Reviews of Physiology, Biochemistry and Pharmacology [1st ed.] 9783030615062, 9783030615079

Table of contents :
Front Matter ....Pages i-vii
CXC Chemokine Receptors in the Tumor Microenvironment and an Update of Antagonist Development (Yang Xun, Hua Yang, Jiekai Li, Fuling Wu, Fang Liu)....Pages 1-40
Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal Dystrophies (Darpan Malhotra, Joseph R. Casey)....Pages 41-81
Promising Anti-atherosclerotic Effect of Berberine: Evidence from In Vitro, In Vivo, and Clinical Studies (Alireza Fatahian, Saeed Mohammadian Haftcheshmeh, Sara Azhdari, Helaleh Kaboli Farshchi, Banafsheh Nikfar, Amir Abbas Momtazi-Borojeni)....Pages 83-110

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Stine Helene Falsig Pedersen  Editor

Reviews of Physiology, Biochemistry and Pharmacology 178

Reviews of Physiology, Biochemistry and Pharmacology Volume 178

Editor-in-Chief Stine Helene Falsig Pedersen, Department of Biology, University of Copenhagen, Copenhagen, Denmark Series Editors Emmanuelle Cordat, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada Diane L. Barber, Department of Cell and Tissue Biology, University of California San Francisco, CA, USA Jens Leipziger, Department of Biomedicine, Aarhus University, Aarhus, Denmark Luis A. Pardo, Max Planck Institute for Experimental Medicine, G€ ottingen, Germany Christian Stock, Department of Gastroenterology, Hannover Medical School, Hannover, Germany Nicole Schmitt, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark Martha E. O’Donnell, Department of Physiology and Membrane Biology, University of California, Davis School of Medicine, Davis, CA, USA

The highly successful Reviews of Physiology, Biochemistry and Pharmacology continue to offer high-quality, in-depth reviews covering the full range of modern physiology, biochemistry and pharmacology. Leading researchers are specially invited to provide a complete understanding of the key topics in these archetypal multidisciplinary fields. In a form immediately useful to scientists, this periodical aims to filter, highlight and review the latest developments in these rapidly advancing fields. 2019 Impact Factor: 4.700, 5-Year Impact Factor: 6.000 2019 Eigenfaktor Score: 0.00067, Article Influence Score: 1.570 More information about this series at http://www.springer.com/series/112

Stine Helene Falsig Pedersen Editor

Reviews of Physiology, Biochemistry and Pharmacology

Editor Stine Helene Falsig Pedersen Department of Biology University of Copenhagen Copenhagen, Denmark

ISSN 0303-4240 ISSN 1617-5786 (electronic) Reviews of Physiology, Biochemistry and Pharmacology ISBN 978-3-030-61506-2 ISBN 978-3-030-61507-9 (eBook) https://doi.org/10.1007/978-3-030-61507-9 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Acknowledgements

Contributions to this volume have partly been personally invited, with kind support of the series editors D.L. Barber, E. Cordat, J. Leipziger, M.E. O’Donnell, L. Pardo, N. Schmitt, C. Stock.

v

Contents

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of Antagonist Development . . . . . . . . . . . . . . . . . . . . . . . Yang Xun, Hua Yang, Jiekai Li, Fuling Wu, and Fang Liu

1

Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal Dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Darpan Malhotra and Joseph R. Casey Promising Anti-atherosclerotic Effect of Berberine: Evidence from In Vitro, In Vivo, and Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Alireza Fatahian, Saeed Mohammadian Haftcheshmeh, Sara Azhdari, Helaleh Kaboli Farshchi, Banafsheh Nikfar, and Amir Abbas Momtazi-Borojeni

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Rev Physiol Biochem Pharmacol (2020) 178: 1–40 https://doi.org/10.1007/112_2020_35 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2020 Published online: 21 August 2020

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of Antagonist Development Yang Xun, Hua Yang, Jiekai Li, Fuling Wu, and Fang Liu

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 CXCR1 and CXCR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Cell Signaling Induced by CXCR1 and CXCR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 CXCR1 and CXCR2 in Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 CXCR1 and CXCR2 in Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Antagonist Development Targeting CXCR1 and CXCR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 CXCR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 CXCR3 Structure and Signaling Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Autocrine and Paracrine Mechanisms of CXCR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Antagonists Targeting CXCR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 CXCR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 CXCR4 Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 CXCR4 Expression and Signaling in Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Association Between CXCR4 and Leukocyte Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Antagonists Targeting CXCR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 CXCR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 CXCR5 Expression Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 CXCR5 in Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 CXCR5 in Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Absence of Antagonists Targeting CXCR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 CXCR6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Expression of CXCR6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 CXCR6–TM-CXCL16 and CXCR6–sCXCL16 Axes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 CXCR6 Expression by Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Association Between CXCR6 and Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Yang Xun and Hua Yang contributed equally to this work. Y. Xun, H. Yang, J. Li, and F. Liu (*) Department of Basic Medicine and Biomedical Engineering, School of Stomatology and Medicine, Foshan University, Foshan, Guangdong Province, China e-mail: [email protected] F. Wu Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China

3 4 4 6 6 10 13 13 13 15 16 16 16 17 17 18 18 19 19 20 20 20 20 21 22

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6.5 Absence of Antagonist Targeting CXCR6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 CXCR7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 CXCR7 Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 CXCR7 in Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 CXCR7 and Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Crosstalk of CXCR7 with CXCR3 and CXCR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Antagonist Development Targeting CXCR7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22 23 23 23 24 24 25 25 29

Abstract Chemokine receptors, a diverse group within the seven-transmembrane G protein-coupled receptor superfamily, are frequently overexpressed in malignant tumors. Ligand binding activates multiple downstream signal transduction cascades that drive tumor growth and metastasis, resulting in poor clinical outcome. These receptors are thus considered promising targets for anti-tumor therapy. This article reviews recent studies on the expression and function of CXC chemokine receptors in various tumor microenvironments and recent developments in cancer therapy using CXC chemokine receptor antagonists. Keywords CXC antagonists · CXC chemokine receptors · Tumor microenvironment

Abbreviations ADAM10 Akt AML CCRCC CLL CRPC CTL CXCR DLBCL ECM EGFR EMT ERK HCC HER2 HIV IgG IL JNK MAPK MDSC

Disintegrin-like metalloproteinase 10 Protein kinase B Acute myeloid leukemia Clear cell renal cell carcinoma Chronic lymphocytic leukemia Castrated-resistant prostate cancer Cytotoxicity T-cell CXC chemokine receptor Diffuse large B-cell lymphoma Extracellular matrix Epidermal growth factor receptor Epithelial-to-mesenchymal transition Extracellular signal-regulated kinase Hepatocellular carcinoma Epidermal growth factor receptor type 2 Human immunodeficiency virus Immunoglobulin G Interleukin c-Jun N-terminal kinases Mitogen-activated protein kinase Myeloid-derived suppressor cells

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of. . .

MMP NHERF1 NHL NK NKT NSCLC PD-1 PDAC PI3K PMN PTEN sCXCL16 STAT TAK1 TAM Tfh TIL TM-CXCL16 TNBC Treg Trm VEGF

3

Matrix metalloproteinases Na+/H+ exchanger regulatory factor 1 Non-Hodgkin’s lymphoma Natural killer Natural killer T-cell Non-small cell lung cancer Programmed death-ligand 1 Pancreatic ductal adenocarcinoma Phosphoinositide 3-kinase Polymorphonuclear Phosphatase and tensin homolog Soluble CXCL16 Signal transducers and activators of transcription Transforming growth factor-β-activated kinase 1 Tumor-associated microglia/macrophage T-follicular helper cells Tumor-infiltrating lymphocyte Transmembrane CXCL16 Triple-negative breast cancer Regulatory T-cells Resident memory T-cells Vascular endothelial growth factor

1 Introduction Tumor development is strongly dependent on factors in the surrounding matrix or tumor microenvironment, including anchored and diffusible signaling factors such as hormones, growth factors, and cytokines. These factors collectively constitute a complex molecular network, regulating tumor cell proliferation, motility, and survival. The relationship between tumor cells and the microenvironment has been equated to that between seed and soil, as the seed can only grow in congenial soil (Kenny et al. 2007). Nowadays, tumor microenvironment has been intensively studied as a hot spot in aiding the development of anti-tumor drugs, among which the roles of chemokines and their receptors have attracted many attentions. The chemokine receptors constitute a large and diverse subgroup within the seven-transmembrane G protein-coupled receptor superfamily. Based on the sequence of conserved cysteine residues at the N-terminal of the ligand molecule, these receptors are divided into four subtypes, CR, CCR, CXCR, and CX3CR, where C is cysteine and X is an arbitrary amino acid. Chemokine receptors bind specific ligands and transmit extracellular information into cells through structural changes of the G protein to regulate cell growth, differentiation, and apoptosis among other functions under physiological and pathological conditions (Fig. 1).

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Fig. 1 Chemokine ligand–receptor regulatory mechanisms. Chemokine ligands (red) bind to receptors (blue) to induce signal transduction pathways that activate a myriad of physiological and pathological processes

Chemokine receptors are expressed in many tumors and are strongly linked to malignancy. For example, CCR7 promotes lung cancer cell proliferation and lymph node metastasis (Zhang et al. 2013), CX3CR1 expression is associated with poor prognosis of colorectal cancer (Erreni et al. 2010), and CCR2 contributes to the changes in CDb/Gr myelogenous cell number and influences tumor load in colorectal cancer liver metastasis (Zhao et al. 2013). CXC chemokine receptors (CXCRs) (Table 1) in the tumor microenvironment act as seminal regulators of tumor progression by binding to unique ligands or subgroups of ligands (Fig. 2). This review focuses on the expression and function of CXCRs in various tumor microenvironments as well as on the development of CXCR antagonists as anti-tumor drugs.

2 CXCR1 and CXCR2 2.1

Cell Signaling Induced by CXCR1 and CXCR2

CXCR1 and CXCR2 are closely related receptors, also known as interleukin-8receptor A and B, respectively, as both bind interleukin (IL)-8 (CXCL8). These receptors share 77% sequence homology and an ELR motif immediately adjacent to the CXC motif, and both receptors bind CXCL6 and CXCL8, while only CXCR2 binds CXCL1–3, CXCL5, and CXCL7. Two conserved domains at the CXCR2 carboxyl end (ILXLL and PDZ-ligand domains) are critical for signal transduction (Baugher and Richmond 2008). The three dimensional structures of monomeric

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of. . .

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Table 1 Basic information of CXC chemokine receptors in human (Homo sapiens)

Name CXCR1 CXCR2

CXCR3A CXCR3B CXCR4

CXCR5 CXCR6 CXCR7

Alias IL8RA, IL8R1, CD181, CD128, CKR-1, CMKAR1 IL8RB, IL8R2, CD182, CDw128b, CMKAR2

Location 2q35

Number of amino acids 350

2q35

360

40,759

CD183, CKR-L2, CMKAR3, GPR9, IP10-R, Mig-R

Xq13

368

40,659

CD184, D2S201E, FB22, HM89, HSY3RR, LAP3, LCR1, LESTR, NPY3R, NPYR, NPYRL, NPYY3R, WHIM CD185, BLR1, MDR15 CD186, BONZO, STRL33, TYMSTR ACKR3, RDC1, CMKOR1, GPR159

2q21

415 360

45,523 40,607

11q23 3p21

372 342

41,925 39,280

– –

2q37

362

41,478

–

Protein size 39,771

Structure solved Full monomeric structure C-terminal CXCR2NHERF1 PDZ2 complex structure Fragmental structure – CXCR4– CXCL12 complex structure

“–” indicates unavailable structural information

Fig. 2 CXC chemokine receptors (blue) and their corresponding CXC chemokine ligands (gray)

CXCR1 in phospholipid bilayers and the C-terminal of CXCR2 in complex with the Na+/H+ exchanger regulatory factor 1 (NHERF1) PDZ2 domain have been described (Park et al. 2012; Lu et al. 2013), providing crucial information on CXCR1/2-ligand interaction sites for antagonist design. Due to the high level of homology between CXCR1 and CXCR2, the two receptors are usually investigated together.

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CXCR1 and CXCR2 in Tumor Cells

High expression levels of CXCR1 and/or CXCR2 are usually associated with accelerated tumor development (Fig. 3). Both CXCR1–CXCL8 and CXCR2– CXCL8 signaling axes promote lymphatic and distant metastasis of liver cancer (Bi et al. 2019). Overexpression of CXCR1 and CXCR2 also promotes metastasis and chemoresistance of breast cancer cells via epithelial-to-mesenchymal transition (EMT) and neutrophil infiltration, processes mediated by epidermal growth factor receptor type 2 (HER2), protein kinase B (Akt), and cyclooxygenase-2 among other signaling factors (Kaunisto et al. 2015; Xu et al. 2018a; Shah and Osipo 2016). The CXCR2–CXCL8 axis stimulates the growth and proliferation of prostate cancer cells by activating cyclin D via phosphoinositide 3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways (MacManus et al. 2007). In addition, the CXCR2–CXCL7 axis promotes angiogenesis in metastatic colorectal cancer, and high expression of CXCR2 is predictive of poor survival (Desurmont et al. 2015). The CXCR2–CXCL5 axis is a key inducer of EMT and metastatic colonization of bone from breast cancer via Raf/MAPK/extracellular signalregulated kinase (ERK)/Snail signaling (Romero-Moreno et al. 2019) and liver cancer metastasis via PI3K/Akt/GSK-3β/Snail signaling (Zhou et al. 2015). CXCR2 interacts with different CXC ligands to mediate IL-17A-induced vascular endothelial growth factor (VEGF)-independent tumor angiogenesis and endothelial chemotaxis in liver cancer (Liu et al. 2019a). Hypoxia-induced upregulation of CXCR1 and CXCR2 by malignant melanoma cells also enhances CXCL8dependent tumor cell proliferation, invasion, and angiogenesis (Gabellini et al. 2009).

2.3

CXCR1 and CXCR2 in Immune Cells

Both CXCR1 and CXCR2 are expressed on immune cells and facilitate recruitment to the tumor microenvironment, thereby influencing tumor progression (Table 2). In multiple tumor types, CXCR1+ Foxp3+ CD4+ regulatory T-cells (Tregs) promote tumor cell migration toward a CXCL8 gradient upon IL-6 stimulation (Eikawa et al. 2010). In liver cancer and breast cancer, CXCR2 expressed on neutrophils regulates infiltration into tumoral and peri-tumoral stroma, which in turn promotes tumor progression and metastasis mediated by the CXCR2–CXCL1 axis (Li et al. 2015). Further, this infiltration can be stimulated by TNFα-activated mesenchymal stromal cells (Yu et al. 2017). A secretion loop is formed in colorectal cancer when tumor suppressor gene SMAD4 stimulates CXCL1 and CXCL8 secretion to recruit CXCR2+ neutrophils, which produce more CXCL1 and CXCL8, resulting in further CXCR2+ neutrophil infiltration to the tumor microenvironment (Ogawa et al. 2019). Such infiltration at the tumor nest subsequently induces the secretion of inflammatory and angiogenic factors, such as matrix metalloproteinases (MMPs) and VEGF.

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of. . .

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Fig. 3 Expression of CXC chemokine receptors in various normal and tumor tissues. The X-axis represents the expression levels of CXCRs on a log2 scale. The Y-axis represents different normal (blue), tumor (red), and metastatic tumor (purple) tissues. ACC adrenocortical carcinoma, BLCA bladder cancer, BRCA breast cancer, CESC cervical squamous cell carcinoma, CHOL cholesterol tumor, COAD colon adenocarcinoma, DLBC Diffuse large B-cell lymphoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LAML acute myeloid leukemia, LGG brain lower grade glioma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, MESO

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Fig. 3 (continued) mesothelioma, OV ovarian serous cystadenocarcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma, PRAD prostate adenocarcinoma, READ rectum adenocarcinoma, SARC Sarcoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, TGCT testicular germ cell tumors, THCA thyroid carcinoma, THYM thymoma, UCEC uterine corpus endometrial carcinoma, UCS uterine carcinosarcoma, UVM uveal melanoma. The analysis was performed by our group

In addition, elevated CXCR1 and CXCR2 expression levels have been detected in tumor polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSCs), which are known to reduce T-cell infiltration and thus worsen clinical outcome (Zhao et al. 2015; Najjar et al. 2017; Sun et al. 2019). Knockdown of CXCR1 and/or CXCR2 can effectively inhibit tumor development in vitro and in vivo. For instance, CXCR2 knockout in mice significantly suppresses neutrophil infiltration, tumor growth, angiogenesis, and metastasis of

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of. . .

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Table 2 CXCRs expressed on immune cells and their impacts on tumor progression

CXCR2

CXCR+ immune cells Foxp3+ CD4 Treg cells Neutrophils

CXCR1/ CXCR2

MDSC cells

CXCR3

NK cells

CXCR CXCR1

CXCR4

CXCR5

Tumor Lung cancer, malignant mesothelioma, melanoma Colorectal cancer, HCC, pancreatic cancer, breast cancer HCC, renal cell carcinoma, oral carcinoma, lung carcinoma Lymphoma

Dendritic cells Treg cells

Gastric cancer

B-cells

HCC

MDSC cells NK cells

Osteosarcoma

CD4+ T-cells CD8 T-cell

NSCLC

Tfh cells

CRC

Glioma

Thyroid cancer, HCC, follicular lymphoma, colorectal cancer, pancreatic cancer HCC

MDSC cells

Gastric cancer

CD4 T-cells

DLBCL

Outcomes Elevate tumor cell migration

Ref. (Eikawa et al. 2010)

Induce secretion of inflammatory and angiogenic factors, reduce T-cell infiltration, poor prognosis Reduce T-cell infiltration, poor clinical outcomes

(Li et al. 2015; Yu et al. 2017; Ogawa et al. 2019; Nywening et al. 2018) (Zhao et al. 2015; Najjar et al. 2017; Sun et al. 2019)

Increase NK cell infiltration, anti-tumor effects Increase TIL infiltration, anti-tumor effects Induce cancer initiating cell production Induce cytokine production and reduce antitumor T-cell response Reduce CTL activity and anti-tumor effects Increase anti-tumor effects Increase tumor cell migration Increase cytotoxicity and anti-tumor effects

(Wendel et al. 2008)

Increase IL-21 secretion and anti-tumor effects Inhibit T-cell expansion and promote tumor growth Upregulate IL-10 and downregulate IL-21 to promote tumor progression

(Chen et al. 2018) (Yang et al. 2011) (Wei et al. 2019)

(Jiang et al. 2019b) (Muller et al. 2015) (Wald et al. 2006) (Zhou et al. 2018b; Jin et al. 2017a; Chu et al. 2019; Bai et al. 2017; Jifu et al. 2018) (Duan et al. 2015) (Ding et al. 2015)

(Cha et al. 2017)

(continued)

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Table 2 (continued)

CXCR CXCR6

CXCR+ immune cells NK cells

Tumor Breast cancer

CD4+ T-cells CD8+ T-cells

Colorectal cancer, NSCLC Colorectal cancer, NSCLC

Outcomes Increase anti-tumor effects – –

Ref. (Yoon et al. 2016) (Lofroos et al. 2017; Oja et al. 2018) (Lofroos et al. 2017; Oja et al. 2018)

The functions of tumor-associated CXCR7+ immune cells have not been investigated in detail and thus are not listed in the table. “–” is an indication for unavailable information

several cancers (Keane et al. 2004; Singh et al. 2009a; Lee et al. 2012). In addition, CXCR1 knockdown effectively reduced CXCL8 expression and Akt signaling in osteosarcoma (Han et al. 2015). Thus, both receptors may be promising targets for anti-tumor therapy.

