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Lymphatic Structure and Function in Health and Disease [1 ed.]
 0128156457, 9780128156452

Table of contents :
Cover
LYMPHATIC STRUCTURE
AND FUNCTION IN HEALTH
AND DISEASE
Copyright
Dedication
Contributors
About the Editors
1
Introduction/overview
2
Ontogeny and phylogeny of lymphatics: Embryological aspect
Historical background
Ontogenic point of view
Phylogenetic point of view
Vasculogenetic point of view
Conclusion
References
Further reading
3
Lymphatic pumping and pathological consequences of its dysfunction
The lymphatic system
Origin and mechanisms of lymphatic pumping
Electrical activity and pacemaker of the lymphatic muscle
Mechanisms of lymphatic contraction
Regulation of lymphatic pumping
Mechanical regulation
Chemical regulation
Lymphatic alterations during disease
Lymphadenopathy
Lymphangiogenesis
Lymphangiectasia
Lymphatic pumping in diseases
Primary lymphedema
Secondary lymphedema
Changes during chronic inflammation
Arthritic diseases
Inflammatory bowel disease
Metabolic syndrome and diabetes
Cancer
Targeting the lymphatic pump for therapeutic benefits
Conclusions
References
4
Hydrodynamic regulation of lymphatic vessel transport function and the impact of aging
Concepts of intrinsic and extrinsic lymph pumps
Intrinsic lymph pump
Extrinsic lymph pump
Modulation of the lymphatic vessel contractility by pressure/stretch
Modulation of the lymphatic vessel contractility by intrinsic and extrinsic flows
Histamine as endothelium-derived relaxing factor in mesenteric lymphatic vessels
Influences of aging
References
5
CNS lymphatics in health and disease
Lymphatics of the central nervous system
The perivascular pathway
The glymphatic pathway
The meningeal pathway
The role of the lymphatic system in diseases of the CNS
Alzheimer's disease
Multiple sclerosis
Cerebral ischemic injury and microinfarcts
Aging
Exploiting the CNS lymphatics for drug discovery
Maximizing drug delivery into the lymphatics
Summary
Acknowledgments
References
6
Defective development of the peripheral lymphatic system: Lymphatic malformations
General aspects
Embryological aspects
Clinical aspects
Genetic considerations (see Table 1)
Management
References
7
Cardiac lymphatics and cardiac lymph flow in health and disease
Cardiac lymphatics
Flow of lymph in the heart
Lymphatics within different cardiac structures
Cardiac inflammation and lymphatics
Valves in lymphatic vessels
Cardiac conditions that may be exacerbated by lymphatic obstruction, lymphangitis, or obliteration
Effects of experimental ablation of cardiac lymphatics
Experimental manipulation of lymphatics as a form of cardiac therapy
Summary
References
Further reading
8
Lymphatic reconstruction
Introduction
History of surgical approaches for lymphatic reconstruction
Current options for lymphatic reconstruction
Lymphaticovenular anastomosis
Vascularized lymph node transfer
Lympholymphatic graft
Conclusion
References
Index
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Back Cover

Citation preview

LYMPHATIC STRUCTURE AND FUNCTION IN HEALTH AND DISEASE

LYMPHATIC STRUCTURE AND FUNCTION IN HEALTH AND DISEASE Edited by

FELICITY N.E. GAVINS Department of Life Sciences, Brunel University London, Uxbridge, United Kingdom

J. STEVE ALEXANDER Department of Molecular & Cellular Physiology, LSU Health Sciences Center, Shreveport, LA, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. Published in cooperation with The International Society for Neurovascular Disease. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-815645-2 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Nikki Levy Acquisition Editor: Melanie Tucker Editorial Project Manager: Kristi Anderson Production Project Manager: Paul Prasad Chandramohan Cover Designer: Alan Studholme Typeset by SPi Global, India

Dedication This volume is dedicated to Dr. D. Neil Granger who has been our steadfast friend and guiding mentor throughout our careers where he had led the Department of Molecular and Cellular Physiology at LSUHSC-Shreveport for over 30 years. As a leading pioneer in both divisions of the blood and lymphatic vascular system, we would like to recognize Dr. Granger and dedicate this edition to him.

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Contributors J. Steve Alexander Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA, United States M. Amore Phlebology and Lymphology Unit, Cardiovascular Surgery Division, Central Military Hospital; III Chair of Anatomy, Buenos Aires University, Buenos Aires, Argentina Felix Becker Clinic of General, Visceral and Transplantation Surgery, University Hospital M€ unster, M€ unster, Germany Anatoliy A. Gashev Department of Medical Physiology, College of Medicine, Texas A&M University Health Science Center, Temple, TX, United States Felicity N.E. Gavins Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA, United States; Brunel University London, Kingston Ln, London, Uxbridge, United Kingdom B.B. Lee Vascular surgery, George Washington University Hospital, Washington, DC, United States Annika Mohr Clinic of General, Visceral and Transplantation Surgery, University Hospital M€ unster, M€ unster, Germany Daniel Palmes Clinic of General, Visceral and Transplantation Surgery, University Hospital M€ unster, M€ unster, Germany Matthew Stephens Department of Physiology & Pharmacology, Inflammation Research Network, Snyder Institute of Infection, Immunity & Inflammation, University of Calgary, Calgary, AB, Canada H Suami Australian Lymphoedema Education, Research and Treatment, Macquarie University, Sydney, Australia Pierre-Yves von der Weid Department of Physiology & Pharmacology, Inflammation Research Network, Snyder Institute of Infection, Immunity & Inflammation, University of Calgary, Calgary, AB, Canada J. Winny Yun Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA, United States David C. Zawieja Department of Medical Physiology, College of Medicine, Texas A&M University Health Science Center, Temple, TX, United States

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About the Editors Professor Felicity N.E. Gavins is a pharmacologist whose research interest focuses on developing antiinflammatory strategies that promote resolution of inflammation following ischemia reperfusion injury (I/RI) in cardiovascular and cerebrovascular diseases. She is specifically interested in the role of the microvasculature as a dynamic interface between circulating blood and immune cells, lymphatics, and tissue. Her group studies how circulating cells communicate, adhere, and migrate across endothelial borders, along with investigating how circulating and resident cells can render systemic inflammatory responses and alter local inflammatory and thrombotic states. More recently, Professor Gavins has expanded her research to also cover microvascular dysfunction in the setting of organ transplantation, as evidenced by enhanced solute barrier function failure, neutrophil recruitment, and endothelial damage. Professor J. Steve Alexander is a cardiovascular biologist working on how small blood vessels become injured during the process of inflammation. Several diseases including stroke, cancer, Crohn’s disease, and diabetes are associated with disturbances in how blood vessels restrict exchange of their contents, leading to the development of edema. His current models indicate the dysregulation of several classes of junctional

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About the Editors

molecules (tight or occludens junctions, adherens junctions, and complexus adherens junctions) and mediate the elevated exchange of solute proteins and inflammatory cells during active inflammation and in transplantation injury. A new and important direction in this area of research is the development of models that consider the lymphatic system and the growth of lymphatic endothelial cells that is known to participate in not only tumor cell metastasis but also acute and chronic inflammation.

C H A P T E R

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Introduction/overview a

Felicity N.E. Gavinsa,b, J. Steve Alexandera Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA, United States b Brunel University London, Kingston Ln, London, Uxbridge, United Kingdom

The lymphatic system consists of the vascular network of tissues and organs (the lymph nodes, thymus, spleen, tonsils, and appendix) that drain interstitial fluid (lymph) from the peripheral tissue back to the blood circulation. The lymphatic system forms part of the well-described vascular system but is not a circulatory system as it is a group of one-way conduits. This incredibly complex network of vessels not only collects lymph, which is an astounding feat at the level of physics, but also plays a crucial role in our immune system; it is the least well characterized and often maligned and marginalized vascular system reflecting the great difficulty in visualizing lymphatics outside of the confines of a fat-meal consumption, which necessitated the identification of many molecular markers that distinguish lymphatic endothelial cells from the blood vascular endothelium. Consequently, there has been more recently an explosion of research into the lymphatic system with the annual number of publications on lymphatics climbing from 2093 in 1990 to 5519 in 2015. The literature is proceeding so quickly that we thought that this area of study deserved at this point a summary of how lymphatics and lymphatic endothelial cells contribute to the functioning of several different organ systems and to health and disease. This text is therefore devoted to the evaluation of how the lymphatic organization and supply in several organ systems may contribute to how these organs function and how their disturbances in

Lymphatic Structure and Function in Health and Disease https://doi.org/10.1016/B978-0-12-815645-2.00001-0

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Copyright © 2020 Elsevier Inc. All rights reserved.

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1. Introduction/overview

structure, patterning, or function may lead to organ stress. The multifaceted roles that lymphatics play in health and disease include contributions gathered from leading experts around the world who are actively working in the rapidly expanding field of lymphatic research. The lymphatic system was first described by Hippocrates of Kos (c.460–377 BC), and as such, B.B. Lee and H. Suami have focused on the evolution and the embryonic development of the lymphatic system in Chapter 2. This team also brings together the recent advance in the field of lymphangiogenesis, highlighting both the physiological and pathological roles played by lymphatic development. As mentioned earlier, the lymphatic system plays a key role in the immune system. In Chapter 3, M. Stephens and Pierre-Yves von der Weid focus on the role that lymphatic vessels and lymphatic contractions play in edema resolution and immune cell trafficking, especially in the context of the intestine and inflammatory processes associated with the gastrointestinal tract. Stephens and von der Weid discuss the implications and significance of lymph node swelling (lymphadenopathy), which occurs in inflammation, infection, and diseases. Finally, this chapter discusses the dysfunction of lymphatic vessel function in a variety of pathologies (often associated with chronic inflammation) such as rheumatoid arthritis and osteoarthritis, elephantiasis, inflammatory bowel diseases, metabolic syndrome and diabetes, and cancer, coupled with discussions regarding the consequences of targeting lymphatic pump for therapeutic benefits. Next, we move to the hydrodynamic regulation of lymphatic vessel transport and the impact that aging has on these events (Chapter 4). In this chapter, A.A. Gashev and D.C. Zawieja discuss the concepts of intrinsic and extrinsic lymph pumps, along with the job that they play in modulating lymphatic vessel contractility. They will discuss the novel regulatory function of histamine as an endothelium-derived relaxing factor (EDRF). Additionally, while the important role that the aging process plays in and the impact it has on the vascular system (termed vascular aging) are recognized, very little is known with respect to lymphatic system and agingassociated changes in the active lymph pumps. As such, A.A. Gashev and D.C. Zawieja will discuss these issues in detail, bringing together their own work and that of the scientific community.

1. Introduction/overview

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The brain is responsible for 25% of the body’s metabolism, even though it only encompasses 2% of the body’s total mass. As with the rest of the body, metabolism leads to the generation of waste products that need to be cleared, and it is particularly important when it comes to the brain that this clearance mechanism is efficient and effective. Therefore, Chapter 5 concentrates on the lymphatic system in the context of the central nervous system (CNS). J.W. Yun et al. discuss the three different brain lymphatic clearance pathways that are currently described, that is, (i) the perivascular pathway, (ii) the glymphatic pathway, and (iii) the meningeal pathway. These different pathways and the role that they play in diseases and conditions of the CNS, such as Alzheimer’s disease (AD), multiple sclerosis (MS), and cerebral ischemia (stroke), are reviewed at length. As discussed in the previous chapter (Chapter 4), aging is a significant contributing factor to peripheral immune responses; additionally, it also plays a considerable role in central immune responses, incorporating both the glymphatic system and brain lymphatic vasculature. Chapter 5 will deliberate aging in the context of the CNS and the lymphatic system and how the CNS lymphatics can be exploited for drug discovery programs to identify new therapeutic targets for the treatment of neurovascular and neurodegenerative disorders. Moving on from the CNS, in the next chapter (Chapter 6), B.B. Lee and M. Amore weigh up the defective development of peripheral lymphatic system (termed lymphatic malformation (LM)), which is one of the various forms of congenital vascular malformations (CVMs) but only affects the lymphatic circulation. It is known that LMs can present clinically as a solitary/independent lesion or coexist with other CVMs, often as a hemolymphatic malformation (HLM). Not only do the authors discuss LMs in detail, but also they provide clinical and management options for patients with, for example, KlippelTrenaunay syndrome. In Chapter 7, the lymphatics of the heart are reviewed by J.S. Alexander and J.W. Yun. The heart is a dynamic organ in terms of its mechanical propulsion of the blood flow, and as such. Its lymphatic system has long remained enigmatic. J.S. Alexander and J.W. Yun discuss the origins and development of cardiac lymphatics, patterning, and functioning, placing particular emphasis on the roles that the cardiac lymphatics may play in clinical

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conditions ranging from surgically induced lymphatic distress, to myocardial infarction, to heart transplantation. In the final chapter of our book (Chapter 8), A. Mohr et al. focus on the concept of the surgical approach of lymphatic reconstruction, which is commonly used for patients with destructed or obstructed lymphatic vessels who are suffering from secondary lymphedema. A main advantage of this surgical approach is that it allows for additional treatment options for the condition of secondary lymphedema especially if patients are resistant to conservative therapy. However, as with every intervention, there are pros and cons, which A. Mohr et al. discuss in detail in this chapter, along with the history of surgical approaches for lymphatic reconstruction. We hope this volume serves as a starting point for readers both scientifically and medically minded alike at the level of students to more advanced readers. Here, we have begun to consider how this often nearly invisible, parallel sister to the circulatory system supports the blood vasculature, accomplishing so many diverse functions that are only recognized when lymphatic dysfunction occurs. The recognition that lymphatics may contribute to many clinical conditions opens vast new vistas for the development of therapies that exploit new findings, mechanisms, and reagents acting on the lymphatic vasculature. Finally, we would like to thank Kristi and Melanie at Elsevier for their patience, encouragement, and support. In addition, we thank all the authors and reviewers without whom this book would not have been possible.

C H A P T E R

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Ontogeny and phylogeny of lymphatics: Embryological aspect a

B.B. Leea, H Suamib

Vascular Surgery, George Washington University Hospital, Washington, DC, United States b Australian Lymphoedema Education, Research and Treatment, Macquarie University, Sydney, Australia

Historical background The lymphatic system was first described by Hippocrates in 460–377 BC as “white blood” based on observation of “chyle,” a type of lymph formed in the digestive system that is transported through specialized lymph vessels known as lacteals [1,2]. He identified the lymphatic system correctly as one of the two major circulatory systems together with the blood vascular system. This newly discovered lymphatic system was further confirmed by Herophilus (335–280 BC) and Erasistratus (304–250 BC) through systematic dissections of human cadavers [3,4]. However, such historical discovery was largely ignored until Gasparo Aselli rediscovered the system in 1627, describing mesentery lymphatic vessels in canines as the “venae albae et lacteae (milky veins)” [5]. Additionally, critical structural components such as the collecting lymphatic vessels and the thoracic duct were also identified for the first time as additional anatomical structures in the lymphatic system. However, this system has been largely neglected for three

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Copyright © 2020 Elsevier Inc. All rights reserved.

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2. Ontogeny and phylogeny of lymphatics: Embryological aspect

centuries, due primarily to limited knowledge about its true value and the technical difficulties involved in identifying its component parts. Thus, the lymphatic system has been considered less important and the often-invisible auxiliary to the blood vascular system. Until recent years, the lymphatic system has been largely ignored, while the blood vascular system has been extensively studied, despite both systems sharing so many functional, structural, and anatomical similarities. For example, the embryonic origin of the lymphatic system was initially investigated more than a century ago [6], but it only attracted proper scientific attention during the era of molecular biology in the last decade, with the result that the mechanisms of mammalian lymphatic development have finally become better understood [7–9]. Indeed, advanced experimental models to study various aspects of lymphatic biology, both in vitro and in vivo, facilitated tremendous progress in the research into the development and function of the lymphatic system; together with various newly discovered molecular markers to distinguish between blood and lymphatic vessels, this research changed the misconception of the lymphatic system as secondary to the more essential blood vascular system for the first time in two decades. Recent advances in our understanding of the embryonic development of the lymphatic vasculature include the molecular mechanisms mediating lymphangiogenesis and the role of lymphangiogenesis in chronic inflammation and lymphogenous cancer metastasis [10]. Indeed, the growing evidence about how the lymphatic system contributes to various inflammatory disorders and lymphedema as well as cancer metastasis has boosted basic lymphatic research on this second vascular system in higher vertebrates beyond its basic function in the absorption of dietary fat in the intestine, immune surveillance, and the regulation of tissue pressure.

Ontogenic point of view Like all vascular structures, the lymphatic conduits arise from endothelial cell aggregates to become an integral element of the mammalian circulation through the coordinated process of

Ontogenic point of view

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lymphvasculogenesis and lymphangiogenesis (Fig. 1). Different theories have been proposed about the origin of the lymphatic system, and it has remained a controversial issue since the early 20th century. There are two competing theories, the “centrifugal” and the “centripetal” models, and some areas of contention remain unresolved [11–18]. The “centrifugal” model is based on the hypothesis that the lymphatic system is derived from the blood vascular system during its early development, while the “centripetal” model claims that

FIG. 1 Step-by-step development of the mammalian embryonic lymphatic vasculature. Development of the lymphatics is composed of two steps: lymphangiogenesis and lymphvasculogenesis. Some of the cardinal vein endothelial cells commit to the lymphatic lineage (second left). The primitive lymph sac is formed by budding off the vein (bottom left). Lymphatic endothelial cells sprout and migrate from the lymph sac to form the lymphatic vascular network (right).

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mesenchymal cell-derived lymphangioblasts form the primitive lymphatic plexuses first before establishing the connections to the embryonic vein. The “centrifugal” model explains that the primary lymph sacs arise from the endothelial cell population of the embryonic veins early in fetal development and that isolated primitive lymph sacs then continue to progress through “endothelial sprouting” to spread as peripheral lymphatic vessels into the surrounding tissues and organs where local lymphatic capillaries form. In the human embryo, the lymphatic vessels appear at 6–7 weeks, substantially later than the blood vascular structures [11] and nearly 1 month after the appearance of the first blood vessels [12]. The paired structures of jugular lymph sacs adjacent to the jugular section of the cardinal vein are the earliest identifiable precursors of embryonic lymphatics. However, the origin of these lymph sacs and their relationship to the adjacent cardinal vein have remained controversial until recently [10,13]. Although Sabin FR demonstrated with experiments on ink injection into the veins of pig embryos that the lymphatic system is derived from the early embryonic vein [14,15], the presence of lymphangioblasts as lymphatic progenitor cells and the critical role they play have also been validated in embryonic development of the nonmammalian lymphatic system [16–18]. In contrast, the “centripetal” model explains the process independently of the veins, postulating that the lymph sacs are developed from lymphangioblasts, the mesenchymal precursor cells [19,20]. Huntington and McClure et al. [20] claimed that the primitive lymphatic vessels arise independently from the lymphangioblasts in the mesenchyme with no connection to the veins until they fuse with the lymph sacs through centripetal growth to establish venous connections as a secondary process. This theory was further supported by the findings of experimental work in the quail-chick chimera system on lymphangioblasts in the avian wing bud before the emergence of the jugulo-axillary lymph sacs [21,22]. The distal wing buds of 3.5-day-old chick embryos were grafted into the same place in 3–3.5-day-old quail embryos to determine the origin of the lymphatic endothelium. The vascular endothelial growth factor receptor-3 (VEGFR-3) and QH1 double staining of the 10-old-day chimeric wings revealed that lymphatics were formed from both chick and quail endothelial cells [22]. This work shows that the early

Phylogenetic point of view

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lymph sacs appear to arise as sprouts from adjacent veins, while at the same time, mesenchymal lymphangioblasts also grow toward those lymph sacs. There is much evidence to support both centrifugal and centripetal theories, but the centrifugal model appears to more closely mirror the process in higher mammals. The studies in Prox1-deficient mice gave crucial support to Sabin’s centrifugal model [23,24] and have subsequently been confirmed by others [25, 26]. However, mesenchymal cells expressing CD31 and CD45 along with lymphatic endothelial markers (Prox1 and LYVE-1) observed in mouse embryos suggest that these cells might serve as lymph endothelial precursors [27]. This gives ongoing support to the centripetal hypothesis [17,28], particularly in the tissue-specific brain, cardiac, intestinal, visceral, and dermal lymphatics [9], despite the fact that Sabin’s claims that lymphatic endothelial cells are exclusively derived from the endothelium of the venous system has been the most widely accepted historically, with further support by Wigle et al. [14,15,24].

Phylogenetic point of view The cardiovascular circulatory system of blood vessels and the heart develops much earlier than the linear conduit system of the lymphatic vessels. However, there is some evidence that this first vascular system included some lymphatic functions to provide all living organisms with crucial defense mechanisms irrespective of their size [29]. For example, macrophages appear first in the circulation of the lympho-hematic system in insects before erythrocytes develop, demonstrating that lymphatic function is primary. Indeed, the early blood vessels in vertebrates have some structural characteristics of lymphatics in that they express the lymph endothelial receptor flt-4, which represents VEGFR-3. This unique characteristic remains until the definitive lymphatics develop later, at which time flt-4 is restricted to those structures alone, which are formed either from mesodermal lymphangioblasts or from the primary vessels [29]. In addition, the heart is not the only organ to pump cells for circulation. “Lymph hearts” are present in lower vertebrates and also develop in some birds transiently. Even in human beings, there is a

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vestigial form of the contractile system for the lymphatic circulation, defined as the “lymphangion,” which circulates immune cells. Hence, the blood vascular system in mammals can develop from the primitive lymphatic system of lymph hearts before the definitive lymphatics develop. Lymphatic vessels independent of the blood vascular system are identified firstly in cartilaginous fish. In bony fish, arteries and veins are connected by blood capillaries and form a closed blood vascular system and are completely separate from the lymphatics [30]. Recently, a primitive lymphatic system was discovered in zebra fish [26,31] that demonstrates evolutionarily preserved structural and cellular features and has become extremely valuable in various genetic studies. The lymphatic vessels are well developed in tailed amphibians such as salamanders and newts. In amphibians without tails such as frogs and toads, the superficial lymphatics fuse during metamorphosis to form large subcutaneous lymph sacs in the adult animals [30,32]. Amphibians, reptiles, and flightless birds have developed a specialized lymph heart for lymph drainage and transport through the lymphatic system (Fig. 2). However, the lymphatic system of

FIG. 2

Schematic diagram illustrating the lymphatics and lymph hearts (arrows) in salamander, frog, and avian embryos.Salamanders have pairs of lymph hearts located segmentally on each side of the lateral lymph duct. Frogs have a pair of anterior and posterior lymph hearts. Avian embryos temporarily develop posterior lymph hearts. Based on Kotani M. The lymphatics and lymphoreticular tissues in relation to the action of sex hormones. Arch Histol Cytol 1990;53:1–76.

Vasculogenetic point of view

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flying birds and mammals lost the lymph heart through the further evolution of the lymphatic system and acquired lymph nodes for immune functions instead. Avian and reptilian lymphatics only differ from mammals in that the jugular lymph sac in mammals is more highly developed as an anterior/cervical veno-lymphatic heart [26,27]. Therefore, the lymphatic system is believed to have appeared first in vertebrates [33] as an essential component of the immune system and to maintain fluid homeostasis within the body.

Vasculogenetic point of view Through the late 1990s, the identification of lymphatic endothelial cell (LEC)-specific VEGFR-3 [34] gave momentum to the study of lymphvasculogenesis study using lymphatic-specific molecular and cellular markers. Although VEGFR-3 expression is detectable in a majority of vascular endothelial cells during early development, VEGFR-3 is the first lymphatic-specific marker restricted to lymphatic plexuses at later stages of development and post development [34]. VEGF-C (a ligand for VEGFR-3)-deficient mice are unable to form rudimentary lymphatic vessels even though the endothelial cells remain committed to the lymphatic lineage. This mutant phenotype can be rescued by VEGF-C and VEGF-D, another VEGFR-3 ligand showing VEGFR-3 specificity [35]. The tremendous increase in knowledge of lymphatic development at the molecular level over the last two decades allowed lymphvasculogenesis to be redefined according to newly discovered tissue markers. Lymphvasculogenesis is now further verified through four distinctively different stages: lymphatic competence, commitment, specification, and vascular coalescence and maturation [13]. Lymphatic/LEC competence to respond to the initial induction signals for lymphatic vascular differentiation [36] is represented by the cellular expression of flt-4 gene that encodes for VEGFR-3 [10] together with lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) [19,37]. They represent crucial molecular signaling pathways to prime LECs to initiate lymphatic development [38,39]. For example, mouse embryos that lack VEGFR-3 expression die without lymphatic development. VEGF-C/VEGFR-3 signaling also

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plays a key role across multiple stages of lymphatic vascular development because VEGF-C binding to VEGFR-3 transduces signals that promote lymphatic endothelial cell survival, proliferation, and migration [40,41] so that mouse embryos that lack VEGF-C do not develop lymph sacs [42]. Lymphatic commitment is represented by the expression of Prox1, which has a central role in explaining the centrifugal mechanism that is a homolog of the Drosophila homeobox transcription factor prospero 7 and serves as a master regulator of lymphatic development. Prox1 expression is the exclusive nuclear transcription to cells of committed lymphatic lineage [19]. This expression represents a clear-cut shift in commitment of the venous endothelial cells to a lymphatic lineage [20]. The expression of Prox1 within the embryonic endothelial cell is sufficient for lymphatic commitment. Prox1-positive lymphatic endothelial cells subsequently bud and migrate away from the veins [9] to form an initial lymphatic plexus and further develop the lymph sacs [43–45]. However, the initiation of Prox1 expression in venous endothelial cells is dependent on the transcription factors Sox18 [46] and Nr2f2/Coup-TFII [47] in mammals [9]. Prox1 is a homolog of the Drosophila homeobox transcription factor prospero 7 and serves as a master regulator of lymphatic development. Lymphatic endothelial cell specification involves the mandatory expression of the molecular markers of LEC identity that lead to the unique lymphatic endothelial phenotype. These include podoplanin and VEGFR-3 and neuropilin 2 [43–46]. When the newly developing LECs leave the veins, the lymphatic cell population establishes complete autonomy in the venous microenvironment through these unique developmental steps. Peripheral migration occurs, and budding and migration precede the formation of primary lymph sacs. The cells form capillaries in a centrifugal fashion, establishing the lymphatic vasculature [10]. Vascular coalescence and maturation. The embryonic peripheral lymphatic vasculature goes through the process of substantial maturation and remodeling, including the development of valve apparatus. FOXC2, which is highly expressed in adult lymphatic valves, specifies a collecting lymphatic vessel phenotype [48,49]. Valve development is also dependent on GATA2 [50]. Selective knockout of GATA2 in the murine lymphatic endothelium leads to major defects in valve structure and causes distended collecting lymphatic

References

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vessels. BMP [51], Notch [52], and semaphorin3a-neuropilin-1 [53] are also known to play an important role in valvular development as additional signaling pathways. The ephrins and the angiopoietins also contribute to lymphatic vascular maturation. Faulty expression of ephrinB2 would result in hyperplasia of the collecting lymphatics and absent valve formation [54]. EphB4 also has a role in signaling in lymphatic vessel valve development [55]. Angiopoietin 1 and 2 (Ang1 and Ang2) also participate in the maturation of the lymphatic vasculature [56–58]. All of these developmental events are interrelated and complex. New molecular participants in the process continue to be identified.

Conclusion Even 400 years after the rediscovery of the lymphatic system by Gasparo Aselli, the lymphatic system remained misunderstood as a secondary vascular system to support the blood vascular system. However, this has now changed. Our understanding of the lymphatic system has significantly advanced over the last two decades, with many landmark discoveries in lymphatic research, especially in the areas of cellular and molecular biology of LECs. These new discoveries have brought to light the molecular control of physiological and pathological lymphangiogenesis and subsequently changed the paradigm to re-evaluate the lymphatic system’s essential role in the human circulatory system in a brand new way.

References [1] Grotte G. The discovery of the lymphatic circulation. Acta Physiol Scand Suppl 1979;463:9–10. [2] Chikly B. Who discovered the lymphatic system. Lymphology 1997;30:186–93. [3] Lord RS. The white veins: conceptual difficulties in the history of the lymphatics. Med Hist 1968;12:174–84. [4] Leeds SE. Three centuries of history of the lymphatic system. Surg Gynecol Obstet 1977;144:927–34. [5] Asellius G. De lactibus sive lacteis venis. Mediolani, Milan,1627. [6] Kanter MA. The lymphatic system: an historical perspective. Plast Reconstr Surg 1987;79:131–9.

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[7] Nakamura K, Rockson SG. Biomarkers of lymphatic function and disease: state of the art and future directions. Mol Diagn Ther 2007;11:227–38. [8] Nakamura K, Rockson SG. Molecular targets for therapeutic lymphangiogenesis in lymphatic dysfunction and disease. Lymphat Res Biol 2008;6:181–9. [9] Kazenwadel J, Harvey NL. Morphogenesis of the lymphatic vasculature: a focus on new progenitors and cellular mechanisms important for constructing lymphatic vessels. Dev Dyn 2016;245:209–19. [10] Cueni LN, Detmar M. The lymphatic system in health and disease. Lymphat Res Biol 2008;6:109–22. [11] Witte MH, Jones K, Wilting J, Dictor M, Selg M, McHale N, et al. Structure function relationships in the lymphatic system and implications for cancer biology. Cancer Metastasis Rev 2006;25:159–84. [12] van der Putte S. The development of the lymphatic system in man. Adv Anat Embryol Cell Biol 1975;51:3–60. [13] Rockson SG. Embryology of the lymphatic system and lymphangiogenesis. [chapter 4]. Section II. Embryology, anatomy, & histology. In: Lee B-B, Stanley G, Bergan J, editors. Lymphedema: a concise compendium of theory and practice. 2nd ed. Springer International Publishing AG; 2018. p. 47–55. [14] Sabin FR. On the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig. Am J Anat 1902;1:367–91. [15] Sabin FR. On the development of the superficial lymphatics in the skin of the pig. Am J Anat 1905;3:183–95. [16] Wilting J, Tomarev SI, Christ B, Schweigerer L. Lymphangioblasts in embryonic lymphangiogenesis. Lymphat Res Biol 2003;1:33–40. [17] Wilting J, Aref Y, Huang R, Tomarev SI, Schweigerer L, Christ B, Valasek P, Papoutsi M. Dual origin of avian lymphatics. Dev Biol 2006;292:165–73. [18] Wilting J, Papoutsi M, Othman-Hassan K, Rodriguez-Niedenf€ uhr M, Pr€ ols F, Tomarev SI, Eichmann A. Development of the avian lymphatic system. Microsc Res Tech 2001;55:81–91. [19] Huntington GS, Mc Clure CFW. The anatomy and development of the jugular lymph sac in the domestic cat (Felis domestica). Am J Anat 1910;10:177–311. [20] Huntington GS. The genetic interpretation of the development of mammalian lymphatic system. Anat Rec 1908;2:19–46. [21] Le Douarin NM. Particularites du noyau interphasique chez la caille japonaise (Coturnix coturnix japonica). Utilisation de ces particularite`s comme ‘marquage biologique’ dans les recherches sur les interactions tissulaires et les migrationes cellulaires au cours de l’ontogenese. Bull Biol Fr Belg 1969;103:435–52. https://doi.org/10.1111/j.0021-8782.2004.00288.x. [22] Schneider M, Othman-Hassan K, Christ B, Wilting J. Lymphangioblasts in the avian wing bud. Dev Dyn 1999;216:311–9. https://doi.org/10.1111/j.00218782.2004.00288.x. [23] Wigle JT, Chowdhury K, Gruss P, Oliver G. Prox1 function is crucial for mouse lens-fibre elongation. Nat Genet 1999;21:318–22. [24] Wigle JT, Harvey N, Detmar M, Lagutina I, Grosveld G, Gunn MD, et al. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J 2002;21:1505–13.

