPARC gene (secreted protein acidic and rich in cysteine) binds to calcium, copper, collagen, albumin, thrombospondin, PDGF, and cell membranes. It abounds in mineralized organs. It regulates crosslinking of matrix proteins by transglutaminases. 32 πoιησις: fabrication, creation, production, poiesis [creative production]. This suffix indicates that the messenger stimulates production, growth, and proliferation of certain cell types, such as red blood capsules (erythropoietin), platelets (thrombopoietin), and endotheliocytes, angiopoietin2 produced by endotheliocytes countering angiopoietin-1 synthesized in smooth myocytes and pericytes that promotes angiogenesis. 33 From Latin regulo: to control, direct, handle, govern, manage. Amphiregulin, epiregulin, and neuregulin are members of the EGF superfamily. Amphiregulin and epiregulin are coexpressed. Crosscommunication either stimulates or inhibits cell proliferation. Epiregulin binds to the receptor HER1 (EGFR) and HER4 and primes their heterodimerization with HER2 and HER3, thereby contributing to inflammation, organ healing and repair, and angiogenesis. Neuregulins Nrg1 and Nrg2 binds to HER3 and HER4 and concomitantly recruits HER1 and HER2, activating the MAPK module and PKB1. 34 From Latin statuo: to cause to stand, fix upright, erect, set up, station, position, place, set, locate. Cystatins (Csts) are competitive inhibitors of cysteine peptidases. They can be categorized into three families: (1) intracellular unglycosylated stefins StfA and StfB (CstA–CstB); (2) extracellular cystatins (CstC, CstD, CstE, CstF, CstM, CstS, CstSA, and CstSN), which are glycosylated or not; and (3) glycosylated kininogens (kininogen-1, fetuin-A and -B, and histidine-rich glycoprotein [HRGP]). Enterostatin is a pentapeptide (Ala–Pro–Gly–Pro–Arg) released from cleavage of procolipase by trypsin in the gastrointestinal tract during lipid digestion. It decreases fat intake and insulin secretion and increases energy expenditure in brown adipose tissue during high-fat feeding. Somatostatin (stagnation in a body) is synthesized in and secreted by neurons such as those of the hypothalamus, from which it is transported through portal vessels in the pituitary gland, in addition to δ cells of the islets of Langerhans in the pancreas and gut mucosa. This inhibitory hormone as well as auto- and paracrine regulator and neurotransmitter inhibits the activity of cells in the central nervous system, hypothalamus, and anterior pituitary gland, gastrointestinal tract, exocrine and endocrine pancreas, and immune system. In the anterior pituitary, somatostatin prevents release of growth hormone (GH), or somatotropin, and thyrotropin-stimulating hormone (TSH). This neurotransmitter inhibits voltage-gated Ca2+ channels in hippocampal pyramidal neurons as well as precludes activity of neurons in the locus ceruleus and medial amygdala. This neuromodulator influences serotonin release in the hypothalamus and facilitate dopamine release in the nucleus accumbens and striatum. Somatostatin participates in regulating body temperature, blood pressure, and satiety. Somatostatin suppresses gastric acid secretion and activity of certain pancreatic and gastrointestinal hormones. Follistatin antagonizes myostatin (GDF8), a member of the TGFβ set, which limits the size of developing skeletal muscles in cooperation with others factors. Follistatin can increase muscle mass and strength. It also suppresses synthesis in the pituitary gland and secretion of folliclestimulating hormone (FSH). 35 From Latin tensio: stretching (τ νων: tendon, ligament). Neurotensin is an enteric and cerebral peptide; It is produced by endocrine cells (N cells) of the intestine (primarily the ileum) and the myenteric plexus as well as in the central and peripheral nervous systems, heart, adrenal glands, pancreas, and respiratory tract. It stimulates pituitary hormone release (ACTH, GH, FSH, LH,

Notation Rules: Abbreviations, Aliases, and Symbols

785

(e.g., bestrophin, cardiotrophin, dystrophin, and neurotrophin),36 can be written using capital letters for the initial letter of both the compounds and above-mentioned suffixes to highlight its presence (e.g., BdK, CCK, AdpN, FN, VN, AngPt, EPo, TPo, AReg, EReg, NRg, CSt, ESt, FSt, SSt, AgT, and NTs) or, to avoid an inner mixture of upper- and lowercase letters in an alias of a single noun, using a majuscule followed only by minuscules (e.g., Bdk, Cck, Adpn, Fn, Vn, Angpt, Epo, Tpo, Areg, Ereg, Nrg, Cst, Est, Fst, Sst, Agt, and Nts). The latter writing rule is selected in the absence of confusion (e.g., GaL [galectin] and Gal [galanin]). (12) Heavy and pedantic designation of protein isoforms based on Roman numerals has been avoided and replaced by Arabic numerals (e.g., angiotensin-2 rather than angiotensin-I I ). The character I can mean either letter I or number 1 without obvious discrimination at first glance (e.g., GAPI stands for Ras GTPase-activating protein GAP1, but the abbreviation GAPI can be used to designate a growth-associated protein inhibitor). However, the molecular categories are defined by Roman numerals. For example, sirtuins can be classified in group I (SIRT1–SIRT3), which have robust deacetylase activity, whereas those of group II (SIRT4), III (SIRT5), and group IV (SIRT6–SIRT7) have weak deacetylase activity (but SIRT6 is a Lys depalmitoylase and demyristoylase and SIRT5 a deglutarylase, demalonylase, and desuccinylase). Many types of cellular membrane proteins can be defined. • Type-I transmembrane proteins have their C-termini in the cytoplasm and N-termini outside the cell. • Type-I I transmembrane proteins have their N-termini in the cytoplasm and their C-termini outside the cell. • Type-I I I transmembrane proteins cross the plasma membrane more than once, the single polypeptide having multiple transmembrane domains. • Type-I V transmembrane proteins possess several different polypeptides assembled together in a transmembrane channel. • Type-V membrane proteins are anchored to the lipid bilayer via covalently linked lipids such as glycosylphosphatidylinositol (GPI)-anchored proteins. • Type-V I proteins have both a transmembrane domain and lipidic anchors.

and prolactin) and pancreatic, gastric, and intestinal secretion and provokes vasodilation, whereas angiotensin is a potent vasoconstrictor. 36 τρoφη: nourishment, food; τρoφητικoς: concerning maintenance; τρoφικoς: nursing; τρoφιoν: aliment, maintenance; τρoφις: well-fed, stout (heavy build, strong); τρoφoς: feeder; τρoφω: nurse. Bestrophin forms calcium-sensitive chloride channels permeable to bicarbonate. Cardiotrophin favors cardiomyocyte hypertrophy. Dystrophin anchors the extracellular matrix to the actin cytoskeleton, stabilizing the plasma membrane and supporting synaptic transmission. Neurotrophic factors, or neurotrophins, include brain-derived neurotrophic factor (BDNF) and neurotrophins NTF3 and NTF4, which control differentiation, survival, and function of neurons.

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Notation Rules: Abbreviations, Aliases, and Symbols

Nevertheless, types can also be denoted with simple Hindu–Arabic numerals (e.g., type-1 and -2 apoptotic cells linked to mitochondrion-independent and -dependent apoptosis). Another categorization of cellular membrane proteins states that, among transmembrane proteins, type-1 to -3 integral polytopic proteins are singlepass molecules and type-4 are multiple-pass molecules, whereas integral monotopic proteins are only attached to a membrane side and peripheral membrane proteins are temporarily attached to the cell membrane. Molecular complex subtypes are also defined by Roman numerals. The electron transport chain (ETC) complexes comprise proton pumps that transfer electrons and protons, which include the ETC complex-I (NADH– ubiquinone reductase), ETC complex-I I I (ubiquinol–cytochrome-C reductase, which links the electron transport chain to the tricarboxylic acid cycle), and ETC complex-I V (cytochrome-C oxidase), and an electron transfer element, the ETC complex-I I (succinate–ubiquinone reductase). Coagulation (or clotting) factors represent exceptions (e.g., FI I is prothrombin and FI I a is activated prothrombin, or simply thrombin; FI fibrinogen; FI I I tissue factor; and FI V calcium). As an example, in the extrinsic pathway of the coagulation cascade, FV I I is activated by FXI I , FXI a, FXa, and FI I a. On the other hand, complement components are abbreviated Ci (i: natural number from the Indo–Arabic numeral system), whereas complement factor-X (X: any majuscule) is denoted CFx (x: corresponding minuscule). (13) Unnecessary hyphenation in substance aliases, such as those used to define a protein subtype either encoded by a gene or corresponding to a splice variant, is avoided, except for aliases defined by portmanteau words (e.g., the autophagic initiator beclin-1 and histone deacetylase sirtuin-1), considered as full molecular names (e.g., ubiquitin-binding sequestosome-1 [Sqstm1]), and having common nouns and adjectives (e.g., notch-1 [notch: indentation] and jagged-1 [jagged: rough, barbed, spiked surface]). This rule enables the multiplication of hyphens in compound nouns to be obviated (e.g., RNF8-associated chromatin remodelers and SIRT5-regulated target [instead of sirtuin-5–regulated target]). In any case, the Notation sections serve not only to define aliases, but also, in some instances, as disambiguation pages. A space rather than a hyphen is used in (1) structural components on the picoscale (e.g., P loop), nanoscale (e.g., G protein [G standing for guanine nucleotide-binding]), microscale (e.g., H zone, M line, A band, I band, and Z disc of the sarcomere and T tubule of the cardiomyocyte); (2) process stages (e.g., M phase of the cell division cycle); and (3) cell types (e.g., B and T lymphocytes). When these terms are used as adjectives, a hyphen is then employed (e.g., P-loop Cys–X5 –Arg (CX5 R) motif, G-protein-coupled receptor, Z-disc ligand, M-phase enzyme, and T-cell activation). In terms incorporating a Greek letter, similarly, a space is used in (1) structural components (e.g., α and β chains and subunits); (2) cellular organelles (e.g., α granule); and (3) cell types (e.g., pancreatic glucagon-secreting α,

Notation Rules: Abbreviations, Aliases, and Symbols

787

insulin-releasing β, and somatostatin-freeing δ cells). On the other hand, terms are hyphenated when they refer to (1) structural shape (e.g., α-helix and α [β]sheet) and (2) molecule subtype (e.g., α-actinin, β-glycan, and γ-secretase). Numeral subscripts have been used to designate molecular receptors, which localize mainly to the plasma membrane of cells (e.g., endocannabinoid receptors CB1 and CB2 [or CBR1 and CBR2] and fatty acid receptors FFA1 to FFA3 [FFAR1–FFAR3]) to obviate confusion with their cognate ligand isoforms. (14) The alias of a molecule belonging to a large set, such as basic helix–loop– helix (bHLH) transcription factors (e.g., class-2 group-A bHLH transcription factors involved in cardiogenesis, bHLHa26 and bHLHa27, and autophagyrelated proteins Atg6, Atg14, Atg17, Atg18, and Atg38) is preferred to the one associated to its recommended name (transcription factors heart and neural crest derivative-expressed proteins HAND2 and HAND1 and autophagyrelated proteins beclin-1, barkor, RB1-inducible coiled-coil protein RB1CC1 [or 200-kDa FAK family kinase-interacting protein FIP200], WD repeat domain-containing phosphoinositide-interacting protein [WIPI], and nuclear receptor-binding factor NRBF2, respectively), as its function is straightforward.

Suffixes of Protein Subtypes In general, protein subtypes or isoforms are abbreviated with a suffix based on arabic numerals, which are represented by the integer symbol i, without hyphen as separator. Subtypes of a given molecule are denoted not only by a natural number, but also by a Greek letter (symbol ξ), or an uppercase letter (symbol X), its abbreviation suffix being written by a lowercase letter (symbol x). However, the capital letter X also defines a subgroup, means unknown function, stands for many cell types, symbolizes an exchanger (the majuscule letter E is also used; for example, NHX/NHE designates the sodium–hydrogen [Na+ –H+ ] exchanger, that is, a solute carrier family-9 member SLC9ai), or refers to the chromosome-X. Sets, groups, classes, families, and types as well as coagulation (or clotting) factors are defined by a Latin (Roman) numeral (the symbol  standing for I to X or more). Type-1 and -2 actin-related protein isoforms, which were formerly abbreviated ARP2 and ARP3, build the core of the ARP complex (ARPC) upon exposure to nucleation-promoting factor, which localizes to cytosolic regions of actin filament assembly. The so-called ARP2–ARP3 complex, which participates in the control of actin polymerization, consists of 7 subunits. In addition to ARP2 and ARP3, which are encoded by the ACTR2 and ACTR3 gene, ARPC components include ARPC1a, ARPC1b, ARPC2 to ARPC5, and ARPC5L (ARPC subunit-5-like protein).

788

Notation Rules: Abbreviations, Aliases, and Symbols

Muscular α-actin, cellular β-actin, and actin-related proteins share an ATPbinding sequence. In the budding yeast Saccharomyces cerevisiae, ARP1 to ARP3 and ARP10 lodge in the cytoplasm, ARP4 to ARP9 reside in the nucleus.37 Cytoplasmic ARPs contribute to regulating cytoskeletal structures. Nuclear ARPs and actin participate in nucleosome displacement and histone tail acetylation. Actin-related protein-T1 (ARPt1) and -T2 (ARPt2) are components of the calyx, a cytoskeletal component of the perinuclear theca of the sperm head. They are synthesized only in the testis. ARPt1 inhibits hedgehog signaling. Many ARPs are subunits of complexes. The centrosome-associated actin homologs, actin-related protein-1A and -1B (ARP1a–ARP1b), which are encoded by the ACTR1A and ACTR1B gene and also dubbed α- and β-centractin (Cntr1– Cntr2), constitute the dynactin complex involved in microtubule-based vesicular motion and dynein-based microtubule–membrane interactions. Dynactin (Dctn) is a cofactor for the microtubule retro- and anterograde nanomotors dynein and kinesin2, respectively. In fact, the dynactin filament is composed of a minifilament made up of β-actin and nine ARP1 subunits (ARP1a–ARP1i), which interact with Dctn1 to Dctn3, and ARP11, which is encoded by the ACTR10 gene and serves as a platform for Dctn4 to Dctn6.38 Microtubules scaffold organelles, such as the Golgi body and endoplasmic reticulum. Within the nucleus, the chromatin-remodeling complexes are also made up of ARPs. The nucleosome remodeler, the brahma-related gene BRG1 (SMARCa4)associated factor complex (BAF) comprises ActL6a (BAF53a) and ActL6b (BAF53b), which are thus involved in gene transcription regulation.39 Protein abbreviations should match the gene symbol approved by the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC). For example, the new short name ActRPi (old abbreviation ARPi) denotes type-i actinrelated protein, ACTRi being the HGNC approved gene symbol. However, certain alternative unambiguous abbreviations do not rely on short names approved by the HUGO Gene Nomenclature Committee. For example, the alias PCx stands for pyruvate carboxylase instead of the HGCN approved symbol “PC”, which is also used to designate phosphatidylcholine (also abbreviated PtdCho) and polycystin, not only to avoid ambiguity, but also for homogeneity, other types of carboxylases being abbreviated as “Cx” such as γ-glutamyl carboxylase, the HGCN approved gene symbol being GGCX, the protein being abbreviated in this textbook γGCx.

37 Oma Y, Harata M (2011) Actin-related proteins localized in the nucleus.

From discovery to novel roles in nuclear organization. Nucleus 2:38–46. 38 Urnavicius L, Zhang K, Diamant AG, Motz C, Schlager MA, Yu M, Patel NA, Robinson CV, Carter AP (2015) The structure of the dynactin complex and its interaction with dynein. Science 347:1441–1446. 39 In humans, the ATP-dependent chromatin-remodeling complex INO80 consists of actin, ARP3b/ ARP4, ARP5, and ARP8; SRCAP of actin, ARP3b, and ARP6; and BAF and TIP60 of actin and ARP3b.

Notation Rules: Abbreviations, Aliases, and Symbols

789

Symbols + and − Expression state of a given molecule is defined by the plus (+) or minus sign (−) to indicate its presence or absence. For example, the expression “POMC+ neuron” stands for neuron producing proopiomelanocortin; “CD4+ T lymphocyte” for T cell expressing type-4 cluster of differentiation molecule on its surface; “Atg8f+ membrane” for membrane containing the autophagy-related protein-8F, which corresponds to microtubule-associated protein-1 light chain-3β (MAP1LC3β); and “GluT4+ vesicle” for intracellular vesicle possessing the cargo glucose transporter4 isoform. The symbol + also defines a cation (e.g., K+ , Na+ , and Ca2+ ) and an extended molecule set (e.g., the AAA+ hyperset of ATPases associated with diverse cellular activities) as well as a type of bacteria identified by a staining technique proposed by the Danish bacteriologist HC Gram (1853–1938) in 1884, bacteria being treated with crystal and then flushed with an iodine solution, gram+ bacteria remaining purple because of their thick wall that is not easily penetrated by the solvent. On the other hand, the expression “Lnp1− endoplasmic reticulum three-way junction” means that the ER tubule interconnection does not possess the lunapark stabilizer, thereby remodeling more easily. The term “CD25− lymphocyte” signifies that this T cell is devoid of interleukin-2 receptor chain IL2Rα. − 3− The symbol − also designates an anion (e.g., Cl− , HCO− 3 , NO2 , and PO4 ) as SMC −/− well as genetic depletion, the term “ FBLN4 ” signifying the specific removal of the fibulin-4 gene in vascular smooth myocytes, the expression “CXCL4 Tlr4−/− mice” referring to platelet-specific deletion of the mouse Tlr4 gene, platelet being identified by platelet factor-4, which corresponds to the CXCL4 chemokine. Double deletion of a given gene is denoted as “•−/− ”, whereas ablation of the gene in a single allele, hence keeping a single functional copy of the gene (heterozygous alteration or haploinsufficiency)40 is written as “•+/− ”.

MicroRNAs MicroRNAs regulate the expression of transcripts by translational repression and degradation of messenger RNAs, thereby controlling signaling cascades involved in cell-type-specific function and intercellular communication. MicroRNAs are identified by numbers, avoiding useless hyphenation in aliases (microRNA-i, but miRi, where i is a numeral). The numbering of miRNAs is sequential; it is assigned according to the time of discovery, regardless of the organism type, even when miRs differ by a single nucleotide, although it may be redefined over time according to the updated knowledge. 40 Haploinsufficiency

refers to the presence of a single functioning copy of a gene which requires two functional copies to produce its normal effect.

790

Notation Rules: Abbreviations, Aliases, and Symbols

A prefix can be added when dealing with several species, the first three letters being related to the organism (e.g., hsa-miR stands for a human miR species [Homo sapiens]). For example, hsa-miR101 in humans and mmu-miR-101 in mouse (Mus musculus) are orthologous. The founding members of microRNAs are the Lin4 and Let7 gene products originally discovered in the nematode Caenorhabditis elegans (Lin: abnormal cell lineage; Let: lethal). These aliases are kept in mammals as homologs. Mature miR sequences generated from distinct precursors and genomic loci within a species have the same number and an additional numbered or lettered suffix, according to whether they are identical (miRi-1 and miRi-2) or closely related (miria and mirib; i.e., paralogous sequences from which derived mature miRs differ at only one or two positions).41 The mature microRNA is processed from a precursor with a characteristic stem– loop sequence (hairpin-like structure; alias: pre-miR) excised from a longer primary transcript (pri-miR; another hairpin-like structure). The mature sequences are assigned alias miRi for the predominant product (derived from the principal pre-miR strand) and miRi when originated from the opposite arm of the precursor (auxiliary passenger pre-miR strand). When the predominant sequence cannot be determined, aliases take into account the type of precursor arms, miRi-3p arising from the 3 arm and miRi-5p from the 5 arm (instead of using miRi and miRi ). A mature miRNA is designated by miR, its gene by mir, and a family of miRs by MIR (capitalization is usually restricted to plant miRs that are not incorporated in the present textbook).

Symbols for Physical Variables Unlike substance aliases, symbols for physical quantities are most often represented by a single letter of the Latin42 or Greek43 alphabet (i: current; J : flux; L: length; m: mass; p: pressure; P: power; T : temperature; t: time; u: displacement; v: velocity; x: space; λ: wavelength; μ: dynamic viscosity; ρ: mass density; etc.). These symbols are further specified using sub- and superscripts (cp and cv : heat capacity at constant pressure and volume, respectively; DT : thermal diffusivity; Gh : hydraulic conductivity; GT : thermal conductivity; αk : kinetic energy coefficient; αm : momentum coefficient; etc.).

41 Ambros

V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T (2003) A uniform system for microRNA annotation. RNA 9:277–279. 42 Either lower- or uppercase Latin letter. 43 Lowercase Greek letter.

Notation Rules: Abbreviations, Aliases, and Symbols

791

A physical quantity associated with a given point in space at a given time can be (1) a scalar uniquely defined by its magnitude; (2) a vector characterized by a magnitude, a support, and a direction represented by an oriented line segment defined by a unit vector; and (3) a tensor specified by a magnitude and a few directions. Tensors may be classified according to their rank or order (i.e., according to the number of components in the three-dimensional space). Hence, in a three-dimensional space: • A tensor of order 0 has a single component and is a scalar. • A tensor of order 1 has three components and is a vector. • A tensor of order 2 has nine components and can be represented by a matrix. Scalars, vectors, and tensors are represented by single symbols. To ensure a straightforward meaning of symbols used for scalar, vectorial, and tensorial quantities, boldface upper- (T) and lowercase (v) letters are used to denote a tensor and a vector, respectively, whereas both Roman (plain, upright) upper- and lowercase letters denote a scalar. Indices identify components of vectors and tensors. Both mechanical stress and strain are symmetrical tensor quantities in the absence of external moments represented by a 3 × 3 matrix with nine components in the three-dimensional space, the matrix rows corresponding to planes normal to the coordinate axes and columns to the direction of forces according to the coordinate axes. Indicial notation, or component notation, is associated with abbreviation rules, such as commas for partial derivatives and Einstein’s summation convention. The Cartesian coordinate system with an origin, coordinates ({xi }3i=1 ), and basis vectors { ei }3i=1 ) is commonly utilized. Length and volume elements are denoted by d (dx) and dV . The bounded domain over which a process is described by a partial differential equation is, in general, denoted by Ω and its boundary by partialΩ or Γ. Physical quantities are linked by operators of mathematical physics, such as · for scalar (dot) product, × for vectorial (cross) product, ⊗ for tensorial product, and ∇ for gradient and divergence (algebraic notation). Matrix notation is similar to the previous notations, but entities are expressed according to matrix operations. In particular, mechanical stress is a symmetric second-order tensor that can be defined as a 6-vector for matrix-oriented programming languages and mechanical solvers. In the full-form notation every term is spelled out. For example, the scalar product between two physical vectors v1 : (v11 , v12 , v13 ) and v2 : (v21 , v22 , v23 ) can be written: • • • •

In the direct notation as v1 · v2 In full-form notation as v11 v21 + v12 v22 + v13 v23 In the indicial notation as v1i v2i In the matricial notation as v1T · v2

792

Notation Rules: Abbreviations, Aliases, and Symbols

Symbols represent concepts, instructions, relations, and operations, in addition to conventional representations of an abstract object, element, quantity, function, or process. Among common mathematical symbols, the definitional equality symbol def = (alternately :=) means “is equal by definition to,” the universal quantifier symbol ∀ stands for “for all values (elements, positions, or time),” the existential quantifier symbol ∃ means “it exists an element such that,” the implication sign ⇒ “implies that,” the belonging sign ∈ “is an element of,” the inclusion sign ⊂ “this set is a subset of,” the union sign ∪ “elements in either set,” the intersection sign ∩ “common elements among sets,” and the infinity symbol ∞ denotes an arbitrarily large magnitude, in addition to, when used as a subscript, a more or less remote value such as that at a remote boundary of the fluid domain, either far from the entry cross section of a duct or at a distance from the growing boundary layer in the core flow, where the fluid flow is generally assumed to be inviscid. The triple bar symbol (≡) is used as a symbol of an identical notation in addition to the equivalence relation between two elements of a subset to define the symbol on the left-hand side of the relation and to differentiate relations from equations in which the terms on both sides are defined. On the other hand, the equal symbol (=) means that the left and right terms belong to the same species (scalar, vector, or tensor) and have the same magnitude. The equality sign is used as a statement (e.g., to define the position and instant [t = 0: initial time]), and to express an equivalence (e.g., force balance and mass conservation equation). The mathematical symbol ∝ indicates proportionality between two quantities. In classical mechanics, derivatives are ubiquitous. In mathematics, a differential operator is related to differentiation with respect to space (x) and time (t): ∂i ≡

∂ ; ∂xi

∂i,j ≡

∂vi ; ∂xj

∂t ≡

∂ . ∂t

The nabla symbol (∇) is the vectorial gradient operator, which, in the Cartesian coordinate system, is defined in terms of partial derivatives as ∇:

∂ ∂ ,··· , ∂x1 ∂x3

=

3  i=1

 ei

∂ . ∂xi

(0.1)

It is used to calculate not only the gradient, but also divergence, curl, and Laplacian of variables. The scalar Laplace operator ∇ 2 can be applied to scalar (e.g., pressure and temperature), vector (e.g., velocity), or tensor fields: n  ∂2 ∇ = . ∂xi2 i=1 2

(0.2)

Dimensional analysis is a basic tool in mechanics, especially in the selection of dominant terms in the equation. The symbol order of magnitude O written using powers of 10 is then employed. An order-of-magnitude estimate of a variable, the

Notation Rules: Abbreviations, Aliases, and Symbols

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precise value of which is unknown, is rounded to the nearest power of ten, the order of magnitude being defined in terms of the decimal logarithm. For crude approximations, for example, approximations of the terms of an equation with respect to the length, time, velocity, and pressure scales (symbol ), or characteristic values of the length (L ), time (t ), velocity (V ), and pressure (p ), ) and diffusion (t ) the symbol ∼ is utilized. For example, the convection (tconv diff time scales of a flow are: Tconv ∼

L ; V

2

Tdiff ∼

L . ν

(0.3)

In the absence of motion, transport is done by molecular diffusion. The diffusion equation for heat states that: ∂t T = D T ∇ 2 T .

(0.4)

Hence for heat diffusion, the dimensional analysis shows that 2

2

ΔT /t ∼ DT ΔT /L ,

therefore, t ∼

L . DT

(0.5)

When only few accurate statements can be made and the coefficients associated with each term of the equation remain unknown, the analysis makes use of relations rather than equations, and the symbol “is similar or equal” () is involved. For example, in a turbulent flow, the ratios of vortex diffusivity (or eddy diffusivity; Dω ) to momentum diffusivity (ν) and to thermal diffusivity (DT ) are comparable, as diffusion by a random motion is very fast with respect to molecular diffusion: Dω Dω  . ν DT Because the time scale of vortex diffusion (turbulent mixing) is similar to the convection time scale, the dimensional analysis gives rise to the following approximations: 2

L L ∼ ; V Dω

hence, Dω ∼ L V and

Dω ∼ Re, ν

where Re is the Reynolds number, a dimensionless flow governing parameter that 2 2 represents the ratio of inertia (∼ ρV /L ) to viscous forces (∼ μV /L ) and ν = μ/ρ the kinematic viscosity (μ: dynamic viscosity; ρ: mass density). The n dash is used rather than the hyphen to distinguish a double-barreled name from cases for which two different researcher’s names and their derived adjectives (e.g., Newtonian) are joined up to define equations (e.g., Kedem–Katchalsky, Navier–Stokes, and Stefan–Maxwell equations), laws (e.g., Boyle–Mariotte law),

794

Notation Rules: Abbreviations, Aliases, and Symbols

chemical reactions (e.g., Michaelis–Menten enzyme kinetics), model types (e.g., Mitchell–Schaeffer model), effects (e.g., Fahraeus–Lindqvist effect), and numerical procedures (e.g., arbitrary Lagrangian–Eulerian formulation, Chorin–Temam projection scheme, and Dirichlet–Neumann domain decomposition algorithm).

Measurement Unit Abbreviations The International System of Units (SI)44 is the basic system of measurement based on the meter–kilogram–second system of units (MKS), rather than the previous centimeter–gram–second system (CGS). The SI 7-base units include the meter for length, kilogram for mass, second for time, ampere for electric current, kelvin for temperature, mole for the amount of a substance, and candela for luminous intensity. The magnitude value of a quantity is written as a number followed by a small space and a unit symbol, excluding the percentage sign (%) and the symbol for temperature degree using the degree Celsius scale (◦ C). Standard prefixes are employed for multiples and fractions of these unit (e.g., kilo [103 ; symbol: k] and milli [10−3 ; symbol: m]), commonly every three orders of magnitude. A prefix is part of the unit and its symbol is prepended to the unit symbol without a separator (e.g., MPa and GHz). Unit symbols are written in upright (Roman) type (e.g., m for meter), thereby distinguishing them from the italic type, which can be utilized for quantities (m for mass). The derived units in the SI are formed by powers, products, or quotients of the base units. Symbols for derived units formed by multiplication are joined with a single vertically centered dot (· [•]). According to Springer rules, symbols for derived units formed by division are joined with a slash (avoiding multiple slashes) rather than given as a negative exponent, which nevertheless may be clearer. Measurement units symbols that are abbreviations using the initial of a researcher name are uppercase (e.g., A [A.M. Ampere], C [C.A. Coulomb],45 F [M. Faraday],46 J [J.P. Joule],47 K [W. Thomson – Kelvin], N [I. Newton],48 Ω [G.S. Ohm],49 V [A. Volta],50 and W [J. Watt],51 among others). Less often, the two

44 From

French Système International d’Unités. coulomb is the SI unit of electric charge, i.e., the charge transported by a constant current of one ampere in one second. 46 The farad is the unit of electrical capacitance. 47 The joule is the unit of energy or work. 48 The newton is the unit of force. 49 The ohm is the unit of electrical resistance. 50 The volt is the unit for electric potential difference (voltage). 51 The watt is the unit of power. 45 The

Notation Rules: Abbreviations, Aliases, and Symbols

795

first letters of the researcher name are kept, then, the first letter is uppercase and the following one lowercase (e.g., Hz [H.R. Hertz],52 and Pa [B. Pascal].53

52 The 53 The

hertz is the unit of frequency. pascal is the unit of pressure.

796

Notation Rules: Abbreviations, Aliases, and Symbols

Initials of the measurement units of volume that do not pertain to the SI, liter and its compounds, are lowercase, though uppercase are more common in American English (l and ml rather than L and mL), thereby avoiding adding an exception to the above-mentioned rule. In a multidisciplinary compendium, a given abbreviation can have multiple meanings that, in general, are disambiguated by the context. For example, APC stands for “adenomatous polyposis coli ubiquitin ligase” in biochemistry and “antigen-presenting cell” in immunology, and ARP for “actin-related protein” in molecular biology and “absolute refractory period” in physiology. Some abbreviations have evolved with respect to those used from the previous volumes of the book series, not only to avoid duplicated abbreviations, but also to eliminate the mixture of upper- and lowercase letters. In the Lists of Notations, new abbreviations precede the old version included in parentheses. In general, the new abbreviation of a given protein is aimed at matching the gene symbol approved by the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC).

List of Molecule Shortened Abbreviations and Chemical Symbols

A A: actin-binding site Ai : type-i adenosine (P1) G-protein-coupled receptor αKG: α-ketoglutarate αLA: α-linolenic acid α2MG: α2-macroglobulin AA: amino acid AA: arachidonic acid AAA: ATPase associated with diverse cellular activities AAAP: aneurysm-associated antigenic protein AADC: aromatic amino acid decarboxylase AAK: adaptin-associated kinase AAMP: angio-associated migratory cell protein AAP: ATPase-activating protein aaRS: aminoacyl-transfer ribonucleic acid (tRNA) synthetase AATF: apoptosis-antagonizing transcription factor AATK: apoptosis-associated tyrosine kinase ABC: ATP-binding cassette transporter (transfer ATPase) Abhd: abhydrolase domain-containing protein AbI: Abelson kinase interactor Abl: Abelson leukemia viral proto-oncogene product (NRTK) ABLIM: actin-binding LIM domain-containing protein ABP: actin-binding protein

ABR: active breakpoint cluster region (BCR)-related gene product (GEF and GAP) ACi: adenylate cyclase isoform-i ACAA: acetylCoA acyltransferase ACADH: acylCoA dehydrogenase ACADHL (lcACADH): long-chain fatty acylCoA dehydrogenase (ACADL gene) ACADHM (mcACADH): medium-chain fatty acylCoA dehydrogenase (ACADM gene) ACADHS (scACADH): short-chain fatty acylCoA dehydrogenase (ACADS gene) ACADHVL (vlcACADH): very-long-chain fatty acylCoA dehydrogenase (ACADVL gene) ACAP: ArfGAP with coiled-coil, ankyrin repeat, PH domains ACACATi (ACATi): type-i acylCoA– cholesterol acyltransferase (SOATi) ACATi: type-i acetylCoA acetyltransferase (mitochondrial ACAT1; cytosolic ACAT2) ACBD: (translocator-associated) acyl-coenzyme-A-binding domaincontaining protein ACCx (ACC): acetyl coenzyme-A carboxylase ACD: adrenocortical dysplasia protein homolog ACE: angiotensin-converting enzyme

© Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0

797

798

List of Molecule Shortened Abbreviations and Chemical Symbols

ACF: ATP-using chromatin assembly and remodeling factor (ACF1-composed complex) ACh: acetylcholine acinus (Acin1): apoptotic chromatin condensation inducer in the nucleus ACK: activated CDC42-associated kinase ACKR: atypical chemokine receptor ACL: ATP–citrate lyase ACMS: α-amino β-carboxymuconate -semialdehyde ACMSD: ACMS decarboxylase Aco: aconitase ACoT: acylCoA thioesterase ACOx: peroxisomal acylCoA oxidase ACP1: acid phosphatase-1, soluble (lmwPTP) ACS: acetyl-coenzyme-A synthase ACSL: long-chain fatty acylCoA synthase ACSS: short-chain fatty acylCoA synthase ACTH: adrenocorticotropic hormone F actin: filamentous actin G actin: monomeric globular actin ActRPi (ARPi): type-i actin-related protein (ACTRi gene) ActSP (ASP): actin-severing protein AcvR: activin receptor (TGFβ receptor superfamily) Ad: adrenaline adam (ADAM): a disintegrin and metallopeptidase (adamalysin) adamts (ADAMTS): adamalysin with thrombospondin-1 motif ADAP: adhesion and degranulation-promoting adaptor protein ADAP: ArfGAP with dual PH domains ADAR: adenosine deaminase acting on RNA ADCF: adipocyte-derived constricting factor ADCY: adenylate cyclase (gene) ADF: actin-depolymerizing factor (cofilinrelated destrin) AdGRxi: adhesion GPCR-Xi (X: A–G, L; i: integer) ADH: antidiuretic hormone (vasopressin) ADHi: type-i alcohol dehydrogenase (ADH1–ADH7) ADHAPR: acyldihydroxyacetone phosphate reductase Adm: adrenomedullin ADMA: asymmetric dimethylarginine ADNF: activity-dependent neurotrophic factor ADNP: activity-dependent neuroprotective protein ADP: adenosine diphosphate Adpn (AdpN): adiponectin (ADIPOQ gene)

AdpnR: adiponectin receptor (ADIPOR gene) ADPRH (ARH): ADP ribosylhydrolase ADPRT (ART): ADP ribosyltransferase ADRF: adipocyte-derived relaxing factor ADRP: adipose differentiation-related protein (lipid droplet coat) aDuSP: atypical dual specificity phosphatase AE: anion exchanger AEA: N arachidonoyl ethanolamine (anandamide) AEF: adenine nucleotide (ADP-to-ATP)exchange factor AEN: apoptosis-enhancing nuclease AFAP: ArfGAP with phosphoinositide-binding and PH domains AFMR: autocrine motility factor receptor (Ub ligase RNF45) AGA: aspartyl glucosaminidase AGAP: ArfGAP with GTPAse, ankyrin repeat, and PH domains AGE: advanced glycation end product AGF: autocrine growth factor AGFG: ArfGAP with FG repeats Ago: argonaute protein AGPAT: acylglycerol phosphate O acyltransferase AgRP: agouti-related protein homolog AGS: activator of G-protein signaling Agt: angiotensin Agtg: angiotensinogen AHR: aryl hydrocarbon receptor AIF: apoptosis-inducing factor AIMP: aminoacyl-tRNA synthetase-interacting multifunctional protein AIP: actin-interacting protein AIR: antisense Igf2r RNA AIRAP: arsenic-inducible 19S proteasomal regulator-associated protein AIRAPL: AIRAP-like protein AIRe: autoimmune regulator AKAP: A-kinase (PKA)-anchoring protein AKR: aldo–keto reductase AlaRS: alanyl tRNA synthetase ALaS: aminolevulinate synthase Alb: albumin ALCAT: (MAERM-resident) acylCoA– lysocardiolipin acyltransferase (cardiolipin acyltransferase) Ald: aldosterone AlDH: aldehyde dehydrogenase Aldo: (fructose bisphosphate) aldolase AldR: aldose reductase ALIX: apoptosis-linked gene-2-interacting protein X

List of Molecule Shortened Abbreviations and Chemical Symbols ALK: anaplastic lymphoma kinase ALKi: type-i activin receptor-like kinase AlkbH: AlkB demethylase homolog ALOx5: arachidonate 5-lipoxygenase ALOx5AP: ALOx5 activation protein ALP: actinin-associated LIM protein (PDLIM3) alP: alkaline phosphatase alsin: amyotrophic lateral sclerosis protein (portmanteau) ALT: alternative lengthening of telomere ALX: adaptor in lymphocytes of unknown function (X) Aly (Aly/REF): ally of AML1 and LEF1 AMAP: A multidomain ArfGAP protein ambra: activating molecule in beclin-1regulated autophagy protein AMFR: autocrine motility factor receptor (Ub ligase) AMHR: anti-Müllerian hormone receptor (TGFβ receptor superfamily) AMP: adenosine monophosphate AMPAR: α-amino 3-hydroxy 5-methyl 4-isoxazole propionic acid receptor AMPK: AMP-activated protein kinase AMS: α-amino β-muconate -semialdehyde AMSH: associated molecule with SH3 domain (deubiquitinase) AmyR: amylin receptor ANCAAV: antineutrophil cytoplasmic antibody-associated vasculitis ancRNA: activating nonprotein-coding RNA Angpt (AngPt): angiopoietin AngptL (AngPtL): angiopoietin-like protein Ank: ankyrin ANP: atrial natriuretic peptide ANPep: alanyl aminopeptidase [APn(m)] ANPR (NP1 ): atrial natriuretic peptide receptor (guanylate cyclase) ANRIL: antisense noncoding RNA in the INK4 locus ANT: adenine nucleotide transporter Anx: annexin AOC: amine oxidase copper-containing protein AP: (clathrin-associated) adaptor proteic complex APase (AP): aminopeptidase AP4 A: diadenosine tetraphosphate APAF: apoptotic peptidase-activating factor APAP: ArfGAP with PIX- and paxillin-binding domains APC: adenomatous polyposis coli Ub ligase