2.4

Antagonist Development Targeting CXCR1 and CXCR2

Over the last few years, tumor therapeutic strategies targeting CXCR1 and CXCR2 have shown promising results (Table 3). The dual CXCR1/CXCR2 small-molecule antagonist SCH-527123, also known as navarixin or MK-527123, has been shown to inhibit melanoma cell proliferation, migration, and invasion via blockade of PI3K/ Akt and MAPK/ERK pathways (Shang and Li 2018; Singh et al. 2009b) and also to inhibit CXCR2 signal transduction through deactivation of NF-κB/MAPK/Akt signaling, leading to colorectal cancer cell apoptosis (Ning et al. 2012). Application of SCH-527123 together with another dual receptor antagonist, SCH-479833, has been investigated for the treatment of spontaneous liver metastasis of colon cancer (Varney et al. 2011). In preclinical colon cancer models, combination of SCH-527123 with oxaliplatin, a standard therapy for colorectal cancer, exhibits stronger inhibition of tumor cell proliferation and angiogenesis compared with using either therapy alone (Ning et al. 2012). At present, a clinical phase 2 trial (NCT03473925) is investigating the efficacy and safety of SCH-527123 combined with pembrolizumab, a humanized antibody targeting the inhibitory T-cell receptor programmed death-ligand 1 (PD-1), for treatment of non-small cell lung cancer (NSCLC), castrated-resistant prostate cancer (CRPC), and microsatellite stable of colorectal cancer. Reparixin, another dual antagonist that reduces tumor volume and metastasis, augments the therapeutic effects of paclitaxel (Schott et al. 2017) or 5-fluorouracil (Wang et al. 2016) for treatment of metastatic breast cancer and gastric cancer, respectively. Apart from these dual functional antagonists, antagonists specifically targeting CXCR2 have been designed, including SB225002 (Manjavachi et al. 2010), which

CXC Chemokine Receptors in the Tumor Microenvironment and an Update of. . .

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Table 3 Summary of antagonists targeting CXC receptors and corresponding outcomes

Targets CXCR1/ CXCR2

Antagonist SCH-527123

SCH-479833

CXCR2

Type of cancers Melanoma, colorectal cancer, pancreatic cancer, NSCLC, CRPC Melanoma, colorectal cancer, pancreatic cancer

Outcomes Reduced tumor growth, metastasis, angiogenesis

Reduced tumor growth, metastasis, and angiogenesis

SB225002

Prostate cancer, ovarian cancer, esophageal cancer, pancreatic cancer, nasopharyngeal cancer, CCRCC, intrahepatic bile duct cell cancer, breast cancer

Reduced tumor growth and metastasis

AZD-5069 SB332235

Prostate cancer Esophageal cancer, AML

AZ10397767

Type 2 alveolar epithelial adenocarcinoma

CXCR3

AMG 487

Breast cancer, osteosarcoma, colon cancer

– Reduced tumor invasion, viability and clonogenic capacity Reduced chemotaxis of neutrophils to cancer cells, reduced tumor proliferation and microvascular formation Reduced tumor metastasis

CXCR4

AMD3100

Osteosarcoma, colon cancer, lung cancer, prostate cancer, ovarian cancer, NHL, multiple myeloma, B acute lymphoblastic leukemia

Inhibited tumor migration and metastasis

Ref. (Singh et al. 2009b; Varney et al. 2011; Purohit et al. 2016) (Singh et al. 2009b; Varney et al. 2011; Purohit et al. 2016) (Xu et al. 2018b; Yung et al. 2018; Wang et al. 2006; Sueoka et al. 2014; Saintigny et al. 2013; Matsuo et al. 2009; Lo et al. 2013; Ijichi et al. 2011; Grepin et al. 2014; Erin et al. 2015) – (Schinke et al. 2015; Shrivastava et al. 2014)

In vitro or in vivo Both

Both

Both

– In vitro

(Tazzyman et al. 2011)

Both

(Cambien et al. 2009; Zhu et al. 2015a; Pradelli et al. 2009) (Randhawa et al. 2016; Liao et al. 2015; Reeves et al. 2017; Zhu et al. 2019; Bachelerie et al. 2014; Li et al. 2017a)

In vivo

Both

(continued)

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Table 3 (continued)

Targets

CXCR7

Antagonist AMD070

Type of cancers Oral cancer, acute lymphoblastic leukemia, pancreatic cancer

Outcomes Reduced tumor invasiveness and metastasis

Ref. (Uchida et al. 2018; Morimoto et al. 2016; Parameswaran et al. 2011)

MDX-1338

AML, CLL, multiple myeloma

IT1t

Triple-negative breast cancer

(Kashyap et al. 2016; Kuhne et al. 2013) (Tulotta et al. 2016)

POL6326 BKT140

Breast cancer AML, multiple myeloma, NSCLC, NHL, prostate cancer, pancreatic cancer

Induced apoptosis and inhibited proliferation Reduced formation of early metastasis – Inhibited tumor proliferation and metastasis

X4-136

Melanoma

CCX771

Prostate cancer, liver cancer, breast cancer

CCX662

Glioblastoma multiforme

Inhibited tumor growth Reduced tumor growth, migration, and angiogenesis –

– (Fahham et al. 2012; Beider et al. 2013, 2014; Beider et al. 2011; Peng and Kopecek 2014) (Saxena et al. 2019) (Lin et al. 2014; Luo et al. 2018; Qian et al. 2018) –

In vitro or in vivo Both

Both

In vivo

In vivo Both

In vivo Both

In vivo

inhibit the expression of invasion-related proteins, such as bovine seminal plasma, osteopontin, MMPs, and αvβ3 by prostate cancer cells. This leads to blockade of Akt and mTOR activation and reduction of tumor cell proliferation and invasion (Xu et al. 2018b). In addition, SB225002 significantly attenuated the carcinogenic and metastatic potential of ovarian cancer both in vitro and in vivo, likely by inhibiting transforming growth factor-β-activated kinase 1 (TAK1)/NF-κB signal transduction (Yung et al. 2018). In deltaNp63+ triple-negative breast cancer (TNBC) patients, application of SB225002 reduced MDSC recruitment and tumor metastasis (Kumar et al. 2018). A recent study also reported that the CXCR2 antagonist AZD5069 aids in activating CXCR2+ tumor-associated macrophages, which promote an anti-tumorigenic state upon infiltration (Di Mitri et al. 2019). Combination of AZD5069 with anti-PD-1 antibody prolonged survival of mice with pancreatic ductal adenocarcinoma (PDAC) by improving T-cell infiltration (Steele et al. 2016). Another such CXCR2 antagonist, AZ10397767, developed to specifically inhibit CXCR2-mediated neutrophil chemotaxis, reduced neutrophil infiltration into

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xenografts of type 2 alveolar epithelial adenocarcinoma, thereby indirectly suppressing tumor cell proliferation and angiogenesis (Tazzyman et al. 2011).

3 CXCR3 3.1

CXCR3 Structure and Signaling Mechanisms

CXCR3, a predominantly IFN-γ-driven CXC chemokine receptor, interacts with CXCL9–11 to regulate immune cell proliferation, cytokine secretion, and migration (Nakajima et al. 2002). In addition to the structures conserved across all CXCRs, CXCR3 has two different activation domains, one targeting the C-terminal domains of CXCL9 and CXCL10 and the other targeting the third intracellular ring of CXCL11 (Colvin et al. 2004). Structural analysis has revealed three CXCR3 variants, CXCR3A, CXCR3B, and CXCR3-ALT (Lasagni et al. 2003), with distinct intracellular signaling functions. Variant CXCR3A functions as the classical CXCR3, while CXCR3B binds to CXCL9–11 as well as CXCL4 to inhibit endothelial cell growth, which may contribute to suppression of tumor angiogenesis (Lasagni et al. 2003; Puchert et al. 2019), and CXCR3-ALT is a 101-amino acidtruncated CXCR3 that mainly mediates CXCL11 signaling (Lasagni et al. 2003). The CXCR3A variant (also referred to as simply CXCR3) has been the major focus of research because of its known contributions to tumor development. CXCR3 is expressed not only by immune cells, including macrophages, T-cells, natural killer (NK) cells, and dendritic cells but is also detected in various tumor cells (Fig. 3) (Ma et al. 2009; Zhou et al. 2016; Winkler et al. 2011; Cambien et al. 2009; Maekawa et al. 2008; Rezakhaniha et al. 2016).

3.2

Autocrine and Paracrine Mechanisms of CXCR3

Signaling by CXCR3 can be either autocrine or paracrine (Fig. 4). CXCR3 overexpression in cancer cells tends to elevate tumor proliferation, migration, metastasis, and angiogenesis through autocrine signaling, resulting in poor clinical outcomes, while immune cells expressing CXCR3 are recruited to the tumor nest through paracrine signaling, leading to either anti-tumor or pro-tumor effects depending on the recruited population (Table 2). Autocrine signaling: Overexpression of CXCR3 by breast cancer cells is associated with tumor progression and poor survival (Bronger et al. 2017). Binding to CXCL9 or CXCL10 elevates the expression of cathepsin B, a protease that degrades chemokine ligands. This creates a negative feedback loop that disrupts the chemokine gradient at the tumor nest and suppresses CXCR3+ lymphocyte infiltration, resulting in tumor progression (Bronger et al. 2017). High CXCR3 expression also induces MMP and IL-6 activation, resulting in degradation of extracellular matrix

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Fig. 4 Functions of CXCR3 in the tumor microenvironment. CXCR3 signals through autocrine and paracrine mechanisms to either promote or suppress tumor development. CXCR3 expressed on tumor cells interacts with tumor-derived chemokines via autocrine signaling to trigger tumor cell proliferation, migration, and metastasis. CXCR3+ leukocytes are differentiated and recruited toward tumor sites via paracrine signaling to induce or suppress tumor growth

(ECM) proteins, which ultimately leads to vascular invasion, lymph node metastasis, and poor survival (Zhou et al. 2016; Yang et al. 2016; Shin et al. 2010). Paracrine signaling: In a mouse model of lymphoma, secretion of CXCL10 by tumor cells increased CXCR3+ CD27+ NK cell infiltration to tumor sites through CXCR3–CXCL10 signaling, prolonging survival (Wendel et al. 2008). Similarly, paracrine signaling recruits CXCR3+ dendritic cells and tumor-infiltrating lymphocytes (TILs) to gastric cancer tissues, resulting in anti-tumor activity (Chen et al. 2018). High expression of CXCR3 is also correlated with low pro-tumor M2 infiltration into gastric cancer (Chen et al. 2019a); however, detailed mechanisms

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were not clarified. In contrast, elevated CXCR3 expression promotes CXCR3+ Foxp3+ IL-17 Treg cell infiltration toward high CXCL11 gradient colorectal tumor tissues, which then triggers IL-17-dependent production of cancer initiation cells (Yang et al. 2011). In hepatocellular carcinoma (HCC), CXCR3 expressed on B-cells interacts with CXCL10 secreted by macrophages upon CD4+ T-cell activation, causing CXCR3+ B-cells to produce immunoglobulin G (IgG), leading to elevated cytokine expression by macrophage and a reduced anti-tumor T-cell response (Wei et al. 2019). Due to the bidirectional function of CXCR3 in tumor promotion and suppression, CXCR3 knockdown may either suppress or stimulate tumor progression and metastasis. In mouse metastatic breast cancer and melanoma models, CXCR3 depletion effectively reduces tumor progression and metastasis (Zhu et al. 2015a; Kawada et al. 2004). Alternatively, CXCR3 deficient mice exhibited macrophage polarization toward the M2 phenotype and IL-12/IFN-γ/NO axis disruption, resulting in a tumor-promoting microenvironment (Oghumu et al. 2014; Chheda et al. 2016). In addition, although CXCR3B has been reported to inhibit tumor angiogenesis via CXCR3B–CXCL10 signaling (Proost et al. 2001), tumor occurrence is more likely when the expression of CXCR3A is dominant over CXCR3B (Kawada et al. 2007; Goldberg-Bittman et al. 2004). Recently, Saahene et al. (Saahene et al. 2019) suggested that CXCR3B interaction with CXCL4 may be an indicator of poor prognosis. Thus, CXCR3 has diverse functions in the tumor microenvironment that can enhance or suppress tumor progression.

3.3

Antagonists Targeting CXCR3

The fragmental structures of CXCR3–CXCL complexes (Palladino et al. 2012; Trotta et al. 2009) have greatly assisted in the development of antagonists targeting CXCR3 for anti-tumor therapy (Table 3). The small-molecule antagonist of all CXCR3 variants AMG487 exhibits significant inhibitory effects on tumor progression and metastasis and improves host anti-tumor immunity. Experiments on a rat breast cancer model revealed that this anti-tumor mechanism involves reduction of tumor cell migration and improvement of both myeloid cell function and T-cell response (Zhu et al. 2015a). AMG487 also prevented CXCR3-induced migration of colorectal cancer cells in mouse model of colon carcinoma and lung metastasis (Cambien et al. 2009) and blocked MMP-2 and MMP-9 activity induced by the CXCR3–CXCL10 axis, thereby suppressing lung metastasis of osteosarcoma (Pradelli et al. 2009). Other CXCR3 antagonists include SCH-546738 (Du et al. 2017), Takeda 779 (Takama et al. 2011), and NBI-74330 (Gao et al. 2018). However, the anti-tumor effects of these agents have not been assessed.

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4 CXCR4 4.1

CXCR4 Signaling

CXCR4 was first reported as a highly efficient lymphocyte chemoattractant in 1996 (Bleul et al. 1996). In 2001, Muller et al. (Muller et al. 2001) demonstrated elevated expression of CXCR4 in human breast cancer, which influenced tumor cell migration and metastasis through exclusive interaction with CXCL12. Since these seminal reports, many studies of CXCR4 influences on tumor progression have been conducted, making it the most thoroughly investigated CXC chemokine receptor. To date, CXCR4 expression has been reported in more than 20 tumor types (Fig. 3). Elevated expression of CXCR4 and CXCR4–CXCL12 signaling has diverse effects (Cambien et al. 2009; Muller et al. 2001; Scala et al. 2005; Kaifi et al. 2005; Lu et al. 2014; Oliveira Frick et al. 2011) but is generally a biomarker for poor prognosis (Furusato et al. 2010; Du et al. 2019). CXCR4 is also expressed in immune cells, including macrophages, neutrophils, and T and B lymphocytes, where it regulates infiltration and thus the tumor microenvironment. As there are a number of quality reviews available on CXCR4 functions in the tumor microenvironment (Chatterjee et al. 2014; Walenkamp et al. 2017), this review presents a selection of the most recent studies.

4.2

CXCR4 Expression and Signaling in Tumor Cells

Overexpression of CXCR4 was recently identified as a prognostic biomarker for gastrointestinal cancer, acute myeloid leukemia, and tongue squamous carcinoma in addition to many other tumor types (Du et al. 2019; Jiang et al. 2019a; Ciuca et al. 2019). In ovarian cancer, CXCR4 promotes tumor cell invasion by stimulating MMP production and inhibiting expression of the Rho-activating protein ARHGAP10 via upregulation of VEGF/VEGFR2 signaling (Luo et al. 2019). High expression of CXCR4 in glioblastoma multiforme tissue and glioma-initiating cells modulated by Notch1 through Akt/mTOR signaling promotes stem maintenance and migration of glioma-initiating cells (Yi et al. 2019). Combined inhibition of CXCR4 signal transducers and activators of transcription 3 (STAT3), a transcription factor that converts cytokines from anti-tumor to oncogenic, reduced breast cancer lung metastasis by increasing CD8 T-cell infiltration and downregulating MMPs and VEGF (Li et al. 2019a). In addition, blocking B-cell receptor/PI3K signaling upregulated CXCR4–CXCL12-induced chemotaxis in B-cell receptor-dependent diffuse large B-cell lymphoma (DLBCL), suggesting that CXCR4 expression level may explain tumor sensitivity to B-cell receptor inhibitors (Chen et al. 2019b).

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17

Association Between CXCR4 and Leukocyte Trafficking

Signaling through CXCR4 is a crucial controller of tumor leukocyte trafficking and thus a major influence on tumor progression. In osteosarcoma, CXCR4+ MDSCs migrate toward a CXCL12 gradient at the tumor nest, where these cells activate the Akt pathway. In turn, Akt activation reduces MDSC cell apoptosis, forming a positive feedback loop that leads to inhibition of cytotoxic T-cell anti-tumor effects (Jiang et al. 2019b). An in vitro study examining infiltration of NK cells expressing genetically modified CXCR4 found an augmented cytotoxic response against glioma cells via CXCR4–CXCL12 (Muller et al. 2015). In addition, high plasmacytoid dendritic cell infiltration was detected, which stimulated CXCR4 expression via TNF-α/NF-κB signaling. Overexpression of CXCL12 at tumor sites attracts more CXCR4+ cells, forming a positive feedback cycle leading to lymph node metastasis of breast cancer (Gadalla et al. 2019). Alternatively, blocking CXCR4 effectively suppressed tumor metastasis and improved the therapeutic efficacy of an immune checkpoint blocker via upregulation of Treg infiltration, downregulation of tumorassociated fibroblasts, and immunosuppression (Chen et al. 2019c).

4.4

Antagonists Targeting CXCR4

Since elucidation of the CXCR4–CXCL12 complex structure (Wu et al. 2010), a number of antagonists have been developed to target the CXCR4–CXCL12 signaling axis (Table 3). Among all the antagonists available, AMD3100, also known as plerixafor, is the best studied and so far the only one approved by the US Food and Drug Administration for clinical use in treating non-Hodgkin’s lymphoma (NHL) and auto-transplantation of multiple myeloma. AMD3100 was originally designed to antagonize human immunodeficiency virus (HIV) (Donzella et al. 1998) and its potential anticancer efficacy subsequently explored based on studies showing the importance of the CXCR4–CXCL12 axis in tumor progression. Indeed, AMD3100 has been shown to inhibit the migration and adhesion of acute B lymphoblastic leukemia cells (Randhawa et al. 2016), the survival and metastasis of osteosarcoma by blocking c-Jun N-terminal kinase (JNK) and Akt signaling pathways (Liao et al. 2015), and ovarian cancer cell proliferation, while improving the therapeutic efficacy of low-dose paclitaxel and reducing recurrence of ovarian cancer (Reeves et al. 2017), and enhance cellular radiosensitivity and primary tumor response in mouse models of TNBC (Zhou et al. 2018a) and cervical cancer in radiation therapy (Lecavalier-Barsoum et al. 2018). In addition, AMD3100 suppressed EMT and prostate cancer cell migration by inhibiting the CXCR4–CXCL12α pathway (Zhu et al. 2019). It also reduced lung cancer cell proliferation and invasion and suppressed brain metastasis by diminishing the expression levels of CXCR4, VEGF, and MMPs (Li et al. 2017a).

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Combination of CXCR4 antagonist and immune checkpoint inhibitor has been reported to improve anti-tumor immune response by reshaping the tumor microenvironment. Dual blockade of CXCR4–CXCL12 and PD-1–PDL-1 pathways using AMD3100 and anti-PD-1 antibody significantly promoted anti-tumor immunity in PDAC (Seo et al. 2019), osteosarcoma (Jiang et al. 2019b), and ovarian cancer (Zeng et al. 2019). An in vitro study showed that immunotherapy alone failed to effectively induce anti-tumor activity in PDAC due to geographic sequestration of T-cells from the tumor site. This can be reversed by CXCR4 antagonist which greatly enhances anti-tumor CD8+ T-cell infiltration in the tumor microenvironment (Seo et al. 2019). In a mouse model of osteosarcoma, application of AMD3100 showed synergistic effect with immunotherapy by diminishing the immunosuppressive CXCR4+ MDSCs within tumor tissues and thus promoting effector T-cell infiltration (Jiang et al. 2019b). Similarly, AMD3100 improved the efficacy of anti-PD-1 and prolonged survival of ovarian tumor-bearing mice by increasing memory and CD8 + T-cell infiltration, increasing conversion from Treg cells into helper T-cells, and decreasing MDSCs (Zeng et al. 2019). Other CXCR4 antagonists include AMD070, MDX-1338, IT1t, BKT140, POL6326, USL-311, T22, and TG-0054. AMD070, also named AMD11070/X4P001, was reported to suppress CXCR4–CXCL12 signaling-dependent migration and invasion of oral cancer cells and to significantly inhibit lung metastasis (Uchida et al. 2018), while MDX-1338, also known as Medarex, was found to inhibit F-actin aggregation and chronic lymphocytic leukemia (CLL) cell migration and induce tumor cell death by producing reactive oxygen species in CLL cells (Kashyap et al. 2016). Further, IT1t was reported to suppress CXCR4 signaling and reduce early metastasis of TNBC (Tulotta et al. 2016), while BKT140 (BL-8040) was shown to improve sensitivity of tumor cells to chemotherapeutic drugs in NSCLC (Fahham et al. 2012), NHL (Beider et al. 2013; Burger et al. 2011), and pancreatic cancer (Bockorny et al. 2020).

5 CXCR5 5.1

CXCR5 Expression Patterns

CXCR5, also known as Burkitt’s lymphoma receptor 1, exclusively interacts with B-cell chemoattractant (CXCL13). CXCR5 is expressed in all mature peripheral B-cells, a small number of T-cell subtypes, mature dendritic cells, and tumor cells (Qi et al. 2014).