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[25] Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, Tsai MJ, et al. Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Genes Dev 2007;21:2422–32. [26] Yaniv K, Isogai S, Castranova D, Dye L, Hitomi J, Weinstein BM. Live imaging of lymphatic development in the zebrafish. Nat Med 2006;12:711–6. [27] Buttler K, Kreysing A, von Kaisenberg CS, Schweigerer L, Gale N, Papoutsi M, et al. Mesenchymal cells with leukocyte and lymphendothelial characteristics in murine embryos. Dev Dyn 2006;235:1554–62. [28] Pudliszewski M, Pardanaud L. Vasculogenesis and angiogenesis in the mouse embryo studied using quail/mouse chimeras. Int J Dev Biol 2005;49:355–61. [29] Wilting J, Papoutsi M, Becker J. The lymphatic vascular system: secondary or primary? Lymphology 2004;37:98–106. [30] Kotani M. The lymphatics and lymphoreticular tissues in relation to the action of sex hormones. Arch Histol Cytol 1990;53:1–76. [31] Karpanen T, Schulte-Merker S. Zebrafish provides a novel model for lymphatic vascular research. Methods Cell Biol 2011;105:223–38. [32] Hoyer H. Das lymphgefasssystem der wirbeltiere vom standpunkte der vergleicheden anatomie. Mem Acad Polon Sci Lett Med 1934;1:1–205. [33] Rusznyak I, Foeldi M, Szabo G. Lymphatics and lymph circulation. Oxford: Pergamon Press; 1967. [34] Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH, Dumont D, Breitman M, Alitalo K. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A 1995;92:3566–70. [35] Choi IH, Lee SJ, Hong YK. The new era of the lymphatic system: no longer secondary to the blood vascular system. Cold Spring Harb Perspect Med 2012;2. [36] Oliver G. Lymphatic vasculature development. Nat Rev Immunol 2004;4:35–45. [37] Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 2000;60:203–12. [38] Hogan BM, Bos FL, Bussmann J, Witte M, Chi NC, Duckers HJ, et al. Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting. Nat Genet 2009;41:396–8. [39] Bos FL, Caunt M, Peterson-Maduro J, Planas-Paz L, Kowalski J, Karpanen T, et al. CCBE1 is essential for mammalian lymphatic vascular development and enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Circ Res 2011;109:486–91. [40] Makinen T, Veikkola T, Mustjoki S, Karpanen T, Catimel B, Nice EC, et al. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J 2001;20:4762–73. [41] Salameh A, Galvagni F, Bardelli M, Bussolino F, Oliviero S. Direct recruitment of CRK and GRB2 to VEGFR-3 induces proliferation, migration, and survival of endothelial cells through the activation of ERK, AKT, and JNK pathways. Blood 2005;106:3423–31.

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[42] Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova TV, et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat Immunol 2004;5:74–80. [43] Yang Y, Garcia-Verdugo JM, Soriano-Navarro M, Srinivasan RS, Scallan JP, Singh MK, et al. Lymphatic endothelial progenitors bud from the cardinal vein and intersomitic vessels in mammalian embryos. Blood 2012;120:2340–8. [44] Francois M, Short K, Secker GA, Combes A, Schwarz Q, Davidson TL, et al. Segmental territories along the cardinal veins generate lymph sacs via a ballooning mechanism during embryonic lymphangiogenesis in mice. Dev Biol 2012;364:89–98. [45] Hagerling R, Pollmann C, Andreas M, Schmidt C, Nurmi H, Adams RH, et al. A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy. EMBO J 2013;32:629–44. [46] Francois M, Caprini A, Hosking B, Orsenigo F, Wilhelm D, Browne C, et al. Sox18 induces development of the lymphatic vasculature in mice. Nature 2008;456:643–7. [47] Srinivasan RS, Geng X, Yang Y, Wang Y, Mukatira S, Studer M, et al. The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells. Genes Dev 2010;24:696–707. [48] Norrmen C, Ivanov KI, Cheng J, Zangger N, Delorenzi M, Jaquet M, et al. FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1. J Cell Biol 2009;185:439–57. [49] Petrova TV, Karpanen T, Norrmen C, Mellor R, Tamakoshi T, Finegold D, et al. Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med 2004;10:974–81.39. [50] Kazenwadel J, Betterman KL, Chong CE, Stokes PH, Lee YK, Secker GA, et al. GATA2 is required for lymphatic vessel valve development and maintenance. J Clin Invest 2015;125:2979–94. [51] Levet S, Ciais D, Merdzhanova G, Mallet C, Zimmers TA, Lee SJ, et al. Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation. Blood 2013;122:598–607. [52] Murtomaki A, Uh MK, Choi YK, Kitajewski C, Borisenko V, Kitajewski J, et al. Notch1 functions as a negative regulator of lymphatic endothelial cell differentiation in the venous endothelium. Development 2013;140:2365–76. [53] Jurisic G, Maby-El Hajjami H, Karaman S, Ochsenbein AM, Alitalo A, Siddiqui SS, et al. An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation. Circ Res 2012;111:426–36. [54] Makinen T, Adams RH, Bailey J, Lu Q, Ziemiecki A, Alitalo K, et al. PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev 2005;19:397–410. [55] Zhang G, Brady J, Liang WC, Wu Y, Henkemeyer M, Yan M. EphB4 forward signaling regulates lymphatic valve development. Nat Commun 2015;6:6625. [56] Dellinger M, Hunter R, Bernas M, Gale N, Yancopoulos G, Erickson R, et al. Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice. Dev Biol 2008;319:309–20.

Further reading

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[57] Gale N, Thurston G, Hackett S, Renard R, Wang Q, McClain J, et al. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by angiopoietin-1. Dev Cell 2002;3:411–23. [58] Shimoda H, Bernas MJ, Witte MH, Gale NW, Yancopoulos GD, Kato S. Abnormal recruitment of periendothelial cells to lymphatic capillaries in digestive organs of angiopoietin-2-deficient mice. Cell Tissue Res 2007;328:329–37.

Further reading [59] YK H, Detmar M. Prox1, master regulator of the lymphatic vasculature phenotype. Cell Tissue Res 2003;314:85–92. Epub 2003 Jul 22.

C H A P T E R

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Lymphatic pumping and pathological consequences of its dysfunction Matthew Stephens, Pierre-Yves von der Weid Department of Physiology & Pharmacology, Inflammation Research Network, Snyder Institute of Infection, Immunity & Inflammation, University of Calgary, Calgary, AB, Canada

The lymphatic system The lymphatic system is a unidirectional element of the circulation, which is indispensable in human physiology. Composed of a network of vessels connecting all tissue and interstitial space within the body to the cardiovascular circulatory system, the lymphatic system is composed of blunt-ended initial lymphatic vessels and smooth muscle-surrounded collecting vessels that drain to lymph nodes widely distributed around the body. The primary purpose of the lymphatics is to drain excess fluid in the form of lymph and in the same breath transport proteins, lipids, and cells around the body in a tightly orchestrated form of immune surveillance. Without a central pump (such as the heart for blood circulation), the lymphatic system is solely reliant on the intrinsic phasic contractions of lymphatic muscle and on the extrinsic skeletal force to propel lymph back toward the thoracic duct and subsequently back into the circulation. Transporting a substantial volume of interstitial fluid (which can amount to 5–6 L/day) means that there are drastic consequences if the flow is disrupted. Failure to clear

Lymphatic Structure and Function in Health and Disease https://doi.org/10.1016/B978-0-12-815645-2.00003-4

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Copyright © 2020 Elsevier Inc. All rights reserved.

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3. Lymphatic pumping and pathological consequences of its dysfunction

lymph results in localized inflammation, lymphedema, hypoxia, and necrosis caused through stagnation of this protein-rich interstitial fluid. The initial lymphatics, also termed terminal, peripheral lymphatics, or lymphatic capillaries, are composed of a single layer of flattened, nonfenestrated endothelial cells [1]. Typically, significantly larger than blood capillaries, initials are commonly asymmetrical in shape [2–4] and connected to neighboring endothelial cells by button-like junctions, composed of vascular endothelial (VE)-cadherin and tight junction proteins including occludin, zonula occludens (ZO)-1, endothelial cell-selective adhesion molecule (ESAM), claudin-5, and junctional adhesion molecule-A (JAM-A) [5]. Specialized intestinal lymphatics (lacteals) are responsible for a vast majority of fat absorption [6]. They are surrounded by mucosal capillaries in a cagelike lattice, with the base lying just below the basement membrane of enterocytes, thereby providing a strategically beneficial position for the drainage of fluid escaping from the capillaries, as well as absorbed through the mucosa. A pivotal relationship between fat and lymphatics however may exist beyond the gut, as fat deposition is a defining clinical characteristic of lymphedema and a large proportion of the associated swelling is not only fluid but also lipids. It is therefore an up-and-coming area of research looking into the role the fat plays in lymphatic dysfunction and pathogenesis of associated diseases. The lymph content of the initial lymphatics empties into larger collecting lymphatics. The most important difference between initial lymphatics and collectors is the presence of surrounding lymphatic muscle within the vessel wall, which is responsible for the spontaneous contraction of the vessel. The efficiency of these contractions is promoted by the specific design of the collectors, which are segmented into lymphatic chambers or lymphangions, thanks to one-way valves interspersed along the vessels. Constrictions of the lymphangions propel the lymph in a unidirectional fashion toward the thoracic duct. Normal lymphatic contractile function is determined by the intrinsic properties of the lymphatic muscle and the regulation of pumping. Deficiencies in lymphatic contractility, barrier function, or valve defects are typical among pathologies of lymphatic system including inherited and acquired forms of lymphedema, inflammation, lipedema, obesity, metabolic

The lymphatic system

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syndrome, and inflammatory bowel disease. In addition to proteins, lipids, and antigenic material that it transports, lymph also contains a resident and transient population of cells. These immune and stromal cells have the potential to drive, alter, and sustain lymphatic pumping through the secreted content of cytokines, chemokines, adipokines, lymphokines, and extracellular matrix molecules. Measures of these various parameters can be a novel and key indicator to lymphatic health and, by proxy, the surrounding tissue. Disruption with the flow of lymph within a healthy being can result in an inflammatory response within the affected tissue, which otherwise would have occurred within the lymph node. This demonstrates further the importance of correct lymph flow within the body as the dysfunction can lead to not only fluid imbalance and accumulation but also aberrant local immune responses. Spread along the lymphatic vessel network, lymph nodes act as effector venues for adaptive immunity initiation. The number of lymph nodes can vary between people, although the current reason behind the phenomenon and outcomes is still being debated. Within the GI tract, there are numerous nodes acting as draining basins from organs or subproportions of the intestine. Subdivided into three major regions, the lymph node is fed by infiltrating lymph through one or more collecting afferent lymphatic vessels. These feed into the subcapsular sinus where recognition of circulating antigen is primarily done by resident macrophages, before the flow is diverted toward the medullary sinus where, through the efferent lymphatic, screened fluid leaves. Lymph distribution is localized to the lymphatic system via afferent lymphatics, the blood circulation through HEVs, and the parenchyma, which is divided into B-cell follicles and T-cell areas that form the cortex and medulla, respectively. The makeup and structural composition of the lymph node is essential for the correct distribution and movement of antigen, metabolites, and cells throughout the structure so as to create an environment conducive for a strong adaptive immune response. The primary purpose of the lymphatic system is fluid homeostasis. While blood circulation successfully transports a huge volume of blood around the body each day, fluid leakage into the tissue space is not recovered. Retrieval is the lymphatic purpose as around 5 L of interstitial fluid per day is recovered and recycled back into the circulation. Included within this balancing act is the recovery of essential

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3. Lymphatic pumping and pathological consequences of its dysfunction

metabolites, the removal of toxins, and a key method of cellular transport. As mentioned earlier, to promote a strong and correct immune response, antigenic material from pathogens must be transported efficiently to the lymph node for presentation and subsequent adaptive immune response. Flow is initially generated by a hydrostatic gradient; however to overcome increasing osmolarity, the lymph must be physically propelled whether by extrinsic forces such as skeletal contraction and respiratory pressures or by the intrinsic lymphatic pump. It is for that reason that the collecting lymphatic pumping is a critical feature in the lymphatic system and loss of such can have substantial consequences explored within this chapter.

Origin and mechanisms of lymphatic pumping Key to fluid homeostasis, the lymphatic pump is the main driver of lymph propulsion through the body. Without a central pump, such as the blood circulation, the lymphatic system relies heavily on the action of the lymphatic muscle cells surrounding the endothelial layer comprising the collectors. These muscle cells have the intrinsic ability to spontaneously and rhythmically contract and relax. The intrinsic mechanism, called lymphatic pumping, is complemented by extrinsic forces provided by skeletal muscle contractions or respiratory movements, which, by transiently compressing the lymphatic vessels, help lymph propulsion. Efficient lymph drainage also depends on the synchronized contraction wave along the length of the vessel, promoted by the lymphangion compartmentalization stability and function of intraluminal valves to prevent backflow by forming a tight pressure seal. Thanks to these morphological characteristics and the combination of intrinsic and extrinsic forces, lymph can be transported against a hydrostatic pressure gradient in most regions of the body for successful clearance and recycling into the circulation.

Electrical activity and pacemaker of the lymphatic muscle Spontaneous lymphatic pumping is a property of most collectors and is intrinsic to the vessel wall. Lymphatic contraction in human and mammals is driven by electrical changes of the muscle membrane potential exemplified by action potentials [7–12].

Origin and mechanisms of lymphatic pumping

23

The electrophysiological properties of lymphatic muscle are thus critical to its contractile function. As in most smooth muscles, action potentials and constrictions in lymphatics depend heavily on extracellular Ca2+ ions and can be inhibited by voltage-gated Ca2+ channel blockers dihydropyridines [8,13,14]. Involvement of the L-type Ca2+ channel, via its main isoform Cav 1.2, in action potentials was confirmed in rat, mouse, and human mesenteric vessels [15–18]. The voltage dependence of L-type Ca2+ channels and the regularity of the action potentials during lymphatic pumping suggest the involvement of an electrical event that transiently depolarizes the muscle membrane potential (pacemaker potential) to a level where the action potentials can be evoked, leading to calcium entry and phasic contractions [19,20]. Although the origin of cellular events involved in the lymphatic pacemaker remains currently unknown, advancements in lymphatic electrophysiology have allowed a couple of theories to be proposed. Pioneer studies by McHale and his team in bovine and then sheep mesenteric lymphatics showed that the initiation and regularity of the lymphatic action potential bear time and voltage-gated similarities with that observed in the heart and that a hyperpolarizationactivated inward current with properties similar to the sinoatrial node “funny” current If (HCN channels) was functionally present in mesenteric vessels, suggesting that like in the heart, HCN channels may also underlie the lymphatic pacemaker [9,21,22]. A more recent study reported the expression of all four HCN channel isoforms in rat diaphragmatic lymphatics [23]. Importantly, in these studies, pharmacological inhibitors of HCN channels markedly decreased but never totally abolished the frequency of spontaneous constrictions, strongly suggesting that other ion channels are critically involved. Indeed, other currents known to play a role in cardiac pacemaking, such as T-type Ca2+ current, Ca2+-activated Cl current, and fast voltage-activated Na+ current, demonstrated to be expressed in sheep, rat, and human mesenteric lymphatic vessels [15,24,25], have been suggested to also participate to the lymphatic pacemaking. However, similar to HCN channels, pharmacological inhibition of these channels led partial or very limited inhibition of lymphatic pumping, further supporting the idea that other ion conductances are involved.

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3. Lymphatic pumping and pathological consequences of its dysfunction

By studying smaller, more segmented vessels of the guinea pig mesentery, van Helden consistently recorded with a sharp microelectrode small spontaneous transient depolarizations (STDs) of the lymphatic muscle membrane potential. Because, either individually or through summation, STDs underlie the larger action potentials and muscle contractions, he proposed them as the lymphatic pacemaker [26]. This hypothesis is supported by a number of observations reported in this original paper and subsequent studies [20,27–30]. STDs reflect the opening of Ca2+-activated inward current carried by Cl ions upon the “packeted” release of Ca2+ from IP3-sensitive stores within the muscle cells [25,29]. This hypothesis that ClCa channels underlie STDs and participate in the lymphatic pacemaking is also in agreement with the growing body of evidence showing that ClCa channels are involved in agonist-induced smooth muscle contraction [31,32]. The identity of ClCa channels has only recently been determined as anoctamin-1 (ANO1), also known as TMEM16a. ANO1 has been shown to be expressed in human, mouse, and rat lymphatics [33] (von der Weid unpublished), and ANO1 current recorded from lymphatic muscle cells acutely dissociated from mouse inguinalaxillary collectors (SD Zawieja and MJ Davis, personal communication). The important role of ANO1 in lymphatic pumping was further demonstrated by the strong reduction of lymphatic contraction frequency and a lack of response to increase in transmural pressure after selective deletion of the Ano1 gene in mouse lymphatic muscle cells (SD Zawieja and MJ Davis, personal communication). However, action potentials were still occurring, although at a much slower rate, again suggesting that other conductances, such as those discussed earlier, are likely involved in the lymphatic pacemaker. While it is important to keep in mind that different tissue preparation or animal species may account for the variation in ion channel contribution to the pacemaker mechanism, differences in experimental conditions should also be considered. Notably, to allow successful recordings of membrane potential and the pharmacological modulation of STD activity with sharp microelectrodes, experiments were mostly performed on quiescent, unstimulated vessel segments, displaying what we can consider true “spontaneous contractile activity.” On the other hand, assessment of the effects of these blockers on lymphatic pumping was typically

Origin and mechanisms of lymphatic pumping

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performed on isolated vessels under pressure/stretch conditions that may activate a pacemaker mechanism requiring activation of a different set of ion channels than spontaneous pacemaking. It is well known that the degree of distension or stretch of the lymphatic vessel wall is the most important determining factor of the intrinsic lymphatic pumping [34,35]. STD frequency was shown to increase with stretch in rat mesenteric lymphatics mounted on a wire myograph [36], and with intraluminal flow [20,37], other ion channels may underlie the response to stretch or pressure.

Mechanisms of lymphatic contraction In the absence of characterization of the contractile and regulatory elements of the lymphatic muscle contractile apparatus, it had been generally thought that lymphatic muscle cells were smooth muscle cells. However, in a seminal study, Muthuchamy et al. [38] revealed that lymphatic muscle contains both striated and smooth muscle contractile elements and displays differences in both contractile function and contractile machinery when compared with blood vessels, demonstrating the uniqueness of the lymphatic muscle. Specifically, mesenteric lymphatic muscle cells exhibit genotypic and phenotypic characteristics of vascular, cardiac, and visceral myocytes, as they express SMB smooth muscle myosin heavy chain (with both SM1 and SM2 isoforms detected) and messages encoding the fetal cardiac/skeletal slow-twitch muscle-specific β-MHC, and four different actin isoforms, including cardiac α-actin, vascular α-actin, enteric γ-actin, and skeletal α-actin, have also been detected [38]. Preliminary data also demonstrate that lymphatic muscles express striated muscle regulatory components, such as cardiac troponin C and cardiac troponin T (MM Muthuchamy, personal communication). This particular muscle phenotype likely provides the strong contractile ability of the mesenteric vessels. Other functional differences between lymphatic and vascular smooth muscles have been demonstrated, including myofilament Ca2+ sensitivity and cooperativity [39] and lower active tension development [40]. Our understanding of the molecular mechanisms regulating tonic and phasic contractions of lymphatic muscle also improved over the last decade. Wang et al. [41] showed that while phosphorylation of the myosin light chain 20 (MLC20), which triggers cross-bridge

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3. Lymphatic pumping and pathological consequences of its dysfunction

cycling and force development, primarily modulates mesenteric lymphatic tonic contraction, it does not significantly affect the amplitude of phasic contractions suggesting that tonic contraction strength and phasic contraction amplitude of the lymphatics are differentially regulated. Lymphatics exhibit a significant decrease in tone in response to elevated pressure; in rat thoracic duct, it correlated with a decrease in phosphorylation of MLC20 at both of its phosphorylation sites [42]. Using pharmacological tools, Hosaka et al. [43] demonstrated the involvement of Rho kinase and myosin phosphatase in lymphatic tonic contraction but maybe also in phasic contractions (see also reviews by von der Weid and Muthuchamy [44] and Chakraborty et al. [45]). Critically, mesenteric lymphatics exhibit myogenic responses to changes in intraluminal pressures and rate-sensitive contractile responses to stretch [46,47]. Thus, thanks to these unique mechanical and contractile characteristics, composed of both smooth and striated muscle regulatory mechanisms, the lymphatic muscle can accomplish its essential function in the maintenance of the normal lymph transport and in protection against the formation of gross edema and other pathological conditions.

Regulation of lymphatic pumping Mechanical regulation Regulation of the lymphatic pump is important to maintain fluid balance in response to altered fluid load. Under basal condition, an increased interstitial and intraluminal pressure results in a corresponding rise in contraction frequency to a maximum, beyond which flow drops due to a loss in stroke volume. As such, typical profiles of lymphatic pumping in response to pressure appear as bell-shaped curves and are observed in many animal species. All lymphatics have an optimal pumping condition at relatively low transmural pressures [34]. These pressures tend to be higher in more peripheral lymphatic vessels, suggesting that more peripheral lymphatics can develop much higher pressures to prevail over the greater outflow resistance [48]. For instance, mesenteric lymphatics have the highest fractional pumping (6–8 volumes/min at optimal pressure levels) [34].

Origin and mechanisms of lymphatic pumping

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Lymph flow also affects the contraction frequency of lymphatic vessels. Benoit et al. [34], measuring lymph flow in situ in rat mesenteric lymphatics by elevating lymph formation, found increased parameters of active lymph contractility during periods of increased lymph flow, while the pressure in the lymphatic network became less pulsatile at high lymph flow states. Although such experiments give important information on lymphatic contractile behavior, it is difficult to clearly separate the effects of increased flow from the effects of increased transmural pressure. A study on rat iliac microlymphatics showed that increases in intraluminal perfusion correspond to an increase in contraction frequency and a decrease in the amplitude of contractions [49]. But during increases in imposed flow in rat lymphatics, inhibition of both amplitude and frequency of contraction was observed [50,51]. However, it is difficult to conclude that such imposed flow-dependent inhibition of the active lymph pump decreases total lymph flow in vivo. At high levels of lymph formation, passive lymph flow could become a greater driving force to move lymph than the active lymph pump. Flowdependent inhibition of the active lymph pump in such situations could be a reasonable physiological mechanism to save metabolic energy by temporarily decreasing or stopping contractions during the time when the lymphatic does not need it. Inhibition of the lymph pump under these circumstances could also reduce lymph outflow resistance, as a result of a net increase in average lymphatic diameter when contractions are inhibited. This reduction in outflow resistance could ease the removal of fluid from the affected compartment that is producing high lymph flows and thereby facilitate the resolution of edema [50,52].

Chemical regulation Lymphatic pumping is heavily modulated by factors present in the lymph or in fluid surrounding the vessel, whether they are released from nerves, cells present in the interstitium, the vessel itself or from blood. These factors include endothelium-derived mediators, neurotransmitters, and many circulating hormones and inflammatory mediators (reviewed in von der Weid [53], von der Weid and Rehal [54], and Pal et al. [55].

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Like blood vessels, lymphatic behavior is regulated by endothelium-derived factors such as nitric oxide (NO), prostaglandins, and histamine [56–59]. Both lymphatic tone and spontaneous contractions are inhibited by NO produced as a result of shear stress on the endothelium [51]. During the contraction-relaxation cycle, shear stress increases with lymph flow during the contraction phase of the cycle. This causes a rapid and transient production and release of NO, which contributes to interrupt the contraction and promotes vessel dilation [60]. NO inhibits the spontaneous contraction through arresting calcium release associated with the pacemaker, STDs [30], and activated ATP-sensitive K+ channels to hyperpolarize the lymphatic muscle [61]. Prostaglandins are among the most important regulators of lymphatic vessel function. Although they can be produced by surrounding tissues, these metabolites of arachidonic acid are also synthesized by the lymphatic endothelium, which expresses cyclo-oxygenase 1 (COX1) [58,62–64]. Histamine has more recently been demonstrated to be also produced by the lymphatic endothelium and participate to the flow-dependent inhibition of pumping [59]. Depending on species and vessel location, histamine increases the frequency of lymphatic pumping via activation of H1 receptors located on smooth muscle cells or decreases contraction frequency via H2 receptor stimulation or via an indirect effect mediated by the endothelium [27,65,66]. Histamine is also predominant in mast cell granules and is the main mediator of increased lymphatic pumping caused by mast cell activation, as demonstrated in a milk-sensitized animal model, a model of food allergy [67]. As will be discussed in “Changes during chronic inflammation” section, other cells, such as macrophages or mast cells, present in the vicinity of lymphatics in inflammatory situations, produce and release NO, prostaglandins, and histamine to potentially alter lymphatic contractility [68]. Even if sparse in comparison with neighboring blood vessels, innervation contributes to the regulation of lymphatic contractility [69,70], and several neurotransmitters and neuromodulators have been shown to modulate lymphatic contractility. Localized adrenergic and cholinergic nerve fibers were identified around initial and collecting guinea pig mesenteric lymphatic vessels [70,71]. Norepinephrine generally increases contraction frequency. This effect, mediated via excitatory α-adrenoreceptor (both α1 and α2 receptor

Lymphatic alterations during disease

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subtypes), can be modulated by an inhibitory action via β-adrenoreceptor (β1 and β2) stimulation, depending on the class of adrenergic receptors expressed in the tissue [69,72–74].

Lymphatic alterations during disease Many diseases present an inflammatory component. A subset of these inflammatory diseases also have observable changes within lymphatic structures that can have slight or drastic effects on the affected party. Preclinical and clinical studies have shown changes in the lymphatic vasculature in several disease types including those affecting the skin, gut, and cancer microenvironments to mention but a few. The manifestation of change can occur at any point within the lymphatic circulatory system including structural changes to initial lymphatics, collecting lymphatics, or even the draining lymph node.

Lymphadenopathy Lymphadenopathy is one of the most obvious changes to occur to the lymphatic system during inflammation, infection, or disease. Swelling of the lymph node is characterized by the infiltration of peripheral immune cells to the lymph node cortex, expansion and proliferation of the resident T cells and B cells within the node, and fluid accumulation as a result of lymphatic net-outflow disruption [75]. The feature itself is a sign of an immune response, as antigen surveillance, and response to such is the primary purpose of the lymph node [76]. However, in chronic inflammatory conditions, systemic inflammation, such as that found in murine models of rheumatoid arthritis (RA), the lymphadenopathy can become uncontrolled and result in node exhaustion and collapse [77]. The regulation of lymph flow to the lymph node via the afferent lymphatic and away from the node through the efferent lymphatic is regulated by fluid dynamics and pumping characteristics. It has been demonstrated that insufficient flow to the lymph node through dysfunctional pumping not only reduces the incidence of lymphadenopathy but also can result in localized tissue inflammation resultant in inflammatory phenotypes such as elephantiasis [78].

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3. Lymphatic pumping and pathological consequences of its dysfunction

Transport of antigenic material to the lymph node “effector venue” is paramount for a correct immune response. With the lymphatic pump being an important part of this process, a correct flow is critical in the system.

Lymphangiogenesis Lymphangiogenesis, or proliferation of new lymphatic vessels, is another feature of many tissues that are undergoing development or have undergone an inflammatory insult (Reviewed in Halin and Detmar [79]). Sprouting of lymph vessels within a tissue is driven by vascular endothelial growth factor C (VEGFC) and its receptor vascular endothelial growth receptor 3 (VEGFR3) that, expressed on lymphatic endothelium, promotes proliferation and wound healing [80,81]. The promotion of lymphatic architecture expansion is presumably in aid of fluid drainage and clearance of cells and materials from the affected site although, functionally, this seldom occurs. While the blood capillary form of angiogenesis is short lived within a site, any lymphangiogenesis that occurs within the area is long lived. The reason and effect of its presence are still debated to be beneficial or detrimental, such as the long-last lymphatic alterations seen within the small intestinal inflammation resultant from DSS treatment in mice [82]. The analysis of lymphangiogenesis within different tissues demonstrates a wide array of phenotypes depending on how the system responds. While it is known that all new lymphatic vessels branch from preexisting endothelium, the organization and distribution of these new vessels are notably poor, leading many to wonder whether they are actually beneficial or even functional. With the discovery of markers of lymphatic vessel subtypes, differentiation of initial and specialized lymphatic structures can be identified through antibody labeling of LYVE-1, Prox1, VEGFR3, and podoplanin, further allowing investigation into the effects and purpose of lymphangiogenesis within a disease.

Lymphangiectasia As described earlier, collecting lymphatics propel large volumes of fluid, cells, and metabolites, thanks to phasic contraction of the muscle cells embedded in their wall. Inhibition of

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this activity leads to lymph stasis, which is typically accompanied with dilation of the vessels. This dysfunction, known as lymphangiectasia, can have many consequences on the surrounding tissue. Dilation of the vessels can be caused by many means, as described in “Chemical regulation” section (see also von der Weid and Muthuchamy [44]), but an important contributor is NO. This gaseous molecule dilates lymphatic vessels within a matter of seconds and halts pumping when present in high enough concentrations [44,83]. Leaky vessels, along with the failure of lymphatic contractility, primarily result in interstitial fluid accumulation, which has many indirect consequences. Due to a reduction in fluid flow to the lymph node, antigen transport from a sight of damage or injury is dampened leading to an insufficient immune response [84]. In the latter study, Liao et al. further demonstrated that iNOS-expressing CD11b+ Gr1+ cells, while reliant on the lymphatics to propel them toward the lymph node, also produce the NO responsible for vessel dilation and pumping inhibition, thereby impeding the immune response. The movement of fluid is also key in catabolite removal and nutrient recycling; lymphangiectasia, therefore, can cause a toxic accrual of metabolites promoting sterile inflammation. The presence of sterile inflammation has been demonstrated to improve lymphatic function after lymph node transfer in a tolllike receptor-dependent fashion, providing a new area of research into the regulation of contraction through innate immune cell activation [85,86].

Lymphatic pumping in diseases As mentioned, disturbed lymph flow can result in impaired immune response, pathogen, and metabolite clearance. It is therefore feasible to suggest that the reduction in lymph clearance, seen in many diseases, could be a contributing factor to its pathogenesis. The loss of lymphatic pumping can occur in many diseases, some of which have obvious signs of a reduced lymph clearance, such as lymphedema, while others present less obvious signs but are inherently still important.

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Primary lymphedema In primary lymphedema (congenital/hereditary), fluid collects as a consequence of obstruction, malformation, or underdevelopment of various lymphatic components. These dysfunctions can occur within the initial lymphatics, the collecting lymphatics, or the lymph node. Lymphedema usually affects regional drainage from extremities in the upper and lower limbs and can lead to swelling, skin thickening and hardening, and pain. All features of lymphatic alterations can be seen within lymphedema patients, including lymphadenopathy, lymphangiectasia within the affected limb/s, and lymphangiogenesis within the tissue, specifically the skin dermis. Research in the past two decades used advanced high-resolution magnetic imaging to monitor changes within the lymphedematous tissues of lymphedema patients, demonstrating many cutaneous alterations within the dermis [87]. Almost 20 years later, M Detmar’s group discovered that not only was prominent lymphatic hyperplasia evident but so was immune cell infiltration and accumulation within the dermis of the murine tail-model of lymphedema and was associated with tissue fibrosis, swelling, and reduced wound healing efficacy, common phenomenon observed in human counterparts [88]. Lymphedema itself can result from mutations in a variety of genes including prox1, vegfr3, gata2, foxc2, and flt4, leading to malformations of the vessels, valve formation, or cell-cell adhesion [89–92]. FOXC2 having been identified in high incidence within lymphedema-distichiasis has been shown to alter interactions between LECs and pericytes resulting in the valve-defect phenotype [93]. The VEGFR3 mutation prevalent in hereditary primary lymphedema such as Milroy’s disease is an amino-acid shift mutation that inactivates the receptor preventing correct interaction and activation with VEGFC and VEGFD, key players in lymphatic growth [94]. As the flow of lymph could be disrupted on many levels, the consequence of any of these mutations alone or in collaboration could be detrimental to fluid flow and lead to tissue fluid accumulation. Little is documented about the morphological changes within the tissue prior to the established disease; however, what is clear is a prominent alteration in collecting lymphatic pumping efficacy [95–97]. Collecting lymphatics play a central

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role in the onset of lymphedema, although the changes observed can include occlusion of the vessel, dilation, loss of functional lymphatics, or fibrosis acting as a benign blockader of efficient contraction [98,99], a phenomenon that can now be observed within patients through the use of indocyanine green fluorescence lymphography [100].