799

aPC: activated protein-C APC/C: anaphase-promoting complex or cyclosome (Ub ligase) APE: apurinic–apyrimidinic endonuclease (apex1/2 genes) APH: anterior pharynx defective phenotype homolog aPKC: atypical protein kinase-C APLF: aprataxin- and PNKP-like factor Apln: apelin AplnR: apelin receptor APN: base excision repair AP (apurinic// apyrimidinic) endonuclease (apex) Apo: apolipoprotein ApoER: apolipoprotein-E receptor (ApoER1 and 2 are LRP1 and 8) APPL: adaptor containing phosphoTyr interaction, PH, and Leu zipper domain APS: adaptor with a PH and SH2 domain APx: type-X aminopeptidase (e.g., APa and APn) Aqp (AqP): aquaporin AR: adrenergic receptor (adrenoceptor) (TF)AR: transcription factor androgen receptor (nuclear receptor NR3c4) ARAP: ArfGAP with RhoGAP, ankyrin repeat, PH domains ARCC: arachidonic acid-regulated Ca2+ channel Areg (AReg): amphiregulin (EGF superfamily member) ARF: ADP-ribosylation factor ArfRP: ARF-related protein ARFTS: CKI2A-locus alternative reading frame tumor suppressor (ARF or p14ARF ) Argi: type-i arginase ARH: aplysia Ras-related homolog ARH: autosomal recessive hypercholesterolemia adaptor (low-density lipoprotein receptor adaptor) ArhGEF: RhoGEF ARID: AT-rich-interactive domain-containing protein ARK: adrenergic receptor (GPCR) kinase ARL: adpribosylation factor-like protein arno (ARNO): ARF nucleotide site opener ARNT: aryl hydrocarbon nuclear receptor translocator ARPC: actin-related protein ActRP2–ActRP3 complex

800

List of Molecule Shortened Abbreviations and Chemical Symbols

ARPCi: type-i ARPC subunit ARPP: cAMP-regulated phosphoprotein Arr: arrestin ArS: arylsulfatase ART: arrestin-related transport adaptor (α-arrestin) Artn: artemin AS (AktS): Akt (PKB) substrate ASAgP (ASAP): artery-specific antigenic protein ASAP: apoptosis- and splicing-associated protein ASAPC (ASAP): acinus-containing splicing-associated proteic complex ASAPD (ASAP): ArfGAP with SH3, ankyrin repeat, PH domains ASB: ankyrin repeat and SOCS box-containing protein ASCC: activating signal cointegrator complex ASDN: aldosterone-sensitive distal nephron (CT and CD) ASF1: antisilencing factor-1 homolog ASIC: acid-sensing ion channel ASiP: agouti signaling protein ASK: apoptosis signal-regulating kinase ASP: acylation stimulating protein (C3adesArg) AspT: aspartate transaminase ASS: argininosuccinate synthetase AT: antithrombin ATAD: ATPase family, AAA domaincontaining, member ATF: activating transcription factor Atg: autophagy-related protein ATGL: adipose triacylglycerol lipase ATL: aspirin-triggered lipoxin Atl: atlastin (dynamin-like GTPase) ATMK: ataxia telangiectasia-mutated kinase Atox (AtOx): antioxidant protein (metallochaperone) ATP: adenosine triphosphate ATPase: adenosine triphosphatase ATR (AT1/2 ): angiotensin receptor atrap: AT1 receptor-associated protein ATRK: ataxia telangiectasia- and Rad3-related kinase ATRKIP: ATRK-interacting protein Atxn: ataxin AUBP: AU-rich element (AURE)-binding protein AUF: AU-rich element RNA-binding factor AUP: ancient ubiquitous protein AurK: aurora kinase aUTR: alternative untranslated region

AVP (or ADH): arginine vasopressin Avp: propressophysin, AVP/ADH precursor gene (vasopressin–neurophysin-2– copeptin)

B BAAT: bile acid–CoA:amino acid N acetyltransferase BACE: β-amyloid precursor protein-converting enzyme (or membrane-associated aspartic peptidase memapsin) BACE1as: BACE1 (memapsin-2) antisense RNA BACS: bile acid coenzyme A (CoA) synthetase BABAM: brisc and BrCa1a complex member (docker and stabilizer) bach (BACH): BTB and CNC homolog BAD: BCL2 antagonist of cell death (BCL2L8) BAF: BRG1-associated factor BAG: BCL2-associated athanogene (chaperone regulator) BAI: brain-specific angiogenesis inhibitor (adhesion GPCR) BAIAP: brain-specific angiogenesis inhibitor1-associated protein (insulin receptor substrate) BAIF (BAF): barrier-to-autointegration factor BAK: BCL2-antagonist killer (BCL2L7) bambi (BAMBI): BMP and activin membranebound inhibitor homolog bank (BAnk): B-cell scaffold with ankyrin repeats BAP: B-cell receptor (BCR)-associated protein bard (BARD): BrCa1 (BRCC1)-associated RING domain-containing protein barkor: beclin-1-associated autophagy-related key regulator (Atg14L) BATi: human lymphocyte antigen HLAB associated transcript-i BATF: basic leucine zipper ATF-like transcription factor (B-cell-activating transcription factor) BAX: BCL2-associated X protein (BCL2L4) BAZ: bromodomain adjacent to zinc finger domain-containing protein BBC: BCL2-binding component BBP: bilin-binding protein BBSi: Bardet–Biedl syndrome (BBS) protein-i BBIP: bbsome-interacting protein bbsome: BBS coat complex (transport of membrane proteins into cilium) BCAA: branched-chain amino acid

List of Molecule Shortened Abbreviations and Chemical Symbols bcam (bCAM): basal cell adhesion molecule (Lutheran blood group glycoprotein) BCAP: B-cell receptor-associated protein BCAP (formerly): B-cell adaptor for phosphatidylinositol 3-kinase (PI3KAP1) BCAR: breast cancer antiestrogen resistance docking protein BCKA: branched-chain α-keto acid BCKADH: BCKA dehydrogenase (branchedchain 2-oxoacid dehydrogenase [BCOADH]) BCKADHC: BCKADH complex BCL: B-cell lymphoma (leukemia) protein BCL2Li: type-i BCL2-like protein BCR: B-cell receptor BCRGAP/GEF (BCR): breakpoint cluster region protein BCRUb (BCR): BTB–Cul3–RBx1 Ub ligase complex Bdk (BdK): bradykinin BDNF: brain-derived neurotrophic factor beclin (BECN gene): BCL2-interacting protein (Atg6) Best: bestrophin BET: bromodomain and extraterminal domain-containing reader BFUe: burst-forming unit-erythroid BFUmeg: burst-forming unit-megakaryocyte BGP: bone γ-carboxyglutamate acid (Gla)-containing protein (osteocalcin) BGT: betaine–GABA transporter BH4 : tetrahydrobiopterin (enzyme cofactor) bHLH: basic helix–loop–helix protein BID: BH3-interacting domain death agonist BIG: brefeldin-A-inhibited GEFs for ARFs BIK: BCL2-interacting killer (BCL2L11) BIM: BH3-containing protein BCL2-like 11 (BCL2L11) BIn: bridging integrator BIRC: baculoviral IAP repeat-containing protein BK: high-conductance, Ca2+ -activated, voltage-gated K+ channel BLK: B-lymphoid tyrosine kinase Blm: Bloom syndrome, RecQ DNA helicase-like protein BLnk: B-cell linker protein BLOC: biogenesis of lysosome-related organelle complex BMAL: brain and muscle ARNT-like protein (gene Bmal) BMF: BCL2-modifying factor

801

BMI1: B-cell-specific Moloney murine leukemia virus integration site protooncogene product-1 (polycomb group RING finger-containing protein PCGF4 and RNF51) BMP: bone morphogenetic protein (TGFβ superfamily) BMPR: bone morphogenetic protein receptor BMX: bone marrow protein Tyr kinase gene in chromosome-X product BNIP: BCL2/adenovirus E1B 19-kDa protein-interacting protein BNP: B-type natriuretic peptide BOC: brother of CDO BOK: BCL2-related ovarian killer (BCL2L9) BORG: binder of Rho GTPase bp: base pair BPDE: benzo[a]pyrene (7,8)-diol (9,10)epoxide BPS: branch point sequence BRAG: brefeldin-resistant ArfGEF BrCa: breast cancer-associated (susceptibility) protein BrCa1a(b,c): type-A(B,C) BrCa1-forming complex BRCC: BrCa1–BrCa2-containing complex subunit BRCC1A: UIMC1–BRCC3–BABAM1– BRCC4–Fam175a complex BRCC1B: BrCa1–BrIP1 complex BRCC1C: BrCa1–RBBP8–MRN complex BRCT: breast cancer susceptibility BrCa1 C-terminal domain BrD: bromodomain-containing protein BrD4 (CAP): bromodomain-containing protein-4 (mitotic chromosomeassociated protein) BRG: brahma (SMARCa2)-related gene product BRISC: BRCC3 isopeptidase complex (deubiquitinase, regulator of mitotic spindle assembly) BrIP1: BrCa1-interacting protein (helicase) BrK: breast tumor kinase BrMS: breast cancer metastasis suppressor BrPF: bromodomain and PHD fingercontaining protein BrSK: brain-selective kinase BSP: bone sialoprotein BSEP: bile salt export pump BTF: basic transcription factor BTG: B-cell translocation gene product BTK: Bruton protein Tyr kinase BUB: budding uninhibited by benzimidazoles

802

List of Molecule Shortened Abbreviations and Chemical Symbols

BVES: blood vessel epicardial substance (adhesion molecule) Bvht: braveheart (lncRNA) BVR: biliverdin reductase bZip: basic leucine zipper (transcription factors containing a basic DNA-binding motif followed by a leucine zipper region for dimerization

C C-terminus: carboxy (carboxyl group COOH) terminus Ci: complement component-i C1P: ceramide 1-phosphate C1QTNF: group of genes encoding members of the CTRP (C1q–TNF-related) superfamily C1qBP: mitochondrial matrix complement component-1 Q-subcomponent-binding (glyco)protein C3G: Crk SH3-binding GEF C/EBP: CCAAT/enhancer-binding protein Cx: type-X chemokine C (γ chemokine type) CA: cholic acid (bile acid) CAi: carbonic anhydrase isoform-i Ca: calcium CaV : voltage-gated Ca2+ channel CaV 1.i: L-type high-voltage–gated Ca2+ channel CaV 2.i: P(Q)/N/R-type Ca2+ channel CaV 3.i: T-type low-voltage-gated Ca2+ channel CAAT: cationic amino acid transporter cables (CAblES): CDK5 and Abl enzyme substrate CACT: carnitine–acetylcarnitine transferase CAF: chromatin assembly factor CAK: CDK-activating kinase (pseudokinase) Calc: calcitonin calcoco: calcium-binding and coiled-coil domain-containing protein Calr (Crt): calreticulin Cam: calmodulin (calcium-modulated protein) Cam2K: calmodulin-dependent kinase kinase CamK: calmodulin-dependent kinase CaML (CaMLg): calcium-modulating cyclophilin-B (PPIb) ligand CAMP: cathelicidin antimicrobial peptide cAMP: cyclic adenosine monophosphate camsap: calmodulin-regulated spectrinassociated protein Canx: calnexin CAP: adenylate cyclase-associated protein

CAPK: ceramide-activated protein kinase (PI3K, PKCs) CAPN: calpain gene capon (CaPON): C-terminal PDZ ligand of NOS1 (NOS1AP) CAPP: ceramide-activated protein phosphatase (PP1 or PP2) CAPPA (CAP): carboxyalkylpyrrole protein adduct CAR: constitutive androstane receptor (NR1i3) CAR: coxsackievirus and adenovirus receptor CaSR (CaR): calcium-sensing receptor CARD: caspase activation and recruitment domain-containing protein carl: cardiac apoptosis-related lncRNA carma (CARMA): CARD and membraneassociated guanylate kinase-like (MAGuK) domain-containing protein CARP: cell division cycle and apoptosis regulatory protein CART: cocaine- and amphetamine-regulated transcript-derived peptide CAS: cellular apoptosis susceptibility protein CAS: CRK-associated substrate (or P130CAS and BCAR1) CAs: cadherin-associated protein CASK: calcium–calmodulin-dependent serine kinase (pseudokinase) CASL: CRK-associated substrate-related protein (CAS2) CASP: cytohesin-associated scaffold protein Casp: caspase (cysteine-dependent aspartatespecific peptidase) CAT: carnitine acetyltransferase Cat: catalase (CAT gene) Cav: caveolin Cav–actin: caveolin-associated F actin CβS: cystathionine β-synthase (H2 S production) CBC: nuclear RNA cap-binding complex CBCN: CBC–NEXT complex CBCS: CBC–Srrt complex CBCSP: CBC–Srrt–Phax complex CBF: core-binding factor CBL: Casitas B-lineage lymphoma adaptor and Ub ligase Cbl: cobalamin CBLb: CBL-related adaptor CBP: cap-binding protein CBS: cystathionine β-synthase (gene) CBx: chromobox gene product homolog CCx: type-X chemokine CC (β chemokine type) CCAAT: binding motif in gene promoters

List of Molecule Shortened Abbreviations and Chemical Symbols CCBE: collagen- and calcium-binding EGF domain-containing protein CCDC: coiled-coil domain-containing protein CCE: capacitative Ca2+ entry channel (SOC channel) CCG: clock-controlled gene CCICR: calcium channel-induced Ca2+ release Cck (CCK): cholecystokinin CCK4: colon carcinoma kinase-4 (PTK7) CCL: chemokine CC-motif ligand CCN: CyR61, CTGF, and NOv family CCNi: type-i CCN family member (CCN1–CCN3) CcnX: type-X cyclin CcnX–CDKi: type-X cyclin–type-i cyclindependent kinase dimer CCNA: constitutive centromere-associated network CcOx: cytochrome-C oxidase CcOx17: cytochrome-C oxidase copper chaperone CCPg: cell cycle progression protein CCR: chemokine CC motif receptor CcR: cytochrome-C reductase CCS: copper chaperone for superoxide dismutase CCT: chaperonin containing T-complex protein TCP1 complex (cytosolic chaperonin [cCpn]) CD: cluster determinant plasmalemmal protein (cluster of differentiation) identifying leukocyte types. CDase: ceramidase CDC: cell division cycle (control) protein CDCA: chenodeoxycholic acid (bile acid) CDCAi: type-i cell division cycle-associated protein CDF: cation diffusion facilitator CDH: CDC20 homolog Cdh: cadherin CDK: cyclin-dependent kinase CDKN2Bas: antisense long nonprotein-coding RNA in the CDKN2B gene locus Cdm: caldesmon CDO: cell adhesion molecule-related/ downregulated by oncogenes (CDON gene) cdr1as: cerebellar degeneration-related protein-1-related (circular) antisense lncRNA CdS: coding sequence (of mRNA) CDT: CDC10-dependent chromatin licensing and DNA replication transcript product

803

CDY: Y-linked chromodomain-containing protein CE: cholesteryl ester CELF: CUGBP ELAV-like family member (pre-mRNA splicing) CELSR: cadherin, EGF-like, LAG-like, and seven-pass receptor CenP: centromeric protein CEP: carboxyethylpyrrole CeP: centrosomal protein CEPT: choline and ethanolamine phosphotransferase Cer: ceramide CerK: ceramide kinase ceRNA: competing endogeneous RNA CerS: ceramide synthase CerT: ceramide transfer protein CETP: cholesterol ester transfer protein CFT: chromosome transmission fidelity factor homolog CFI (I I ): type-I (I I ) cleavage factor (CPA subcomplex) CFx: complement factor-X CFhR: complement factor-H-related protein CFLAR: caspase-8 and FADD-like apoptosis regulator (casper [caspase-8-related protein or caspase-like apoptosis regulator]) CFTR: cystic fibrosis transmembrane conductance regulator CG: chorionic gonadotropin cGAMP: cyclic GMP–AMP dinucleotide cGAS: cGAMP synthase Cgb: cytoglobin (CYGB gene) cGK: cGMP-dependent protein kinase (protein kinase-G) cGMP: cyclic guanosine monophosphate CGRP: calcitonin gene-related peptide chaer: cardiac hypertrophy-associated epigenetic regulator (lncRNA) ChAFi: chromatin assembly factor subunit-i chanzyme: ion channel and enzyme chast: cardiac hypertrophy-associated transcript (lncRNA) chemokine: chemoattractant cytokine CHD: chromodomain helicase DNA-binding protein (complex) ChFR: ubiquitin ligase checkpoint with forkhead-associated and RING finger domains Chgx (Cgx): type-X chromogranin (CHGA–CHGB genes) Chi3L: chitinase-3-like enzyme ChiA: acidic chitinase

804

List of Molecule Shortened Abbreviations and Chemical Symbols

CHIP: C-terminus heat shock cognate-70interacting protein Chit: chitinase CHK: CSK homologous kinase ChKi: type-i checkpoint kinase ChKξ: type-ξ choline kinase Chn: chimerin (GAP) CHOP: C/EBP homologous protein CHRAC: chromatin accessibility complex CHREBP: carbohydrate-responsive elementbinding protein chrf: cardiac hypertrophy-related factor (lncRNA) ChS (CS): chondroitin sulfate ChT: choline transporter ChTOP: chromatin target of PRMT1 ChTF: chromosome transmission fidelity factor CHX (CHE/CHA): Ca2+ –H+ exchanger (antiporter) CICR: calcium-induced calcium release CIDE: cell death-inducing DFFA-like effector Cin: chronophin CIP: CDC42-interacting protein CIP2a: cancerous inhibitor of protein phosphatase-2A CIPC: CLOCK-interacting protein, circadian CIS: cytokine-inducible SH2-containing protein CISD: CDGSH iron–sulfur domain-containing protein cited: CBP/P300-interacting transactivator with glutamic (E) and aspartic acid (D)-rich C-terminus-containing protein CIZ: CKI1a-interacting zinc finger-containing protein CK: creatine kinase CkAP: cytoskeleton-associated protein CsnKi (CKi): type-i casein kinase (a misnomer) CKI: cyclin-dependent kinase inhibitor CL: cardiolipin CLAsP: CLiP-associated protein (microtubule binder) ClASP: clathrin-associated sorting protein ClBX (ClBE): chloride–bicarbonate (Cl− –HCO− 3 ) exchanger CLC: cardiotrophin-like cytokine ClC: voltage-gated chloride channel ClCa: calcium-activated chloride channel ClCn: chloride channel Cldn: claudin CLEAR: coordinated lysosomal expression and regulation gene network CLec: C-type lectin

ClHX (ClHE): Cl− –H+ exchanger ClIC: chloride intracellular channel CLINT: clathrin-interacting protein located in the trans-Golgi network CLIP: corticotropin-like intermediate peptide CLiP: cytoplasmic CAP-Gly domaincontaining linker protein CLK: CDC-like kinase CLN: ceroid lipofuscinosis neuronal protein ClNS: Cl− channel nucleotide-sensitive CLOCK: circadian locomotor output cycles kaput CLP: caseinolytic peptidase ClP: pre-mRNA cleavage complex-I I protein CrLS (CLS): cardiolipin synthase CLS: ciliary localization signal Clspn: claspin CM: chylomicron CMR: chylomicron remnant CNC: Cap and (shortened by ‘n’) Collar (group of transcription factors of the ATF–CREB category) CNG: cyclic nucleotide-gated channel CNKSR (CnK): connector enhancer of kinase suppressor of Ras CNOT: CNONC component (NOT: negative regulator of transcription) CNOTC: CCR4 (CNOT6)–NOT-like (CNOT1) transcriptional complex (CCR4: carbon catabolite repressor protein-4 homolog) CNTi: concentrative nucleoside transporter (SLC28ai) CNTF: ciliary neurotrophic factor CntnAP: contactin-associated protein CoA: coenzyme-A CoQ: coenzyme-Q (Q: quinone) CoQOR: CoQ (ubiquinone [ubiquitous quinone]) oxidoreductase CoBl: cordon-bleu homolog (actin nucleator) Col: collagen CoLec: collectin ColF: collagen fiber COMP: cartilage oligomeric matrix protein (Tsp5) COOL: cloned out of library (RhoGEF6/7) CoP: coat protein CoP/RFWD2: constitutive photomorphogenic Ub ligase (RING finger- and WD repeat domain-containing protein-2) CoPS: signalosome CoP9 homolog subunit CORM: carbon monoxide (CO)-releasing molecule coSMAD: common (mediator) SMAD (SMAD4)

List of Molecule Shortened Abbreviations and Chemical Symbols COx: cyclooxygenase (PGhS) CP: proteasomal core particle CP4H: collagen prolyl 4-hydroxylase CPA: cleavage and polyadenylation complex CPase (CP): carboxypeptidase CPC: chromosomal passenger complex CPEB: cytoplasmic polyadenylation element-binding protein CpG: cytidineP –guanosine oligodeoxynucleotide (motif) CMe pG: methylated CpG dinucleotide CpGI: CpG island cPKC: conventional protein kinase-C CPSF: cleavage and polyadenylation specificity factor (hexamer; CPA subcomplex) CPSFi: type-i CPSF complex subunit CPT: carnitine palmitoyltransferase (acyltransferase) CPTP: ceramide 1-phosphate transfer protein CPVT: catecholaminergic polymorphic ventricular tachycardia Cpx: complexin CR: complement component receptor Cr: creatine cRABP: cellular retinoic acid-binding protein CRAC: Ca2+ release-activated Ca2+ channel CRACR: CRAC regulator CRADD: Casp2 and RIPK1 domain-containing adaptor with death domain CrAT: carnitine acetyltransferase cRBP: cellular retinol-binding protein CREB: cAMP-responsive element-binding protein CREB3L: cAMP responsive element-binding protein-3-like protein CRF: corticotropin-releasing factor (family) CREG: cellular repressor of E1A-stimulated genes CRH: corticotropin-releasing hormone CRIB: CDC42/Rac interactive-binding protein CRIK (Cit): citron, Rho-interacting, protein Ser/Thr kinase (STK21 [CIT gene]) CRK: chicken tumor virus CT10 regulator of kinase CRKL: CRK avian sarcoma virus CT10 homolog-like CRLi: type-i cullin–RING Ub ligase complex CRLR: calcitonin receptor-like receptor CrlS: cardiolipin synthase CROT: carnitine O octaniltransferase CRP: bacterial carbohydrate (C)-reactive protein (Ptx1) CRTC: CREB-regulated transcription coactivator

805

CRU: Ca2+ release unit (couplon or dyad) CRUMBS (Crb): crumbs homolog polarity complex Cry: cryptochrome CS: cholesterol CSase (CS): citrate synthase CSBP: cytokine-suppressive anti-inflammatory drug-binding protein CSE (Cth): cystathionine γ-lyase (cystathionase; H2 S production) CSF1: monocyte colony-stimulating factor (mCSF) CSF2: granulocyte–macrophage colonystimulating factors (gmCSF and sargramostim) CSF3: granulocyte colony-stimulating factors (gCSF and filgrastim) CSH: chorionic somatomammotropin hormone CSHL: chorionic somatomammotropin hormone-like hormone CSK: C-terminal Src kinase CSN: constitutive photomorphogenic homolog signalosome CoP9 CSPG: chondroitin sulfate proteoglycan Csq: calsequestrin (CASQ gene) CSR: class-switch recombination (during antibody formation) CSS: candidate sphingomyelin synthase Cst (CSt): cystatin CSTF: cleavage stimulation factor (hexamer; CPA subcomplex) CStFi: type-i CTSF complex subunit CSTI: cytosolic and mitochondrial cholesterol transduceosome and importer CSx: Cockayne syndrome protein-X CT: cardiotrophin CTBP: C-terminal-binding protein Ctbs: diN acetylchitobiase CTCF: CCCTC motif-binding zinc finger factor (transcriptional repressor) CTCFL: CTCF-like factor CTen: C-terminal tensin-like protein CTF: C-terminal fragment CTGF: connective tissue growth factor Cth: cystathionase (cystathionine γ-lyase) CTIF: CBP80–CBP20 [NCBP1–NCBP2]dependent translation initiation factor CTL: cytotoxic T lymphocyte CTLA: cytotoxic T-lymphocyte-associated protein Ctnn: catenin CTr: copper transporter CtR: calcitonin receptor

806

List of Molecule Shortened Abbreviations and Chemical Symbols

CTRC: CREB-regulated transcription coactivator CTRP: C1q and TNF-related protein (adiponectin paralog) Cts: cathepsin CTSBP (CBP): C-terminal Src kinase-binding protein CUGBP: cytidine–uridine–guanidine (CUG) triplet repeat RNA-binding protein Cul: cullin cuoxLDL: copper-oxidized LDL CUT: cryptic unstable transcript cUTR: constitutive untranslated region CWC: complexed with CDC5 (Cef1) homolog Cx: connexin CXCLi: type-i CXC (C–X–C motif; α) chemokine ligand CXCRi: type-i CXC (C–X–C motif; α) chemokine receptor CX3 CLi: type-i CX3 C (δ) chemokine ligand CX3 CRi: type-i CX3 C (δ) chemokine receptor Cy CK (cyCK): cytosolic creatine kinase CyB5: cytochrome-B5 CyB5R: CyB5 reductase CyC: cytochrome-C CyFIP: cytoplasmic FMR1 (or FMRP)interacting protein Cyld: cylindromatosis tumor suppressor protein (deubiquitinase USPL2) CYP: cytochrome-P450 superfamily member CyP: cytochrome-P450 Cyp: cyclophilin (PPI) CyPOR: cytochrome-P450 oxidoreductase (POR gene) CyR61: cysteine-rich angiogenic inducer (CCN1 or IGFBP10) CysCts: cysteinyl cathepsin (CtsB–CtsC, CtsF, CtsH, CtsK–CtsL, CtsO, CtsS, CtsV–CtsW, and CtsX) CysLTR: cysteinyl leukotriene (LTc4 –LTe4 ) receptor

D DA: dopamine DAAM: disheveled-associated activator of morphogenesis Dab: disabled homolog DAD: defender against cell death DAG: diacylglycerol DAGK: diacylglycerol kinase DAMP: damage-associated molecular pattern DAOx: D amino acid oxidase DAP: death domain-associated protein

DAP: dipeptidyl aminopeptidase DAPC: dystrophin-associated protein complex DAPK: death-associated protein kinase DARC: Duffy antigen receptor for chemokine DAT: dopamine active transporter DAX: dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X (NR0b1) DAXX: death domain-associated protein (DAP6) DBC: deleted in breast cancer protein DBF: dumbbell formation kinase (in Saccharomyces cerevisiae; e.g., DBF2) DBP: albumin D-box element-binding protein (PARBZIP set) DCA: deoxycholic acid (bile acid) DCAF: DDB1- and Cul4-associated factor DCC: deleted in colorectal carcinoma (netrin receptor) DCLK: doublecortin and calcium–calmodulindependent protein kinase-like kinase DCLRe1: DNA cross-link repair nuclease (DCLRe1c and DCLRe1b being artemis and apollo) DCP: dipeptidyl carboxypeptidase (i.e., ACE1, ACE2, and DPP4) DCp: mRNA decappase Dctn: dynactin DDAH: dimethylarginine dimethylaminohydrolase DDB: damage-specific DNA-binding protein DDC: DOPA decarboxylase DDEF: development and differentiationenhancing factor (ArfGAP) DDIi: type-i DNA-damage inducible protein DDI1 homolog DDITi: type-i DNA-damage-inducible transcript-derived protein DDK: Dbf4 (MAP3K5)-dependent kinase (CDC7L–MAP3K5 kinase complex) DDOST: dolichyl diphosphooligosaccharide– protein glycosyltransferase DDOx: D aspartate oxidase DDR: discoidin domain receptor DDSR: DNA damage-sensitive RNA DDx: DEAD (Asp–Glu–Ala–Asp) boxcontaining protein DEC: deleted in esophageal cancer DECi: differentially expressed in chondrocytes, DEC1 and DEC2 corresponding to bHLHe40 and bHLHe41, bHLHb2 and bHLHb3, or HRT2 and HRT1

List of Molecule Shortened Abbreviations and Chemical Symbols DEG: delayed-early gene degron: degradation signal DENND: differentially expressed in normal and neoplastic cell domain-containing protein (RabGEF) deoxyHb: deoxygenated hemoglobin deptor: DEP domain-containing TOR-binding protein (DEPDC6 gene) DERL: degradation in endoplasmic reticulum-like protein (derlin) DES: dihydroceramide desaturase DeS (DS): dermatan sulfate DFCP: double FYVE-containing protein DGAT: diacylglycerol acyltransferase DGC: dystrophin–glycoprotein complex DGCR: DiGeorge syndrome critical region gene product (Pasha) DH: Dbl homology DHA: docosahexaenoic acid DHAP: dihydroxyacetone phosphate DHAPAT: DHAP acyltransferase dhCer: dihydroceramide DHDH: dihydrodiol dehydrogenase DHDP(E): dihydroxydocosapentaenoic acid DHEA: dehydroepiandosterone DHEE: dihydroxyeicosaenoic acid DHET(rE): dihydroxyeicosatrienoic acid DHETE: dihydroxyeicosatetraenoic acid DHETrE: dihydroxyeicosatrienoic acid DHh: desert hedgehog DHODE: dihydroxyoctadecadienoic acid DHOME: dihydroxyoctadecamonoenic (dihydroxyoctadecenoic) acid dhSph: dihydrosphingosine DHx: DEXH (Asp–Glu–X–His) boxcontaining protein Dia: diaphanous diablo: mitochondrial direct IAP-binding protein with low pI Dio: iodothyronine deiodinase DIRAS: distinct subgroup of the RAS hyperset member DISC: death-inducing signaling complex Dkk: dickkopf DLg: disc large homolog DLL: delta-like (notch) ligand DLx: distal-less homeobox-derived (homeodomain-containing) protein DMi: type-i double minute Dm: Drosophila melanogaster DMG: dimethylglycine DMM: DNA methylation modulator DMPK: myotonic dystrophy-associated protein kinase

807

DNA: deoxyribonucleic acid DNA2: DNA synthesis-defective protein-2 (DNA replication and repair helicase and nuclease) DNAPK: XRCC5–XRCC6–DNAPKc kinase complex DNAPKc : DNA-dependent protein kinase catalytic subunit DNM: dynamical network marker DNMT: DNA methyltransferase DNMT1AP (DMAP): DNA methyltransferase1-associated protein DNPep: aspartyl aminopeptidase DMPK: myotonic dystrophy-associated protein kinase DMR: differentially methylated region (imprinting) DMT: divalent metal transporter DMTF: cyclin-D-binding MyB-like transcription factor DNA: deoxyribonucleic acid DNAPK: DNA-dependent protein kinase complex DNAPKcs: DNAPK catalytic subunit DNA Pol: DNA polymerase Dnase: deoxyribonuclease dNDP: deoxynucleoside disphosphate dNTP: deoxynucleoside triphosphate DOC: deoxycorticosterone DoC2: double C2-like domain-containing protein DOCK: dedicator of cytokinesis (GEF) DOHH: deoxyhypusine hydroxylase DOK: downstream of protein Tyr kinase docking protein DOPA: (3,4)-dihydroxyphenylalanine DOR: δ-opioid receptor DPepi: type-i dipeptidase (DPep1–DPep3) DPG: diphosphoglyceric acid DPP: dipeptidyl peptidase DPt: dermatopontin DRAK: death receptor-associated proteinrelated apoptotic kinase DRAM: lysosomal DNA damage-regulated autophagy modulator DRF: diaphanous-related formin (for GTPase-triggered actin rearrangement) Drl: derailed DRP1: dynamin-related protein-1 (dynamin-1like protein [DNM1L gene]) Dsc: desmocollin DSCC: defective in sister chromatid cohesion protein dsDNA: double-stranded DNA

808

List of Molecule Shortened Abbreviations and Chemical Symbols

Dsg: desmoglein DSK: dual-specificity kinase dsRNA: double-stranded RNA Dst: dystonin Dtl: denticleless homolog DUb: deubiquitinase DuOx: dual oxidase DuSP: dual-specificity phosphatase Dvl (Dsh): Disheveled (cytoplasmic Wnt phosphoproteic mediator)) dworf: lncRNA-encoded dwarf ORF DXO: decappase and exoribonuclease dynactin: dynein activator DYRK: dual-specificity protein Tyr (Y) phosphorylation-regulated kinase

E E1 : estrone (a single hydroxyl group in its molecule) E2 : estradiol (2 hydroxyl groups), or 17β-estradiol E3 : estriol (3 hydroxyl groups) E-box: enhancer box sequence of DNA E2: ubiquitin conjugase E3: ubiquitin ligase EAAT: excitatory amino acid (glutamate– aspartate) transporter EAP: ESPL1-associated protein EAR: v-ErbA-related nuclear receptor (NR2f6) EB: end-binding protein EBF: early B-cell factor ECC: EloBC–Cul2–CIS Ub ligase complex ECE: endothelin-converting enzyme ECS: EloBC–Cul2(5)–SOCS1(3) Ub ligase complex EcircRNA: exonic circular RNA ECPAS: Ecm29 proteasomal adaptor and scaffold ecRNA: extracellular RNA ED1L (edil): EGF-like repeat- and discoidin-1 (I)-like domain-containing protein EDC: enhancer of mRNA decappase homolog EDEM: ER degradation-enhancing αmannosidase-like protein EDGR: endothelial differentiation gene receptor EDHF: endothelial-derived hyperpolarizing factor EDME: ER degradation enhancer and mannosidase eDMR: differentially methylated regions of environmental origin EDN: endothelin gene (ET[Edn]1/2/3)

EdNaC: endothelial Na+ channel EDP(E): epoxydocosapentaenoic acid EEA: early endosomal antigen EEE: epoxyeicosaenoic acid EED: epoxyeicosadienoic acid EETE (EEQ): epoxyeicosatetraenoic acid EET(rE): epoxyeicosatrienoic acid EF-Tu: elongation factor Tu EFA6: exchange factor for ARF6 (ArfGEF) eFGF: endocrine FGF (FGF19/21/23) Efn: ephrin (EPH receptor interactor) EG: epoxyeicosatrienoylglycerol EGF: epidermal growth factor EGFL: EGF-like domain-containing protein EGFR: epidermal growth factor receptor EGFTM7: EGF 7-span transmembrane group of AdGRs EgLN: egg laying-9 EGo: exit from rapamycin-induced growth arrest EGR: early growth response transcription factor EHD: C-terminal EGFR substrate-15 homology domain-containing protein EHHADH: enoylCoA hydratase/3hydroxyacylCoA dehydrogenase EIcircRNA: exon–intron circular RNA EJC: between-exon junction complex EJCSR: EJC–SR multimer EKODE: epoxyketooctadecenoic acid EL: endothelial lipase ELAM: endothelial–leukocyte adhesion molecules ELAVL: embryonic lethal, abnormal vision protein-like (ELAV)-like RNA-binding protein ELk: ETS-like transcription factor (ternary complex factor [TCF] subfamily) ElMo: engulfment and cell motility adaptor Eln: elastin elncRNA: enhancer-associated long nonprotein-coding RNA ElnF: elastin fiber Elo: fatty acid elongase EloX: elongin-X (X: A, B, C) EMI: early mitotic inhibitor EMR: EGF-like module-containing, mucin-like, hormone receptor-like GPCR EMRe: essential mtCU regulator (Smdt1 gene) En: engrailed ENA–VASP: enabled homolog and vasoactive (vasodilator)-stimulated phosphoprotein family

List of Molecule Shortened Abbreviations and Chemical Symbols ENaC: epithelial Na+ channel EnaH: enabled homolog ENC1: BTB-like domain-containing actinbinding ectodermal–neural cortex protein-1 endo-siRNA: endogenous small interfering RNA Eng: endoglin (CD105) ENOSC (eNoSC): energy-dependent nucleolar silencing complex ENPep: glutamyl aminopeptidase [APa] ENPP: ectonucleotide pyrophosphatase– phosphodiesterase Ens: endosulfine ENT: equilibrative nucleoside transporter ENTPD: ectonucleoside triphosphate diphosphohydrolase EnY: transcription and mRNA export factor enhancer of yellow EODE: epoxyoctadecadienoic acid EOME: epoxyoctadecamonoenic acid EPA: eicosapentaenoic acid EPAC: exchange protein activated by cAMP EPAS: endothelial PAS domain protein ePCR: endothelial activated protein-C (aPC) receptor EpETE: epoxyeicosatetraenoic acid Epgn: epigen (EGF superfamily member) EPH: erythropoietin-producing hepatocyte receptor kinase or pseudokinase (EPHa10 and EPHb6) Epo (EPo): erythropoietin EPS: epidermal growth factor receptor pathway substrate EpStI: epithelial–stromal interaction protein EPT: ethanolamine phosphotransferase ERξ: type-ξ estrogen receptor (ξ: α or β; NR3a1/2) eRas: embryonic stem cell-expressed Ras (or hRas2) ErbB: erythroblastoma viral gene product-B (HER) ERC: elastin receptor complex ERCCi: excision repair cross-complementing repair deficiency complementation group i Ereg (EReg): epiregulin (EGF superfamily member) Erg: erythroblastosis virus E26 proto-oncogene product homolog (transcriptional regulator)

809

ERGIC: endoplasmic reticulum–Golgi intermediate compartment ERK: extracellular signal-regulated protein kinase ERLec: endoplasmic reticulum lectin ERM: ezrin–radixin–moesin ERMES: ER–mitochondrion-encountered structures ERMIC: endoplasmic reticulum transmembrane insertase complex ERMIONE: ER–mitochondria organizing network ERN: endoplasmic reticulum to nucleus signaling endoribonuclease and kinase eRNA: enhancer-derived nonprotein-coding RNA ERO1ξ: type-ξ endoplasmic reticulum oxidoreductin-1 (thiol oxidase; ERO1A–ERO1B genes) ERP: endoplasmic reticulum-resident protein ERRξ: type-ξ estrogen-related receptor (NR3b1–NR3b3) ERV: endogenous retrovirus (retrotransposon) ERV1: essential for respiration and viability protein-1 esco: establishment of cohesion-1 homolog (acetyltransferase) ESCRT: endosomal sorting complex required for transport ESE: exonic splicing enhancer ESL: E-selectin ligand ESM: endotheliocyte-specific molecule ESPL: extra spindle poles-like protein esRNA: exosomal shuttle RNAs ESRP: epithelial splicing regulatory protein ESS: exonic splicing silencer eSyt: extended synaptotagmin ET: endothelin ETC: electron transport chain ETF: electron transfer flavoprotein ETFDH: ETF dehydrogenase EtnK: ethanolamine kinase ETR (ETA/B ): endothelin receptor ETS: E-twenty six transformation-specific sequence (transcription factor; erythroblastosis virus E26 protooncogene product homolog) ETV: ETS-related translocation variant EvaL: EVI1: ecotropic virus integration site-1 protein homolog Exo: exocyst subunit

810

List of Molecule Shortened Abbreviations and Chemical Symbols

Exo1: exonuclease-1 ExosC: exosome component Ext: exostosin (glycosyltransferase) EZH: enhancer of zeste homolog (histone Lys methyltransferase)

F F: coagulation (clotting) factor- F6P: fructose 6-phosphate FA: fatty acid FAAH: fatty acid amide hydrolase FAAP: Fanconi anemia-associated protein FABP: fatty acid-binding protein FACAP: F actin complex-associated protein FACoA: fatty acylCoA FACS: fatty acylCoA synthase FACT (FaCT): heterodimeric histone chaperone that facilitates chromatin transcription FActBP (FABP): filamentous actin-binding protein FAD: flavine adenine dinucleotide FADD: FasR (TNFRSF6a)-associated death domain-containing protein FADS: fatty acid desaturase FAK: focal adhesion kinase FAKIP (FIP): focal adhesion kinase family-interacting protein Famix: type-X family-i member Fanc: Fanconi anemia protein FAN: Fanconi anemia (FancD2)-associated nuclease FAPP: phosphatidylinositol four-phosphate (PI4P) adaptor protein FAS: fatty acid synthase (FASN gene) FAST: forkhead activin signal transducer FASTK: Fas (TNFRSF6)-activated protein Ser/ Thr kinase FATP: fatty acid transport protein (SLC27a) FBG: F-box- and G-domain-containing protein Fbln (Fibl): fibulin Fbn: fibrillin FBP: fructose bisphosphatase FBSi: type-i F-box-containing protein that recognizes sugar chains (FBGi/FBxOj ) FBx: F-box-containing protein FBxL: F-box- and leucine-rich repeat (LRR)-containing protein FBxO: F-box-containing protein with another or no other motif (F-box only protein) FBxO7 (FBx): F-box only ArfGEF FBxO8 (FBS): (Ccn)F-box- and Sec7 domain-containing ArfGEF