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19

CXCR5 in Tumor Cells

In CXCR5-expressing tumor cells, the CXCR5–CXCL13 axis activates divergent cell signaling cascades to affect tumor progression and lymph node metastasis, thereby influencing prognosis. The CXCR5–CXCL13 axis was reported to promote the growth of clear cell renal cell carcinoma (CCRCC) (Zheng et al. 2018) and colorectal cancer (Zhu et al. 2015b) via activation of PI3K/AKT/mTOR signaling. This was accompanied by activation of MMP cascades for ECM degradation, thereby enhancing tumor metastasis (Zhu et al. 2015b). Overexpression of CXCR5 upregulates ERK signaling in breast and prostate cancer, thus reducing tumor cell apoptosis (El Haibi et al. 2010; Xu et al. 2018c), and promotes tumor cell proliferation via JNK signaling in prostate cancer (El Haibi et al. 2010; Singh et al. 2009c). In addition, loss of the oncogenic protein PKCε and the tumor suppressor phosphatase and tensin homolog (PTEN) can induce prostate cancer progression through NF-κB-mediated upregulation of CXCR5–CXCL13 signaling (Garg et al. 2017). In contrast, suppressing CXCR5 in colorectal cancer reduces tumor growth and liver metastasis (Meijer et al. 2006). Taken together, these findings suggest that targeting the CXCR5–CXCL13 axis and related signaling pathways may be an effective antitumor strategy.

5.3

CXCR5 in Immune Cells

The main functions of CXCR5–CXCL13 signaling in the immune response are the regulation of B-cells, T-cells, and dendritic cells in secondary lymphoid organs (Schiffer et al. 2015). In turn, the balance of these influences determines tumor suppression or progression. High CXCR5+ CD8 T-cell infiltration was detected at tumor nests and tumor-involved lymph nodes in thyroid cancer (Zhou et al. 2018b), HBV-related HCC (Jin et al. 2017a), follicular lymphoma (Chu et al. 2019), colorectal cancer (Jifu et al. 2018), and pancreatic cancer (Bai et al. 2017). Compared to CXCR5 CD8 T-cells, CXCR5+ CD8 T-cells generally exhibit higher proliferation potency, granzyme B production, PD-1 and TIM-3 expression levels, and cytotoxicity against tumors. In addition, CXCL13 was reported to recruit CXCR5+ tumorinfiltrating T-follicular helper (Tfh) cells into HCC sites, which in turn promoted section of the anti-tumor cytokine IL-21(Duan et al. 2015). In contrast, the CXCR5– CXCL13 axis can also promote escape of tumor cells from immune surveillance. For instance, CXCR5+ CD40+ MDSCs migrate and accumulate at gastric tumor sites via CXCR5–CXCL13 signaling to suppress T-cell expansion and promote tumor growth (Ding et al. 2015). In DLBCL as well, CXCR5+ CD4+ T-cells promote survival and proliferation of tumor cells through upregulation of IL-10 and downregulation of IL-21 (Cha et al. 2017).

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Absence of Antagonists Targeting CXCR5

Little progress has been made in the development of antagonists that directly target CXCR5 due to the lack of CXCR5 structural information, so a more common inhibitory strategy is blockade of the ligand CXCL13. To date, only a few methods are available to target CXCR5 directly for anti-tumor activity. For example, CXCR5::CD3, a trifunctional antibody that targets both CXCR5 and CD3, has been developed to suppress NHL progression via stimulation of CD4+ and CD8+ T-cells, cytokine secretion, and CXCR5+ B-cell lysis (Panjideh et al. 2014). Nevertheless, CXCR5 antagonist development still holds greater promise for anti-tumor therapy.

6 CXCR6 6.1

Expression of CXCR6

CXCR6, originally known as orphan receptor BONZO (or STRL33) as it is the only receptor for CXCL16, is expressed in both normal and tumor tissues (Fig. 3). Investigations of CXCR6 in the tumor microenvironment have only started recently. In 2004, the CXCR6–CXCL16 signaling axis was reported to be significantly downregulated in colon cancer (Wagsater and Dimberg 2004; Wagsater et al. 2004). Since this initial discovery, many studies have investigated CXCR6 functions in regulating the tumor microenvironment.

6.2

CXCR6–TM-CXCL16 and CXCR6–sCXCL16 Axes

Immune signaling by CXCR6 is unique in that this receptor binds to both a full length transmembrane (TM)-ligand (TM-CXCL16) and a secreted soluble (s) ligand (sCXCL16), a shorter version generated by disintegrin-like metalloproteinase 10 (ADAM10). The CXCR6–TM-CXCL16 axis has been reported to inhibit renal and breast cancer progression (Chung et al. 2017; Gutwein et al. 2009), possibly by enhancing immune cell infiltration into tumor sites (Hojo et al. 2007), while CXCR6–sCXCL16 signaling may promote or suppress tumor progression, depending on expression site (Chung et al. 2017; Lang et al. 2017) (Fig. 5).

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Fig. 5 CXCR6 interactions with transmembrane (TM) or soluble (s) CXCL16. TM-CXCL16 undergoes cleavage by ADAM10 to form sCXCL16. (a) CXCR6 interacts with TM-CXCL16 to suppress tumor progression via leukocyte recruitment into the tumor nest. (b) CXCR6 interacts with sCXCL16 to promote or suppress tumor progression via downstream pro-tumor signaling pathways or by leukocyte trafficking, respectively

6.3

CXCR6 Expression by Tumor Cells

CXCR6 is overexpressed in multiple tumor types (Fig. 3), where enhanced signaling promotes tumor cell proliferation, invasion, metastasis, malignant transformation, reoccurrence, advanced clinical stage, and lower patient survival (Mir et al. 2019; Ma et al. 2017; Ke et al. 2017; Chang et al. 2017; Jin et al. 2017b; Hong et al. 2018). Activation of the CXCR6–CXCL16 axis is mainly associated with PI3K/Akt and NF-κB signaling pathways. For example, overexpression of CXCR6 promotes

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tumor cell proliferation, invasion, and metastasis through upregulation of Akt signaling in osteosarcoma and gastric cancer (Ma et al. 2017; Jin et al. 2017b), through the PI3K/Akt pathway in ovarian cancer (Hong et al. 2018), and via the NF-κB pathway in prostate cancer (Kapur et al. 2019). This pro-tumor activity is usually associated with elevated EMT and expression levels of MMPs, ADAM10, and CXCL16 (Mir et al. 2019; Ma et al. 2017; Jin et al. 2017b; Hong et al. 2018; Kapur et al. 2019). High expression of CXCR6 is also associated with Ezrin activation and αvβ3 integrin clustering, which promotes gastric cancer by reduced cellular adhesion and concomitant mobilization (Singh et al. 2016). Alternatively, blockade of CXCR6 significantly inhibits tumor growth and potentiates drug efficacy against prostate cancer (Kapur et al. 2019) and HCC (Xu et al. 2014) by suppressing the CXCR6–CXCL16 and VEGF signaling.

6.4

Association Between CXCR6 and Immune Cells

Similar to other CXCR signaling pathways, CXCR6–CXCL16 signaling can recruit immune cells to tumor sites and modulate immune cell phenotype transformation to influence tumor progression. Elevated CXCL16 upon irradiation enhances CXCR6+ NK cell infiltration into tumor sites and promote anti-tumor activity in breast cancer (Yoon et al. 2016). Both CD4+ and CD8+ T-cells also express CXCR6, and the proportions of CXCR6+ CD4+ and CD8+ T-cells differ between normal tissue and colorectal cancer, suggesting that T-cell recruitment to tumors may involve distinct mechanisms (Lofroos et al. 2017). Elevated CXCR6 expression was also detected in CD103+ CD4+ and CD13+ CD8+ T-cells from NSCLC tissues and served as a novel biomarker for resident memory T-cells (Trm), which prevent secondary infection by releasing IFN-γ, TNF-α, and IL-2 (Oja et al. 2018). Immune responses mediated by natural killer T-cells (NKT) and CD4+ T-cells, which are critical for hepatocyte senescence recognition and hepatocarcinogenesis suppression, appear to be CXCR6-dependent. CXCR6-deficient mice exhibit reduced NKT and CD4+ T-cell numbers and concomitantly greater tumor burdens (Mossanen et al. 2019). In glioma, CXCL16 released by tumor cells drives tumorassociated microglia/macrophage (TAM) polarization toward the anti-inflammatory (pro-tumor) phenotype. These cells then infiltrate and stimulate glioma development via the CXCR6–CXCL16 axis. Indeed, CXCR6 knockout greatly suppressed this effect and prolonged survival in mice (Lepore et al. 2018). Unfortunately, the cellspecific functions of CXCR6 are still largely unknown.

6.5

Absence of Antagonist Targeting CXCR6

Overall, the CXCR6–CXCL16 axis presents a promising target for anti-tumor therapy. Although there are several different methods to block CXCR6–CXCL16

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activity, such as by gene silencing or inhibition of related signaling pathways, no information is yet available on CXCR6 antagonist development. Elucidation of CXCR6–CXCL16 structure should greatly aid antagonist design.

7 CXCR7 7.1

CXCR7 Signaling

CXCR7, better known as ACKR3, is an atypical chemokine receptor as it contains an Asp-Arg-Tyr-Leu-Ser-Ile-THR (DRYL-SIT) sequence instead of the classical chemokine receptor motif Asp-Arg-Tyr-Leu-Ala-Ile-Val (DRYLAIV). Due to this alteration, CXCR7 signaling is not mediated through conventional G proteins but rather through β-arrestin2 upon ligand interaction (Naumann et al. 2010). CXCR7 is expressed in normal tissues such as activated endothelial cells (Thelen and Thelen 2008), as well as in the tumor microenvironment upon ligand binding. CXCR7 is a second receptor for CXCL11 and CXCL12 and binds to CXCL12 at a tenfold higher binding affinity compared to CXCR4 (Wang et al. 2018a).

7.2

CXCR7 in Tumor Cells

Both CXCR7–CXCL11 and CXCR7–CXCL12 signaling axes have been reported to participate in tumor progression. Overactivation of the CXCR7–CXCL11 axis upon estrogen stimulation promotes EMT and ECM remodeling, thereby contributing to ovarian cancer progression (Benhadjeba et al. 2018). Significant overexpression of CXCR7 and CXCL12 was also found in lung metastasis of colorectal cancer (Wang et al. 2018b) and breast cancer (Al-Toub et al. 2019) and was associated with elevated cell adhesion to fibronectin and laminin (Qian et al. 2018), triggering tumor metastasis. There is accumulating evidence demonstrating that CXCR7 elevation can induce tumor growth and angiogenesis mainly by facilitating VEGF secretion. This enhanced VEGF secretion is mediated via Akt/MAPK signaling in HCC (Chen et al. 2016) and colon cancer (Li et al. 2019b), the MAPK pathway in gastric cancer (Shi et al. 2017), and the CXCR7–Src kinase axis in melanoma (Xu et al. 2019). In addition, lipopolysaccharide-induced CXCR7 expression promotes gastric cancer cell proliferation and migration via transmembrane Toll-like receptor 4 (TLR4)/myeloid differential protein-2 (MD-2) signaling (Li et al. 2019c). Alternatively, downregulation of CXCR7 suppresses migration and invasion of HCC-derived tumor endothelial cells by inhibiting MMP2 and VEGF via STAT3 signaling (Wu et al. 2018). In addition to interacting with cognate CXC ligands, CXCR7 also binds directly to the epidermal growth factor receptor (EGFR) to trigger activation of MAPK and Akt pathways, leading to tumor cell proliferation in prostate cancer (Singh and Lokeshwar 2011), breast cancer (Salazar et al. 2014), and NSCLC

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(Liu et al. 2019b). Downregulation of CXCR7 or dual EGFR/CXCR7 inhibition disrupted this interaction, diminished EGFR and ERK1/2 activities, and suppressed tumor cell proliferation (Salazar et al. 2014; Liu et al. 2019b).

7.3

CXCR7 and Drug Resistance

In recent years, the contributions of CXCR7 signaling to drug resistance have attracted widespread attention. In CRPC, high CXCR7 expression confers resistance to enzalutamide through activation of MAPK/ERK signaling (Li et al. 2019d) and induction of M-phase cell cycle genes through PI3K/Akt signaling upon association with macrophage migration inhibitory factor (Rafiei et al. 2019). Both mechanisms lead to prostate cancer progression. Overexpression of CXCR7 in NSCLC promotes resistance to the EGFR TKI (a class of EGFR inhibitor) and EMT upregulation via MAPK signaling. The combination of CXCR7 depletion and EGFR inhibition diminished ERK activation and improved drug activity (Becker et al. 2019). The expression level of CXCR7 may also correlate with imatinib resistance of chronic myelogenous leukemia through upregulation of ERK signaling (Li et al. 2017b), cisplatin resistance of esophageal squamous cell carcinoma via IL-6-mediated NF-κB signaling (Qiao et al. 2018), and Taxotere resistance of breast cancer (Rong et al. 2013). Collectively, these findings indicate that CXCR7 may serve as a valuable biomarker for drug-resistant cancers.

7.4

Crosstalk of CXCR7 with CXCR3 and CXCR4

Although CXCR7 shares ligands with CXCR3 and CXCR4, downstream signaling is frequently independent, even when these receptors are co-expressed by the same cell. For instance, while CXCR7 co-expresses with CXCR3 or CXCR4 in CRPC, breast cancer, colon cancer, and lung cancer cell lines, activities appear independent (Puchert et al. 2019; Rafiei et al. 2019). In contrast, CXCR7 and CXCR4 co-overexpressed in colorectal cancer cells form heterodimers that induce histone demethylation and promote transcription of oncogenes and inflammatory factors, leading to tumorigenesis (Song et al. 2019). Double knockout of CXCR7 and CXCR4 in TNBC model mice resulted in significantly stronger suppression of tumor cell proliferation, migration, and invasion than either CXCR7 or CXCR4 single knockout (Yang et al. 2019).

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Antagonist Development Targeting CXCR7

There have been a series of reports on CXCR7-targeted antagonists, such as CCX771 and CCX662 as well as their prototypes CCX733 and CCX754 (Bachelerie et al. 2014). The competitive antagonist CCX771 was found to reduce activation of CXCR7-related downstream signals and subsequently inhibit tumor proliferation and angiogenesis (Qian et al. 2018; Luo et al. 2018). Further, CCX771 diminished EGFR expression in breast cancer cells both in vitro and in vivo (Qian et al. 2018). Combined use of CCX771 and enzalutamide effectively reduced activation of EGFR, Akt, and VEGF in prostate cancer xenografts (Luo et al. 2018). The antagonist CCX733 simultaneously suppressed Akt and ERK signaling pathways, resulting in significantly reduced CXCR7-induced EMT and thus greater therapeutic efficacy against bladder cancer compared to combined use of the ERK inhibitor U0126 and Akt inhibitor LY294002 (Hao et al. 2012). It has been reported that CXCR7 is susceptible to CXCR4 regulation (Wang et al. 2008), and the CXCR4 antagonist AMD3100 also interacts with CXCR7 (Kalatskaya et al. 2009). However, combination therapy using enzalutamide plus AMD 3100 appeared to be less effective than enzalutamide plus CCX771 for inhibition of prostate cancer cell growth and migration (Luo et al. 2018). Finally, CCX662 has been applied for treatment of glioblastoma multiforme (Walters et al. 2014). However, no further information on CCX662 has been reported since 2014.

8 Concluding Remarks Chemokine receptors are expressed by both cancerous cells and immune cells to regulate tumor growth, invasion, metastasis, and angiogenesis through a variety of chemical signals in the tumor microenvironment. Intensive research over the past two decades has revealed the detailed structure, function, and signaling mechanisms of many chemokine receptors. The human seven-transmembrane CXCRs are mid-sized proteins of 350–415 amino acids and 39,280–45,523 Da (Table 1). However, resolving the molecular structures of the transmembrane CXCRs has proven challenging, and to date only the full structures of CXCR1 (Park et al. 2012) and CXCR4 (Wu et al. 2010) have been revealed. This lack of protein structure has greatly hindered investigations on molecular signaling mechanisms and the development of selective chemical modulators such as antagonists. Nonetheless, evidence to date indicates that CXCRs are involved in a variety of tumorigenic processes, including cell proliferation, migration, invasion, metastasis, and angiogenesis, mediated by numerous downstream signaling pathways (Fig. 6), and thus are promising biomarkers and molecular targets for anti-tumor therapy. Elevated expression of CXCRs in tumor cells is usually associated with tumor progression, while CXCR+ tumor-infiltrating immune cells can be anti- or pro-tumorigenic (Table 2). For example, CXCR1+ CD4 Treg cells, CXCR2+

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Fig. 6 Network diagram representing the associations between CXCR1–CXCR7 and different signaling pathways

neutrophils, CXCR1+ and CXCR2+ MDSC cells, CXCR3+ Treg cells, CXCR3+ B-cells, CXCR4+ MDSC cells, CXCR4+ CD4+ T-cells, CXCR5+ MDSC cells, and CXCR5+ CD4+ T-cells are generally associated with tumor progression, while CXCR3+ NK cells, CXCR3+ dendritic cells, CXCR4+ NK cells, CXCR5+ CD8+ T-cells, CXCR5+ Tfh cells, and CXCR6+ NK cells are usually reported to have antitumor effects. Studies by Yu (Yu and Zhang 2019) and Lofroos (Lofroos et al. 2017) examining the frequencies of gastric cancer cells expressing CXCR1–7 and lymphocytes expressing CXCR3–6 in colorectal cancer revealed substantial variation in the proportions of different T-cells (CD4+ and CD8+) and receptors in the same tumor type. These findings indicate that the expression profile of different CXCRs, as well as the recruitment of CXCRs+ tumor cells and CXCRs+ TILs into the tumor microenvironment, is highly regulated. However, detailed molecular mechanism of such regulation is not fully understood. Among CXCR members, CXCR4 is the most intensively studied for its functions in tumor regulation, and the CXCR4 antagonist AMD3100 is the only CXCRtargeted drug approved for clinical use (De Clercq 2019). In contrast, only a few anti-tumor antagonists have been developed for CXCR7, and no antagonists yet exist for CXCR5 or CXCR6 due to the lack of protein structural information, despite numerous studies implicating these receptors in tumorigenic processes (Table 3). It is noteworthy that CXCL16 has both transmembrane and secreted forms with

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opposing functions in tumor progression (Gutwein et al. 2009; Lang et al. 2017). CXCL16 may even act in an “inverse signaling” mechanism, where sCXCL16 directly interacts with TM-CXCL16 to induce downstream signaling without involving CXCR6 activity (Hattermann et al. 2016). Therefore, caution is required when considering the clinical use of antagonists targeting the CXCR6–CXCL16 axis, as this may lead to unpredictable outcomes among tumor types. Similarly, CXCR3A and CXCR3B have both pro- and anti-tumor effects, respectively, so application of the nonselective. CXCR3 antagonist AMG487 may suppress or promote tumor growth depending on context (Puchert et al. 2019). Further investigation may be required when using AMG487 to treat tumors expressing both CXCR3A and CXCR3B. CXCR–CXCL signaling affects a variety of immune cells, leading to a strong rationale to develop translational treatment combining the CXCR targeting with radio-, chemo-, or immunotherapy (Table 4). Some CXCR antagonists have been shown to improve sensitivity of tumor cells to radiotherapy or chemotherapy drugs and reduce tumor recurrence compared with single agent administration (Ning et al. 2012; Reeves et al. 2017; Zhou et al. 2018a; Lecavalier-Barsoum et al. 2018; Fahham et al. 2012; Beider et al. 2013; Bockorny et al. 2020). CXCR antagonist together with immunotherapy also provides novel anticancer strategies by interfering and reshaping the tumor microenvironment. Combination of CXCR2 or CXCR4 antagonist with PD-1–PD-L1 blockade has been shown to promote anti-tumor immunity in various cancer types (Steele et al. 2016; Jiang et al. 2019b; Seo et al. 2019; Zeng et al. 2019), mainly by enhancing T-cell infiltration and removing or inhibiting immunosuppressive cells such as Treg cells and MDSC infiltration, creating an anti-tumor microenvironment. To perform such combination therapies, it is important to understand the function of CXCR–CXCL signaling in immune cells and the effects of CXCR blockade on the immune response to tumor cells. Subtle associations have been discovered among CXCRs. For example, CXCR3 was reported to elevate surface expression of CXCR4 on colorectal cancer cells and enhance CXCR4 activity by forming a heterodimer, thereby promoting cancer invasion (Jin et al. 2018). A similar interaction was reported between CXCR7 and CXCR4 in colorectal cancer progression, with coupling to CXCR7 enhancing the pro-tumor activity of CXCR4 (Song et al. 2019). This implies that application of a CXCR3 antagonist, CXCR7 antagonists, dual antagonist targeting CXCR3 and CXCR4, or a dual antagonist targeting CXCR7 and CXCR4 may be a more effective way to suppress tumorigenesis than a CXCR4 antagonist alone. In addition, CXCR7 is subject to CXCR4 regulation (Wang et al. 2008), so the CXCR4 antagonist AMD3100 also acts on CXCR7 as an allosteric agonist (Kalatskaya et al. 2009) but with less efficiency for suppressing tumor migration (Luo et al. 2018). Furthermore, CXCL12 not only inhibits the expression of CXCR4 by negative feedback but also inhibits the expression of CXCR5 (Chan et al. 2003). While this helps keep the internal environment stable, it also affects the clinical efficacy of anti-tumor antagonists. In the development of CXCR antagonist-based therapies, it is also important to consider that autocrine and paracrine signaling mechanisms in the tumor