Secondary lymphedema While the etiology of primary forms of lymphedema can be linked to lymphatic-associated genetic mutations, secondary lymphedema, the more common forms of lymphedema, arises from insults to the lymphatic architecture at the initial or collecting lymphatic level, or removal/disruption of the lymph node. It is common for patients presenting with secondary lymphedema to have enlarged, weak collecting vessels in the affected limb when compared with the healthy bilateral controls suggesting issues in fluid drainage and associated lymphangiectasia. However, whether lymphedema precedes or follows, lymphatic dysfunction is still debated as both lymph vessel ablation and fluid overload can both affect function. While histological changes within human disease states display altered lymphatic capillary size and density, transitional studies in animal have been hampered by a lack of models able to replicate all aspects of the disease and more specifically its chronicity. However, while lasting only a few weeks, the edema caused in the mouse tail by surgical ablation of a ring of skin, one of the only useable and current model of animal lymphedema, certainly displays lymphangiectasia and many feature of the human condition [101,102]. Lymphedema itself can affect all limbs in the upper and lower body hemispheres and can quickly become a burden to those afflicted, limiting movement. Visceral lymphatics can also be affected although the occurrence is lower than peripheral. The causality of the disease can be a result of many anatomic changes, including lymphatic hypoplasia or the absence of functional valves through mutations in associated genes such as FOXC2 and others mentioned earlier.

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Changes during chronic inflammation Defined by swelling, redness, and heat, inflammation is a burden of the medical world that has a number of initiators and as such can be treated in a variety of manners. Lymphatic dysfunction is a key mediator of inflammation as, through stagnation of fluid, cellderived molecules and infiltrated peripheral immune cells can be exacerbated by and result in lymphatic dysfunction and further inflammatory insult. Lymphatic contractile dysfunction is often associated with tissue inflammation, a phenomenon first described during endotoxin infusion in sheep where, through the hypothesized actions of prostaglandins, edema formation was seen [103]. More recent work has focused on the role of NO by regional iNOS positive cells regulating contractile function within collecting lymphatics [84]. These iNOS positive cells include the resident populations of immune cells such as macrophages, dendritic cells, or mast cells that reside on and within the vessel wall and can respond to lymphatically trafficked antigens [104,105]. These resident cells can modulate contraction through mechanisms other than NO including the release of prostaglandins [68], histamine [67], and cytokines such as IL-1β [106] and TNFα [107], which negatively impacts upon the vessel pumping and could also alter its barrier function [108]. There is mounting evidence that interactions between lymphatic vessel stromal cells and immune cells exist influencing localized effects, migration, and adaptive immune stimulation. Active lymphangiogenesis occurring during inflammation is a process dominated by the production of lymphangiogenic growth factors secreted from tissue-resident macrophages [109–111]. In addition to their paracrine function, macrophages are also able to induce neolymphangiogenesis through transdifferentiation into lymphatic endothelial cells incorporating themselves as such and into the lymphatic vessels [112,113], a phenomenon also observed by Wilting’s group using transgenic mice during development [114]. During inflammation, this transdifferentiation event is prominent, with many groups recording macrophage to lymphatic endothelial transitions in LPS-induced peritonitis [115] and even diabetes [116]. Although the contribution of these resident immune cells in modulation of the lymphatic pump remains to be further investigated,

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it is reasonable to hypothesize that during inflammation, these populations of immune cells within the vessels may alter pumping through chemotaxis of peripheral cells, production of inflammatory mediators, and dedifferentiation processes within the vessels and surrounding tissue altogether promoting inflammation. These cells could also directly impact the flow of lymph through vessel relaxation, reduce contraction frequency, and permeate the endothelial layer through modulation of tight junction proteins. Leakage of lymph into the peripheral tissue could further drive inflammation through promotion of a localized immunogenic response a feature commonly seen in many chronic inflammatory conditions.

Arthritic diseases Many inflammatory joint diseases are associated with lymphatic alterations. Swelling, pain, joint remodeling, and endpoint surgical intervention are common to many forms of arthritic diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). However, in such highly inflammatory and degenerative diseases, the changes in lymphatic structures are widely overlooked yet present. While the literature yields low numbers of studies linking lymphedema and inflammatory rheumatological diseases, new approaches to treatments have greatly improved the lives of those suffering. Lymphedema within this disease group is primarily isolated to the highly inflammatory pathologies such as RA [117] but is observed in smaller numbers in psoriatic arthritis [118] and ankylosing spondylitis [119]. Studies demonstrating a possible link between joint disease and lymphatic function occurred through the use of the transgenic TNFα-overexpressing mice, which showed that the popliteal lymph node enlarges during the prearthritic phase, subsequently collapsing during active joint disease [120]. The prearthritic phase was associated with contractile dysfunction, assumedly through the buildup of inflammatory mediators, after which the pumping pressure of the afferent lymphatics is diminished following lymph node collapse resulting in severely compromised lymph flow [121]. In recent years, the involvement of the lymphatic system, primarily the drainage function, has been identified as a potential model for the treatment of human RA. The inflamed synovium (pannus)

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creates a highly localized inflammatory environment packed with proinflammatory cytokines, degenerative enzymes, and immune cells, which can all have direct effects upon the lymphatic architecture. It is thought that through chronic disruption of the collecting lymphatics, fluid accumulates within the joint leading to localized swelling. In the context of our focus, chronic damage to the lymphatic endothelium and surrounding muscle cells leads to loss of lymphatic vessel contraction and subsequent lymph node collapse. As such, does the reduced lymphatic drainage result in the observed synovitis and joint erosion? In theory, restoration or increased lymphatic drainage from the affected site facilitates the removal of catabolic factors, cytokines, and inflammatory cells from the pannus. Mouse models of osteoarthritis (OA), such as the meniscalligamentous injury model or the transforming growth factor β (TGFβ) type II knockout mice, have associated lymphatic maladies [122]. The severe cartilage loss and joint destruction, evident in the disease, appear in tandem with capillary and mature lymphatic vessel changes. It has been documented that a majority of lymphangiogenesis occurs within the soft tissues surrounding the joint. Within patients, lymphatic alterations can be noted to differentiate early to mild OA, where lymphangiogenesis is rife with increased density of vessels, to severe OA that shows a distinct loss in mature lymphatics and increased swelling, due to decreased lymph clearance from the afflicted joint [122]. Treatment targeting inflammation and degradation within the joints of OA patients may also affect associated lymphatics as nonsteroidal antiinflammatory drugs (NSAIDS) such as ketoprofen, by blocking the action of COXs impeding lymphatic contraction through the regulation of spontaneous smooth muscle contraction [123]. As a multifactorial manifestation, lymphedema within arthritic disease is incredibly complicated to dissect and study. The multifaceted disease itself proves to wax and wane in a cyclic rhythm of chronicity in a manner common to many inflammatory diseases, interestingly corresponding with the occurrence of lymphatic dysfunction. Mounting evidence suggests these changes include an initial compensatory expanding phase, in which the lymph node expansion and contraction of collecting lymphatics are proficient. Soon after, however, intrinsic lymphatic contraction fails, and the lymph node decreases in volume entering a collapsed stage

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corresponding with disease flare. This new information provides hope for the targeting of lymphatics as a treatment during arthritis-associated inflammation, whereby it appears that until the lymphatic collapse, the joint swelling and acute phase of the disease has not taken hold. Stalling in this phase could be a poignant treatment for many sufferers of RA although more studies need to be performed.

Inflammatory bowel disease Lymphatic vessels are not usually considered key players in any disease whose pathologies are not directly linked, such as inflammatory bowel disease (IBD). While not evidently visible, because revealed only after surgical intervention, overloaded lymphatics, lymphangiogenesis, and lymph node remodeling are hallmark features of these diseases (reviewed in von der Weid et al. [124]). Crohn’s disease is described as a “regional ileitis” whereby the disease can affect anywhere along the GI tract but primarily manifests at the terminal ileum [125]. Interestingly, regional lymphatic dysfunction around the sites of tissue inflammation is regularly reported (reviewed in D’Alessio et al. [126]). This observation led to the suggestion that lymphatic vessel obstruction and ineffective relocation of the immune response to the mesenteric lymph nodes perpetuated the chronic localized inflammation. Through artificial damage or blockade of the mesenteric lymphatics with silica particles, sutures, or cauterization, researchers were able to mimic many of the phenotypes of Crohn’s disease ([127,128]. However, not all features are conserved, and the use of such an artificial system questions the accuracy of the models and will remain under scrutiny by peers for years to come. Other than benign obstruction of lymphatics, chemical treatment such as surgical instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in the guinea pig ileum causes an inflammation recapitulating many common effects to the lymphatic dysfunction, such as loss in contractions and dilation of vessels [129]. Similar results have been demonstrated within the rat TNBS model of colitis whereby an altered lymphatic microenvironment resulted in sustained inflammation within the ileum [105]. Unfortunately, due to the lack of pumping within the mouse mesenteric collecting lymphatics, no data are available in relation to

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contractile function changes in genetic or treatment models of Crohn’s disease in this animal. However, mouse models do provide valuable insights into lymphatic alteration during associated GI inflammation. For example, a vast array of lymphatic alterations has been observed in the mesentery of the TNFdeltaARE model of Crohn’s disease [130] and in the dextran sodium sulfate (DSS)induced colitis model [130]. Lymphatic vessel dilation and associated edema seen within IBD patients undergoing surgery are theorized to be a consequence of lymphatic obstruction, impaired lymphatic contraction, or a combination of both [124,127,131,132]. Tissue fluid accumulation would suggest issues in fluid drainage, which can lead to local mucosal hypoxia, systems commonly associated with CD [133]. Furthermore, the lack of clearance of other particulate matter, such as macromolecules, dead cells, bacterial components, and antigen-bearing innate immune cells, can promote site-directed infection and a delay in adaptive immune response [134]. However, the exact mechanisms behind lymphatic contractile dysfunction are still not fully characterized. Regardless of the mechanism, fluid buildup ultimately results in lesions within the intestinal mucosa such as those seen in IBD [135]. Although lymphatic dysfunction was observed in human cases of IBD, it was rarely thought of as a causative agent, especially where more aggressive symptoms lay upstream. Granuloma formation within the lamina propria, along with potent lymphangiogenesis, is seen as more targetable areas of therapy in prevention of primary lesions in CD and UC [136].

Metabolic syndrome and diabetes In recent years, metabolic syndrome and diabetes have become one of the largest global health issues with the International Diabetes Federation (IDF) estimating 25% of the world’s population present with metabolic syndrome [137]. Chronic inflammation underlying these diseases drives remodeling of collecting lymphatics and impairs their functions, which ultimately results in inadequate lymph flow. While the effect upon blood microvasculature had been associated with metabolic syndrome, it was only recently that Zawieja’s group noted lymphatic alterations in a rat model of metabolic syndrome and suggested their possible contribution to disease pathogenesis. They demonstrated severely

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reduced lymphatic capacity within rats suffering from metabolic syndrome and a reduced diameter of prenodal collecting lymphatics under pressurization resulting in a significantly reduced ability to generate pumping force [138]. With the apparent disturbance of the lymphatic system within the mesentery, the contribution of surrounding adipose tissue and resident cells has come under scrutiny. Notably, high-fat diet-fed mice have been shown to display functional impairment of collecting lymphatics consistent with remodeling of the associated tissue lymphatics [139]. It therefore draws into question whether diet-induced obesity and involved conditions such as metabolic syndrome and diabetes are drivers of lymphatic dysfunction or rather a consequence. Additionally, with the importance of the lymphatic system in the transport of dietary fats and lipid mediators such as LDL, what effect could these alterations have on the body at distant sites? An exemplar study in support of this concept occurred through the use of LDL receptor knockout mice, which suggested that the accumulation of cholesterol in arteries and skin from mice was a resultant factor of poor lymphatic drainage, a phenomenon re-established by the restoration of lymphatic flow [140]. Scallan et al. also revealed that NO contributes to collecting lymphatic vascular integrity disturbance in a type II diabetic model of mice. Through a novel advanced imaging technique, the lab demonstrated that the leptin receptordeficient (db/db) obese mice had “leakier” collecting lymphatic vessels than wild-type counterparts [141]. It was also noted that the decreased function and enhanced permeability were due to a low bioavailability of NO, a factor as previously mentioned, to be important in the regulation of pumping.

Cancer Metastasis, described as the development of secondary tumors at a distance from the site of primary cancer establishment, is the most common reason for fatality in many forms of cancer [142]. The movement of cancerous material throughout the body primarily occurs through the lymphatic vasculature and is evident by the secondary sites of metastasis being most often the peripheral lymph nodes. Primarily occurring through the lymphatic system, cancer metastasis can elicit effects upon the system itself, modulating many factors that can result in impaired immune responses, susceptibility

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to infection, and modulation of cancer growth. Tumor microenvironment-derived growth factors such as VEGFC and VEGFD promote lymphangiogenesis within and close to the primary tumor, thereby accelerating its potential to spread (detailed in Wang et al. [143]). It is seen that the expansion of initial and collecting lymphatics around solid tumors requires key remodeling of smooth muscle cells, a process that can ultimately result in rapid expansion of cancer correlated to altered lymphatics [144]. The generation of new lymphatic vessels and modification of preexisting lymphatic structures are thought by many to be an important step in metastasis due to the prevalence of incidence in numerous forms of cancer. However, it is a sign of poor prognosis as the cancer has more than likely spread and colonized within sentinel lymph nodes and distal organs. With such an apparent involvement of the lymphatics in cancer metastasis, it has been suggested that modulating the changes within lymphatics may be beneficial to prevent cancer progression. Lymphangiogenesis at the primary tumor site is a common phenomenon in many forms of primary human cancers such as melanoma [145]. Originally, it was thought that tumors were devoid of lymphatics and that the importance of blood vasculature was the preferred method of transport of shedding tumor cells into the periphery, a theory only debunked within the last 15 years [146]. However, it has become apparent that tumorderived growth factors including VEGFC and VEGFD drive the formation of intratumoral lymphatic vessels, an important process in cancer establishment and metastasis seen in many tumor types and an observable clinical feature. VEGFC was noted to be a predictive marker for aggressive metastasizing cancers over a decade ago with even low-grade intratumoral lymphangiogenesis being predictive of the progression of laryngopharyngeal carcinoma, promoting its use as a predictive diagnostic tool [147]. Later, the analysis of solid tumors within gastric cancer, by Ikeda and colleagues, demonstrated that lymphangiogenesis correlated to increased incidence of metastasis and a significantly lower survival rate [148]. While all cancers may not present with lymphangiogenesis, the importance of the system in the context of cancer proliferation and metastasis makes it a hot topic of research to this day additionally displaying the importance of lymphatics in the context of cancer. Beyond the primary tumor, distant collecting lymphatics undergo substantial remodeling. While many studies implicating

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VEGFC and VEGFD with tumor lymphangiogenesis focus primarily on the tumor microenvironment and structural alterations held therein, the knock-on effects this has on the lymphatic vasculature as a whole are still being discovered. Observations in altered antigen uptake, flow, and presentation during tumor metastasis have directed studies into focusing on antitumor immunity in the context of being lymphatically modulated (reviewed in Lund [149]). As described in “Lymphangiogenesis” section, lymphatic dysfunction in the form of cancer-acquired secondary lymphedema is one of the most common secondary conditions affecting patients that have undergone surgical intervention. One of the most commonly acquired forms of lymphedema is a consequence of breast cancer treatment, where, after surgical excision of the axillary lymph nodes, a classical treatment during breast cancer surgery, fluid accumulates. The accumulation of fluid, while not lethal, can be debilitating, and due to the stretching of dermal layers, prolonged presence of immune cells and chronic inflammation is susceptible to infection such as cellulitis [150]. Prominent lymphatic alterations are an observed clinical phenomenon within cancer patients. Whether this be through lymphangiogenesis at the sites of inflammation, failure in collector pumping, or localized infection as a product of failed antigen clearance, the disturbed lymphatic system can have many detrimental effects. Ongoing research into this area and novel treatments and surgical interventions such as lymph node transplant has been somewhat successful although not universally effective. Confounding these results is the evidence suggesting that the incidence of secondary lymphedema within the patient cohort is not direct, and as such, the exact cause and importance of the lymphatic system are still debated [151].

Targeting the lymphatic pump for therapeutic benefits The potential for the manipulation of the lymphatic system as a therapeutic approach has been a dream of many in the research community. Current treatments for lymphedema are insufficient and usually limited to resolving symptoms rather than targeting the cause. Skin care, manual lymph drainage, compression, and exercise remain the frontline therapies for all suffering from lymphedema

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despite the underlying cause. In the treatment of inflammatory diseases such as RA, the introduction of disease-modifying drugs does not always resolve the lymphatic alterations and is therefore commonly seen as an unlinked phenomenon. Interestingly, however, several studies into the use of anti-TNF therapy within the treatment of rheumatological diseases may be effective for lymphedema treatment with etanercept significantly reduced lymphedematous swelling in RA patients with associated inflammation [152–154]. The therapy is thought to act by reducing inflammation within the inflamed joint and thereby the adjacent inflammation within affected lymphatics, thus reducing fluid accumulation. However, in the same manner as many RA patients, the efficacy of this treatment is poor and can aggravate other underlying issues with many having reported the paradoxical effects of anti-TNF therapies promoting underlying psoriasis, suggesting that its use should be carefully considered. For IBD sufferers, the use of biologic treatments is limited due to the varying efficacy on the resolution of symptoms. At the time of writing this, six biologic agents are approved for the treatment of IBD, four that target TNFα (infliximab, golimumab, and certolizumab pegol) while the other two that target integrins (natalizumab and vedolizumab). The full spectrum of effects that these treatments have upon the lymphatics has not been characterized although with the known effect of TNFα on lymphatic pumping modulation and the involvement of α4β7 integrins in endothelial cell migration and lymph node B-cell adherence, there could be significant overlap. Time and further analysis will demonstrate the efficacies of these treatments upon IBD progression, although they may only regulate the inflammatory symptoms of the disease rather than the underlying cause, suggested by many to be lymphatically modulated. Currently, a base of research aims at resolving disease through the restoration of correct lymphatic flow identifying it as the primary source of pathogenicity. Several groups are investigating this avenue at the moment through the modulation of lymphangiogenesis and lymphatic pumping with varying results. While the outcomes of these studies have been globally inconclusive, the information gleamed from them has developed ideas into treatments and new targets. Selective drug delivery through lipophilic

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agents and regulation/restoration of the lymphatic flow through pumping modulation are but some of the ideas [155]. In many inflammatory conditions, the accumulation of fluid through lymphatic disruption creates many symptoms not associated with the actual primary disease manifestation. Chronic damage, matrix remodeling, and tissue necrosis are all common side effects to several inflammatory diseases such as IBD, RA, OA, and cancer, and targeting the lymphatics to aid in the resolution of the condition is an innovative novel strategy for therapeutic intervention. However, the purpose of lymphatic remodeling within these diseases is still not fully understood; as such, interfering with the natural process of events may further exacerbate disease pathogenesis. Therefore, fully understanding the consequences of lymphangiogenesis, lymphangiectasia, and other such lymphatic changes is paramount for researchers and clinicians alike. It is through developing imaging techniques such as near-infrared lymphoscintigraphy that the efficacy of lymphatic pumping-targeted treatments can be monitored in a clinical scenario further allowing us to understand the importance of this system, which until recently has been widely ignored. With the recent recognition of the interstitium as an organ [156], the development of research involving lymphatics are sure to expand even further, relating to the close interplay of the system in almost every facet of disease establishment and progression.

Conclusions Divided into three major components, the lymphatic system is seemingly rather simplistic. Collection of fluid through initial lymphatics is driven by hydrostatic pressure gradients, after which transport to the lymph node is modulated by pumping of collecting lymphatics where the fluid can be screened by the innate resident cells and any antigen-presenting cells can promote an adaptive response. However, if any step of this process is altered, the effects can be unfavorable. The collection of lymph and transport of its components are undoubtedly the most important step in this structurally hierarchical system as without flow, the system is obsolete and would solely rely on cellular migration and diffusion. During diseases of lymphatic involvement, a common feature is a failure

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of this pumping system, which in turn disrupts fluid balance, immune defense, and even the uptake of dietary material. The advent of better imaging systems and techniques has allowed for real-time visualization of lymphatic pumping down to the cellular level. With the information from these techniques, key players have been identified to modulate lymphatic contractility, whether through genetic susceptibility, physical obstruction, and remodeling of lymphatic architecture, such as that found during cancer progression, or novel involvement of the lymphatics in chronic inflammatory disease features, such as those found in IBD and arthritic disease. Unfortunately, despite the advancements, a vast majority of interactions, consequences, and etiologies remain unknown. Continuing work and novel ideas will undoubtedly answer some of these questions and aid in the development of tools for the management of lymphatic-associated diseases.

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Hydrodynamic regulation of lymphatic vessel transport function and the impact of aging Anatoliy A. Gashev, David C. Zawieja Department of Medical Physiology, College of Medicine, Texas A&M University Health Science Center, Temple, TX, United States

Concepts of intrinsic and extrinsic lymph pumps There are several driving forces, varying by their origin, that support lymph flow. Historically these forces are divided into two groups by reference to their sources: inside or outside of the spontaneous contracting lymphangions (the sections of lymphatic vessels between adjacent valves) [1–3]. The term “active” or “intrinsic” lymph pump describes the lymph driving force that is generated by the active spontaneous contractions of lymphangions themselves. The term “passive” or “extrinsic” lymph pump summarizes together the influences of all other forces that are not generated by active phasic and tonic contractions of lymphatic muscle cells across lymphatic vessel wall. These latter forces may support more or less the lymph flow in different regions of body.

Intrinsic lymph pump There is no single pump in the lymphatic system similar to the cardiovascular system where the energy of heart contractions is enough to move blood through the whole blood circulation. Due to the fact that the beginnings of lymphatic capillaries (initial

Lymphatic Structure and Function in Health and Disease https://doi.org/10.1016/B978-0-12-815645-2.00004-6

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lymphatics) do not normally have direct connections with blood capillaries, the energy of contractions of the heart cannot be used directly to create a pressure gradient in the lymphatic system sufficient enough to propel lymph centripetally all way toward the central veins. Moreover, a negative net pressure gradient exists in the lymphatic system, as lymph pressures closer to their final output (central veins) are higher than the basal intralymphatic pressures near the lymphatic capillaries [4] (described in more details later). Therefore, the lymphatic system possesses its own chains of serially arranged intrinsic pumps—lymphangions, the contraction energy of which is absolutely necessary for lymph flow in humans and majority of mammals. Since the driving force created by one such small pump (i.e., a single lymphangion) is not enough to propel lymph all way down through the lymphatic system, lymphatic vessels are organized in chains of these pumps. Coordinated contraction of these serial pumps appears important to effective lymph flow as there are numerous reports that have demonstrated the coordinated propagation of peristaltic-like contractile waves along lymphatic vessels [5, 6], while there is little to no evidence that either simultaneous contraction of the lymphatic vessel occurs momentarily along all of its length from capillaries toward the local “output” of this regional net nor that the lymphangion contractions are completely independent of one another. For some of regional lymphatic nets (like in lower limbs), the presence of interrupted fluid column in lymphatic vessels was demonstrated during the normal contractile activity of lymphangions [7–9], and for such situations, adjacent lymphangions contract in counterphase fashion [9]. As such, each lymphangion can be principally described as a short-distance local intrinsic pump whose primary “task” is to drive bolus of lymph only to fill next one long or few shorter lymphangion pumps. However, altogether, the chains of such pumps are capable of maintaining effective long-distance transport of lymph. During the active contractions of lymphangions, the lymphatic muscle cells create an increase of intralymphatic pressure and form a local positive pressure gradient to propel lymph centripetally. As a result of such intrinsic spontaneous pumping activity of lymphangions, a positive (supportive to lymph flow) pressure gradient occurs near

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the downstream front of propagating contraction zone in lymphatic vessels (whether this zone includes one or several lymphangions contracting at the same time is unknown). At the upstream edge of contracting zone, a negative (opposing to lymph flow) pressure gradient exists between contracting lymphangions and upstream relaxing lymphangions. This gradient generates the short-lasting local reversed flow and following valves closure at the input edge of the contracting zone in lymphatic vessels [5,10,11]. The majority of lymphangions’ systolic contractile force is used to produce an increase of intraluminal pressure and propel lymph. However, some part of energy generated by lymphatic contraction will be utilized to produce deformation of collagen and elastic fibers in lymphatic wall. During diastole, these fibers will release their remaining tension and lead lymphatic wall to expand. To provide experimental evidence of this hypothesis, measurements of the pressure inside single isolated bovine mesenteric lymphangions have been performed in conditions when diastolic pressure inside lymphangions was set to 0 cm H2O [12,13]. The pressure tracings obtained in this study demonstrated the development of negative fluctuations of pressure inside these lymphangions of up to 5 cm H2O during diastole. It was concluded that at low levels of lymphangions filling (seen during the relatively low lymph formation states), such negative pressure waves may produce a suction such that the energy of active lymphatic pump will be used not only for emptying of the lymphangion during systole but also for the lymphangion refilling during diastole. More detailed measurements of intraluminal pressure performed recently [11] strengthen these conclusions on the existence of “suction pressure” in collecting lymphatic vessels, which manifests as a transient drop in pressure downstream of the inlet valve following systolic contraction. This suction opens the inlet valve and is required for filling in the presence of low upstream pressure. A positive transmural pressure is required for this suction, providing the energy required to reopen the vessel. Alternatively, external vessel tethering can serve the same purpose when the transmural pressure is negative. Suction is transmitted upstream, allowing fluid to be drawn in through initial lymphatics from interstitial space [11].

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Extrinsic lymph pump The unique feature of the lymph dynamics is that the driving forces generated by the active lymphatic pump are greatly influenced by the action of different extralymphatic forces. These forces can sometimes have a greater impact on lymph flow than active pump does, and the sum vector of these forces is not always favorable to lymph flow. The action of variable extralymphatic forces complicates pressure and flow patterns in the lymphatic network and may vary dramatically [14,15], even in the same region of the body depending on the level of activity of tissues and organs surrounding lymphatic vessels. The term “passive” or “extrinsic” lymph pump combines all extralymphatic forces that influence lymph flow. The origin of these forces is not connected with the active contractions of muscle cells in lymphatic vessel wall. But the use of the term “passive” is correct only in referral to the active contractions of lymphangions and is not completely correct from the point of view that all of these forces are generated by different active processes that are just nonrelated to the lymphangion contractions. Extrinsic lymph driving forces include driving forces of lymph formation (historically also called as “vis a tergo”); influences of the surrounding tissues (affected by cardiac and arterial pulsations, contractions of skeletal muscles adjacent to the lymphatic vessels, gastrointestinal peristalsis, and respiration); and forces affecting outflow/afterload, that is, central venous pressure fluctuations. All of these forces may affect the hydrostatic gradients in the lymphatic network that could effectively propel lymph, even sometimes in the absence of lymphatic vessels contractions. Some of these extrinsic lymph driving forces, such as influences of lymph formation and central venous pressure fluctuations, affect lymph flow in the whole body; influences of others are more or less localized. Lymph flow directly depends on the intensity of lymph formation. In general, lymphatic capillaries have their own capacity limits. Therefore, the faster the volume of recently formed lymph rises inside the certain regional lymphatic capillary network, the quicker excessive lymph volumes will move downstream into corresponding collecting lymphatic vessels. Other extralymphatic forces may influence the emptying of lymphatic capillaries and the pumping

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lymphatic vessels too. In gastrointestinal lymphatic network, intestinal peristalsis has a great impact on lymph formation and transport. An increase in intraluminal pressure in the gut directly increases the rates of lymph formation and pressure in intestinal initial lymphatics with subsequent increase in lymph flow through a network of mesenteric collecting lymphatics [16]. In the thoracic cavity, cardiogenic and respiratory tissue motions have a direct influence on lymphatic pressure by the generation of rhythmic expansions and compressions of lymphatic wall [14,15,17]. Many lymphatic vessels are affected by contractions of adjacent or surrounding skeletal muscles, which cause alternate compression/ re-expansion of the lymphatics and create additional forces to empty and refill the lymphatic vessels. That has been demonstrated through the direct correlation of the local values of lymphatic pressure and levels of lymph flow with the intensity of skeletal muscle activity in legs [18]. Thus, the values of intraluminal pressure and its gradients in lymphatic networks depend upon the contractile activity of lymphangions and actions of extrinsic forces. Pressure measurements in the different parts of lymphatic system demonstrated the presence of pressure fluctuations that are connected with contraction of lymphangions. Although it was demonstrated that increments of pressure of 1–1.5 cm H2O is sufficient to open a closed valve, lymphatic vessels generate pressure peaks about 5–10 cm H2O higher than baseline diastolic intralymphatic pressure [12,19–21]. As there are tremendous difficulties to measure pressures in different parts of lymphatic system in situ, only a few observations can present a comparatively complete range of pressures along lymphatic system or along some of its comparatively long parts. Until now, the most inclusive representation of pressure patterns was done by Szabo and Magyar [4]. These authors published the results of their systematic measurements of pressure in major lymphatic trunks in dogs that were obtained during cannulations of different lymphatic vessels. The authors demonstrated that mean intralymphatic pressure in thoracic duct was 5.11 mmHg (6.95 cm H2O) and in right lymphatic trunk was 2.13 mmHg, while jugular venous pressure was 5.83 mmHg. In more peripheral lymphatic vessels, the mean intralymphatic pressure were as follows: left jugular trunk 0.85 mmHg, efferent lymphatic trunk of heart 2.91 mmHg,

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bronchomedial trunk 2.09 mmHg, hepatic trunk 3.43 mmHg, intestinal trunk 3.60 mmHg, left lumbar trunk 2.72 mmHg, and femoral lymph vessel 0.51 mmHg. This study was the first in which detailed evidence has been presented for the fact that there is no constant positive (supportive to lymph flow) pressure gradient along the entire lymphatic network. The data of this study confirmed that the action of so-called vis a tergo (or force of lymph formation) is limited probably by the initial capillary segments of lymphatic net and by the actual levels of lymph formation there. Majority of the time, the lymphangions in collecting lymphatic vessels are faced to produce the extra force needed to prevail over the usual negative pressure gradient between the segments. These pressure differences are dependent on the action of gravitational forces, the wide variability of influences of skeletal muscles contractions, and the presence of competent valves along the lymphatic vessels [4]. In bipedal humans, the negative pressure gradient along lymphatic net may be even much higher than in animals. Olszewski and Engeset [18,22,23] performed lymphatic cannulations using T-shape tubes to measure the lateral pressures. At rest, the mean lateral systolic pressure in the leg lymphatic vessels with free lymph flow (measurements of side-pressure) was 13.5  8.01 mmHg, the lymph pulse amplitude was 8.8  4.6 mmHg, and contraction frequency was 2.42  1.88/min. All these values rose sequentially from a horizontal rest position, to a horizontal position with flexing of the foot, to an upright rest position, to upright with rising toe position. In the last case, these values were as follows: peak pressure 23.8  6.15 mmHg, the lymph pump pulse amplitude 9.67  4.08 mmHg, and contraction frequency 5.5  1.04/min. The authors calculated mean lymph flow by measuring the movement of a minute air bubble introduced into tubing inserted into both ends of leg lymph vessel. It was 0.25  0.04 mL/h (0.004 mL/min) in the horizontal position at the rest and 0.76  0.26 mL/h (0.012 mL/min) in the upright position rising on toes. Lymph flow was only observed during the pump pulse waves, and there was no flow in between the pulses, even when massaging was applied. Several other groups performed measurements of lymphatic pressures in the human legs, and these data presented in general the same order of magnitudes of intralymphatic pressure in this region of body. In 1991, Krylov et al. [24] published

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measurements of lymphatic pressure before lymphography when they catheterized leg lymphatic vessels and immediately measured the end-lymphatic pressure in them. The values of these pressures measured on dorsal surface of foot were low, 0.86–1.1 mmHg. Then, during the catheterization of more downstream lymphatic vessels in the leg, lymph pressure pulses have been recorded about 2–20 mmHg in amplitude from basal levels of 8–17 mmHg. The authors visibly correlated the elevations of lymph pressure with the contractions of lymphatic vessel. Zaugg-Vesti and others published [25] measurements of lymphatic capillary pressure in healthy volunteers. Pressure was measured using the servo-null technique in the distal forefoot proximal to the base of the first and second toes. Mean lymph capillary pressure was 7.9  3.4 mmHg, and pressure fluctuations of more than 3 mmHg were found. In another study, this group described the influences of postural changes on cutaneous lymph capillary pressure at the dorsum of the foot [26]. Mean lymph capillary pressure was 9.9  3.0 mmHg in the sitting and 3.9  4.2 mmHg in the supine position. In general, the comparatively high values of resting intralymphatic pressures and peak pressure fluctuations described earlier reflect much higher outflow resistance for the leg lymphatic network in humans and the physiological demands for local lymphangions to develop much stronger contractions than in animals. Pressure measurements in the human thoracic duct are much more limited, and some of them represent the tracings only of endlymphatic pressure [27] with pressure fluctuations of about 5–10 mmHg in amplitude from a basal level near 30 mmHg. But even in those pressure measurements that could be considered as measurements of side-pressure [28], the majority of thoracic duct pressures fluctuated between 14 and 22 mmHg on average. These levels are much higher than the basal resting pressures in leg lymphatic network and represent the outflow lymph pressures within the lymphatic network. However, during inspiration, the intralymphatic pressure in the thoracic duct may drop to slightly negative or slightly positive values [28], which could temporarily make the net pressure gradient favorable to lymph flow. However, it is not completely clear if these short temporal patterns of pressure changes in thoracic duct directly influence lymph flow only locally in thoracic cavity, but not in the legs which lymphatic

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vessels located as far as on almost all height of human body and many times possess interrupted fluid column in them [7–9].