FBxW: F-box- and WD (Trp–Asp) repeatcontaining protein FCHO: FCH domain only protein FcαR: Fc receptor of IgA FcγR: Fc receptor of IgG Fc R: Fc receptor of IgE FcμR: Fc receptor of IgM FCP: TF2F-associating C-terminal domain phosphatase FeCh: ferrochelatase (mitochondrial heme synthase) FEn: flap endonuclease (FEn1 is Dnase4) fendrr: FoxF1 adjacent enhancer-associated noncoding developmental regulatory RNA (lncRNA) FeR: FeS-related protein Tyr kinase FERM: four point-1, ezrin–radixin–moesin domain FeS: feline sarcoma kinase FFA: free fatty acid Fgξ: type-ξ fibrinogen polypeptidic chain FGF: fibroblast growth factor FGFR: fibroblast growth factor receptor FGR: viral feline Gardner–Rasheed sarcoma oncogene homolog kinase FHL: four-and-a-half LIM-only protein FHoD: formin homology domain-containing protein (FmnL) FIH: factor inhibiting HIFα (asparagyl hydroxylase) FIP: family of Rab11-interacting protein FIP1L1: factor interacting with polyadenylating polymerase FIP1-like protein-1 Fis: mitochondrial fission protein FITM: fat storage-inducing transmembrane protein FKBP: FK506-binding protein FLI: friend leukemia virus integration proto-oncogene product homolog (transcription factor) FlIP: flice-inhibitory protein FLK: fetal liver kinase Flcn: folliculin Fln: filamin fMLP: formyl methionyl leucyl phenylalanine (fMet–Leu–Phe) FMO: flavin-containing monooxygenase FMR1 (FMRP): fragile X mental retardation protein Fn (FN): fibronectin Fn: fibrin FnBP: formin-binding protein Fng: fibrinogen

List of Molecule Shortened Abbreviations and Chemical Symbols XFng: fringe homolog (X: L [lunatic], M [manic], and R [radical]) FnIP: folliculin-interacting protein FOS: Finkel–Biskis–Jinkins murine osteosarcoma virus sarcoma protooncogene product Fox: forkhead box-containing transcription factor FOxRed: FAD-dependent oxidoreductase domain-containing protein FPC: replication fork protection complex Fpn: ferroportin FRK: Fyn-related kinase FrmD: FERM domain-containing adaptor FRNK: FAK-related nonkinase FRS: fibroblast growth factor receptor substrate Fru(1,6)P2 : fructose (1,6)-bisphosphate Fru(2,6)P2 : fructose (2,6)-bisphosphate FSH: follicle-stimulating hormone Fst (FSt): follistatin FstL (FStL): follistatin-like protein Fucα1: tissular αL fucosidase Fum: fumarase FunDC: Fun14 domain-containing protein Fus: RNA-binding fused in sarcoma (hnRNPp2) FXR: farnesoid X receptor (NR1h4) FXR (FXRP): FMR1 autosomal homolog Fyn: SRC family (SFK) membrane-associated nonreceptor protein Tyr kinase FyTTD: forty-two–three domain-containing protein Fzd (Fz): frizzled (Wnt GPCR) G GSH (GSH): sulfhydryl glutathione (“reduced form”) GSS (GSSG): glutathione disulfide (“oxidized form”) G protein: guanine nucleotide-binding protein (Gαβγ trimer) G3BP1: Ras GTPase-activating proteinbinding protein-1 G6PDH (G6PD): glucose 6-phosphate dehydrogenase γGCx: γ-glutamyl carboxylase Gα: α subunit (signaling mediator) of G protein Gα12/13 (G12/13): Gα subunit class 12/13 Gαgust : gustducin, G protein α subunit (Gi/o) of taste receptor Gαi (Gi): inhibitory Gα subunit Gαi/o (Gi/o): Gα subunit class

811

Gαolf : G protein α subunit (Gs) of olfactory receptor Gαq/11 (Gq/11): Gα subunit class Gαs (Gs): stimulatory Gα subunit GαsXL : extralarge Gs protein Gαt (Gt): transducin, Gα subunit of rhodopsin GαTc , cone transducin GαTr : rod transducin Gβγ: dimeric subunit (signaling effector) of G protein GβA: acid β-glucosidase (glucosylceramidase) Gaα: lysosomal α-glucosidase Ggust : (G protein) Gα subunit gustducin GAB: GRB2-associated binder GAD: glutamic acid decarboxylase (GABA synthase) GABA: γ-aminobutyric acid GABAA : GABA ionotropic receptor (Cl− channel) GABAB : GABA metabotropic receptor (GPCR) GABARAP: GABAA receptor-associated protein (Atg8a) GABARAPL: GABARAP-like protein (Atg8b–Atg8d) GABAT: GABA transaminase GaBP: globular actin-binding protein GADD: growth arrest and DNA-damageinduced protein gadd7: growth-arrested DNA damageinducible transcript-7 (lncRNA) GADD45γIP: GADD45γ-interacting protein gadkin: γ1-adaptin and kinesin interactor GAG: glycosaminoglycan GAK: cyclin G-associated kinase GaL: galectin (β-galactoside-binding lectin) Gal: galanin GalC: galactocerebrosidase (galactosylceramidase) GalCer: galactosylceramide GalNS: N acetylgalactosamine 6-sulfatase GAP: GTPase-activating protein GAPDH: glyceraldehyde 3-phosphate dehydrogenase GARP: Golgi body-associated retrograde protein complex GAS: growth arrest-specific noncoding, single-stranded RNA gas5: growth arrest-specific noncoding lncRNA GAT: γ-aminobutyric acid transporter GAsK: Golgi body-associated kinase GATA: (A/T)GATA(A/G) DNA sequence in the regulatory region of gene

812

List of Molecule Shortened Abbreviations and Chemical Symbols

GATA: DNA sequence GATA-binding transcription factor gator: GTPase-activating protein toward Rag gator1: NPRL2–NPRL3–DEPDC5 complex gator2: milos–WDR24–WDR59–SEHL1L– SEC13 complex GBF: Golgi body-associated brefeldin-Aresistant guanine nucleotide-exchange factor GBP: glycan-binding protein GBPi: type-i (interferon-induced) guanylatebinding protein (GTPase) GCAP: guanylate cyclase-activating protein GCC: Golgi coiled-coil domain-containing protein Gcg: glucagon GcgR: glucagon receptor GCK: germinal center kinase GCKR: GCK-related kinase GCL: (heterodimeric) glutamate cysteine ligase (catalytic [GCLc ] and modifier subunit [GCLm ]) GCN: general control of amino acid synthesis GCNF: germ cell nuclear factor (NR6a1) GCN2: general control nonderepressible-2 (pseudokinase) GCS: glutamylcysteine synthase Gcs: glucosidase gCSF: granulocyte colony-stimulating factor (CSF3) GD: disialoganglioside GDP: guanosine diphosphate GDF: (Rab)GDI displacement (dissociation) factor GDFi: type-i growth and differentiation factor (TGFBSF member) GDI: guanine nucleotide-dissociation inhibitor gDMR: differentially methylated regions of genetic origin GDNF: glial cell line-derived neurotrophic factor GEF: guanine nucleotide (GDP-to-GTP)exchange factor gemin: nuclear Gemini body (Gem)-associated protein GF: growth factor GFAP: glial fibrillary acidic protein (intermediate filament) GFL: GDNF family of ligands GFPT: glutamine–fructose 6-phosphate aminotransferase GFR: growth factor receptor GFRαi: type-i GDNF family receptor-α

GFRαL: GDNF family receptor-α-like receptor (GDF15 receptor) GGA: Golgi body-localized γ-adaptin ear-containing ARF-binding protein GGT (GGT1): γ-glutamyltransferase (gene) GH: growth hormone GHR: growth hormone receptor GHRH: growth hormone-releasing hormone Ghrl: ghrelin GHRP: growth hormone-releasing peptide (or GHS) GHS: growth hormone secretagogue (or GHRP) GHSR: (ghrelin and) growth hormone secretagogue receptor GIF: gastric intrinsic factor GILZ: glucocorticoid-induced leucine zipper protein GINS: go–ichi–ni–san (Japanese numbers 5–1–2–3); DNA replication-involved heterotetramer GIP: gastric inhibitory peptide GIPR: gastric inhibitory peptide receptor GIP: GPCR-interacting protein GIRK: Gβγ-regulated inwardly rectifying K+ channel GIT: GPCR kinase-interacting protein GKAP/DLgAP1: guanylate kinase-associated protein (disc large homolog-associated protein-1) GKAP/DuSP12: glucokinase-associated dual specificity phosphatase GKAP1: (PK)G-kinase-anchoring protein-1 Glα (GLA gene): galactosidase-α Glβ: galactosidase-β GlcNAc (GlcNAc): N acetylglucosamine O GlcN Ac: βN acetyl D glucosamine Gli: GLI family zinc finger-containing transcriptional regulator GLK: GCK-like kinase glLDL: glycated low-density lipoprotein gl–oxLDL: glycated and oxidized LDL GLP: glucagon-like peptide GLP1R: glucagon-like peptide-1 receptor Gls: glutaminase GLTP: glycolipid transfer protein GluDH: glutamate dehydrogenase GluK: ionotropic glutamate receptor (kainate type) GluL: glutamate–ammonia ligase (glutamine synthetase) GluN: ionotropic glutamate receptor (NMDA type)

List of Molecule Shortened Abbreviations and Chemical Symbols GluR: ionotropic glutamate receptor (AMPA type) GluT: glucose transporter GlxR (GlyR): glyoxylate reductase homolog (glyoxylate reductase/hydroxypyruvate reductase [GRHPR]) glycam (GlyCAM): glycosylation-dependent cell adhesion molecule GlyR: glycine receptor (channel) GlyT: glycine transporter GM: monosialoganglioside gmCSF: granulocyte–monocyte colonystimulating factor (CSF2) GMP: guanosine monophosphate GNAT: GCN5 (KAT2a)-related N acetyltransferase GNPT: UDP–N acetylglucosamine 1phosphotransferase GnRH: gonadotropin-releasing hormone GNRH1(2): GnRH-encoding (progonadoliberin-1[2]) gene GNS: N acetylglucosamine 6-sulfatase GP: glycoprotein GPAT: glycerol phosphate O acyltransferase Gpc: glypican GPD1L: glycerol 3-phosphate dehydrogenase1-like protein GPDH1: cytosolic glycerol 3-phosphate dehydrogenase (GPD1 gene) GPER: G-protein-coupled estrogen receptor GPI: glycosylphosphatidylinositol anchor gpiAP: GPI-anchored protein GPCR: G-protein-coupled receptor GPOx: glutathione peroxidase GQ: quadrisialoganglioside GR: glucocorticoid receptor (NR3c1) GRAP: GRB2-related adaptor protein (or GAds) GRB: growth factor receptor-bound protein GRC: growth factor-regulated, Ca2+ permeable, cation channel (TRPV2) GRd: glutathione reductase (GSR gene) GRdx: glutaredoxin Grem: gremlin GrhL: grainyhead-like transcription factor GRK: G-protein-coupled receptor kinase GRP: G-protein-coupled receptor phosphatase GRP: γ-carboxyglutamate acid (Gla)-rich protein (UCMA) GRPEL: bacterial GrpE-like protein (GrpE: glucose-regulated protein of Escherichia [E.] coli) GSK: glycogen synthase kinase Gsdm: gasdermin

813

GSNOR: S nitrosoglutathione (GSNO ) reductase (ADH5) GRd (GSR): glutathione disulfide reductase GsS: glutathione (GSH ) synthase GST: glutathione S transferase GT: trisialoganglioside GTF: general transcription factor GTP: guanosine triphosphate GTPase: guanosine triphosphatase GuCy: guanylate cyclase (CyG) Gusβ: glucuronidase-β GyS: glycogen synthase H HA: histamine HAAT: heterodimeric amino acid transporter HAc (HA): hyaluronic acid (hyaluronan; anionic hyaluronate) HAD: haloacid dehalogenase HADH: hydroxyacylCoA dehydrogenase HAg (HA): hemagglutinin HAND: heart and neural crest derivatives expressed protein HAOx: hydroxyacid oxidase HAP: huntingtin-associated protein HAPLn: hyaluronan and proteoglycan linker HaS: hyaluronan synthase HAT: histone acetyltransferase Hb (Hgb): hemoglobin HbSN O : S nitrosohemoglobin HBS1L: HSP70 subfamily B suppressor-like translational GTPase HBEGF: heparin-binding EGF-like growth factor hcam: homing cell adhesion molecule (epican/ ABCb1) hcg22: HLA complex group-22 lncRNA HCK: hematopoietic cell kinase HCLS: hematopoietic lineage cell-specific Lyn substrate protein HCN: hyperpolarization-activated, cyclic nucleotide-gated K+ channel HCNP: hippocampal cholinergic neurostimulatory peptide Hcrt: hypocretin (orexin) HCS: holocarboxylase synthase HCTL: homocysteine thiolactone HCys: homocysteine HDAC: histone deacetylase complex HDGF: hepatoma-derived growth factor HDGFL1: HDGF-like protein-1 (or PWWP1) HDL: high-density lipoprotein

814

List of Molecule Shortened Abbreviations and Chemical Symbols

HDLApoA1 : apolipoprotein-A1-containing HDL HDLCs : HDL–cholesterol HDLCsE : HDL–cholesteryl ester HDLTG : triglyceride-enriched HDL HDM: human double minute (Ub ligase) HDHE: hydroxydocosahexaenoic acid HeBP: heme-binding protein HECT: homologous with E6-associated protein C-terminus domain-containing Ub ligase HEET: hydroxyepoxyeicosatrienoic acid hemin: heme oxygenase-1 inducer HEPE: hydroxyeicosapentaenoic acid HERG: human ether-a-go-go–related gene HER: human epidermal growth factor receptor (HER3: pseudokinase) HES: hairy and enhancer of split HeS (HS): heparan sulfate HETE: hydroxyeicosatetraenoic acid HETEE: HETE ethanolamide Hex: hexosaminidase HExo: histone mRNA 3 -end-specific exoribonuclease HGF: hepatocyte growth factor HGFA: hepatocyte growth factor activator (serine peptidase) HGFR: hepatocyte growth factor receptor HGS: HGF-regulated protein Tyr kinase substrate (HRS) HhIP: hedgehog-interacting protein HHT: hydroxyheptadecatrienoic acid HIB: hydroxyisobutyrate HIF: hypoxia-inducible factor HIP: huntingtin-interacting protein hif1as: antisense transcript of HIF1α (lncRNA) HIP: HSPa8-interacting protein (Fam10a1, suppression of tumorigenicity ST13, and aging-associated protein AAG2) HIP1R: HIP1-related protein HIPK: homeodomain-interacting protein kinase HIRA (HiRa): histone cell cycle regulation homolog-A complex His: histamine HJuRP: holliday junction recognition protein Hjv: hemojuvelin HK: hexokinase HL: hepatic lipase HLA: human leukocyte antigen HLAa(b,c): MHC class-I peptides HLAdp(dm,doa,dob,dq,dr): MHC class-I I peptides HLB: histone locus body

HLF: hepatic leukemia factor (PARBZIP set) HMG: high-mobility group protein HMGB: high-mobility group box-containing protein HMGCoA: 3-hydroxy 3-methylglutaryl coenzyme-A HMGCoAL: HMGCoA lyase (HMGCL gene) HMGCoAR: HMGCoA reductase (HMGCR gene) HMGCoAS: HMGCoA synthase (HMGCS gene) HMT: histone methyltransferase HMWK: high-molecular–weight kininogen HNDC: hypokinetic nondilated cardiomyopathy HNF: hepatocyte nuclear factor (NR2a1/2) HNP: human neutrophil peptide hnRNP: heterogeneous nuclear ribonucleoprotein HODE: hydroxyoctadecadienoic acid (hydroxylinoleic acid) HOP: HSP70–HSP90 complex-organizing protein (stress-induced phosphoprotein StIP1) HoPS: homotypic fusion and vesicle protein sorting complex hotair: HOX transcript antisense intergenic RNA (lincRNA) HOTrE: hydroxyoctadecatrienoic acid hottip: HOXA transcript at the distal tip (lincRNA) HOx: heme oxygenase (HMOX gene) Hox: homeobox DNA sequence (encodes homeodomain-containing transcriptional regulators and morphogens) HpCa: hippocalcin HPETE: hydroperoxyeicosatetraenoic acid HPETEE: HPETE ethanolamide HPK: hematopoietic progenitor kinase (MAP4K) HPODE: hydroperoxylinoleic acid (hydroperoxylinoleic acid) hpRNA: long hairpin RNA Hpse: heparanase hRas: Harvey Ras homolog Hrk: BH3-interacting domain-containing activator of apoptosis harakiri HRM: hypoxia-regulated microRNA hRNP: heterogeneous ribonucleoprotein HRS: hepatocyte growth factor-regulated protein Tyr kinase substrate HRT: HES-related transcription factor Hs: Homo sapiens

List of Molecule Shortened Abbreviations and Chemical Symbols HSC: heat shock cognate HSDH: hydroxysteroid dehydrogenase HSER: heat stable enterotoxin receptor (guanylate cyclase-2C) HSF: heat shock transcription factor HSP: heat shock protein (chaperone) HSPG: heparan sulfate proteoglycan HSR: heat shock (lnc)RNA HTMSIC: high-threshold mechanosensitive ion channel HTR: high temperature requirement endopeptidase Htt: huntingtin huFA: highly unsaturated fatty acid hulc: highly upregulated in liver cancer lncRNA HUNK: hormonally upregulated Neuassociated kinase HuR: human antigen-R (mRNA stabilizer ELAVL1) HUWE: HECT, UBA, and WWE domaincontaining protein Hyal: hyaluronidase hyperlnc: hypoxia-induced endoplasmic reticulum stress-regulating lncRNA

I I2C: importin-α/β–CBC iAAA: mitochondrial intermembrane space ATPase associated with diverse cellular activities peptidase IAP: inhibitor of apoptosis protein IBABP: intestinal bile acid-binding protein IBDH (IBCADH): isobutyrylCoA dehydrogenase icam (ICAM): intercellular adhesion molecule (IgCAM member) ICDH: isocitrate dehydrogenase (IDH gene) IcircRNA: intronic circular RNA IgCAM: immunoglobulin-like cell adhesion molecule ICL: DNA interstrand crosslink ICLR: ICL repair iCliP: intramembrane-cleaving peptidase (that clips) ICR: imprinting control region ID: inhibitor of DNA binding (inhibitor of differentiation; bHLHb24–bHLHb27) IDL: intermediate-density lipoprotein IDmiR: immediately downregulated microRNA IDO: indoleamine (2,3)-dioxygenase

815

IDOL: inducible degrader of LDL receptor (Ub ligase MyLIP) IdS: iduronate 2-sulfatase Iduα: αL iduronidase IEG: immediate-early gene IfIH: interferon-induced with helicase-C domain-containing protein iFGFi: type-i intracellular FGF (FGF11– FGF14) Ifn: interferon IfITM: interferon-inducible transmembrane protein IfnAR: interferon-α/β/ω receptor IF4eNIF: (e)IF4e nuclear import factor IFT: intraflagellar transport complex IFTi: intraflagellar transport protein-i Ig (IG): immunoglobulin IGF: insulin-like growth factor IGFBP: IGF-binding protein IgHC: immunoglobulin heavy chain IgLC: immunoglobulin light chain iGluR: ionotropic glutamate receptor IHh: Indian hedgehog IK: intermediate-conductance Ca2+ -activated K+ channel IκB: inhibitor of NFκB IKK: IκB kinase IL: interleukin ILiR: interleukin-i receptor iLBP: intracellular lipid-binding protein ILK: integrin-linked (pseudo)kinase ILKAP: integrin-linked kinase-associated protein Ser/Thr phosphatase-2C IMAC: inner mitochondrial membrane anion channel IMMP: IMM peptidase complex IMP: impedes mitogenic signal propagation Imp: importin IMPa: inositol monophosphatase INADl: inactivation no after-potential Dprotein inava: innate immunity activator InCenP: inner centromere protein InF: inverted formin Ino80: inositol-requiring DNA helicase (chromatin-remodeling complex) InPP5: inositol polyphosphate 5-phosphatase insig (InsIG): insulin-induced gene product (ER anchor) Ins: insulin (INS gene) InsL: insulin-like peptide InsR (IR): insulin receptor InsRR: insulin receptor-related receptor IP: inositol phosphate

816

List of Molecule Shortened Abbreviations and Chemical Symbols

IP3 : inositol (1,4,5)-trisphosphate IP3 R: IP3 receptor (IP3 -sensitive Ca2+ -release channel) IP4 : inositol (1,3,4,5)-tetrakisphosphate IP5 : inositol pentakisphosphate IP6 : inositol hexakisphosphate IPCEF: interaction protein for cytohesin exchange factor Ipo: importin (IPO/KPN set) IPOD: (perivesicular) insoluble protein deposit IPP: ILK–PINCH–parvin complex IPPP (IPP): inositol polyphosphate phosphatase IQGAP: IQ (Ile–Gln) motif-containing GTPase-activating protein IRAK: IL1 receptor-associated kinase (IRAK2: pseudokinase) IRAP: insulin-regulated membrane aminopeptidase (AT4 ) IRE: inositol-requiring enzyme (endoplasmic reticulum-to-nucleus signaling kinase and endoribonuclease [ERN]) IRF: interferon-regulatory protein (transcription factor) IRFF: interferon-regulatory factor family IRG: immunity-related GTPase IRP: iron regulatory protein IRS: insulin receptor substrate IS: indoxylsulfate (PBUT) ISCu: mitochondrial iron–sulfur cluster assembly scaffold homolog ISE: intronic splicing enhancer ISG: interferon-stimulated gene product Isl: islet (insulin gene enhancer protein) iSMAD: inhibitory SMAD (SMAD6 or SMAD7) isoLG: isolevuglandin isoP: isoprostane Irx: Iroquois-related homeobox gene product (homeodomain-containing protein) ISS: intronic splicing silencer IWS1 (IWS1L): homolog that interacts with Spt6; ISwi: imitation switch ITAM: immunoreceptor tyrosine-based activation motif itch: itchy homolog (Ub ligase) ItFG: integrin-α phenylalanyl–[glycyl]2 – alanyl–prolyl (FG–GAP) repeat-containing protein Itg: integrin ITIM: immunoreceptor tyrosine-based inhibitory motif ITK: interleukin-2-inducible T-cell kinase

ITPK (IP3 3K): inositol trisphosphate kinase IVCDH: mitochondrial isovaleryl CoA dehydrogenase J Jag: jagged (notch ligand) JaK: Janus (pseudo)kinase JAM: junctional adhesion molecule JAMM: JAB1/MPN/Mov34 set deubiquitinase JAMP: JNK1-associated membrane protein JARID: jumonji AT-rich interactive domaincontaining protein JbtSi: JbtS protein-i JHDM: JmjC domain-containing histone demethylase JIP: JNK-interacting protein (MAPK8IP1 and -2) JmjC: jumonji catalytic domain-containing histone demethylase JmjD: jumonji domain-containing protein JMy: junction-mediating and regulatory protein JNK: Jun N-terminal kinase (MAPK8– MAPK10) JNKBP: JNK-binding protein JNKK: JNK kinase JPh (JP): junctophilin JSAP: JNK/SAPK-associated protein Jun: avian sarcoma virus-17 proto-oncogene product (Japanese juunana: seventeen [17]; TF) JUNQ: juxtanuclear quality-control compartment

K KATP : ATP-sensitive K+ channel KCa 1.i: BK channel KCa 2/3/4.i: SK channel KCa 5.i: IK channel KIR : inwardly rectifying K+ channel KV : voltage-gated K+ channel KADH (BCKADH): ketoacid dehydrogenase KAP: kinesin-associated protein Kap (KaP): karyopherin KAT: lysine (K) acetyltransferase KAT3a/CBP: CREB-binding protein katna1: katanin P60 ATPase-containing subunit-A1 KBTBD: kelch repeat and BTB domaincontaining protein

List of Molecule Shortened Abbreviations and Chemical Symbols KCC: K+ –Cl− cotransporter KChAP: K+ channel-associated protein KChIP: KV channel-interacting protein kcnq1ot1: KCNQ1 opposite strand [antisense] transcript-1 (lncRNA) KCTD: K+ channel tetramerisation domain-containing protein KDELR: KDEL (Lys–Asp–Glu–Leu) endoplasmic reticulum retention receptor KDM: histone Lys demethylase KeS (KS): keratan sulfate KDR: kinase insert domain receptor KDS: 3-ketodihydrosphingosine KDSR: KDS reductase keap (KEAP): kelch-like enoylCoA hydratase (ECH)-associated protein (KlhL19; NFE2L2 inhibitor, Ub ligase in redox stress response) KGDH: ketoglutarate dehydrogenase (Ogdh gene) KGDHC: KGDH complex KHC: kinesin heavy chain KHSRP: KH-type splicing regulatory protein KHX (KHE): K+ –H+ exchanger kicstor: Kptn–ItFG2–C12orf66–SzT2 complex KIF: kinesin family KIFi: KIF member-i KIR: killer cell immunoglobulin-like receptor KIT: cellular kinase in tyrosine (SCFR) Kk: plasmatic kallikrein (serine peptidase) KLC: kinesin light chain KLF: Krüppel-like factor KlhL: kelch-like protein KlhDC: kelch domain-containing protein Klk: tissular kallikrein-related serine peptidase KLR: killer cell lectin-like receptor KMN: KNL1–MIS12–NDC80 decamer KMOS: set of KLF4, MyC, Oct4, and Sox2 transcription factors that trigger genomic reprogramming KMT: histone Lys methyltransferase KNl1: kinetochore-null protein-1 (PP1r55 ) KOR: κ-opioid receptor Kpn: karyopherin Kptn: actin-binding kaptin kRas: Kirsten Ras homolog KRIT1: Kirsten sarcoma virus Ras-revertant (KRev)-interaction trapped protein-1 (Krev1 being Rap1a) Krt: keratin KSR: kinase suppressor of Ras (adaptor; pseudokinase)

817

L L3MBTL: lethal-3 malignant brain tumor-like protein LA: linoleic acid LAB: linker of activated B lymphocyte LAd: LCK-associated adaptor LAL: lysosomal acid lipase (LipA) Lam: laminin LAMP: lysosome-associated membrane protein lamtor: late endosomal and lysosomal adaptor, MAPK and TOR activator LANP: long-acting natriuretic peptide LAP: nuclear lamina-associated polypeptide LAPi: type-i latency-associated peptide (LAP1–LAP4) LAPFi (LAPi): leucine-rich repeat and PDZ domain-containing protein family member-i (LRRC7/LAP1, erbin/LAP2, and scribble homolog/LAP4) LAPTM: transmembrane lysosome-associated protein LAR: leukocyte common antigen-related receptor (PTPRF) LAS1L: lethal in the absence of SSD1 homolog LAT: linker of activated T lymphocytes LaTS: large tumor suppressor LAX: linker of activated X cells (both B and T cells) LBP: lysosomal lipid-binding protein LBR: lamin-B receptor LBRC: lateral border recycling compartment LCA: lithocholic acid (bile acid) lcACADH (lcADH): long-chain acylCoA dehydrogenase LCAT: lecithin–cholesterol acyltransferase lcFA: long-chain fatty acid (10–16 carbon atoms) lcsFA: long-chain saturated fatty acid lcFACS: lcFA–CoA synthase (ligase) lcHADH (lcHACADH): long-chain 3-hydroxyacylCoA dehydrogenase LCK: leukocyte-specific cytosolic protein Tyr kinase LCL: lysocardiolipin LCLAT: LCL acetyltransferase LCP: lymphocyte cytosolic protein (adaptor SLP76) LDH: lactate dehydrogenase LDL: low-density lipoprotein LDLApoB : apolipoprotein-B-containing LDL LDLCS : LDL–cholesterol Li LDL: LDL subfraction-i MDA LDL: malondialdehyde-modified LDL

818

List of Molecule Shortened Abbreviations and Chemical Symbols

LDLR: low-density lipoprotein receptor LDLRAP: LDLR adaptor protein LEF: lymphoid enhancer-binding transcription factor Lep: leptin LepR: leptin receptor LETM: leucine zipper- and EF hand-containing transmembrane protein (CHX and KHX) LeuRS: leucyl-tRNA synthetase (LARS gene) lexis: liver-expressed LXR-induced sequence (ncRNA) LFng: lunatic fringe LGalS: lectin, galactoside-binding, soluble cell adhesion molecule LGIC: ligand-gated ion channel LGL: lethal giant larva protein LH: luteinizing hormone LHx: LIM and homeobox-derived domaincontaining transcription factor LIF: leukemia-inhibitory factor LIFR: leukemia-inhibitory factor receptor Lig4: ATP-dependent DNA ligase-4 LIMA: LIM domain and actin-binding protein LIME: LCK-interacting molecule LIMK: Lin1, Isl1, and Mec3 motif-containing kinase LIMS: LIM and senescent cell antigen-likecontaining domain protein LINC: linker of nucleoskeleton and cytoskeleton (complex) lincMD1: long intergenic ncRNA muscle differentiation-1 lincRNA: large intergenic nonprotein-coding RNA (encoded intergenically) LINE: long interspersed nuclear element (retrotransposon) LipC: type-C lipase (hepatic lipase) LipD: type-D lipase (lipoprotein lipase) LipE: type-E lipase (hormone-sensitive lipase) LipG: type-G lipase (endothelial lipase) LipH: lipase-H (membrane-associated phosphatidic acid-selective mPAPLA1α, or PLA1b) LipL; lipase-like enzyme liprin: LAR PTP-interacting protein LIR: leukocyte immunoglobulin-like receptor LIS: lissencephaly protein LKb: liver kinase-B LLTC: large latent TGFβ complex LMan: lectin, mannose-binding LMO: LIM domain-only-7 protein Lmod: leiomodin (actin nucleator) Lnc: lipocalin

lncRNA: long (large) nonprotein-coding RNA lncang: angiotensin-2-regulated lncRNA Lnp (Lnpk): lunapark (ER stabilizer) LNX: ligand of numb ubiquitin–protein ligase-X LNXD: LAS1L–Nol9–XRN2–DXO complex LOx: lipoxygenase LP: lipoprotein LPA: lysophosphatidic acid LPa: lipoprotein-A (subcategory) LPAAT: LPA acyltransferase LPC: lysophosphatidylcholine LPCAT: lysophosphatidylcholine acyltransferase LPE: lysophosphatidylethanolamine LPEAT: LPE acetyltransferase LPG: lysophosphatidylglycerol LPGAT: LPG acetyltransferase Lphn: latrophilin (adhesion GPCR) LPI: lysophosphatidylinositol LPIAT: LPI acyltransferase LPL: lipoprotein lipase LPLasei (LyPLai): type-i lysophospholipase LPLip (LPL): lysophospholipid LPP: lipid phosphate phosphatase LPR: lipid phosphatase-related protein LPS: lipopolysaccharide LPSer (LPS): lysophosphatidylserine LRAT: lecithin–retinol acyltransferase LRG: leucine-rich glycoprotein LRH: liver receptor homolog (NR5a2) LRO: lysosome-related organelle LRP: LDL receptor-related protein LRR: leucine-rich repeat (regulatory function) LRRTM: leucine-rich repeat-containing transmembrane protein LSK: Lin−, SCA1+, KIT+ cell LSM: LSm-made heteroheptamer (LSm1 to LSm7), LSm: RNA- and U6 snRNA-associated LSm domain-containing protein (or mRNA P body-assembly factor; Sm proteins being common components of the U1, U2, U4/U6, and U5 snRNPs) LST: lethal with Sec-thirteen LT (Lkt): leukotriene LTBP: latent TGFβ-binding protein LTCC: L-type Ca2+ channel (CaV 1) LTK: leukocyte tyrosine kinase LTMSIC: low-threshold mechanosensitive ion channel Ltn: listerin LTR: long terminal repeat

List of Molecule Shortened Abbreviations and Chemical Symbols LUBAC (LUbAC): linear ubiquitin chain assembly complex Lx: lipoxin LXR: liver X receptor (NR1h2/3) LyVE: lymphatic vessel endothelial hyaluronan receptor LZTFL: leucine zipper-containing transcription factor-like protein

M MAC: maxi anion channel MAC: membrane attack complex MACF: microtubule–actin crosslinking factor mAChR: muscarinic acetylcholine receptor (metabotropic; GPCR) MaCoA: malonylCoA MAD: mitotic arrest-deficient protein MAD2L1(2): MAD2-like protein-1(2) MAD2L1IP: MAD2L1-associated protein madcam (MAdCAM): mucosal vascular addressin cell adhesion molecule MAF: musculoaponeurotic fibrosarcoma proto-oncogene homolog (TF) MAFa(b): large MAF subtype-A(B) MAFf(g,k): small MAF subtype-F(G,K) MAG: monoacylglycerol MAGAT: acylCoA–monoacylglycerol acyltransferase MAGI: membrane-associated guanylate kinase-related protein with inverted domain organization MAGL: monoacylglycerol lipase magoh: protein mago (short form of mago nashi) homolog (from Japanese mago nashi: none grandchildren) MAGP: microfibril-associated glycoprotein MagT: magnesium transporter maguk (MAGuK): membrane-associated guanylate kinase MALT1: mucosa-associated lymphoid tissue lymphoma translocation peptidase-1 (paracaspase) MamL: mastermind-like coactivator MAML: MamL1–MamL3 complex ManβA: lysosomal β-mannosidase Manixj : class-iX α-mannosidase-j Man2b1: lysosomal α-mannosidase MAO: monoamine oxidase MAP: microtubule-associated protein MAP1LC3: microtubule-associated protein-1 light chain-3 (LC3; Atg8e–Atg8g and Atg8j) MAPK: mitogen-activated protein kinase

819

MAP2K: MAPK kinase MAP3K: MAP2K kinase MAP3K7IP: MAP3K7-interacting protein MAPKAPK: MAPK-activated protein kinase MAPT: microtubule-associated protein tau Mar: maresin march: membrane-associated RING and CH finger-containing protein MARCKS: myristoylated alanine-rich C kinase substrate MaRCo: macrophage receptor with collagenous structure (ScaRa2) MARK: microtubule affinity-regulating kinase MARVEL: MAL and related proteins for vesicle trafficking and membrane link (tetraspanning protein domain) marveld: MARVEL domain-containing protein MASP: mannan-binding lectin-associated serine peptidase MASTL: microtubule-associated protein Ser/ Thr kinase-like protein MAT: ménage à trois MATK: megakaryocyte-associated protein Tyr kinase MaU2: maternal uncoordinated protein-2 (SCC4 homolog) MAVS: mitochondrial antiviral signaling protein MAX: MyC-associated factor-X (bHLHd4– bHLHd8) MAZ: MyC-associated zinc finger protein Mb (Mgb): myoglobin MBDi: methylated CpG-binding domain (MBD)-targeting protein-i (MBD2 demethylase) MBln: muscleblind-like protein MBP: myosin-binding protein MBTPSi: membrane-bound transcription factor peptidase, site i mcACADH (mcADH): mitochondrial medium-chain acylCoA dehydrogenase MCAK: mitotic centromere-associated kinesin mcam (MCAM): melanoma cell adhesion molecule MCC: mitotic checkpoint complex MCC: monocarboxylate carrier MCD: malonylCoA decarboxylase mcFA: medium-chain fatty acid (6–12 carbon atoms) MCH: melanin-concentrating hormone MCL1: BCL2-related myeloid cell leukemia sequence protein-1 (BCL2L3) MCLC: stretch-gated Mid1-related chloride channel

820

List of Molecule Shortened Abbreviations and Chemical Symbols

McoLn: mucolipin (late endosome and lysosome unselective cation channel) MCM: minichromosome maintenance protein MCP: monocyte chemoattractant protein mCSF: macrophage colony-stimulating factor (CSF1) MCT: monocarboxylate–proton cotransporter MCUR: mitochondrial calcium uniporter regulator MDA: malondialdehyde MDA LDL: malondialdehyde-modified LDL MDC: mediator of DNA damage checkpoint MDH: malate dehydrogenase MDM: mitochondrial distribution and morphology protein MDR: multiple drug resistance (ABC transporter) ME: NADP+ - (Cy ME1 and Mt ME3) or NAD+ -dependent (Mt ME2) malic enzyme mEcircRNA: multiexonic circular RNA MeCP: methylated CpG-binding protein MEF: myocyte enhancer factor meg3: maternally expressed gene transcript (lncRNA) megCSF: megakaryocyte colony-stimulating factor MELK: maternal embryonic leucine zipper kinase mepe (MEPE): matrix extracellular phosphoglycoprotein MerTK: Mer proto-oncogene product receptor protein Tyr kinase MET: mesenchymal–epithelial transition factor (proto-oncogene; HGFR) MetAP: methionine aminopeptidase metHb: methemoglobin MetS: methionine synthase (MTR gene) MetTL: (N6-methyladenosine [mN6 A]) methyltransferase-like protein MFF: mitochondrial fission factor Mfn: mitofusin MFng: manic fringe MFO: mixed-function oxidase MFS: major facilitator superfamily MGAT: mannosyl (α1,3)-glycoprotein (β1,2)N acetylglucosaminyl transferase MGAT: monoacylglycerol acyltransferase MGIC: mechanogated ion channel MGL: monoacylglycerol (monoglyceride) lipase mGluR: metabotropic glutamate receptor

MGP: matrix γ-carboxyglutamate acid (Gla)-containing protein MGST: microsomal glutathione S transferase MHC: major histocompatibility complex MHCc: MHC class- (molecule) mhrt: myosin heavy chain-associated RNA transcript (myheart lncRNA) MIA: mitochondrial intermembrane space import and assembly machinery miai: MIA complex component-i miat: myocardial infarction-associated transcript (lncRNA) MIB: mitochondrial intermembrane space bridging complex Mic: micos (minos) complex constituent MICOS (MiCOS): mitochondrial contact site and cristae organization system micu (MiCU): mitochondrial Ca2+ uptake protein MiD: mitochondrial dynamics protein Mid: midline MIF: macrophage migration-inhibitory factor (nuclease) MIM: mitochondrial import machinery mimi: MIM complex subunit-i MInK: MAPK-interacting protein Ser/Thr kinase MinK: misshapen-like kinase (MAP4K6) minK: minimal potassium channel subunit minos: mitochondrial inner membrane organizing system protein miR: microRNA miRNP: microribonucleoprotein MiRP: MinK-related peptide MIRR: multichain immune-recognition receptor MIS: MIND kinetochore complex subunit MIS: Müllerian-inhibiting substance MIST: mastocyte immunoreceptor signal transducer MiTF: microphthalmia-associated transcription factor (bHLHe32) MIZ: Myc-interacting zinc finger protein MJD: Machado–Joseph disease protein domain-containing peptidase (DUb) MKL: megakaryoblastic leukemia-1 fusion coactivator MKnK: MAPK-interacting protein Ser/Thr kinase (MnK) MKP: mitogen-activated protein kinase phosphatase MkSi: Meckel syndrome protein-i MKKS: McKusick–Kaufman syndrome protein