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Table 4 Combination of CXC receptor antagonist and other drugs for anti-tumor treatment

Targets CXCR1/ CXCR2

Antagonist SCH527123

Combinatory drugs SCH-479833 Oxaliplatin Pembrolizumab

Reparixin

CXCR4

AZD5069 AMD3100

Phase 2

Anti-PD-1 antibody Paclitaxel

PDAC

Both

Ovarian cancer

In vitro

Radiotherapy

TNBC, cervical cancer

Both

Anti-PD-1 antibody Anti-PD-1 antibody

PDAC

In vitro

Ovarian cancer, osteosarcoma

Both

Metastatic pancreatic cancer (NCT04177810) Hand and neck cancer (NCT04058145) Relapsed multiple myeloma (NCT00903968) NSCLC

Phase 2

(Jiang et al. 2019b; Zeng et al. 2019) –

Phase 2

–

Phase 2

–

Both

(Fahham et al. 2012)

NHL

Both

Pancreatic cancer (NCT02826486)

Phase 2

(Beider et al. 2013) (Bockorny et al. 2020)

Paclitaxel

Cemiplimab

Pembrolizumab

Bortezomib

BKT140

Both

Ref. (Varney et al. 2011) (Ning et al. 2012) –

NSCLC, CRPC, and colorectal cancer (NCT03473925) Metastatic breast cancer (NCT02370238) Gastric cancer

5-fluorouracil CXCR2

Targeted cancer types Liver metastasis of colon cancer Colon cancer

In vitro/ in vivo, or clinical studies In vitro

Radiotherapy, cisplatin, or paclitaxel Rituximab Pembrolizumab

Phase 2

Both

(Wang et al. 2016; Schott et al. 2017) (Wang et al. 2016) (Steele et al. 2016) (Reeves et al. 2017) (Zhou et al. 2018a; LecavalierBarsoum et al. 2018) (Seo et al. 2019)

(continued)

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Table 4 (continued)

Targets

CXCR7

Antagonist AMD070

Combinatory drugs Nivolumab

Targeted cancer types Renal cell carcinoma (NCT02923531)

Pembrolizumab

Advanced melanoma (NCT02823405) Metastatic breast cancer (NCT01837095) Relapsed glioblastoma multiforme (NCT02765165) Prostate cancer

POL6326

Eribulin

USL-311

Lomustine

CCX771

Enzalutamide

In vitro/ in vivo, or clinical studies Phase 2

Ref. –

Phase 1

–

Phase 1

–

Phase 2

–

Both

(Luo et al. 2018)

microenvironment may give rise to divergent outcomes (Wendel et al. 2008; Chen et al. 2018). Moreover, some CXCR antagonists have strong side effects, such as cardiotoxicity, so development of safer alternatives is required (Gimenez-Arnau et al. 2007). Development of antagonists with milder toxicity and both greater efficiency and receptor specificity is essential. In conclusion, the effects of CXCR signaling on tumor progression warrant continued study to define novel therapeutic targets, including anti-tumor CXCR antagonists. Acknowledgments This study was supported by National Key Research and Development Program (No.2018YFA0902702) and National Science and Technology Major Project (No.2018ZX10731301-003), National Natural Science Foundation of China (No.81570202), Guangdong Medical Research Fund (No.A2019396), basic and applied basic research project of Guangdong Province (No.2019A1515110495), and Research Start-up Foundation for Lingnan Scholar of Foshan University (directed by Fang Liu). Conflict of Interest The authors declare no conflicts of interest regarding this report.

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Rev Physiol Biochem Pharmacol (2020) 178: 41–82 https://doi.org/10.1007/112_2020_39 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2020 Published online: 14 August 2020

Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal Dystrophies Darpan Malhotra and Joseph R. Casey

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 The Cornea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Descemet’s Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Roles of Corneal Endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Supplying Corneal Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Maintenance of Corneal Transparency by the Endothelial Pump . . . . . . . . . . . . . . . . . . . . . 3 Corneal Dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Endothelial Corneal Dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Fuchs Endothelial Corneal Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Congenital Hereditary Endothelial Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Harboyan Syndrome (HS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Endothelial Corneal Dystrophies Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Molecular Mechanisms of Endothelial Corneal Dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Sex and Environmental Influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 RNA Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Mitochondrial Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Epithelial-Mesenchymal Transition (EMT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Cell Adhesion Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 ER Stress and the Unfolded Protein Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D. Malhotra Department of Biochemistry, University of Alberta, Edmonton, AB, Canada Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada J. R. Casey (*) Department of Biochemistry, University of Alberta, Edmonton, AB, Canada Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada Department of Physiology, University of Alberta, Edmonton, AB, Canada Department of Ophthalmology and Visual Science, University of Alberta, Edmonton, AB, Canada e-mail: [email protected]

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5.8 Water Pump Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9 Altered Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 70 70 71

Abstract The cornea, the eye’s outermost layer, protects the eye from the environment. The cornea’s innermost layer is an endothelium separating the stromal layer from the aqueous humor. A central role of the endothelium is to maintain stromal hydration state. Defects in maintaining this hydration can impair corneal clarity and thus visual acuity. Two endothelial corneal dystrophies, Fuchs Endothelial Corneal Dystrophy (FECD) and Congenital Hereditary Endothelial Dystrophy (CHED), are blinding corneal diseases with varied clinical presentation in patients across different age demographics. Recessive CHED with an early onset (typically age: 0–3 years) and dominantly inherited FECD with a late onset (age: 40–50 years) have similar phenotypes, although caused by defects in several different genes. A range of molecular mechanisms have been proposed to explain FECD and CHED pathology given the involvement of multiple causative genes. This critical review provides insight into the proposed molecular mechanisms underlying FECD and CHED pathology along with common pathways that may explain the link between the defective gene products and provide a new perspective to view these genetic blinding diseases. Keywords Cell adhesion · CHED · COL8A2 · Corneal dystrophy · Corneal endothelium · Descemet’s membrane · FECD · Molecular mechanism · SLC4A11 · TCF4

1 Introduction 1.1

The Cornea

The cornea, the outer portion of the eye, is convex, transparent, avascular, with two principal functions: (1) primary structural, protective barrier for the rest of the eye and (2) providing a significant fraction of the eye’s focusing power. The cornea is the most densely innervated tissue of the body and most of its nerves are sensory (Labetoulle et al. 2019). Human cornea consists of five layers: three cellular layers: epithelium, stroma and endothelium, and two acellular connective tissue structures: Bowman’s layer and Descemet’s membrane (DM) (Fig. 1). This review centers on two specific posterior corneal dystrophies (PCDs), caused by defects in the endothelium. We will thus only discuss the endothelium and adjacent regions contributing to PCDs.

Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal. . .

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Choroid Vitreous chamber Vitreous humor Sclera Retina Conjunctiva Optic Nerve Posterior chamber Anterior chamber Aqueous Humor

A

Iris Lens Cornea Ciliary body

B Epithelium Bowman’s Layer

Stroma

Keratocytes

Descemet’s Membrane

20 µm

Endothelium

Fig. 1 Structure of the human eye and the cornea. (a) Schematic representation of the human eye with outside on the right. The outermost layer consists of cornea and the sclera, which is covered by a transparent mucous layer of conjunctiva. The middle layer consists of the lens (that receives the light and focuses it to the retina in the inner layer), ciliary body, iris, and the choroid. The inner layer consists of the retina, the sensory region that receives the light and transmits the signal to the brain through the optic nerve. (b) Stained cross-section of the cornea’s five layers, courtesy of Dr. Mathieu Thériault, University of Laval. Epithelium is the outermost layer of the cornea. Going toward the interior is the Bowman’s layer, the stroma, DM, and the endothelium

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Stroma

Stroma, the central corneal layer which is approximately 450-μm thick in humans, comprises about 90% of the total corneal volume (Meek and Knupp 2015). Major components of stroma are collagen fibers, composed of collagen I and V, which form heterodimers with a narrow diameter. These fibers are surrounded by proteoglycans (chondroitin sulfate and keratan sulfate), which maintain corneal hydration (see section “Maintenance of corneal transparency by the endothelial pump”). These collagen fibers precisely arrange into parallel bundles called fibrils that further pack as parallel layers (lamellae) to sustain stromal architecture and transparency. Stroma is comprised of 200–250 lamellae arranged at right angle to the fibers in adjacent lamellae, resulting in reduced light scattering and corneal transparency (Meek and Boote 2004; Torricelli and Wilson 2014). Keratocytes, the resident cells of the stroma, secrete collagens and glycosamines and are located throughout stroma. Stromal avascularity critically contributes to corneal transparency. Moreover, corneal architecture and its hydration state are essential to maintain its refractive index and clarity.

1.3

Descemet’s Membrane

DM is a basement membrane, composed of extracellular matrix (ECM) proteins secreted by the corneal endothelium on which endothelial cells adhere. DM’s collagenous and non-collagenous proteins have a more complex organization than other basement membranes in the body. DM, first described by Descemet in 1758 (Johnson et al. 1982), is secreted throughout life by the endothelium. DM has an anterior banded layer and a posterior non-banded layer. In the anterior banded layer, collagen fibrils have a lattice-like organization with continuous banding pattern at 110 nm intervals. Unlike other basement membranes in which collagen IV is common, collagen VIII is the specific major structural component of DM. Other DM proteins include collagen I, collagen IV, collagen XII, collagen XVIII, fibronectin (FNC), laminins, osteonectin, and versican (Schlotzer-Schrehardt et al. 2011). While FNC and vitronectin concentrate on the stromal side of DM, other proteins are prominent facing the endothelium. Two collagen VIII proteins in DM, collagen VIII alpha chain 1 (COL8A1) and collagen VIII alpha chain 2 (COL8A2), form trimers of two COL8A1 and one COL8A2 that further arrange into hexagonal lattices in the DM (Kapoor et al. 1986). DM provides a structural support for the endothelium (Eghrari et al. 2015).

Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal. . .

1.4

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Endothelium

Corneal endothelium, a single flat monolayer of highly specialized cells [corneal endothelial cells (CEC)], adheres to DM (Fig. 1). CEC, which are 5 μm deep and 20 μm in diameter, arrange in hexagonal lattices forming a honeycomb-like mosaic. Humans have a significant reserve of CEC at birth, about 5,000–7,000 cells/mm2 (Fig. 2). CEC density declines rapidly, by 45% in the first year and then it continues to decrease until the mid-twenties (Wilson and Roper-Hall 1982; Sherrard et al. 1987). CEC density declines more gradually between 20 and 80 years of age, at a constant rate of 0.5% per year (Tuft and Coster 1990; Sanchis-Gimeno et al. 2005). The endothelium maintains corneal hydration state, which is critical for clear vision. CEC have ion-transport systems that form an “endothelial pump,” continuously moving fluid from stroma to aqueous humor to maintain stroma hydration state at 78% (% water in stroma by weight) (Fig. 3). Homeostatic control of this corneal deturgescence is essential to maintain precise collagen fibril organization, essential for clear vision (Willoughby et al. 2010). Since CEC do not proliferate (Joyce 2012), cell damage leads to irreplaceable CEC loss. CEC do, however, enlarge (polymegathism), change shape (pleomorphism), and migrate to fill the space left by cell death to maintain an intact Healthy FECD CHED

CEC density/mm2

6000 5000 4000 3000 2000

Symptomatic Threshold

1000 0

0

10

20

30

40

50

60

70

80

90 100

Age (years) Fig. 2 CEC density declines with age in healthy individuals, FECD and CHED patients. CEC density profiles from several studies were analyzed to present cell density with age in healthy individuals, FECD and CHED patients (Wilson and Roper-Hall 1982; Sherrard et al. 1987; Syed et al. 2017; McLaren et al. 2014; Iovino et al. 2018; Sultana et al. 2007; Kenyon and Antine 1971; Abdellah et al. 2019; Arici et al. 2014; Duman et al. 2016; Ewete et al. 2016; Angmo et al. 2018; Mittal et al. 2011). CEC density of approximately 6,000 cells per mm2 at birth declines rapidly in the first few years reaching a steady rate in adulthood. Decline in CEC density below a critical threshold (symptomatic threshold) (Syed et al. 2017; McLaren et al. 2014; Iovino et al. 2018) may compromise the endothelial monolayer integrity which further compromises the endothelial pump, resulting in corneal edema. FECD patients see a faster decline in CEC density approaching the critical threshold faster than healthy individuals. CHED patients are born with 5–10 times lower CEC density than healthy individuals, leaving them close to the symptomatic threshold

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Epithelium Bowman’s Layer

Collagen Lamellae

Stroma

Keratocytes Osmotic Leak

Descemet’s Membrane

Endothelial Pump

Endothelium

Aqueous Humor

Stroma

H2O, + H+/OH- H

Na+ 2HCO3- Na+

NBCe1

SLC4A11

NHE1

H+

Basolateral H+ Lactate

MCT1

Na+

Kir2.1

NKCC1

K+

3Na+

Na-K ATPase 2K+

HCO3AE2 Cl-

TJ

Cl- or HCO3-

CACC1

Aqueous Humor

CFTR

H 2O

AQP1

H+

Lactate

Endothelial Pump

Osmotic Leak

H+

K+ 2Cl-

TJ

MCT4

Apical

Fig. 3 The Corneal Endothelial Pump maintains corneal fluid balance and transparency. Upper panel presents cornea structure with five distinct layers. Stroma features collagen lamellae organized in a distinct fashion to prevent light scattering, and keratocytes with vital roles in stromal biology. The osmotic leak represents the endothelial leaky barrier, which allows nutrient diffusion to the stroma. Fluid osmotically leaks into the stroma and is pumped back to the aqueous humor by the endothelial pump as shown. The lower panel is a zoomed view of the CEC with basolateral side facing the stroma and the apical side facing the aqueous humor. Proteins that contribute to the endothelial pump include basolateral components NBCe1, NHE1, SLC4A11, MCT1, NKCC1, AE2, Kr2.1 and Na+-K+ ATPase and apical proteins CACC1, CFTR, AQP1, and MCT4. TJ represents tight junctions. HCO3
transport by basolateral NBCe1 and AE2 and lactate transport by MCT1 into the cell drives fluid secretion. Apical CACC1 and CFTR move HCO3
and MCT4 transports lactate across the apical surface to drive fluid movement into the aqueous humor. Basolateral SLC4A11 and apical AQP1 form a water permeation pathway from stroma to aqueous humor. Adapted from (Bonanno 2012; Vilas et al. 2013)

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endothelial monolayer. With constant loss in number, increased polymegathism and pleomorphism arise in CEC, accompanied by reduced ability of CEC to deturgesce the cornea. Decline in CEC count below 500–700 cells/mm2 disturbs the endothelial monolayer integrity (Fig. 2), compromises the endothelial pump, and causes corneal edema (DelMonte and Kim 2011). The focus of this review is on endothelial corneal dystrophies (ECDs) that arise from compromised endothelial physiology.

2 Roles of Corneal Endothelium The cornea forms an optically clear protective layer by precise organization of collagen stromal fibrils to ensure light transmission to the retina. While stroma is the unique structural component of the cornea, the endothelium crucially maintains healthy corneal physiology.

2.1

Supplying Corneal Nutrition

Corneal avascularity is required for corneal transparency, but lack of blood supply presents a challenge to provide nutrients to stromal keratocytes and corneal epithelial cells. The endothelium, therefore, provides a pathway to provide nutrients to these cells from the aqueous humor. Corneal epithelium receives oxygen, calcium, magnesium, potassium, and sodium chloride through the tears (Bourne 2003). In contrast, other nutrients, including glucose, are virtually absent in tear fluid. The adjacent limbal vasculature provides about 20% of the glucose demand to the cornea, yet ablation of this source does not disrupt corneal physiology (Sweeney et al. 1998). Aqueous humor is the main source of glucose and other essential nutrients. This supply is achieved by the “leaky” nature of the endothelium. Corneal endothelium acts as a partially leaky barrier to allow diffusion of these solutes from the aqueous humor into the stroma through the tight junctions (Fig. 3) (Bonanno 2012). Blocking this supply route induces stromal and epithelial degeneration (Bourne 2003).

2.2

Maintenance of Corneal Transparency by the Endothelial Pump

Stromal hydration is maintained by several factors, including endothelial tight junctions, endothelial pump function, and the ability of CEC to accommodate for the continual cell loss by altering their shape and size. CEC are polarized cells with their basolateral surface facing DM and apical surface toward aqueous humor.

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Movement of material between CEC (paracellular pathway) is limited by the connections between cells (Barry et al. 1995; Petroll et al. 1999). The endothelial “pump” is a two-step process to move excess fluid from stroma to aqueous humor against its osmotic gradient (Fig. 3), which requires substantial energy expenditure (Bonanno 2012). Solutes are moved against their electrochemical gradients to promote the exit of water along a local osmotic gradient. CEC proteins include SLC4A11, Na+/K+-ATPase, Na+/H+ exchanger 1 (NHE1), Cl
/ HCO3
anion exchanger 2 (SLC4A2 or AE2), electrogenic Na+/HCO3
cotransporter (SLC4A4 or NBCe1), Na+:K+:2Cl
cotransporter (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR), calcium-activated chloride channels (CaCC1), monocarboxylate transporters 1 and 4 (MCT1 and 4), and aquaporin 1 (AQP1) (Vilas et al. 2013; Jalimarada et al. 2013; Bonanno 2003). Models have been proposed to explain the endothelial pump mechanism (Bonanno 2003, 2012; Jalimarada et al. 2013). Movement of ions across the basolateral and apical surface of CEC may generate localized osmotic gradients, sometimes driven by negative membrane potential, which leads to the movement of fluid from the stroma back to aqueous humor (Bonanno 2003, 2012; Fischbarg 2012; Schey et al. 2014). SLC4A11, at the basolateral surface of CEC (facing stroma), facilitates the direct movement of water into the cells, which could be further moved into the aqueous humor by apical AQP1 (facing aqueous humor) (Vilas et al. 2013). The combined leaky barrier function and the fluid pump, referred to as the pump-leak mechanism, is essential to maintain healthy corneal physiology and transparency. Failures of CEC pumping mechanism give rise to stromal edema and visual haziness by distorting the stromal collagen fibril array (Meek and Boote 2004).

3 Corneal Dystrophies Corneal dystrophies are classified by their anatomical location in the layer where the defect lies: (1) superficial or anterior corneal dystrophies caused by defects in the epithelium, basal lamina or the Bowman’s layer, (2) stromal corneal dystrophies caused by the defects in the corneal stroma, and (3) posterior or endothelial corneal dystrophies caused by defects in the DM or the endothelium. Most follow a Mendelian pattern of inheritance [(autosomal dominant (AD), autosomal recessive (AR), or X-linked (XLD)] and have a variable clinical presentation with different ages of onset (Vincent 2014). This review focuses on diseases arising from defects in the DM or the endothelium and hence the ECD. More particularly, this review centers on two ECD: Congenital Hereditary Endothelial Dystrophy (OMIM #217700) and Fuchs Endothelial Corneal Dystrophy (OMIM #136800). Patients present with corneal edema, variable corneal opacity, and compromised visual acuity (Klintworth 2009). In some CHED patients (see “Harboyan syndrome” below) hearing deficits are present and hearing loss occurs at a high frequency in FECD patients than the general population (Stehouwer et al. 2011). Symptoms beyond cornea and hearing have not been reported in corneal dystrophy patients.