Modulation of the lymphatic vessel contractility by pressure/stretch Transmural pressure is an important physical factor of lymph dynamics, which influences the contractile activity of lymphangions causing inotropic (changes in the strength of contraction) and chronotropic (changes in the contraction frequency) effects. Transmural pressure is defined as the pressure gradient across vessel wall and is affected by intralymphatic and extralymphatic forces. In collecting lymphatics, two main forces may produce increases in intravascular (intraluminal) pressure and cause lymphangion filling and distension of lymphatic wall. These forces are the driving force of lymph formation and pressure pulses generated by contractions of upstream lymphangions. The influences of several extralymphatic forces on the lymphatic wall may help to expand lymphatics but in other situations may lead to vessel compression. Since the studies of Florey [29,30], Smith [31], and Horstmann [32,33], it has been postulated that the generation and distribution of lymphatic contractions depend exclusively on mechanical stimuli. Traditional paradigms postulate that distension of the lymphatic wall activates the lymphatic contraction, which generates a pressure pulse sufficient to propel lymph to the next lymphatic segment. Later in numerous studies performed both in vivo and in vitro [5,34–41], it was shown that increases in transmural pressure caused positive inotropic and chronotropic effects in lymphatic vessels. Lymphatics from different tissues and species reach the maximums of their pumping at the different values of intravascular pressure. These values found for most tissues are comparatively low and vary in different species and different regions between 3 and 15 cm H2O. Further increase of intraluminal pressure causes overdistension of the lymphatic wall and diminishes pumping. McHale and Roddie particularly demonstrated [5] that isolated bovine mesenteric lymphatic segments that contained five to seven lymphangions were able to increase frequency of contractions and stroke volumes during the increase of transmural pressure from

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1 to 4 cm H2O. These lymphatic vessels reached their maximums of pumping at transmural pressures of about 4–5 cm H2O. Further increase in transmural pressure led to a decrease in stroke volume. Frequency of lymphatic contractions continued to rise, but this positive chronotropic effect of the increase in transmural pressure did not compensate for the negative inotropic influences of continuing distension and flow felt at transmural pressures 6 cm H2O and higher. Principally, the same patterns of lymphatic contractile behavior in response to increased transmural pressure were observed by Ohhashi et al. [36] Isolated one- or two-lymphangion segments of bovine mesenteric lymphatic vessels with outer diameters 0.5–3 mm had their pumping maximums between 5 and 10 cm H2O of intraluminal end-diastolic pressure. Typical bell-shaped curves of the pressure/pumping relationship was shown for different regions and for different species. But it is important to mention that for smaller lymphatic vessels located more peripherally, the maximums of lymphatic pumping occurs at higher values of transmural pressure. Such was demonstrated for the rat mesentery there: the lymphangions located nearer the intestinal wall had five times greater lymph propulsion ability thanlymphangions nearer the outflow from the mesentery [42]. For another example, in the maximum pumping in sheep prenodal popliteal lymphatic vessels, the greatest pumping ability was observed at values of transmural pressure near 18–26 cm H2O, and greater than 50% pumping occurred between 12- and 43-cm H2O [41]. Recent studies demonstrated the same tendency in the bovine mesenteric lymphatic network [43]. Bovine prenodal mesenteric lymphatic vessels are able to increase contractility during the increases in transmural pressure and reach the pumping maximums at transmural pressures 6–9 cm H2O. During further increases in transmural pressure, their contractility and pumping were not significantly depressed up to 15 cm H2O, whereas pumping in bovine postnodal mesenteric lymphatic vessels is typically depressed at transmural pressures higher than 10 cm H2O. Thus, when comparing the pumping capacities of pre- and postnodal lymphatic vessels, the bovine prenodal mesenteric lymphatic pumps appear to be adapted to overcome the fluid resistance of the lymphatic nodal architecture. Together, these data appear to indicate that more peripheral lymphatic vessels may develop higher pressures

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to prevail over the greater outflow resistance in their particular location, and the conditions for their optimal pumping are shifted toward higher values of intraluminal/transmural pressures accordingly. Later, new evidence was obtained to demonstrate the regional variations in pressure-induced changes in lymphatic contractility; studies were performed on lymphatic vessels taken from four different regions of one species—rat [44]. The local differences in pressure sensitivities and pumping ability were determined for thoracic duct, cervical, mesenteric, and femoral lymphatic vessels. All these lymphatics were able to increase their pumping during moderate increases in transmural pressure up to some pumping maximum. The largest pump productivity was observed at 3 cm H2O transmural pressure for all lymphatics except mesenteric lymphatics, where maximum pumping occurred at a pressure of 5-cm H2O. Moreover, detailed analysis demonstrated that all these lymphatics had a range of transmural pressures over which there were no significant large differences in pumping. Experimental data demonstrate that these ranges of pressure were as follows: 2–4-cm H2O for the thoracic duct, 2–8-cm H2O for cervical lymphatics, 2–7-cm H2O for mesenteric vessels, and 2–9-cm H2O for femoral lymphatics. These data reveal that all selected lymphatic vessels have their optimal pumping conditions at comparatively low levels of transmural pressure that were comparable with typical in situ lymph pressures [39] and that these pressure levels have a tendency to be higher in more peripheral lymphatic vessels. The highest pumping at the optimal pressure levels (6–8 volumes/min) was demonstrated for mesenteric lymphatics, and the lowest pumping (2 volumes/min) was seen in the thoracic duct in the rat. Recently, similar studies of regional lymphatic contractile heterogeneity were performed in mice [45]. These authors identified an important regional dichotomy in the mouse lymphatic pump function dependent on the location of the lymphatic collectors. They observed that lymphatic vessels isolated from five different subcutaneous/peripheral regions; cervical, popliteal, inguinal, axillary, and internodal inguinal axillary exhibited strong lymph pumping (with maximal ejection fractions of 50%–80%) similar to those described earlier in rat and other larger mammals, while lymphatic vessels isolated from the deep/visceral lymphatic

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network; mesenteric, thoracic duct, and iliac were not effective lymph pumps (with maximal EFs < 15%). This lack of an effective lymph pump agrees with the data we have also seen in mouse visceral lymphatics [46]. To evaluate what the difference between the active pumping subcutaneous and nonpumping visceral mouse lymphatic function was due to, the authors assessed the membrane potential and L-type calcium channels in the muscle cells of those vessels. While they observed some differences in the electrical and L-type calcium channel activities, they concluded that “it is unlikely that the lack of robust spontaneous contractions in visceral vessels is explained entirely by L-type calcium channels.” The question of what these important differences in lymphatic contractile pumping activity of the mouse mean in terms of its physiological and pathological consequences remains to be determined. However, the authors conclude that “care must also be taken in understanding the limitations of using lymphatic vessels from within the visceral cavity of the mouse” [45] in studies of lymphatic contractile function in physiological and pathophysiological states. Due to the importance of pressure stimuli for lymphatic contractility, the idea that distension stimuli are mandatory to generate lymphatic contractions has dominated the literature for many decades. But in several studies, it was reported that lymphatic vessels could contract in a coordinated fashion without significant distension stimuli [5,21,34,47]. Moreover, experiments performed on lymphatics from different tissues and species showed a high percentage of cases in which the contractile wave propagates along the vessel in a direction retrograde to flow [3,6,39,48,49]. At low or normal levels of lymph formation, in many tissues, lymphangions at the end of contractions are nearly empty [9]. Due to the presence of highly competent valves in lymphatics, the stretchdependent activation of several upstream lymphangions in such situations is very unlikely. Particularly, Mislin and Rathenow noted [3] that the contractile wave could propagate in the retrograde direction through several lymphangions unconnected with the increase of the local transmural pressure. More recent studies [12,13,50] demonstrated for 80% of lymphangions poor or no correlation between experimentally generated fluctuations of their intraluminal pressure and lymphatic contractions. Moreover, it was also shown [12,13,50] that isolated bovine and rat mesenteric lymphatics can have stable

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long-lasting spontaneous contractility at 0-cm H2O intraluminal pressure and in the absence of significant radial or axial distensions. These data lead to the reasonable conclusion that the distension of the lymphatic wall by intraluminal/transmural pressure is an important factor to modulate the contractile activity in lymphatic vessels but is not a mandatory factor for pacemaking.

Modulation of the lymphatic vessel contractility by intrinsic and extrinsic flows As discussed earlier, lymph flow is generated as a resulting complicated combination of influences of active and passive lymph driving forces/pumps. As the peaks of the actions of passive lymph pumps are often not synchronized with intrinsic contractile activity of lymphangions, flow profiles in lymphatic nets are extremely variable and complex. Only the presence of valves in lymphatic vessels prevents the existence of extended periods of backflow and supports net unidirectional lymph flow. On other hand, the presence of lymphatic valves additionally complicates the lymph flow profile. During the lymphangion systole, the pressure difference between contracting and relaxing lymphangions causes the temporal lymph backflow and leads to closure of downstream valve(s) [5,10,36]. The bulblike shape of valve sinuses (the portion of lymphangions immediately downstream to the valve) that exist in a majority of lymphatic vessels is a structural factor that could promote the formation of local transitional/nonlaminar flows during the lymphatic valve closure [10]. The unique shape of lymphatic valves is another structural factor that by itself could complicate lymph flow profile locally during the lymphangion diastolic filling. The space between two valve leaflets is much narrower than lymphatic lumen in nonvalve areas of lymphangions. During lymphatic filling, the narrow valve-containing section between lymphangions could be a factor that causes local temporal accelerations and nonaxial lymph flow. Moreover, although lymphatic valves more commonly contain two leaflets, observations of up to five-leaflet valves are also described in literature [51]. Our own, yet unpublished, observations demonstrated that in rat mesenteric lymphatic vessels, the adjacent lymphatic valves could be oriented by 90 degrees to each other,

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which may be an additional factor to form nonlaminar flow profiles near the valves as well extended along the lymphatic vessel. Such variability in lymphatic valve structure and position predetermines even much more unpredictability in lymph flow profiles in lymphatic vessels. Comparatively recently data were obtained on lymph flow velocities and values of shear stress in lymphatic vessels [52–54]. A highspeed video system was used to capture multiple contraction cycles in rat mesenteric lymphatic preparations in situ. The images were analyzed to determine fluid velocity, volume flow rate, wall shear stress, and retrograde flow. It was determined that immune cell density and flux varied from 326 to 35,500 cells/μL and 206 to 2030 cells/min, respectively. Lymphatics contracted phasically, with a mean diameter of 91  9.0 μm and amplitudes of 39%, and lymph velocity in them varied with the phasic contractions in both direction and magnitude with an average of 0.87  0.18 and peaks of 2.2–9.0 mm/s. The velocity was 180 degrees out of phase with the lymphatic contractile cycle. The average lymph flow was 13.95  5.27 μL/h with transient periods of reversed flow. This resulted in an average shear of 0.64  0.14 with peaks of 4–12 dynes/cm2. These studies confirmed that shear rate in mesenteric lymphatics is low but had large variations in magnitude compared with that seen in blood vessels and common reversal of flow to close the valves. Importantly, in this work, the authors actually measuring the velocity fluctuations in contracting mesenteric lymphatics and more importantly approximating the wall shear stresses that occur in situ validated that the velocities experimentally induced in the isolated vessels experiments were physiologically relevant. Historically, researchers started to investigate the influences of different lymph flows/velocities on lymphatic pumping, creating the different levels of shear in experiments with steady increases in lymph flow rates. Particularly, Benoit et al. [39] increased lymph flow in rat mesenteric lymphatics by elevating lymph formation and accordingly lymph flow rates as a result of plasma dilution. They found the increased parameters of active lymph contractility in mesenteric lymphatics during the periods of increased lymph flow. But they also mentioned that pressure in the lymphatic network became less pulsatile in high lymph flow states. Of course, such kinds of experiments with increased lymph formation in situ give important

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information on lymphatic contractile behavior in situations similar to those that happen during different phases of tissue and organ activities. The studies on influences of imposed flow on contractile activity of rat isolated lymphatic vessel were presented by Gashev et al [44,55] These authors performed studies on isolated and perfused lymphatic vessels from four different regions of the body from the same species—rat that allowed comparisons of the imposed flow-induced modulations of lymphatic contractility in different regional lymphatic beds. In all of these studies, the inflow pressures were changed simultaneously with changes of outflow pressures made on the same level but in opposite direction to maintain the mean transmural pressure equal during the periods of increased imposed flow. Parameters of lymphatic contractions were evaluated immediately after imposed flow was set or changed and have been monitored during the next 5 min with an analysis of timedependent changes in 1-min intervals. In some series of experiments, authors changed the direction of imposed flow in isolated mesenteric lymphatic segments to retrograde. Using these experimental approaches, the potent imposed flowdependent inhibition of the active lymph pump has been found in mesenteric lymphatics and in the thoracic duct [55] and later in femoral and cervical lymphatic vessels [44]. Imposed flow gradient caused reductions in the frequency and amplitude of lymphatic contractions. As a result of these negative chronotropic and inotropic effects, the active pumping of lymphatics was greatly diminished. However, it is difficult to conclude that such imposed flowdependent inhibition of the active lymph pump decreases the total lymph flow in vivo. Because total lymph flow is the sum of passive and active flows, it is likely that the increase in imposed (or passive) flow would overwhelm any decreases in active pump lymph flow. A potentially important overriding factor would be an enhanced rate of lymph formation. At high levels of lymph formation, passive lymph flow could become a greater driving force to move lymph than the active lymph pump. Imposed flow-dependent inhibition of the active lymph pump in such situations could be a reasonable physiological mechanism to save metabolic energy by temporarily decreasing or stopping contractions during the time when the lymphatic vessel does not need it. An additional outcome of the

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inhibition of the lymph pump under these circumstances would be a reduction in lymph outflow resistance. This reduction in outflow resistance is the result of the net increase in average lymphatic diameter that occurs when contractions are inhibited. For example, complete cessation of the mesenteric active lymph pump (at zero imposed flow and 5-cm H2O transmural pressure gradient) would result in a net increase in the time averaged diameter by about 23%, thus theoretically reducing resistance by approximately 56% [55]. This reduction in the outflow resistance could ease the removal of fluid from the affected compartment that is producing the high lymph flows and facilitate the resolution of edema. Therefore, during these periods, when “passive” lymph flow [56,57] is elevated, such mechanisms maintain lymphatic vessel contractile function in an energy-saving mode [55] by diminishing phasic pumping contractions and tone, thereby converting lymphatic functional behavior from acting primarily like a pump to acting more like a conduit [58]. Imposed flow-induced inhibition of the lymph pump followed two temporal patterns [55]. The first is the rapidly developing inhibition of contraction frequency. Upon imposition of flow, the contraction frequency immediately fell and then partially recovered over time during continued flow. This effect was dependent on the magnitude of imposed flow but was not dependent on the direction of flow. The effect also depended upon the rate of change in the direction of flow. The second pattern was a slowly developing reduction of the amplitude of the lymphatic contractions, which increased over time during continued flow. The inhibition of contraction amplitude was dependent on the direction of the imposed flow but independent of the magnitude of flow. Therefore, the chronotropic and inotropic imposed flow-induced inhibitory responses appear somewhat different. In the first minute of the initiation of imposed flow, the lymphatic response to imposed flow occurs primarily through a rapid inhibition of the contraction frequency. In vivo, short periods of increased flow occur very often due to the contractions of upstream lymphangions. It is possible that a fast chronotropic response of lymphatics is an important shortterm regulatory reaction to rapid but short-lasting periods of increased flow. At high rates of lymph formation, which can be present in the mesenteric lymphatic bed in vivo, long periods of

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increased flow may occur. The slow inotropic effect, which develops in lymphatics in minutes, could be an important long-term regulatory reaction to slow but long-lasting periods of increased flow. This slowly developing flow-induced inhibition of lymphatic contractility could conserve energy in lymphatics when there are sufficient passive forces to move lymph without the active lymph pump and decrease local outflow resistance. Additionally, the imposed flow-induced inhibition of contraction frequency does not depend on direction of flow, whereas imposed flow-induced inhibition of amplitude of lymphatic contractions does. Long-term inhibition of lymphatic contractility is stronger during orthograde flow, which occurs in lymphatics more often in comparison with long periods of retrograde flow, which rarely occur because of the competency of the lymphatic valves. This evidence allows proposing that the mechanisms of the rapidly developing chronotropic and the slowly developing inotropic lymphatic responses to flow could be different. As mentioned earlier, lymphangions work in a short-distance pump fashion, and the force of their phasic contractions generates flow and shear in the lymphatics by themselves. Recently, it was demonstrated that the flow generated during phasic contractions in the rat thoracic duct itself plays an important self-regulatory role in the lymphatic contractile cycle in a shear-dependent manner [59]. In this study, the thoracic duct was chosen as a vessel sensitive to an imposed flow and for its variable contractile behavior [28,44,60–64]. In many cases contractions may occur in one part of the thoracic duct but do not propagate between different segments [28,44,60–64]. Contractile waves often do not propagate along this vessel, and many times, the phasic contractions develop locally, while adjacent parts of the duct are not contracting [65]. This feature of the thoracic duct was used to design the experiments to evaluate the importance of flow and shear generated by lymphatic phasic contractions in the regulation of the lymphatic contractile cycle. Two types of segments of thoracic duct were taken into this study: phasically active segments and phasically inactive segments. Close attention was paid to maintain the input and output pressures at the same level therefore excluding any imposed flow. Thus, in the phasically active lymphatic segments, flow and shear occurred only as a result of their inherent contractions. In phasically inactive

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segments, flow and shear did not take place. As a result, experimental conditions that allowed investigating the influences of flow and shear, generated solely by the phasic lymphatic pump, on the contractile function without any extra imposed flow were used. The authors found [59] that lymphatic resting tone in phasically active segments of the thoracic duct was 2–2.6 times lower than in phasically inactive segments. The only difference between the experimental conditions of the active and inactive segments was the existence of actively generated flow in the contracting lymphatic segments. Hence, in this study, a relaxation in the thoracic duct, which was connected exclusively with its spontaneous phasic pumping activity, was discovered. Investigating the possible mechanisms of this relaxation, it was demonstrated that blockade of nitric oxide synthase by L-NAME completely abolished this difference in lymphatic tone between the phasically active and nonactive segments. Therefore, this evidence indicates that the contraction-generated reduction of lymphatic tone in the thoracic duct is mediated only by nitric oxide (NO). Based on this fact, authors determined what happened with active lymphatic pumping if the mechanism of the contractiongenerated reduction of tone will be completely blocked. Authors found that the reduction of tone in lymphatic segments generated by the phasic contractions improves their diastolic filling (enhanced lusitropy—lowering half relaxation time—indicates the speed of diastolic filling), makes lymphatic contractions stronger (enhanced inotropy—higher contraction amplitudes), and propels more fluid forward during each contraction (elevated ejection fraction) while decreasing contraction frequency (reduced chronotropy). After NO-synthase blockade, the lymphatic segment must contract more often (higher contraction frequency) to maintain the minute productivity (fractional pump flow) appropriate to the existing level of preload (transmural pressure). Thus, the reduction in lymphatic tone due to the flow/shear generated by phasic contractions is a regulatory mechanism that maintains lymphatic pumping in an energy-saving efficient mode (stronger but fewer contractions per minute). Importantly, the flow-mediated relaxation can exist in any phasically contracting lymphatic. But the lymph flow profile is a complicated and variable sum of different forces, not only the result of phasic lymphatic contractions. When discussing the flow conditions in a single lymphangion, it is reasonable to divide the flow pattern

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into two components: “intrinsic flow” (meaning the flow that is a result of the contractions of that lymphangion) and “extrinsic flow” (meaning the flow that is a result of all influences from outside that single lymphangion, predominantly influences from upstream of that single lymphangion but some influences from downstream as well). From the experiments with imposed flow, it was known that as the imposed flow was increased, the degree of inhibition of lymphatic pumping increased [13,44,55]. In the thoracic duct, it was observed [44] that during periods of high imposed flow (transaxial pressure gradient of 5-cm H2O), the diastolic diameter increased, resulting in a 57% reduction in resting tone (in comparison with the absence of imposed flow at the same transmural pressure level). On the other hand, at this level of imposed flow, the spontaneous contractions of the thoracic duct were almost completely abolished [44]. This leads to the conclusion that in situ, where the extrinsic flow varies dramatically and is dependent on many factors, the lymphangions are constantly operating under a combination of intrinsic and extrinsic flows. When extrinsic flow is not enough to move lymph downstream, the maintenance of low lymphatic tone by the extrinsic flow (demonstrated in [13,44,55]) is supported or completely replaced by the reduction in lymphatic tone mediated by intrinsic flow during the pumping-effective phasic contractions (demonstrated in [59]). When extrinsic flow is high enough to propel lymph by itself, spontaneous contractions may be inhibited to save energy, and only the lowering of tone by the extrinsic flow will exist. Currently available data support the idea that contractile activity of the lymphatic vessels in situ constantly adjusts to the local “need” to propel variable volumes of lymph by a continuous interplay between the influences of extrinsic and intrinsic flows. At low levels of inflow in the transporting lymphatics, the influences of intrinsic flow will dominate, and NO release due to the phasic contractions will maintain the effective energy-saving lymphatic pumping patterns. As soon as the levels of lymph formation and accordingly inflow are increased in the transporting lymphatics, the influences of extrinsic flow will dominate, leading to a large NO release that will temporarily inhibit the intrinsic contractility of transporting lymphatics. Later studies with direct measurements of NO in

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mesenteric lymphatic vessels confirmed these conclusions about the functional role on the NO for lymphatic contractility [66,67]. In conclusion, it is important to recognize that knowledge of basic principles of physiological adaptive reactions to the changes in main physical factors of their environment (pressure and flow) is far from complete. It was thought for many decades that the adaptive reactions of lymphatic vessels to the increases in their volume loads are simple and could be explained by simple “bell-shaped” pressure-dependent regulation. Recent finding described earlier demonstrated that the contractions of lymphangions are constantly influenced by complicated sum of different factors of lymph dynamics: pressure/stretch, generated by active and passive lymph pumps, and subsequent combination of intrinsic and extrinsic flows/shears. Moreover, lymphangions react differently to changes in these factors depending on species and also on their location in the whole body and inside the regional lymphatic nets. However, due to the natural limitations and difficulties in possibilities of scientists to investigate lymphatic contractility, the picture of all possible adaptive contractile events in lymphatic vessels is far from complete. To ease the first steps in the investigation of the basic regulation of lymphatic contractility, the researchers artificially separated the studies on pressure and flow effects on it, but in vivo lymphangions exist in conditions of simultaneously changes in both pressure and flow. The next step of the research efforts on basic principles of lymph flow must be focused on a combined evaluation of interaction between pressure/stretch- and flow/shear-dependent adaptive reactions in lymphangions. As an example, someone may raise here a reasonable question: do the flow-dependent regulatory mechanisms act similarly or differently at different levels of lymphatic wall distension by intraluminal diastolic lymphatic pressure, because more likely the increases of extrinsic flow will follow by the increases in the lymphangions filling pressure? Obtaining the answers on such principle question on functioning of lymphatic vessels not only will promote our abilities to create the commonly accepted theory of physiology of lymph flow but also will help to precisely target the clinical efforts toward the correction of the main “driving engine” of lymph flow altered during lymphatic diseases— partially or completely incompetent lymphatic muscle cells unable to generate sufficient flow in lymphatic nets.

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Histamine as endothelium-derived relaxing factor in mesenteric lymphatic vessels As summary of previous notions presented earlier, studies in the last decades demonstrated the importance of nitric oxide (NO) for the regulation of lymphatic contractility as a crucial driving force maintaining effective lymph flow [55,59,66,68–71]. Considered a classical endothelium-derived relaxing factor (EDRF) for blood vasculature [72], NO plays an important role as an EDRF in lymphatic vessels through the inhibitory/relaxing effect of its increased basal level during the periods of increased imposed/passive flow [55,59], while oscillatory phasic fluctuations of NO at conditions of absent/minimal diastolic flow support an effective diastolic filling in the largest lymphatic trunk of the body, the thoracic duct [59]. Pump-conduit duality of lymphatic contractile behavior [55,58] in part can be explained by short-term partial diastolic NO-driven lymphatic relaxation at low levels of basal NO [59] and by long-term permanent NO-driven relaxation at higher levels of basal NO [55]. However, regional heterogeneity of lymphatic contractile behavior [44], complicated patterns of NO release in vivo during combined effects of increased stretch and shear [66], and differences in lymphatic contractility in various species [46] have led to conclusions concerning existence of controversy in current literature data on the functional importance of the NO molecule in regulating the active lymph pump [71]. Consideration of the differences between the roles of phasic release of NO at low/moderate levels of basal lymph flow and high levels NO at high levels of basal flow taken together with new knowledge of differences between functional behavior of peripheral and visceral lymphatic vessels in mice [45] appears to clarify the earlier mentioned controversy in comparisons between mice and rat lymphatic vessels [71] and supports the current view on role of NO in the regulation of lymphatic vessels contractile behavior presented earlier. Studies performed by others [73] provided further developments of discussed concepts. In contrast to the extensive literature on the predominant but not exclusive role of NO in the regulation of tone in the blood vasculature, there are only sparse literature reports indicating the potential existence of other endothelium-dependent mechanisms regulating lymphatic contractility and tone in a shear-dependent manner

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linked to measured changes in flow inside of lymphatic vessels [59,69,74,75]. At the same time, in our past experiments, we found that endothelial NO blockade, either by Nω-methyl-L-arginine acetate (L-NMMA) [55] or by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) [76], was not able to completely eliminate all endothelium-dependent relaxation induced by increases in imposed flow in rat mesenteric lymphatic vessels (MLVs). Our recent studies [76] directly suggested the existence of a yet undiscovered, shear-dependent, but NO-independent regulatory mechanism in rat MLVs. Later, in experiments with immunohistochemical characterization of the phenotype of the mast cells located in close proximity to MLVs [77], we identified the presence of a signal for the histamine-producing enzyme histidine decarboxylase (HDC), not only in mast cells but also in the walls of lymphatic vessels. This intriguing finding, at that moment, together with literature evidence of the role of histamine as an EDRF in some blood vasculature compartments [78–82] and the potential of high but still physiological doses of externally added histamine to relax lymphatic vessels [83], moved us toward the idea of evaluating the role of histamine as another potential lymphatic endothelium-derived relaxing factor in MLVs. Recently, we, for the first time, demonstrated role of histamine as an additional endothelium-derived relaxing factor in MLVs [84]. The detection of HDC protein in lymphatic endothelial cells suggests the ability of these cells to synthesize histamine locally within the vascular wall. HDC is the only enzyme responsible for the formation of histamine in mammalian tissues and thus represents the fundamental regulatory entity for histamine production [85,86]. In this study [84], we not only detected the HDC protein by immunohistochemical labeling and western blot analysis but also reconfirmed its presence in lymphatic endothelium by specific vivo-morpholino silencing. We also verified the presence of histamine within lymphatic vessels (which in many cases is more concentratedby the lymphatic valves). The reason for this should be determined in follow-up studies but could be reasonably linked to the more complicated flow patterns and higher lymph flow velocities in the areas of lymphatic valves [67,87–89] and/or the presence of more lymphatic endothelium locally due to the valve leaflets. Combined, our findings significantly expand knowledge of the

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biology of lymphatic vessels by identifying histamine as an additional lymphatic EDRF that together with NO contributes to the fine-tuning of lymphatic contractility. Due to the lack of knowledge, in our opinion, it is too premature to discuss in detail the timing and the mechanisms of the potential interaction between NO and histamine in providing the relaxation of MLVs. However, the potential of high but still physiological doses of externally added histamine to relax lymphatic vessels has been demonstrated recently [83,90]. Such findings [83,90] appear to be highly supportive of the idea that NO and histamine realize their relaxatory effects on lymphatic muscle through the same molecular targets. At lower doses of externally added histamine increases frequency and amplitude of lymphatic contractions [83,91] while higher doses of externally added histamine relax lymphatic vessels [83,90]. We propose that the effects of endothelium-dependent release of internal histamine correspond to its relaxatory effects observed in other studies [83,90]. We believe that further in-depth investigations are necessary to understand the mechanisms of combined effects of NO and histamine in lymphatic vessels including the potential switch from predominant relaxatory role of short-living NO molecule to long-term steady flowdependent relaxation induced by prestored long-living histamine molecule during long (hours) postprandial increases in basal lymph flow in MLVs.

Influences of aging Until recently, there were no published systematic studies on the aging-associated changes in the active lymph pumps. Due to the profound difficulty of measuring lymph flow in vivo, there are only a few reports demonstrating the measurements of reduced lymph flow in aged animals [92–94]. In particular, it was reported [94] that aging significantly decreases lymph flow from the main mesenteric lymph duct in rats—by 60% between ages of 3- and 22 months. During the last decade, we obtained important functional and molecular evidences of the aging-associated alterations of contractility in lymphatic vessels, which already widened our knowledge on the biology of aged lymph flow. In particular, we performed experiments with analysis of the contractile activity of the isolated aged

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rat thoracic duct segments and compare these data with those obtained from their adult counterparts [95]. We found various signs of the age-related alterations of active pump in the rat thoracic duct (TD). The transmural pressure-/stretch-dependent modulation of lymphatic contractility is one of the principle regulatory mechanisms of lymphatic pumping, which serves a goal to adapt lymphatic transport to the different lymphatic preloads [5,13,39,40,44,96]. The alterations in stretch-related regulatory mechanisms in 24-month-old segments indicate that both pacemaking and contractile machinery are involved in age-related changes of active lymph pump in the rat TD. Lowered lymphatic tone in aged TD segments together with decreased lymphatic contraction amplitude may be considered an indicator of age-related weakening of muscle cells and their diminished ability to create enough force to maintain the level of tone and contractile force appropriate to the lymphatic preloads: at a comparatively low pressure level of 1-cm H2O, the contraction amplitude is moderately lower in aged segments of TD. At higher pressure levels, the contraction amplitude is diminished in greater degree in the aged TD and reached the statistically significant difference levels between 9- and 24-month-old specimens. This negative agerelated inotropy in thoracic duct was accompanied with alteration in function of lymphatic pacemaking: the frequency of lymphatic contraction was diminished, especially at high levels of transmural pressure (5-cm H2O). Such negative age-related chronotropy in the TD together with negative inotropy led to a greatly decreased functional pump flow indicating the diminished pumping ability of the aged TD. The differences between stretchinduced responses in adult and aged animals are greater at higher levels of transmural pressure, also suggesting a diminished ability of the aged TD to adapt its contractility to increased preloads. Thus, aging in the TD leads to decreases in its functional reserves to adapt the contractility and pumping to the increased levels of lymph inflow in it. At higher levels of preload in the aged TD, its possibility to serve the increased demand in pumping through the duct will be diminished. Consequently, partial or complete failure to provide the adequate transport of lymph through the duct may occur.