List of Molecule Shortened Abbreviations and Chemical Symbols MLC: myosin light chain MLCK: myosin light chain kinase MLCL: monolysocardiolipin MLCLAT: MLCL acyltransferase MLCP: myosin light chain phosphatase MLHi: DNA mismatch repair protein MutL homolog-i MLK: mixed lineage kinase MLKL: mixed lineage kinase-like pseudokinase MLL: mixed lineage (myeloid–lymphoid) leukemia factor (trithorax homolog) MLLT: mixed lineage leukemia translocated protein MLN: metastatic lymph node protein MLP: muscle LIM protein mmCK: myofibrillar creatine kinase MMACHC: methylmalonic aciduria and homocystinuria-related type-C protein MMADHC: methylmalonic aciduria and homocystinuria-related type-D protein MMCMut: methylmalonyl coenzyme-A mutase (MUT gene) MME: membrane metalloendopeptidase MMM: maintenance of mitochondrial morphology protein MMP: matrix metallopeptidase MMS: methyl methane sulfonate MNAm: methylnicotinamide MNAR: modulator of nongenomic activity of estrogen receptor MO: mouse protein MOR: μ-opioid receptor MoSPD: motile sperm domain-containing protein MOTrp: 5-methoxytryptophan MP: MAPK partner MPAS: mitochondrial precursor protein accumulation stress MPC: mitochondrial pyruvate carrier MPF: mitosis (maturation)-promoting factor (CcnB–CDK1 complex) MPG: N methylpurine (N methyladenine)-DNA glycosylase MPGF: major proglucagon fragment MPOx: myeloperoxidase MPP : membrane protein, palmitoylated MPP: M-phase phosphoprotein MPP: mitochondrial processing peptidase MPS: multipolar spindle protein MPST: mercaptopyruvate sulfurtransferase MR: mineralocorticoid receptor (NR3c2) mRas: muscle Ras (or rRas3)

821

MRC: mediator of DNA replication checkpoint (claspin) MRCK: myotonic dystrophy kinase-related CDC42-binding kinase MRe11: meiotic recombination homolog-11 MRGd: Mas-related G-protein-coupled receptor-D MRN: MRe11–Rad50–NBS1 complex (DDB detection) MRPLi: type-i large mitoribosomal (subunit) protein MRPSi: type-i small mitoribosomal (subunit) protein MRX: MRe11–Rad50–XRS2 complex mRNA: messenger RNA mRNP: messenger ribonucleoprotein MRTF: myocardin-related transcription factor MSH: melanocyte-stimulating hormone MSHi: DNA mismatch repair protein MutS homolog-i MSIC: mechanosensitive ion channel MSK: mitogen- and stress-activated protein kinase MST: mammalian sterile-twenty-like kinase MSt1R: macrophage-stimulating factor-1 receptor (RON) Mstn: myostatin MT: metallothionein Mt AAA (mAAA): mitochondrial matrix ATPases associated with diverse cellular activities peptidase Mt CK (mtCK): mitochondrial creatine kinase Mt CU: mitochondrial (IMM) Ca2+ (uptake) uniporter (MCU gene) Mt CUC: Mt CU complex Mt CUR: mitochondrial calcium uniporter regulator Mt DAMP: mitochondrial alarmin Mt DNA: mitochondrial DNA Mt ETC: mitochondrial electron transport chain Mt neet: mitochondrial protein containing the amino acid sequence NEET (Asn–Glu–Glu–Thr) Mt TE: mitochondrial thioesterase Mt TerF: mitochondrial transcription termination factor MTHFDH (MTHFD): methylene tetrahydrofolate dehydrogenase MTHFR: methylene tetrahydrofolate reductase MTHHCMT: methyltetrahydrofolate– homocysteine S methyltransferase (MTR gene) MTM: myotubularin (myotubular myopathyassociated gene product)

822

List of Molecule Shortened Abbreviations and Chemical Symbols

mtiMMP: type-i membrane-type MMP MTMR: myotubularin-related phosphatase MTr: methyl tetrahydrofolate–homocysteine methyltransferase (methionine synthase) MTr: mRNA transport mediator MTrR: methyl tetrahydrofolate–homocysteine methyltransferase reductase (methionine synthase reductase) MTTP (MTP): microsomal triglyceride transfer protein MTVR: mammary tumor virus receptor Mtx: metaxin Muc: mucin muFA: monounsaturated fatty acid mulan: mitochondrial ubiquitin ligase activator of NFκB MUM: melanoma-associated antigen (mutated) MUM1L: MUM1-like protein MuRF: muscle-specific RING finger (Ub ligase) MuSK: muscle-specific kinase MUTL: MLH1–PMS2 heterodimer MUTSα: MSH2–MSH6 heterodimer MUTSβ: MSH2–MSH3 heterodimer MutYH: MutY homolog MyB: myeloblastosis viral oncogene homolog (cellular counterpart; TF) MyBPc: myosin-binding protein-C MyC: myelocytomatosis viral oncogene homolog (TF) MyD88: myeloid differentiation primary response gene product-88 MyDGF: myeloid-derived growth factor MyF: myogenic factor MYH: myosin heavy chain gene MyHC (MHC): myosin heavy chain MyHC: myosin heavy chain MYL: myosin light chain gene MyLC: myosin light chain MyLIP: myosin regulatory light chaininteracting protein (Ub ligase) Myocd: myocardin (MYOCD gene) MyPT: myosin phosphatase targeting subunit MYST: MOZ (MYST3), Ybf2/Sas3, Sas2, and Tip60 (KAT5) MyT: myelin transcription factor

N N-terminus: amino (amine group NH2 ) terminus NA: neuraminidase (sialidase) NAAD: nicotinic acid adenine dinucleotide

NAADP: NAAD phosphate NAC: nascent chain-associated complex NACC1: BEN and BTB (POZ) domain-containing nucleus accumbensassociated transcriptional repressor nAChR: nicotinic acetylcholine receptor (ionotropic; LGIC) NAD+ : oxidized form of nicotinamide adenine dinucleotide NAd: noradrenaline NADH: reduced form of nicotinamide adenine dinucleotide NADHDH: NADH dehydrogenase NADK: NAD+ kinase NADP+ : oxidized form of nicotinamide adenine dinucleotide phosphate NADPH: reduced form of nicotinamide adenine dinucleotide phosphate NADS: NAD+ synthetase NAF: nutrient-deprivation autophagy factor NAGα: N acetylgalactosaminidase-α NAGlu: αN acetylglucosaminidase NAIP: NLR family apoptosis inhibitory protein NALP: NACHT domain-, C-terminal leucine-rich repeat-, and N-terminal PYD-containing protein NAm: nicotinamide NAmMT: nicotinamide methyltransferase NA(m)MN (NMN): nicotinic acid (nicotinamide) mononucleotide NA(m)MNAT (NMNAT): NMN adenylyltransferase (uses both substrates, NAMN and NAmMN with the same efficiency) NAmNT (NNT): nicotinamide nucleotide transhydrogenase NAmPRT (NamPT): nicotinamide phosphoribosyltransferase NAmR: nicotinamide riboside NAmRK (NRK): NAmR kinase nanog: ever young (Gaelic) NAP (NCKAP): NCK-associated protein NAP1Li: type-i nucleosome assembly protein-1-like histone chaperone NAPE: N acylphosphatidylethanolamine NASP: histone-binding nuclear autoantigenic sperm protein NAT: natural antisense transcript NATi/SLC23ai: type-i nucleobase–ascorbate transporter NAT1 (SLC6a2): noradrenaline transporter NaV : voltage-gated Na+ channel NBC: Na+ –HCO− 3 cotransporters

List of Molecule Shortened Abbreviations and Chemical Symbols − NBCX (NBCE): Na+ –2 HCO− 3 –Cl exchanger NBR: neighbor of BRCA1 gene product (autophagic adaptor) NBS1: Nijmegen breakage syndrome protein-1 (nibrin) ncam (NCAM): neural cell adhesion molecule NCAP: nonSMC (structural maintenance of chromosomes) condensin-I (I I ) complex subunit NCBP: nuclear cap-binding protein (RNA cap-binding complex subunit) NCC: Na+ –Cl− cotransporter Ncdn: neurochondrin NCEH: neutral cholesterol ester hydrolase NCK: noncatalytic region of protein Tyr kinase adaptor NCKX: Na+ –Ca2+ –K+ exchanger (SLC24a1– SLC24a5) NCLX: Na+ –Ca2+ –Li+ exchanger (SLC24a6/ SLC8b1) NCoA: nuclear receptor coactivator NCOAT: nuclear and cytoplasmic oglcnacase and acetyltransferase NCOR: NCoR complex NCoR: nuclear receptor corepressor NCR: natural cytotoxicity-triggering receptor ncRNA: nonprotein-coding (noncoding) RNA NCS: neuronal calcium sensor NCX: Na+ –Ca2+ exchanger (SLC8a1– SLC8a3) NDC: nuclear division cycle NDCBE: Na+ -dependent Cl− –HCO− 3 exchanger NDPKi (NDKi): type-i nucleoside diphosphate kinase NDR: nucleosome-depleted region NDST: heparan sulfate glucosaminyl N deacetylase and N sulfotransferase NDUF: NADH dehydrogenase (ubiquinone)1α (NADH–ubiquinone oxidoreductase) neat (NEAT): nuclear-enriched paraspeckle assembly transcript (lncRNA) NecL: nectin-like molecule NEDD: neural precursor cell expressed, developmentally downregulated NDFIP: NEDD4 family-interacting protein NEF: nucleotide-exchange factor NeF (NF): neurofilament protein (intermediate filament) neFA: nonesterified fatty acid (FFA) NeK: never in mitosis gene-A (NIMA)-related kinase NElFx: negative elongation factor-X

823

NELF: NElFa–NElFb–NElFc–NElFe complex NES: nuclear export signal NESK: NIK-like embryo-specific kinase nesprin: nuclear envelope spectrin repeat protein (SyNE1–SyNE4) Neu: N acetyl α-neuraminidase (sialidase) NeuroD: neurogenic differentiation protein NExT: notch extracellular truncation domain NEXT: nuclear exosome-targeting complex NF: neurofibromin (RasGAP) NFAT: nuclear factor of activated T cells NFE2: erythroid-derived nuclear factor-2 NFE2L: nuclear NFE2-related factor NFH: neurofilament, heavy polypeptide NFκB: nuclear factor κ light chain enhancer of activated B cells (NFKB gene) NFIL3: nuclear factor interleukin-3-regulated protein NFL: neurofilament, light polypeptide NFM: neurofilament, medium polypeptide NFy: nuclear transcription factor-Y NGAL: neutrophil gelatinase-associated lipocalin (lipocalin-2) Ngb: neuroglobin NGF: nerve growth factor Ngn: neogenin (netrin receptor) NHERF: NHE regulatory factor (SLC9a3R1– SLC9a3R2) NHR: nuclear hormone receptor NHX (NHE/NHA): Na+ –H+ exchanger (antiporter (SLC9a1–SLC9a9, SLC9b1–SLC9b2, SLC9c1/SLC9a10– SLC9c2/SLC9a11) NiAc: nicotinic acid NIc: nucleoporin-interacting protein NIK: NFκB-inducing kinase NIK: NCK-interacting kinase NipBL: nipped-B-like protein (SCC2 homolog) NKCC: Na+ –K+ –2Cl− cotransporter NKG: NK receptor group NKx2: NK2 transcription factor-related homeobox-derived (homeodomaincontaining) protein NLK: nemo-like kinase NLR: NOD-like receptor NLRC: NACHT-, LRR-, and CARDcontaining protein NLRP: NACHT-, LRR-, and PYD-containing protein NLS: nuclear localization signal NMx: type-X neuromedin NMDAR: N methyl D aspartate receptor NMM2: nonmuscle myosin-2

824

List of Molecule Shortened Abbreviations and Chemical Symbols

NMVOC: nonmethane volatile organic compound NO: nitric oxide (nitrogen monoxide) NOx : nitrogen oxides NOD: nucleotide-binding oligomerization domain-containing protein Nogo: neurite outgrowth inhibitor (reticulon-4) Nol: nucleolar protein NonO: nonPOU domain-containing octamerbinding protein NoP: nucleolar protein NOR: neuron-derived orphan receptor (NR4a3) NOS: nitric oxide synthase NOS1: neuronal NOS NOS1AP: NOS1 adaptor protein NOS2: inducible NOS NOS3: endothelial NOS nosip: nitric oxide synthase (NOS3)-interacting protein nostrin: nitric oxide synthase (NOS3) traffic inducer novlnc: novel lncRNA NOx: NAD(P)H (NADH/NADPH) oxidase noxa: damage (Latin) NP: natriuretic peptide NPAT: nuclear protein ataxia–telangiectasia locus NPAS: neuronal PAS domain-containing transcription factor NPC: nuclear pore complex NPc: Niemann–Pick disease type-C protein NPc1L: Niemann–Pick protein-C1-like NPepPS: puromycin-sensitive aminopeptidase NPF: actin nucleation-promoting factor Nphpi: nephrocystin-i nPKC: novel protein kinase-C NPL: nuclear protein localization homolog Npm (NPm): nucleoplasmin (Npm1: nucleophosmin) NPY: neuropeptide-Y NQO1: NADPH:quinone oxidoreductase-1 (NAD[P]H dehydrogenase quinone) NR: nuclear receptor (TF) NRAMP: natural resistance-associated macrophage protein (manganese symporter) NRAP: nebulin-related actinin-binding protein NRARP: notch-regulated ankyrin repeatcontaining protein nRas: human neuroblastoma Ras NRBP (NRBF): nuclear receptor-binding protein (factor) NRdx: nucleoredoxin NRF: nuclear respiratory factor

Nrg (NRg): neuregulin (EGF superfamily member) Nrgn: neuroligin NRIP: nuclear receptor-interacting protein Nrp: neuropilin (VEGF-binding molecule; VEGFR coreceptor) NRPTP: nonreceptor protein Tyr phosphatase NRSTK: nonreceptor protein Ser/Thr kinase NRTK: nonreceptor protein Tyr kinase Nrxn: neurexin NSC: nonselective cation channel NSD: nuclear receptor-binding SET domain-containing protein NSF: N ethylmaleimide-sensitive factor NSLTP: nonspecific lipid-transfer protein NT: neurotrophin nt: nucleotide NT5E: ecto-5 -nucleotidase NTCP: sodium–taurocholate cotransporter polypeptide NTF: N-terminal fragment NTP: nucleoside triphosphate NTPase: nucleoside triphosphate hydrolase NTPase: nucleoside triphosphate hydrolase superfamily member NTRK: neurotrophic tyrosine receptor kinase (TRK) NTRKR: neurotrophic protein Tyr receptor kinase-related protein (ROR(RTK) ) Nts (NTs): neurotensin NuAK: nuclear AMPK-related kinase NuFIP: cytoplasmic FMR1 (or FMRP)interacting protein numbL: numb homolog-like protein NuP: nucleoporin (nuclear-pore complex protein) NURD (NuRD): nucleosome remodeling and histone deacetylase complex nurf (NuRF): nucleosome remodeling factor NuRR: nuclear receptor-related factor (NR4a2) nWASP: neuronal WASP NXF1: nuclear RNA export factor-1 NXT1: nuclear transport factor NuTF2-like export factor-1

O Obscn: obscurin OCRL: oculocerebrorenal syndrome of Lowe phosphatase Oct: octamer-binding transcription factor ODC: ornithine decarboxylase ODFi: type-i outer dense fiber protein OGA: O GlcN Acase (βN acetylglucosaminidase)

List of Molecule Shortened Abbreviations and Chemical Symbols OGDH (KGDH):: oxoglutarate (αketoglutarate) dehydrogenase Olfm (OlfM): olfactomedin OMA: overlapping activity with mAAA (AAA+ peptidase active at the matrix side of the IMM) homolog omm: OMM protein oORF: overlapping open reading frame OPn: osteopontin (secreted phosphoprotein SPP1) OpA: dynamin-like IMM optic atrophy protein (GTPase) ORC: origin recognition complex ORF: open reading frame Orm: orosomucoid OrmDL: Orm1-like protein ORP (OSBPL): OSBP-related (-like) protein OSBP: oxysterol-binding protein OsM (OSm): oncostatin-M OsMR (OSmR): oncostatin-M receptor OSR (OxSR): oxidative stress-responsive kinase OSTC: oligosaccharyltransferase complex OTK: off-track (pseudo)kinase OTU: ovarian tumor superfamily peptidase (deubiquitinase) Otub: otubain (Ub thioesterase of the OTU superfamily) OTUD: OTU domain-containing protein oxFA: oxidized fatty acid oxLDL: oxidized low-density lipoprotein Oxm: oxyntomodulin oxPC: oxidized phosphatidylcholine oxPL: oxidized phospholipid OXT: preprooxytocin gene (oxytocin– neurophysin-1) Oxt: oxytocin oxyHb: oxygenated hemoglobin

P Pi : inorganic phosphate (free phosphate ion [HPO2− 4 ]) P1: adenosine G-protein-coupled receptors P2X: ionotropic nucleotide (purinergic) ligand-gated channel P2Y: metabotropic nucleotide G-proteincoupled receptor P3Hi: type-i prolyl 3-hydroxylase (leprecan and leprecan-like-1/2) P4Hi: type-i prolyl 4-hydroxylase C P4Hi: type-i collagen prolyl 4-hydroxylase HIF P4Hi: type-i HIF prolyl 4-hydroxylase (EglN1–EglN3 and PHD1–PHD3)

825

P53: 53-kDa (full-length) transcription factor P53AIP: P53-regulated apoptosis-inducing protein P53BP: P53-binding protein P53I: P53-inducible protein P63: P53-related 63-kDa (full-length) transcription factor P73: P53-related 73-kDa (full-length) transcription factor P75NTR : pan-neurotrophin receptor (TNFRSF16) PA: phosphatidic acid PAAT: proton–amino acid transporter PACS: phosphofurin acidic cluster sorting protein PABPi : type-i (mRNA) polyadenylated motif-binding protein PABPC: cytoplasmic PABP PABPN: nuclear PABP PADI: peptidyl arginine deiminase PAF: platelet-activating factor PAF1: RNA polymerase-2-associated factor PAFAH: platelet-activating factor acetylhydrolase PAG: phosphoprotein associated with glycosphingolipid-enriched microdomain PAH: phenylalanine 4-hydroxylase PAHC (PAH): polycyclic aromatic hydrocarbon (pollutant) PAI: plasminogen activator inhibitor PAK: P21 (CKI1a)-activated kinase palRNA: promoter-associated long RNA PALS: protein associated with Lin-7 PAM: preprotein translocase-associated motor (mitochondrial importer) pami: PAM module subunit-i PAMOx (PAM): peptidylglycine amidating monooxygenase PAMP: pathogen-associated molecular pattern PAMP: proadrenomedullin peptide pancRNA (pncRNA): promoter-associated nonprotein-coding RNA PAOx: polyamine oxidase PAP: phosphatidic acid (phosphatidate) phosphatase PAPα: (mRNA) polyadenylating polymerase-α (PAPOLA gene) PAPC: palmitoyl arachidonoyl glycerophosphorylcholine PAQR: progestin and adipoQ receptor PAR: polyADP ribose PARi : type-i peptidase-activated receptor Par: partitioning defective protein

826

List of Molecule Shortened Abbreviations and Chemical Symbols

PARbZip: proline and acidic amino acid-rich basic leucine zipper parc: P53-associated parkin-like cytoplasmic protein PARG: polyADP ribosyl glycosidase park (Park): Parkinson’s disease-related protein PARL: mitochondrial intramembrane cleaving presenilin-associated rhomboid-like peptidase PARN: polyadenylate-specific ribonuclease PARP: poly[ADP ribose] polymerase pasRNA: promoter-associated short RNA PATJ: protein (PALS1) associated to tight junctions PATL: protein associated with topoisomerase-2 homolog Pax: paxillin Paxi: paired box-containing transcription regulator-i PaxIP: paired box protein Pax2 transactivation activation domain-interacting protein PAXT: polyadenylated tail exosome targeting trimer PBIP: Polo box-interacting protein PBUT: protein-bound uremic toxin PBZ: polyADP ribose-binding zinc finger domain PC: polycystin Pc (PC): protein-C PCi: type-i positive cofactor (PC1–PC4) PCh (PC/PtdCho): phosphatidylcholine PCAF: P300/CBP-associated factor PCBPi: type-i polyribocytidylic acid-binding protein PCF: protein of (pre-mRNA) cleavage factor PcG: polycomb group protein PcGF: polycomb group RING fingercontaining protein PcL: polycomb-like protein PCNA: proliferating cell nuclear antigen PcP (Pc): polycomb protein PCr: phosphocreatine pCS: p-cresyl sulfate (PBUT) PCSK: proprotein convertase subtilisin/kexin PCTP: phosphatidylcholine transfer protein PcyT: phosphate cytidylyltransferase (phosphocholine [PcyT1] or phosphoethanolamine [PcyT2]) PCx (PC): pyruvate carboxylase PdCD: programmed cell death protein PdCD6IP: PdCD-6-interacting protein PdCD1Lg: programmed cell death-1 ligand

PDE: cyclic nucleotide phosphodiesterase PDGF: platelet-derived growth factor PDGFR: platelet-derived growth factor receptor PDH: pyruvate dehydrogenase PDHC: PDH complex PDHKi: type-i mitochondrial PDH kinase (PDHK1–PDHK4) PDHPi: type-i mitochondrial PDH phosphatase (PDHP1–PDHP2) PDI: protein disulfide isomerase PDPK: phosphoinositide-dependent protein kinase PDRG: P53 and DNA damage-regulated gene product PDS: regulator of sister chromatid cohesion maintenance homolog PDx: pancreatic and duodenal homeoboxderived transcription factor PE (PtdEtn): phosphatidylethanolamine pear (PEAR): platelet endothelial aggregation receptor PEBP: phosphatidylethanolamine-binding protein pecam (PECAM): platelet–endotheliocyte adhesion molecule PEDF: pigment epithelium-derived factor (serpin-F1) PELP: proline-, glutamic acid-, and leucine-rich protein PEMTi: type-i phosphatidylethanolamine methyltransferase (PEMT1 in the ER; PEMT2 in MAERMs). PEn2: presenilin enhancer-2 PEPCK: phosphoenolpyruvate carboxykinase Per: period homolog PERK: protein kinase-like endoplasmic reticulum kinase PERP: P53 apoptosis effector related to peripheral myelin protein PMP22 PesS: serine peptidase (known as PrsS [protease, serine]) PesS8/TMPesS4/14 (CAP): transmembrane serine channel-activating peptidase Pex: peroxin PF: platelet factor PFDN (PFD): prefoldin complex PFDNL (PFDL): prefoldin-like complex Pfdni: type-i prefoldin (PFDN/PFDNL complex subunit) PFK: phosphofructokinase (PFK1) PFKM: phosphofructokinase muscle type gene (encoding SkM PFK1)

List of Molecule Shortened Abbreviations and Chemical Symbols PFKFB: 6-phosphofructo 2-kinase/fructose (2,6)-biphosphatase (both PFK2 and FBPase) pFGF (cGFG): paracrine (or autocrine) canonical FGFs pFRG: parafacial respiratory group PG: prostaglandin PGaM: phosphoglycerate mutase family member-5 (mitochondrial protein Ser/ Thr phosphatase) PGAM5: phosphoglycerate mutase PGC: PPARγ (NR1c3) coactivator pGC: particulate guanylate cyclase PGDH: phosphogluconate dehydrogenase (PGD gene) PGEA: prostaglandin ethanolamide PGF: paracrine growth factor PGG: prostaglandin glycerol ester PGhS: prostaglandin-G/H synthase (COx) PGi2 : prostacyclin PGK: phosphoglycerate kinase PGP: permeability glycoprotein PGP: phosphatidylglycerolphosphate PGS (PGPS): phosphatidylglycerolphosphate synthase PGx: type-X (D, E, F, H, I) prostaglandin PGxS: type-X prostaglandin synthase Phax: phosphorylated adaptor of RNA export Phb: prohibitin PHD: prolyl hydroxylase domain-containing protein PHF: PHD finger-containing protein PhK: phosphorylase kinase PHLPP: PH domain and Leu-rich repeatcontaining protein phosphatase PI (PtdIns): phosphatidylinositol (phosphoinositide) PI(4)P: phosphatidylinositol 4-phosphate PI(i)Pj K: phosphatidylinositol i-phosphate j -kinase (i, j : integers) PI(i, j )P2 : phosphatidylinositol (i, j )bisphosphate (PIP2 ) PI(3,4,5)P3 : phosphatidylinositol (3,4,5)trisphosphate (PIP3 ) PI3K: phosphatidylinositol 3-kinase PI3KG : group- PI3K PI3KAP: PI3K adaptor protein PI3KG3: group-I I I PI3K-based complex (PI3Kc3 complex) PIiK: phosphatidylinositol i-kinase PIAS: protein inhibitor of activated STAT (sumo ligase) PIC: preinitiation complex PICK: protein that interacts with C-kinase

827

PIDD: P53-induced protein with a death domain PIH1D: protein interacting with HSP90 (PIH1) domain-containing protein PIKE: phosphoinositide 3-kinase enhancer (GTPase; ArfGAP) PIKK: phosphatidylinositol 3-kinase-related kinase (pseudokinase) PIM: provirus insertion of Moloney murine leukemia virus gene product PIN: peptidyl prolyl isomerase interacting with NIMA PINCH: particularly interesting new Cys–His protein (or LIMS1) pink1 (PInK1): PTen-induced kinase-1 PIP: phosphoinositide monophosphate PIPiK: phosphatidylinositol phosphate i-kinase PIP2 : phosphatidylinositol bisphosphate PIP3 : phosphatidylinositol trisphosphate PipOx: pipecolic acid oxidase PIPP: proline-rich inositol polyphosphate 5-phosphatase PIR: paired immunoglobulin-like receptor piRNA: P-element-induced wimpy testisinteracting (PIWI) RNA PIRT: phosphoinositide-interacting regulator of TRP channels PITP: phosphatidylinositol-transfer protein Pitx: pituitary (or paired-like) homeoboxderived (homeodomain-containing) transcription factor PIX: P21-activated kinase (PAK)-interacting exchange factor (RhoGEF6/7) PK: pyruvate kinase PKA: protein kinase-A PKB: protein kinase-B PKC: protein kinase-C PKD: protein kinase-D PKG: protein kinase-G PKL: paxillin kinase linker PKM: pyruvate kinase muscle isozyme (gene) PKMYT (MYT): membrane-associated protein Tyr/Thr kinase PKNi: type-i (PKC-related) protein kinase novel Pkp: plakophilin PKZ: Z-DNA-binding domain-containing kinase protein PL: phospholipase PLA: phospholipase-A PLC: phospholipase-C PLD: phospholipase-D PLd: phospholipid

828

List of Molecule Shortened Abbreviations and Chemical Symbols

PlekH: pleckstrin homology domaincontaining protein PlekHxi: pleckstrin homology and RUN domain-containing family-X member-i Plg: plasminogen PlGF: placental growth factor PLin: perilipin PLK: polo-like kinase Plm: phospholemman PLn (Pln): phospholamban Pln: plasmin PLPP: phospholipid phosphatase PLOx: protein lysine (lysyl) oxidase PLTP: phospholipid transfer protein Plxn: plexin PM: prostamide (prostaglandin ethanolamide) PMAIP1: phorbol myristate acetate-induced protein-1 (caspase activator) PMCA: plasma membrane Ca2+ ATPase PML: promyelocytic leukemia protein PMME: phosphatidylmonomethylethanolamine PMRT: protein arginine methyltransferase PMS: DNA mismatch repair endonuclease postmeiotic segregation increased homolog PNKP: bifunctional polynucleotide DNA 5 -kinase and 3 -phosphatase Pnn: pinin (auxiliary EJC component) PnPLA: patatin-like phospholipase-A domain-containing protein POFuT: protein O fucosyltransferase PoG: proteoglycan POGluT: protein O glucosyltransferase polyQ: polyglutamine tract (10–100 Gln-containing sequence related to nucleotide repeat expansions in protein-coding genes) PoM: pore membrane protein POMC: proopiomelanocortin PoMP: proteasome maturation protein POn: paraoxonase POPx: partner of PIX Porcn: porcupine homolog POSH: scaffold plenty of SH3 domains Postn: periostin POT: protection of telomeres (single-stranded telomeric DNA-binding protein) PP: protein phosphatase PPARξ: type-ξ peroxisome proliferatoractivated receptor (NR1c1–NR1c3) PPI: peptidyl prolyl isomerase PPIP: monopyrophosphorylated inositol phosphate

(PP)2 IP: bispyrophosphorylated inositol phosphate PPK: PIP kinase PPM: phosphomannomutase PPM: protein phosphatase (magnesiumdependent) PPR: pathogen-recognition receptor PPRC: PPARγ coactivator PGC1-related coactivator PPT: palmitoyl protein thioesterase PPTC: protein phosphatase T-cell activation (TAPP2c) PPy (PP): pancreatic polypeptide PR: progesterone receptor (NR3c3) PREB: prolactin regulatory element-binding protein PRC: polycomb repressive complex PRCK (PRC): protein regulator of cytokinesis PRDIBF: positive regulatory domain I-binding factor PRDm: PR domain-containing protein PRdx: peroxiredoxin (PRDXi genes) pre-miR: precursor microRNA preKk: prekallikrein PRELI: protein of relevant evolutionary and lymphoid interest PRELID: PRELI domain-containing protein PREx: PIP3 -dependent Rac exchanger (RacGEF) PRG: plasticity-related gene product PRH: prolactin-releasing hormone pri-miR: primary microRNA PRL: phosphatase of regenerating liver Prl: prolactin PrlR: prolactin receptor PRMT: protein arginine (R) N methyltransferase Prompt (PrompT): promoter upstream transcript PROS: PIK3CA gene-related overgrowth spectrum protor: protein observed with rictor PROX: prospero homeobox-containing gene Prox: PROX gene product (homeodomaincontaining transcription factor) PrP: processing protein PRPF: pre-mRNA processing factor PRPK: P53-related protein kinase PR/RR (PRR): prorenin and renin receptor PRR: pattern recognition receptor PrRC: proline-rich coiled-coil protein Prx: paired mesoderm homeobox-containing gene product Ps (PS): protein-S PSD: postsynaptic density adaptor

List of Molecule Shortened Abbreviations and Chemical Symbols PSeni (PSi): type-i presenilin PSer (PS, PtdSer): phosphatidylserine PSGL: P-selectin glycoprotein ligand PSIP: PC4 and SFRS1-interacting protein (histone mark reader) PSKh: protein serine kinase-H Psm: proteasome subunit PSTPIP: protein Pro–Ser–Thr phosphataseinteracting protein PTA: plasma thromboplastin antecedent Ptc: patched receptor (hedgehog signaling) PtcH: patched hedgehog receptor PtdSSi: type-i phosphatidylserine synthase PTen: phosphatase and tensin homolog deleted on chromosome ten (phosphatidylinositol 3-phosphatase) PTFE: polytetrafluoroethylene PtgERi: type i prostaglandin-E receptor (EPi) PtgIR: prostacyclin (PGi2 ) receptor (IP) PTGS1(2): prostaglandin endoperoxide synthase PtgS1(2) (COX1/2 genes) PTH: parathyroid hormone PTHRP: parathyroid hormone-related protein PTIP: Pax transactivation domain-interacting protein PTK: protein Tyr kinase PTK7: pseudokinase (RTK) PTP: protein Tyr phosphatase PTPIP: PTP-interacting protein PTPni: protein Tyr phosphatase, nonreceptor, type i PTPR: protein Tyr phosphatase receptor PTRF: RNA polymerase-1 and transcript release factor Ptx: pentraxin PUbC: polyubiquitin chain puFA: polyunsaturated fatty acid puma (PUMA): P53-upregulated modulator of apoptosis PVF: PDGF- and VEGF-related factor PWWPi: PWWP domain-containing protein-i Px: pannexin PXR: pregnane X receptor (NR1i2) pycard (PYCARD): PYD and CARD domain-containing adaptor PYDCi: type-i pyrin domain-containing protein pyhin (PYHIN): PYD and DNA-binding HIN domain-containing protein PYK: proline-rich tyrosine kinase PYM: partner of Y14–magoh (RBM8a– magoh) PYY: peptide tyrosine–tyrosine

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Q QK: RNA-binding KH domain-containing Quaking homolog QSOx: quiescin sulfhydryl oxidase

R R2TP: Rvb1, Rvb2, TAH1 (TPR motifcontaining protein associated with HSP90) and PIH1 (protein interacting with HSP90) in yeasts (RuvBL1, RuvBL2, RPAP3, and PIH1D1 [R3P] in humans) R3P/PFDNL: chaperone complex composed of the R3P module, PfdnLs+ and Pfdns+ module, WDR92, and RPB5 Rab: Ras from brain Rab11FIP: Rab11 family-interacting protein Rac: Ras-related C3-botulinum toxin substrate RACC: receptor-activated cation channel RACK: receptor for activated C-kinase (PKC) RAD: recombination protein-A (RecA)homolog DNA-repair protein Rad: radiation sensitivity protein (radiationsensitive prosurvival factor upon exposure to UV beam and ionizing radiation) Raf: rapidly accelerated fibrosarcoma oncogene homolog (MAP3K) Rag: Ras-related GTP-binding protein RAGE: receptor of advanced glycation end products ragulator: Rag and TORC1 regulator complex Ral: Ras-related protein RAlBP: retinaldehyde-binding protein RalGDS: Ral guanine nucleotide-dissociation stimulator RAM: rapidly adapting mechanoreceptor RAMP: (calcitonin receptor-like) receptor activity-modifying protein Ran: Ras-related nuclear protein RanBP: Ran-binding protein RAP: receptor-associated protein RAP: telomeric repressor and activator protein Rap: Ras-proximate (Ras-related) protein raptor: regulatory associated protein of TOR RAR: retinoic acid receptor (NR1b2/3) RARRes: retinoic acid receptor responder Ras: rat sarcoma viral oncogene homolog (small GTPase; retrovirus-associated DNA sequence) RasA: Ras p21 protein activator rasiRNA: repeat-associated small interfering RNA (PIWI)

830

List of Molecule Shortened Abbreviations and Chemical Symbols

RASR: rapidly adapting stretch receptor RASSF: Ras interaction/interference protein RIN1, afadin, and Ras association domain-containing protein family member RB: retinoblastoma protein RB1CC1: RB1-inducible coiled-coil protein (Atg17) RBBP: RB-binding protein RBFox: RNA-binding Fox homolog RBM: RNA-binding motif-containing protein RBP: retinoid-binding protein RBPJκ: recombination signal-binding protein for immunoglobulin κJ region RBR: RING between RING finger-containing protein group RBx: RING box-containing protein RC (RyR): ryanodine-sensitive calcium channel RCan: regulator of calcineurin RCC: regulator of chromosome condensation RDM: regulator of microtubule dynamics REDD: regulated in development and DNA-damage response gene product Rec: meiotic recombination protein homolog RecQL: RecQ homolog (recombination and repair of damaged DNA helicase) REEP: [GPCR] receptor [cell surface] expression-enhancing protein REF: mRNA export factor ReF: redox factor Rel: reticuloendotheliosis proto-oncogene product (TF; member of NFκB) ReNTi: type-i regulator of nonsense transcript RENTC: ReNT1–ReNT2–ReNT3 surveillance complex REP: Rab escort protein reptor: repressed by TOR ReR: renin receptor (PRR) REST: repressor element RE1-silencing transcription factor restin: Reed–Steinberg cell-expressed intermediate filament-associated protein (CLiP1) ReT: rearranged during transfection (RTK) RetnL: resistin-like protein Rev1L: DNA repair Rev1-like protein RFc: replication factor-C RFng: radical fringe RFC: replication fork complex RFHC: replication fork helicase complex RFPC: replication fork protection complex RGC: RalGAP complex (RGC1–RGC2 heterodimer)

RGL: Ral guanine nucleotide-dissociation stimulator-like protein (GEF) RGS: regulator of G-protein signaling RHEB: Ras homolog enriched in brain Rho: Ras homologous GTPase RhXG: Rhesus type-X glycoprotein (X: A–C; ammonium transporters SLC42a1–SLC42a3) RIAM: Rap1GTP -interacting adaptor molecule RIBP: RLK- and ITK-binding protein RIC: ribosomal initiation complex RICH: RhoGAP interacting with CIP4 homolog RICK: receptor for inactive C-kinase rictor: rapamycin-insensitive companion of TOR RIDD: regulated IRE1 (ERN1)-dependent degradation RIF: Rho in filopodium RIF1: Rap1-interacting factor-1 RIG1: retinoid-inducible gene product-1 (RARRES3 gene) RIN: Ras-like protein expressed in neurons (GTPase) RIn: Ras and Rab interactor (RabGEF) RING: really interesting new gene motif RIPK: receptor-interacting protein kinase RISC: RNA-induced silencing complex RIT: Ras-like protein expressed in many tissues RIZ: retinoblastoma protein-interacting zinc finger-containing protein RKIP: Raf kinase inhibitor protein RLC: RISC-loading complex RLK: resting lymphocyte kinase (TXK) RLR: retinoic acid-inducible gene RIG1-like receptor RMDn: regulator of microtubule dynamics protein RMI1: RecQ-mediated genome instability homolog-1 RNA: ribonucleic acid RNABP (RBP): RNA-binding protein RNA Pol: RNA polymerase Rnase (RNase): ribonuclease RnBP: renin-binding protein rNDP: ribonucleotide diphosphate RNF: RING finger-containing protein (Ub ligase) RNP: ribonucleoprotein RNPep: arginyl aminopeptidase [APb] RNPS: RNA-binding protein with a serine-rich domain RNR: ribonucleotide reductase

List of Molecule Shortened Abbreviations and Chemical Symbols RNS: reactive nitrogen species RNU: spliceosomal RNA RO: Ro ribonucleoproteic complex (Y RNA) robo: roundabout ROC: receptor-operated channel rock: Rho-associated, coiled-coil-containing protein kinase ROMK: renal outer medullary potassium channel romo: ROS modulator RONS: reactive oxygen and nitrogen species RORξ: type-ξ RAR-related orphan receptor (NR1f1–NR1f3) ROR(RTK) : receptor protein Tyr kinase-like orphan receptor ROS: reactive oxygen species Ros: ros UR2 sarcoma virus proto-oncogene product (RTK) RP: proteasomal regulatory particle (cap) RPa: replication protein-A RPAP: RNA polymerase-2-associated protein RPGRIP1L: retinitis pigmentosa GTPase regulator-interacting protein-1-like protein RPIP: Rap2-interacting protein RPLi: type-i large ribosomal (subunit) protein RPN: proteasomal regulatory particle non-ATPase Rpn: ribophorin RPSi: type-i small ribosomal (subunit) protein RPS6: ribosomal protein S6 RRPi: type-i ribosomal RNA processor RPT: proteasomal regulatory particle AAA (triple A) ATPase RPTOR: regulatory protein of TOR RPTP: receptor protein Tyr phosphatase RQC: ribosome-associated quality control rRas: related Ras rRNA: ribosomal RNA RSC: remodeling of the structure of chromatin complex RSF: remodeling and spacing factor (RSF1-composed dimer) RSK: P90 ribosomal S6 kinase (P90 RSK) RSKL: ribosomal protein S6 kinase-like (pseudokinase) rSMAD: receptor-regulated SMAD (SMAD1– SMAD3, SMAD5, and SMAD9) RSpo: R-spondin RSR: replication stress response RSTK: receptor protein Ser/Thr kinase RTF: replication termination factor domaincontaining protein RTK: receptor protein Tyr kinase

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RTMP: resting transmembrane potential RTR: DNA damage repair RecQL–Top3–RMI1 complex RTT: regulator of Ty1 transposition rubicon: RUN domain and Cys-rich domain-containing, beclin-1-interacting protein RUNDC: RUN domain-containing protein RUSC: RUN and SH3 domain-containing protein RUTBC: RUN and TBC1 domain-containing RabGAP (SGSM) Runx: Runt-related transcription factor RuvBLi: type-i AAA+ ATPase homologous to the bacterial RuvB DNA helicase RXRξ: type-ξ retinoid X receptor (NR2b1– NR2b3) RYBP: RING1- and YY1-binding protein RYK: receptor-like (or related to receptor) protein Tyr (Y) kinase (pseudokinase) RyR: ryanodine receptor (ryanodine-sensitive Ca2+ -release channel)