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4 Endothelial Corneal Dystrophies ECDs are characterized by defects in the corneal endothelium or the DM secreted by the endothelium. Disruptions of endothelial monolayer compromise the endothelial pump, resulting in excessive fluid in the stroma, causing corneal edema and a decline in visual acuity. The three prominent diseases discussed in this review are Fuchs Endothelial Corneal Dystrophy, Congenital Hereditary Endothelial Dystrophy, and Harboyan Syndrome (HS) (Table 1).

4.1

Fuchs Endothelial Corneal Dystrophy

FECD arises due to a complex combination of genetic factors with two different ages of onset in patients. Early onset happens around the second to third decade of life and is well characterized, but rare. Late onset occurs around the fourth to fifth decade and accounts for the majority of patients, with heterogeneous genetics and physiological etiology (Biswas et al. 2001) (Table 1). FECD, which arises due to defects in the corneal endothelium, has a regionally varied prevalence of 4–9% in individuals over 40 years of age (Soh et al. 2020) and is one of the leading causes for corneal transplants globally (Feizi 2018). FECD arises from AD inheritance of several Table 1 Genes established to cause or implicated in CHED and FECD Endothelial corneal dystrophy CHED/FECD

Gene SLC4A11

Gene function H2O/H+, NH3 transport, cell adhesion

CHED/FECD

MPDZ

FECD

SLC4A11

Protein–protein interactions stabilization of intercellular connections H2O/H+, NH3 transport, cell adhesion

FECD

COL8A2

FECD

COL17A1

FECD

LAMC1

FECD FECD FECD FECD

ATPB1 LOXHD1 TCF4 ZEB1 (TCF8) AGBL1 DMPK

FECD FECD

Collagen-Descemet’s membrane component Collagen-Descemet’s membrane component Laminin-Descemet’s membrane component Na+/K+-ATPase subunit Protein trafficking Transcription factor Transcription factor Glutamate decarboxylase Dystrophia myotonia protein kinase

Possible disease mechanisms Water pump/mitochondria ROS/cell adhesion Water pump Water pump/mitochondria ROS/cell adhesion Cell adhesion/ER stress Cell adhesion/ER stress Cell adhesion/ER stress Water pump/ER stress ER stress RNA toxicity RNA toxicity RNA toxicity RNA toxicity

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different genes (see below). Age of onset and degree of penetrance may be influenced by environmental factors and the specific disease allele inherited and is characterized by a progressive decline in CEC density, CEC polymegathism, and pleomorphism in patients (Feizi 2018; EBAA 2017) (Fig. 4). In FECD patients, CEC density declines until the endothelial pump fails (symptomatic threshold), which compromises corneal deturgescence (Fig. 2). Stromal fluid accumulation increases corneal thickness and cloudiness, which compromises visual clarity. Patients also have bead-like deposits of ECM on DM, called guttae. Non-confluent guttae on the peripheral DM are normal with progressing age (Hassall–Henle warts), but FECD patients have guttae in the central cornea that enlarge to grow confluent as the disease progresses (Eghrari and Gottsch 2010) (Fig. 4). FECD typically progresses in four stages: Stage I is asymptomatic but non-confluent guttae are present in the central cornea. In stage II, guttae become confluent with loss of CEC, polymegathism, and pleomorphism. In stage III, the endothelial pump is compromised, and corneal edema ensues. By stage IV, corneal edema severity causes corneal scarring, diminishing visual acuity (Feizi 2018). With the compromised endothelial pump, stromal fluid accumulation produces a bluegray stromal haze. Eventually, this fluid accumulation increases corneal thickness and gives the cornea a ground glass-like appearance. Ultimately, excess fluid may accumulate between epithelial cells and sub-epithelium. These bubbles burst through the epithelium causing painful corneal erosions, bullous keratopathy (BK). Symptoms of epithelial edema subsequently reduce but visual acuity continues to decline as the epithelium secretes connective tissue to repair itself (Klintworth 2009). FECD begins in the central cornea and radially progresses to peripheral cornea with extensive DM wrinkling. In advanced FECD, abnormalities arise in all corneal layers, but consistent changes are observed in DM and the endothelium. CEC are progressively lost over the guttae. Abnormal CEC are significantly larger in size, have widened intercellular spaces, swollen mitochondria, and dilated rough endoplasmic reticulum (ER). Some CEC also develop fibroblast-like morphology. DM thickness increases to about fourfold due to excessive ECM deposition. Size and confluence of guttae also increase in the central DM where they may protrude into the anterior chamber (Elhalis et al. 2010).

4.2

Congenital Hereditary Endothelial Dystrophy

CHED is a rare ECD with symptoms present in infancy or within the first few years of life, although sometimes symptoms are not reported until age 5–10 (Brejchova et al. 2019). CHED has an AR mode of inheritance and is thus most prevalent in countries with high incidence of consanguineous marriages (Vithana et al. 2006). CHED is characterized by corneal opacification in infancy with twofold to threefold increase in corneal thickness. Patient corneas have a ground glass-like appearance and present with extensive stromal edema. Patients are born with significantly decreased CEC density, with a fibrotic morphology. CHED patients also have

Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal. . .

A

B

C

D

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Fig. 4 Clinical presentation of CEC loss in FECD and CHED patients. Representative images of specular microscopy examination of ECD patient eyes reveal the change in CEC shape, density and appearance of DM. (a) Healthy corneal endothelium with hexagonal shaped CEC arranged tightly in monolayer. (b, c) Loss of CEC is visible along with polymegathism, pleomorphism, and exposed underlying DM (black). (d) Significant CEC loss in advanced stages of FECD exposes the underlying DM (black), which disrupts corneal endothelium integrity and the endothelial pump. (Procured and modified with consent from Konan Medical, CA, USA)

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thickened DM, but guttae are not usually evident. DM has a normal anterior banded layer and a posterior non-banded layer, but with an additional 20–40 nm thick fibrous layer at the posterior surface consisting of a mixture of ECM-like fibrous material. CHED is not a progressive disease with symptoms that remain stable throughout the patient’s life (Klintworth 2009).

4.3

Harboyan Syndrome (HS)

HS or Corneal Dystrophy and Perceptive Deafness (CDPD) is a sensorineural hearing disorder associated with some cases of CHED, although HS is also seen as a manifestation of CHED. Infants with HS are born with a compromised visual acuity, but they develop progressive hearing deficits usually around 10–15 years of age (Desir and Abramowicz 2008). A single pedigree revealed that CHED patients eventually develop HS, although their hearing is not routinely examined, which may be due to lack of information and infrastructure in developing countries (Siddiqui et al. 2014). In some cases, parents of CHED patients are affected by FECD and are carriers of disease alleles, indicating that homozygous inheritance of FECD alleles causes CHED, but a single allele may suffice to cause FECD (Kim et al. 2015). The presence of all three diseases in the same pedigree suggests changes in clinical practices to monitor for HS in all CHED patients and for FECD in their parents (Siddiqui et al. 2014). The lack of reports of symptoms beyond vision and hearing may reflect the prevalence of CHED/HS patients in developing countries where parents may not report non-corneal symptoms due to lack of information/infrastructure. More thorough investigations in patients are needed to detect possible disease manifestation beyond vision and hearing.

4.4

Endothelial Corneal Dystrophies Genes

Amongst ECD, FECD has an AD mode of transmission, although sporadic cases are more prevalent. Multiple genetic and environmental factors underlie the FECD pathology. The genetic basis of FECD is heterogeneous with incomplete penetrance and variable expressivity. Mutations in FECD (Vedana et al. 2016; Weiss et al. 2015) have been found in genes (Table 1) including: COL8A2 (Biswas et al. 2001), SLC4A11 (Vithana et al. 2008), TCF4 (Baratz et al. 2010), ZEB1 (formerly called TCF8) (Riazuddin et al. 2010), AGBL1 (Riazuddin et al. 2013a), LOXHD1 (Riazuddin et al. 2012), and MPDZ (Moazzeni et al. 2019). Genome-wide association studies (GWAS) implicated KANK4, LAMC1, and ATPB1 in the pathogenesis of FECD (Afshari et al. 2017). Thus far, CHED has been found to arise only from AR inheritance of SLC4A11 (Vithana et al. 2006) and possibly MPDZ (Moazzeni

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et al. 2019) mutations. How can the pathology of FECD and CHED be rationalized by the involvement of this set of genes? COL8A2 COL8A2 codes for collagen VIIIA2 protein which is the principal component of DM and secreted by CEC. It associates with collagen VIIIA1 to form heterotrimers organized into highly ordered three-dimensional arrays in DM (Greenhill et al. 2000). Two COL8A2 mutations, p.Leu450Trp (Gottsch et al. 2005) and p.Gln455Lys (Biswas et al. 2001), cause abnormal intracellular accumulation and distort DM arrangement. Knock-in mouse models of both Col8a2 mutations mimic the early onset FECD phenotype in mice (Meng et al. 2013; Jun et al. 2012). COL8A2 mutations also cause a type of posterior polymorphous corneal dystrophy (PPCD) (OMIM #609140 (Biswas et al. 2001). Since this finding has not been confirmed, PPCD has similarities to FECD, and members of the index family had FECD, this finding may represent a special presentation of FECD rather than PPCD (Biswas et al. 2001). SLC4A11 SLC4A11 is a large (891 amino acids, Accession no. NP_114423.1) multi-domain integral membrane protein encoded by SLC4A11 gene. Identified in 2001 as BTR1 or Bicarbonate Transporter 1, SLC4A11 belongs to the SLC4 bicarbonate transporter gene family (Parker et al. 2001). SLC4A11 is expressed in most body tissues with high expression in cornea, cerebral cortex, epididymis, gallbladder, small intestine, testes, kidneys, breast, cerebellum, lungs, ovaries, pancreas, and stomach (THPA 2019). SLC4A11 localizes at the basolateral surface of CEC where its extracellular region faces the DM (Vilas et al. 2013; Jalimarada et al. 2013). It is one of the most abundantly expressed proteins of the corneal endothelium (Frausto et al. 2014; Chng et al. 2013). Three N-terminal variants of human SLC4A11 have been reported in the genome database, due to differential splicing of the mRNA: SLC4A11 Variant 1 (v1) which encodes for a 918 amino acid protein (NCBI Reference Sequence: NP_001167561.1), SLC4A11 Variant 2 (v2) which encodes for an 891 amino acid splice form 2 (NP_114423), and SLC4A11 Variant 3 (v3) which encodes for an 875 amino acid splice form 3 (NP_001167560). These variants have also been respectively called SLC4A11A, -B, and -C (Kao et al. 2015). These variants differ in their N-termini by 20–60 amino acids. SLC4A11 v2 was the first variant to be cloned in 2001 when the protein was discovered (Parker et al. 2001) and is the most widely used variant in in vitro studies. In human cornea SLC4A11 v1 is not expressed, whereas v2 is highly expressed relative to v3 (Malhotra et al. 2019a; Hara et al. 2019). Multiple transport roles have been attributed to SLC4A11. The plant ortholog of SLC4A11, BOR1, transports borate, and an initial functional assessment indicated that the human protein also transports borate (Park et al. 2004; Takano et al. 2002). Borate cross-links vicinal diol components of cell wall and is critical to their structural integrity in bacteria, plants, and fungi (O'Neill et al. 2004). Mammals do not have cell wall and insignificant to no borate in their blood. Hence, the borate transport function of SLC4A11 is unclear from a physiological standpoint. Later studies found that indeed SLC4A11 is not a borate transporter (Vilas et al. 2013;

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Jalimarada et al. 2013). SLC4A11 is a member of the SLC4 family, whose other members are all bicarbonate transporters, yet SLC4A11 does not transport bicarbonate (Vilas et al. 2013; Jalimarada et al. 2013). Human SLC4A11 v2 was reported to function as an amiloride-sensitive Na+-OH
(H+) transporter (Ogando et al. 2013). Evidence is converging to indicate that both mouse and human SLC4A11 act as amiloride-insensitive Na+-independent electrogenic H+ (OH
) permeation pathways (Myers et al. 2016; Kao et al. 2020). Moreover, SLC4A11 H+ transport function is regulated by pH (Quade et al. 2020). SLC4A11 also facilitates NH3 transport either alone (Loganathan et al. 2016) or by cotransport with H+ (Kao et al. 2020; Zhang et al. 2015). SLC4A11 also facilitates movement of water (Vilas et al. 2013). In addition to its membrane transport function, SLC4A11 was recently found to serve as an attachment site between CEC and DM (Fig. 5) (Malhotra et al. 2019b).

Basolateral

Integrins

SLC4A11

Descemet’s Membrane

COL8A1

COL8A2

COL1

COLIV

Vitronectin

Tenasin

Fibronectins

Laminins

Tight Junction

Fig. 5 Endothelial cell adhesion to Descemet’s membrane. CEC cell–cell and cell-DM attachment maintains the endothelial monolayer and efficient endothelial pump. SLC4A11 and integrins promote CEC-DM attachment and tight junctions maintain cell–cell interactions and endothelial polarity

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Until recently SLC4A11 was the only gene reported as responsible for CHED (Vithana et al. 2006). Now MPDZ mutations (see below) have been suggested as responsible for one case of CHED (Moazzeni et al. 2019). While SLC4A11 mutations cause CHED when inherited recessively, it appears that carrier parents may also develop FECD (Kim et al. 2015; Chaurasia et al. 2020). TCF4 TCF4 or transcription factor 4 encodes the transcription factor 4 that belongs to the E-protein family of class I basic helix-loop-helix (bHLH) transcription factors (Baratz et al. 2010). A CTG (cytosine–thymine–guanine) trinucleotide repeat expansion in the third intron of TCF4 causes the majority of FECD (Baratz et al. 2010). ZEB1 Zinc-finger E-box binding Homeobox 1 protein (ZEB1) is a transcription factor, also known as TCF8 (transcription factor 8). Five late-onset FECD-causing mutations have been identified in TCF8 (Riazuddin et al. 2010). Interestingly, a different set of ZEB1 mutations cause a disease similar to FECD, AD posterior polymorphous corneal dystrophy (OMIM #609141), which has been attributed to ZEB1 haploinsufficiency (Krafchak et al. 2005). AGBL1 AGBL1 mutations have been reported to cause some cases of FECD (Riazuddin et al. 2013a; Zhang et al. 2019). ATP/GTP binding-protein like 1 (AGBL1) is a metallo-carboxypeptidase that mediates removal of glutamyl groups from polyglutamylated proteins. The most notable biological effect of protein glutamylation is to destabilize tubulin (Wloga et al. 2017). The link between tubulin and mechanisms of ECDs is unclear. The role of AGBL1 mutations in FECD should be viewed cautiously as only three cases have been identified. Two of these mutations are predicted to have low pathogenicity, yet one is protein truncation mutant. LOXHD1 LOXHD1 gene (Lipoxygenase Homology Domain-containing 1 protein) is a rare FECD gene whose mutations also cause hearing loss (Riazuddin et al. 2012). This cytosolic protein, which has received too little attention, is composed of 15 PLAT (polycystin/lipoxygenase/α-toxin) domains, which likely are Ca++ binding sites (Grillet et al. 2009). PLAT domains have a role in plasma membrane targeting of client proteins via protein–protein interactions (Grillet et al. 2009). Five LOXHD1 mutations were recently identified to also cause autosomal recessive non-syndromic hearing loss (Bai et al. 2020). KANK4 KANK4 gene encodes KN motif and ankyrin repeat domain containing protein 4 or KANK4 which is a cytoplasmic protein involved in actin polymerization and control of cytoskeletal formation (Kakinuma et al. 2009). A genome wide association study (GWAS) identified KANK4 as an FECD locus, but whether it represents a modifier gene or a disease locus is not yet established (Afshari et al. 2017). LAMC1 LAMC1 encodes Laminin gamma-1 protein (LAMC1), an ECM protein of DM. GWAS identified LAMC1 as an FECD locus, but no point mutations have yet been identified (Afshari et al. 2017).

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ATPB1 ATPB1 encodes the beta subunit of the Na+/K+ ATPase. GWAS identified ATBP1 as a locus in FECD, however, its correlation to the disease pathophysiology remains unclear (Afshari et al. 2017). Since the Na+/K+ ATPase is central to the maintenance of the membrane potential and the Na+ and K+ gradients across the plasma membrane of mammalian cells, ATPB1 defects could readily be understood to affect the water pump function of the endothelium. COL17A1 COL17A1 encodes collagen 17A1. Homozygous p.Pro1185Leu mutation has been reported in one FECD patient (Zhang et al. 2019). MPDZ Multi-PDZ domain protein (MPDZ, also known as MUPP1) was identified as homozygously mutated in one CHED patient who lacked any SLC4A11 defect (Moazzeni et al. 2019). One parent of the child with CHED carried an MPDZ mutation and had FECD, suggesting that MPDZ mutations cause CHED when homozygous and FECD when heterozygous (Moazzeni et al. 2019). MPDZ is a cytosolic scaffold protein containing 13 PDZ protein–protein interaction domains that interact with proteins, including those of the tight junction (Feldner et al. 2017). Expressed in the choroid plexus, some MPDZ mutations cause autosomal recessive non-syndromic hydrocephalus (HYC2, OMIM #615219) (Feldner et al. 2017). The mpdz
/
mouse has increased paracellular permeability arising from defective intercellular junctions in the choroid plexus (Yang et al. 2019).

5 Molecular Mechanisms of Endothelial Corneal Dystrophies Complex pathophysiology and genetics challenge our understanding of molecular mechanisms underlying ECD. In healthy corneal physiology, endothelial monolayer integrity, its association with the DM (Fig. 5), and pumping function (Fig. 3) are essential to prevent ECD. Factors that compromise the endothelial pump can explain symptoms of corneal edema. Healthy individuals experience declining CEC density at an average rate of 0.5% per year (Wilson and Roper-Hall 1982). When cell density falls below 1,000–1,200 cells/mm2, corneal edema (symptomatic threshold) may develop (Fig. 2) (Syed et al. 2017; McLaren et al. 2014; Iovino et al. 2018). Molecular mechanisms that increase the rate of CEC loss will thus accelerate the development of ECD. In addition, defects in genes integral to the endothelial fluid pump may also promote ECD development. This section highlights the underlying molecular mechanisms that disrupt the endothelial integrity and pump function to explain ECD symptoms.

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Sex and Environmental Influences

Women have a threefold increased risk of FECD for reasons that are unclear (Musch et al. 2011). Amongst the other factors, smoking has been linked to increased guttae in patients. Smoking in female FECD patients increases the risk of progressing to advanced stages, perhaps due to increased oxidative stress (see below) (Zhang et al. 2013). Diabetes in FECD patients also increases central corneal thickness, which is independent of FECD. FECD patients have a fourfold increase in myocardial infarction, angina pectoris, and cardiac insufficiency compared to control individuals, but the underlying reason remains unexplained (Vedana et al. 2016).