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Aging also alters the contraction-initiated self-regulatory mechanism in TD that serves to maintain lymphatic contractions in efficient energy-saved mode [95]. Even though administration of L-NAME in the aged TD caused increases of lymphatic tone to the same values observed in adult specimens, the contraction amplitude and contraction frequency did not demonstrate the regular patterns of changes after NO-synthase blockade as we observed for 9-month-old segments in F-344 rats or as we have previously shown in TD segments obtained from adult Sprague-Dawley rats [95]. Such alterations in the contraction-initiated self-maintained regulation in TD may also have an impact on its pumping ability and responsible for diminishing of its contractile reserves. The fact that fractional pump flow (pumping) was slightly increased in the aged thoracic duct in the presence of L-NAME may indicate the presence of inhibitory influences of the constant release of nitric oxide in the aged thoracic duct independent of the phasic contractions. The findings described earlier correlate well with the data obtained in experiments with increased imposed flow in thoracic duct. In these experiments, the 9-month-old TD segments demonstrated the regular [13,44,55,97] pattern of imposed flow-induced inhibition. Administration of L-NAME completely abolished this inhibition. Fractional pump flow in 9-month-old specimens remained unchanged even at high levels of imposed flow after NO-synthase blockade. At the same time, TD segments from 24-month-old rats behaved differently during the increases in imposed flow. The TD segments from the aged group did not exhibit any significant imposed flow-dependent inhibition of the parameters of the active lymph pump, which remained unchanged during the imposed flow elevations. We concluded that the eNOSmediated imposed flow-dependent regulatory mechanism was completely depleted in 24-month-old TD segments. Therefore, the ability of aged TD to adapt its pumping to the different levels of extrinsic lymph flow was severely altered. Moreover, the administration of L-NAME moderately increased lymphatic pumping in 24-month-old TD segments independent of the value of imposed flow. These findings indicated the presence of a flow-independent but NO-dependent inhibition of lymphatic contractions in aged TD. We propose that this inhibition exists in aged lymphatic segments due to the activation of iNOS. To confirm the functional data

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on pressure- and flow-independent NO-dependent inhibition of the active lymph pump in the aged TD, we performed Western blot analyses of eNOS and iNOS in samples isolated from the 9- and 24-month-old TD. Data obtained in these experiments clearly indicated that the relative levels of eNOS were decreased in the 24-month-old thoracic duct when compared with that of the 9-month-old thoracic duct, whereas iNOS levels were dramatically increased in the 24-month-old thoracic duct. The detailed functional tests of aged TD described earlier clearly demonstrated that it is too simplistic to attribute the diminished contractility only to the sclerosis of the aged TD [98] and/or to atrophy of muscle cells in its wall [98,99]. In the next aging study, we evaluated the aging-associated changes in pumping of mesenteric lymphatic vessels in adult (9-month-old) and aged (24-month-old) Fisher-344 rats [76]. These data demonstrated a weakening of the lymphatic pump in aged MLVs. The data also suggest that the imposed flow gradientgenerated, shear-dependent relaxation does not exist in aged rat MLVs and the sensitivity of both adult and aged MLVs to such shear cannot be eliminated by blockade of NO synthases. The objectives of another study [100] were to evaluate the aging-associated changes, contractile characteristics of mesenteric lymphatic vessels (MLV), and lymph flow in vivo in male 9- and 24-month-old Fischer-344 rats. Lymphatic diameter, contraction amplitude, contraction frequency and fractional pump flow, lymph flow velocity, wall shear stress, and minute active wall shear stress load were determined in MLV in vivo before and after L-NAME application at 100 μM. The active pumping of the aged rat mesenteric lymphatic vessels in vivo was found to be severely depleted, predominantly through the aging-associated decrease in lymphatic contractile frequency. Such changes correlate with enlargement of aged MLV, which experienced much lower minute active shear stress load than adult vessels. At the same time, pumping in aged MLV in vivo may be rapidly increased back to levels of adult vessels predominantly through the increase in contraction frequency induced by NO elimination. These findings support the idea that in aged tissues surrounding the aged MLV, the additional source of some yet unlinked lymphatic contraction-stimulatory metabolites is counterbalanced or blocked by NO release. The comparative

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analysis of the control data obtained from experiments with both adult and aged MLV in vivo and from isolated vessel-based studies clearly demonstrated that ex vivo isolated lymphatic vessels exhibit identical contractile characteristics to lymphatic vessels in vivo. In this study, we also performed, for the first time, a detailed evaluation of the contractile activity of MLV and lymph flow in vivo in 9- and 24-month-old F-344 rats [100]. In the first part of this study, we investigated, in detail, the contractile characteristics of MLV and lymph flow in adult (9-month-old) and aged (24-month-old) animals under control conditions. We found that in aged animals, the lymphatic vessels diameters from the same location (group III by Benoit [42]) are significantly larger than in adult animals. The end-diastolic diameters and end-systolic diameters in 24-month-old MLV were, respectively, 71% and 79% greater than in their adult counterparts. At the same time, we observed only minor, nonsignificant, lowering of the contraction amplitude in aged MLV versus adult vessels under control conditions (20% and 25% of diameter changes during the contractions respectively). The aging-associated negative chronotropy was observed in all aged MLV: we noted threefold decrease in their contraction frequency compared with adult MLV. As a result of the described agingassociated changes in lymphatic contractile force (contraction amplitude) and pacemaking (contraction frequency), the minute active lymphatic pumping was significantly lower in aged animals. Both indices of the lymphatic pumping, AFP (amplitude frequency product) and FPF (fractional pump flow), were significantly diminished in the aged group with AFP 76% lower (4.2-fold lower) and FPF—77% lower (4.3-fold lower) than the adult group. In addition, we analyzed the aging-associated differences in the characteristics of lymph flow in rat mesentery using selected single contraction cycles of “diastole-systole” with suitable contractile cycles during each experimental condition for the 9-month-old animals and for the 24-month-old animals. While the diastolic lymph flow velocity was slightly, but not significantly, higher in aged MLV, the maximal systolic lymph flow velocity was significantly (43%) lower in aged animals. Correspondingly, we did not find any aging-associated changes in calculated diastolic (resting) wall shear stress, but during the phasic contractions, the lymphatic

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endothelial cells in aged MLV experienced a approximately threefold reduction of the maximal systolic wall shear stress. We also compared the rate of change of the phasic contractiongenerated (i.e., active) wall shear stress in both adult and aged groups and found a dramatic 6.2-fold aging-associated decrease in 24-month-old MLV in comparison with the 9-month-old group. The phasic contraction-generated (active) minute wall shear stress “load” that the lymphatic endothelial cells experienced a minute was 9.7-fold lower in aged MLV. Due to of the importance of the NO molecule released by lymphatic endothelium for the regulation of lymphatic contractility and flow in adult [57,59,66,69,101–103] and aged [76,95,104] lymphatic vessels, in this study, we implemented in vivo local NO-synthase blockade induced by topical administration of 100-μM of L-NAME [100]. We compared the contractile behavior of MLV and lymph flow in adult and aged groups before and after the L-NAME administration. We found that the NO-synthase blockade with a duration of 15 min induced slight, but not significant, constriction in both adult and aged MLV. Only end-systolic diameter in aged MLV was significantly decreased by 25% after 15 min of the L-NAME application. During these small changes in lymphatic diameters, after the NO-synthase blockade in MLV of both aged groups, the difference between the contraction amplitude in adult and aged MLV was reversed by L-NAME from slightly negative (20% lower in aged group) to positive (50% higher in aged group). The greatest observed influence of the NO-synthase blockade was its chronotropic effect. While in adult MLV the contraction frequency was significantly increased (2.1-fold) 15 min after the L-NAME application, in the aged MLV, this increase in the lymphatic contraction frequency was over of 3.5-fold. The average contraction frequency of the aged MLV treated even only 5 min by L-NAME was 25% (although not statistically significant) higher than the contraction frequency in adult lymphatic vessels under control conditions. The main paradox of the influence of the NO-synthase blockade in aged MLV was found when analyzing the indices of their minute productivity, AFP and FPF, which increased in both age groups as consequence of chronotropic and inotropic influences of the L-NAME administration. In adult vessels, L-NAME application was able to increase significantly

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both of these indices by 57% and 59%, respectively. In aged MLV, the influence of the NO-synthase blockade was remarkably greater. Specifically, AFP was increased by 538% and FPF by 511% compared with the control conditions after 15 min of the L-NAME administration. Such influence of the elimination of NO on aged MLV not only was able to compensate for the observed 4.2-fold aging-associated depletion in minute productivity in the aged MLV lymph pump in vivo but also, after only 5 min of the L-NAME application, was able to maintain this productivity at the same level as the L-NAME-treated adult lymphatic vessels. Another important result for this study [100] was in our findings that the diastolic lymph flow velocity remained unchanged during the L-NAME application in both selected aged groups. At the same time that we observed a moderate, nonsignificant, increase in the maximal systolic lymph flow velocity in the adult group, in aged MLV, the maximal systolic lymph flow velocity was more than tripled after 15 min of the L-NAME treatment (3.6-fold increase), thus being higher than in the adult MLV under the same experimental conditions. Correspondingly, we did not find any aging-associated changes in calculated diastolic (resting) wall shear stress after the NO-synthase blockade in both selected age groups, while the maximal systolic wall shear stress was significantly increased in MLV of both selected ages (139% increase in adult and 452% increase in aged MLV). Although the absolute difference (roughly 7 dynes/cm2) is similar, the percentage as a reflection of the basal control level of the maximal systolic wall shear stress does matter. By underlying the degree of changes, we are underlying here the general tendency of lymphatic pumping in aged MLV to be increased dramatically after NO elimination toward values observed in adult animals at the same conditions. Additionally, we compared effects of the L-NAME treatment on the rate of change of the phasic contraction-generated (i.e., active) wall shear stress in both adult and aged groups. We found its dramatic 4.8-fold increase in 24-month-old L-NAME-treated MLV as compared with aged MLV under control conditions. Such changes after L-NAME administration nearly matched the rate of change in active wall shear stress for the NOS-blocked aged MLV versus the NOS-blocked adult vessels, for which the rate did not change

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significantly from control during the L-NAME treatment. As a reflection of these changes, the phasic contraction-generated (active) minute wall shear stress “load,” which the aged lymphatic endothelial cells experienced a minute after NO-synthase blockade, showed the ASFP to increase by 25.3-fold in comparison with aged MLV in control conditions. However, the ASFP did not change significantly over the NO-synthase blockade in adult MLV during the same period of time. Furthermore, in this study, we performed comparative data analysis of the contractile parameters of rat MLV obtained both in vivo and in isolated vessels experiments [100]. We found that in control groups of 9- and 24-month-old MLV, all three investigated contractile characteristics, namely, contraction amplitude, contraction frequency, and fractional pump flow, obtained in isolated vesselbased experiments were not significantly different from those obtained in vivo. Moreover, after L-NAME administration, as described in this study for the in vivo experiments and previously [76] for isolated vessels, all three parameters of contractile activity of the 9-month-old MLV were not significantly different between in vivo and isolated vessel data groups with the same degrees and directions of changes both in vivo and under isolated vessel conditions. The primary intriguing difference we found was in contractile chronotropy of the aged MLV in vivo after L-NAME administration as compared with the isolated vessel experiments with the same aged MLV under the same experimental conditions (NO-synthase blockade). The contraction frequency of the 24-month-old MLV situated in vivo increased 3.5-fold in comparison with the frequency of the same vessels under control conditions. As a result of this positive L-NAME-induced chronotropy and the 50% (but nonsignificant) increase of the contraction amplitude, the minute pumping (FPF) of the L-NAME-treated aged MLV was 6.1-fold higher than in the same aged vessels under control conditions. In other words, the NO-synthase blockade of aged MLV in vivo was able to not only compensate the aging-associated deficiency of minute pumping in aged vessels under control conditions but also enhance their minute pumping all the way up to the levels of NOS-treated adult 9-month-old MLV. In isolated aged MLV, we observed much weaker, nonsignificant, positive chronotropy and a much smaller increase in pumping after L-NAME administration [76].

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Therefore, we performed a detailed evaluation of the parameters of the contractility of aged mesenteric lymphatic vessels in vivo and characterized lymph flow in the aged mesenteric lymphatic network [100]. Results were compared with the same characteristics of lymphatic contractility and flow in adult mesenteric lymphatic vessels. We performed these evaluations under control conditions and after a nitric oxide synthases blockade of 100-μM L-NAME. This allowed us to make important conclusions about the comparative roles of the NO-dependent regulatory mechanisms, which control the lymphatic contractility and lymph flow in vivo in the adult and aged body. Importantly, we performed, for the first time, the direct detailed comparison of the characteristics of the lymphatic contractility in vivo and contractile characteristics of the isolated lymphatic vessels exteriorized from the same location for both adult and aged MLV under both control conditions and after NOS synthases blockade. On the other hand, a careful analytical comparison of the influences of the NO-synthase blockade in the aged MLV in vivo and in isolated vessel-based studies allowed us to determine the important differences (discussed later) between functioning of the lymphatic vessels under isolated conditions and functioning of the same vessels under the additional influence of the aged tissue microenvironment. In a follow-up study, we evaluated the aging-associated changes in the functional role of histamine as an EDRF in aged MLVs [105]. We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat mesenteric lymphatic vessels (MLVs) obtained from 9- to 24-month-old Fischer-344 rats under control conditions and after pharmacological NO blockade by Nω-nitro-Larginine methyl ester hydrochloride (L-NAME, 100 μM) or/and blockade of histamine production by α-methyl-DL-histidine dihydrochloride (α-MHD, 10 μM). We also quantitatively compared results of immunohistochemical labeling of the histamine-producing enzyme histidine decarboxylase (HDC) in adult and aged MLVs. This study [105] demonstrated that aging introduces significant changes in wall shear stress-dependent modulation of lymphatic contractility related to the increased functional role of histamine as an EDRF in aged MLVs and simultaneous effective loss of the NO as a molecular player in such regulatory reactions. These findings

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correlate with the increased expression of HDC, the histamineproducing enzyme, in aged MLVs. At the same time, we confirmed that transmural pressure-/stretch-dependent regulatory reactions of contracting MLVs are not affected by intralymphatic-borne histamine, neither in adult nor in aged MLVs. Such is not true for effects of histamine delivered from sources external to lymphatic vessel on the lymphatic contractility. In particular, data from the literature suggest that histamine is a potent dose-dependent modulator of lymphatic contractility [83,90,91,106–111]. At lower concentrations, histamine acts synergistically with the lymphatic-relaxing effects of NO, but at higher concentrations, histamine acts predominantly antagonistically to NO and stimulates lymphatic contractility [83,90,91,106–116]. Such findings illustrate the clear necessity of further in-depth discovery-driven research efforts to establish complicated interrelations between histamine-producing lymphatic vessels and histamine-producing cellular elements of perilymphatic tissue compartments. The findings of the current study arose from the experiments, which clearly illustrate the continued necessity of describing the many unknown features of lymphatic vessels to gain insight into the origin and pathogenesis of diseases of the lymphatic system, for example, lymphedema. As we mentioned earlier [84], histamine, as a known chemoattractant of mast cells [117], may be responsible for increased density of mast cells observed near MLVs [77]. In light of our current findings, an observed further increase of mast cell density in aged perilymphatic mesenteric tissues (compared with adult tissues) [77] directly correlates with the increased expression of HDC in aged MLVs demonstrated in this study. Increased levels of histamine in aged MLVs, with corresponding increased diffusion of this molecule out of lymphatic vessels, are very likely important for increased recruitment and potentially for chronic basal activation of perilymphatic mast cells in the elderly [77,118]. Lastly, recent data demonstrate that the aging-associated basal activation of perilymphatic mast cells maintains chronic histaminedependent activation of NF-κB signaling in aged perilymphatic mesenteric tissues [119]. The mast cell/histamine/NF-κB axis through a myriad of the NF-κB-controlled vasoactive cytokines induces and maintains aging-associated alterations of the contractility of neighboring MLVs. However, the underlying molecular mechanisms have not yet been discovered but without a doubt are

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complex and require detailed investigations of aged lymphatic contractility through careful discovery-driven research.

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[37] Hayashi A, Johnston MG, Nelson W, Hamilton S, McHale NG. Increased intrinsic pumping of intestinal lymphatics following hemorrhage in anesthetized sheep. Circ Res 1987;60(2):265–72. [38] Reddy NP, Staub NC. Intrinsic propulsive activity of thoracic duct perfused in anesthetized dogs. Microvasc Res 1981;21(2):183–92. [39] Benoit JN, Zawieja DC, Goodman AH, Granger HJ. Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress. Am J Physiol 1989;257(6 Pt 2):H2059–69. [40] Gashev AA. The pump function of the lymphangion and the effect on it of different hydrostatic conditions [In Russian]. Fiziol Zh SSSR Im I M Sechenova 1989;75(12):1737–43. [41] Eisenhoffer J, Lee S, Johnston MG. Pressure-flow relationships in isolated sheep prenodal lymphatic vessels. Am J Physiol 1994;267(3 Pt 2):H938–43. [42] Benoit JN. Relationships between lymphatic pump flow and total lymph flow in the small intestine. Am J Physiol 1991;261(6 Pt 2):H1970–8. [43] Gashev AA, Wang W, Laine GA, Stewart RH, Zawieja DC. Characteristics of the active lymph pump in bovine prenodal mesenteric lymphatics. Lymphat Res Biol 2007;5(2):71–9. [44] Gashev AA, Davis MJ, Delp MD, Zawieja DC. Regional variations of contractile activity in isolated rat lymphatics. Microcirculation 2004;11(6):477–92. [45] Zawieja SD, Castorena-Gonzalez JA, Scallan J, Davis MJ. Differences in L-type calcium channel activity partially underlie the regional dichotomy in pumping behavior by murine peripheral and visceral lymphatic vessels. Am J Physiol Heart Circ Physiol 2018;. [46] Gashev AA, Davis MJ, Gasheva OY, et al. Methods for lymphatic vessel culture and gene transfection. Microcirculation 2009;16(7):615–28. [47] Mawhinney HJ, Roddie IC. Spontaneous activity in isolated bovine mesenteric lymphatics. J Physiol 1973;229(2):339–48. [48] McHale NG, Meharg MK. Co-ordination of pumping in isolated bovine lymphatic vessels. J Physiol 1992;450:503–12. [49] Crowe MJ, von der Weid PY, Brock JA, Van Helden DF. Co-ordination of contractile activity in guinea-pig mesenteric lymphatics. J Physiol 1997;500 Pt 1: 235–44. [50] Gashev AA, Zawieja DC. Lymphatic contractions: the role of distension mechanisms. FASEB J 1999;13:A11. [51] Mazzoni MC, Skalak TC, Schmid-Schonbein GW. Structure of lymphatic valves in the spinotrapezius muscle of the rat. Blood Vessels 1987;24 (6):304–12. [52] Dixon JB, Gashev AA, Zawieja DC, Moore Jr JE, Cote GL. Image correlation algorithm for measuring lymphocyte velocity and diameter changes in contracting microlymphatics. Ann Biomed Eng 2007;35(3):387–96. [53] Dixon JB, Greiner ST, Gashev AA, Cote GL, Moore JE, Zawieja DC. Lymph flow, shear stress, and lymphocyte velocity in rat mesenteric prenodal lymphatics. Microcirculation 2006;13(7):597–610. [54] Dixon JB, Zawieja DC, Gashev AA, Cote GL. Measuring microlymphatic flow using fast video microscopy. J Biomed Opt 2005;10(6).

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[71] Scallan JP, Davis MJ. Genetic removal of basal nitric oxide enhances contractile activity in isolated murine collecting lymphatic vessels. J Physiol 2013;591 Pt 8: 2139–56. [72] Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288(5789):373–6. [73] Kunert C, Baish JW, Liao S, Padera TP, Munn LL. Mechanobiological oscillators control lymph flow. Proc Natl Acad Sci U S A 2015;112(35):10938–43. [74] Tsunemoto H, Ikomi F, Ohhashi T. Flow-mediated release of nitric oxide from lymphatic endothelial cells of pressurized canine thoracic duct. Jpn J Physiol 2003;53(3):157–63. [75] Gasheva OY, Gashev AA, Zawieja DC. Imposed flow-dependent inhibition in rat thoracic duct is not dependent on K channel blockade. FASEB J 2007;21(5): A485. [76] Nagai T, Bridenbaugh EA, Gashev AA. Aging-associated alterations in contractility of rat mesenteric lymphatic vessels. Microcirculation 2011;18(6): 463–73. [77] Chatterjee V, Gashev AA. Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels. Am J Physiol Heart Circ Physiol 2012;303(6):H693–702. [78] Hollis TM, Rosen LA. Histidine decarboxylase activity of bovine aortic endothelium and intima-media. Proc Soc Exp Biol Med 1972;141(3):978–81. [79] Hollis TM, Ferrone RA. Effects of shearing stress on aortic histamine synthesis. Exp Mol Pathol 1974;20(1):1–10. [80] DeForrest JM, Hollis TM. Shear stress and aortic histamine synthesis. Am J Physiol 1978;234(6):H701–5. [81] Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J 1989;3(9):2007–18. [82] Benedito S, Prieto D, Nielsen PJ, Nyborg NC. Histamine induces endotheliumdependent relaxation of bovine retinal arteries. Invest Ophthalmol Vis Sci 1991;32(1):32–8. [83] Petunov SG, Egorova AA, Orlov RS, Nikitina ER. Effect of histamine on spontaneous contractions of mesenteric lymphatic vessels and lymph nodes of white rats: endothelium-dependent responses. Dokl Biol Sci 2010;432:176–80. [84] Nizamutdinova IT, Maejima D, Nagai T, et al. Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels. Microcirculation 2014;21(7):640–8. [85] Hocker M, Zhang Z, Koh TJ, Wang TC. The regulation of histidine decarboxylase gene expression. Yale J Biol Med 1996;69(1):21–33. [86] Moya-Garcia AA, Medina MA, Sanchez-Jimenez F. Mammalian histidine decarboxylase: from structure to function. Bioessays 2005;27(1):57–63. [87] Davis MJ, Rahbar E, Gashev AA, Zawieja DC, Moore Jr JE. Determinants of valve gating in collecting lymphatic vessels from rat mesentery. Am J Physiol Heart Circ Physiol 2011;301(1):H48–60.

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[105] Nizamutdinova IT, Maejima D, Nagai T, Meininger CJ, Gashev AA. Histamine as an endothelium-derived relaxing factor in aged mesenteric lymphatic vessels. Lymphat Res Biol 2017;15(2):136–45. [106] Ohhashi T, Kawai Y, Azuma T. The response of lymphatic smooth muscles to vasoactive substances. Pflugers Arch 1978;375:183–8. [107] Johnston MG, Kanalec A, Gordon JL. Effects of arachidonic acid and its cyclooxygenase and lipoxygenase products on lymphatic vessel contractility in vitro. Prostaglandins 1983;25(1):85–98. [108] Unthank JL, Hogan RD. The effect of vasoactive agents on the contractions of the initial lymphatics of the Bat’s wing. Blood Vessels 1987;24:31–44. [109] Dobbins DE, Buehn MJ, Dabney JM. Constriction of perfused lymphatics by acetylcholine, bradykinin and histamine. Microcirc Endothelium Lymphatics 1990;6(6):409–25. [110] Plaku KJ, von der Weid PY. Mast cell degranulation alters lymphatic contractile activity through action of histamine. Microcirculation 2006;13(3):219–27. [111] Kurtz KH, Moor AN, Souza-Smith FM, Breslin JW. Involvement of H1 and H2 receptors and soluble guanylate cyclase in histamine-induced relaxation of rat mesenteric collecting lymphatics. Microcirculation 2014;21(7):593–605. [112] Mislin H. The contractile properties of lymphatic vessels. Angiologica 1971;8(3–5):207–11. [113] Orlov R, Lobov G. Ionic mechanisms of the electrical activity of the smoothmuscle cells of the lymphatic vessels. Fiziol Zh SSSR Im I M Sechenova 1984;70(5):712–21. [114] Ferguson MK, Shahinian HK, Michelassi F. Lymphatic smooth muscle responses to leukotrienes, histamine and platelet activating factor. J Surg Res 1988;44(2):172–7. [115] Watanabe N, Kawai Y, Ohhashi T. Dual effects of histamine on spontaneous activity in isolated bovine mesenteric lymphatics. Microvasc Res 1988;36(3): 239–49. [116] Pan’kova MN, Lobov GI, Chikhman VN, Solnyshkin SD. Effects of histamine on contractile activity of lymphatic node capsules. The NO role. Ross Fiziol Zh Im I M Sechenova 2011;97(6):633–40. [117] Keles N, Yavuz Arican R, Coskun M, Elpek GO. Histamine induces the neuronal hypertrophy and increases the mast cell density in gastrointestinal tract. Exp Toxicol Pathol 2012;64(7–8):713–6. [118] Chatterjee V, Gashev AA. Mast cell-directed recruitment of MHC class II positive cells and eosinophils towards mesenteric lymphatic vessels in adulthood and elderly. Lymphat Res Biol 2014;12(1):37–47. [119] Nizamutdinova IT, Dusio GF, Gasheva OY, et al. Mast cells and histamine are triggering the NF-kappaB-mediated reactions of adult and aged perilymphatic mesenteric tissues to acute inflammation. Aging (Albany NY) 2016;8(11): 3065–90.

C H A P T E R

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CNS lymphatics in health and disease a

J. Winny Yuna, J. Steve Alexandera, Felicity N.E. Gavinsa,b Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA, United States b Brunel University London, Kingston Ln, London, Uxbridge, United Kingdom

Lymphatics of the central nervous system The lymphatic vasculature, a secondary transport system that parallels the blood vasculature, is responsible for clearing interstitial fluid (ISF), immune cells (lymphocytes, antigen-presenting cells), and extracellular proteins carried in it, which maintains overall tissue water and solute balance, homeostasis, and immunity [1]. Such vessels have remained elusive in the context of the brain, and as such, compartments of the central nervous system (CNS) have been historically believed to be devoid of a “conventional” lymphatic system. This has remained a perplexing conundrum not only because the brain is exposed to levels of waste products reflecting the brain’s high metabolic rate, which critically depends on such clearance, but also because resident cells such as neurons are extremely sensitive to their ionic changes. Recently, it has been proposed that in the brain, the clearance of extracellular fluids and extracellular proteins take on alternative mechanisms that differ from the rest of the organs of the body. To date, three sets of brain lymphatic clearance pathways have been described: (i) the perivascular pathway, (ii) the

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glymphatic pathway, and (iii) the meningeal pathway. Each of these pathways are discussed in the succeeding texts.

The perivascular pathway The Weller-Carare group [2] has outlined the perivascular pathway of CNS lymphatic clearance using tracer studies. CNS lymphatics are responsible for draining two types of extracellular fluid (ECF): cerebrospinal fluid (CSF) and ISF; these each drain via distinct routes from the brain [3] (Fig. 1). The lymphatic drainage of CSF represents only a minor fraction; studies involving the injection of radiolabeled albumin into rats traced approximately 15% penetrating into the CSF, the other 85% draining out with ISF [4]. Most of the CSF drainage in the adult human brain returns back into the venous blood through arachnoid villi via vacuolar channels that pass through the endothelium, from the abluminal side to the veins [5–7]. Only a minor portion of CSF drains through the lymphatics in humans, which is significantly different from results in experimental animals, where up to 50% of the CSF drains into the lymph nodes [6]. Most recent data from Proulx’s group suggest that

FIG. 1 Drainage of extracellular fluid by the CNS lymphatics. The CNS lymphatics are responsible for draining two types of extracellular fluid (ECF): cerebrospinal fluid (CSF, shown in blue) and interstitial fluid (ISF, shown in purple). The drainage of these fluids is via distinct routes from the brain [3].

Carotid artery

Cervical lymph node

ISF CSF

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lymphatic vessels are predominantly responsible for CSF outflow in mice, and this clearance reduces significantly with increasing age [8]. Injection of Indian ink into the subarachnoid space of rats revealed drainage pathways along the olfactory nerves within nasal lymphatic vessels, which ultimately emptied into the superficial and deep cervical lymph nodes [9]. Similar experiments have revealed CSF lymphatic drainage occurring through nasal lymphatic vessels in several animals including mice, cats, dogs, and rabbits [10–12]. Injection of radioactive albumin into the CSF of nonhuman primates resulted in an accumulation of tracer in the cervical lymph nodes, supporting CSF lymphatic drainage in nonhuman primates [13]. Direct evidence of CSF lymphatic flow in human was demonstrated using Microfil injection into recently deceased cadavers that delineated a remarkably similar pattern to that seen in mouse [5]. Lymphatic drainage of ISF differs not only from that used for CSF but also significantly from lymphatic drainage of other systems. Mouse studies involving the injection of various tracers revealed a pathway for ISF drainage within intracranial arterial walls into the cervical lymph node (LN) in the neck [2, 4]. In particular, ISF and solutes, but not cells, were found to drain along 100–150-nm-wide basement membranes within the walls of these arteries and capillaries [2]. Interestingly, this lymphatic flow of ISF is described to occur in the reverse direction from blood flow in these arteries and capillaries, at a rate similar to lymphatic drainage of other tissues (0.1–0.3 uL/min) [14, 15], and may depend upon rhythmic arterial blood pulsations to provide propulsion through these channels.

The glymphatic pathway A separate lymphatic clearance system has been proposed by the Nedergaard group, based on in vivo two-photon imaging and ex vivo fluorescence imaging with various tracers [16]. They showed that a large proportion of subarachnoid CSF (but not ventricular CSF) enters into the brain parenchyma. This movement was demonstrated to be rapid and size dependent, the size restriction being dictated by astrocytic end-feet that are thought to serve as a size exclusion. This bulk flow of CSF is believed to be responsible for the clearance of fluid and solutes from the brain, and the CSF exchanges with the ISF. The water channel protein aquaporin-4 (AQP4), which is highly polarized to the end-feet of the astroglia, is critical in facilitating the bulk flux of CSF

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and therefore clearance from the brain parenchyma. Contrastenhanced magnetic resonance imaging (MRI) was used to observe live rat brains and to identify the key nodes of influx at the pineal and pituitary recesses. [17]. Owing to the importance of AQP4 in glial cells in this system, it is referred to as the “glymphatic” system. AQP4 antibody-mediated autoimmunity has also been implicated in the pathogenesis of neuromyelitis optica (NMO), an inflammatory disorder of the CNS [18], indicating NMO may potentially be associated with glymphatic system dysfunction.

The meningeal pathway More recently, specific molecular markers for lymphatic endothelial cells have become available and have allowed two independent studies to identify anatomical lymphatic vessels in the brain’s meninges: first by Louveau et al. and then Aspelund et al. [19, 20]. Louveau et al. observed a simple network of narrow lymphatic vessels in the meninges underlying the bony parts of the skull (Fig. 2). These vessels, with a distinct lumen, run parallel to the dural sinuses. Meningeal lymphatics, identified in adult mice, express most of the molecular hallmarks of lymphatic endothelial cells, including the lymphatic vascular endothelial hyaluronic acid receptor (LYVE-1), prospero homeobox protein-1 (Prox1), podoplanin, and vascular endothelial growth factor receptor (VEGFR)-3. These meningeal lymphatics have been shown to not belong to the “cardiovasculature” [20] and are devoid of smooth muscle cells, maintaining only anatomical and molecular features characteristic of “initial lymphatics.” Similar to initial peripheral lymphatics, meningeal lymphatics display a discontinuous basement membrane surrounded by anchoring filaments without intraluminal (secondary) valves [20] (Fig. 3). Aspelund et al. further identified meningeal lymphatics, although relatively scarce, in the superior parts of the skull, becoming more extensive at the skull base [19]. Lymphatics at the base of the skull have intraluminal valves and exit the skull alongside arteries, veins, and cranial nerves. The valves show evidence for the presence of smooth muscle cells in these vessels, suggesting a transition from initial to collecting lymphatics as these vessels exit the brain [19]. Further studies are still needed to examine if these vessels demonstrate collecting vessel structure or display active lymphangion pumping function. Brain ISF and CSF are absorbed into

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FIG. 2

Schematic representation of the pattern of intracranial cerebral spinal fluid flow. (A) Cerebral spinal fluid (CSF) is produced by the choroid plexus, flows through the lateral and third ventricles, and exits through the foramina of Luschka and Magendie, to reach the subarachnoid space over the convexities. (B) Movement of CSF and interstitial fluid (ISF) to and from the subarachnoid space. CSF is also able to diffuse in and out of the brain parenchyma along the perivascular space. From Louveau A, Smirnov I, Keyes TJ, Eccles JD, Rouhani SJ, Peske JD, et al. Structural and functional features of central nervous system lymphatic vessels. Nature 2015;523(7560):337–341, with permission.

the meningeal lymphatics from the subarachnoid space and are transported downstream of deep cervical LNs. Additional studies are also necessary to examine the extent of meningeal lymphatic networks, their roles in CSF clearance, and ultimately how these findings can be translated into clinical applications.

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Perivascular flow of ISF

Perivascular flow of ISF

Blood flow Glymphatics para-arterial inflow of CSF

Brain parenchyma

CSF mixes with ISF

Dura matter

BC< 4

Subarachnoid space

End-feet of astrocytes

Glymphatics para-venous outflow of CSF-ISF

Blood flow Meningeal pathway ISF and CSF flow

Cervical lymph node

FIG. 3 Schematic displaying meningeal lymphatics. The meningeal lymphatics display a discontinuous basement membrane surrounded by anchoring filaments without intraluminal (secondary) valves.