S S1P: sphingosine 1-phosphate S6K: P70 ribosomal S6 kinase (P70 RSK) σR1: σ1 nonopioid intracellular receptor (chaperone) SAa: serum amyloid-A SAC: spindle assembly checkpoint SACCl(K) : stretch-activated Cl− (K+ )-selective channel SAc: suppressor of actin domain-containing 5-phosphatase sAC: soluble adenylate cyclase SACCNS : stretch-activated cation nonselective channel SACM1L: suppressor of actin mutation-1-like SAE: sumo-activating enzyme SAGA: Spt–Ada–GCN5 acetyltransferase (and deubiquitinase) SAIC: stretch-activated ion channel SAM: sorting and assembly machinery sami: SAM complex subunit-i SAMR (SAM): slowly adapting mechanoreceptor SAP: stress-activated protein SAPi: synapse-associated protein i SAPK: stress-activated protein kinase (MAPK) SAR: secretion-associated and Ras-related protein saraf: SOCE-associated regulatory factor

832

List of Molecule Shortened Abbreviations and Chemical Symbols

SARM: sterile-α and TIR motif-containing protein SARNP: SAP domain-containing ribonucleoprotein SASR: slowly adapting stretch receptor SBF: SET-binding factor Sc: Saccharomyces cerevisiae SC4MOL: sterol C4-methyloxidase-like protein (methylsterol monooxygenase) SCA: stem cell antigen scACADH (scADH): short-chain acylCoA dehydrogenase SCAMP: secretory carrier membrane protein SCAP: SREBP cleavage-activating protein (SREBP escort) SCAR: suppressor of cAMP receptor (wave) ScaR: scavenger receptor scaRNA: small Cajal body RNA SCC: sister chromatid cohesion protein SCD: stearoylCoA desaturase SCF: SKP1–Cul1–F-box-containing protein Ub ligase complex SCF: stem cell factor scFA: short-chain fatty acid (4–8 carbon atoms) SCFR: stem cell factor receptor (KIT) Scgi (Sgi): type-i secretogranin (SCG2/3/5 genes) Scgb: secretoglobin SCMH: sex comb on midleg homolog (SCMH1 is SCML3) SCML: sex comb on midleg-like protein SCO: synthesis of cytochrome-C oxidase SCoAS: succinylCoA synthase SCOT: succinylCoA:3-oxoacidCoA transferase SCP (CTDSP): small C-terminal domain (CTD)-containing phosphatase Scp: stresscopin (urocortin-3) scRNA: small cytoplasmic RNA scRNP: small cytoplasmic ribonucleoproteic complex scrib: Scribble polarity protein Sct: secretin SctR: secretin receptor scube: signal peptide CUB and EGF-like domain-containing protein Sdc: syndecan SDCCAg: serologically defined colon cancer antigen SDF: stromal cell-derived factor SDH: succinate dehydrogenase SDLGMD: sarcoglycan-deficient limb-girdle muscular dystrophy

sdRNA: small nucleolar RNA (snoRNA)derived RNA SEF: similar expression to FGF genes (inhibitor of RTK signaling) sEH: soluble epoxide hydrolase SEK: SAPK/ERK kinase Sel1L: suppressor of Lin12-like protein SelE(L,P): selectin-E [endothelial] (L [leukocyte], P [platelet]) SelPx: type-X selenoprotein (encoded by the SELENOPX gene) selncr: smooth myocyte- and endotheliocyte-enriched migration/ differentiation-associated lncRNA sema: semaphorin (Sema, Ig, transmembrane, and short cytoplasmic domain) sencr: smooth myocyte- and endotheliocyte-enriched migration/ differentiation-associated lncRNA SenP: sentrin (sumo)-specific peptidase serca (SERCA): sarco(endo)plasmic reticulum calcium ATPase SERP: stress-associated endoplasmic reticulum protein serpin: serine peptidase inhibitor SerT: serotonin transporter Sesn: sestrin Setx: senataxin SF: splice factor SF/NR5a1: steroidogenic factor SFK: SRC family kinase SFPQ: splicing factor proline and glutaminerich sFRP: secreted frizzled-related protein SftP (SP): surfactant protein sGC: soluble guanylate cyclase SGF: SAGA-associated factor SGK: serum- and glucocorticoid-regulated kinase SGlT: N+ –glucose cotransporter (SLC5a) Sgo: shugoshin (Japanese: guardian spirit) SgoL: Sgo-like protein SgPL (S1PL): sphingosine 1-phosphate lyase SgPP (SPP/S1PP): sphingosine 1-phosphate phosphatase SGSH: N sulfoglucosamine sulfohydrolase (sulfamidase) SGSM: small GTPase (Rap and Rab) signaling modulator SGTξ (i): type-ξ (i) small glutamine-rich tetratricopeptide repeat (TPR) domain-containing protein SH: Src homology domain

List of Molecule Shortened Abbreviations and Chemical Symbols SH3GL: SH3 domain-containing GRB2-like protein (endophilin) SH3P: Src homology-3 domain-containing adaptor protein SH3RF: SH3 domain- and RING fingercontaining protein (Ub ligase) shank (SHAnk): SH3 and multiple ankyrin repeat domain-containing protein sharpin: shank-associated RH domaininteracting protein SHAX: SNF7 (VSP32) homolog associated with ALIX SHB: Src homology-2 domain-containing adaptor SHBG: sex hormone-binding globulin SHC: Src-homologous and collagen-like substrate SH2C (SHC): Src homology-2 domaincontaining transforming protein SHh: sonic hedgehog SHIP: SH-containing inositol phosphatase SHox: short stature homeobox-containing gene product SHP: SH-containing protein Tyr phosphatase (PTPn6/11) SHP/NR0b2: small heterodimer partner shRNA: small (short) hairpin RNA sHSP: small HSP siah (SIAH): seven in absentia homolog (Ub ligase) siglec: sialic acid-binding Ig-like lectin SIK: salt-inducible kinase SiM: single-minded homolog SIn: stress-activated protein kinase-interacting protein SinAP (SAP): Sin3-associated polypeptide SIP: steroid receptor coactivator-interacting protein siRNA: small interfering RNA SIR: specificity influencing residue (in transcription factor) SiRP: signal-regulatory protein SIRT: sirtuin (silent information regulator-2 [two]; histone deacetylase) SIT: SHP2-interacting transmembrane adaptor SITS: sense-induced transsilencing SK: small conductance Ca2+ -activated K+ channel SKi: avian Sloan–Kettering virus protooncogene homolog SKI: superkiller complex linked to RNA exosomes (mRNA quality control) Ski: superkiller protein SKIP: sphingosine kinase-1-interacting protein

833

skIP (SKIP): skeletal muscle and kidneyenriched inositol phosphatase SkiV2L: superkiller viralicidic activity-2 homolog SKP: S-phase kinase-associated protein SLA: Src-like adaptor SLAM: signaling lymphocytic activation molecule SLAMAP (SAP): SLAM-associated protein SLAMF: SLAM family member SLAP: Src-like adaptor protein SLC: solute carrier superclass member SLCO: solute carrier organic anion class transporter SLK: Ste20-like kinase Slmo: slowmo homolog Sln: sarcolipin SLPI: secretory leukocyte peptidase inhibitor SLRPG: small leucine-rich proteoglycan SLSni: SLSn protein-i SLTC: small latent TGFβ complex SM: sphingomyelin SMA: smooth muscle actin SMAD: small (son of, similar to) mothers against decapentaplegia homolog SMAP: stromal membrane-associated GTPase-activating protein (ArfGAP) SMARC (smarc): Swi/SNF-related, matrix associated, actin-dependent regulators of chromatin SMase: sphingomyelinase SMC: smooth myocyte SMCi: type-i structural maintenance of chromosome SMDT: mitochondrial single-pass membrane protein with aspartate-rich tail (Mt CU regulator) SMG: suppressor with morphogenetic effect on genitalia protein SMF: suppressor of mitochondrial import function SMG1C: SMG1–SMG8–SMG9 complex SMN: survival of motor neuron complex SMN1: survival of motor neuron protein-1 (gemin-1) Smo: Smoothened SMPD: sphingomyelin phosphodiesterase SMRT: silencing mediator of retinoic acid and thyroid hormone receptor (NCoR2) SMS: sphingomyelin synthase SMT: suppressor of Mif two homolog smurf (SMURF): SMAD ubiquitination regulatory factor

834

List of Molecule Shortened Abbreviations and Chemical Symbols

SNAAT: sodium-coupled neutral amino acid transporter SNAP: soluble N ethylmaleimide-sensitive factor-attachment protein SnAP: synaptosomal-associated protein snare (SNARe): SNAP receptor Snc: synuclein; SncαIP: Sncα-interacting protein (synphilin-1) sncRNA: short (small) nonprotein-coding RNA SNF7: sucrose nonfermenting (VPS32) SNIP: SMAD nuclear-interacting protein SNo: SKi novel gene product snolncRNA: small nucleolar long nonproteincoding RNA snoRa: first type of snoRNA snoRd: second type of snoRNA snoRNA: small nucleolar RNA snoRNP: small nucleolar ribonucleoprotein SNRK: SNF-related kinase snRNA: small nuclear RNA snRNP: small nuclear ribonucleoprotein snRPx: type-X Sm domain-containing small nuclear ribonucleoproteic polypeptide SNx: sorting nexin SOx : sulfur oxides SOATi: type-i sterol O acyltransferase (ACACATi) SOC: store-operated Ca2+ channel SOCS: suppressor of cytokine signaling protein SOD: superoxide dismutase Sorbs (SorbS): sorbin and SH3 domaincontaining adaptor SOS: son of sevenless (GEF) Sost: sclerostin SostDC: sclerostin domain-containing protein SOX: sex-determining region Y (SRY)related high mobility group (HMG) homeobox-containing gene Sox: SOX gene product (transcription factor) SP1: specificity protein-1 (transcription factor) sparc (SPARC): secreted protein acidic and rich in cysteine (osteonectin) SPase: serine peptidase SPC: sphingosylphosphorylcholine SPCi: type-i spindle pole component SPCA: secretory pathway Ca2+ ATPase SPEG: striated muscle preferentially expressed gene product (kinase) Sph: sphingosine SphKi (SKi): sphingosine kinase-i SPI: spleen focus-forming virus (SFFV) proviral integration proto-oncogene product (transcription factor)

SPInt: serine peptidase inhibitor SPM: specialized proresolving mediator SpRED: sprouty-related protein with an EVH1 domain Spry: sprouty SpRy: dual-specificity kinase SplA and ryanodine receptor domain-containing protein SPSB: SpRy domain and SOCS box-containing protein SpT: suppressor of Ty SPTLC: serine palmitoyltransferase long-chain subunit spurt (SPURT): secretory protein in upper respiratory tract Sqstm1: sequestosome-1 SR: Arg/Ser domain-containing protein (alternative splicing) sra: steroid receptor RNA activator (lncRNA) SRC: steroid receptor coactivator Src: sarcoma-associated (Schmidt–Ruppin A2 viral oncogene homolog) kinase SRCAP: Snf2-related CREBBP activator protein SRd: steroid reductase (SRD5Ai genes) SRdx: sulfiredoxin SREBP: sterol regulatory element-binding protein SRF: serum response factor SRM/SMRS: Src-related kinase lacking regulatory and myristylation sites SRP: stresscopin-related peptide (urocortin-2) SRPK: serine–arginine-rich (SR) domaincontaining protein kinase Srrt: serrate RNA effector molecule homolog SRSF: serine–arginine-rich motif-containing splicing factor SRTRF: SMG1–ReNT1–TRF1–TRF3 complex SRY: sex-determining region Y SSAC: shear stress-activated channel ssDNA: single-stranded DNA SSEA: stage-specific embryonic antigen SSEN: structure-specific endonuclease Ssh: slingshot homolog phosphatase SSI: STAT-induced STAT inhibitor ssRNA: single-stranded RNA SSRP: structure-specific recognition protein Sst (SSt): somatostatin STAGA: SpT3–TAF9–GCN5 histone acetyltransferase and deubiquitinase complex STAM: signal-transducing adaptor molecule

List of Molecule Shortened Abbreviations and Chemical Symbols STAMBP: STAM-binding protein (Ub isopeptidase) star (StAR): steroidogenic acute regulatory protein stardi (StARD): star-related lipid transfer domain-containing protein-i start (StART): StAR-related lipid transfer protein STAT: signal transducer and activator of transduction STEAP: six transmembrane epithelial antigen of the prostate STICK: substrate that interacts with C-kinase stim (StIM): stromal interaction molecule sting (StInG): stimulator of interferon genes STK: protein Ser/Thr kinase STK1: stem cell protein Tyr kinase receptor STLK: protein Ser/Thr kinase-like (pseudo)kinase StRAd: Ste20-related adaptor STRAP: protein Ser/Thr kinase receptorassociated protein StRAP: stress-responsive activator of P300 STUB: stress-induced phosphoprotein StIP1 (HOP) homology and U-box-containing Ub ligase Stx: syntaxin (SNAREQ ) StxBP: Sufu: suppressor of fused Sulfi: type-i sulfatase (Sulf1–Sulf2) SulTi: type-i sulfotransferase (SulT1a1– SulT1a3, SulT1b1, SulT1c2–SulT1c4, SulT1e1, SulT2a1, SulT2b1a– SulT2b1b, SulT4a1, and SulT6b1) sumo (SUMo): small ubiquitin-related modifier SUNi: type-i SUN domain-containing protein SupTiH: suppressor of Ty i homolog (i = 3,. . . , 7, and 16) SUR: sulfonylurea receptor SUT: stable unannotated transcript SVCT: sodium-dependent vitamin-C transporter SVF: slow ventricular filling SVP: synaptic vesicle precursor SwAP70: 70-kDa switch-associated protein (RacGEF) SWI/SNF: switch/sucrose nonfermentable complex Sxc: super sex comb (polycomb group) SYK: spleen tyrosine kinase SyNEi: type-i synaptic nuclear envelope protein (nesprin) Synj: synaptojanin

835

Syvn: synoviolin Syp: synaptophysin Syt: synaptotagmin SzT2: seizure threshold-2 protein

T T3 : triiodothyronine T4 : thyroxin T6SS: bacterial type-6 secretion system TβRi: type-i TGFβ receptor TAB: TAK1-binding protein TAFi: TBP-associated factor-i (transcription initiation; i: integer) TAFj : template-activating factor-j (histone chaperone; j : Roman numeral) TAG: triacylglycerol (triglyceride [TG]) Tagln: transgelin (actin crosslinker [gelling protein]) TAK: TGFβ-activated kinase (MAP3K7) TAldo: transaldolase TALK: TWIK-related alkaline pH-activated K+ channel TAM: translocator assembly and maintenance protein tami: TAM complex subunit-i TAMP: tight junction-associated MARVEL domain-containing protein set TANK: TRAF family member-associated NFκB activator TAP: transporter associated with antigen processing (ABCb2–ABCb3; not the synonym TAP of NTX1) taP: tail-anchored protein TARP: transmembrane AMPA-type ionotropic glutamate receptor regulatory protein TASK: TWIK-related acid-sensitive K+ channel TASR: terminus-associated short RNA Taz: taffazin (transcriptional coactivator with PDZ-binding motif) TBC1D: Tre2 (USP6), BUB2, CDC16 domain-containing RabGAP TBCK: tubulin-binding cofactor kinase (pseudokinase) TBK: TANK-binding kinase TBP: TATA box-binding protein (subset-4F transcription factor) TBPLi (TRFj ): type-i TBP-like (related) factor TBx: T-box-containing transcription factor TCC: tricarboxylate carrier TCEC: transcription elongation complex Tcerg: transcription elongation regulator

836

List of Molecule Shortened Abbreviations and Chemical Symbols

TCF/ELki: ternary complex factor subgroup member (ELk1/3/4) TCF1α/LEF1: T-cell factor (lymphoid enhancer-binding factor-1) TcFi: type-i transcription factor Tchp: keratin filament-binding trichoplein TCP: T-complex protein TCPIC: transcription preinitiation complex TCR: T-cell receptor TDO: tryptophan (2,3)-dioxygenase TDP: transactive response (TAR) DNA-binding protein TEA: transluminal extraction atherectomy TEAD: transcriptional enhancer activator domain-containing factor TEC: protein Tyr kinase expressed in hepatocellular carcinoma TEF: thyrotrophic embryonic factor (PARBZIP set) TEFix (eEFix): type-iX (eukaryotic) translation elongation factor TEK: protein Tyr endothelial kinase Tel (Telo): telomere maintenance homolog Ten: tenascin Tent (TeNT): terminal nucleotidyltransferase TeRC: telomerase RNA component TeRT: telomerase reverse transcriptase TET: ten–eleven translocation dioxygenase TEx: testis-expressed protein TF: transcription factor Tf: transferrin TFI I : transcription factor-I I (general transcription factor GTF2) TF/FI I I : tissue factor TFA: trans-fatty acid TFAP (AP): activating enhancer-binding protein, i.e., transcription factor activator protein TFDP1: transcription factor DP1 (E2F dimerization partner) TFE3: transcription factor binding to IGHM enhancer-3 (bHLHe33) TFeb: transcription factor-EB (bHLHe35) TFec: transcription factor-EC (bHLHe34) TFPI: tissue factor pathway inhibitor TfR: transferrin receptor TFTC: TATA-binding protein-free TAFI I containing histone acetyltransferase and deubiquitinase complex TG (TAG): triglyceride (triacylglycerol) TGm: transglutaminase TGF: transforming growth factor TGFBR: TGFβ receptor gene TGFβRAP: TGFβ receptor-associated protein

TGN: trans-Golgi network TGRL: triglyceride-rich lipoprotein THED: trihydroxyeicosadienoic acid THET: trihydroxyeicosatrienoic acid THIK: tandem pore-domain halothaneinhibited K+ channel THOC: THO complex component THOME: trihydroxyoctadecamonoenic acid ThOP: thimet oligopeptidase THR: thyroid hormone receptor (NR1a1/2) THRAP: thyroid hormone receptor-associated protein TIA1L: RNA-binding T-cell-restricted intracellular antigen TIA1, cytotoxic granule-associated RNA-binding protein-like (nucleolysin) TIAM: T-lymphoma invasion and metastasisinducing protein (RacGEF) TIAR: T-cell intracellular antigen (TIA1)related protein TIE: protein Tyr kinase with Ig and EGF homology domains (angiopoietin receptor; also abbreviating tunica interna endothelial kinase) tie1as: TIE1 antisense transcript (lncRNA) TIEG: TGFβ-inducible early gene product TIF: telomere dysfunction-induced focus TIF1a(b)/TriM24(28): transcription intermediary factor (kinase and Ub ligase; RNF82[96]) TIFix (eIFix): type-iX (eukaryotic) translation initiation factor TIF4eBP1: inhibitory TIF4e (eIF4e)-binding protein tigar (TIGAR): TP53-inducible glycolysis and apoptosis regulator Tim: timeless homolog TIMD: T-cell immunoglobulin and mucin domain-containing protein TIMM: translocase of inner mitochondrial membrane timmi: TIMM complex subunit-i TIMP: tissue inhibitor of metallopeptidase TINF: TRF1-interacting nuclear factor tipin: timeless-interacting protein TIRAP: toll–IL1R domain-containing adaptor protein tiRNA: transcription initiation RNA TKR: protein Tyr kinase receptor TKt: transketolase TLIC: translation initiation complex Tln: talin TLPIC: translation preinitiation complex TLR: toll-like receptor

List of Molecule Shortened Abbreviations and Chemical Symbols TLS: transcript leader sequence (5 -UTR) TLT: TREM-like transcript TLX: tailless receptor (NR2e1) TM: thrombomodulin TMi: transmembrane segment-i of membrane protein TMA: trimethylamine (gut flora product) TMAO: TMA N oxide TMC: twisting magnetocytometry TMem: transmembrane protein tmepai (TMePAI): transmembrane prostate androgen-induced protein TMG: trimethylglycine TMx: thioredoxin-related transmembrane protein TMy: tropomyosin Tnn (TN): troponin Tn: thrombin TNF: tumor-necrosis factor TNFαIP: TNFα-induced protein TNFR: TNF receptor TNFRSF: TNFR superfamily member TNFSF: TNF superfamily member (TNFSF1: TNFα; TNFSF2: TNFβ) TNK: protein Tyr kinase inhitor of NFκB TNRC: trinucleotide repeat containing-protein Tns: tensin TOMM: translocase of outer mitochondrial membrane tommi: TOMM complex subunit-ii Top: DNA topoisomerase TOP2CC: Top2–DNA cleavage complex TopBP: Top-binding protein TOR: target of rapamycin TORC: transducer of regulated CREB activity (a.k.a. CRTC) TORC1(2): target of rapamycin complex-1(2) TP: thromboxane-A2 Gq/11-coupled receptor tPA: tissular plasminogen activator TPIC: transcription preinitiation complex Tpo (TPo): thrombopoietin TpoR (TPoR): thrombopoietin receptor Tpm (TpM): tropomyosin TPP: thiamine (vitamin-B1) pyrophosphate TPP: tripeptidyl peptidase TPPP: tubulin polymerization-promoting protein TPR: tetratricopeptide repeat domain TPST: tyrosylprotein sulftotransferase TPX2: targeting protein for Xenopus plusend–directed centrosomal kinesin-like protein XKLP2 TR: testicular receptor (NR2c1/2)

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TRAAK: TWIK-related arachidonic acid-stimulated K+ channel TRADD: TNFR1-associated death domaincontaining adaptor TRAF: TNFR-associated factor trak: kinesin-binding trafficking adaptor protein TRAM: TRIF-related adaptor molecule TRAMP: Trf4–Air2–Mtr4 polyadenylation complex transceptor: transporter-related receptor TRAP: TNFR-associated protein (HSP75) TraPPi: type-i transport protein particle TRAPPC: transport protein particle complex TRAPPCi: type-i TRAPP gene (encoding TraPPi) TRAT: T-cell receptor-associated transmembrane adaptor Trb: Tribbles homolog (pseudokinase) TRC: transmembrane domain-containing recognition complex component TRdx: thioredoxin TRdxDC: thioredoxin domain-containing protein TRdxIP: thioredoxin-interacting protein TRdxRd: thioredoxin reductase (TXNRDi genes) TRE: trapped in endoderm TREK: TWIK-related K+ channel TREM: triggering receptor expressed on myeloid cells TRESK: TWIK-related spinal cord K+ channel TREX: transcript export complex TRF: double-stranded telomeric DNA-binding repeat-binding factor TRFi (eRFi): type-i (eukaryotic) translation release factor TRH: thyrotropin-releasing hormone TRIAp: TP53-regulated inhibitor of apoptosis TRIC: tailless complex polypeptide TCP1 ring complex TRIF: toll–IL1R domain-containing adaptor inducing Ifnβ TRIM: T-cell receptor-interacting molecule TriM: tripartite motif-containing protein TRIP: TGFβ receptor-interacting protein (eIF3S2) TRIP: thyroid receptor-interacting protein TRK: tropomyosin receptor kinase (NTRK) TRL: triglyceride-rich lipoprotein tRNA: transfer RNA tRNAF: transfer RNA (tRNA)-derived fragment TRP: transient receptor potential channel

838

List of Molecule Shortened Abbreviations and Chemical Symbols

TRPA: ankyrin-like transient receptor potential channel TRPC: canonical transient receptor potential channel TRPM: melastatin-related transient receptor potential channel TRPM6CK: chanzyme type-6 TRPM (TRPM6)-cleaved kinase TRPML: mucolipin-related transient receptor potential channel TRPN: no mechanoreceptor potential C TRPP: polycystin-related transient receptor potential channel TRPV: vanilloid transient receptor potential channel TrrAP: transactivation (transformation)/ transcription domain-associated pseudokinase TRX: trithorax chromatin activator complex Trx: trithorax (histone Lys methyltransferase) TrxG: trithorax group protein TSC: tuberous sclerosis complex TSci: tuberous sclerosis protein-i (i = 1, 2) TSH: thyroid-stimulating hormone TSLP: thymic stromal lymphopoietin Tsp (TSp [Thbs]): thrombospondin (THBS gene) Tspan: tetraspanin Tspo (TsPO): translocator protein of the outer mitochondrial membrane tSNARE: target SNARE tsRNA: tRNA-derived small RNA tssaRNA: transcription start site-associated RNA TTbK: Tau-tubulin kinase TTCi: type-i tetratricopeptide repeatcontaining protein TTFi (eTFi): type-i (eukaryotic) translation termination factor TTFL: transcriptional–translational feedback loop (circadian clock) TTG: tissue transglutaminase TTK: dual-specificity protein Thr/Tyr kinase Ttn: titin (pseudokinase) TTR RBP: mRNA turnover and translation regulatory RNA-binding protein Tubξ: type-ξ tubulin tug (TUG): taurine upregulated gene transcript (lncRNA) TuSC3: tumor suppressor candidate-3 (magnesium transporter) TUT: terminal uridine transferase TWIK: tandem of P domains in a weak inwardly rectifying K+ channel

TxA2 : thromboxane-A2 (thromboxane) TxB2 : thromboxane-B2 (thromboxane metabolite) TXK: protein Tyr kinase mutated in X-linked agammaglobulinemia TxaS: thromboxane-A synthase TxnDC: TRdx domain-containing protein TyK: tyrosine kinase + TP: plus-end-tracking proteins

U Ub: ubiquitin UbC: ubiquitin conjugase UbD: ubiquitin-D UbE2: ubiquitin conjugase (i.e., E2) UbE3: ubiquitin ligase (i.e., E3) UbL: ubiquitin-like protein UOx: urate oxidase Ubqln: ubiquilin UbR: ubiquitin ligase (N degron) recognin UbXD: ubiquitin regulatory X domaincontaining protein uca1 (UCA1): urothelial carcinoma-associated transcript (lncRNA) UCH: ubiquitin C-terminal hydrolase (DUb) UCMA: upper zone of growth plate and cartilage matrix-associated protein (GRP) Ucn: urocortin UCP: uncoupling protein UDP: uridine diphosphate UDP glucose: UDP–glucose UFD1: ubiquitin fusion degradation protein-1-like protein UFM: ubiquitin-fold modifier (UbL) UGGT: UDP glucose–glycoprotein glucosyltransferase UGT: UDP–glucuronyl transferase UK: urokinase ULK: uncoordinated-51-like kinase (pseudokinase, ULK1 corresponding to Atg1) Unc: uncoordinated receptor UNG: uracil–DNA glycosylase uORF: upstream open reading frame uPA: urokinase-type plasminogen activator (urokinase) uPAR: uPA receptor uPARAP: uPAR-associated protein (CLec13e) UP4 A: uridine adenosine tetraphosphate URI: unconventional prefoldin subunit RPb5 interactor-like protein URM: ubiquitin-related modifier (UbL)

List of Molecule Shortened Abbreviations and Chemical Symbols Uro: urodilatin USF: upstream stimulatory factor USP: ubiquitin-specific peptidase (deubiquitinase) UTP: U three protein UTP: uridine triphosphate UTR: untranslated region (of mRNA) UVRAG: ultraviolet wave resistanceassociated gene product UXT: ubiquitously expressed transcript product

V V1A/1B/2 : type-1A/1B/2 arginine vasopressin receptor V1(2)R: type-1(2) vomeronasal receptor VAAC: volume-activated anion channel VACCl(K) : volume-activated Cl− (K+ )selective channel VACamKL: vesicle-associated CamK-like (pseudokinase) VACCNS : volume-activated cation nonselective channel VAChT: vesicular acetylcholine transporter VAIC: volume-activated ion channel VAMP: vesicle-associated membrane protein (synaptobrevin) VanGL: Van Gogh (Strabismus)-like protein VAP: VAMP-associated protein vasp (VASP): vasodilator-stimulated phosphoprotein vaspin: visceral adipose tissue-derived serpin VAT: vesicular amine transporter Ve ATPase (vATPase): vesicular H+ ATPase Vav: GEF named from Hebrew sixth letter vcam (VCAM): vascular cell adhesion molecule VCP: valosin-containing protein (AAA ATPase) VDAC: voltage-dependent anion channel (porin) VDACL: plasmalemmal, volume- and voltage-dependent, ATP-conductive, large-conductance, anion channel VDCC: voltage-dependent calcium channel VDP: vesicle docking protein VEGF: vascular endothelial growth factor VEGFR: VEGF receptor VEZF: vascular endothelial zinc finger protein VGAT: vesicular GABA transporter VGC: voltage-gated channel VgL: vestigial-like protein vGluT: vesicular glutamate transporter

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VHL: von Hippel–Lindau Ub ligase subunit VIP: vasoactive intestinal peptide vlcACADH (vlcADH): very-long–chain acylCoA dehydrogenase vlcFA: very-long-chain fatty acids (17–26 carbon atoms) VLDL: very-low–density lipoprotein VLDLR: VLDL receptor VMAT: vesicular monoamine transporter VMP: vesicular membrane protein Vn (VN): vitronectin vPOx: vascular peroxidase VPS: vesicular protein-sorting–associated protein VRAC: volume-regulated anion channel VRK: vaccinia-related kinase VS: vasostatin vSNARE: vesicular SNAP receptor (SNARE) VSOR: volume-sensitive outwardly rectifying anion channel VSP: voltage-sensing phosphatase vWF: von Willebrand factor

W WAPL: wings apart protein homolog WASF: Wiskott–Aldrich syndrome (WAS) protein family member WASH: WASP and SCAR homolog WaRS: tryptophanyl tRNA synthase WASP: Wiskott–Aldrich syndrome protein wavei (WAVe): WASP family verprolin homolog-i WBSCR: WBS chromosomal region protein WDFY: WD repeat and FYVE domaincontaining protein WDHD: WD repeat-containing and HMG box-derived DNA-binding protein WDPCP: WD repeat-containing and planar cell polarity effector WDR: WD repeat-containing protein wee: small (Scottish) WHAMM: WASP homolog associated with actin, membranes, and microtubules WHSC: Wolf–Hirschhorn syndrome candidate (WHSC1 is a.k.a. NSD2) WHSC1L1: WHSC1-like protein (or NSD3) WIP: WASP (WAS and WASL)-interacting protein WIPF: WASP-interacting protein family protein WIPI: WD repeat-containing phosphoinositideinteracting protein

840

List of Molecule Shortened Abbreviations and Chemical Symbols

wisp: Wnt1-inducible signaling pathway protein wisper: wisp2 superenhancer-associated RNA (lncRNA) WNK: with-no-K (Lys) kinase (Lys-deficient kinase) Wnt: wingless-type WNRRTK: Wnt and neurotrophin receptorrelated receptor protein Tyr kinase (ROR(RTK) ) WRD: tryptophan-rich protein WRS: wave regulatory complex WSB: WD40 repeat and SOCS box-containing protein (Ub ligase) WSTF: Williams syndrome transcription factor WT1: Wilms tumor protein-1 (transcription factor) WWP1: WW domain-containing protein (Ub ligase) WWTR: WW domain-containing transcription regulator (WWTR1 is Taz)

X XABi: type-i xeroderma pigmentosum group-A-complementing (XPa)-binding protein XBP: X-box-binding protein (transcription factor) XDH: xanthine dehydrogenase XFE: XPF–ERCC4 mutation-resulting progeroid syndrome XIAP: X-linked inhibitor of apoptosis protein (Ub ligase) xist (XIST): X-inactive specific transcript (lncRNA) XOR: xanthine oxidoreductase (XDH) XOx: xanthine oxidase (a form of XDH) XPx: xeroderma pigmentosum (XP) groupX-complementing protein (DNA repair protein) XRCC: X-ray repair cross-complementing protein XRNi: type-i exoribonuclease XRS: X-ray-sensitive protein

Y YAF: YY1-associated factor YAP: Yes-associated protein YBP: Y-box-binding protein (transcription factor)

YBx: Y-box-binding transcription factor (gene transcription and pre-mRNA splicing regulator) Yes: Yamaguchi sarcoma viral proto-oncogene protein Tyr kinase homolog YME1L: yeast mitochondrial escape (the catalytic subunit of iAAA [AAA+ peptidase active at the IMS side of the IMM]) YME1-like YTHDC: YTH domain-containing protein (mN6 A reader) YWHA: tyrosine 3-monooxygenase and tryptophan 5-monooxygenase activation protein (14-3-3 protein) YY: yin yang (transcriptional repressor)

Z ZAP70: 70-kDa TCRζ chain-associated protein ZBP: zDNA-binding protein ZBTB: zinc finger and BTB (Broad complex, tramtrack, and bric-à-brac) domain-containing transcription factor ZC3H: zinc finger CCCH domain-containing protein ZCCHC: zinc finger CCHC domain-containing protein ZCWPW: zinc finger CW-type and PWWP domain-containing protein ZDHHC: zinc finger and DHHC motifcontaining palmitoyl acyltransferase ZFC3H1: zinc finger C3H1 domain-containing protein ZFYVE: zinc finger FYVE domain-containing protein ZHX (ZHE): Zn2+ –H+ exchanger ZnHIT: zinc finger HIT domain-containing protein ZiC: zinc finger-containing transcription factor of cerebellum ZKSCAN: zinc finger with KRAB and SCAN domain-containing transcription factor ZMYND: zinc finger MYND-type protein ZnF: zinc finger protein

Miscellaneous 9–1–1: Rad9–Rad1–Hus1 checkpoint sliding clamp complex 2-5A: 5 -triphosphorylated, (2 ,5 )phosphodiester-linked oligoadenylate 2AG: 2-arachidonoyl glycerol 3BP2: Abl Src homology-3 domain-binding adaptor

List of Molecule Shortened Abbreviations and Chemical Symbols 4HNE: 4-hydroxynonenal 5FMC: five friends of methylated ChTOP 5HT: 5-hydroxytryptamine (serotonin) 7TMR: 7-transmembrane receptor (GPCR) 14-3-3: protein eluted on DEAE-cellulose chromatography in the 14th fraction of bovine brain homogenate and localizes to position 3.3 of subsequent starch-gel electrophoresis

Protein Domain—Protein Category ADD: ATRX–DNMT3–DNMT3L domain ADFH: actin-depolymerizing factor homology domain ANK: ankyrin repeats (tandemly repeated modules of about 33 amino acids) ARID: AT-rich interaction domain (DNAbinding module) ARM: repetitive (several tandemly repeated) amino acid sequence from Drosophila β-catenin-like armadillo AWS: associated with SET domain (predominantly in histone methyltransferases) BAH: bromodomain-associated [adjacent] homology domain BAR: bridging integrator BIn1, amphiphysin, and reduced viability on starvation (RVS) domain (phospholipid binding) BEACH: beige or Chediak–Higashi syndrome (CHS) domain BH (BH1–BH4): BCL2 homology domain BIR: baculovirus IAP (inhibitor of apoptosis) repeat bromodomain: brahma (SMARCa2) organization modifier BTB (POZ): broad complex, tramtrack, and bric-à-brac domain BRCT: BrCa1 C-terminal domain (DNA repair and cell cycle regulation) bZIP: basic leucine zipper domain C1: cysteine-rich domain (phospholipid binding) C2: PKC homology domain (phospholipid binding) CALM: clathrin assembly lymphoid myeloid domain CARD: caspase recruitment domain CC: coiled-coils (typically consisting of at least 2 α helices; oligomerization domain) CH (CH1–CH3): calponin homology domain (in cytoskeletal proteins and signaling mediators)

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CHCH: coiled coil helix–coiled coil helix fold chromodomain: chromatin organization modifier CSD: chromo shadow domain (forms stable dimers) CTD: C-terminal domain CUE: homology region shared by the yeast coupling of ubiquitin to ER degradation CUE1 and human tollip (binding of mono- and polyubiquitin) DEAD box: Asp–Glu–Ala–Asp box (RNA helicase) DEAH box: Asp–Glu–Ala–His box (RNA helicase) DD: death domain (involved in apoptotic signal transduction) DED: death effector domain (protein interaction domain regulating caspase activation) DH: Dbl homology (RhoGEF) domain DPF: double PHD finger domain ENTH: epsin N-terminal homology domain EF hand: helix-turn–helix structural motif EH: epidermal growth factor receptor substrate EPS15 homology domain (endocytosis and vesicular transfer) EVH: Ena–Vasp homology domain (control of the actin cytoskeleton and signal transduction) F box: N-terminal amino acid domain and module of adaptor subunit of ubiquitin ligase complex responsible for phosphorylation-mediated ubiquitination FCH: Fes and CIP4 homology domain FERM: band 4.1, ezrin, radixin, and moesin domain FF: phenylalanine–phenylalanine tandem repeats (in transcription and splicing factors) FH (FH2–FH3): formin homology-2(3) domain FHA: forkhead-associated domain (DNA damage response) FYVE; Fab1 (phosphoinositide kinase, FYVE finger-containing PIKFYVE), YGL023 (pumilio homology domain-containing Puf4), VPS27, and EEA1 domain G: six glycine (G)-containing patch of several RNA-associated proteins GAT: homology region found in GGA (Golgi body-localized, γ ear-containing ADP ribosylation factor-binding protein) and Tom1 (target of MyB)

842

List of Molecule Shortened Abbreviations and Chemical Symbols

GEL: gelsolin homology domain, also called gelsolin, severin, and villin homology domain GLUE: GRAM-like ubiquitin-binding in EAP45 (VPS36) domain (phosphoinositide binding) GRAM: glucosyltransferase, Rab-like GTPase activator, and myotularin domain (phosphoinositide binding) GRIP: golgin-97, RanBP2α, golgin integrin and myosin homology protein IMH1, and P230 (golgin-245) domain (interaction with ARF and ARL GTPases) GYF: glycine–tyrosine–phenylalanine domain HEAT: huntingtin, elongation factor-3, 65-kDa PP2 regulatory subunit-α (A), and TOR domain HPD: homeo–prospero domain comprising a homeodomain (HD) and associated prospero domain (PD) needed for sequence-specific DNA binding HECT: homologous with E6-associated protein C-terminus domain HUWE: HECT, UBA, and WWE domain IG (IgV, IgC1–IgC2, and IgI): immunoglobulin-like domain IQ: consensus sequence [I,L,V]Q(AA)3 RG(AA)3 [R,K], a basic unit of about 23 amino acids KASH (domain): klarsicht, Anc1, and SyNE homology KH: K homology domain related to heterogeneous nuclear ribonucleoprotein hnRNPk LIM: Lin11, Isl1, and Mec3 domain, a zinc-binding, cysteine-rich motif consisting of 2 tandemly repeated zinc fingers LRR: leucine-rich repeat MBD: methylated CpG-binding domain MBT: malignant brain tumor repeat (homology region with 2 repeats shared by polycomb group (PcG) protein sex comb on midleg (SCM) and lethal(3) malignant brain tumor L3MBTL) MH (MH1–MH2): MAD homology domain (SMADs have N-terminal MH1 and C-terminal MH2 domains) MIU: motif interacting with ubiquitin NACHT: neuronal apoptosis inhibitor protein (NAIP), MHC class-I I transcription activator (CI I TA), vegetative or heterokaryon incompatibility locus

protein (in the fungus Podospora anserina) HetE, and telomeraseassociated protein TP1 domain NZF: nuclear protein localization NPL4 zinc finger domain OTU: ovarian tumor homology region PAH: paired amphipathic helix motif (e.g., in the MYC family of helix–loop–helix DNA-binding proteins) PAS: Per, ARNT, and Sim domain PAZ: piwi, argonaute, and zwille/pinhead domain PB1: phagocyte oxidase Phox and bud emergence Bem1 domain PBZ: polyADP ribose-binding zinc finger domain PDZ: postsynaptic density PSD85, disc large, and zonula occludens ZO1 domain PH: pleckstrin homology domain (phosphoinositide binding) POLO box: domain exclusively shared by polo-like kinases POU: Pit1 (POU1F1)–Oct1 (POU2F1)–Unc86 domain (set) POZ (BTB): poxvirus and zinc finger domain (set) PTB: phosphotyrosine-binding domain PUF: pumilio protein family domain PWWP: Pro–Trp–Trp–Pro central corecontaining domain PX: phagocyte oxidase Phox homology domain (phosphoinositide binding) PYD: pyrin domain R3H: sequence containing an invariant arginine and histidine separated by 3 residues RBCC: RING finger, B boxes, and coiled-coil domain RGS: regulator of G-protein signaling domain RING finger: zinc finger domain of really interesting new gene (RING)-containing ubiquitin ligase RUN: RUNDC3a (RPIP8), Unc14, and nesca (RUSC1) domain RWD: RING finger and WD repeat-containing protein and DEAD-like helicase domain S1: ribosomal protein S1 motif existing also in numerous RNA-associated proteins SAM: sterile-α motif SAP: scaffold attachment factor SAFa (hnRNPu) and SFAb (HSP27 estrogen response element- and TATA boxbinding protein [HET]), acinus, and protein inhibitor of activated STAT1 (PIAS) motif