5.2

RNA Toxicity

In FECD, about 70% of the disease burden rests on mutations of the transcription factor, TCF4 (Vasanth et al. 2015). The third intron of TCF4 contains a repeated DNA base sequence of CTG that is prone to expansion. When the size of the CTG array expands to beyond 40, odds of developing FECD rise significantly (Mootha et al. 2015; Soh et al. 2019) and FECD severity correlates with the CTG repeat expansion size in FECD patients (Soliman et al. 2015). The proposed disease mechanism was first established for Myotonic dystrophy type 1 (DM1). CUG-expanded RNA transcripts bind and sequester the RNA splicing regulator, MBNL1 (Mankodi et al. 2001). In the case of FECD and TCF4, this sequestration leads to microscopically detectable foci of MBNL1 protein with CUG-expanded TCF4 transcript (Du et al. 2015) (Fig. 6). Altered splicing of MBNL1 client transcripts arises from insufficiency of MBNL1, which has been detected in corneas from FECD patients (Du et al. 2015). Thus, CTG repeat expansion ultimately may cause disease from defective splicing and expression of critical genes in the endothelium (Fig. 6). Additional complexity arises with the observation that MBNL2 protein, related to MBNL1, is also sequestered by CUG-expanded TCF4 transcripts (Zarouchlioti et al. 2018). Over-expression of MBNL1, however, fails to rescue mice with a repeat expansion in their DMPK1 gene and mRNA splicing remains defective (Yadava et al. 2019). This observation suggests that sequestration of MBNL1 does not fully explain the basis for DM1 and by extension for TCF4 in FECD; other factors may be required in combination with MBNL1 to rescue the splicing defects. The authors emphasize that MBNL1, binding over 2,400 target RNAs, has important roles in RNA metabolism (RNA translation, localization, mRNA decay) and binds spliced RNAs, indicating functions beyond being a splice effector (Yadava et al. 2019). An additional explanation is provided by the observation that splicing factor, MBNL2, is sequestered by CUG-expanded transcripts (Zarouchlioti et al. 2018), so some toxicity likely arises from MBNL2 sequestration in addition to MBNL1. Taken together, MBNL1 has complex interactions with RNAs that may require other

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Basolateral

Protein-mRNA aggregates MBNL1/2

CUG CU G C

UG

Fig. 6 TCF4 RNA toxicity. Trinucleotide CUG repeats in the third intron of TCF4 mRNA sequester MBNL1/2, an RNA splicing regulator, which causes accumulation of microscopically detectable foci of mRNA-protein complexes in the CEC nuclei. Loss of MBNL1/2 function due to sequestration could affect the transcriptional splicing of client transcripts. Inability to clear MBNL1/ 2-mRNA complexes causes RNA toxicity that may cause cell death. Halted transcriptional maturation of MBNL1 client transcripts could irreversibly trigger pathways that can additionally contribute to CEC death. This disturbs the endothelial pump and a healthy endothelial physiology leading to FECD onset

factors and binding to CUG-expanded transcripts adversely affects these roles. In addition, inability to clear toxic MBNL-RNA complexes may trigger diseasecausing pathways that cannot be reversed by MBNL overexpression. DM1 arises from CTG repeat expansion in the 30 -untranslated region of the dystrophia myotonia protein kinase gene (DMPK) (Mootha et al. 2017). As is the case with TCF4, the DMPK transcript associates with MBNL1, giving rise to mis-splicing of some MBNL1 target genes. Interestingly, FECD symptoms are tenfold more prevalent in DM1 patients than in the general population (Mootha et al. 2017). Thus, (1) DMPK defects can cause FECD-like corneal symptoms

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(Winkler et al. 2018) and (2) sequestration of MBNL1 leading to aberrant expression of its client genes is the molecular mechanism underlying both TCF4 and DMPK mutations in FECD. FECD mutations in AGBL1 include one nonsense mutation, p.Arg1028X and one missense mutation p.Cys990Ser (Riazuddin et al. 2013b), but their roles in FECD pathology are unclear. AGBL1 binds TCF4 and both identified mutations affect AGBL1-TCF4 interaction. Thus, alterations of the AGBL1-TCF4 complex may contribute to FECD through the same pathway as TCF4 (Riazuddin et al. 2013b). Whereas wild-type AGBL1 localizes to the cytoplasm, truncated p.Arg1028X protein traffics to the nucleus (Riazuddin et al. 2013b). Similarly, ZEB1 (TCF8) and TCF4 modulate each other’s expression (SanchezTillo et al. 2015) and are hypothesized to be a part of the same pathway that causes FECD. A defective step in the transcription cascade containing TCF4, TCF8, and AGBL1 may lead to accumulation of toxic RNA-protein complexes to explain CEC loss in FECD corneas.

5.3

Oxidative Stress

The late onset of FECD suggests disease mechanisms requiring prolonged low-level insults. Indeed, increased rates of CEC loss will lead to FECD. Because of the cornea’s high metabolic activity and exposure to the aerobic atmosphere and ultraviolet light, CEC are especially vulnerable to oxidative damage. Contribution of oxidative mechanisms to CEC health and development of FECD have recently been well summarized (Jurkunas 2018; Vallabh et al. 2017). Oxidative stress in FECD patients is underscored by increased levels of damaged mitochondrial DNA and dysregulation of antioxidant protective pathways (Jurkunas et al. 2010). SLC4A11 is implicated in resistance to oxidative stress. SLC4A11 mutations leave cells more vulnerable to oxidative insults (Roy et al. 2015). Cells with SLC4A11 mutations that cause SLC4A11 protein ER-retention had elevated reactive oxygen species (ROS) levels and impaired mitochondrial function. Antioxidant signalling pathway triggered by nuclear factor erythroid-2 related factor 2 (Nrf2) was impaired by SLC4A11 depletion (Guha et al. 2017). Nrf2 is tightly regulated by KEAP1 (Kelch-like ECH associated protein 1) under physiological conditions where Nrf2 is held in the cytoplasm and constantly degraded by KEAP1-mediated ubiquitination (Kensler et al. 2007; Sun et al. 2007). Under oxidative stress, protein DJ-1 (also known as PARK7 or Parkinsonism-associated deglycase) prevents NRF2-KEAP1 interaction and regulates its nuclear translocation where it activates the antioxidant defense genes (Liu et al. 2014; Bitar et al. 2012). NRF2 (Jurkunas et al. 2010), DJ-1 (Bitar et al. 2012), and SLC4A11 (Gottsch et al. 2003) are downregulated in FECD. TCF4, in osteoblasts, is suggested to be involved in antioxidant defense (Yang et al. 2013) and absence of TCF4 triggers apoptosis (Forrest et al. 2013), suggesting potential transcript regulation of DJ-1 and NRF2 by TCF4 to regulate healthy CEC physiology. Loss of TCF4 expression in most

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cases of FECD can lead to loss of antioxidant defense pathways, which may increase ROS in cells leading to apoptosis. Oxidative stress provides a compelling explanation for cell death, which will contribute to loss of CEC density leading to FECD. Nrf2 is also downregulated in CHED (Guha et al. 2017), which suggests direct or indirect feedback regulation of antioxidative pathway by SLC4A11 as CHED is only caused by SLC4A11 mutations. That said, none of the identified FECD or CHED genes (above) have a clear role in oxidative mechanisms. This suggests that factors causing oxidative stress will contribute importantly to these diseases, but most likely by an environmental, or background factor or as secondary effects resulting from other mechanisms described that alter CEC gene expression.

5.4

Mitochondrial Function

Potential mitochondrial roles in FECD progression have recently been reviewed (Miyai 2018). The CEC water pump function is highly energy intensive, making these cells among the body’s most energy-demanding (Chng et al. 2013). High energy metabolism inevitably causes higher levels of damaging free radical species as a fraction of electrons leak from the respiratory electron transport chain (Miyai 2018). Free radicals can damage nuclear and mitochondrial DNA. Cells may respond to mitochondrial DNA damage by activating the programmed cell death pathway (Miyai 2018), which may underpin some CEC loss observed in ECD. Evidence for mitochondrial dysfunction in FECD patient corneas is, however, somewhat indirect. Menadione, which generates free radicals in cells, thus mimicking the effects of electron leakage in mitochondrial function, induced mitochondrial DNA damage and cytochrome C release (an apoptotic marker) in immortalized CEC models (Halilovic et al. 2016). Cultured, immortalized CEC, derived from FECD patients had lower levels of mitochondria and reduced mitochondrial fitness (as measured by mitochondrial membrane potential) than cells derived from control subjects (Benischke et al. 2017). This would be consistent with oxidative damage culminating in reduced mitochondrial fitness. Potentially then, CEC from FECD patients are poised to undergo apoptosis upon additional oxidative damage. Consistent with this, a comparison of CEC from control and FECD patients revealed increased activation of mitophagy pathway in FECD (Miyai et al. 2019). An additional role for mitochondria in corneal dystrophies centers on SLC4A11 (Ogando et al. 2019). Corneal endothelial cells from slc4a11
/
mice display elevations of superoxide, mitochondrial depolarization, and higher rates of apoptosis than controls (Ogando et al. 2019). This could explain the increased rate of cell loss in patients with SLC4A11 mutations. In this model (Fig. 7), SLC4A11 functions in both plasma membrane and inner mitochondrial membrane as a H+ channel, moving H+ from the extracellular medium into the mitochondrial matrix, following the plasma membrane and mitochondrial membrane potentials (Ogando et al. 2019). SLC4A11-mediated H+ flux in this manner would degrade the mitochondrial membrane potential and thus decrease ATP production by F0-F1-ATPase. In the context

Molecular Mechanisms of Fuchs and Congenital Hereditary Endothelial Corneal. . .

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+

Basolateral

H

+

Mitochondrion

Δψm

H

+

ROS

NH3 NH3

Gln Glu

α-KG

TCA

Fig. 7 Compromised mitochondrial function. CEC use glutaminolysis to produce much of their ATP: Glutamine (Gln) is deaminated to glutamic acid (Glu), which in turn is deaminated to alphaketoglutarate (α-KG), which feeds into the tricarboxylic acid cycle (TCA). Ammonia (NH3) stimulates reactive oxygen species (ROS) production by some component of the mitochondrial electron transport chain (MET), which can induce apoptotic cell death. SLC4A11 (green square) has been proposed to localize to the mitochondrial inner membrane, where it may mediate H+ influx that would slightly depolarize the mitochondrial membrane potential (ΔΨm), resulting in decreased ROS production (Ogando et al. 2019). SLC4A11 could also normally contribute to NH3 efflux from the inner mitochondrial membrane, or plasma membrane. SLC4A11 function may thus reduce ROS production either by reducing matrix NH3 levels or by slightly reducing ΔΨm. ROS levels thus may rise upon loss of SLC4A11 function, leading to apoptotic cell death

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of CEC, however, the heavy use of glutaminolysis for energy production (Zhang et al. 2017) gives rise to mitochondrial hyperpolarization and high NH3 production. NH3 stimulates ROS production by some component of the mitochondrial electron transport chain (Murthy et al. 2001). SLC4A11 is proposed to serve a protective function by reducing ROS production (Ogando et al. 2019). This could occur either by reducing the mitochondrial membrane potential by SLC4A11-mediated transport of H+ into the mitochondrial matrix or by transport of NH3 out of the matrix. Indeed, ROS production is reduced in glutamine-exposed cells expressing SLC4A11 in comparison to cells lacking SLC4A11 (Ogando et al. 2019). Further, supporting a link of SLC4A11 to mitochondria, HEK293 cells expressing WT SLC4A11 were less prone to accumulate ROS and to undergo apoptosis than those expressing corneal dystrophy mutants of SLC4A11 (Roy et al. 2015). Finally, in immortalized cultured human CEC, knockdown of SLC4A11 expression led to increased apoptotic cell death (Liu et al. 2012). Taken together, loss of SLC4A11 function in FECD patients increases their CEC ROS load, which may lead to an increased rate of CEC loss via cell death. The data supporting the role of SLC4A11 in protecting against glutaminolysisassociated cell death are compelling (Ogando et al. 2019), but some caution needs to be exercised in details of the mitochondrial mechanism. Although evidence has been presented for an inner mitochondrial membrane localization for SLC4A11, none of the protein’s splicing variants contain an N-terminal mitochondrial targeting sequence (MTS) recognized by an online analysis (Fukasawa et al. 2015). Further, full sequence analysis (Kumar et al. 2018) does not predict a mitochondrial localization for SLC4A11. Lacking a recognizable N-terminal MTS, SLC4A11 would need to target to the inner mitochondrial membrane via the mitochondrial carrier family pathway, which has been observed for carrier proteins (SLC25) with six transmembrane segments (Ogunbona and Claypool 2019). SLC4A11 with 14 transmembrane segments (Badior et al. 2017) would be extraordinarily hydrophobic to be able to reach the inner membrane. Finally, SLC4A11 does not appear in the list of 1,098 proteins present in mitochondria (Pagliarini et al. 2008), from tissues including kidney where SLC4A11 is expressed (Groeger et al. 2010). Targeting of SLC4A11 to mitochondria may be signaled by high glutamine metabolism or by a CEC-specific factor, but the reported localization of the protein to IMM will require additional study. In addition, a role of SLC4A11 in preventing glutaminolysis-associated cell death could occur through mechanisms that do not require IMM localization. The crux of the glutaminolysis-cell death model is that mitochondrial matrix NH3 promotes ROS production. Removal of NH3 from the matrix would thus reduce ROS and reduce cell death. SLC4A11 acts as either an NH3 or NH3/H+ transporter (Loganathan et al. 2016; Zhang et al. 2015; Kao et al. 2019). Both the mitochondrial membrane potential and H+ gradient (matrix is more alkaline than cytosol) disfavor SLC4A11 transport of NH3/H+ out of the matrix. Similarly, SLC4A11 could not transport NH3/H+ out of the cell as the plasma membrane potential is negative. SLC4A11 does localize to the CEC plasma membrane, where it could facilitate NH3 efflux from the cell. In so doing, it would reduce the cytosolic NH3 concentration,

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which would increase the magnitude of the NH3 concentration gradient from the mitochondrial matrix to the cytosol. Thus, observations of reduced glutaminolysisassociated cell death could also be explained by SLC4A11 plasma membrane NH3 transport function, in addition to the proposed role of SLC4A11 in H+ movement into the mitochondrial matrix (Ogando et al. 2019).

5.5

Epithelial-Mesenchymal Transition (EMT)

Cell polarity and tight cell–cell contacts are essential for proper functioning of the corneal endothelium. These epithelial cell features are lost when cells undergo EMT (Lavin and Tiwari 2020). Analysis of RNA from corneas affected by FECD revealed enrichment of genes promoting EMT (Cui et al. 2018). Further, ZEB1 promotes EMT possibly through its role in downregulating expression of E-cadherin (Iliff et al. 2012). Finally, TCF4 promotes EMT and MBNL1 splicing is important in EMT (Du et al. 2015; Iliff et al. 2012), suggesting that some part of TCF4 pathology arises through promotion of EMT.

5.6

Cell Adhesion Defects

Cell attachment to a basement membrane is a key feature of epithelia, like the corneal endothelium. Cell adhesion sites connect the inside of the cell to the extracellular environment and transduce signals to maintain a healthy physiology (Gumbiner 1996). Cell adhesion complexes include: CAM or receptors, ECM proteins, and the cytoplasmic/ancillary/peripheral membrane proteins. CAMs, which link cytosolic proteins and ECM, are integral membrane proteins in five families: integrins, immunoglobulin cell adhesion molecules (IgCAMs), selectins, cadherins, and proteoglycans (Murray et al. 1999). ECM proteins that constitute the basement membrane to which the cells adhere are usually secreted by these cells themselves. ECM proteins include collagens, laminins, fibronectins (FNCs), and proteoglycans that assemble into macromolecular arrays to create a defined basement membrane arrangement (Khalili and Ahmad 2015). Genes encoding ECM proteins are upregulated in corneas from FECD patients (Du et al. 2015). ECM proteins interact with their plasma membrane receptors to hold the cells tightly in place (Fig. 5). Cytosolic/peripheral membrane proteins connect cytosolic CAM regions to the cytoskeleton to transduce signals to the cytoplasm. Loss of epithelial cell anchorage from ECM triggers programmed cell death called anoikis, a physiological defense mechanism in multi-cellular organisms that prevents growth of detached cells on distal matrices (Gilmore 2005). Thus, cell death observed in corneal dystrophies could arise from defects in cell adhesion. Attachment of CEC to DM is critical to maintain the endothelial barrier and thus corneal transparency. Integrins in the corneal endothelium contribute to CEC-DM

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attachment (Stepp 2006; Carter 2009; Goyer et al. 2018). Integrin αV (ITGAV), Integrin β3 (ITGB3), and integrin β5 (ITGB5), the corneal endothelial integrins, are heterodimers of αVβ3 and αVβ5. They interact with collagens, FNC, and laminins in DM by binding to their Arg-Gly-Asp (RGD) motifs (Stepp 2006). α4β1 and α6β1, discovered recently in CEC, interact with common basement membrane proteins through their large extracellular domains (Goyer et al. 2018; TCGA 2019; Hall et al. 1990; Mould et al. 1994). ECD patients show progressive CEC loss suggesting that defective cell adhesion could be one of the potential underlying causes of the disease (Fig. 8). In FECD, DM thickens and guttae formed from extracellular matrix secreted by CEC. This could be interpreted as an adaptive response to close gaps left by detached CEC to maintain endothelial integrity or that cells sense diminished adhesion to DM and attempt to compensate by secretion of DM components to provide a matrix for cell attachment. Indeed, CEC from FECD patients secrete higher levels of extracellular matrix proteins, including collagens (Xia et al. 2016) and fibronectin (Goyer et al. 2018). A prominent FECD gene is COL8A2 (Biswas et al. 2001), encoding a key component of DM. Normally, COL8A2 forms triple helices, which FECD point mutations are predicted to compromise (Moschos et al. 2019). Unfortunately, since COL8A2 is a large, post-translationally modified, helical, secreted protein, the effects of mutations have not been fully assessed. Col8a2L450W/L450W and col8a2Q455K/Q455K mice, however, exhibit CEC loss due to decrease in DM stiffness as a consequence of mis-organization of collagen fibrils (Leonard et al. 2019). Mis-organization of COL8A2 protein and biomechanical changes in DM could compromise CEC attachment, with consequences for cell survival, although there is no experimental evidence yet to support the idea. Interestingly, a second DM component, laminin γ1 chain (LAMC1), was identified in a GWAS as a FECD gene (Afshari et al. 2017). Specific LAMC1 mutations have not yet been reported, making assessment of the role of LAMC1 in corneal dystrophies somewhat speculative. A role of LAMC1 in cell adhesion has, however, been studied in the context of tumor cell adhesion to basement membranes (Sun et al. 2018). Interestingly, LAMC1 has been connected as an extracellular component of the PI3K/Akt signaling pathway (Sun et al. 2018), in which KANK4 acts as an intracellular effector to regulate actin filament polymerization (Kakinuma et al. 2009). KANK4 also was identified in a GWAS as an FECD gene (Afshari et al. 2017), thus implicating the cell adhesion-cytoskeletal axis as a potential disease mechanism in FECD. Both LAMC1 and KANK4 are associated with tumor phenotypes, promoting cell growth, but in the same vein defects in these proteins could adversely affect CEC growth leading to the increased rates of cell death found in FECD. Recent identification of COL17A1 mutation in FECD (Zhang et al. 2019) is interesting and points toward a cell adhesion defect. As a collagen, COL17A1 likely contributes to DM. The reported site of mutation is located on the COL17A1 surface (Zhang et al. 2019), where the defect is more likely to affect protein–protein contacts than protein folding. Other COL17A1 mutations cause epithelial recurrent erosion

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Loss of Cell Adhesion

Healthy Endothelium

Adhesion Defect

DM Oversecretion

Folding Defect

Diseased Endothelium

Adhesion Mutants

Folding Mutants

COL8A2

COL8A1

Gutatta

Fig. 8 Defective SLC4A11-mediated cell adhesion in FECD and CHED. SLC4A11 contributes to CEC adhesion to DM components (Malhotra et al. 2019b). Mutations in SLC4A11 that affect its cell adhesion role (adhesion mutants) or protein folding that cause ER-retention (folding mutants) contribute to the compromised CEC-DM attachment. CEC, in a failed attempt to retain attachment to DM, may oversecrete DM components which leads to increases in DM thickness and continuous deposition of DM components can lead to guttae on the posterior surface of DM. Compromised SLC4A11 adhesion role, increase in DM thickness and guttae can eventually contribute to loss of CEC in diseased corneas. The severity of the effect of SLC4A11 mutations may contribute to increased rate of cell loss (as in CHED) or delayed CEC detachment along with other physiological factors (as in FECD) that affect CEC-DM physiology

dystrophy (MIM #122400), which could be explained by an epithelial cell adhesion defect. Recently, a role of SLC4A11 in CEC adhesion to DM has been described (Malhotra et al. 2019b). SLC4A11 is among the CEC’s most abundant proteins (Frausto et al. 2014; Chng et al. 2013) and localizes to the basolateral surface of CEC, facing DM (Vilas et al. 2013). Further, SLC4A1 (Band 3, AE1), a member of

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the SLC4 family with SLC4A11, is an abundant erythrocyte membrane protein with a key role in attachment to cytoskeletal proteins in addition to its transport role (Cordat and Casey 2009), suggesting that SLC4 proteins may have membrane adhesion roles apart from transport. Through its large third extracellular loop (EL3), SLC4A11 promotes adhesion of cultured HEK293 cells and human CEC to culture dishes coated with DM extracts (Malhotra et al. 2019b). Transfection of HEK293 cells with SLC4A11 or αV and β3 integrins promoted adhesion to DM to the same degree. Further, an antibody against SLC4A11 EL3 blocked about 25% of cell adhesion to DM by primary cultures of human CEC (Malhotra et al. 2019b). Together, this suggests a significant role of SLC4A11 in maintaining CEC-DM attachment (Fig. 8). SLC4A11 potentially interacts with DM components, COL8A2 and COL8A1. Four FECD-causing mutations in SLC4A11-EL3 compromised the proteins’ ability to bind DM components, without affecting SLC4A11 cell surface trafficking or ability to facilitate a water flux (Malhotra et al. 2019b). Increased rates of cell loss found in FECD patients with SLC4A11 mutations may thus be explained by a reduced strength of CEC-DM adhesion, leading to cell death through anoikis. The majority of CHED-causing SLC4A11 mutations compromise cell surface abundance of SLC4A11 due to its misfolding and subsequent ER-retention (Alka and Casey 2018). CHED-causing SLC4A11 mutations also compromised SLC4A11mediated adhesion to DM (Malhotra et al. 2019b). Reduced abundance or absence of SLC4A11 at CEC surface may compromise adhesion, explaining onset of CHED during infancy. Transcriptional profiling of neural crest cells (NCC), the embryonic cells that migrate into the cleft between surface ectoderm and lens vesicle to form the endothelium, shows SLC4A11 expression (Lumb et al. 2017). CHED may be a developmental disorder due to defective NCC adhesion during migration absence of SLC4A11. Indeed, CHED patients are born with a tenfold lower CEC density compared to healthy infants further consistent with prenatal defects (Sultana et al. 2007; Kenyon and Antine 1971). This strongly suggests cell adhesion as a defective mechanism in FECD and CHED that can explain CEC loss in patients.