The role of the lymphatic system in diseases of the CNS Although the significance of CNS lymphatic drainage has yet to be fully explored, it is likely to play an important part in the CNS physiology, and its disturbances may lead to a number of neuroimmunological diseases, for example, Alzheimer’s disease (AD),

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Parkinson’s disease (PD), and multiple sclerosis (MS). Lymphatic drainage in the brain is especially critical because intracranial pressure is extremely sensitive to the volume and CSF-ISF balance and intracranial pressure therefore elevate rapidly with CSF or ISF imbalance. Impaired CSF drainage can also cause hydrocephalus, where CSF penetrating into white matter causes interstitial edema [21]. Furthermore, elevated intracranial pressure also impairs cerebral blood flow, which can lead to widespread cerebrovascular, neural, and metabolic distress [6].

Alzheimer’s disease Alzheimer’s disease (AD) is characterized by intraneuronal accumulations of tau and the extracellular accumulation of insoluble amyloid-beta plaques in the brain [22]. Amyloid-beta is produced in the brain itself and can serve as an endogenous “tracer” of CNS lymphatic drainage [23]. AD brains typically exhibit increased fibrous thickening, arterial stiffening, and diminished pulsatility, which may in turn limit periarterial lymphatic transport [24]. When lymphatic drainage is impaired, cerebral vessels exhibit increased insoluble amyloid-beta deposits in the arteries and microvessels and may further limit perivascular lymphatic drainage; this is especially evident in cerebral amyloid angiopathy (CAA) [23, 24]. Impaired CNS lymphatic function results in the loss of normal homeostasis in the brain, for example, insufficient removal of Ab and other metabolites. Such accumulation of Ab can result in the formation of toxic forms of Ab that causes neuronal injury [25]. This evidence suggests the importance of CNS lymphatic function in amyloid-beta clearance; an improved understanding of the physiological responses could provide possible targets for drug discovery programs for therapy.

Multiple sclerosis The CNS is considered to be relatively “immunologically privileged.” Nevertheless, immune reactions do occur in the CNS, and its associated lymphatics drain into cervical LN and influence neuroimmunity. Antigen injection into the brain parenchyma (or CSF) has been shown to trigger the formation of antibodies, and

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similarly, myelin antigens have been detected in cervical LNs in MS patients and in experimental autoimmune encephalomyelitis (EAE), “an experimental mouse model for MS” [26, 27]. The pathogenesis of MS and EAE involves T-cell-mediated immune reactions in the brain. These T cells enter the CNS via interactions between specific integrins on the T cells (alpha4beta1 and alpha4beta7) and vascular adhesion molecule (VCAM) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on brain endothelial cells [28]. Cervical lymphadenectomy has been shown to reduce T cell-mediated immune reactions in the brain of EAE and consequently EAE severity, leading to the conclusion that the T lymphocytes may come from cervical LNs [29]. MS pathogenesis involves the drainage of soluble antigens to LN from the CNS leading to the activation of T cells for CNS-specific proteins [30]. Another possibility is molecular mimicry resulting in the activation of T cells for CNS proteins in the periphery [3]. Activated T cells then migrate to cervical LN where they interact with antigens drained from the CNS, which drive autoimmunity [3, 28, 31].

Cerebral ischemic injury and microinfarcts The role of the CNS lymphatic system in the pathogenesis of stroke has been investigated using the cervical lymphatic blockade (CLB) model, in which the cervical lymph nodes are surgically removed after ligating the input and output vessels. This model therefore blocks the cerebral lymphatic drainage, causing excess water and waste retention that can lead to brain edema and intracranial hypertension [1]. The importance of cerebral lymphatic function was demonstrated in the ischemic stroke model in which the middle cerebral artery is occluded; the resulting phenotype (such as edema, oxidative stress injury, and neuronal damage) was dramatically worsened with cervical lymph node blockade [32], and CLB has also been shown to cause an expansion of infarct volume in rats [33]. In terms of secondary injury from ischemia, oxidative injury significantly worsened with CLB, indicated by the increased activity of lactate dehydrogenase in the serum and the decreased activity of superoxide dismutase in brain tissues, supporting the role of CNS lymphatic drainage in the protection of the brain, especially from secondary oxidative injury [34].

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Aging Aging plays a considerable role in both central and peripheral immune responses, incorporating both the glymphatic system and brain lymphatic vasculature. As we age, immunosenescence (or a decline in immune cell functions) sets in [35, 36] leading to increased cytokine and chemokine production, for example, IL-1beta, IL-6, IL-18, and TNF-alpha suggestive of links between CNS hyperimmune activation and advancing age and neuroinflammation. This increased low-grade chronic inflammatory milieu in aged brains is especially relevant and present in neurodegenerative disorders. Several studies have shown that when senescent cells were removed from aged mice artificially, the animals lived longer and were healthier [37, 38]. These findings further support the important role that the glymphatic system and brain lymphatic vasculature play in trying to maintain a healthy environment for brain-resident cells, which prolongs cognitions and preserves brain structure. Aging also increases cancer risk, and with respect to the brain, aging is a strong risk factor for the most aggressive adult brain cancer, glioma. While the mechanisms underlying how aging significantly contributes to the occurrence of gliomas are unclear, recent studies suggest that rather than undergoing proliferation and selfrenewal in response to growth signals, some dysregulated neuronal progenitor cells undergo senescence instead [39, 40].

Exploiting the CNS lymphatics for drug discovery As our knowledge of the lymphatic system has expanded, so has the design of lymphatic delivery systems for drug delivery. Initially, these were simple systems that relied on passive lymphatic access; however, rapid progress in the lymphatic field now means that lymphatic delivery systems are now much more complex involving the use of nanotechnology to mimic endogenous macromolecules and lipid conjugates that “hitchhike” onto lipid transport processes [37]. Additionally, it is now appreciated that the lymphatic system is involved in a number of processes and diseases, which have compounded the need and optimization of drug delivery systems to treat diseases such as inflammatory diseases (e.g., rheumatoid

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arthritis and inflammatory bowel disease [41, 42]), cancer and metastatic disease [43, 44], infections, and autoimmune diseases—such as HIV, hepatitis, and Ebola virus [45–48]—and also issues associated with solid organ transplant rejections [49, 50].

Maximizing drug delivery into the lymphatics Several major advantages for drug discovery programs have focused on routes of lymphatic delivery based on the ability to increase drug exposure and/or enhance therapeutic efficacy (i.e., increased efficacy and lower toxicity) in immunotherapy, vaccination, viral therapy, and cancer metastasis (by targeting lymph nodes) [37]. Rates of flow into the blood capillaries is approximately 100–500-fold higher than lymph flow, and as such, drainage of orally or parenterally delivered small molecules and drugs typically occurs via this route into the interstitial space (of importance, intestinal lymphatic transport bypasses the hepatic first-pass metabolism). In the case of larger molecules such as proteins or large peptides, drainage into capillaries is not an option due to size, and as such, they are always transported directly into the lymphatics. In addition, ALL injected drugs enter the lymphatics first. Thus, drug delivery programs have focused on targeting the lymphatic system for effective drug treatments. Recent developments and advancements in the fields of materials and pharmaceutical sciences have driven the movement into finding additional methods of delivery to facilitate the movement of low-molecular-mass therapeutic directly into the lymphatics; these include the use of synthetic macromolecular carriers such as liposomes, in situ association with endogenous macromolecular constructs such as lipoproteins, and also the use of cells with inherent lymphotropic properties such as leukocytes [37, 51]. Other approaches include targeting of the lymph nodes to deliver effective therapy: for example, Liu et al. [52] elegantly used the albumin hitchhiking approach to molecular vaccines, via the synthesis of amphiphiles (amph-vaccines) [52]. In so doing, they were able to successfully deliver optimized CpG-DNA/ peptide amph-vaccines to mice increasing T-cell priming and antitumor efficacy with reduced toxicity.

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The breakthrough in discovering the glymphatic and lymphatic systems in the brain has opened up the window of opportunity to identify new therapeutic targets for the treatment of neurovascular and neurodegenerative disorders. These newly identified cerebral systems are both able to facilitate the transport and clearance of fluid, drug-like molecules, macromolecules, proteins, and immune cells from the brain [53]. These events have huge implications in drug discovery programs, particularly in the context of clearance of a drug, which may be much less efficient in aging or in neurovascular and neurodegenerative disorders. These are exciting times, and certainly, the drug pipeline (with new or repurposed drugs) will continue to expand as targeting of the glymphatic and lymphatic system continues.

Summary In this chapter, we have reviewed that recent developments in lymphatic vascular biology have led to the understanding that the brain does indeed have an efficient waste disposal system that uses both the glymphatic system and the meningeal lymphatic vessels. These previously unknown mechanisms have spearheaded greater opportunities for drug discovery programs (focusing on drugs, prodrugs, vaccines, and delivery systems) targeting the lymphatics for the treatment of a number of neurovascular and neurodegenerative disorders. Much work is still needed, but the pace progress is increasing and will lead to many future therapies and refinements that target these systems.

Acknowledgments The authors would like to thank Ms. G. Kaur for her help with Figs. 1 and 3.

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C H A P T E R

6

Defective development of the peripheral lymphatic system: Lymphatic malformations a

B.B. Leea, M. Amoreb,c

Vascular surgery, George Washington University Hospital, Washington, DC, United States b Phlebology and Lymphology Unit, Cardiovascular Surgery Division, Central Military Hospital, Buenos Aires, Argentina c III Chair of Anatomy, Buenos Aires University, Buenos Aires, Argentina

General aspects Like other perfusion systems, a defective development can occur in the lymphatic system during the complicated lymphangiogenic development and termed lymphatic malformation (LM). LM is therefore one of the various forms of congenital vascular malformations (CVMs) affecting only the lymphatic conduction system as the outcome of defective development during various stages of lymphangiogenesis [1–4]. LM lesion can present as a solitary independent lesion or coexist with other CVMs often as a hemolymphatic malformation (HLM) [5–8] (Fig. 1). In other words, LM exists with other kinds of CVM as part of a complex birth defect affecting the entire blood circulatory system and lymphatics including arteries, veins, capillaries, and the lymphatic system. This combined form of CVM is often one of the vascular malformation components of “syndromic” entities (e.g., Klippel-Trenaunay syndrome). For example, LM coexists with venous malformation (VM) [9–12] and capillary malformation

Lymphatic Structure and Function in Health and Disease https://doi.org/10.1016/B978-0-12-815645-2.00006-X

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Copyright © 2020 Elsevier Inc. All rights reserved.

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6. Defective development of the peripheral lymphatic system

(A)

(B)

(C)

FIG. 1 Hemolymphatic malformation (KTS: Klippel-Trenaunay syndrome). Photograph showing (A) lymphatic malformation (LM), lymphocutaneous (B) capillary malformation (CM), and (C) venous malformation (VM).

fistula,

(CM) [13,14] as the vascular malformation component of KlippelTrenaunay Syndrome (KTS) [15–18]. When they are further combined with arteriovenous malformation (AVM) [19–22], it is defined as Parkes Weber syndrome (PWS) [23,24]. Hence, LM develops as a sole CVM lesion or one of multiple CVM lesions coexisting with other forms of CVM lesions at the same time, for example, VM, CM, and/or AVM.

Embryological aspects During the embryogenesis, the blood and lymphatic systems in particular encounter extremely strenuous conditions to serve as the logistics to provide necessary support to the rapidly growing embryo: from single fertilized egg to billions of cells to form various tissues and organs. Hence, the embryogenesis of this circulation system goes through an intricate sequential process involving evolution, involution, generation, and degeneration to fulfill such critical role to meet its mandate properly [25–28]. As such, the complicated process of embryological development of the lymphatic system gets involved in anomalous involution to form an abnormal network as a consequence of the persistence of the primitive structure. Therefore, it is quite conceivable the embryonic lymphatic vessels/system would encounter such a high risk of defective development in view of extensive evolutional process till it reaches complete maturation, together with blood circulation system, before birth [29–32].

Embryological aspects

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A defective development of the lymphatic system, therefore, can occur throughout any stage of lymphangiogenesis to result in various conditions of malformed lymphatic system [33–36] For example, developing lymphatic sacs, two jugular and two iliac, can fail to make normal maturation in early eighth week of the gestation to have defective development to remain a sequestration of primitive lymphatic tissue as isolated clusters of amorphous lymphatic tissues (e.g., lymphangioma) [37,38]. When these primitive lymphatic tissues fail to make normal development, they are sequestered as an isolated amorphous cluster of lymphatic tissue in various regions with no direct involvement to the lymphatic transport systems; they continue to dilate to possess a unique multicystic morphology in various extents: macrocystic and microcystic lesion, termed as “cystic/cavernous lymphangioma” [39–42]. This lesion affecting the head and neck has been named “cystic hygroma,” with notorious recurrence by its nature/evolution potential to grow when stimulated by surgical excision, etc. Such malformed lymphatic tissue has a unique characteristic of the mesenchymal cell as a residual embryonic tissue remnant. These “prematured” lymphatic tissues are classified separately as “extratruncular” lesions, based on the embryonic stage when such developmental arrest arise, since its clinical outcome is profoundly different depending upon when such event occurred [43] (Fig. 2).

(B)

(A) FIG. 2

Extratrucular lymphatic malformation/lymphangioma. Photograph showing (A) clinical findings of gluteal lymphangioma, affecting right buttock, and (B) ultrasonography view of macrocystic-type lesion, right gluteal region.

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6. Defective development of the peripheral lymphatic system

LM caused by developmental arrest in the “earlier” stage of lymphangiogenesis is referred to as the extratruncular lesion because this lesion is caused by embryonic tissue remnants from the reticular stage, which maintains “evolutional potential,” a unique embryonic characteristic originated from the mesenchymal cells. Therefore, they steadily grow by various intrinsic and extrinsic factors/stimulations (e.g., menarche, pregnancy, female hormone, trauma, and surgery). In other words, LMs are further classified into two groups: “extratruncular” and “truncular” type, and an extratruncular LM lesion represents the outcome of the defective development following the developmental arrest in the “earlier” stage of “lymphangiogenesis” [44–47], while truncular LM lesion represents the lesion/defective development during the “later” stage of lymphangiogenesis before the completion of fully matured normal lymphatic trunk formation. The extratruncular lesion, better known as “lymphangioma,” continues to grow whenever the condition should be met due to this unique characteristic of mesenchymal cells/lymphangioblasts, while “truncular” lesion cannot continue to grow since it no longer possesses this evolutional potential to grow (cf. extratruncular lesion) since it is the outcome of developmental arrest in the “later” stage. However, when disruption of the lymph vessels and node formation processes occurs along the main lymphatic trunk formation after the ninth week of gestation, various structural abnormalities would develop in the lymphtransporting system [48,49]. Hence, truncular LM lesions give a much more serious impact to overall lymph-transporting function due to the direct involvement to the lymph vessels and/or nodes/system as various structural if not functional defects, known clinically as “primary” lymphedema, since this defective development occurs during lymphatic trunk formation [50,51] (Fig. 3).

Clinical aspects Primary lymphedema represents a clinical manifestation of the defective lymphatic system, occurring in the “late” stage of lymphangiogenesis with direct involvement of lymphatic vessels and nodes. Various defective developments result in hypoplasia,

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Clinical aspects

(A)

(B)

(C)

FIG. 3 Truncular lymphatic malformation/primary lymphedema affecting right lower extremity. Photograph showing (A) anterior view, (B) posterior view, and (C) lymphoscintigraphic findings.

aplasia, numerical hyperplasia, or dilation (lymphangiectasia) of lymphatic vessel trunk with valvular incompetence [52–55]. Lymph node dysplasia, known as “lymphnododysplasia,” is generally involved together with lymphangiodysplasia, but selective lymph node dysplasia alone can cause primary lymphedema [56,57]. However, not all primary lymphedema accompanies such anatomical defects to cause lymph stasis, and some have only defective function with no visible defect. Such conditions that are limited to a functional defect with very little structural derangement are caused by some effect that is molecular in origin (e.g., Milroy’s disease) [58–61], with all primary lymphedemas being genetically derived. In other words, truncular LMs are not always accompanied by morphologically detectable defects of the lymphatic system. “Milroy-Meige syndrome” does not have any apparent structural defects of the lymphatic system but rather has a functional impairment at the capillary lymphatics/initial lymphatics level. Also, “lymphedema-distichiasis syndrome” exhibits impairment of the endoluminal valves, which cause lymphatic reflux. There are 41 syndromes with peripheral primary lymphedema, added to 85 syndromes with primary generalized lymphedema: 1. Noonan syndrome 2. Turner syndrome

114 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

6. Defective development of the peripheral lymphatic system

Yellow nail syndrome Nevo syndrome Aplasia cutis + primary lymphedema (Bronspiegel syndrome) Cholestasis + primary lymphedema (Aagenaes syndrome) Progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PHEO) syndrome Cerebral arteriovenous malformation + primary lymphedema (Avasthey syndrome) Cleft palate + primary lymphedema (Figueroa syndrome) Hypoparathyroidism + primary lymphedema (Dahlberg syndrome) Distichiasis + primary lymphedema Microcephaly + primary lymphedema

These 12 syndromes are most frequently mentioned among many others. Such syndromes are often detected among the newborns and 1-month-old pediatric patients, with primary lymphedema combined with various conditions such as uni- or bilateral Wilms’ tumor, unilateral suprarenal cysts, superficial and deep venous malformations, KTS, PWS, neurofibromatosis, and combined angiodysplastic syndromes. Rarely, such a condition becomes more complicated with the Gorham-Stout syndrome, Haferkamp syndrome, Proteus syndrome, lipodysplasias, exudative enteropathies, and chylous reflux syndromes. The classification of primary lymphedema into three groups of congenital, praecox, and tarda types is based on the age at first clinical manifestation, but they all have similar dysplastic and or functional causes. Instead, primary lymphedema can be graded based on its expression in grades (0–3). In terms of incidence and prevalence statistics, degrees 0–1 are not reported but are the most frequent ones [62,63]. Hence, many believe that “primary” lymphedema is caused either by intrinsic “defects” [64–67] or a malfunction of the lymph conducting elements [68–71] due to a genetically determined abnormality of lymphatic anatomy or function. Therefore, a malformation may require detection or definition in molecular or other functional terms since it may not necessarily provide sufficient structural attributes that can be imaged [54,55,72,73].

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Genetic considerations

Genetic considerations (see Table 1) All primary lymphedema clinically manifesting as a truncular LM with a macroscopic structural abnormality is the outcome of heritable abnormal structural development [74]. Such inherited conditions TABLE 1 Genetic disorders associated with primary lymphedema Single gene disorders associated with primary lymphedema

Chromosome/gene

Milroy disease

VEGFR3

Milroy-like disease

VEGFC

Late-onset 4 limb lymphedema

GJC2

Lymphedema-distichiasis syndrome

FOXC2

Syndromes associated with primary lymphedema Aagenaes syndrome

Not known

Cardiofaciocutaneous syndrome

KRAS, BRAF, MAP2K1,MAP2K2

CHARGE syndrome

CDH7

Choanal atresia-lymphoedema

PTPN14

Ectodermal dysplasia, anhidrotic, immunodeficiency, osteopetrosis, and lymphoedema(OL-EDA-ID syndrome)

IKBKG (NEMO)

Fabry disease

GLA

Hennekam syndrome (generalized lymphatic dysplasia)

CCBE1

Hypotrichosis–lymphoedema–telangiectasia

SOX18

Lymphoedema-myelodysplasia (Emberger syndrome)

GATA2

Microcephaly with or without chorioretinopathy, lymphoedema, and mental retardation (MCLMR)

KIF11

Noonan syndrome

PTPN11, KRAS, SOS1

Chromosomal abnormalities associated with primary lymphedema Turner syndrome

45,X0

Phelan-McDermid syndrome

22q Terminal deletion Ring chromosome 22 Continued

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TABLE 1 Genetic disorders associated with primary lymphedema—cont’d Single gene disorders associated with primary lymphedema

Chromosome/gene

Prader-Willi syndrome

15q11 Microdeletion Or maternal UPD 15

Velocardiofacial syndrome

22q11 Microdeletion

Lymphatic abnormalities associated with a mosaic genetic defect Proteus syndrome

AKT1

Klippel-Trenaunay syndrome CLOVE/Fibroadipose hyperplasia

AKT pathway

Macrocephaly-capillary-malformation (MCM)

AKT pathway (PIK3CA)

Primary lymphedema with unknown etiology but likely to be genetic Meige disease Multisegmental lymphatic dysplasia with systemic involvement From Lee BB, Andrade M, Antignani PL, Boccardo F, Bunke N, Campisi C, Damstra R, Flour M, Forner-Cordero J, Gloviczki P, Laredo J, Partsch H, Piller N, Michelini S, Mortimer P, Rabe E, Rockson S, Scuderi A, Szolnoky G, Villavicencio JL. Diagnosis and treatment of primary lymphedema. Consensus Document of the International Union of Phlebology (IUP)-2013. Int Angiol 2013;32(6):541–574.

are caused by the mutation of any of the genes involved in the lymphatic system development. FLT4 [59,64,75], FOXC2 [60–64], and GJC2 [76,77] are the ones identified in a familial distribution of primary lymphedema as representative genetic mutations. Various genes have been found associated with this pattern of inheritance with variable expression and age at onset [70,73,75] All these forms of genetically determined lymphedema have the lymphedema as primary phenotype as major clinical sign, and an autosomal dominant pattern of inheritance has been also reported among multigeneration families. Milroy’s disease is one model for familial lymphedema caused by an autosomal dominant single gene disorder inherited as a germline mutation at the locus on distal chromosome 5q35.3. This disease represents one subgroup of congenital lymphedema with clinically evident lymphedema at birth (more often among females), lower extremities, and single leg involved. In this condition, FLT4 is the gene that is mutated, which encodes for the vascular endothelial growth factor receptor 3 (VEGFR-3) receptor [73,75,78] Various

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mutations in the VEGFR-3 gene have been further identified confirming the underlying etiology of this disease [79,80]. (Further detailed descriptions in regard to the role of VEGFR-3 is given in Chapter 2.) Milroy’s disease is also known as hereditary lymphedema type 1A representing the group of “early” onset lymphedema. Milroy’s disease is an autosomal dominant condition caused by a mutation in the gene FLT4 associated, which is encoded to VEGFR-3 gene located on the long arm (q) on chromosome 5 (5q35.3). In contrast to Milroy’s disease, Meige disease, also known as hereditary lymphedema type II, represents the majority of primary lymphedema (60%–80%) with onset at ages 1–35, most often around the time of puberty. Meige disease is frequently associated with several other anomalies including yellow nails, distichiasis, vertebral anomalies, extradural cysts, cerebrovascular malformation, and sensorineural hearing loss [81] Meige disease is also an autosomal dominant disease, linked to the mutations of FOXC2 gene, which is located on the long arm of chromosome 16 (16q24.3). Absence of FOXC2 leads to the failure of lymphatic valves formation and lymphatic remodeling; a number of mutations in the FOXC2 gene have been associated with lymphedema-distichiasis syndrome [82,83]. Lymphedema–distichiasis syndrome is a subset of lymphedema praecox, genetically linked to the FOXC2 gene involved in diverse developmental pathways [84,85]; this disease is a rare autosomal dominant disease to cause swollen limbs and double rows of eyelashes [86] FOXC2-deficient mice display lymphatic dysfunction as the results of abnormal mural cell coverage on lymphatic capillaries, defective valves of the collecting lymphatic vessels, and irregular lymphatic patterning [87]. Hypotrichosis–lymphedema–telangiectasia syndrome is another subset of lymphedema praecox associated with hypotrichosis and telangiectasia [88]; the mutations of SOX18, which acts upstream of PROX1, is responsible for both recessive and dominant forms of this disease [89,90]. Two more genes (GJC2 and CCBE1) were recently identified to cause lymphedema. The mutations in the protein encoded in GJC2 gene cause impaired gap junction activities resulting in defective lymphatic flow [74], while the mutations in CCBE1 that plays a role in lymphatic sprouting during zebrafish development were found in Hennekams’ lymphangiectasia-lymphedema syndrome [91,92].

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The group of primary lymphedema with onset after the age of 35, also known as lymphedema tarda, is considered as the third hereditary lymphedema. However, the majority of primary lymphedema belongs to “sporadic” type, and the “hereditary” type is quite rare although both have a genetic basis. Nevertheless, all the truncular LMs are present at birth by definition although postnatal obliterations of lymph collectors and lymph nodes mimic congenital/prenatal pathology. LM patients belonging to several congenital lymphatic disorders such as Down syndrome, Turner syndrome, and Noonan syndrome should receive genetic counseling to become parents, including cytogenetic analysis for chromosomal aneuploidy because aneuploidic conditions can recur in subsequent pregnancies [93–96]. Therefore, correct understanding of the pathophysiology of primary lymphedema as the outcome of defective lymphatic system development is mandated for proper definition on the normal anatomy and physiology of the lymphatic system.

Management However, lymphedema is manageable, with a remarkable response to multidisciplinary treatment, including specific conditions such as head, face and neck lymphedema, genital lymphedema, and lymph leakage. Most patients presenting with lymphedema in adulthood are diagnosed with secondary lymphedema, even without a clear underlying cause. It is likely that many of these patients will have an underlying genetically determined primary lymphatic abnormality that the clinician has not considered. The identification of the molecular abnormality for each subtype of primary lymphedema is crucial because it advances the understanding of the underlying mechanism of the disease. The identification of the genetic causes of primary lymphedema provides a molecular diagnostic test for some subtypes. Patients and families benefit hugely from a molecular diagnosis because it allows the clinician to confidently predict the clinical prognosis and offer screening for family members [97].

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References [1] Lee BB. Lymphedema-Angiodysplasia syndrome: a prodigal form of lymphatic malformation (LM). Phlebolymphology 2005;47:324–32. [2] Lee BB, Laredo J, Lee TS, Huh S, Neville R. Terminology and classification of congenital vascular malformations. Phlebology 2007;22(6):249–52. [3] Lee BB, Laredo J, Lee SJ, Huh SH, Joe JH, Neville R. Congenital vascular malformations: general diagnostic principles. Phlebology 2007;22(6):253–7. [4] Lee BB, Kim YW, Seo JM, Hwang JH, Do YS, Kim DI, Byun HS, Lee SK, Huh SH, Hyun WS. Current concepts in lymphatic malformation (LM). Vasc Endovascular Surg 2005;39(1):67–81. [5] Lee BB, Laredo J. Classification: venous-lymphatic vascular malformation. [chapter 21], Part 3. In: Allegra C, Antignani PL, Kalodiki E, editors. News in phlebology. Turin: Edizioni Minerva Medica; 2013. p. 91–4. [6] Lee BB, Bergan J, Gloviczki P, Laredo J, Loose DA, Mattassi R, Parsi K, Villavicencio JL, Zamboni P. Diagnosis and treatment of venous malformations consensus document of the International Union of Phlebology (IUP)-2009. Int Angiol 2009;28(6):434–51. [7] Lee BB, Baumgartner I, Berlien P, Bianchini G, Burrows P, Gloviczki P, Huang Y, Laredo J, Loose DA, Markovic J, Mattassi R, Parsi K, Rabe E, Rosenblatt M, Shortell C, Stillo F, Vaghi M, Villavicencio L, Zamboni P. Diagnosis and treatment of venous malformations consensus document of the International Union of Phlebology (IUP): updated 2013. Int Angiol 2015;34(2):97–149. [8] Lee BB, Antignani PL, Baraldini V, Baumgartner I, Berlien P, Blei F, Carrafiello GP, Grantzow R, Ianniello A, Laredo J, Loose D, Lopez Gutierrez JC, Markovic J, Mattassi R, Parsi K, Rabe E, Roztocil K, Shortell C, Vaghi M. ISVI-IUA consensus document diagnostic guidelines on vascular anomalies: vascular malformations and hemangiomas. Int Angiol 2015;34 (4):333–74. [9] Lee BB. Venous malformation and haemangioma: differential diagnosis, diagnosis, natural history and consequences. Phlebology 2013;28(Suppl 1):176–87. [10] Lee BB, Laredo J. Venous malformation: treatment needs a bird’s eye view. Phlebology 2013;28:62–3. [11] Lee BB, Baumgartner I. Contemporary diagnosis of venous malformation. J Vasc Diagn 2013;1:25–34. [12] Lee BB. Current concept of venous malformation (VM). Phlebolymphology 2003;43:197–203. [13] Berwald C, Salazard B, Bardot J, Casanova D, Magalon G. Port wine stains or capillary malformations: surgical treatment. Ann Chir Plast Esthet 2006;51(4–5):369–72. [Epub 2006 Sep 26]. [14] Goldman MP, Fitzpatrick RE, Ruiz-Esparza J. Treatment of port-wine stains (capillary malformation) with the flashlamp-pumped pulsed dye laser. J Pediatr 1993;122(1):71–7. [15] Lee BB, Laredo J. Hemo-lymphatic malformation: Klippel-Trenaunay syndrome. Rev Acta Phlebologica 2016;17(1):15–22.

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[16] Lee BB. Klippel-Trenaunay syndrome: is this term still worthy to use? Acta Phlebol 2012;13:1–2. [17] Lee BB, Laredo J, Neville R, Mattassi R. Primary lymphedema and KlippelTrenaunay syndrome. Chapter 52. Section XI—lymphedema and congenital vascular malformation. In: Lee B-B, Bergan J, Rockson SG, editors. Lymphedema: a concise compendium of theory and practice. 1st ed. London: Springer-Verlag; 2011. p. 427–36. [18] Lee BB. A stepwise daily guide to the KTS. Vasculab debate. J Theor Appl Vasc Res 2016;1(2):33–8. [19] Lee BB, Lardeo J, Neville R. Arterio-venous malformation: how much do we know? Phlebology 2009;24:193–200. [20] Lee BB, Baumgartner I, Berlien HP, Bianchini G, Burrows P, Do YS, Ivancev K, Kool LS, Laredo J, Loose DA, Lopez-Gutierrez JC, Mattassi R, Parsi K, Rimon U, Rosenblatt M, Shortell C, Simkin R, Stillo F, Villavicencio L, Yakes W. Consensus document of the International Union of Angiology (IUA)-2013. Current concept on the management of arterio-venous management. Int Angiol 2013;32(1):9–36. [21] Lee BB, Mattassi R, Kim BT, Kim DI, Ahn JM, Choi JY. Contemporary diagnosis and management of venous and AV shunting malformation by whole body blood pool scintigraphy (WBBPS). Int Angiol 2004;23(4):355–67. [22] Lee BB, Mattassi R, Kim BT, Park JM. Advanced management of arteriovenous shunting malformation (AVM) with transarterial lung perfusion scintigraphy (TLPS) for follow-up assessment. Int Angiol 2005;24(2):173–84. [23] Ziyeh S, Spreer J, Rossler J, Strecker R, Hochmuth A, Schumacher M, Klisch J. Parkes Weber or Klippel-Trenaunay syndrome? Non-invasive diagnosis with MR projection angiography. Eur Radiol 2004;14(11):2025–9. [Epub 2004 Mar 6]. [24] Courivaud D, Delerue A, Delerue C, Boon L, Piette F, Modiano P. Familial case of Parkes Weber syndrome. Ann Dermatol Venereol 2006;133(5 Pt 1):445–7. [25] Lee BB. New classification of congenital vascular malformations (CVMs). Rev Vasc Med 2015;3(3):1–5. [26] Lee BB, Laredo J. Pathophysiology of primary lymphedema [chapter 12]. In: Neligan PC, Piller NB, Masia J, editors. Complete medical and surgical management. Boca Raton, FL: CRC Press; 2016. p. 177–88. [27] Suami H, Lee BB, Kim YW, Lee BB, Yakes WF, Do YS, editors. Embryological background of congenital vascular malformations. Chapter 2. Part I. Introduction. In: Congenital vascular malformations—a comprehensive review of current management. Berlin, Heidelberg: Springer-Verlag; 2017. p. 7–14. [28] Lee BB, Laredo J. Obstructive primary truncular venous malformations— general overview. Chapter 4. In: Giaquinta A, Lee BB, Setacci C, Veroux P, Zamboni P, editors. Latest frontiers of hemodynamic, imaging and treatment of large veins. 2018. p. 28–36. [29] Lee BB. Congenital venous malformation: changing concept on the current diagnosis and management. Asian J Surg 1999;22(2):152–4. [30] Lee BB, Bergan JJ. Advanced management of congenital vascular malformations: a multidisciplinary approach. J Cardiovasc Surg 2002;10(6):523–33.