List of Molecule Shortened Abbreviations and Chemical Symbols SH (SH2–SH3): Src homology-2(3) domain SNARE: soluble NSF attachment protein (SNAP) receptor homology domain (membrane fusion) SOCS box: homology region originally identified in suppressors of cytokine signaling at the C terminus, a target for ubiquitination SPRY: sprouty and sprouty-related EVH1 (SpRED) domain SR: serine–arginine-rich domain STARD: star-related lipid transfer (StaRT) domain SUN: Sad1 and Unc84 homology domain (set) SWIRM: Swi3, remodel the structure of chromatin RSC8, and moira domain (implicated in DNA binding and chromatin remodeling) TAD: topologically associating domain TBC: Tre2 (USP6), BUB2, and CDC16 domain TEAD: transcriptional enhancer activator domain TIMD: T-cell immunoglobulin and mucin domain TIR: toll–IL1R domain TMD (TR): transmembrane domain TPR: tetratricopeptide repeat domain TRADD: TNFR1-associated death domain TRAF: tumor-necrosis factor receptorassociated factor domain (cell survival) TRdxD: thioredoxin domain Trr: transactivation (transformation)/ transcription domain TUBBY: tubby and tubby-like protein (TuLP) domain TUDOR: repeat found in Drosophila tudor protein UbA: ubiquitin-associated domain UbX: ubiquitin regulatory X domain UEV: ubiquitin E2 variant domain (ubiquitinbinding motif) UIM: ubiquitin-interacting motif VHL: von Hippel–Lindau protein domain VHS: vacuolar protein sorting VPS27, hepatocyte growth factor-regulated Tyr kinase substrate (HRS/Vps27) and signal transducing adaptor molecule (STAM) domain WD40: sequence with tryptophan and aspartic acid residue and a repeat of approximately 40 amino acids WDFY: WD repeat and FYVE domain

843

WW (WWP): Trp–Trp domain (binding to Pro-rich [(AP)–P–P–(AP)–Y] sequence) WWE: 2-tryptophans and 1 glutamatecontaining domain (in proteins linked to ubiquitination and polyadpribosylation) YTH: Splicing factor YT521b homology domain ZC3H: zinc finger and CCCH domain ZCCHC: zinc finger and CCHC domain ZCWPW: zinc finger, CW-type, and PWWP domain ZDHHC: zinc finger and DHHC motif ZFC3H1: zinc finger and C3H1 domain ZFYVE: zinc finger and FYVE domain ZnF–GATA: zinc finger and DNA sequence [AT]GATA[AG]-binding domain ZnHIT: zinc finger HIT domain ZKSCAN: zinc finger with KRAB and SCAN domain ZMYND: zinc finger and MYND motif ZnF: zinc finger

DNA Sequences in Gene Enhancers and Promoters AARE: amino acid response element AcRE (ARE): activin response element AnRE (ARE): androgen response element AORE (ARE): antioxidant response element AURE (ARE): AU-rich element CARE: C/EBP–ATF response element CHRE: carbohydrate-responsive element CRE: cAMP-responsive element DSAPE: D site of albumin promotor (albumin D-box) element (among site A to site F) EpRE: electrophile response element ERE: estrogen response element ERSRE: endoplasmic reticulum stress (ERS) response element FBE: forkhead-binding element GRE: glucocorticoid response element HifRE: HIF response element HRE: hormone response element ISRE: interferon-stimulated response element MRE: microRNA response element NfkbRE: NFκB response element NRSE: neuron-restrictive silencer element (repressor element RE1) NSRE: nutrient-sensing response element PPRE: PPAR response element PRE: polycomb response element PrlRE (PRE): prolactin regulatory element (pituitary-specific positive transcription

844

List of Molecule Shortened Abbreviations and Chemical Symbols

factor Pit1 (or POU class-1 homeobox domain-containing gene-encoded factor POU1F1)-binding element of prolactin promoter RE1: repressor element-1 RRE: reverse (Rev) erythroblastosis (Erb; NR1d1/2) and RAR-related orphan receptor (ROR; NR1f1/2/3) response element SBE: SMAD-binding element SINE: short interspersed nuclear element (retrotransposon) SRE: sterol regulatory element TRE: TPA (tetradecanoylphorbol acetate)response element (perfect AP1/ CREB-binding site; TGAC[G]TCA) TREL: TRE-like (imperfect AP1) element UPRE: unfolded protein response element VDRE: vitamin-D response element XBE: X-factor-binding element Metabolic Pathways CCM: central carbon metabolism HSP: hexosamine synthesis pathway oxphos: oxidative phosphorylation PPP: pentose phosphate pathway TCAC: tricarboxylic acid cycle DNA and RNA Nucleotides A: adenine, purine derived from adenosine C: cytosine, pyrimidine derived from cytidine G: guanine, purine derived from guanosine T: thymine, DNA pyrimidine derived from thymidine U: uracil, RNA pyrimidine derived from uridine Amino Acids—Triple- and Single-Letter Codes Ala (A): alanine Arg (R): arginine Asn (N): asparagine Asp (D): aspartic acid − AspCOO : aspartate CysSH (C): cysteine (reduced form) CysSS Cys: cystine (oxidized form) Gln (Q): glutamine Glu (E): glutamic acid − GluCOO : glutamate Gly (G): glycine His (H): histidine

Iso, Ile (I): isoleucine Leu (L): leucine Lys (K): lysine Met (M): methionine Orn: ornithine (not encoded by DNA, but use in the urea cycle) Phe (F): phenylalanine Pro (P): proline Ser (S): serine S249P (SNP): Ser at position 249 (wild type) replaced by Pro (mutant) Thr (T): threonine Trp (W): tryptophan Tyr (Y): tyrosine Val (V): valine

Carbohydrates Fuc: fucose Gal: galactose GalNAc: N acetylgalactosamine Glc: glucose GlcA: glucuronic acid (glucuronate) GlcNAc: N acetylglucosamine IdoA: iduronic acid (iduronate) Man: mannose NANA: N acetylneuraminic acid (or sialic acid [Sia]) Xyl: xylose

Ions Asp− : aspartate (carboxylate anion of aspartic acid) ADP3− : ADP anion ATP4− : ATP anion C2 H3 O− 2 : acetate Ca2+ : calcium cation Cl− : chloride anion ClO− : hypochlorite ClO− 2 : chlorite ClO− 3 : chlorate ClO− 4 : perchlorate CN− : cyanide CO2− 3 : carbonate Co2+ : cobalt cation Cu+ : copper monovalent cation Cu2+ : copper divalent cation Fe2+ : ferrous iron cation Fe3+ : ferric iron cation Glu− : glutamate (carboxylate anion of glutamic acid) H+ : hydrogen cation (proton)

List of Molecule Shortened Abbreviations and Chemical Symbols H3 O+ : hydronium (oxonium or hydroxonium) cation HCO− 3 : bicarbonate anion (hydrogen carbonate) HPO2− 4 : hydrogen phosphate divalent anion (inorganic phosphate species) H2 PO− 4 : dihydrogen phosphate monovalent ion (inorganic phosphate species) HS− : hydrosulfide (hydrogen sulfide) anion (sulfanide) HSO− 4 : hydrogen sulfate (bisulfate) K+ : potassium cation Mg2+ : magnesium cation Mg ATP2− : ATP anion Mn2+ : manganese cation MnO− 4 : permanganate Na+ : sodium cation NH+ 4 : ammonium Ni2+ : nickel cation (common oxidation state) O2− : peroxide 2 OH− : hydroxide anion PO3− 4 : phosphate anion (inorganic phosphate species) SeO2− : selenite 3 SeO2− : selenate 4 S2− : sulfide anion S2− n : polysulfide anion SO2− 3 : sulfite anion SO2− 4 : sulfate anion S2 O2− 3 : thiosulfate Zn2+ : zinc cation (common oxidation state)

Atmospheric Pollutants CH4 : methane HNO2 : nitrous acid HNO3 : nitric acid H2 SO4 : sulfuric acid NOx : nitrogen oxides NO2 : nitrogen dioxide O3 : ozone PM10 : inhalable coarse particulate matter (2.5 < size < 10 μm) PM2.5 : fine particulate matter (0.1 < size ≤ 2.5 μm) PM0.1 : ultrafine particulate matter (aerodynamic diameter ≤ 0.1 μm) SOx : sulfur oxides SO2 : sulfur dioxide

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Inhaled and Signaling Gas CO: carbon monoxide (or carbonic oxide; signaling gas and pollutant [air level ∼ 0.1 ppm]) CO2 : carbon dioxide (cell waste) H2 S: hydrogen sulfide (signaling gas) [air level ∼ 0.0001 ppm]) He: helium (inert monatomic gas) N2 : nitrogen (inert diatomic gas) NH3 : ammonia (trihydrogen nitride; trace quantities in air) NO: nitric oxide (or nitrogen monoxide; signaling gas and pollutant [air level ∼ 0.1 ppm]) O2 : oxygen (cell energy producer)

Nitric Oxide Derivatives HNO: protonated nitroxyl anion NO• : free radical form NO+ : nitrosyl or nitrosonium cation NO− : nitroxyl or hyponitrite anion (inodilator) NO− 2 : nitrite anion NO− 3 : nitrate anion Reactive Oxygen and Nitrogen Species CO•− 3 : carbonate radical H2 O2 : hydrogen peroxide HOCl: hypochlorous acid HS• : sulfanyl or hydrosulfide radical N2 O3 : dinitrogen trioxide NO•2 : nitrogen dioxide 1 O : singlet oxygen 2 •− O− 2 : superoxide (O2 ) • − O=C(O )O : carbonate radical OH• : hydroxyl radical (hydroxide ion neutral form) ONOO− : peroxynitrite RO• : alkoxyl RO•2 : peroxyl Moieties (R denotes an organic group) R: alkyl group (with only carbon and hydrogen atoms linked exclusively by single bonds) R–CH3 : methyl group (with 3 forms: methanide anion [CH− 3 ], methylium cation [CH+ 3 ], and methyl radical [CH•3 ])

846 R–CHO: aldehyde group R–CN: nitrile group R–CO: acyl group R–CO–R: carbonyl group R–COO− : carboxylate group R–COOH: carboxyl group R–NC: isonitrile group R–NCO: isocyanate group R–NH2 : amine group R–NO: nitroso group R–NO2 : nitro group R–O: alkoxy group R=O: oxo group R–OCN: cyanate group R–OH: hydroxyl group R–ONO: nitroso–oxy group R–ONO2 : nitrate group

List of Molecule Shortened Abbreviations and Chemical Symbols R–OO–R: peroxy group R–OOH: hydroperoxy group R–S–R: sulfide group R–SH: thiol (or sulfhydryl) moiety R–SN: sulfenyl-amide moiety R–SNO: nitrosothiol (or thionitrite) moiety R–SO: sulfinyl R–SO–R: sulfoxide group R–SO2 : sulfonyl group R–SO2 H: sulfinic acid (sulfinyl moiety) R–SO2 N: sulfonyl-amide moiety R–SO3 H: sulfonic acid (sulfonyl moiety) R–SOH: sulfenic acid (sulfenyl moiety) R–SON: sulfinyl-amide moiety R–SSH: hydropersulfide moiety R–SS–R: disulfide group R–S(S)n S–R: polysulfide

List of Shortened Aliases of Anatomical and Histologic Terms

A ADMSC: adipose tissue-derived mesenchymal stem cell AgPC (APC): antigen-presenting cell AIC: agranular insular cortex AMIS: apical membrane initiation site (lumenogenesis) amivEC: adipose depot blood microvascular endotheliocyte ANS: autonomic nervous system AoV: aortic valve AP: area postrema ArcN: hypothalamic arcuate nucleus aSMC: airway smooth muscle cell AT: adipose tissue AVA: aortic valve area AVN: atrioventricular node AVV: atrioventricular valves

B B lymphocyte (B cell): bone marrow lymphocyte (i)BALT: (inducible) bronchus-associated lymphoid tissue BAT: brown adipose tissue BBB: blood–brain barrier BCSFB: blood–cerebrospinal fluid barrier (choroid plexus) BeAT: beige adipose tissue BECF: brain extracellular (interstitial) fluid BHB: blood–heart barrier BM: basement membrane

bmAT: bone marrow adipose tissue BMSC: bone marrow stromal cell BNST: bed nucleus of the stria terminalis BSCB: blood–spinal cord barrier

C CAC: circulating angiogenic cell CASC: cardiac atrial appendage stem cell CCD: cortical collecting duct (nephron) CD: collecting duct (nephron) CDC: cardiosphere-derived cell cDC: classical dendrocyte CeA: central amygdala cEC: circulating endotheliocyte cemivEC: cerebral microvascular endotheliocyte CEPC: circulating endothelial progenitor cell CFB: cardiofibroblast CFU: colony-forming unit CFUb: CFU-basophil (basophil-committed stem cells) CFUc: CFU in culture (granulocyte precursors, i.e., CFUgm) CFUe: CFU-erythroid CFUeo: CFU-eosinophil CFUg: CFU-granulocyte CFUgm: CFU-granulocyte–macrophage CFUgemm: CFU-granulocyte–erythroid– macrophage–megakaryocyte CFUm: CFU-macrophage CFUmeg: CFU-megakaryocyte CFUs: CFU-spleen (pluripotent stem cells)

© Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0

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848

List of Shortened Aliases of Anatomical and Histologic Terms

CGN: cis-Golgi network CLP: common lymphoid progenitor cmavEC: coronary macrovascular endotheliocyte CMC: cardiomyocyte cmivEC: coronary microvascular endotheliocyte CMLP: common myeloid–lymphoid progenitor CMP: common myeloid progenitor CNS: central nervous system CnT (CT): connecting tubule (distal nephron) CPC: cardiac progenitor cell (aCPC: adult; nCPC: neonatal) CRPC: cardiac resident progenitor cell CS (CoS): coronary sinus CSC: cancer stem cell CSF: cerebrospinal fluid CSF: colony-stimulating factor Csk: cytoskeleton CT: connecting tubule (distal nephron) cTAL: cortical thick ascending limb CVLM: caudal ventrolateral medulla CVS: cardiovascular system

D DC: dendrocyte DCT: distal convoluted tubule (nephron) DETC: dendritic epidermal γδ T cell DMH: dorsomedial hypothalamus dmivLEC: dermal lymphatic microvascular endotheliocyte DMNV (DMV): dorsal motor nucleus of the vagus DN1 (2, 3): double-negative-1 (2, 3) cell DpgcN (dPGi): dorsal paragigantocellular nucleus DRG: dorsal root ganglion DRN: dorsal raphe nucleus DVC: dorsal vagal complex

E eAT: epicardial adipose tissue EC: endotheliocyte ECA: external carotid artery ECANS: extrinsic cardiac autonomic nervous system ECDMV: endotheliocyte-derived microvesicle ECF: extracellular fluid ECFC: endothelial colony-forming cell ECFV: extracellular fluid volume ECM: extracellular matrix

EdMT: endothelial–mesenchymal transition EEL: external elastic lamina eEC: endocardial endotheliocyte ELP: early lymphoid progenitor EMT: epithelial–mesenchymal transition EMTU: epithelial–mesenchymal trophic unit ENS: enteric nervous system EPC: endothelial progenitor cell EpC: epitheliocyte EPDC: epicardial-derived cell epiAT: epididymal adipose tissue epvAT: epicardial perivascular adipose tissue ER: endoplasmic reticulum ER–GB: endoplasmic reticulum–Golgi body couple (intracellular reticular apparatus) ER–Mt: endoplasmic reticulum–mitochondria contact site ER–PM: endoplasmic reticulum–plasma membrane contact site ERES: endoplasmic reticulum exit site ergic: endoplasmic reticulum–Golgi intermediate compartment ERMD: endoplasmic reticulum-associated mitochondrial division ermes: endoplasmic reticulum–mitochondrion encounter structure ERQC: endoplasmic reticulum quality control ESC: embryonic stem cell ESL: endothelial surface layer ERS: ER stress ERSR: ERS response (UPR) ETP: early thymocyte progenitor EV: extracellular vesicle evBAT: epididymal visceral brown adipose tissue

F FB: fibroblast FC: fibrocyte

G GB: Golgi body GBM: glomerular basement membrane GCV: great cardiac vein GIC: granular insular cortex GMP: granulocyte–monocyte progenitor

H HEV: high endothelial venule HSC: hematopoietic stem cell

List of Shortened Aliases of Anatomical and Histologic Terms I ICA: internal carotid artery ICANS: intrinsic cardiac autonomic nervous system ICF: intracellular fluid ICC: (collecting duct) intercalated cell IEL: internal elastic lamina IELc (IEL): intraepithelial lymphocyte ifM: interfibrillar mitochondrion ILC: infralimbic cortex ILc (ILC): innate lymphocyte (lymphoid cell) IMCD: inner medullary collecting duct (nephron) IMJ: intermitochondrial junction IMM: inner mitochondrial membrane IMS (MIMS): intermembrane space of the mitochondrial envelope (between OMM and IMM) ingAT: inguinal adipose tissue INM: inner nuclear membrane IPOD: insoluble protein deposit iPSC: induced pluripotent stem cell IRES: internal ribosome entry site isBAT: interscapular brown adipose tissue iscBAT: inguinal subcutaneous brown adipose tissue ISC: intestinal stem cell ISEMF: intestinal subepithelial myofibroblast IVC: inferior vena cava

J JUNQ: juxtanuclear quality control compartment jSR: junctional sarcoplasmic reticulum

L LA: left atrium LBRC: lateral border recycling compartment LC: locus ceruleus LCA: left coronary artery LCC: left coronary cusp LD: lipid droplet LDSV: lung-derived small vesicle LDTN: laterodorsal tegmental nucleus LEC: lymphatic endotheliocyte LHA: lateral hypothalamic area LMP: lysosomal membrane permeabilization LMPP: lymphoid-primed multipotent progenitor lPA: lateral periaqueductal gray lPBN: lateral parabrachial nucleus

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lpDC: lamina propria dendrocyte LpgcN (lPGi): lateral paragigantocellular nucleus LPT: lateral pontine tegmentum LSV: long saphenous vein LTI: lymphoid tissue inducer cell LTO: lymphoid tissue organizer cell LV: left ventricle

M M/M/DCDMV: monocyte/macrophage/ dendritic cell-derived microvesicle MAERM: mitochondrion-associated endoplasmic reticulum membrane maiT: mucosal-associated invariant T lymphocyte maLT: mucosa-associated lymphoid tissue MBH: mediobasal hypothalamus MBP: myeloid–B-cell progenitor MCD: medullary collecting duct MCS: (organelle) membrane contact site mDC: myeloid dendrocyte MDTN: mediodorsal thalamic nucleus MECC: myoendothelial close contact MEJ: myoendothelial junction MEP: myeloid–erythroid progenitor MEPj (MEP): myoendothelial projection (from Latin projecto: to drive forth; pro-: before, in front of, forth; and jaceo: to be thrown) MIPS: misfolding-induced protein secretion MiV: mitral valve MKEP (MEP): megakaryocyte erythroid progenitor MlPON: medial preoptic nucleus MM: mitochondrial matrix MMDMV: monocyte/macrophage-derived microvesicle MnPON (MPON): median preoptic nucleus Mo: monocyte MOMP: mitochondrial outer membrane permeabilization MPOA: medial preoptic area (hypothalamus) MPP: multipotent progenitor MSC: mesenchymal stem cell MSRA: mesenteric small resistance artery mTAL: medullary thick ascending limb MTOC: microtubule organizing center MTP: myeloid–T-cell progenitor MVB: multivesicular body MVE: multivesicular endosome (MVB)

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List of Shortened Aliases of Anatomical and Histologic Terms

N NAcc: nucleus accumbens naLT: nasal-associated lymphoid tissue NAmb (NA): nucleus ambiguus NB: nuclear body NCC: noncoronary cusp NET: neutrophil extracellular trap NK: natural killer cell NKT: natural killer T cell NOR: nucleolar organizer region NRO: nucleus raphe obscurus NTS: nucleus tractus solitarius (nucleus of the solitary tract)

O OMCD: outer medullary collecting duct (nephron) OMM: outer mitochondrial membrane OMMP: OMM permeabilization ONM: outer nuclear membrane OVLT: organum vasculosum lamina terminalis

P paeAT: periatrial epicardial adipose tissue PAERM: plasma membrane-associated endoplasmic reticulum membrane PAG: periaqueductal gray matter paoAT: periaortic adipose tissue pAT: pericardial adipose tissue PB: mRNA processing body PBC: pre-Bötzinger complex (ventilation frequency) PBN: pontine parabrachial nucleus pceAT: pericoronary epicardial adipose tissue PC: podocyte; principal cell (nephron) PCT: proximal convoluted tubule (nephron) pDC: plasmacytoid dendrocyte PDSV: plasma-derived small vesicle PDMV: platelet-derived microvesicle PEO: proepicardial organ PLO: primary lymphoid organ PLV: (coronary) posterolateral vein PM: plasma membrane pnM: perinuclear mitochondrion PNS: peripheral nervous system PPT: pedunculopontine nucleus PPTN: pedunculopontine tegmental nucleus PQC: protein quality control preBotC: pre-Bötzinger complex pre-cDC: preclassical dendritic cell PSC: pluripotent stem cell PsD: postsynaptic density

PST: proximal straight tubule (nephron) PT: proximal tubule (nephron) PTP (Mt PTP): mitochondrial permeability transition pore PuV: pulmonary valve pvAT: perivascular adipose tissue pveAT: periventricular epicardial adipose tissue PVN: hypothalamic paraventricular nucleus

R RA: right atrium RBC: red blood capsule (cell, or erythrocyte [without nucleus]) RCA: right coronary artery RCC: right coronary cusp rMR: rostral medullary raphe RTN: retrotrapezoid nucleus RV: right ventricle RVLM: rostral ventrolateral medulla RVMM: rostral ventromedial medulla

S SAN: sinoatrial node scAT: subcutaneous adipose tissue SCN: hypothalamic suprachiasmatic nucleus SECV: small extracellular vesicle sER: smooth endoplasmic reticulum SFO: subfornical organ SG: stress granule SLDN: sublaterodorsal nucleus SLO: secondary lymphoid organ SON: hypothalamic supraoptic nucleus SPVZ: hypothalamic subparaventricular zone SR: sarcoplasmic reticulum SRA: small resistance artery ssM: subsarcolemmal mitochondrion SSV: short saphenous vein SVC: superior vena cava

T T lymphocyte (T cell): thymic lymphocyte TC : cytotoxic T lymphocyte (CD8+ effector T cell; CTL) TC1 : type-1 cytotoxic T lymphocyte TC2 : type-2 cytotoxic T lymphocyte TCM : central memory T lymphocyte TConv : conventional T lymphocyte TEff : effector T lymphocyte TEM : effector memory T lymphocyte TFH : follicular helper T lymphocyte

List of Shortened Aliases of Anatomical and Histologic Terms TH : helper T lymphocyte (CD4+ effector T cell) THi : type-i helper T lymphocyte (i = 1/2/9/17/22) TH3 : TGFβ-secreting TReg lymphocyte TL : lung transfer capacity (alveolocapillary membrane) TR1 : type-1, IL10-secreting, regulatory T lymphocyte TReg : regulatory T lymphocyte aTReg : CD45RA−, FoxP3hi , activated TReg cell iTReg : inducible TReg lymphocyte nTReg : naturally occurring (natural) TReg lymphocyte rTReg : CD45RA+, FoxP3low , resting TReg cell TAL: thick ascending limb of the loop of Henle (nephron) tAL: thin ascending limb of the loop of Henle (nephron) TATN: transverse and axial tubular network TC: thrombocyte (platelet) tDL: thin descending limb of the loop of Henle (nephron) TEM: transendothelial migration TJ: tight junction TLO: tertiary lymphoid organ TMN: hypothalamic tuberomammillary nucleus TrV: tricuspid valve U USC: unipotential stem cell V vAT: visceral adipose tissue VAV: ventriculoarterial valve vHpc: ventral hippocampus vlPAG: ventrolateral periaqueductal gray VLPO: ventrolateral preoptic area VLPON: ventrolateral preoptic nucleus VMH: ventromedial hypothalamus VOM: vein of Marshall VP: ventral pallidum VPMpcTN: ventral posteromedial parvicellular thalamic nucleus

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VRC: ventral respiratory column vSMC: vascular smooth myocyte VTA: ventral tegmental area VVO: vesiculovesicular organelle

W WAT: white adipose tissue W/BAT: mixed WAT–BAT W/BeAT: mixed WAT–BeAT WBC: white blood cell

Z ZO: zonula occludens

Dual Notations aaMφ: alternatively activated macrophage ATMφ: adipose tissue macrophage Bφ: basophil caMφ: classically activated macrophage Eφ: eosinophil Lφ: lymphocyte Mφ: macrophage Nφ: neutrophil SNAMφ: sympathetic nerve-associated macrophage c: sympathetic pc: parasympathetic

Cranial Nerves I : olfactory nerve (sensory) I I : optic nerve (sensory) I I I : oculomotor nerve (mainly motor) I V : trochlear nerve (mainly motor) V : trigeminal nerve (sensory and motor) V I : abducens nerve (mainly motor) V I I : facial nerve (sensory and motor) V I I I : vestibulocochlear (auditory-vestibular) nerve (mainly sensory) I X: glossopharyngeal nerve (sensory and motor) X: vagus nerve (sensory and motor) XI : cranial accessory nerve (mainly motor) XI I : hypoglossal nerve (mainly motor)

List of Shortened Aliases of Physiological and Medical Terms

A α1ATD: α1-antitrypsin deficiency (hereditary lung emphysema) AAA: abdominal aortic aneurysm AAN: autoimmune autonomic neuropathy AAS: acute aortic syndrome ACaVCeVE: adverse cardio- and cerebrovascular events ACoS (ACS): acute coronary syndrome (collective term) AD: Alzheimer’s disease AD: autosomal dominant disorder ADHF: acute decompensated heart failure ADHSP: autosomal dominant hereditary spastic paraplegia AF: atrial fibrillation AHD: atherosclerotic heart disease AwHR (AHR): airway hyperresponsiveness ALI: acute lung injury ALS: amyotrophic lateral sclerosis AOA: cerebellar ataxia and oculomotor apraxia APAH: acquired pulmonary arterial hypertension AR: autosomal recessive disorder ARDS: acute respiratory distress syndrome ARSACS: autosomal recessive spastic ataxia of Charlevoix–Saguenay ARVC(D): arrythmogenic right ventricular cardiomyopathy (dystrophy or dysplasia) AS: Angelman syndrome ASCVD: atherosclerotic cardiovascular disease (atherosclerosis)

ASO: arteriosclerosis obliterans AT: ataxia telangiectasia ATAA: ascending thoracic aortic aneurysm AVB: atrioventricular node block AVM: arteriovenous malformation AVMLM: combined arteriovenous and lymphatic malformation AVR: aortic valve regurgitation AVS: aortic valve stenosis

B BAS: biodegradable (bioresorbable) arterial scaffold (stent) BAV: bicuspid aortic valve BBS: Bardet–Biedl syndrome BD: Behçet disease BES: biolimus-eluting stent BHR: bronchial hyperresponsiveness BlmS: Bloom syndrome BMS: bare-metal stent BRBN: blue rubber bleb nevus BRS: bioresorbable scaffold (stent) BthS: Barth syndrome BVS: biodegradable (bioresorbable) vascular scaffold (stent)

C CAA: familial cerebral amyloid angiopathy CABG: coronary artery bypass grafting CACTD: carnitine–acylcarnitine translocase deficiency

© Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0

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854

List of Shortened Aliases of Physiological and Medical Terms

cadasil: cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CAN: cardiovascular autonomic neuropathy CapM: capillary malformation CAPS: cryopyrin (NLRP3)-associated periodic syndrome carasil: cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy CAVD: calcific aortic valve disease CaVD: calcific valve disease CCHS: congenital central hypoventilation syndrome CCM: congenital cardiac malformation CCVM (CVM): combined capillary and venous malformation CDG: congenital disorders of glycosylation CDSP: primary systemic carnitine deficiency CeAD: cerebral artery disease CeAVM: cerebral arteriovenous malformation CeCM (CCM): cerebral cavernous malformation CED: cranioectodermal dysplasia CeVD: cerebrovascular disease (collective term) CGLD: congenital generalized lipodystrophy CHD: congenital heart defect CHIPH: chronic hypoxia-induced pulmonary hypertension CIHD: chronic ischemic heart disease CKD: chronic kidney disease CLM: combined capillary and lymphatic malformation CLVM: combined capillary, lymphatic, and venous malformation CMAVM: combined capillary and arteriovenous malformation syndrome CMVD: coronary microvascular dysfunction CMVM: cutaneomucosal venous malformation CoAD: coronary artery disease COFS: cerebro–oculo–facio–skeletal syndrome CoHD: coronary heart disease COLD: chronic obstructive lung disease COPD: chronic obstructive pulmonary disease CORS: cerebello–oculo–renal syndrome CPT1AD: carnitine palmitoyl transferase-1A deficiency CPT2D: carnitine palmitoyl transferase-2 deficiency CRHD: chronic rheumatic heart disease CRS: cardiorenal syndrome CRT: cardiac resynchronization therapy

CS: Cockayne syndrome CSA: central sleep apnea CSD: cortical spreading depression CSVD: cerebral small vessel disease CTEPH: chronic thromboembolic pulmonary hypertension Ctlni: type-i citrullinemia CTTH: capillary transit time heterogeneity CUA: calcific uremic arteriolopathy (calciphylaxis) CUD: carnitine uptake defect CVD: cardiovascular disease (collective term) CVI: chronic venous insufficiency CVM: congenital vascular malformation

D DAD: delayed afterdepolarization DCA: directional coronary atherectomy DCM: dilated cardiomyopathy DCMA: DCM with ataxia (DNAJC19 [Hsp40] mutations) DES: drug-eluting stent DGS: DiGeorge syndrome DHF: dyssynchronous heart failure DIDMOAD: diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (Wolfram syndrome) DIIR: diet-induced insulin resistance DILR: diet-induced leptin resistance DIO: diet-induced obesity DM: dermatomyositis DMD: Duchenne muscular dystrophy DPLD: diffuse parenchymal lung disease DVT: deep vein thrombosis

E EAD: early afterdepolarization ECC: excitation–contraction coupling EDS: Ehlers–Danlos syndrome EGPA: eosinophilic granulomatosis with polyangiitis EHT: essential hypertension EIEEi: type-i early infantile epileptic encephalopathy ELCA: excimer laser coronary angioplasty EROA: effective regurgitant orifice area ESC: excitation–secretion coupling ESHF: end-stage heart failure ESRD: end-stage renal disease ETC: excitation–transcription coupling EVAR: endovascular aneurysm repair

List of Shortened Aliases of Physiological and Medical Terms F FA: Fanconi anemia FAOD: fatty acid oxidation disorder FHCM: familial hypertrophic cardiomyopathy FHCS: familial hypercholesterolemia FHHCN: familial hypomagnesemia with hypercalciuria and nephrocalcinosis FPAH: familial pulmonary arterial hypertension FPLD: familial partial lipodystrophy FTLD: frontotemporal lobar degeneration

G GCA: giant cell arteritis GOF: gain-of-function mutation GPA: granulomatosis with polyangiitis GRS: genetic risk score GVM: glomuvenous malformation

H HACE: high-altitude cerebral edema HAPE: high-altitude pulmonary edema HCD: hemangiomatosis chondrodystrophica HCM: hypertrophic cardiomyopathy HD: Huntington’s disease HF: heart failure HFlEF: heart failure with low LVEF HFpEF: heart failure with persistently preserved LVEF HFpLVEF: heart failure with preserved left ventricular ejection fraction HFrEF: heart failure with recovered LVEF HFrLVEF: heart failure with reduced left ventricular ejection fraction HGNET: high-grade neuroendocrine tumor HGPS: Hutchinson–Gilford progeria syndrome HHCy: hyperhomocysteinemia HHT: hereditary hemorrhagic telangiectasia HLI: hind limb ischemia HLTS: hypotrichosis–lymphedema– telangiectasia syndrome HOCM: hypertrophic obstructive cardiomyopathy HPAH: heritable pulmonary arterial hypertension HPD (HTVPD): high transvalvular pressure difference HSP: hereditary spastic paraplegia HTHD: hypertensive heart disease HUV: hypocomplementemic urticarial (antiC1q) vasculitis

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I IBDD: IBDH deficiency IgAV: immunoglobulin-A vasculitis ICM: ischemic cardiomyopathy ICSA: intracranial saccular aneurysm IH: intimal hyperplasia IHD: ischemic heart disease ILD: interstitial lung disease IMH: intramural hematoma InsRce: insulin resistance IPAH: idiopathic pulmonary arterial hypertension IPC: ischemic preconditioning IRI: ischemia–reperfusion injury ISGU: insulin-stimulated glucose uptake ISR: in-stent restenosis

J JbtS: Joubert syndrome JHHT: juvenile polyposis–HHT syndrome

K KTWS: Klippel–Trenaunay–Weber syndrome

L LBBB: left bundle branch block LCHADD: lcHADH deficiency LCS: lymphedema–cholestasis syndrome LD: lipodystrophy LDS: lymphedema–distichiasis syndrome LGNET: low-grade neuroendocrine tumor LM: lymphatic malformation LMR: laser myocardial revascularization LPD (LTVPD): low transvalvular pressure difference LQTS: long-QT syndrome LRI: lower respiratory infection LTFR: low transvalvular flow rate LVAD: left ventricular assist device LVH: left ventricular hypertrophy LVM: combined lymphatic and venous malformation LVNC (LVNCCM): left ventricular noncompaction cardiomyopathy

M M/SCHADD: medium- and short-chain L 3-hydroxyacylCoA dehydrogenase deficiency

856

List of Shortened Aliases of Physiological and Medical Terms

MACE: major adverse cardiovascular event MADD: multiple acylCoA dehydrogenase deficiency MCADD: mcADH deficiency MCTD: (systemic autoimmune) mixed connective tissue disease MD: Menkes disease melas: mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes MI: myocardial infarction midas: mitochondrial dysfunction-associated senescence MIS: mini-invasive surgery MIT: mini-invasive therapy MkS: Meckel syndrome MKKS: McKusick–Kaufman syndrome MPA: microscopic polyangiitis MSA: multiple system atrophy mvih: microvascular invasion in hepatocellular carcinoma (lncRNA) MVO: microvascular obstruction MVR: mitral valve regurgitation MVS: mitral valve stenosis

N NAFLD: nonalcoholic fatty liver disease NASH: nonalcoholic steatohepatitis NET: neuroendocrine tumor NICCD: neonatal intrahepatic cholestasis caused by citrin deficiency NIP: neointimal proliferation Nphp: nephronophthisis NMH: neurally mediated hypotension NPD: Niemann–Pick disease NSCLC: nonsmall-cell lung cancer nsTAAD: nonsyndromic familial thoracic aortic aneurysms and dissection NSTEMI: nonST-segment elevation myocardial infarction

O OCRL: oculocerebrorenal syndrome of Lowe OFDS: oral–facial–digital syndrome OHS: obesity hypoventilation syndrome OHT: orthostatic hypotension OI: osteogenesis imperfecta OLEDAID: osteoporosis, lymphedema, and anhydrotic ectodermal dysplasia with immunodeficiency ONARE: obstructive nonapneic respiratory event

ORG: obesity-related glomerulopathy OSA: obstructive sleep apnea OSHAS: obstructive sleep hypopnea–apnea syndrome

P PAA: pulmonary arterial aneurysm PAD: peripheral artery disease PAF: pure autonomic failure PAHT: pulmonary arterial hypertension PAU: penetrating atherosclerotic ulcer PAVM: peripheral arteriovenous malformation PCD: primary ciliary dyskinesia PCH: pulmonary capillary hemangiomatosis PCI: percutaneous coronary intervention PCL: primary congenital lymphedema PD: Parkinson’s disease PD: pharmacodynamics PE: pulmonary embolism PEEP: positive end-expiratory pressure mechanical ventilation PGD: peroxisomal genesis disorder PH (PHT): pulmonary hypertension PHTS: PTEN hamartoma tumor syndrome PIH: pregnancy-induced hypertension (preeclampsia) PK: pharmacokinetics PKD1(2): autosomal dominant polycystic kidney type-1(2) disease (mutations in genes encoding polycystin-1 [TRPP1] and -2 [TRPP2]) PKHD: autosomal recessive polycystic kidney and hepatic disease (mutations in genes encoding fibrocystin) PKU: phenylketonuria PM: polymyositis PML: promyelocytic leukemia PMR: percutaneous (laser) myocardial revascularization POTS: postural orthostatic tachycardia syndrome PP2CM (PPCM): peripartum and postpartum cardiomyopathy PPCM: progressive patchy capillary malformation (angioma serpiginosum) PPHN: persistent pulmonary hypertension of the newborn PTCA: percutaneous transluminal coronary angioplasty PTCRA: PTC rotational burr atherectomy PTES: paclitaxel-eluting stent PTS: postthrombotic syndrome

List of Shortened Aliases of Physiological and Medical Terms PVOD: pulmonary venoocclusive disease PVR: pulmonary valve regurgitation PVS: pulmonary valve stenosis PWS: Prader–Willi syndrome PXE: pseudoxanthoma elasticum

R RA: rheumatoid arthritis RCM: restrictive cardiomyopathy RERA: respiratory effort-related arousal RF: regurgitant fraction RFA: radiofrequency ablation RFl: regurgitant flow RHD: rheumatic heart disease RIPC: limb remote ischemic preconditioning RSA: respiratory sinus arrhythmia RSMCS: robot-supported medical and surgical system RTI: respiratory tract infection RVCL: retinal vasculopathy and cerebral leukodystrophy RVHT: renovascular hypertension

S SAH: subarachnoid hemorrhage SARD: systemic autoimmune rheumatic disease (a group of disorders) SASP: senescence-associated secretory phenotype SAST: stented arterial segment thrombosis SBMA: spinobulbar muscular atrophy SCA: spinocerebellar ataxia (atrophy) SCADD: scADH deficiency SCLC: small-cell lung cancer scLC: squamous-cell lung cancer (NSCLC subtype) SES: sirolimus-eluting stent SjS: Sjögren’s syndrome SLE: systemic lupus erythematosus SLS: Senior–Løken syndrome SMARD: spinal muscular atrophy with respiratory distress SND: sinusal node dysfunction SNP: single-nucleotide polymorphism SQTS: short-QT syndrome SRTD: short-rib thoracic dystrophy SSc: systemic sclerosis (scleroderma) SSEHT: salt-sensitive essential hypertension SSS: sick sinus syndrome

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STEMI: ST-segment elevation myocardial infarction SVAS: supravalvar aortic stenosis