5.7

ER Stress and the Unfolded Protein Response

A consequence of mutations can be defective folding of the corresponding mutated protein. Proteins synthesized in association with the endoplasmic reticulum (ER), integral membrane proteins, and secretory proteins, may be retained in the ER upon misfolding. Prolonged ER retention provides time for misfolded proteins to achieve a native structure. Ultimately, if ER-associated proteins fail to fold, they can accumulate or be targeted for degradation. Accumulation of misfolded proteins in ER can induce the unfolded protein response (UPR) in cells, which can lead to apoptosis (Almanza et al. 2019). Through this mechanism, mutations of corneal dystrophy genes could induce CEC loss. Indeed, increased levels of apoptotic cell death are a feature of FECD endothelium (Borderie et al. 2000) and markers of UPR are elevated in FECD patient corneas (Engler et al. 2010). Thus, corneal dystrophy

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gene products that pass through the endoplasmic reticulum (membrane proteins or secretory proteins) are candidates for causing disease through UPR/apoptosis. These include: LAMC1, ATPB1, SLC4A11, and COL8A2. ATPB1 and LAMC1 were identified in a GWAS screen as FECD loci (Afshari et al. 2017) but point mutants have not been found in the genes. So, it is impossible to assess their potential pathogenic mechanism of their mutations. DM component, COL8A2, is secreted by CEC and its mutations cause some FECD cases (Riazuddin et al. 2009). COL8A2 point mutations have been proposed to cause the protein to accumulate in ER prior to secretion, leading to UPR-associated cell death (Moschos et al. 2019). Most significantly, a transgenic knock-in mouse of the col8a2 p.Gln455Lys FECD allele revealed ER-swelling and increased apoptotic markers (Jun et al. 2012). This provides strong evidence for UPR-induced apoptosis as the disease mechanism in the case of one COL8A2 FECD mutation. LOXHD1 does not have any predicted transmembrane segments and the wildtype protein is cytosolic (Riazuddin et al. 2012), meaning that LOXHD1 does not pass through the ER. Fifteen missense mutations have been identified in LOXHD1, localizing at the protein surface, suggesting a role in protein–protein interactions. Analysis of the explanted cornea of an individual with an p.Arg547Cys mutation revealed increased levels of LOXHD1 expression and evidence of stained protein aggregates (Riazuddin et al. 2012). This would be consistent with protein misfolding leading to LOXHD1 accumulation. Since LOXHD1 is a polyvalent protein binder, with 15 PLAT domains, mutant LOXHD1 could potentially cause aberrant clustering of its client membrane proteins, potentially causing them to accumulate in the ER, leading to UPR. LOXHD1 is associated with late-onset FECD, suggesting that the effect of LOXHD1 results in a slight increase in cell death and late-onset disease. Extensive studies of SLC4A11 point mutants revealed folding defects in 20% of FECD-causing mutants and 61% of CHED-causing mutants (Alka and Casey 2018). SLC4A11 would thus appear to be a candidate to cause disease through the UPR/apoptotic route. The case for increased cell death arising from UPR is not, however, strong. Indeed, knock-down of SLC4A11 expression in human corneal endothelial cells increases apoptotic cell death (Liu et al. 2012), suggesting that SLC4A11 prevents cell death. Expression of misfolded, intracellular-retained SLC4A11 mutants was associated with increased apoptotic cell death, but via increased sensitivity to oxidative stress (Roy et al. 2015). Arguing against increased cell death associated with expression of SLC4A11 mutants, no increase of apoptotic or necrotic markers was observed in cells expressing misfolded SLC4A11 in a cell culture model (Loganathan and Casey 2014). Recent data suggesting that SLC4A11 localizes to the mitochondrial inner membrane (Ogando et al. 2019) lead to the possibility that misfolded SLC4A11 induces cell death via the mitochondrial precursor overaccumulation stress pathway (Coyne and Chen 2019). The observation that slc4a11
/
mice manifest corneal dystrophy symptoms (Vilas et al. 2013; Groeger et al. 2010; Lopez et al. 2009), however, strongly suggests that corneal dystrophy arises from loss of SLC4A11 function rather than a toxic gain of function, as would be the case if SLC4A11 mutants induce UPR/apoptosis.

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5.8

Water Pump Defects

The principal symptom for corneal dystrophy patients is corneal haziness, secondary to corneal edema. Disease mechanisms increasing rates of CEC loss will impair the endothelial water pump, as a result of increased water leakage from impaired continuity of the endothelial layer and because fewer cells will be available to pump fluid out of the stroma. Pleomorphism and polymegathism also reduce the ability of the endothelium to deturgesce the cornea through endothelial pump (DelMonte and Kim 2011; Feizi 2018). In addition, defects in the molecular components of the corneal fluid pump may explain corneal dystrophy in some patients. The endothelial water pump mechanism has been well elaborated (Bonanno 2012) (Fig. 3). While the components of the pump are generally agreed upon, how they work together remains uncertain. The corneal edema observed upon treatment with the Na+/K+-ATPase inhibitor, ouabain, argues persuasively for a central role of this protein (Fig. 9) (Geroski et al. 1984). It also argues that FECD associated with ATPB1 defects are affected by the loss of the protein’s function. While AQP1, found

Stromal Edema Fluid accumulation

Distorted lamellae arrangement

H2O H+

NBCe1

NHE1

SLC4A11

MCT1

Kir2.1

NKCC1

Osmotic Leak

Na-K ATPase 2K+

TJ

TJ

H 2O

CACC1

CFTR

AQP1

Endothelial Pump

3Na+

AE2

MCT4

Aqueous Humor

Fig. 9 Endothelial pump defects in FECD and CHED. An array of proteins contributes to corneal endothelial pump (Fig. 3), which help in maintaining corneal transparency. Defective fluid transport due to SLC4A11 mutations affects the movement of excessive fluid from stroma to aqueous humor via AQP1. Accumulation of fluid in stroma presents as edema while it also distorts the lamellar organization which further compromises visual clarity in FECD and CHED. ATP1B1, a subunit of sodium–potassium ATPase which also contributes to endothelial pump, has also been linked to FECD in GWAS. However, no mutations have been reported that affect the transport role of the protein in FECD or CHED

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in apical CEC surface, is the only AQP protein of CEC (Vilas et al. 2013; Hamann et al. 1998), its role in maintenance of stromal deturgescence appears dispensable as aqp1
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mice have thinned corneal stroma (Thiagarajah and Verkman 2002). Complicating this interpretation, these mice also manifest slowed recovery from corneal swelling induced by hypoosmotic challenge at the corneal epithelial surface (Thiagarajah and Verkman 2002), which suggests a role of AQP1 in recovery from edema. The strongest argument that AQP1 is not essential for water pump function is the lack of corneal phenotype in patients lacking AQP1 in spite of their profound urinary concentrating defect (King et al. 2001). Amongst the corneal dystrophy genes, only two stand out as potentially involved in the endothelial pump: SLC4A11 and ATP1B1. ATP1B1 encodes the beta subunit of the Na+/K+-ATPase. As discussed above, a critical role of Na+/K+-ATPase in maintenance of stromal fluid balance is established, so defects in the protein could readily explain corneal symptoms. Thus far, however, ATP1B1 has only been identified as an FECD gene in a GWAS, which identified a single nucleotide polymorphism (SNPEffect 2019), rs1200114, in an intergenic region near ATP1B1 (Afshari et al. 2017). Since the other gene flanking the SNP is not expressed in cornea, the linkage implies involvement of ATP1B1. No mutations have yet been identified in the ATP1B1 gene of corneal dystrophy patients. Given the critical role of Na+/K+-ATPase in maintaining plasma membrane potential, and Na+, and K+ gradients, mutations profoundly altering the protein’s function would likely manifest in many tissues beyond cornea. SLC4A11 contributes to maintenance of stromal deturgescence by two mechanisms: (1) It provides a water flux pathway from stroma back into endothelial cells and (2) SLC4A11’s H+ transport function has been proposed to have a critical role in the endothelial water pump by countering the alkalinizing action of HCO3
accumulation (Myers et al. 2016). The only characterized mutation that compromises the water flux function without affecting cell surface abundance is p.Arg125His (Vilas et al. 2013). Other SLC4A11 mutations that affect protein trafficking will compromise cell surface abundance of SLC4A11, which in turn will compromise the water flux function in plasma membrane (Loganathan and Casey 2014). Thirty out of 55 missense mutations compromise cell surface trafficking of the protein (Alka and Casey 2018; Loganathan and Casey 2014; Chiu et al. 2015). SLC4A11 defects can compromise the endothelial pump, resulting in corneal edema. The accumulated fluid distorts the arrangement of stromal collagen lamellae resulting in increased scattering of light and compromised visual acuity. Indeed, slc4a11
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mice develop corneal edema and increased central corneal thickness, resembling the clinical presentations of ECD in patients (Vilas et al. 2013; Han et al. 2013). Recent recognition that MPDZ mutations may be responsible for CHED when homozygous and FECD when heterozygous (Moazzeni et al. 2019) suggests an additional component of the endothelial water pump that may cause FECD and CHED. MPDZ mutations cause hydrocephalus (Feldner et al. 2017). In turn hydrocephalus arises from over-accumulation of cerebrospinal fluid, whose production is controlled by the choroid plexus, an endothelium with some similarities to the corneal endothelium. Careful analysis of mpdz-null mice revealed loss of integrity

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of the connections between the cells of the choroid plexus (Yang et al. 2019), giving rise to fluid leak between these cells. If a similar phenomenon happens in the corneal endothelium, then increased leakage of fluid from aqueous humor into stroma would counter the normal pumping action of the corneal endothelial cells. In spite of effective corneal endothelial water pump, stromal edema would ensue because the pump could not overcome the rate of fluid leakage through this paracellular pathway.

5.9

Altered Gene Expression

Products of some ECD-causing genes modulate the expression of other ECD genes. ZEB1 is a transcriptional activator and repressor of several genes of Wnt and TCF4/β-catenin pathway (Aigner et al. 2007; Sanchez-Tillo et al. 2011). ZEB1 binds to the SLC4A11 promoter at its transcription start site to modulate its expression (Zhang and Aldave 2018). TCF4 interacts with ZEB1, converting ZEB1 from a transcriptional repressor to an activator (Sanchez-Tillo et al. 2015). The association between TCF4 and ZEB1 suggests that TCF4 may regulate SLC4A11 expression. ZEB1 acts as a transcription suppressor in the absence of TCF4, suggesting that SLC4A11 may be downregulated under reduced TCF4 levels. Interestingly, SAGE analysis from FECD corneas (which were probably defective in TCF4, since 70% of FECD arises from TCF4 mutations) revealed downregulated SLC4A11 as a common FECD feature (Gottsch et al. 2003). Other CAMs, including NRXN1 (Forrest et al. 2012), CNTNAP2 (Forrest et al. 2012), and integrins (Plantefaber and Hynes 1989) are also regulated by TCF4. TCF4 also binds to non-coding regulatory regions of COL8A2, KANK4, ATP1B1, and LAMC1, suggesting downstream transcriptional regulation (Zhang and Aldave 2018). Reduced ATP1B1 RNA levels are found in FECD corneas (Jalimarada et al. 2013). ECD-causing ZEB1 mutation also leads to downregulation of COL8A2 (Lechner et al. 2013), which further suggests transcriptional regulation of other ECD genes by TCF4 and ZEB1. Together, these data suggest that aberrant gene expression caused by TCF4 and TCF8 deficits could underlay some pathophysiology of corneal dystrophies.

6 Conclusions Endothelial corneal dystrophies are complex, arising from a combination of environmental and genetic factors. All are marked by a decreased density of CEC, giving rise to defective cornea stromal deturgescence, which in turn disrupts collagen fibril packing and greatly impairs vision. Since CEC cannot proliferate, our CEC density can only decline through life. Factors that increase the rate of cell loss will hasten the onset of ECD, marked by the stage when the endothelium can no longer maintain the required level of stromal dehydration. Thus, environmental factors increasing cell

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death, including oxidative stress, interplay with genetic predispositions, culminating in either early onset CHED or requiring decades to ensue in the case of FECD. Endothelial dystrophy genes point toward molecular mechanisms of disease that include RNA toxicity associated with deficiencies in the MBNL1 RNA processing pathway, oxidative stress, mitochondrial dysfunction, the unfolded protein response/ apoptosis associated with misfolded proteins, ammonia toxicity, cell adhesion defects leading to anoikis and defects in the endothelial water pump. As outlined in the review, some genes may cause CHED or FECD through more than one of these mechanisms. Acknowledgments We thank the many researchers who have contributed to this rich field and apologize to those whose work we were unable to cite. We thank Dr. Mathieu Thériault, University of Laval, for providing images used in this manuscript. Canadian Institutes of Health Research supports research in the JRC laboratory.

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Rev Physiol Biochem Pharmacol (2020) 178: 83–110 https://doi.org/10.1007/112_2020_42 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2020 Published online: 14 August 2020

Promising Anti-atherosclerotic Effect of Berberine: Evidence from In Vitro, In Vivo, and Clinical Studies Alireza Fatahian, Saeed Mohammadian Haftcheshmeh, Sara Azhdari, Helaleh Kaboli Farshchi, Banafsheh Nikfar, and Amir Abbas Momtazi-Borojeni

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Berberine Lowers Atherogenic Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 In Vivo Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Clinical Evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Mechanisms Underlying the Cholesterol-Lowering Effect of Berberine . . . . . . . . . . . . . 3 Berberine’s Effects on Atherosclerosis Lesion Progression: In Vitro Evidence . . . . . . . . . . . . 3.1 Berberine Improves Endothelial Cell Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Berberine Modulates Atherogenic Activities of Macrophages . . . . . . . . . . . . . . . . . . . . . . . .

A. Fatahian Department of Cardiology, Cardiovascular Research Center, Mazandaran University of Medical Sciences, Sari, Iran S. M. Haftcheshmeh Department of Medical Immunology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran S. Azhdari Department of Anatomy and Embryology, School of Medicine, Bam University of Medical Sciences, Bam, Iran H. K. Farshchi Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran B. Nikfar (*) Pars Advanced and Minimally Invasive Medical Manners Research Center, Pars Hospital, Iran University of Medical Sciences, Tehran, Iran e-mail: [email protected] A. A. Momtazi-Borojeni (*) Halal research center of IRI, FDA, Tehran, Iran Department of Medical Biotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran e-mail: [email protected]

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3.3 Berberine Blocks Proliferation and Migration of Vascular Smooth Muscle Cells . . 97 4 Berberine’s Effects on Atherosclerosis Lesion Progression: In Vivo Evidence . . . . . . . . . . . 101 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Abstract Elevated levels of plasma cholesterol, impaired vascular wall, and presence of inflammatory macrophages are important atherogenic risk factors contributing to atherosclerotic plaque formation and progression. The interventions modulating these risk factors have been found to protect against atherosclerosis development and to decrease atherosclerosis-related cardiovascular disorders. Nutritional approaches involving supplements followed by improving dietary habits and lifestyle have become growingly attractive and acceptable methods used to control atherosclerosis risk factors, mainly high levels of plasma cholesterol. There are a large number of studies that show berberine, a plant bioactive compound, could ameliorate atherosclerosis-related risk factors. In the present literature review, we put together this studies and provide integrated evidence that exhibits berberine has the potential atheroprotective effect through reducing increased levels of plasma cholesterol, particularly low-density lipoprotein (LDL) cholesterol (LDL-C) via LDL receptor (LDLR)-dependent and LDL receptor-independent mechanisms, inhibiting migration and inflammatory activity of macrophages, improving the functionality of endothelial cells via anti-oxidant activities, and suppressing proliferation of vascular smooth muscle cells. In conclusion, berberine can exert inhibitory effects on the atherosclerotic plaque development mainly through LDL-lowering activity and suppressing atherogenic functions of mentioned cells. As the second achievement of this review, among the signaling pathways through which berberine regulates intracellular processes, AMP-activated protein kinase (AMPK) has a central and critical role, showing that enhancing activity of AMPK pathway can be considered as a promising therapeutic approach for atherosclerosis treatment. Keywords AMP-activated protein kinase · Atherosclerosis · Berberine · Cholesterol · Endothelial cells · LDL-C · Macrophage dysfunction · Vascular smooth muscle cell

Abbreviations 30 -UTR ABCA1 AMP AMPK EMMPRIN eNOS ERK HMG-CoA

30 -untranslated region ATP-binding membrane cassette transport protein A1 Adenosine monophosphate AMP-activated protein kinase Exhibited a decreased expression of MMPs and extracellular MMP inducer endothelial nitric oxide synthase Extracellular receptor-activated kinase 3-hydroxy-3-methyl-glutaryl-coenzyme A

Promising Anti-atherosclerotic Effect of Berberine: Evidence from In Vitro, In. . .

HNF1α HUVECs IL-6 JNK LDL LDL-C LDLR LOX 1 LXRα lysoPC MAP MCP-1 MIP-1α MMPs NADPH NAFLD NOD PCSK9 PCSK9 ROS SR-BI SREBP2 TG VCAM-1 VSMCs

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hepatocytes nuclear factor 1α Human umbilical vein endothelial cells Interleukin 6 c-Jun N-terminal kinase Low-density lipoprotein LDL cholesterol LDL receptor Low-density lipoprotein receptor 1 Liver X receptor α Lysophosphatidylcholine Mitogen-activated protein Monocyte chemoattractant protein-1 Macrophage inflammatory Protein 1 alpha Matrix metalloproteinases Nicotinamide adenine dinucleotide phosphate Non-alcohol fatty liver disease Nonobese diabetic Proprotein convertase subtilisin kexin 9 Proprotein convertase subtilisin/kexin type 9 Reactive oxygen species Scavenger receptor class B type I sterol regulatory element-binding protein 2 Triglyceride Vascular cell adhesion molecule-1 Vascular smooth muscle cells

1 Introduction Berberine is an isoquinoline alkaloid presented as the principal bioactive ingredient in stem, bark, rhizome, and roots of several plants, including barberry (Berberis vulgaris), Coptis (Coptis chinensis), goldenseal (Hydrastis canadensis), tree turmeric (Berberis aristata), and Oregon grape (Berberis aquifolium) (Singh and Mahajan 2013; Srivastava et al. 2015). Evidence from traditional and modern medicine shows that berberine exerts polytrophic pharmacological effects, including lipid-lowering, anti-diabetic, anti-tumor, anti-inflammatory, anti-diarrheal, and antimicrobial activities (Ayati et al. 2017; Imanshahidi and Hosseinzadeh 2008; Li et al. 2014; Singh et al. 2010; Zhang et al. 2019). A growing body of research confirms the anti-atherogenic properties of berberine, although there is no enough report showing its direct effect on atherosclerotic plaque formation and progression. Herewith, to exhibit the possible protective effect of berberine on atherosclerotic plaque progression, the present review was aimed to gather underlying mechanisms ascribed to the anti-atherogenic effects of berberine.

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As discussed in the following sections, there are documented findings demonstrating that berberine has potential anti-atherosclerotic effects through protecting or lowering hypercholesterolemia, modulating the atherogenic activity of inflammatory macrophages, and improving abnormal functions of vascular smooth muscle and endothelial cells.