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[49] Krutsiak VN, Polianskiı˘ II. Development of the thoracic duct in the prenatal period of human ontogeny. Arkh Anat Gistol Embriol 1983;85(11):79–84. [50] Gloviczki P, Duncan AA, Kalra M, Oderich GS, Ricotta JJ, Bower TC, et al. Vascular malformations: an update. Perspect Vasc Surg Endovasc Ther 2009;21(2):133–48. [51] Lee BB, Laredo J. Classification of congenital vascular malformations: the last challenge for congenital vascular malformations. Phlebology 2012;27(6):267–9. [52] Lee BB, Villavicencio JL. Primary lymphedema and lymphatic malformation: are they the two sides of the same coin? Eur J Vasc Endovasc Surg 2010;39:646–53. [53] Lee BB, Andrade M, Bergan J, Boccardo F, Campisi C, Damstra R, Flour M, Gloviczki P, Laredo J, Piller N, Michelini S, Mortimer P, Villavicencio JL. Diagnosis and treatment of primary lymphedema—consensus document of the International Union of Phlebology (IUP)-2009. Int Angiol 2010;29(5):454–70. [54] Lee BB, Andrade M, Antignani PL, Boccardo F, Bunke N, Campisi C, Damstra R, Flour M, Forner-Cordero J, Gloviczki P, Laredo J, Partsch H, Piller N, Michelini S, Mortimer P, Rabe E, Rockson S, Scuderi A, Szolnoky G, Villavicencio JL. Diagnosis and treatment of primary lymphedema. Consensus document of the International Union of Phlebology (IUP)-2013. Int Angiol 2013;32(6):541–74. [55] Lee BB, Antignani PL, Baroncelli TA, Boccardo FM, Brorson H, Campisi C, Damstra RJ, Flour M, Giannoukas AD, Laredo J, Liu NF, Michelini S, Piller N, Rockson SG, Scuderi A, Szolnoky G, Yamamoto T. IUA-ISVI consensus for diagnosis guideline of chronic lymphedema of the limbs. International Angiology 2014 [EPUB ahead of print]. [56] Lee BB, Laredo J, Neville R. Primary lymphedema as a truncular lymphatic malformation. Chapter 51, section XI—lymphedema and congenital vascular malformation. In: Lee B-B, Bergan J, Rockson SG, editors. Lymphedema: a concise compendium of theory and practice. 1st ed. London: Springer-Verlag; 2011. p. 419–26. [57] Papendieck CM. Lymphangiomatosis and dermoepidermal disturbances of lymphangio-adenodysplasias. Lymphology 2002;35:478–85. [58] Witte MH, Erickson R, Bernas M, et al. Phenotypic and genotypic heterogeneity in familial Milroy lymphedema. Lymphology 1998;31:145–55. [59] Brice G, Child AH, Evans A, Bell R, Mansour S, Mortimer P, et al. Milroy disease and the VEGFR-3 mutation phenotype. J Med Genet 2005;42:98–102. [60] Erickson RP. Lymphedema-distichiasis and FOXC2 gene mutations. Lymphology 2001;34(1):1–5. [61] Brice G, Mansour S, Bell R, Collin JRO, Child AH, Mortimer P, et al. Analysis of the phenotypic abnormalities in lymphoedema-distichiasis syndrome in 74 patients with FOXC2 mutations or linkage to 16q24. J Med Genet 2002;39:478–83. [62] Papendieck CM, Amore M. An atlas of neonatal and infantile lymphedema. [chapter 62]. In: Lee B-B, Rockson SG, Bergan J, editors. Lymphedema: a concise compendium of theory and practice. 2nd ed. Berlin, Heidelberg: SpringerVerlag; 2018. p. 777–96.

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C H A P T E R

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Cardiac lymphatics and cardiac lymph flow in health and disease J. Steve Alexander, J. Winny Yun Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA, United States

Cardiac lymphatics Like most organs, the heart has a dense lymphatic supply with lymphatic plexuses found in all layers of the heart, within the subepicardium, myocardium, and subendocardium. These networks fulfill similar functions seen in other organ systems including the collection of interstitial fluid, fat and cholesterol reverse transport, and immune surveillance. In most species, the distribution of lymphatics through these layers is fairly consistent [1, 2] although in mice and rabbits, the subepicardial and myocardial plexuses may be denser than the endocardium, with some anatomists even reporting no lymphatics in the endocardium. The ontogeny of the cardiac lymphatic vasculature in the heart is also becoming better understood [3]. Embryologically, the layers of the myocardium and endocardium are derived from lateral plate mesodermal tissue. However, several other critical components of the heart, including the epicardium and cardiac blood vessels, have extra cardiac origins in the proepicardium [3a]. In the mouse, cardiac lymphatic development is prenatally restricted to the subepicardium. After birth, lymphatics expand from the subepicardium through the myocardial wall, with progressive development of lymphatics starting at the base of the heart gradually moving toward the apex of the heart.

Lymphatic Structure and Function in Health and Disease https://doi.org/10.1016/B978-0-12-815645-2.00007-1

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Copyright © 2020 Elsevier Inc. All rights reserved.

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Lymphatics were also found to become associated with the major branches of cardiac veins in the subepicardium [4]. Early on in cardiac development, cells expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), which is commonly employed as a molecular marker for lymphatic endothelial cells [4a], can be seen in some epicardial cells. Additionally, just prior to the septation of the cardiac chambers, a sheet of prospero homeobox 1 (Prox1)—expressing cells—is found on the aorta and pulmonary trunk, an event that is seen in avian and murine embryos [4b]. Prox1 is a transcription factor typically referred to as “the master regulator” of lymphatic vasculature because it regulates the expression of downstream lymphatic endothelial— specific proteins, such as LYVE-1 and vascular endothelial growth factor receptor-3 (VEGFR-3) [4c]. VEGFR-3 responds to its ligands vascular endothelial growth factor (VEGF)-C and (VEGF)-D to maintain an important feedback loop with Prox1 that maintains the lymphatic identity and undergoes lymphangiogenesis, the process of lymphatic vessel formation from preexisting lymphatic vessels [4d]. Once LYVE1+ PROX1+ VEGFR3+ lymphatic vessels appear at the sinus venosus, they progressively spread over both ventricles, eventually ensheathing the entire epicardium. Although lymphatic and venous networks do show early interactions with these networks, this is progressively lost as development progresses. Hemangioblasts that give rise to blood vessels in the heart are contributed by the proepicardium; however, it seems that cardiac lymphatics may be formed at least in part by invading CD31+/CD45+/LYVE-1+ lymphangioblasts, which progressively colonize the subepicardial space from the base to the apex of the growing heart during development. Studies using mice bearing markers of vascular endothelial cells (Tie2-Cre and PDGFRβ-CreERT2) support cardiac lymphatics originating from a mixed pool of progenitors including venous and putative hemogenic endothelial origins [3]. Ultimately, Karunamuni described three types of lymphatic endothelial-like lineages in the heart: Prox1 + cells from an extracardiac source (possibly mesothelium) that migrate into outflow trunks of the epicardium, LYVE1 + cells found in the epicardium that may merge into Prox1+ lymphatic vessels, and LYVE1+ cells in the myocardium that remain Prox1

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negative even in mature heart tissues [4b]. We have reported that mesothelial cells also express lymphatic markers including VEGFR3, LYVE1, and Prox1. Therefore, these may be a source of some of these epicardial cells [5], which also concurs with findings by Wada et al. [6] and Perez-Pomares et al. [7]. Lymphatics are found in the ventricular septum that appear to communicate with the atrioventricular node in the human [8], which may coordinate information about the immune or inflammatory stress within the septal interstitium, thereby modulating atrioventricular (AV) nodal conduction. The lymphatic system of the heart was probably recognized originally by the Swedish anatomist Olaus Rudbeck in 1653 (and J.F. Cassebohm), who noted subepicardial lymphatics in dogs. This was later described in humans by Eberth and Belajeff [9] in 1866 (summarized in an excellent historical review on lymphatics by [9a]). Lymphatics in the myocardium appear to have been first recognized by Leyh in 1859; he later described lymphatic vessels in myocardial interstitial tissue [9b].

Flow of lymph in the heart Lymph is formed in all layers of the heart and is moved progressively outward from the subendocardium through the myocardium and ultimately into the subepicardium. Lymph outflow from the subepicardium is collected by the left and the right cardiac lymphatic trunks. These collectors ultimately transport lymph into the left coronary trunk and then to the right venous angle and to the left venous angle via the right coronary trunk. These lymphatic collectors contain unidirectional valves, which prevent lymph reflux and are arranged in pairs, which parallel the coronary arteries in the subepicardium [3a]. Like in most organs, both active lymphangion pumping and passive lymphatic compression contribute to lymph propulsion in the heart. Phasic pumping can occur within lymphangions, which are literally lymph “hearts.” Lymphangions are segments of lymphatics bounded by one-way valves, whose rhythmic contractions force lymph forward toward the heart. The rate and forcefulness of these contractions have been proposed to be regulated both by

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intraluminal and extraluminal pressure [10] that determine stretch on the muscle in the wall as well as inflammatory cytokines, nitric oxide, and prostaglandins. By comparison, passive tissue compressive force also provides a continuous squeezing on these structures, which also propels lymph toward the heart [11, 12]. The degree to which each contributes to lymph propulsion varies anatomically often reflecting the capacity for the development of passive compressive force. Due to the fact that the heart is such a dynamic organ, which undergoes significant changes in tissue pressures in its different regions (endocardium and myocardium) during the distinct phases of the cardiac cycle, passive lymph conduction likely represents the dominant mechanism through which lymph is removed from the heart. Furthermore, because the frequency of the cardiac cycle is often higher than that reported for lymphangion pumping, active pumping, at least in the endo- and myocardium, might be difficult to coordinate in terms of matching pressures. However, this might be a possibility in the epicardium [13–15]. With respect to this, cardiac pacing, which decreases the time spent in diastole, reduced cardiac lymph flow, despite a slight increase in cardiac blood flow (which might be expected to increase cardiac lymph flow) [16]. Cardiac lymph flow is usually continuous and high with respect to several other organs. The efferent outflow from the heart is 5–27 mg/min 10 kg. Miller reported that cardiac lymph flow varies between 35 and 189 mg/min in dogs 70–388 mg/min in dogs or a rate of formation between 4.2 and 23 mL/h. Miller has also reported 3.2 mL/h under basal conditions [17]. Michael et al. [18] more recently reports this figure as 0.45–5.6 mL/h. With respect to coordination between the cardiac cycle and passive lymphatic compression to drive lymph movement through the heart, Kampmeier [19] stated that in diastole, pressure of the blood filling the ventricles forces lymph outward from the subendocardial lymphatics into lymphatic vessels in the myocardium. By comparison, during systole, myocardial contractility squeezes lymph from the myocardium into lymphatics in the subepicardium. Lastly, at the end of diastole, pericardial pressure helps to move lymph from these vessels into lymphatic trunks exiting heart tissue. However, Cui points out that surgeries that involve the removal of the pericardium have little effect on cardiac edema; thus, this may only be a minor component with most of the force deriving from

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subepicardial compressive forces. Similarly, uncoordinated muscular compression in the subepicardium could lead to retention on lymph and lymphedema under some circumstances [20, 21]. The ventricles have more extensive lymphatic networks than those found in the atria [22]. Since ventricular force development is greater in the ventricles than in the atria, tissue compressive forces are likely to be lower in the atria. Therefore, the physics supporting lymph flow out of the atria favor a net lower flow based on the product of vessel density and compressive forces, which may render the atria more susceptible to forces that may oppose lymph flow out of the atria (compared with that in ventricles). Consequently, structures in the atria, for example, the sinoatrial (SA) node, may experience stresses produced by impaired lymph conduction if any forces opposed the exit of lymph. The lymphatics within the subepicardium have been extensively studied through injection of tracers [22a] and have been shown to merge to several lymphatic trunks that cumulatively collect cardiac lymph passing it to the mediastinal lymphatics of the right lymphatic duct and the thoracic duct. Ventricular myocardial contractions are assumed to squeeze lymph from lymphatic vessels within the myocardium into lymphatic vessels in the subepicardium. It has been proposed that the distention of the heart at the conclusion of diastole would create pressure at the surface of the epicardium resisting the fluid pressure from the pericardium, which would provide the force necessary to force lymph in the subepicardial vessels into the lymphatic trunks exiting the heart. However, Cui [20, 21] points out that this assumption has not been tested and that it would be expected that in the absence of a pericardium, the heart should have an increased risk of developing lymphedema. However, the surgical removal of the pericardium has few such clinical effects in experimental or clinical cardiac procedures. Cui proposes that instead, the final propulsion can be simply mediated by the forceful contractions of subepicardial muscle alone.

Lymphatics within different cardiac structures In 1866, Eberth and Belajeff first reported lymphatics in human cardiac valves; Johnson and Blake [22b] later also reported the presence of lymphatics in human mitral valves, which were located

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more superficially (closer to the valve surface) than the blood vessels supplying the valves. Indeed, in 1966, Johnson and Blake (using hydrogen peroxide to reveal lymphatics) described “dense networks” of small (15–20 μm) lymphatic vessels without valves under the subepicardium of both ventricles and septum, small lymphatic vessels of relatively uniform caliber (15–20 μm in diameter), which ran perpendicularly to the muscle fibers. Although AV valves are nearly devoid of blood capillaries (except in the proximal region), there are extensive lymphatics draining these valves with the most abundant lymphatic plexus located in the anterior cusp of the mitral valve. In 1973, Bradham [23] described lymphatics in the atrioventricular valves (but possibly not within arterial valves) (according to Refs. [22a, 22b, 23a]). There also appears to be a reticular network of lymphatics within the interventricular septum and within papillary muscles [22]. The SA node has lymphatics, which discharge their contents via subepicardial lymphatics [23b]. Similarly, lymph flow from the AV node and the associated bundle complex drains to the subendocardial lymphatics near the tricuspid and then back to the left lymph trunk finally reaching the inferior tracheobronchial lymph nodes [23c]. Noguchi [23d] also described lymphatics in the mitral and tricuspid valves, which communicated with lymphatic networks within the subendocardium at the atrial side of the valves. Lymphatic vessels have also been described within papillary muscles, where a lymphatic channel oriented with the long axis of the muscle is described, which eventually merges with lymphatics in the myocardium and epicardium; there is only minor drainage near the chordae tendineae [22b]. There also appears to be lymphatics in the wall of coronary arteries [23e, 23f], which connect to the adventitia and the outer annuli of the media [23e, 23g], which Eliska et al. [23h] disagree with this, and describe a denser supply of lymphatics coronary arteries in the myocardium than in epicardial arteries. The recent description of lymphatic biomarkers including Prox1, LYVE-1, VEGFR-3, podoplanin, and forkhead box C2 (FOXC2) probably justifies an immunoanatomical review of these findings based on protein expression of lymphatic

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markers rather than ultrastructure. In any case, the effective clearance of materials, which penetrate into the arterial matrix by lymphatics present in the wall, represents an important and understudied mechanism for the development of atherosclerosis, particularly those within the aorta and coronary vessels. There appears to be a subendothelial lymphatic network in the aorta (in pigs) originally described in cats by Lee [23i]. Due to the fact that lymphatics function in the reverse transport of cholesterol [24, 25] via the scavenger receptor class B type I (SRB1) expressed on lymphatic endothelial cells [24], the obstruction or obliteration of lymphatics in the arterial walls in atherosclerosis might represent an important connection, which may lead to intensification or acceleration of plaque development in atheromatous vessels (arterial lymphatics have recently been extensively reviewed by Csanyi and Singla [26]). Conversely, it has been reported that hypercholesteremia can also provoke lymphatic failure, perhaps by exhausting the reverse transport capacity of this system as a vicious cycle [27]. This paradigm has found additional support in studies, which show that mice, which are deficient in PCSK9, have enhanced lymphatic vessels; similarly, atheroprone low-density lipoprotein receptor knockout (LDLR / )mice (a model of human atherosclerosis) exhibited lymphatic disturbances before the development of atherogenesis, and this atheromatous phenotype could be relieved by administration of VEGFR-3 agonist VEGF-C152S, which expands lymphatic networks [28]. Therefore, one of the more important functions that lymphatics may play in the heart could be to maintain the clearance of materials, for example, cholesterol, reaching the arterial intimamedia which if not adequately removed drives atheroma development. This concept originated by GM Lemole in 1981 and reiterated in 2016 [29, 30] (summary by Bland [31]) suggested a cardiac lymphatic drainage model for the origin of (cardiac) vascular atherosclerosis, which was supported by Lim et al. [24]. Therefore, factors and conditions, which may impair the perivascular or vasa vasorum-integrated lymphatics, may represent an important and underrecognized contributor to atherogenesis, both in coronary vessels and in the communicating arterial tree.

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In addition to the roles that lymphatic vessels play in the reverse transport of cholesterol, the lymphatic failure, specifically leakiness produced by Prox1 heterozygosity (in mice), leads to a chylothorax (fat/lipid accumulation in the thorax), and the development of adult onset obesity [32] and restoration of Prox1 can reverse obesity in Prox1+/ mice [33]. Interestingly, adipocytes persistently express Prox1 with omental fat showing net greater expression compared with subcutaneous Prox1 expression [34]; removal of omental fat does not provide any benefit for correcting insulin sensitivity [35]. Indeed, it is the obesity itself, rather than the consumption of fat, which interferes with lymphatic functioning, suggesting that fat-laden adipocytes produce factors, which impair lymphatics and produce pathologies stemming from lymphatic failure [36]. Prox1 deficiency, which suppresses lymphatics, increases leptin expression to enhance satiety; conversely, lymphatic expansion may promote feeding behaviors. It is also worth noting that there is an increased binding and fat tissue burden of immune cells in high-fat diet/obese C57BL/6J mice, which had significantly more perilymphatic CD3+ and CD11b+ cells (threefold) more than controls [36]. It is interesting and important to note that exercise can correct lymphatic dysfunction that is caused by obesity [37], an observation that has important implications in not only cardiovascular disease but also many other pathologies. Similarly, weight loss normalizes monocyte immune functioning [38], which may also dysregulate lymphatic functions. The plethora of pathological changes, which accompany metabolic syndrome, including those of the heart, stems in part from excessive ingestion of fats, which need to be transported by lymphatics. Interestingly, the disturbances in lymphatic function produced by fat burdens may differ depending on the various fat depots affected. Similarly, the consumption of excessive salt in salt-sensitive hypertension may increase the retention of sodium in subcutaneous tissues where lymph flow is increased; lymph flow is also increased in muscular tissues (skeletal). This increased lymph flow was apparently dependent upon monocyte/macrophages as the depletion of these cells using clodronate (a calcium sparing bisphosphonate) reduces lymph flow [38a]. This effect of salt on lymphatic networks is produced by the activation of the tonicity-responsive enhancer binding protein (“tonEBP”) within monocytes in subcutaneous

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tissues, which drive vascular endothelial growth factor-C (VEGFR-C) expression that binds to VEGFR-3 to caused lymphangiogenesis and expands these networks. It is unclear how this may directly affect the heart itself as increased lymph flow in the heart could be protective but might also represent a response to increased sodium burden in muscle tissues. For example, Yang et al. [39] showed that VEGF-C participated in the observed cardiac lymphangiogenesis seen during high salt-mediated left ventricular cardiac remodeling [39] and that VEGF-C overexpression might be beneficial in high salt-mediated ventricular edema and remodeling, possibly establishing a link between interstitial fluid balance and maladaptive cardiac expansion. Cardiac lymph nodes were first described by Mouchet in 1909 (in dogs and horses), but he did not find these in human hearts. The mediastinal lymph nodes associated with the brachiocephalic lymph nodes located above the aorta and inferior tracheobronchial lymphatics near the exit of the aorta from the heart merging into the right coronary trunk. The enlargement of mediastinal lymph nodes during left heart disease with pulmonary edema suggests that fluid decompensation may intensify lymph node enlargement, but this enlargement seems to be noninflammatory and regresses rapidly when the underlying heart disease is treated [40]; a similar expansion and retraction over the course of a month has been reported [41]. Therefore, conditions that increase lymph flow or impede its return to the circulation could expand lymph node volume and potentially influence immune surveillance within lymph nodes. Ultimately, lymph is returned to the circulatory system by the thoracic and right lymphatic ducts. The thoracic duct proper communicates 75% of the lymph collected by the lymph trunks back to the heart and anatomically begins above the cisterna chyli. Anatomically, the thoracic duct proceeds vertically in front of the vertebral column between the aorta and the azygous vein and by the level of the fifth thoracic vertebra has become a single channel. This duct ends at left brachiocephalic vein at the junction of the left subclavian vein and the left internal jugular vein. The right lymphatic duct carries lymph derived from the right side including the upper limb, right side of thorax, and right side of the head and neck. The right lymphatic duct assumes several patterns of communication with the

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right subclavian vein and right internal jugular vein and carries 25% of the lymph. At the terminus of the duct, two valves prevent blood from refluxing into the duct, which could trigger coagulation and damage the lymphatic system.

Cardiac inflammation and lymphatics Influence of inflammation on the contractility of cardiac lymphatic muscle. Lymphatic muscular contraction can be negatively impacted by prostaglandins, nitric oxide, and inflammatory cytokines that may influence the abundance of these mediators. (Conversely, Johnstson and Feuer [42] suggested that inhibition of prostanoid formation may also impair phasic contraction of lymphangions.) Lymphatic disturbances in the heart not only can provoke myocardial edema but also lead to alterations in development of the normal sinus rhythm and signal propagation through the atrioventricular node and downstream tracts [17, 43]. Okada et al. [44] showed that experimentally induced lymphostasis caused injury to perivascular muscle cells and altered vascular electrolyte disturbances leading to disturbances in sinus rhythm. We have recently described that lymphatics within the epicardium are invested with muscle and that the contractility of these lymphatic muscle cells can be depressed by exposure to inflammatory cytokines such as interleukin-1 beta (IL-1 β) and tumor necrosis factor-alpha (TNF-α), which cooperatively drive a cyclooxygenase-2 (COX-2) and apparently prostaglandin E2 PGE2-dependent vasodilation in lymphatic muscle, which is EP4-receptor dependent; this same mechanism has been observed in intestinal lymphatic muscle and in experimental inflammatory bowel disease [44a, 45]. In some models, this cytokine-dependent depression of lymphatic pumping is nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)/inducible nitric oxide synthase (iNOS) and nitric oxide dependent [46]. It is not clear whether the contraction of such lymphatics actively propels lymph centripetally to the lymphatic trunks, or if changes in the tonic contraction of these vessels influence their ability to passively conduct lymph in response to the continuous rhythmic pulsations of the heart, which provide passive force to massage lymph through these vessels.

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Miller has described increased lymph flow even during periods of cardiac stress, for example, myocardial infarction, even though there may actually be a decrease in the compressive forces, which are assumed to drive most lymph propulsion in the heart (A. J. Miller, personal communication). While expansion of lymphatic networks is a typical response to acute inflammation [47–49], the failure to mobilize lymphatics in experimental myocarditis represents an interesting contrast. Recently, Omura et al. have reported that lymphatic marker expression was decreased in experimental Theiler’s murine encephalomyelitis virus (TMEV)-induced myocarditis [49a]. It is intriguing to speculate that an inflammation-associated failure to clear lymph might prevent or delay the spread of virus and inflammatory mediators within and from the heart; however, this might also lead to an accumulation of interstitial fluid and inflammatory cytokines, which locally depress normal cardiac lymphatic structure and function.

Valves in lymphatic vessels Initially, cardiac is lymph formed as it enters initial lymphatic capillaries in the heart that are lined with oak leaf-shaped lymphatic endothelial cells with unique button-like junctional configuration, in contrast to zipper-like junctions composed of both continuous and discontinuous zones of vascular endothelial cadherin (VE-cadherin) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and tight junctional proteins including occludin, claudin-5, and zona occludens-1 (ZO-1) that are also the characteristic of blood vascular endothelial cells, but in blood vascular endothelial cells, these are usually continuous. Interstitial fluid collected from the initial lymphatics flows into precollector and collector lymphatic vessels. Lymphatic vessels under physiological conditions maintain unidirectional flow via bicuspid valves, which prevent lymph reflux. Lymphatic valve formation is controlled by the transcription factors, FOXC2, Sox18, and GATA 2 [50, 50a], which are expressed at high levels by valve-forming lymphatic endothelial cells [51]. Sox18 is also known to be involved in the development of cardiac valves and of cardiac septum [50a]. Mice lacking FOXC2 expression (FOXC2 / ) have defective lymphatic remodeling that includes failure of valve formation, which is considered to be the hallmark feature of mature collecting vessels [52].

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FIG. 1

The drainage pattern of the heart and components of the pathway. The posterior interventricular lymphatic trunk (PVT) flows into the right coronary channel (RCC). The anterior interventricular trunk (AVT) joins the obtuse marginal lymphatic trunk (OMT) to form the left coronary channel (LCC). The right and left coronary lymphatic channels merge into a single main supracardiac channel (MSC), which flows into the cardiac lymph node (CLN), which is situated between the superior vena cava (SVC) and the brachiocephalic artery. The outflow of the node continues to the right lymphatic duct (RLD) that empties into the right angulus venosus formed by the junction of the right subclavian vein (RSV) and the internal jugular vein (IJV). The sinoatrial node (SA) and atrioventricular node (AV) may be influenced by factors, which accumulate in these areas when cardiac lymphostasis occurs. Based on diagrams in Miller AJ. Lymphatics of the heart, New York: Raven Press; 1982. 382 p.

This effectively highlights the importance of FOXC2 in controlling valve formation and maturation in the lymphatic vasculature. Additionally, patients with lymphedema-distichiasis, caused by a mutation in the FOXC2 gene, display lymph reflux and lymphedema associated with the absence (or failure) of lymphatic valves [52], which interestingly is also associated with congenital heart disease [53].

Cardiac conditions that may be exacerbated by lymphatic obstruction, lymphangitis, or obliteration Patients undergoing the Fontan (or Fontan-Kreutzer) procedure are usually neonates or pediatric patients who exhibit with cyanosis or congestive heart failure [54]. This procedure is used to correct

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defects in patients that have only a single functional ventricle, reflecting the absence or degeneration of either a tricuspid or mitral valve, which depresses the pumping capacity of the heart (hypoplastic left or right heart syndrome) or complex heart defects where biventricular repair cannot be accomplished. In these conditions, the ventricle has to accomplish double the work of the normal heart as it must support both the pulmonary and systemic circulations. The Fontan procedure to repair univentricular hearts provokes protein-losing enteropathy and peripheral edema that involve the lymphatic circulation. Patients with repaired Fontan circulation have an impaired lymphatic return capacity and morphologically enlarged thoracic ducts, measured using T2-weighted magnetic resonance imaging (MRI) [54a] and by ultrasonography [55]. This suggests that the lymphatic vasculature is stressed in the Fontan circulation and may play a role in the pathogenesis and complications associated with Fontan patients. As lymphatic vessels are so close anatomically to features of the heart, they are compressed or cut during surgery, and the thoracic duct is highly susceptible to traumatic injury during heart surgeries. Savla et al. [55a] described three forms of postoperative chylothorax associated with operations for congenital heart disease: traumatic leak from the thoracic duct, pulmonary lymphatic perfusion syndrome, and central lymphatic flow disorder. In most cases, these cases are resolved by lymphatic embolization to prevent leakage. Central lymphatic flow disorder, unfortunately, did not respond well to this approach, and additional therapies are being sought. Onoda et al. have reported the development of unilateral limb edema (leg) due to iatrogenic thoracic duct injury/stenosis [56]. Similarly, because the sinoatrial node lymphatic collectors and the right lymphatic trunk are merged within the fat pad of the ascending aorta, near the aortic root, damage to this complex can provoke atrial fibrillation [57], alter signal propagation, and diminish myocardial contraction that has also been reported [20, 21].

Effects of experimental ablation of cardiac lymphatics The experimental termination of lymphatic communication back to the main circulation leads to the accumulation of interstitial fluid and mediators normally cleared by this pathway, which has

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devastating effects on heart function. The interference with the normal clearance of lymph by obstruction of lymph outflow from the heart leads to progressive disturbances in cardiac performance, which begin within 30 min. These studies extended to 2 weeks where several important changes in heart structure and function were observed. Within 5 days, lymphatic obstruction provoked ST-depression with T-wave flattening. Lymphatic obstruction also caused problems with impulse generation (presumably sinoatrial nodal disturbances) and atrioventricular node conduction blockade [57a]. Solti et al. [58] showed that evidence for cardiac lymphostasis was observed during open-heart surgical procedures and in hearts exhibiting long-lasting tachycardiac attacks. Therefore, at least two nonobstructive clinical conditions may impair lymph communication leading to altered sinus nodal signal conduction disturbances; this does not even consider physical obstructive phenomena. Myocardial contractility (assessed by preload recruitable stroke work) was decreased significantly over 3 h following experimental obstruction of cardiac lymphatic vessel. This manipulation slightly (albeit significantly) increased cardiac edema (wet/dry weight ratio) with extensive lymphatic swelling (lymphangiectasis) and showed expansion of the interstitial spacing compared with controls [59]. Therefore, conditions, which lead to cardiac lymphostasis, are likely to depress heart function. Solti et al. also reported that the interstitial edema produced by lymphostasis has negative influences on tissue perfusion, with capillary perfusion decreasing and arteriovenous shunts assuming a fraction of the flow [60]. While not yet clear, we have observed remarkable increases in cytokines and chemokines in tissues where experimental interruption of lymph flow has been imposed, which may lead to increased inflammatory signaling (as mentioned in the section earlier “Cardiac inflammation and lymphatics”). Chaitanya et al. found that TNF-α, IL-1β, and IFN-g increased lymphatic endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), mucosal vascular addressing cell adhesion molecule-1 (MAdCAM-1), and E-selectin on mouse lymphatic endothelial cells (SV-LECs). In that report, we found that in human lymphatic endothelial cells (HMEC-1a), TNF-α, IL-1 beta, and IFN-gamma induced all ECAMs except for MAdCAM-1. IL-1 β increased, while IFN-gamma and TNF-α reduced lymphatic

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endothelial proliferation. TNF-α, IL-1 β, and IFN-gamma each reduced capillary formation in mouse and human, and TNF-α and IL-1 β reduced barrier function in mouse and human lymphatic endothelium. Therefore, in the setting of inflammation, inflammatory cytokines appear to dysregulate lymphatic growth, activation, and barrier function, which disturbs lymphatic clearance increasing tissue edema in vivo. Additionally, the evolution of IL-1 beta and TNF-a in the setting of cardiac inflammation may also lead to the induction of C-C chemokine receptor type 7 (CCR7) on monocytes, allowing them to penetrate into lymphatics via C-C motif ligand 21 (CCL21), an effect that is PGE2 and cyclic adenosine monophosphate (cAMP) dependent [61]. There has been some suggestion that TNF receptors are more abundant on blood endothelial cells than on lymphatics [48]. Conversely, TNF blockade using antibody therapeutics [62] has been shown in some models to enhance lymphangiogenesis. These differences may be tissue, species, and model specific. Interestingly, such changes could lead to lymphatic obstruction, which may in turn provoke fibrotic changes in the heart. Following the termination of experimental lymphatics in the heart (in rabbits), myocardial expression of collagen type I and III was increased at the mRNA levels, and plasma levels of C-terminal propeptide type I procollagen (PICP) and N-terminal propeptide type III procollagen (PIIINP) myocardial collagen type I and III mRNA increased for 30 days, eventually normalizing at 60 days [63]. These experiments, carried out in otherwise healthy animals, demonstrate the capacity of the heart to restore structure and function in response to lymphostasis. However, more long-standing stresses could provoke proportionately intensified levels of fibrosis, which might lead to persistent and pathological remodeling. Additionally apelin, a bioactive peptide released under hypoxia induced the expression of sphingosine1-phosphate (S1P) kinase and the sphingosine transporter SPNS2 to enhance the formation and export of S1P where it may also enhance lymphatic endothelial barrier function following myocardial ischemia [64]. With respect to fibrosis, for example, that might be seen following myocardial infarction, Kinashi et al. [65] suggested that in the setting of fibrosis, transforming growth factor-beta (TGF-β) might oppose lymphatic proliferation to suppress lymphatics. In the setting of tissue recovery, this may delay or interfere with healing; conversely, in the setting of transplantation, this may be

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beneficial in delaying the transmission of antigen presenting cells, which can drive rejection.