T T1DM: type-1 diabetes mellitus (severe autoimmune diabetes) T2DM: type-2 diabetes mellitus (severe insulin-deficient diabetes) T3DM: severe insulin-resistant diabetes T4DM: mild obesity-related diabetes T5DM: mild age-related diabetes TA: Takayasu arteritis TAA: thoracic aortic aneurysm TAAD: familial thoracic aortic aneurysm and dissection TACE: transarterial chemoembolization TCFA: thin-cap fibroatheroma TTS: takotsubo syndrome TVR: tricuspid valve regurgitation TVS: tricuspid valve stenosis

U UARE: upper airway resistance episode URI: upper respiratory infection

V VCF: velocardiofacial syndrome VF: ventricular fibrillation VHD: valvular heart disease VLCADD: vlcADH deficiency VM: venous malformation VTE: venous thromboembolism (collective term)

W WAS: Wiskott–Aldrich syndrome WBS: Williams–Beuren syndrome WD: Wilson disease WfS: Wolfram syndrome WHS: Wolf–Hirschhorn syndrome WPWS: Wolff–Parkinson–White syndrome WS: Williams syndrome WT: wild type

X XFE: XPF–ERCC4 mutation-resulting progeroid syndrome XP: xeroderma pigmentosum

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List of Shortened Aliases of Physiological and Medical Terms

Hemodynamics—Physical and Physiological Parameters AC: atrial contraction APl: action potential ARP: absolute refractory period AP: arterial pressure BAFMD: brachial artery flow-mediated dilation BP: blood pressure C: chronotropy CeBF: cerebral blood flow CFR: coronary flow reserve CFV: cardiac frequency variability CMRGlc : cerebral metabolic rate of glucose consumption CMRO2 : cerebral metabolic rate of oxygen consumption CO: cardiac output CoBF: coronary blood flow CVMR: cerebral vasomotor reactivity CVP: central venous pressure D: dromotropy DBP: diastolic blood pressure DPTI: diastolic pressure time interval EDV: end-diastolic volume EFCV: extracellular fluid volume ERP: effective refractory period ESV: end-systolic volume fC : cardiac frequency FFR: fractional flow reserve FR: flow ratio GFR: glomerular filtration rate HABR: hepatic arterial buffer response HPV: hypoxic pulmonary vasoconstriction HRV: heart rate variability I: inotropy IC: isovolumetric contraction IR: isovolumetric relaxation mAP: mean arterial pressure MBF: myocardial blood flow MVO2 : myocardial oxygen consumption NTFR: normal transvalvular flow rate NVC: neurovascular coupling PVR: pulmonary vascular resistance rAP: renal arterial pressure RIHP: renal interstitial hydrostatic pressure RVF: rapid ventricular filling RSE: rapid systolic ejection SBP: systolic blood pressure SE: systolic ejection SPN: supernormal period SPTI: systolic pressure time interval SSE: slow systolic ejection SV: stroke volume

SVR: systemic vascular resistance SW: stroke work TCG: transcoronary concentration gradient TR: time to wave reflection Vc : pulmonary capillary blood volume in alveolar walls VCt: vasoconstriction VDt: vasodilation VF: ventricular filling VR: venous return

Clinical Indices ABI: ankle–brachial index AIx: augmentation index AVAI: aortic valve area index (AVA/BSA [dimensionless]) BMI: body mass index BSA: body surface area CI: cardiac index DI: desaturation index DT: deceleration time HI: hemolysis index IMR: index of microvascular resistance MHR: monocyte-to-high–density lipoprotein ratio NHI: normalized hemolysis index OEF: oxygen extraction fraction OSI: oscillatory shear index PHT: pressure half-time PISA: proximal isovelocity surface area PTT: pulse transit time PWS: pulse wave speed QTI: QT index (QT/QTp × 100; QTp = 656/(1 + fC /100) RDI: respiratory disturbance index RHI: reactive hyperemia index RI: arterial resistivity (resistance) index SEVR: subendocardial viability ratio SV R: surface area-to-volume ratio UAREI: upper airway resistance episode index

Other Physiological Quantities and Entities APIN: acute pressure-induced natriuresis batSNA: brown adipose tissue sympathetic nerve activity BW: body weight DIT: diet-induced thermogenesis NREMS: nonrapid eye movement sleep REM: rapid eye movement REMS: REM sleep RMR: resting metabolic rate rSNA: renal sympathetic nerve activity

List of Shortened Aliases of Physiological and Medical Terms SNA: sympathetic nerve activity sSNA: splanchnic sympathetic nerve activity SSR: sympathetic skin response TGF: tubuloglomerular feedback TMP: transmembrane potential (u(t)) TRT: total recording time TST: total sleep time

Sleep and Arousal Indices AAI: autonomic arousal index AHI: apnea–hypopnea index BAI: behavioral arousal index BRAI: breathing-related arousal index CAI: central apnea index CHI: central hypopnea index MAI: microarousal index OAI: obstructive apnea index OHI: obstructive hypopnea index PLMAI: periodic leg movement arousal index RAI: respiratory arousal index SAI: spontaneous arousal index SPS: sleep pressure score TAI: total arousal index VAI: vegetative arousal index

Signaling Axes and Associated Machineries HPAA: hypothalamic–pituitary–adrenal axis KKA: kallikrein–kinin axis RAA: renin–angiotensin axis RAAA: renin–angiotensin–aldosterone axis UPA: ubiquitin–proteasome axis

DNA and RNA Sites APA: alternative polyadenylation site BE: boundary element CFS: common fragile site DSE: DNA double-strand end ERFS: early-replicating fragile site LE: DNA loop extrusion PAS: proximal polyadenylation site RF: replication fork RFS: rare fragile site SS: splice site TAD: topologically associated domain TFBS: transcription factor-binding site TFPD: trifunctional protein deficiency TSS: transcription start site

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DNA Damage Repair BER: base excision repair DDR: DNA damage response DDT: DNA damage tolerance DSB: DNA double-strand break HR: homologous recombination (DNA DSB repair) MMR: (DNA) mismatch repair NER: nucleotide excision repair NHEJ: nonhomologous end-joining (DNA DSB repair) PRR: postreplication restoration SSA: single-strand annealing SSB: DNA single-strand break SSBR: SSB repair TLS: DNA translesion synthesis

Gene Mutations GOF: gain-of-function mutation LOF: loss-of-function mutation

Cellular Stresses ERS: endoplasmic reticulum stress ERSR: ERS response GBS: Golgi body stress GBSR: GBS response ISR: integrated stress response MSR: metabolic stress response PSR: proteotoxic stress response RSR: replication stress response SDPR: serum deprivation protein response UPR: unfolded protein response

Autophagy and Cell Death ACD: accidental cell death ALA: autophagy–lysosome axis CMA: chaperone-mediated autophagy IMD: integrin-mediated death PCD: programmed cell death RCD: regulated cell death

Quality Control, Proteolysis, and mRNA Decay ERAD: endoplasmic reticulum-associated protein degradation ERQC: endoplasmic reticulum quality control

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List of Shortened Aliases of Physiological and Medical Terms

NGD: NoGo mRNA decay NMD: nonsense-mediated mRNA decay NSD: nonstop mRNA decay PQC: protein quality control QA: quality assurance QC: quality control RIDD: regulated IRE1-dependent degradation (also REDD: regulated ERN1dependent decay) RIP (RIMP): regulated intramembrane proteolysis SMD: Staufen-1-mediated mRNA decay

Other Cellular Events and Processes CICR: calcium-induced calcium release DICR: depolarization-induced Ca2+ release FAO: fatty acid oxidation GluO: glucose oxidation NICR: NO-induced Ca2+ release RCT: reverse cholesterol transport REDD: regulated ERN1-dependent decay (mRNA degradation) RIRR: ROS-induced ROS release RQC: ribosome-associated quality control SOCE: store-operated Ca2+ entry STIC: spontaneous transient inward current STOC: spontaneous transient outward current TICE: transintestinal cholesterol excretion (enterocyte cholesterol efflux)

Medical Imaging CA: computed angiography CCA: coronary computed angiography CT: computed tomography CVUS: compression venous ultrasonography DICOM: digital imaging and communication for medicine DUS: Doppler ultrasound EBCT: electron beam CT HCT: helical CT IVUS: intravascular ultrasonography MPSPECT: myocardial perfusion singlephoton emission CT MRI: (nuclear) magnetic resonance imaging MSSCT: multislice spiral CT OCT: optical coherence tomography PCMRV: phase-contrast MR velocimetry PET: positron emission tomography ROI: region of interest SPECT: single-photon emission computed tomography STE: speckle-tracking echocardiography

TDI: tissue Doppler imaging USI: ultrasound imaging

Complementary Examination ECG: electrocardiogram GWAS: genome-wide association study Ht: hematocrit S: hemoglobin saturation of a given gas species (%; e.g., SaO2 ]S) IRE: irreversible electroporation NLR: neutrophil-to-lymphocyte ratio PAT: pulse amplitude tonometry PPG: photoplethysmography TFM: traction force microscopy

Breathing Parameters and Lung Function Testing DV: dead space volume ERV: expiratory reserve volume fR : breathing frequency FEFf : forced expiratory flow at a fraction (f [%]) of forced expiration (FEF25 , FEF50 , and FEF75 ) FEV1 : volume expired at the end of the first second of forced expiration FEVτ : forced expiratory volume at time τ (fraction of a second over which volume exhaled as fast as possible starting from full inspiration is measured) FRC: functional residual capacity (lung volume at the end of rest expiration) IC: inspiratory capacity (IRV + VT ) IRV: inspiratory reserve volume MBC: maximum breathing capacity (per mn of effort) MVV: maximal voluntary ventilation (volume of air breathed in a specified period during repetitive maximal exercise) PEF: peak expiratory flow RR : respiratory quotient (V˙CO2 /V˙O2 ) RV: residual volume TLC: total lung capacity VA : alveolar gas volume VD : dead space volume VL : lung volume VT : tidal volume V˙ : total ventilation (air volume exhaled par mn) V˙A : alveolar ventilation (fR (VT − VD )) V˙O2 : oxygen consumption V˙CO2 : carbon dioxide production

List of Shortened Aliases of Physiological and Medical Terms VC: vital capacity (air volume quietly expelled from full inspiration) Diet HCD: high-carbohydrate(–content) diet HCHFD: high-carbohydrate, high-fat diet HFD: high-fat diet HFHSD: high-fat high-sucrose diet HPD: high-protein diet HSD: high-salt diet

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HSHFD: high-sucrose, high-fat diet LFD: low-fat diet SD: standard diet

Pollution PM: particulate matter (particulates or particle pollution) POP: persistent organic pollutant VOC: volatile organic compound

List of Chemical, Mathematical, and Physical Symbols

A A: Avogadro number A(p): area–pressure relation

A: Almansi strain tensor A: cross-sectional area A: surface area-to-volume ratio a: acceleration a: activation rate (or term) a: major semiaxis ALE: arbitrary Eulerian Lagrangian AR: area ratio AW: analysis window

B B: Biot–Finger strain tensor B: bulk modulus B: bilinear form B: binding rate b: minor semiaxis b: body force  b: unit binormal b: birth rate BC: boundary condition Be: Bejan number BEM: boundary element method Bo: Boltzmann constant (1.38×10−23 J/K) Br: Brinkman number

C C: stress tensor C : consumption rate C: compliance, capacitance

C: heat capacity C: content CD : drag coefficient Cf : friction coefficient CL : lift coefficient Cp : pressure coefficient CB : Biot stress tensor CC : Cauchy stress tensor CK : Kirchhoff stress tensor CN : nominal stress tensor CPK1 : first Piola–Kirchhoff stress tensor CPK2 : second Piola–Kirchhoff stress tensor CVM : van Mises stress c: stress vector cτ : shear stress cw : wall shear stress cS : concentration of species S c(p): wave speed cp : isobar heat capacity cv : isochor heat capacity CFD: computational fluid dynamics

D D: vessel distensibility D : diffusion coefficient DT : thermal diffusivity D: deformation rate tensor d: displacement vector D: flexural rigidity D: differentiation rate D: demobilization function (from proliferation to quiescence) DO2 : oxygen delivery

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864 DRBC : damage rate Df : fractal dimension d: death, decay, degradation rate d: distance d: duration dh : hydraulic diameter Da: Damköhler number De: Dean number Deb: Deborah number

E E: strain tensor E: electric field E: elastic modulus E: elastance E : energy E: efflux rate (or term) e: strain vector e: specific free energy { ei }3i=1 : basis Ec: Eckert number

F F : Faraday constant F: transformation gradient tensor F : fraction of gas F : function fraction of proliferating cells F: erythrocytic rouleau fragmentation rate f: surface force  f: fiber direction unit vector f : binding frequency f : friction shape factor fv : head loss per unit length fS : molar fraction of gas component S FDM: finite difference method FEM: finite element method FSI: fluid–structure interaction FVM: finite volume method

G G: Green–Lagrange strain tensor G: shear modulus G : storage modulus G

: loss modulus G : Gibbs function G: conductance Gp : pressure gradient Gb : perfusion conductivity Ge : electrical conductivity Gh : hydraulic conductivity GT : thermal conductivity

List of Chemical, Mathematical, and Physical Symbols g: gravity acceleration g: physical quantity g: gravity g: detachment frequency g: free enthalpy GFP: geodesic front propagation Gr: Graetz number H H : height H: history function H: dissipation H: Henry parameter (solubility) h: head loss h: thickness h: specific enthalpy hm : mass transfer coefficient hT : heat transfer coefficient I I: identity tensor I: influx rate (or term) i: current IVP: initial value problem J J : flux Jmb : cell surface current density K K: conductivity tensor K: bending stiffness K: reflection coefficient Kd : dissociation constant (index of ligand– target affinity: ([L][T])/[C]; [L], [T], [C]: molar concentrations of the ligand, target, and created complex, respectively) KM : Michaelis constant (chemical reaction kinetics) Km : material compressibility KR : resistance coefficient k: cross-section ellipticity kATP : myosin ATPase rate kB : Boltzmann constant (1.38×10−23 J/K) kc : spring stiffness ki : kinetic coefficient km : mass transfer coefficient kP : Planck constant Kn: Knudsen number Kr: Krogh’s diffusion coefficient

List of Chemical, Mathematical, and Physical Symbols L L: velocity gradient tensor L: inertance L: length Le : entry length LDV: laser Doppler velocimetry LHS: equation left-hand side

M M: molar mass M: metabolic rate M: moment m: mass Ma: Mach number MWSS: maximal wall shear stress

N N : sarcomere number  n: unit normal vector n: mole number n: actomyosin filament density with elongation x n: myosin head density Nu: Nusselt number

O ODE: ordinary differential equation

P P : permeability

P: power P: cell division (proliferation) rate P (X): probability of event X p: production rate p: pressure pS : partial pressure of gas component S PDE: partial differential equation Pe: Péclet number PIV: particle image velocimetry Pl: Planck constant (6.62606957 × 10−34 J · s) Pr: Prandtl number PSEF: pseudo-strain energy function

Q Q: material quantity Qe : electric current density QT : thermal energy (heat) q: flow rate

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qT : transfer rate of thermal energy (power) qmet : metabolic heat source

R R: rotation tensor R: resistance R: local reaction term Rh : hydraulic radius Rg : gas constant RR : respiratory quotient R: recruitment function (from quiescence to proliferation) r: position vector r: cell renewal rate r: electrical resistivity r: radial coordinate Re: Reynolds number RHS: equation right-hand side

S S: pure deformation (strain) tensor SCGr : right Cauchy–Green strain tensor (or Green deformation tensor) SCGl : left Cauchy–Green strain tensor (dilation) SEA : Euler–Almansi deformation tensor SGL : Green–Lagrange deformation (strain) tensor Sr : right stretch tensor Sl : left stretch tensor s: arclength s: entropy s: sarcomere length s: sieving coefficient s⊥ : normal strain s : shear strain s: evolution speed s: solubility Sc: Schmidt number SD: standard deviation SEF: strain-energy function SEM: standard error of the mean Sh: Sherwood number SMD: standardized mean difference St: Strouhal number Sto: Stokes number

T T: extrastress tensor T: transition rate from a cell cycle phase to the next

866

List of Chemical, Mathematical, and Physical Symbols

T : temperature T : transport parameter TL : transfer capacity of the alveolocapillary membrane for gas species T: molecular turnover Ts : surface tension  t: unit tangent vector t: time U U: displacement gradient tensor U : (oxygen) uptake u: displacement vector u: electrochemical command, electrical potential u: specific internal energy US: ultrasound V V: left stretch tensor V : volume V : porosity (void fraction) Vq : cross-sectional average velocity Vs : specific volume v: fluid velocity vector v: recovery variable vX : volume of gas component X W W: vorticity tensor W : strain energy density W : work, deformation energy w: weight w: computational grid velocity WMD: weighted mean difference WSS: wall shear stress WSSTG: WSS transverse gradient X X : trajectory

X: reactance X: Lagrangian position vector x: position vector {x, y, z}: Cartesian coordinates Y Y: admittance coefficient Z Z: impedance

Miscellaneous 1D: one-dimensional 2D: two-dimensional 3D: three-dimensional 3DR: three-dimensional reconstruction Greek Symbols α: volumic fraction α: convergence/divergence angle α: attenuation coefficient αk : kinetic energy coefficient αm : momentum coefficient β: inclination angle βg : gas g solubility {βi }21 : myocyte parameters βT : coefficient of thermal expansion : domain boundary L : local reflection coefficient G : global reflection coefficient γ : (specific) heat capacity ratio (adiabatic index) γ : activation factor γG : amplitude ratio (modulation rate) of G γ˙ : shear rate •: difference, drop δ: boundary layer thickness T : emissivity (thermal energy radiation) e : electric permittivity : strain ε: dimensionless small quantity ζ : singular head loss coefficient ζ : transmural coordinate {ζj }31 : local coordinate η: azimuthal spheroidal coordinate θ: circumferential polar coordinate θ: (ˆex , ˆt) angle κ: wall curvature κc : curvature ratio κd : drag reflection coefficient κf : frictional sieving coefficient κh : hindrance coefficient κo : osmotic reflection coefficient κr : reflection coefficient κs : size ratio {κk }9k=1 : tube law coefficients κe : correction factor Λ: head loss coefficient λL : Lamé coefficient λ: stretch ratio λ: wavelength λA : area ratio λa : acceleration ratio λL : length ratio

List of Chemical, Mathematical, and Physical Symbols λLd : length-to-diameter ratio λp : molecule radius-to-pore radius ratio λq : flow rate ratio λt : time ratio λv : velocity ratio μ: dynamic viscosity μ0 : near-zero–shear rate viscosity μ∞ : high-shear rate viscosity μL : Lamé coefficient ν: kinematic viscosity νP : Poisson ratio : osmotic pressure ρ: mass density τ: time constant τ : space curve torsion : potential φ(t): creep function ϕ: phase χ : Lagrangian label χi : molar fraction of species i χi : wetted perimeter ψ(t): relaxation function #: porosity Ω: computational domain ω: angular frequency

Mathematical Notations T: boldface capital letter means tensor v: boldface minuscule letter means vector S, s: upper- or lowercase, lightface (italic typeface) letter means scalar Δ•: difference δ•: increment d • /dt: time gradient ∂t : first-order time partial derivative ∂tt : second-order time partial derivative ∂i : first-order space partial derivative with respect to spatial coordinate xi ∇: gradient operator ∇u: displacement gradient tensor ∇v: velocity gradient tensor ∇·: divergence operator ∇ 2 : Laplace operator | |+ : positive part | |− : negative part •˙ : time derivative •¨ : second-order time derivative •¯ : time mean •˘ : space averaged •ˇ : conduit generation averaged $•%: ensemble averaged •t |x : time average at a given point of a turbulent flow variable

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•s |t : space average at a given time of a turbulent flow variable •e |(x,t) : ensemble average at a given point and time of a turbulent flow variable •˜ : dimensionless •+ : normalized (∈ [0, 1]) •ˆ : peak value •∼ : modulation amplitude •p : power •T : transpose det(•): determinant cof(•): cofactor tr(•): trace O[value unit]: magnitude order Time Units d: day h: hour mn: minute mo: month s: second wk: week yr: year SI-Based and Non-SI Units of Quantity kDa: kiloDalton (Da: atomic or molecular mass unit) l: liter mmHg: millimeter of mercury (133.322 Pa [∼ 0.1333 kPa]) mmol, nmol, μmol: milli-, nano-, micromoles (amount of a chemical species, one mole containing about 6.02214078×1023 molecules) mosm: milliosmole (osm: number of moles of a osmotically active chemical compound) ppm: parts per million Temperature and Pressure Conditions ATPS: ambient temperature and pressure, saturated with water at body temperature, i.e., at 37◦ C, pH2 O = 6.27 kPa (47 mmHg) BTPS: body temperature and ambient pressure, saturated with water STPD: standard temperature (0◦ C) and pressure (101 kPa [760 mmHg]), dry air 273 + 37 p − pH2 O VBTPS = VATPS × × 273 + T p − 47 273 p − pH2 O VSTPD = VATPS × × 273 + T 760

Subscripts and Superscripts

Latin Subscripts ac : acid app : apparent atm : atmospheric ax : axial (• ) b : bound form of a molecule c : contractile c : center c : curvature c : point-contact cf : circumferential cl : closed co : core (flow) cond : conduction (velocity) conv : convection crit : critical value D : Darcy (filtration) D : dead space (airway) dias : diastolic diff : diffusion down : downstream, distal dyn : dynamic eff : effective ed : end diastolic es : end systolic E : expiration, Eulerian e : external e : extremum syst : systolic se : systolic ejection f : free form of a molecule f : fluid fast : fast (inward current) g : grid

h:

heat hydraulic he : hyperemic hea : healthy state I : inspiration i : internal in : (ionic) influx inc : incremental ion : sum of transmembrane ionic currents L : Lagrangian l : limit  : line-contact M : macroscopic m : mass (e.g., qm mass flow rate) m : mean max : maximum mb : membrane md : mesodiastolic mea : measured met : metabolic min : minimum ms : mesosystolic op : open out : (ionic) outflux p : parallel p : particle por : pore pd : protodiastolic ps : protosystolic q : quasi-ovalization r : radial ref : reference refr : refractory (time) regur : regurgitant h:

© Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0

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870 relative value at rest S : systemic, S phase s : solute s : serial si : sink slow : slow (inward current) so : source sten : stenosis stim : external stimulus syst : systolic T : tidal (breathing) tr : transition value t : turbulence t : stream division t : time derivative of order 1 tt : time derivative of order 2 td : telediastolic tors : torsional tot : total tf : related to mass transfer ts : telesystolic ung : ungated up : upstream, proximal v : volume (e.g., qv volumetric flow rate) w : wall w : water (solvent)

Subscripts and Superscripts

rel :

P:

rest :

pa :

Histology-Related Latin Subscripts A : alveolar, atrial A : mixed alveolar a : arterial a : mixed arterial ACM : alveolocapillary membrane Ao : aortic aA : transthoracopulmonary ao : airway opening (mouth or nose) aw : airway B : transbronchial, Bowman’s capsule b : blood cap : capillary coat : stent polymeric coating CW : chest wall (transthoracic) ECF(M) : extracellular fluid (medium) G : renal glomerulus H : heart ib : intrabronchial ICF(M) : extracellular fluid (medium) int : interstitial L : lung (transpulmonary) m : muscle, mouth musc : muscular

pulmonary pulmonary arterial pl : plasmatic, pleural pv : pulmonary veinous sa : systemic arterial sv : systemic venous tis : tissue V : ventricular Vv : valvular v : systemic venous blood v : pulmonary (mixed) venous blood vregur : regurgitant valve vcomp : competent valve wes : wetted endothelial surface

Greek Subscripts  : boundary θ : azimuthal μ : microscopic ω : vortical

Miscellaneous Subscripts + : positive command − : negative command ∗ : at interface 0 : reference state (unstressed) ∞ : high value Latin Superscripts a : active state e : elastic f : fluid fs : fluid–solid h : hypertensive M : mutant form N : normal (wild-type) form n : normotensive p : passive state s : solid v : viscoelastic

Miscellaneous Superscripts scale ∗ : complex variable (z∗ = 'm z + ı(e z) · : first component of complex elastic and shear moduli ·

: second component of complex elastic and shear moduli $ : static, stationary, steady variable :

Subscripts and Superscripts Chemical Notations X (x): upper- and lowercase letters correspond to gene or transcript (mRNA) and corresponding protein or conversely (e.g., Fes, FES, and fes designate protein, a proto-oncogene product that acts as a kinase, corresponding gene or transcript, and oncogene product, respectively) [X]: concentration of species X • : radical (unpaired electron[s]) Xx (x: single letter): splice variants X1: human form (ortholog) Xi: type-i isoform (paralog or splice variant; i: integer) Xi/j (i,j: integers): refers to either both isoforms (i.e., Xi and Xj, such as ERK1/2) or heterodimer (i.e., Xi–Xj, such as ARP2/3) Xi : type-i isoform of the receptor of ligand X (i: integer) XRi: receptor isoform i of ligand X (i: integer) X+: expressed molecule X (X-positive) X−: absent molecule X (X-negative) (X1 –X2 )i : oligomer made of i complexes constituted of molecules X1 and X2 (e.g., histones) a, c, nX: atypical, conventional, novel molecule X (e.g., PKC) al, ac, nX: alkaline, acidic, neutral molecule X (e.g., sphingomyelinase) cX: cellular, constitutive (e.g., cNOS), or cyclic (e.g., cAMP and cGMP) molecule X dX: deoxyX GPX: glycoprotein (X: molecule abbreviation or assigned numeral) hX: human form (ortholog); hormone-like isoform (FGF) iX: inhibitory mediator (e.g., iSMAD) or intracellular (e.g., iFGF) or inducible (e.g., iNOS) isoform lpX: lipoprotein-associated molecule X (e.g., lpPLA2) nX: neutral X; neuronal type (e.g., nWASP) oxX: oxidized molecule X (e.g., oxLDL) PI(i)P, PI(i,j)P2 , PI(i,j,k)P3 : i,j,k (integers): position(s) of phosphorylated OH groups of the inositol ring of phosphatidylinositol mono-, bis-, and trisphosphates rX: receptor-associated mediator or receptorlike enzyme; also regulatory type of molecular species (e.g., rSMAD)

871 tX: target type of X (e.g., tSNARE); tissue type (e.g., tPA) tmX: transmembrane type of X ubX: ubiquitous isoform

Chemical Notations with Right Subscripts Xalt : alternative splice variant XFL : full-length protein X Xh(l,m)MW : high (low, mid)-molecular-weight isotype XL(XL,S,XS) : long (extra long, short, extra short) isoform (splice variants) Xox : oxidized form Xred : reduced form XSp : spliced form XUSp : unspliced form Xc : catalytic subunit Xi : number of molecule or atom (chemical formula; i: integer)

Chemical Notations with Right Superscripts X+ : cation; also intermediate product X of oxidation (loss of electron) from a reductant (or reducer) by an oxidant (electron acceptor that removes electrons from a reductant) X− : anion; also intermediate product X of reduction (gain of electron) from an oxidant (or oxidizer) by a reductant (electron donor that transfers electrons to an oxidant) ΔNT : truncated form without the N-terminal domain ΔCT : truncated form without the C-terminal domain XA : activator form of molecule X Xa : active form of molecule X XCT : molecule X C-terminus XECD : soluble fragment corresponding to the ectodomain of molecule X after extracellular proteolytic cleavage and shedding (possible extracellular messenger or sequestrator) XGTP(GDP) : GTP (GDP)-loaded (activated [inactivated]) molecule X small GTPaseGTP(GDP) : active (inactive) form of small (monomeric), regulatory guanosine triphosphatase XHC : molecule X heavy chain Xhigh : molecule X produced at high levels XICD : soluble fragment correponding to the intracellular domain of a molecule

872 X after intracellular proteolytic cleavage (possible messenger and/or transcription factor; e.g., NotchICD : intracellular Notch fragment) Xlow : molecule X produced at low levels XLC : molecule X light chain XNT : molecule X N-terminus XR : repressor form of molecule X XS : soluble form

Chemical Notations with Left Subscripts carboxy (carboxyl group COOH [C])terminal cleaved part of molecule X L,Ac X: lysosomal, acidic molecule X (e.g., sphingomyelinase) m X: membrane-bound molecule X NT X: amino (amine group NH2 [N])-terminal cleaved part of molecule X S X: secreted form of molecule X S,Ac X: secreted, acidic molecule X (e.g., sphingomyelinase) s X: strong activation domain-containing (active) isoform t X: truncated isoform CT X:

Chemical Notations with Left Superscripts D(L) X: D (L)-stereoisomer of amino acids and carbohydrates (chirality prefixes for dextro- [dexter: right] and levorotation [lævus: left]), i.e., dextro(levo)rotatory enantiomer Epi X: epigenetic regulation-involved molecule X (e.g., lncRNA) G X: globular form of molecule X F X: fetal isoform F(G) actin: polymeric, filamentous (monomeric, globular) actin Imm X: immature form of molecule X Mat X: mature form of molecule X N : attachment of a molecule to a nitrogen atom (amide nitrogen of asparagine residue) of a protein1

1 For

Subscripts and Superscripts O:

attachment of a molecule to an oxygen atom in an amino acid residue of a peptide or protein2 P X: precursor form of molecule X PC X: phosphatidylcholine-preferring enzyme X (e.g., PLC) PI X: phosphoinositide-preferring enzyme X (e.g., PLC) S : attachment of a molecule to a sulfur atom in an amino acid residue of a peptide or protein3 SC X: single-chain molecule X WT X: wild-type molecule X

Oligomers and Polymers 1 X: monomeric molecule X 2 X: dimeric molecule X 3 X: trimeric molecule X 5 X: pentameric molecule X 6 X: hexameric molecule X mono X: monomeric molecule X olig X: oligomeric molecule X poly X: polymeric molecule X

Posttranslational Modifications XAc : acetylated molecule X XAr : adpribosylated molecule X XBi : biotinyladpribosylated molecule X XBu : butyrylatlated molecule X XCa : carbamylated molecule X XCi : citrullinated molecule X XCo : carboxylated molecule X XCr : crotonylated molecule X XFo : formylated molecule X XGl : glycosylated molecule X XGs : glutathionylated molecule X XHCy : homocysteinated molecule X XHy : hydroxylated molecule X XMa : malonylated molecule X XMe : methylated molecule X XMy : myristoylated molecule X XOx : oxidized molecule X XPa : palmitoylated molecule X

example, glycan in N-linked glycosylation, in which the carbohydrate group is covalently connected to a peptide or protein via the side chain nitrogen of asparagine. 2 For example, O-linked glycosylation, in which a carbohydrate group is covalently attached to a peptide or protein via the hydroxyl group of serine or threonine residues. 3 For example, S-linked glycosylation, in which a carbohydrate is covalently tethered to the sulfur atom of a cysteine residue.

Subscripts and Superscripts XPh : phosphorylated molecule X XPiso : proline isomerized molecule X XPr : propionylated molecule X XRed : reduced form of molecule X XSc : succinylated protein X XSNO : S nitrosylated molecule X XSSG : S glutathionylated molecule X XSu : sumoylated molecule X XUb : ubiquitinated protein X Organ-Related Left Superscripts of the arcuate nucleus AT : related to the adipose tissue BAT : related to brown adipose tissue BBB : of the blood–brain barrier Br : brain-specific (cerebral subtype) CCD : of the cortical collecting duct CD : of the collecting duct (nephron) circ : circulating cell pool CNS : of the central nervous system CnT : of the connecting tubule (nephron) DCT : of the distal convoluted tubule (nephron) Ep : epidermal subtype He : heart-specific (cardiac subtype) HT : hypothalamus-specific Il : ileal subtype In : intestinal subtype Ki : kidney-specific (renal subtype) LHA : of the lateral hypothalamic area Li : liver-specific (hepatic subtype) Lu : lung-specific (pulmonary subtype) Mu : muscle isoform NTS : of the nucleus tractus solitarius PCT : of the proximal convoluted tubule (nephron) PT : of the proximal tubule (nephron) pvAT : related to perivascular adipose tissue PVN : of the paraventricular nucleus RVLM : of the rostral ventrolateral medulla SkM : skeletal muscle-specific TAL : of the thick ascending limb of the loop of Henle (nephron) Te : testis-specific tr : tissue-resident cell pool VMH : of the ventromedial hypothalamic area VTA : of the ventral tegmental area WAT : related to white adipose tissue Arc :

873 Cell-Related Left Superscripts AC : astrocytic AdC : adipocytic CFB : cardiofibroblastic CMC : cardiomyocytic CSk : cytoskeletal EC : endotheliocytic EnC : enterocytic FB : fibroblastic FC : fibrocytic HC : hepatocytic KC : keratinocytic LC : leukocytic Mφ : macrophagic Mo : monocytic Ne : neuronal pΣN : belonging to parasympathetic neuron Pl : belonging to platelet RBC : belonging to red blood capsule ΣN : belonging to sympathetic neuron SMC : smooth myocytic SMN : belonging to sensory-motor nerve

Organelle-Related Left Superscripts Cy : cytosolic ER : endoplasmic reticulum type GB : Golgi body-resident IMM : of the inner mitochondrial membrane IMS : of the mitochondrial intermembrane space Lu : luminal (organelle lumen) Ly : lysosomal MAERM : of ER–mitochondrion contact site Mb : organelle membrane MM : of the mitochondrial matrix Mt : mitochondrial Nu : nuclear OMM : of the outer mitochondrial membrane Pe : peroxisomal PM : plasmalemmal Pr : proteasomal SG : related to stress granule Ve X: vesicular molecule X (e.g., Ve SNARE, Ve GluT2 [glutamate transporter]), and Ve ATPase)

Pathway-Related Left Superscripts of the electron transport chain FAO : of fatty acid oxidation TCAC : of the tricarboxylic acid cycle ETC :

Index

Symbols Cβ S, 658 P300 (KAT3b), 560 H2 S, 65, 478 h19 lncRNA, 175 H+ –K+ ATPase, 267, 268 CaV , 48, 307, 630 H+ ATPase, 266, 268 P38MAPK, 117 P53, 36, 121, 132–134, 321, 389, 599, 601 P63, 141, 602 P73, 602 S1PR, 587 S1P, 348 S6K, 311, 333, 539 S100, 194, 306, 397 nACh receptor, 572 Na+ –Pi co-transporter, 227 Na+ –HCO− 3 symporter, 239 Na+ –K+ ATPase, 48, 57, 324, 380 Na+ –H+ exchanger, 209, 239, 259, 291 Na+ –K+ –2Cl− co-transporter, 209, 243, 380 Na+ –Cl− co-transporter, 263 Na+ –Ca2+ exchanger, 48, 49 Na+ –H+ exchanger, 48, 57 NaV , 48 7SK RNA, 120

A aaRS, 115 ABC, 7, 107, 143, 338, 346, 347, 480, 481, 484, 486, 530, 599, 639, 645, 648, 649, 663

Abl, 141 ACADH, 630 ACE, 139, 278, 660 Acetyl-CoA carboxylase, 452, 455 ACS, 565 ACSL, 145 actin, 622 acyl-CoA thioesterase, 436 adam, 188, 193, 227, 279, 398, 413, 476, 656 adamts, 177, 432, 666 AdCyAP, 502 Addison disease, 46 adenosine, 251, 277 adhesion GPCR, 81, 639 adhesion molecule, 112 adipocyte, 312, 314, 322, 326, 390, 394, 395, 432, 433, 460, 462, 463, 465, 473, 475, 541 adipocyte dysfunction, 325, 382, 404, 430, 433, 434 adipogenesis, 429 adipokine, 442, 443 adiponectin, 361, 367, 369, 376, 390, 394, 399, 425, 428, 444, 493, 577 adiponectin receptor, 360 adipose tissue, 9 adiposopathy, 366 adipsin, 418, 459 ADMA, 140 ADNF, 503 ADNP, 503 AdpnR, 493 ADPR cyclase/cADPR hydrolase, 564 adrenal gland, 9

© Springer International Publishing AG, part of Springer Nature 2018 M. Thiriet, Vasculopathies, Biomathematical and Biomechanical Modeling of the Circulatory and Ventilatory Systems 8, https://doi.org/10.1007/978-3-319-89315-0

875

876 adrenaline, 569 adrenomedullin, 470 ADRF, 423 AGE, 33, 329 Aging, 15, 21, 34, 36, 48, 94, 146, 230, 285, 291, 299, 303, 317, 370, 378, 381, 403, 409, 418, 430, 470, 475, 564, 565, 568–570, 575 AgRP, 455, 458, 459, 472, 486, 509, 532, 543 Agt2 receptor, 378 AlDH, 630 Air pollution, 101 AKAP, 308 alarmin, 35, 44 albumin, 178 aldehyde reductase, 12 aldo–keto reductase, 12 aldose reductase, 12, 328 aldosterone, 6, 200, 204, 208, 209, 211, 216, 225, 242, 260, 264, 269, 270, 274, 289, 293, 378, 452 aldosteronism, 289 α cell, 530, 535, 536, 539 ALK, 78, 85, 347, 351, 665 aminopeptidase, 279, 511, 517 AMPK, 46, 50, 141, 144, 170, 171, 258, 279, 293, 311, 315, 316, 333, 394, 411, 447, 448, 452, 455, 456, 458, 460, 464, 530, 577, 582, 583, 590 amygdala, 402, 507, 512, 540 amygdala central nucleus, 504 amylin, 500, 529, 531 androgen, 5, 470 aneurysm, 7, 9, 41, 79, 178, 571, 622, 664 aneurysm–osteoarthritis syndrome, 668 angiogenesis, 7, 8, 20, 62, 63, 77, 89, 117, 122, 159, 169, 286, 297, 329, 388, 427, 433, 446, 455, 460, 463, 466, 474, 541, 630, 639, 647, 650, 664, 665 angioma, 40 angioma serpiginosum, 75 angiotensin, 138, 139, 141, 204, 209, 225, 265, 269, 270, 274, 284, 296, 308, 367, 379, 427, 452, 460, 470, 624, 634 angiotensinogen, 276, 470 angiotensin receptor, 660 AngptL, 359, 361, 373, 380, 434, 442, 524, 590, 647, 648, 650 anthocyanidin, 580 aortic dissection, 622 AP, 51, 141, 329 apelin, 279, 460 ApoA , 67, 353, 356

Index ApoA1, 7, 97, 152, 338, 339, 345, 348, 353, 397, 483, 645 ApoA2, 348 ApoA4, 532 ApoA5, 359, 655 ApoB100, 67, 144, 337, 340, 342, 344, 346, 348, 353, 356, 643, 645, 654, 660 ApoC1, 359 ApoC2, 359, 484, 643, 645 ApoC3, 340, 353, 359, 646, 655, 660 ApoD, 359, 463 ApoE, 7, 335, 340, 353, 358, 359, 484, 650, 656, 660, 674 ApoJ, 353 ApoL1, 65 apolipoprotein, 359 ApoM, 348 apoptosis, 137, 138, 146 apoptotic body, 167, 187 aquaporin, 255 arachidonic acid, 464 Arcuate nucleus, 4, 491, 505, 508, 532, 534, 540, 542, 543, 546 Area postrema, 498, 504, 531, 536 ARF, 341, 438 ArfGEF, 439 arginase, 425 AR (NR3c4), 8, 556 arrhythmogenic RV cardiomyopathy, 47 arterial tortuosity syndrome, 667 arteriogenesis, 86 arteriosclerosis, 43 arteriovenous fistula, 69 arteriovenous malformation, 69 arteritis, 305 ASiP, 472, 509 aspalathin, 317, 582 ASP (C3), 418, 431, 473 asprosin, 461 AspRS (DaRS), 116 asthma, 568 ataxin, 629 ATF, 324, 599 ATGL, 331, 347, 385, 409, 473 atherosclerosis, 7, 8, 19, 42, 93, 97, 102, 103, 107, 144, 157, 163, 176–178, 183, 184, 193, 307, 328, 338, 344, 350, 360, 369, 376, 463, 466, 475, 568, 571, 582, 600, 621, 645, 648, 650, 655, 656, 663 AtoH, 525 ATP, 36, 209, 261 Atrial fibrillation, 47 Atxn3 deubiquitinase, 37

Index AUP, 341 Autoimmune thrombotic antiphospholipid syndrome, 633 Autophagy, 137, 440 Axl (RTK), 192

B bach, 662 BAF (Swi/SNF) complex, 166, 611, 615 Bannayan–Riley–Ruvalcaba syndrome, 74 baroreflex, xv, 306 Bartter syndrome, 259 batokine, 412 bcar4 lncRNA, 321 BCL2, 137, 138, 141 Beige adipose tissue, 415 β-adrenoceptor, 63, 277, 409, 415, 417, 424, 634 β-AR, 259 β-catenin, 80 β cell, 297, 530, 531, 538 berberin, 317 Berberine, 400 Biased agonism, 505 Bile acid, 346, 406, 478, 482, 486 Biotinylation, 560 Blood–brain barrier, 80, 311, 532, 537, 540, 541, 543 Blood–retina barrier, 80 Blue rubber bleb nevus syndrome, 72 B lymphocyte, 434, 476, 594 BMP, 41, 78, 79, 85, 88, 90, 139, 366, 411, 524 BMPR, 598, 632 Bombesin, 533 Bowman capsule, 244 Bradykinin, 270, 272, 282, 477 Bradykinin receptor, 282 Brain, 544 Brain–gut axis, 498 Brainstem, 535, 536 Bronchoconstriction, 564 Brown adipose tissue, 404

C Cadherin, 447 caff1, 163 Calcific valve disease, 66 calciphylaxis, 65 calcitonin, 233 Calcium, 370 Calcium-sensing receptor, 212, 230, 255 calgranulin, 397

877 calmodulin, 279 Cam2K, 315 cAMP, 63, 559 Cancer, 39, 43, 93, 122, 143, 166, 168, 175, 183, 190–192, 321, 356, 552, 572, 573, 592, 628, 639, 650, 664 Cannabinoid receptor, 546 Capillary–arteriovenous malformation syndrome, 69 Capillary malformation– arteriovenous malformation syndrome, 75 CAP (PesS), 292 carbonic anhydrase, 234, 242, 260 carboxypeptidase, 281, 509, 517, 520 Cardiac dysfunction, 65 Cardiac fibrosis, 50, 64, 152, 153, 175, 196, 380, 388, 429 Cardiac hypertrophy, 48, 50, 64, 95, 149, 153, 175, 380, 471, 602, 613 cardiomyokine, 335 cardiomyopathy, 307, 329, 434, 603 cardiorenal syndrome, 65 cardiotrophin, 381 carl lncRNA, 174 CAR (NR1i3), 557 catalase, 29, 575 catechin, 580, 582 cathepsin, 279, 297 caveolin, 378 CBP (KAT3a), 560 CBS, 660 CD2AP, 246 cdennd4c, 163 cdkn2bas lncRNA, 167, 663, 664 CDO, 64 cdr1as lncRNA, 175 C/EBP, 323, 346, 379, 394, 430, 431, 449, 457, 464, 656 Celiac disease, 633 Cell cycle, 562 Central nervous system, 9 ceramidase, 360 ceramide, 167, 332, 360, 363, 365 cerebrospinal fluid, 546 CETP, 346, 349, 417, 485, 660 CFTR, 262 cGMP, 624 CGRP, 272, 422 chaer lncRNA, 175 chast lncRNA, 175 CHD complex, 612 chemerin, 462 chemokine, 443

878 Chemoreceptor, xiii Chemoreceptor reflex, xiii cholecystokinin, 529, 533 Cholesterol, 397 Cholesterol desmolase, 11 chrac complex, 612 CHREBP, 367 chrf lncRNA, 174 Chromogranin, 520 Chronic kidney disease, 65 chymase, 282, 288 cinnamon, 582 circActa2 lncRNA, 164 circadian rhythm, 327, 511, 517, 549 circHipk3, 157 circRNA, 143 CK, 96, 146, 179, 475 CKI, 660, 664, 668 class-7 (group E) bHLHe, 525 Claudin, 226, 230, 232, 254 ClC, 629 Cl− –HCO− 3 exchanger, 239, 266, 268 CMC, 177, 194, 324, 326–328, 330, 448, 449, 455–457, 461, 471, 560, 583 CMC dysfunction, 361 Coagulation factor, 582, 660 Coatomer, 438 Cohesin, 596 Collectrin, 279 Complement, 428, 455, 457, 459, 473 Complement receptor, 459 Connexin, 76, 89 COP complex, 439 Corin, 624 CoroMarker lncRNA, 172 Coronary artery disease, 3 COUPTF (NR2f1/2), 77, 79 Cowden syndrome, 74 C-reactive protein, 112 Creatine, 402 CREG, 275 CRL complex, 432 CRP (Ptx1), 99, 176, 178, 182, 351, 376, 474, 569 CRTC, 553 Cryptic, 100 CS desmolase (lyase), 9 CSF2, 102, 158 CS25H, 132 CSK, 629, 630 CsnK, 293, 412, 447, 454, 559, 560, 570 csrsf4, 163 CTCF, 649 CTGF, 380

Index cthsd1, 163 CTRP, 450 Cullin, 73 Cup cell, 523 Cushing disease, 46 Cutaneomucosal venous malformation, 72 Cyanidin, 582 Cyclophilin, 197 Cyclosporin, 132 CyP, 7, 572, 628, 630, 660 CyR61 (CCN1), 180 cytotoxic T cell, 402 cznf292, 163

D DAG, 332 D cell, 529, 536 deep vein thrombosis, 96 DEG–ENAC, 292 dendrocyte, 183, 188, 201, 462 deoxyribonuclease, 17 desmosine, 35 DGAT, 347 Diabetes, 3, 15, 23, 93, 138, 139, 142, 143, 301, 574, 583 Diabetic cardiomyopathy, 46, 361, 387 Diaphragm, 270 Dicer, 121, 420 Dickkopf, 100 Dilated cardiomyopathy, 45, 603 DLL, 79 DNAPK, 316 DNMT, 315 Dopamine, 217, 229, 297 Dorsal vagal complex, 505, 507 Dorsomedial hypothalamus, 491 DPP, 511, 530, 541 Drosha, 121 dysbacteriosis, 587 dyslipidemia, 332, 550 dyslipoproteinemia, 343, 642

E EBF, 411 EC, 67, 103, 104, 113, 183, 197, 298, 307, 311, 326, 327, 329, 347, 359, 433, 434, 446, 449, 452, 455, 457, 462, 463, 465, 468, 474, 476, 536, 538, 551, 583 EC dysfunction, 8, 353, 363, 369 ectopic calcification, 41, 66, 184, 599 EDHF, 424, 427 EEPD, 485

Index EGF, 104, 233 EGR, 51, 131, 135, 142, 330, 397, 553 Ehlers–Danlos syndrome, 667, 669 eIF2α K3 (PERK), 118, 270 EKODE, 379 ELk/TcF, 129, 132, 139, 150 ENaC, 292 Endocannabinoid, 546, 591 Endoglin, 87, 88 Endorphin, 528 Endothelial cell dysfunction, 333 Endothelial dysfunction, 65, 68, 102, 104, 122, 177, 192, 301, 307, 322, 325, 329, 427, 449, 472, 474 Endothelin, 270, 293, 296, 427, 466, 624 Endothelin receptor, 296 ENPP, 374 enterochromaffin cell, 529 enterocyte, 335, 523 entero-endocrine cell, 523, 528 Enterohepatic circulation, 480 Enterostatin, 534 eosinophil, 390, 401, 434 EPH receptor, 71, 83 ephrin, 71, 78, 83 epican (CD44), 52 Epithelial–mesenchymal transition, 123 EPRS, 116, 333 ER–LD contact, 439 ERK, 117, 125, 138–142, 164, 184, 197, 293, 311, 381, 456, 457, 460, 539 ER (NR3a1/2), 5, 556 ERR (NR3b1/2/3), 556 ER stress, 449, 468, 494, 581, 583, 663 Erythritol, 366 Estradiol, 5, 141 Estrogen, 4, 229, 266, 276, 470, 654 Estrogen GPCR, 654 ETC, 156 ETS, 129, 275 Exercise, 21, 288, 370 Exocyst, 348 Exosome, 120, 143, 159, 161, 167, 187, 188, 190

F FABP, 62, 365, 394, 428, 459, 460, 472, 481 FAK, 31, 321 Fam20, 229 Fam198, 229 FAR, 631 FATP, 62, 333, 347, 359, 367, 459 FBxL Ub ligase, 560

879 Feedback, 307, 459, 463, 473, 524, 528, 543, 551, 553, 554, 559–561, 564, 631, 634, 639 fendrr lncRNA, 173 FFA GPCR, 526 FGF, 121, 134, 205, 229, 335, 354, 388, 396, 408, 410, 412, 414, 628 FGFR, 205 FHL, 129 Fibrinogen, 112, 177 Fibroblast, 433, 455, 456, 558 Fibronectin, 133 Fibrosis, 196, 199, 273, 297, 324, 329, 368, 372, 382, 399, 404, 418, 426, 434, 455, 456, 471 Flavanol, 580 Flavanone, 580 Flavone, 580 Flavonol, 580 Flotillin, 188 Fos, 633, 634 FoxC, 76, 79, 82, 84 FoxF, 173 FoxO, 36, 63, 129, 137, 138, 140, 312, 313, 319, 326, 391, 394, 460, 542, 543, 547 Frizzled, 80 Furin, 283, 292, 458 FXR (NR1h4/5), 101, 144, 478, 557, 591, 645

G GABA, 561 Galectin, 191, 297, 332, 395 gas5 lncRNA, 172 Gastrin, 528 Gastrin-releasing peptide, 554 GATA, 123, 457 GcgR, 504, 505, 537 GDF, 540 GFRα, 540 GGT, 184 GH, 462, 508 ghrelin, 486, 493, 503, 509, 512, 528, 529, 534 GHRH, 203, 508 Giant cell arteritis, 67 GIP, 508, 529, 530, 536 GIPR, 505, 537 Gli, 321 Glicentin, 521, 529, 535 Gliocyte, 539 GliS, 433 Glomerulomegaly, 366 Glomerulosclerosis, 366 Glomulin, 73

880 Glomuvenous malformation, 73 GLP, 341, 505, 507 GLP1, 460, 521, 529, 530, 535, 579, 589, 590, 592, 601 GLP2, 521, 529, 538, 589, 591, 592 GLP1R, 505, 601 Glucagon, 261, 324, 504, 520, 521, 529, 537, 539 glucocorticoid, 229, 361, 445, 469, 494 glucose 6-phosphatase, 453, 458 glucotoxicity, 363 GluT, 320, 455, 462, 467 glutaminase, 148 glutathione peroxidase, 21, 30, 576, 583, 632 Glycocalyx, 124 Glycolysis, 320 Glycosylation, 328 Glypican, 134 Goblet cell, 523 GPBAR GPCR, 536 GPCR, 498, 505 GPD, 646 GPER1, 5 GPIHBP1, 646, 654 GPR119 (GPCR2), 587 G protein, 74 GPR132, 587 Granulomatosis with polyangiitis, 15 Granzyme, 631, 639 GRB, 142 Gremlin, 524 GRK, 63, 293 Grönblad–Strandberg syndrome, 669 GR (NR3c1), 290, 556 GRP, 533, 536 GRPP, 521 GSK, 311, 449, 556, 560 GTF2d (TFI I D) complex, 616, 632 GuCy, 624 Gut flora, 100, 393, 514, 517, 584

H Hamartoma, 74 HAND, 137, 150 HAT, 315 HCA GPCR, 319 HDAC, 123, 132, 137, 323, 431 HDL, 345, 346 Heart, 538 Heart failure, 45, 64, 145–147, 175, 179, 183, 192, 194, 203, 270, 273, 275, 279, 571 Hedgehog, 77, 81, 305

Index Helper T cell, 67, 402, 434 hemangioendothelioma, 40 hemangioma, 40, 72 hemangiomatosis chondrodystrophica, 72 heme oxygenase, 270 Henry law, xii Heparanase, 61, 125 Heparin, 135 Heparinase, 124 Hepatocyte, 312, 314, 335, 339, 395, 453, 458, 459, 476 Hepatosteatosis, 581, 591 Hereditary hemorrhagic telangiectasia, 69 Hereditary lymphedema, 76 HER/EGFR, 52, 378, 538 HES, 78, 525 HETE, 231, 261, 298 HETrE, 581 Hexosamine axis, 327, 329 HGFR, 73, 192 HIF, xvii, 31, 146, 149, 150, 171, 321, 427, 434, 455, 558, 584 hif1as lncRNA, 172 HIF prolyl hydroxylase, 338 HIPK, 639 Hippocampus, 534 Hippo (STK3/4), 321, 636 Histamine, 424, 426, 528 HMGB1, 397 HMGCR, 145 HNF1/3, 649 HNF4 (NR2a1/2), 557, 577, 650 HODE, 359 Homocysteine, 93, 96, 113, 177, 178 Homocystinuria, 658 Hotair lncRNA, 167, 175, 614 Hox, 415 HRT, 78, 129 HSDH, 11, 12, 291, 469 HSP, 189 HSPG, 80, 124, 374, 645 hulc lncRNA, 175 Hyaluronidase, 52 Hydrogen peroxide, 31, 197, 328, 425, 477 Hydroxyisobutyrate, 395 Hypercalcemia, 230 Hypercalciuria, 259 Hypercapnia, 242 Hyperchemerinemia, 462 Hypercholesterolemia, 332, 337, 644, 650, 653, 656 Hyperchylomicronemia, 642, 655 Hyperglucagonemia, 538

Index Hyperglycemia, 92, 141, 146, 301, 325, 333, 362, 370, 417, 426, 459, 463, 464, 474, 550, 581 Hyperhomocysteinemia, 94 Hyperinsulinemia, 322, 367, 386, 417, 427, 464 Hyperkalemia, 217, 225, 238, 290 Hyperleptinemia, 463 Hyperlipidemia, 332 Hyperlipoproteinemia, 343 Hyperlnc, 167 Hyperplasia, 430, 432, 622, 638 Hypertension, 3, 4, 8, 15, 48, 92, 113, 179, 194, 195, 199, 230, 297, 370, 426, 457, 461, 569, 575, 600, 624, 625, 630, 632, 634 hypertension, 113 Hypertriglyceridemia, 332, 340, 341, 581, 644, 645, 655 Hypertrophic cardiomyopathy, 46, 173, 175 Hypertrophy, 324, 430, 449, 457, 635 Hyperuricemia, 644 Hypoadiponectinemia, 449 Hypocretin, 410, 545, 546 Hypoglycemia, 322 Hypokalemia, 217, 236, 238, 259, 289 Hypophysis, 520 Hypoplasia, 273 Hypotension, 581 Hypothalamus, 297, 311, 317, 363, 411, 443, 455, 458, 460, 462, 467, 486, 491, 507, 534–536, 541, 544, 546, 551, 552, 577 Hypothyroidism, 399 Hypotrichosis–lymphedema– telangiectasia syndrome, 76 Hypoxia, 104, 122, 145, 146, 163, 168, 169, 190, 197, 279, 300, 370, 372, 386, 427, 557, 650, 664 I icam, 462, 669 I cell, 529, 533 ID, 88, 132 IDH, 72, 565 IDL, 107 IDO, 7, 572 IGF, 142, 206, 412, 445, 508, 538 igf2as lncRNA, 169 IGFBP, 627 IGF1R, 366 IGF2R, 471 IKK, 76, 293, 311, 476, 494 IL, 185, 306, 317, 443, 444, 455, 463, 474, 475, 494, 569, 582, 632, 660, 669

881 il21ras1 lncRNA, 172 IL receptor, 194, 669, 672 Immunity, 297, 443, 462, 541, 568, 570, 584, 629, 631, 633, 659, 661 Incretin, 529 Inflammaging, 35 Inflammasome, 327, 581 Inflammation, 4, 7, 8, 18, 34, 67, 102–105, 111, 112, 178, 183, 196, 199, 332, 363, 369, 382, 433, 434, 446, 455, 462–465, 468, 472–474, 476, 528, 568, 569, 571, 592, 600, 624, 633, 634, 639, 655, 656, 660, 664 Innate-like T lymphocyte, 274 INO80 complex, 612 insig, 599 InsR, 311 Insulin, 177, 191, 196, 206, 270, 293, 308, 332, 333, 341, 348, 349, 363, 367, 371, 375, 383, 387, 407, 420, 424, 431, 433, 434, 445, 449, 454, 456, 458–460, 463, 467, 486, 493, 520, 529, 531, 537, 541, 543, 547, 576 Insulinopenia, 459 Insulin receptor, 386, 395, 476 Insulin resistance, 192, 313, 325, 332, 337, 341, 366, 374, 383, 387, 395, 427, 434, 444, 446, 458, 460, 464–466, 468, 472, 475, 476, 494, 542, 543, 575, 583 Integrin, 524, 660 Interferon, 44, 393 Interleukin, 102, 176 Intervening peptide, 521 Intestine, 480, 482, 484, 521, 522, 528 Intimal hyperplasia, 125, 132, 135, 137, 139–141, 163, 164, 428, 456, 463, 470, 478, 656 IRAG, 624 IRF, 44, 365, 385, 393 irisin, 44, 335, 411 IRS, 138, 191, 311, 386, 467, 476, 542 Ischemia, 44, 94, 149, 177, 299, 340, 434, 455, 571 Ischemic cardiomyopathy, 46 Ischemic preconditioning, 537 ISCu, 150 Isoprostane, 33

J Jagged, 67 JaK, 197 JaK–STAT axis, 542 Janus kinase, 542

882 JNK, 138, 311, 458 Juxtaglomerular apparatus, 249

K KAT, 132, 380, 392 KATP , 48, 311 KCa (BK), 624 K cell, 529 KIR , 633 KCC co-transporter, 239, 266 KCR Ub ligase, 583 KDM, 378 Kennedy pathway, 437 Ketone, 146 Kidney, 274, 386 Kinesin, 341 KLF, 38, 90, 123, 129, 131, 139, 179, 374, 458, 599 Klippel–Trenaunay–Weber syndrome, 72 Klk, 279, 292 klotho, 205, 227, 229 Kupffer cell, 186

L Lands cycle, 437 Late-onset lymphedema, 76 Lateral hypothalamus, 491, 543 LCADH, 565 LCAT, 336, 346, 348, 645 L cell, 529, 535, 536, 541 LDH, 149 LDL, 7, 107, 343, 350 LDLR, 7, 67, 347, 350–353, 357, 359, 360, 368, 484, 485, 645, 672 LepR, 493 Leptin, 281, 367, 376, 379, 410, 428, 443, 462, 486, 493, 509, 531, 541, 546 Leptin receptor, 541 Leptin resistance, 463, 494, 542, 543, 547 Leukotriene, 191, 332 Lexis ncRNA, 168 LHx, 415 LIF, 350 lincMD1, 175 lincrnap21, 172 Linoleic acid, 365, 379, 536, 581 lipase-A , 337 lipase-A (LAL), 643 lipase-C (HL), 340, 648, 655 lipase-D (LPL), 63, 339, 340, 359, 417, 448, 485, 643, 645, 654, 656, 660

Index lipase-E (HSL), 62, 326, 331, 365, 385, 400, 409, 417, 440, 473 lipase-G (EL), 347, 648 Lipcar lincRNA, 171, 175 Lipid droplet, 341, 434 Lipin phosphatase, 454 Lipocalin, 365, 428, 444, 463 Lipodystrophy, 418 Lipokine, 332 Lipoprotein-A, 67, 93, 335, 356 Lipotoxicity, 363 Lipoxygenase, 23, 137 Livedoid vasculitis, 15 Liver, 366, 386, 447, 458, 462, 465, 466, 468, 474, 478, 484, 486, 536, 540, 547 LKb1 (STK11), 50, 315, 447 lncAng362, 141 lncarsr, 192 lncRNA, 2, 115, 165 Locus ceruleus, 495 Loeys–Dietz syndrome, 667, 669 LPL, 672 LRP, 80, 351, 357, 359, 671, 672 LRP1/8 (ApoER1/2), 428, 654, 656 LRRC8, 262 Lupus erythematosus, 68 LXR (NR1h2/3), 36, 132, 144, 478, 557, 645 Lymphangiogenesis, 77, 122, 140 Lymphedema, 75 Lymphedema–cholestasis syndrome, 76

M mACh receptor, 536, 537 Macrophage, 104, 107, 158, 183, 186, 193, 338–340, 344, 346, 351, 354, 360, 377, 382, 385, 390, 395, 401, 433, 434, 449, 462, 463, 465, 473, 475, 485, 572, 582 MAERM, 58, 326 MAF, 649, 662 Malformation, 39 Malondialdehyde, 33 mantis lncRNA, 169 MAO, 385 MAPK, 139, 193, 312, 322, 542, 635 MAPKAPK, 630, 639 Marfan syndrome, 665, 669 Marker, 92, 99, 103, 144, 178, 452, 455, 475, 568, 569, 599, 634, 646, 660 Mastocyte, 20, 282, 434, 572 Maxi anion channel, 251

Index maxiCl channel, 262 MBTPS, 283 MCADH, 565 M cell, 523, 529 MCU, 371 Mechanical stress, 190, 197, 209, 624 Mechanotransduction, 87, 135 Medial preoptic nucleus, 508 Mediator complex, 454 Medulla, 507 MEF, 76, 123, 632 Megalencephaly capillary malformation syndrome, 73 meg3 lncRNA, 168, 169 Melanocortin, 508 Melanocortin receptor, 346, 509 Melatonin, 462 Mepe, 229 Meprin, 355 M–ER contact, 409 Metabolic syndrome, 144, 317, 325, 337, 427, 449, 467, 474, 644 Metabolon, 234 Metallothionein, 442 Methylglyoxal, 327–329 MGL, 331, 417 MHC, 189 mhrt lncRNA, 174 miat lncRNA, 169 MicroRNA, 2, 118, 120, 143, 153, 183, 186, 190, 191, 277, 279, 307, 322, 330, 413, 416, 418, 432, 559, 627 Microsomal triglyceride transfer protein, 144 Microvascular dysfunction, 371 Microvesicle, 103, 143, 167, 185, 187, 192, 375 Mitochondrial dysfunction, 359, 361, 363 Mitochondrial transcription factor, 316 Mitochondrion, 9, 42, 145, 170, 315, 370, 392, 406, 566 Mitochondrogenesis, 411 Mitofusin, 396, 408 Mitophagy, 415 MKnK, 126 M–M contact, 409 MME, 279, 355, 511 MMP, 18, 135, 189, 193, 365, 370, 398, 463 Monocarboxylate transporter, 359 Mönckeberg media sclerosis, 43 Monocyte, 102–104, 158, 183, 463, 465 Mononuclear phagocyte system, 158 Motilin, 529 Moyamoya syndrome, 676 MPGF, 521 MR (NR3c2), 290, 378

883 MRTF, 132, 139, 196 MSH, 546 MSK, 126 Mt ETC, 22 MTHFR, 628, 629, 659, 660 MTr, 660 MTrR, 660 muFA, 369 MyADM, 140 MyBPc, 179, 180 MyC, 321, 599, 669 MycBP2 Ub ligase, 556 myeloperoxidase, 24 MyLIP Ub ligase, 484 Myocardial infarction, 43, 44, 101, 114, 143, 150, 172, 174, 179, 183 Myocardin, 137, 139, 196, 665 Myocyte, 312, 395 MyoD, 137 Myokine, 335 Myosin, 622

N NAD+ , 563 NAFLD, 342, 365, 393, 398, 453, 547 NAmPT, 469, 564, 565 NASH, 436 Natriuretic peptide, 96, 177, 178, 184, 211, 293, 325, 354, 410, 422, 428, 445, 624, 627, 629, 634 NCoA coactivator, 430 N cell, 529 NDPK, 63 neat2 lncRNA, 166, 168, 169 NEDD Ub ligase, 293 NeK, 75 Nephrin, 246 Nephron, 210 Nesfatin, 507 NETosis, 17, 327 Neuregulin, 52, 61, 164 Neuromedin-B, 533 Neuromedin-C, 536 Neuron, 455, 459, 528, 539, 564, 623 Neuropeptide LEN, 544 Neuropeptide PEN, 544 Neuropeptide-Y, 422, 455, 459, 486, 510, 532, 543 Neuropilin, 82 Neurotensin, 529, 530 Neutrophil, 102, 183, 401, 434, 463 Nevus anemicus, 75 Nevus comedonicus, 74

884 Nevus flammeus, 74 Nevus roseus, 75 NFAT, 319, 329, 390 NFE2L, 27, 270, 318, 328, 377, 582, 583 NFκB, 36, 107, 113, 118, 139, 150, 159, 185, 327–329, 350, 369, 381, 397, 447, 460, 464, 468, 471, 476, 524, 568, 582 Nicotinic acid, 356 Nitric oxide, 197, 478 Nitrosative stress, 449 NK cell, 104, 463 NKT cell, 390, 434 NMDA GluR, 673 NMT, 639 NO, 60, 113, 133, 209, 242, 251, 264, 295, 297, 360, 363, 370, 371, 374, 431, 446, 452, 460, 463, 474, 476, 539, 580, 623, 673 Nocturnin, 560 Nodose ganglion, 537 Noggin, 524 Non-alcoholic fatty liver disease, 144 Nono, 562 Nonsyndromic familial thoracic aortic aneurysm, 668 Noonan syndrome, 636 Noradrenaline, 407 Norrin, 80 NOS, 6, 16, 61, 197, 297, 321, 327, 329, 349, 371, 425, 428, 456, 460, 464, 466, 538, 634, 656, 660, 666 Notch, 67, 78, 86, 89, 525 NOx, 6, 22, 51, 107, 114, 146, 185, 197, 275, 285, 322, 326, 327, 370, 380, 385, 394, 425, 427, 471, 583 NPc, 346, 351 NPR, 624 NPy receptor, 511 NR4a1, 553 NR4a3, 142 NRF, 316 Nt5E, 374 Nucleosome, 595 Nucleus accumbens, 507, 510, 534 Nucleus of the solitary tract, 272, 497, 504, 507, 533, 534, 536

O obesity, 3, 92, 306, 325, 334, 369, 462, 467, 468, 474–476, 573, 583, 592 Oct, 599 OGA, 329 O-GlcNAcylation, 328 OGT, 328

Index OLEDAID syndrome, 76 Oleic acid, 365, 369 Olfaction, 366 Olfactomedin, 129 Omentin, 464 OpA1, 396 Opsin, 553 Orexin, 410, 546 OSR1 kinase, 258 Ouabain (EOLC), 49 Ovary, 9 Overweight, 95, 302, 334, 368, 467, 469 oxLDL, 137, 138, 183 Oxphos, 371, 566 Oxylipin, 35 Oxyntomodulin, 521, 529, 540

P PAI, 177, 297, 377, 443, 444, 474, 582, 660 Palmitic acid, 332, 365, 385 Palmitoleate, 581 Palmitoleic acid, 536 Pancreatic polypeptide, 510, 529 Paneth cell, 523, 563 PAQR, 447 PAR, 436 Parabrachial nucleus, 507, 540 Paraoxonase, 348, 660 Parasympathetic, 210, 537 Paraventricular nucleus, 272, 488, 491, 501, 543 PARP, 327 Pattern-recognition receptor, 35 PBUT, 65 PCSK, 95, 96, 283, 351, 458, 519, 520, 535, 543, 624, 645, 650, 653, 654, 663 PCTP, 436 PDE, 625 PDGF, 133, 665 PDGFR, 366 PDHK, 149 PDK1, 542 PDx1, 538 Pentraxin, 182 PEPCK, 453, 458, 467 Peptide-YY, 510, 529, 535, 541, 579, 589, 590 PeptidylArg deiminase, 327 Perforin, 639 Pericyte, 366 Perilipin, 326, 439 Period, 540 Periodontitis, 178 Peripheral artery disease, 7

Index Perivascular adipose tissue, 385, 420 Perlecan, 134 Peroxiredoxin, 29, 568, 576 Peroxynitrite, 448 PesS (PrsS), 292 PGC, 316, 371, 461 PGhS, 113, 141, 449 PGi2 , 136, 374 Phacomatosis pigmentovascularis, 75 Phosphatidic acid, 438 Phospholemman, 49 Photorelaxation, 553 PI3K, 72, 74, 293, 312, 348, 386, 539, 542, 599 PIM, 137, 339 PK, 118 PKA, 258, 293, 308, 486 PKB, 87, 117, 136, 152, 191, 193, 197, 293, 311, 349, 388, 453, 456, 539, 582 PKC, 135, 293, 308, 311, 329, 378, 381 PKD, 293 PLA, 176, 357 Plasmin, 292 Platelet, 104, 113, 143, 159, 163, 177, 183, 192, 197, 330, 463, 474, 551 PLC, 628, 634 Pleiotrophin, 432 PlekHa, 630 Plexin, 135 PlgRkt, 357 PLTP, 650, 672 P/O ratio, 320 PMCA, 630 PnPLA, 399, 438, 473 Podocalyxin, 246 Podocin, 246 Polyol axis, 327, 328 Polyphenol, 580 POMC, 408, 472, 520, 532, 542, 543, 546 PP1, 624, 633 PPAR (NR1c1/2/3), 47, 180, 192, 273, 326, 369, 379, 389, 430, 431, 452, 457, 462, 556, 565, 577, 582, 613, 632 PRC complex, 166, 525 PRDm, 411, 414, 429, 430 pre-eclampsia, 270, 297 presenilin, 87 pressure natriuresis, 208 PRMT, 649 Procyanidin, 580 Prolactin, 297 (pro)renin receptor, 471 Prostacyclin, 330 Prostaglandin, 233, 261, 277 Protein-C, 342

885 Proton-activated GPCR, xv PRR, 182 PrRx, 132 PTen, 74, 311, 411, 542 PTEN hamartoma tumor syndrome, 74 PtgER, 587 PtgIR, 136, 587 PTH, 226, 233, 261 PTPn1, 311 PTPn11/SHP2, 31, 542 PTPn6/SHP1, 311 PTPRo, 633 Pulmonary arterial hypertension, 189, 598 Pulmonary embolism, 96 Pulmonary fibrosis, 51 Pulmonary hypertension, 68, 190, 388 PXR (NR1i2), 478, 557

Q Quercetin, 582 Quinone, 195

R Rab, 439 Rac, 185, 378 Raf, 635 RAGE, 33, 306, 329, 397 Ral, 348 Rap, 135 raphe, 495 RAR (NR1b1/2/3), 123, 132, 466, 557 Ras, 294, 635 RasA GAP, 75 RasGAP, 71 Raynaud syndrome, 68 RBBP, 315 RBC, 163 RBP4, 428, 444, 466 RBPJκ, 78 Redoxome, 148 Redox stress, 34, 68, 102–105, 107, 111, 113, 122, 138, 146, 148, 170, 173, 179, 183, 195, 197, 285, 307, 318, 322, 330, 359, 363, 369, 381, 425, 426, 433, 448, 449, 456, 575, 660, 662 Regulatory T cell, 401, 434 Remote conditioning, 537 Renal corpuscle, 244 Renal dysfunction, 65 Renal glomerulus, 244 Renin, 177, 178, 194, 204, 212, 249, 251, 259, 272, 276

886 Renin–angiotensin–aldosterone axis, 153, 378 Renin–angiotensin axis, 6, 274, 377 Renorenal reflex, 272 Replication stress, 15 Resistin, 444, 465, 494, 569 Restenosis, 183 Restrictive cardiomyopathy, 606 Resveratrol, 317, 583 Retina, 80 Retinoid, 410 ReT receptor, 540 RevERb (NR1d1/2), 557 Reverse cholesterol transport, 346 Rheumatoid vasculitis, 67 Rho, 312, 348, 460, 634 RIPK, 17 Risk factor, 92 Risk marker, 92 RNA editing, 621 RNS, 32 Rock, 195, 380, 381, 425, 448 ROMK, 264 ROS, 6, 27, 51, 114, 122, 137, 141, 146, 148–150, 156, 185, 193, 197, 204, 274, 318, 322, 326, 327, 329, 330, 359, 360, 370, 377, 463, 474, 477, 568, 583, 673 Rostral ventrolateral medulla, 574 Rostral ventromedial medulla, 272 RSC/SMARC complex, 615 RSF dimer, 612 RSK, 125 Runx, 129, 206, 436 RXR (NR2b1/2/3), 123, 466, 486, 557, 645 RyR, 48, 55

S SAa, 353, 397, 475 SAGA, 616 Saposin, 351 ScaR, 7, 107, 144, 192, 346–348, 353, 357, 367, 433, 448, 459, 466, 467 SCD1, 364 S cell, 529 SCFR, 141 SCF Ub ligase, 454, 560, 583 Scleroderma, 68 SctR, 501 Secretin, 498, 501, 529 Secretogranin, 520 Seipin, 439 Selectin, 462, 661 Semaphorin, 135 Sencr lncRNA, 169

Index Senescence, 193, 285, 370, 475, 665 SERCA, 48, 55, 330 Serotonin, 529, 530 Serpin, 474 Sestrin, 29 Sex steroid, 356, 411 SF1 (NR5a1), 557 sFRP, 80, 229, 472 SGK, 293 SHC adaptor, 307 Shox, 415 SIK, 311 Single-nucleotide polymorphism, 193 Sirtuin, 35, 49, 206, 279, 285, 307, 369, 389, 394, 475, 554, 559, 561, 564, 583 Skeletal muscle, 366, 370, 386, 462, 465 SKi, 135 SLC, 239, 323, 324, 333, 400, 460, 480–482, 486, 530, 558, 560, 567, 650 Sleep apnea, 39 SMAD, 79, 85, 88, 89, 665 SMC, 449 SMC dysfunction, 137 smoking, 571, 593 smooth myocyte dysfunction, 65 Snai, 126 SNP, 600, 620, 624, 627, 656 SOCS, 323, 542, 543 SOD, 28, 370 SoD, 285, 661 Sodium nitroprusside, 427 Somatostatin, 528, 529, 537 Sorbitol, 328 Sortilin, 656 Sox, 76, 78, 80, 599, 668 SP, 129, 131, 144, 321, 547 SP1, 139 Spinal cord, 272 Spliceosome, 618 Src, 190, 293 SREBP, 367, 430, 431, 454, 484, 565, 577, 589 SRF, 139, 196, 197, 397, 665 Star, 11 Starvation, 313, 574 STAT, 542, 546, 664, 669 Stearic acid, 365 Stem cell, 190, 524 Stenosis, 41, 95 Steroid aromatase, 11 Steroid hydroxylase, 11 sting, 385 STK3, 140 STK39 (SPAK), 258, 632 Stress fiber, 197, 650

Index Stroke, 3, 43, 44, 94, 102, 114, 160, 305, 353, 500 STUB Ub ligase, 294 Subcutaneous adipose tissue, 405, 418 Substance-P, 20, 272, 422, 528 Sulfiredoxin, 568 Sulforaphane, 582 Sulfotransferase, 14 Superoxide, 31, 370, 477 Superoxide dismutase, 583 Suprachiasmatic nucleus, 508, 551 Sympathetic, 63, 153, 184, 208, 271, 281, 291, 366, 367, 408, 413, 422, 463, 593, 623, 624 Syndecan, 134, 190, 357 Syntenin, 190 Syvn1 Ub ligase, 294, 583

T TAFI, 377 Tangier disease, 653 TASK channel, xv TBK1, 348 TBx, 415, 630 TCAC, 371 T1DM, 15, 349, 412 T2DM, 15, 104, 138, 142, 144, 169, 334, 339, 342, 349, 464, 467–469, 473, 475, 538 TEAD, 249, 664 Telangiectasia, 69, 88 Testis, 9 Testosterone, 8, 470 Tetraspanin, 80, 189, 631 TFPI, 374 TGF, 51, 73, 78, 85, 90, 134, 139, 164, 369, 381, 664, 665, 668 TGFR, 85, 639, 667, 669 TH, 4 Thioredoxin, 299 Thrombin, 582 Thrombocytopenic purpura, 177 Thrombomodulin, 374 Thrombopoietin, 306 Thrombosis, 104, 114, 143, 192, 196, 305, 463, 486, 581, 600, 656, 659 Thrombospondin, 373 Thromboxane, 113, 136, 330 Thrombus, 2, 17, 96, 113, 197 Thyroid hormone, 229, 356, 400, 411, 412, 462 TIE, 72, 73, 599 Tight junction, 254 TIMP, 159

887 Tissue factor, 377 Tissue transglutaminase, 206 TLR, 332, 380, 393, 433, 452, 466, 467, 485 T lymphocyte, 103, 104, 199, 337, 390, 434, 463, 476 TMAO, 100 TMem, 351 TMem100, 86 TMPesS (TMPrsS), 292 TNFSF, 7, 104, 184, 315, 326, 350, 359, 431, 443–445, 455, 463, 474, 476, 569, 581, 661 TOMM, 11 TOR, 171, 175, 312, 319, 320, 333, 487, 539 TORC1, 175, 379, 454, 488 TORC2, 379 tPA, 135, 582 TRAF Ub ligase, 466 Transintestinal CS excretion, 347 TRAPP complex, 439 Tricarboxylic acid cycle, 156, 567 TRIF adaptor, 365 Troponin, 179 TRP, 40, 86, 206, 232, 246, 302, 370, 613, 673 Trx complex, 166 trypsin, 292 TSH, 400 Tspo, 11 Tubuloglomerular feedback, 250 Tuft cell, 523 Tug1 lncRNA, 168 Turner syndrome, 665 Twinkle, 43 Twist, 126, 430

U uca1 lncRNA, 175 UCP, 45, 316, 371, 379, 406 ULK, 630 uPAR, 357 UPR, 117, 599 Uridine, 547 Uromodulin, 271, 634 USF, 454

V Vagus nerve, 497, 498, 511, 513–515, 534, 536, 537 Vasa recta, 254 Vasculitis, 2, 183 Vasculogenesis, 77 Vasoconstriction, 113, 199, 307, 330

888 Vasodilation, 197, 297, 307, 325, 329, 356, 434, 460, 553, 580, 581, 624 Vasopressin, 208, 211, 216, 225, 233, 261, 264, 269, 270, 274, 293, 554 Vaspin, 467 Vault, 119 Vcam, 76, 123 VDAC/porin, 148, 262 VDR (NR1i1), 229 VEGF, 62, 76, 79, 82, 104, 347, 361, 372, 373, 388, 410 VEGFR, 76, 347, 372 Venous malformation, 599 Venous thromboembolism, 102 Ventral tegmentum, 507, 534 Ventromedial hypothalamus, 4, 491 Vimentin, 639 VIP, 503, 528, 554 Visceral adipose tissue, 405, 418 Visfatin, 469, 564 Vitamin-D, 226, 266 VLDL, 145, 341 VLDLR, 357 von Willebrand disease, 177 VRAC, 262 vSMC, 183, 196, 197, 298, 329, 369, 433, 446, 463, 465, 474, 572, 622 vSMC dysfunction, 142 VSOR, 262 vWF, 177

Index W Wall remodeling, 382 Warburg effect, 320 Warburg micro syndrome, 440 WDTC, 432 White adipose tissue, 404 Williams–Beuren syndrome, 659 Wisper lncRNA, 175 WNK, 216, 221, 226, 294, 632 Wnt, 64, 80, 206, 372, 380, 472, 524, 562

X XBP, 599 Xist lncRNA, 175, 622 XOx, 23

Y YAP, 249, 321 YRNA, 119, 143 YY, 129

Z ZEB, 123, 126, 141 Zeitgeber, 551 ZiC, 415 Zinc, 370