Experimental manipulation of lymphatics as a form of cardiac therapy Treatment of lymphangitis-like sequelae is produced by myocardial infarction. Cui describes how hyaluronidase and CLS2210, a benzenesulfonate, two “lymphagogues” enhanced lymph flow [66] and reduced injury following experimental occlusion of coronary arteries in dogs. This suggests that lymphostasis may result from myocardial infarction and that this is beneficially influenced by agents, which maintain lymph flow. Whether lymph flow might also be beneficially influenced during myocarditis or other cardiac inflammatory conditions is not clear but could be considered as a mechanism-based therapy to preserve interstitial fluid balance. Because lymphangiogenic growth factors have recently been identified, which may drive the expansion of lymphatic networks, it has been suggested that the use of such modulators could increase the net cardiac lymphatic drainage [21]; however, the formation of these vessels may occur at the expense of tissue structure and might need to be cautiously regulated (Fig. 1). Conversely, suppression of signaling through VEGFR-3 using a soluble VEGFR-3 decoy leads to an intensified injury in the setting of experimental myocardial ischemia, consistent with adequate or enhanced lymphatic drainage network acting to maintain tissue integrity following hypoxic stress [67]. We have also described improved tissue outcomes following administration of VEGF-D [67a]. Nonetheless, cardiac lymphatic expansion is apparently not beneficial in the setting of cardiac transplant [68, 69] where additional lymphatics may intensify immune surveillance leading to graft failure. In keeping with this concept, although lymphatic ablation can lead to deterioration of cardiac functioning, it is worth noting that cardiac lymphatics cannot be easily if ever reanastomosed following heart transplantation, although this does not appear to impede recovery. Lymphatics do however expand in the heart immediately after transplantation but usually diminish at later times (in models using cyclosporine immunosuppression) appearing to

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be associated with graft survival. With respect to lymphatic expansion, Nyk€ anen et al. [70] showed that VEGFR-3-driven CCL21 represents an important mechanism, which drives heart allograft rejection by communication of antigen presenting cells into secondary lymphoid organs where they activate immune responses, indicating that inhibition of VEGFR-3 signaling may be a valuable target in heart transplantation (Fig. 1). Consequently, blockade of lymphatics may be injurious, but allowing lymph to be passively released from the transplanted heart into the thorax does not drive injury. In the setting of endocarditis, increased lymphangiogenesis has been reported in heart valves where an intense >20-fold increase in lymphatic density was observed, with a fivefold increase in diameter [71]. Such a profound response indicates that lymphatic expansion must be adaptive in maintaining valve structure/function, which may be a possible therapeutic target in endocarditis. It is also interesting that yellow nail syndrome, a type of idiopathic lymphedema, was observed following a mitral valve replacement [72]; this condition eventually abated but suggests that complications from valve replacement, for example, thoracic duct impairment, might actually lead to the development of forms of lymphedema. Similarly, yellow nail syndrome has also been described following pacemaker placement, suggesting that ectopic/iatrogenic damage to the cardiac lymphatics may produce systemic lymphedema [73]. The appropriate investment of such vessels with muscular components might be required to beneficially enhance lymph flow. It has been reported by Viera et al. [74] that VEGF-C improves outcomes in experimental myocardial infarction, while LYVE-1 knockout mice (which ordinarily exhibit only a minor phenotype with abnormal lymphatic capillary vessel morphology in the liver and intestines and increased intradermal interstitial-lymphatic flow) show enhanced injury and a decreased ability to resolve cardiac injury. As LYVE-1 is often thought to overlap functionally with CD44, these studies show an important contribution of LYVE-1 lymphatics and lymphangiogenic growth factors in the recovery from myocardial infarction. Outside of growth factors, adrenomedullin and verapamil have been suggested as mediators, which stabilize interendothelial junctions, which may help restitute cardiac structure function following myocardial infarction through induction of connexin 43. Due to the fact that

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connexin 43 also participates in the patterning of lymphatic valves, connexin 43 may also represent a target for normalizing valve structure to prevent or lessen lymphostasis produced by lymphatic valve dysfunction [75]. Lastly, because lymphatic leakage may represent a target for clinical intervention in lymphatic failure, adrenomedullin has been shown to organize lymphatic junctions via Rap1A/B GTPases and might be able to limit lymphatic leakage, particularly in organs like the heart, which are particularly vulnerable to the effects of lymph reflux or stasis [76].

Summary The heart is clearly well supplied with lymphatics and like other organs can increase the flow of lymph during acute inflammation. However, it is still far less clear how the failure of lymphatic conduits to adequately drain cardiac interstitial fluid contributes to forms of heart injury, like myocardial infarction, myocarditis, and transplantation. While studies on experimental interruption of lymph flow show the central importance of cardiac lymphatics in maintaining normal heart rhythm and propagation as well as contractility, this same conduit mediates the transfer of dendritic cells, which sensitize the immune system to the engrafted heart and may be a significant risk, which could be managed therapeutically.

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[69] Soong TR, Pathak AP, Asano H, Fox-Talbot K, Baldwin WM. Lymphatic injury and regeneration in cardiac allografts. Transplantation 2010;89:500–8. [70] Nyk€anen AI, Sandelin H, Krebs R, Ker€ anen MA, Tuuminen R, K€ arp€ anen T, Wu Y, Pytowski B, Koskinen PK, Yl€ a-Herttuala S, Alitalo K, Lemstr€ om KB. Targeting lymphatic vessel activation and CCL21 production by vascular endothelial growth factor receptor-3 inhibition has novel immunomodulatory and anti-arteriosclerotic effects in cardiac allografts. Circulation 2010;121(12):1413–22. https://doi.org/10.1161/CIRCULATIONAHA.109. 910703. Epub 2010 Mar 15. PubMed PMID: 20231530. [71] Niinim€aki E, Mennander AA, Paavonen T, Kholova´ I. Lymphangiogenesis is increased in heart valve endocarditis. Int J Cardiol 2016;219:317–21. https:// doi.org/10.1016/j.ijcard.2016.06.049. Epub 2016 Jun 14. PubMed PMID: 27344132. [72] Sarmast H, Takriti A. Yellow nail syndrome resulting from cardiac mitral valve replacement. J Cardiothorac Surg 2019;14(1):72. https://doi.org/10. 1186/s13019-019-0903-1. PubMed PMID: 30971303. [73] Suzuki T, Tokuda Y, Kobayashi H. The development of yellow nail syndrome after the implantation of a permanent cardiac pacemaker. Intern Med 2017; 56(19):2667–2669. https://doi.org/10.2169/internalmedicine.8769-16. Epub 2017 Sep 6. PubMed PMID: 28883248; PubMed Central PMCID: PMC5658537. [74] Vieira JM, Norman S, Villa Del Campo C, Cahill TJ, Barnette DN, GunadasaRohling M, Johnson LA, Greaves DR, Carr CA, Jackson DG, Riley PR. The cardiac lymphatic system stimulates resolution of inflammation following myocardial infarction. J Clin Invest 2018;128(8):3402–3412. https://doi. org/10.1172/JCI97192. Epub 2018 Jul 9. PubMed PMID: 29985167; PubMed Central PMCID: PMC6063482. [75] Munger SJ, Davis MJ, Simon AM. Defective lymphatic valve development and chylothorax in mice with a lymphatic-specific deletion of Connexin43. Dev Biol 2017;421(2):204–218. https://doi.org/10.1016/j.ydbio.2016.11.017. Epub 2016 Nov 27. PubMed PMID: 27899284; PubMed Central PMCID: PMC5217530. [76] Xu W, Wittchen ES, Hoopes SL, Stefanini L, Burridge K, Caron KM. Small GTPase Rap1A/B is required for lymphatic development and adrenomedullin- induced stabilization of lymphatic endothelial junctions. Arterioscler Thromb Vasc Biol 2018;38(10):2410–2422. https://doi.org/10.1161/ ATVBAHA.118.311645. PubMed PMID: 30354217; PubMed Central PMCID: PMC6206876.

Further reading [77] Al-Kofahi M, Becker F, Gavins FN, Woolard MD, Tsunoda I, Wang Y, Ostanin D, Zawieja DC, Muthuchamy M, von der Weid PY, Alexander JS. IL-1β reduces tonic contraction of mesenteric lymphatic muscle cells, with the involvement of cycloxygenase-2 and prostaglandin E2. Br J Pharmacol 2015; 172(16):4038–51. https://doi.org/10.1111/bph.13194. Epub 2015 Jul 6. PubMed PMID: 25989136; PubMed Central PMCID: PMC4543611.

Further reading

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[78] Becker F, Yi P, Al-Kofahi M, Ganta VC, Morris J, Alexander JS. Lymphatic dysregulation in intestinal inflammation: new insights into inflammatory bowel disease pathomechanisms. Lymphology 2014; 47(1):3–27. Review. PubMed PMID: 25109166. [79] Machnik A, Neuhofer W, Jantsch J, Dahlmann A, Tammela T, Machura K, Park JK, Beck FX, M€ uller DN, Derer W, Goss J, Ziomber A, Dietsch P, Wagner H, van Rooijen N, Kurtz A, Hilgers KF, Alitalo K, Eckardt KU, Luft FC, Kerjaschki D, Titze J. Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism. Nat Med 2009;15(5):545–52. https://doi.org/10.1038/nm.1960. Epub 2009 May 3. PubMed PMID: 19412173. [80] Trincot CE, Xu W, Zhang H, Kulikauskas MR, Caranasos TG, Jensen BC, Sabine A, Petrova TV, Caron KM. Adrenomedullin induces cardiac lymphangiogenesis after myocardial infarction and regulates cardiac edema via Connexin 43. Circ Res 2019;124(1):101–113. https://doi.org/10.1161/ CIRCRESAHA.118.313835. PubMed PMID: 30582443; PubMed Central PMCID: PMC6318063. [81] Udink Ten Cate FEA, Tjwa ETTL. Imaging the lymphatic system in Fontan patients. Circ Cardiovasc Imaging 2019;12(4):e008972. https://doi.org/10. 1161/CIRCIMAGING.119.008972. PubMed PMID: 30943768. [82] von der Weid P-Y, Muthuchamy M. Regulatory mechanisms in lymphatic vessel contraction under normal and inflammatory conditions. Pathophysiology 2010;17(4):263–76. [83] Yang GH, Zhou X, Ji WJ, Zeng S, Dong Y, Tian L, Bi Y, Guo ZZ, Gao F, Chen H, Jiang TM, Li YM. Overexpression of VEGF-C attenuates chronic high salt intake-induced left ventricular maladaptive remodeling in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 2014;306(4):H598–609. https://doi.org/10.1152/ajpheart.00585.2013. Epub 2013 Dec 13. PubMed PMID: 24337460.

C H A P T E R

8

Lymphatic reconstruction Annika Mohr, Daniel Palmes, Felix Becker Clinic of General, Visceral and Transplantation Surgery, University Hospital M€ unster, M€ unster, Germany

Introduction Lymphatic reconstruction as a surgical approach is mostly utilized for patients with destructed or obstructed lymphatic vessels who are suffering from secondary lymphedema [1]. The accumulation of abnormal fluid in the interstitium results in an enlargement of the affected area [2]. Due to continued lymphatic stasis, inflammatory activation bay immune cells, cytokines, and microorganisms might lead to inflammation, gradual fibrosis, and thus a decrease in the number of functional lymphatic channels [1]. Underlying reasons for the disruption of the lymphatic drainage system are mostly posttraumatic, postinfectious, or postinflammational due to surgery, radiotherapy, or cancer [3]. However, clinical manifestation may be delayed after the initial incidence by months or even years. Nevertheless if once occurred, adjusted treatment is initiated for providing symptomatic improvement and deceleration of the progressive disease with conservative therapy representing the today’s gold standard [4]. Although for most patients beneficial, major disadvantages are the required compliance of the patient, as well as the lifelong commitment necessary for a prosperous therapy. Thus, surgical treatment options represent an additional option when conservative therapy is unable to control the symptoms of

Lymphatic Structure and Function in Health and Disease https://doi.org/10.1016/B978-0-12-815645-2.00008-3

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lymphedema [1]. Specifically, patients with insufficient reduction by medical and physical therapy (less than 50%) and those dissatisfied by the results of conservative methods and willing to proceed with surgical options might benefit from surgical reconstruction approaches of the lymphatic system [5].

History of surgical approaches for lymphatic reconstruction Surgical approaches for lymphatic reconstruction are performed since the 1960s. Niebulowisz et al. performed a lymphdraining anastomosis between a lymph node and a neighboring vein in patients with secondary lymphedema of the lower limb [6]. Subsequent research about the mechanisms of the development of secondary lymphedema mostly due to improvement of imaging systems led to adjustments of surgical methods. Thus, spontaneous lymphatic pulse, stagnation of tissue fluid, and the presence of bacterial flora could be visualized [7–9]. In addition to that, the improvement of microsurgical equipment (e.g., microscope and atraumatic sutures) helped to facilitate lymphatic reconstruction [10,11].

Current options for lymphatic reconstruction Lymphaticovenular anastomosis First anastomoses between lymph vessels and veins were performed in the 1960s due to the introduction of dissection microscopes into surgery [10]. The procedure should imitate the natural communication between lymphatics and veins, thus redirecting the accumulated lymph fluid into the venous system. Since the first approaches, several studies have been conducted leading to a variation of surgical techniques (e.g., end to end, end to side, and side to end) and heterogeneous evidence of treatment effectiveness. Major problems when utilizing this technique are high venous blood pressure and consequently the risk of thrombus formation due to blood coagulation [12].

Current options for lymphatic reconstruction

157

To enhance long-term outcome, the measurement of endolymphatic pressure, lymphatic flow rate, and venous pressure is recommended before the performance of microsurgical anastomosis [3]. Thus, optimal conditions could be created for the best approach. Moreover, whenever possible, lymphatic vessels with contracting ability should be chosen for the anastomosis [13]. In addition to that, patency of the selected vessels should be proven prior surgery [14]. It has also been shown that the absence of blood inside of the venous branch utilized for the anastomosis decreases the risk of thrombosis [15,16]. Most successful results could be accomplished in patients with local, segmental obstructions in the proximal lymph vessels [17]. First results in patients suffering from secondary lymphedema were promising indicating adequate decompression of the limb [18,19]. However, long-term results were less effective in the proximal compared with the distal part of the extremity [18]. The authors concluded the higher degree of destruction of the proximal lymphatic system was responsible for this phenomenon suggesting surgery at an earlier time when lymphatic disruption is less advanced. Consequently, it is generally recommended to treat lymphedema with lymphaticovenular anastomosis as early as possible [15,16], before the development of tissue fibrosis or lymphatic damage due to increasing tissue pressure or infection. Nevertheless, the impact of duration and severity of lymphedema is classified differentially with some surgeons claiming to have found no influence of the length of the condition of the patient on the outcome after surgery [11,20]. Regarding the technique of anastomosis (e.g., end to end, end to side, and side to end), most authors indicated their choice was based on anatomical conditions at the site of operation after isolating lymphatics and veins [3,15,16]. In contrast to the other options, side-to-end anastomosis preserves the original flow of the lymphatic fluids and offers the possibility for further operations at the same vessels if an obstruction of the first anastomosis occurs [21]. Nevertheless, this procedure is more complex, which is why a novel method was introduced to facilitate side-to-end anastomosis. With expanding the lymphatic vessel, shortly before performing the anastomosis, the creation of a lateral window in the lymphatic wall is facilitated, thus improving the success rate

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in the performed study [22]. Obstacles still present small lymphatic vessels with a diameter below 0.35 mm where side-toend anastomosis is not recommended even after expansion due to a relatively high failure rate. Follow-up studies indicated beneficial results after lymphaticovenular anastomosis with immediate and long-term significant improvements [11,12,14–16,20,21,23]. In conclusion, lymphaticovenular anastomosis represents a promising approach for decreasing secondary lymphedema. Nevertheless, postoperative compressive therapy still has to be performed. Additionally, it has to be noted that the aforementioned studies utilized different methods and follow-up periods. For the determination of patency, methods varied from circumference measurement to volumetry, lower extremity lymphedema (LEL) index, or lymphoscintigraphy. Thus, a direct comparison between the studies appears to be challenging. Yet, objective and subjective results are indicating a place in the treatment of secondary lymphedema especially when conservative therapy is failing.

Vascularized lymph node transfer Vascularized lymph node transfer represents a relatively novel method where a transfer into regions with dissected lymph nodes (e.g., cancer treatment) or into distal regions of limbs with lymphedema is performed to reestablish lymphatic drainage function [24]. Starting in animal studies, vascularized lymph node transfer showed a complete preservation of the original histological structures [25] in contrast to what has been seen in a vascularized lymph node transfers [26,27]. First attempts in clinical studies in 1982 showed a stable reduction of lymphedema after transferring inguinal lymph nodes to the contralateral inguinal region [28]. Similar results have been reported for the upper extremity with a transfer either into the axillary region [29] or into the wrist area [30]. In general, all studies performed on vascularized lymph node transfer showed favorable outcomes for the patients after surgery including reduction of lymphedematous limb volumes, infections, and improved quality of life [31–34]. Lymph node flap harvest is performed from different areas of the donor including groin,

Conclusion

159

submental, supraclavicular, lateral thoracic, gastroepiploic, jejunal, appendicular, and ileocecal with no general ideal recommendation until now. For better outcomes, lymph nodes may be transferred with a skin paddle containing lymph capillaries, thus facilitating recanalization of the lymphatic system [24]. It has to be noted that only few long-term results are available for vascularized lymph node transfers until now. Moreover, the interaction between transferred lymph node and the lymphatic system in the recipient area still needs to be elucidated. In addition, the risk of causing lymphedema in the region of the donor lymph node might be heightened [35–37]. Despite the promising technique, additional excisional procedures are recommended after vascularized lymph node transfers in patients suffering from secondary lymphedema.

Lympholymphatic graft In contrast to lymphaticovenular anastomosis and vascularized lymph node transfer, only few studies have been conducted evaluating lymphatic grafts in lymphedema treatment. First attempts of harvesting autologous lymphatic grafts to bypass damaged lymphatic vessels resulted in not only volume reduction of 80% but also high risk of donor site morbidity [38]. The bypass should be carried out prior permanent damage of the remainder lymphatic vessels due to back pressure or infection [39]. Alternatively, vein grafts have been used in patients with chronic lymphedema to obtain a lymphatic-venous-lymphatic bypass [15,16] with the result of improvement of both limb function and edema. Most recent studies aimed to develop tissue-engineered grafts to reestablish lymphatic circulation [40], and first studies under culturing conditions offered promising results for future clinical application and as a potential new treatment option for secondary lymphedema [41].

Conclusion Nevertheless, until now, and despite the promising results regarding the surgical approaches of lymphatic reconstruction, it has to be noted that lymphatic reconstruction is still a palliative

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procedure. The creation of a bypass system for lymphatic system offers no elimination of the etiologic factor; thus, obstructions or destructions of the lymphatic vessel and infections leading to decreased transport capacity of lymphatic fluid are still present. In addition to that, in most cases, conservative therapy is still required even though surgical reconstruction was performed. Further improvement of the surgical techniques and a consensus in standardized protocols and reporting of outcome is finally needed to enhance and consolidate lymphatic reconstruction in secondary lymphedema.

References [1] Kung TA, Champaneria MC, et al. Current concepts in the surgical Management of Lymphedema. Plast Reconstr Surg 2017;139(4). [2] Ozturk CN, Ozturk C, et al. Free vascularized lymph node transfer for treatment of lymphedema: a systematic evidence based review. J Plast Reconstr Aesthet Surg 2016;69(9):1234–47. [3] Campisi C, Boccardo F. Frontiers in lymphatic microsurgery. Microsurgery 1998;18(8):462–71. [4] Becker C, Vasile JV, et al. Microlymphatic surgery for the treatment of iatrogenic lymphedema. Clin Plast Surg 2012;39(4):385–98. [5] Boccardo F, Fulcheri E, et al. Lymphatic microsurgery to treat lymphedema: techniques and indications for better results. Ann Plast Surg 2013;71(2):191–5. [6] Nielubowicz J, Olszewski W. Surgical lymphaticovenous shunts in patients with secondary lymphoedema. Br J Surg 1968;55(6):440–2. [7] Olszewski WL. Episodic dermatolymphangioadenitis (DLA) in patients with lymphedema of the lower extremities before and after administration of benzathine penicillin: a preliminary study. Lymphology 1996;29(3):126–31. [8] Olszewski WL. Contractility patterns of human leg lymphatics in various stages of obstructive lymphedema. Ann N Y Acad Sci 2008;010. [9] Olszewski WL, Engeset A, et al. Lymph flow and protein in the normal male leg during lying, getting up, and walking. Lymphology 1977;10(3):178–83. [10] Jacobson II JH, Suarez EL. Microvascular surgery. Dis Chest 1962;41:220–4. [11] Koshima I, Inagawa K, et al. Supermicrosurgical lymphaticovenular anastomosis for the treatment of lymphedema in the upper extremities. J Reconstr Microsurg 2000;16(6):437–42. [12] Winters H, Tielemans HJP, et al. The efficacy of lymphaticovenular anastomosis in breast cancer-related lymphedema. Breast Cancer Res Treat 2017;165(2):321–7. [13] Campisi C, Bellini C, et al. Microsurgery for lymphedema: clinical research and long-term results. Microsurgery 2010;30(4):256–60.

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[14] Demirtas Y, Ozturk N, et al. Supermicrosurgical lymphaticovenular anastomosis and lymphaticovenous implantation for treatment of unilateral lower extremity lymphedema. Microsurgery 2009;29(8):609–18. [15] Campisi C, Boccardo F, et al. Long-term results after lymphatic-venous anastomoses for the treatment of obstructive lymphedema. Microsurgery 2001;21(4):135–9. [16] Campisi C, Boccardo F, et al. The use of vein grafts in the treatment of peripheral lymphedemas: long-term results. Microsurgery 2001;21(4):143–7. [17] Olszewski WL. The treatment of lymphedema of the extremities with microsurgical lympho-venous anastomoses. Int Angiol 1988;7(4):312–21. [18] O’Brien BM, Mellow CG, et al. Long-term results after microlymphaticovenous anastomoses for the treatment of obstructive lymphedema. Plast Reconstr Surg 1990;85(4):562–72. [19] O’Brien BM, Shafiroff BB. Microlymphaticovenous and resectional surgery in obstructive lymphedema. World J Surg 1979;3(1):3–15. [20] Koshima I, Nanba Y, et al. Long-term follow-up after lymphaticovenular anastomosis for lymphedema in the leg. J Reconstr Microsurg 2003;19(4):209–15. [21] Maegawa J, Yabuki Y, et al. Outcomes of lymphaticovenous side-to-end anastomosis in peripheral lymphedema. J Vasc Surg 2012;55(3):753–60. [22] Yamamoto T, Yoshimatsu H, et al. Side-to-end lymphaticovenular anastomosis through temporary lymphatic expansion. PLoS One 2013;8(3):25. [23] Cornelissen AJM, Kool M, et al. Lymphatico-venous anastomosis as treatment for breast cancer-related lymphedema: a prospective study on quality of life. Breast Cancer Res Treat 2017;163(2):281–6. [24] Ito R, Suami H. Overview of lymph node transfer for lymphedema treatment. Plast Reconstr Surg 2014;134(3):548–56. [25] Shesol BF, Nakashima R, et al. Successful lymph node transplantation in rats, with restoration of lymphatic function. Plast Reconstr Surg 1979;63(6):817–23. [26] Jaffe HL, Richter MN. The regeneration of autoplastic lymph node transplants. J Exp Med 1928;47(6):977–80. [27] Tobbia D, Semple J, et al. Experimental assessment of autologous lymph node transplantation as treatment of postsurgical lymphedema. Plast Reconstr Surg 2009;124(3):777–86. [28] Clodius L, Smith PJ, et al. The lymphatics of the groin flap. Ann Plast Surg 1982;9(6):447–58. [29] Becker C, Assouad J, et al. Postmastectomy lymphedema: long-term results following microsurgical lymph node transplantation. Ann Surg 2006;243(3):313–5. [30] Lin CH, Ali R, et al. Vascularized groin lymph node transfer using the wrist as a recipient site for management of postmastectomy upper extremity lymphedema. Plast Reconstr Surg 2009;123(4):1265–75. [31] Cheng MH, Huang JJ, et al. A novel approach to the treatment of lower extremity lymphedema by transferring a vascularized submental lymph node flap to the ankle. Gynecol Oncol 2012;126(1):93–8. [32] Ciudad P, Maruccia M, et al. The laparoscopic right gastroepiploic lymph node flap transfer for upper and lower limb lymphedema: technique and outcomes. Microsurgery 2017;37(3):197–205.

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[33] De Brucker B, Zeltzer A, et al. Breast Cancer-related lymphedema: quality of life after lymph node transfer. Plast Reconstr Surg 2016;137(6):1673–80. [34] Gratzon A, Schultz J, et al. Clinical and psychosocial outcomes of vascularized lymph node transfer for the treatment of upper extremity lymphedema after breast Cancer therapy. Ann Surg Oncol 2017;24(6):1475–81. [35] Pons G, Masia J, et al. A case of donor-site lymphoedema after lymph nodesuperficial circumflex iliac artery perforator flap transfer. J Plast Reconstr Aesthet Surg 2014;67(1):119–23. [36] Sulo E, Hartiala P, et al. Risk of donor-site lymphatic vessel dysfunction after microvascular lymph node transfer. J Plast Reconstr Aesthet Surg 2015;68(4):551–8. [37] Viitanen TP, Maki MT, et al. Donor-site lymphatic function after microvascular lymph node transfer. Plast Reconstr Surg 2012;130(6):1246–53. [38] Baumeister RG, Siuda S. Treatment of lymphedemas by microsurgical lymphatic grafting: what is proved? Plast Reconstr Surg 1990;85(1):64–74. [39] Ho LC, Lai MF, et al. Microlymphatic bypass in obstructive lymphoedema. Br J Plast Surg 1988;41(5):475–84. [40] Kanapathy M, Patel NM, et al. Tissue-engineered lymphatic graft for the treatment of lymphedema. J Surg Res 2014;192(2):544–54. [41] Kanapathy M, Kalaskar D, et al. Development of a tissue-engineered lymphatic graft using nanocomposite polymer for the treatment of secondary lymphedema. Artif Organs 2016;40(3):30.

Index Note: Page numbers followed by f indicate figures, and t indicate tables.

A

Chemokine production, 101 Cholesterol, reverse transport, 133 Circulatory system, 1, 4 Congenital vascular malformations (CVMs), 3, 109 Contractility, pressure/stretch, 62–66 Cyclooxygenase-2 (COX-2), 136 Cystic/cavernous lymphangioma, 111 Cytokines, 101

Aging CNS lymphatics in, 98–101 influences of, 76–86 Alzheimer’s disease (AD), 99 Angiopoietins, 13 Anoctamin-1 (ANO1), 24–25 Arthritic diseases, 35–37 Atrioventricular (AV) nodal conduction, 129

C

D

Diabetes, 38–39

Cancer, 39–41 Cardiac lymphatics characteristics, 127–128 within different cardiac structures, 131–136 experimental ablation effects, 139–142 experimental manipulation, cardiac therapy, 142–144 flow of lymph in heart, 129–131 Fontan procedure, 138–139 inflammation and, 136–137 valves in lymphatic vessels, 137–138 in ventricular septum, 129 Central nervous system (CNS) lymphatics aging, 98–101 Alzheimer’s disease, 99 cerebral ischemic injury, 100 characteristics, 93–97 in diseases, 96–97 drug delivery, 102–103 for drug discovery, 101–102 glymphatic pathway, 95–96 meningeal pathway, 96–97 microinfarcts, 100 multiple sclerosis, 99–100 perivascular pathway, 94–95 Centrifugal model, 7–9 Centripetal model, 6–9 Cerebral ischemic injury, 100

E

EphB4, 13 Ephrins, 13 Extratrucular lymphatic malformation, 111f, 112 Extrinsic lymph pump, 58–62

F

Fontan-Kreutzer procedure, 138–139 FOXC2, 32–33

G

Glymphatic pathway, 95–96

H

Heart, flow of lymph in, 129–131. See also Cardiac lymphatics Hemolymphatic malformation (HLM), 3, 109–110, 110f Histamine, endothelium-derived relaxing factor, 74–76

I

Inducible nitric oxide synthase (iNOS), 136 Inflammation, 19–20, 29–30 arthritis-associated, 36–37 and cardiac lymphatics, 136–137 changes during chronic, 34–35

163

164

Index

Inflammation (Continued) within ileum, 37–38 in RA patients, 41–42 sites of, 41 sterile, 31 Inflammatory bowel disease (IBD), 37–38 Interleukin-1 beta (IL-1 β), 136 Intraluminal pressure, 59 Intrinsic lymph pump, 55–57

K

Klippel-Trenaunay syndrome, 3, 110f

L

Lymphadenopathy, 2, 29–30 Lymphangiectasia, 30–31, 33 Lymphangioblast, 7–9 Lymphangiogenesis, 6–7, 7f, 30 Lymphangioma cystic/cavernous, 111 gluteal, 111f Lymphangion, 9–10, 20–21, 55–57 Lymphangitis cardiac conditions, 138–139 treatment of, 142 Lymphatic commitment, 12 Lymphatic endothelial cells (LECs), 7f, 9, 12 competence, 11–12 specification, 12 Lymphatic malformation (LM), 3 clinical aspects, 112–114 definition, 109 embryological aspects, 110–112 extratruncular lesion, 112 genetic considerations, 115–118 hemolymphatic, 110f management, 118 molecular diagnostic test, 118 truncular lesion, 112 Lymphatic muscle, 19–22 contractile apparatus, 25 electrical activity and pacemaker, 22–25 electrophysiological properties, 22–23 Lymphaticovenular anastomosis, 156–158 Lymphatic pumping action potentials during, 23 ANO1 role, 24–25 chemical regulation, 27–29 in diseases, 31–41 intrinsic, 24–25 mechanical regulation, 26–27

mechanisms, 22–29 origin, 22–29 spontaneous, 22–23 for therapeutic benefits, 41–43 Lymphatic reconstruction clinical manifestation, 155 lymphaticovenular anastomosis, 156–158 lympholymphatic graft, 159 secondary lymphedema, 155 surgical approaches, 156 vascularized lymph node transfer, 158–159 Lymphatic system and aging-associated changes, 2 characteristics, 1 collecting vessels, 19–20 distribution, 21 historical background, 2, 5–6 initial lymphatics, 20 initial lymphatic vessels, 19–20 lymph nodes, 19–20 mechanisms of lymphatic contraction, 25–26 ontogenic point of view, 6–9 peripheral, 3 purpose, 21–22 step-by-step development, 7f structural characteristics, 9 vasculogenetic point of view, 11–13 Lymphatic vessel afferent, 21 contraction frequency, 27 dilation, 38 distension/stretch of, 24–25 initial, 19–20 mesenteric, 23 network, 21 peripheral, 26 subtypes, 30 Lymphatic vessel contractility by intrinsic and extrinsic flows, 66–73 by pressure/stretch, 62–66 Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), 128–129 Lymphedema clinical characteristic, 20 primary, 32–33 secondary, 33 Lymph flow impact, 58 Lymph hearts, 9–10, 10f

Index

Lymph node dysplasia, 113 Lympholymphatic graft, 159 bypass, 159–160 Lymphostasis, 136, 138f cardiac, 139–140 induced, 136 interstitial edema, 139–140 from myocardial infarction, 142 structure and function restoration, heart, 141–142 Lymph pump extrinsic, 58–62 intrinsic, 55–57

M

Meningeal pathway, 96–97 Mesenteric lymphatic vessels, 74–76 Metabolic syndrome, 38–39 Microinfarcts, 100 Multiple sclerosis, 99–100

N

Neuroinflammation, 101 Nitric oxide (NO), 71, 74 Nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB), 136

O

Ontogenic point of view, lymphatic system, 6–9 Osteoarthritis (OA), 36

P

Pacemaker, 22–25 Perivascular pathway, 94–95 Primary lymphedema causes, 114 classification, 114 definition, 112 genetic disorders associated with, 115–116t

165

peripheral, 113–114, 113f Prospero homeobox 1 (Prox1), 128–129, 134

R

Rheumatoid arthritis (RA), 35

S

Scavenger receptor class B type I (SRB1), 133 Secondary lymphedema, 155–156, 159

T

Theiler’s murine encephalomyelitis virus (TMEV)-induced myocarditis, 137 TMEM16a, 24–25 Tonicity-responsive enhancer binding protein (“tonEBP”), 134–135 Transmural pressure, 62 Transplantation heart, 142–143 setting of, 141–142 Truncular lesion, 111–112 Truncular lymphatic malformation, 113f. See also Lymphatic malformation (LM) Tumor necrosis factor-alpha (TNF-α), 136

V

Vascular aging, 2 Vascular coalescence and maturation, 12–13 Vascular endothelial growth factor receptor-3 (VEGFR-3), 128–129 Vascularized lymph node transfer, 158–159 Vascular system, 1–2 Vasculogenetic point of view, lymphatic system, 11–13 VEGFR-3 expression, 11 Vis a tergo, 58