2 Berberine Lowers Atherogenic Lipids Atherogenic lipids, especially low-density lipoprotein (LDL) cholesterol (LDL-C), have a casual effect on atherosclerotic plaque development and progression (Ference et al. 2017). Various LDL-lowering medications, such as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, have been found to reduce the risk of atherosclerosis-related cardiovascular disorders proportional to the absolute reduction of LDL-C in numerous randomized trials (Ference et al. 2017; Ridker et al. 2017; Sabatine et al. 2017; Sahebkar and Watts 2013). Hence, an LDL-lowering agent may have the potential to exert protective and therapeutic effects against atherosclerotic plaque progression. Although statin therapy is the most commonly used approach to treat hypercholesterolemia, there might be a relatively large number of patients who are statin-resistant or statin-intolerant and unable to achieve optimal LDL-C levels despite intensive statin therapy (Toth et al. 2018; Ward et al. 2019). Therefore, exploring complementary or alternative LDL-lowering agents to prevent or treat atherosclerotic lesions is important. There are several in vivo, clinical, and mechanistic studies that show berberine can efficiently reduce increased levels of atherogenic lipids, mainly LDL-C, suggesting that this natural compound has the strong potential to protect against atherosclerotic plaque development.

2.1

In Vivo Evidence

A large number of experimental studies on rodent models of diet-induced hypercholesterolemia have been conducted to determine the cholesterol-lowering effects of the various doses and administration routes of berberine (Table 1). Since an early report introduced berberine as a new cholesterol-lowering agent (Kong et al. 2004), a growing body of experimental studies has further investigated and confirmed the therapeutic potential of berberine on hypercholesterolemia in recent years (Briand et al. 2013; Brusq et al. 2006; Chang et al. 2012; He et al. 2016; Hu and Davies 2010; Hu et al. 2012; Jia et al. 2008; Kong et al. 2004; Li et al. 2011; Wang et al. 2013, 2014; Xiao et al. 2012). As reviewed elsewhere, berberine can modulate the dysregulated lipid profile through reducing plasma levels of TC, LDL-C, and TG as well as increasing plasma HDL-C in various rodent models of hyperlipidemia (Wang and Zidichouski 2018). These results are supported by the recent animal

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Table 1 In vivo lipid-lowering effect of berberine in various animal models Animal model Male ApoE/ mice

Route and time of administration Daily gavage for 13 weeks

Effect on plasma lipid profile Decreasing TC and LDL-C Increasing HDL-C

Ref. Wu et al. (2020)

Daily gavage for 2 weeks

TC (28%) LDL-C (51%)

100 mg/kg/ day

Daily gavage for 4 weeks

Significantly decreasing TC and LDL-C Significantly increasing HDL-C

30 mg/kg/ day

Daily gavage for 4 weeks

TC (10.5) LDL-C (9.8%)

50, 100, 150 mg/ kg/day 100 mg/kg/ day

Daily gavage for 8 weeks

TC (29%, 33%, 33%), non-HDLC (31%, 41%, 38%), at 50, 100, and 150 mg/kg/day, respectively No effect on both TC and non-HDL-C. but (31%) plasma TG levels

Singh and Liu (2019) Kim et al. (2019) Zhu et al. (2018) Wang et al. (2014) Jia et al. (2008) Chang et al. (2012) Brusq et al. (2006) Briand et al. (2013) Xiao et al. (2012) Hu et al. (2012) Kong et al. (2004) He et al. (2016) Abidi et al. (2006) Wang et al. (2011)

Berberine dose 50 mg/kg/ day 100 mg/kg/ day 200 mg/kg/ day

Male C57BL/6 mice SpragueDawley male rats Female C57BL/ 6 J mice SpragueDawley male rats SpragueDawley male rats SpragueDawley male rats Male hamsters

200 mg/kg/ day

Daily gavage for 16 weeks

TC (28%) and HDL-C (41%)

100 mg/kg/ day

Inhibited both cholesterol and TG synthesis

Male hamsters

150 mg/kg/ day

Female C57BL/ 6 J mice SpragueDawley rat Female hamsters

10 and 30 mg/kg/ day 500 mg/kg

Orally twice a day for 10 days Daily injection for 2 weeks Daily gavage for 4 weeks

Hamsters

50 and 100 mg/kg/ day 46.7 mg/kg/ day

Daily gavage for 6 weeks

3 times a day gavage for 12 weeks Daily gavage for 10 days

LDL-C (35%), TG (34%)

TC (42%, 56%) and TG (37%, 47%) at 10 and 30 mg/kg/day, respectively T-C (9%) and TG (34.7%)

LDL-C (26%, 42%), at 50 and100 mg/kg/day, respectively

Daily gavage for 140 days

TC (19%) and HDL-C (11.34%)

Male hamsters

1.8 mg/kg/ day

Daily gavage for 24 days

TC (30%), TG (34%) and LDL-C (9.2%)

Male rats

7, 60, and 300 mg/kg/ day

Daily gavage for 12 weeks

Decreased TC and LDL-C, but increased HDL-C

(continued)

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Table 1 (continued) Animal model Rats

Berberine dose 200 mg/kg/ day

Male hamsters

50 and 100 mg/kg/ day

Route and time of administration Daily gavage for 16 weeks Daily gavage for 10 days

Effect on plasma lipid profile T-C (29%) and HDL-C (41%)

TC (44%, 70%,), TG (34%, 51%), and LDL-C (47%, 71%) at 50 and100 mg/kg/day, respectively

Ref. Chang et al. (2010) Li et al. (2008)

study that showed the oral administration of berberine (100 mg/kg/day) via gavage feeding or dietary supplementation can ameliorate severe hypercholesterolemia in hyperlipidemic rats (Kim et al. 2019). It is consistent with another study that demonstrated the oral gavage of berberine at both high dose (100 mg/kg/day) and low dose (50 mg/kg/day) could effectively and dose-dependently reduce elevated levels of plasma TC and LDL-C in ApoE/ mice fed with a high-fat diet, which the effect of high dose was stronger than that of the low dose (Wu et al. 2020). Further study revealed that berberine treatment (200 mg/kg/day) could decrease plasma levels of TC and LDL-C by 28% and 51% in wild-type mice fed a high cholesterol diet (Singh and Liu 2019). It was also found that intraperitoneal (i.p.) injection of berberine (1.5–5 mg/kg/day) could decrease plasma cholesterol to a similar amount as compared to oral administration; however, the therapeutic dose was decreased by 10 to 100-folds in i.p. route (Abidi et al. 2006; Hu and Davies 2010; Kim et al. 2009; Wu et al. 2010). Although i.p. injection of berberine shows similar cholesterollowering effects at a lower dose, it is not an acceptable route for administration in humans because of its inconvenience and invasiveness. The cholesterol-lowering potential of berberine is further supported by the study that showed oral administration of berberine (90 mg/kg/day) and simvastatin (6 mg/ kg/day) exerted similar reductions of LDL-C (27% and 28%, respectively) in rats fed with a high-cholesterol diet. Of note, a combination of berberine with simvastatin decreased plasma LDL-C by 46%, which was significantly higher than that of either berberine or simvastatin monotherapy, resulting in the reduction of simvastatin dose by 50% that still exerts similar lipid-lowering effect (Kong et al. 2008). It was also reported that combination therapy of berberine (orally, 30 mg/kg/day) with resveratrol (orally, 20 mg/kg/day) in hyperlipidemic mice decreased plasma levels of total cholesterol by 27% and LDL-C by 31.6%, which was significantly greater than that of berberine (10.5% and 9.8%) or the resveratrol (8.4% and 6.6%) monotherapy (Zhu et al. 2018). Therefore, berberine can be considered as an add-on therapy to improve lipid-lowering effects and decrease the therapeutic dosage. Besides, the cholesterol-lowering effect of berberine is also documented by other studies where it has been shown that administration of berberine markedly reduced the plasma level of LDL-C in animal models of type 2 diabetes mellitus fed with high-fat diet (Zhang et al. 2008, 2011, 2014). Interestingly, in nonobese diabetic (NOD) mice, berberine supplementation (50, 150, and 500 mg/kg/day) significantly

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decreased the ratio of LDL-C/TC in a dose-dependent manner (Chueh and Lin 2011). Moreover, studies carried out in animal models of hyperlipidemia and non-alcohol fatty liver disease (NAFLD) have investigated lipid-modulating effects of berberine. In a good agreement with the previous findings, the results of these studies have also confirmed that berberine (alone or in combination with other natural compounds such as curcumin) can facilitate the treatment of hyperlipidemia and NAFLD through its LDL-lowering effects (Feng et al. 2018; Kou et al. 2016; Zhou et al. 2017). To sum up, berberine in combination with lipid-lowering drugs can efficiently ameliorate hypercholesterolemia, whereby it can reduce therapeutic doses of such drugs, like statins, and thereby might improve treatment of patients who are statin intolerance or statin resistance even at high-dose therapy.

2.2

Clinical Evidence

The beneficial cholesterol-lowering effect of berberine has been verified in numerous clinical trials conducted on patients with mild to moderate hypercholesterolemia in various populations (Table 2). Recently, a systematic review and meta-analysis of 16 randomized clinical trials with a total of 2,147 participants was directed to evaluate the efficacy and safety of berberine in patients with dyslipidemia. The results indicate that berberine can improve plasma lipid profile in dyslipidemia with satisfactory safety, in which significant reduction of TC and LDL-C as well as no significant incidence of adverse events is evident (Ju et al. 2018). A placebocontrolled clinical trial investigating the cholesterol-lowering efficacy of berberine in patients with mild hyperlipidemia showed that oral administration of berberine capsule (900 mg/day, for 3 months) effectively improved the plasma levels of TC and LDL-C (Wang et al. 2016b). Comparably, a clinical trial conducted on Chinese population indicated that oral administration of berberine (1 g/day, for 3 months) decreased the plasma levels of TC by 29% and LDL-C by 35% in patients with hypercholesterolemia suffering from TC levels more than 200 mg/dL (Doggrell 2005). Similarly, another clinical trial conducted on the Caucasian population has demonstrated that berberine treatment (1 g/day, for 3 months) decreased TC by 11% and LDL-C by 16%, without any adverse effects, in hypercholesterolemic patients with low cardiovascular risk (Derosa et al. 2013). Moreover, it was shown that berberine treatment could lower LDL-C (20–30%) (Johnston et al. 2017; Koppen et al. 2017) at a percentage range close to those achieved by statin therapy (30–50%) (Dong et al. 2013). Cholesterol-lowering effects of berberine have been also indicated by the other studies that investigated the effects of berberine in combination with lipid-lowering nutraceuticals and/or drugs in hypercholesterolemic patients. Of note, berberine has poor intestinal absorption and low oral bioavailability, which limits its therapeutic efficacy. Silymarin, which is a flavonolignan-rich complex extracted from the milk thistle Silybum marianum (L.), has been found to possess hepato- and cardio-

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Table 2 Clinical trials evaluating cholesterol-lowering effect of berberine in hypercholesterolemic patients

Subject Prediabetic patients

Patients with mild-tomoderate hypercholesterolemia Hyperlipidemic patients

Moderately hypercholesterolemic patients Caucasians with low cardiovascular risk

Treatment group Berberine-containing polyherbal dietary supplement, n ¼ 40 Berberine-containing food supplement, n ¼ 90; placebo, n ¼ 90 Berberine-containing nutraceuticalsa, n ¼ 30 (15/15, M/F); placebo, n ¼ 9 (3/6, M/F) AP-1b, n ¼ 51 (18/33, M/F); placebo, n ¼ 51 (14/37, M/F) Berberine, n ¼ 71 (35/36, M/F); placebo, n ¼ 70 (35/35, M/F)

Does, frequency, duration 500 mg/day, once daily, 12 weeks 500 mg/day, once daily, 4 weeks 0.2 g/d, once daily, 12 weeks 500 mg/d, once a day, 12 weeks 1 g/d, twice daily, 3 months

AP-1, n ¼ 29 (20/9, M/F); placebo, n ¼ 30 (18/12, M/F) AP-1, n ¼ 152 (62/90, M/F); compared to baseline

500 mg/d, once daily, 18 weeks 500 mg/d, once daily, 6 months

Menopausal women with moderate dyslipidemia

Berberine + isoflavones, n ¼ 60; compared to baseline

Dyslipidemic patients

AP-1, n ¼ 933 (416/518, M/F); placebo, n ¼ 818 (384/434, M/F)

Berberine and isoflavones combination, 12 weeks 500 mg/d, once daily, 16 weeks

Elderly (>75 years) hypercholesterolemic patients Hypercholesterolemic patients

AP-1, n ¼ 40 (21/19, M/F); placebo, n ¼ 40 (20/20, M/F) AP-1, n ¼ 25 (13/12, M/F); placebo, n ¼ 25 (13/12, M/F)

500 mg/d, once daily, 12 months 500 mg/d, once daily, 6 weeks

Patients with metabolic syndrome Hypercholesterolemic patients

Effect on plasma lipid profile Decreasing TC and LDL-C LDL-C (26%) Non-HDL-C (15%), LDL-C (19%) TC (5%), LDL-C (7.8%) T-C (11.6%), LDL-C (16.4%), HDL-C (+9.1%) T-C (15%), LDL-C (23%) TC (24%), LDL-C (32%), non-HDL-C (30%), TG (20%) T-C (14%), LDL-C (12%), TG (19%) T-C (10%), LDL-C (13%), TG (7%), HDL-C (+8%) T-C (20%), LDL-C (31%) T-C (17%), LDL-C (23%)

Ref. Feinberg et al. (2019) D’Addato et al. (2017) Spigoni et al. (2017) Sola et al. (2014) Derosa et al. (2013)

Affuso et al. (2012) Pisciotta et al. (2012)

Cianci et al. (2012)

Trimarco et al. (2011)

Marazzi et al. (2011) Affuso et al. (2010) (continued)

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Table 2 (continued)

Subject Hypercholesterolemic patients

Treatment group Berberine, n ¼ 24; compared to baseline (no sex ratio provided)

Does, frequency, duration 1 g/d, twice daily, 2 months

Moderate dyslipidemic subjects

Berberine, n ¼ 20 (8/12, M/F); AP-1, n ¼ 20 (8/12, M/F)

500 mg/d, once daily, 4 weeks

Hypercholesterolemic patients

Berberine, n ¼ 63 (35/28, M/F); placebo, n ¼ 28 (17/11, M/F)

1 g/d, twice daily, 3 months

Effect on plasma lipid profile TC (21.8%), LDL-C (23.8%), TG (22.1%) T-C (16%), LDL-C (20%), TG (22%), HDL-C (+7%); berberine and AP-1 did not differ T-C (29%), LDL-C (25%), TG (35%)

Ref. Kong et al. (2008)

Cicero et al. (2007)

Kong et al. (2004)

a

Berberine-containing nutraceutical: berberine 200 mg, chitosan 10 mg, monacolin K 3 mg, and CoQ10 10 mg b AP-1: 1 tablet contains berberine 500 mg, red yeast rice extract 200 mg (equivalent to 3 mg monacolins), policosanol 10 mg, CoQ10 2 mg, folic acid 0.2 mg, and astaxanthin 0.5 mg

protective activities and optimize intestinal berberine absorption (Guarino et al. 2017). A meta-analysis of 19 controlled and cross-sectional trials showed that a combination of berberine with silymarin could significantly improve the cholesterollowering effect of berberine (Bertuccioli et al. 2019). Results from a randomized double-blind, placebo-controlled clinical trial on patients with moderate hypercholesterolemia (LDL-C up to 130–190 mg/dL) without cardiovascular disease demonstrated that, after 2 months treatment, berberine combined with a dry extract of artichoke, a lipid-lowering agent, could exert a significant reduction in plasma TC by 19%, LDL-C by 16%, and non-HDL-C by 19%, with no significant side effect (Cicero et al. 2019). Furthermore, it has been shown that the oral administration of berberine alone (500 mg/day) or with a combination of lipid-lowering nutraceuticals (consisting policosanols, red yeast extract, folic acid, coenzyme Q10, and astaxanthin) for 4 weeks effectively reduced the plasma levels of LDL-C (by 20% and 25%, respectively) and total cholesterol (by 16% and 20%, respectively) in 40 subjects with moderate dyslipidemia (Cicero et al. 2007). A multicenter, randomized, doubleblind, placebo-controlled trial on patients with mild-to-moderate hypercholesterolemia showed that 4 weeks treatment with a daily oral dose of berberine-continuing nutraceutical supplement (red yeast rice, coenzyme Q10, and hydroxytyrosol) had a favorable efficacy and safety profile, wherein LDL-C was decreased by 26%

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(D’Addato et al. 2017). Another double-blind placebo-controlled study showed that 6 weeks oral administration of the mentioned combination of berberine and nutraceuticals could decrease plasma levels of LDL-C by 23% and TC by 19% without adverse effects in 25 hypercholesterolemic patients (Affuso et al. 2010). Moreover, 12-month treatment with this combination was found to reduce the plasma levels of TC by 20% and LDL-C by 31% in elderly hypercholesterolemic patients (more than 75 years) who were statin intolerance (Marazzi et al. 2011). The combination containing berberine was also compared with ezetimibe drug in hypercholesterolemic subjects who were intolerant or refusing to take statin; the combination therapy was more effective in decreasing LDL-C (32% vs  25%) and TC (24% vs  19%) (Pisciotta et al. 2012). Several clinical trials have also investigated the effects of berberine combined with statins in hypercholesterolemic patients. A recent comprehensive meta-analysis indicates that berberine treatment could synergistically increase cholesterol-lowering potential of statins, while the incidence of statin-related adverse reactions, such as elevation of transaminase and muscle aches, was markedly decreased (Zhang et al. 2019). Cholesterol-lowering efficacy of berberine is further verified by the other clinical trials in dyslipidemic subjects with various disease conditions, such as prediabetic (Feinberg et al. 2019) and diabetic patients (Bertuccioli et al. 2019; Yin et al. 2008; Zhang et al. 2010), patients with metabolic syndrome (Affuso et al. 2012; PérezRubio et al. 2013), and post-menopause subjects (Affuso et al. 2012). These findings confirm that berberine improves plasma levels of cholesterol in dyslipidemic conditions, and since dyslipidemia, particularly high level of plasma LDL-C, is an independent risk factor for promotion and progression of atherosclerotic lesions, this natural compound possesses potential preventive and/or therapeutic effects for treating atherosclerosis. Hence, given that atherosclerosis is a chronic disorder, further rigorous clinical studies are needed to better determine the longterm efficacy of berberine and its effects on the progression of atherosclerotic lesions.

2.3

Mechanisms Underlying the Cholesterol-Lowering Effect of Berberine

Berberine is known to reduce plasma cholesterol through modulating cholesterol metabolism and hemostasis. Berberine has been shown to suppress cholesterol biosynthesis through inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase via activating AMP-activated protein kinase (AMPK) in hepatocytes. The activated AMPK phosphorylates the rate-limiting enzyme HMG-CoA reductase and leads to the inactivation of cholesterol biosynthesis (Brusq et al. 2006).

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Fig. 1 Schematic view of mechanisms underlying LDLR-dependent cholesterol lowering effects of berberine. (a) Berberine can enhance LDLR expression through elevating LDLR mRNA stability via inducing ERK signaling activity that suppresses binding of heterogeneous nuclear ribonucleoprotein 1 (hnRNP I) to AREs, leading to inhibition of RNA degradation machinery. (b) Berberine can also induce LDLR expression through inducing the degradation of two cellular trans-activators, hepatocytes nuclear factor 1α (HNF1α) and sterol regulatory element-binding protein 2 (SREBP2)

Moreover, intestinal absorption of dietary cholesterol has an important role in cholesterol hemostasis, and berberine has been shown to inhibit various processes involved in intestinal absorption, including intraluminal cholesterol micellization, cholesterol uptake by enterocytes, and cholesterol esterification in the enterocytes via reducing expression of acyl-coenzyme A cholesterol acyltransferase-2 (Wang et al. 2014). Plasma cholesterol is mainly removed from the blood circulation by the liver LDL receptor (LDLR). LDLR deficiency increases plasma cholesterol and accelerates atherosclerosis (Umans-Eckenhausen et al. 2002; Xu and Weng 2020). Recently, an in vivo study showed that berberine treatment failed to decrease the plasma levels of cholesterol in hypercholesterolemic LDLR-deficient mice (Ldlr/), providing direct evidence that supports cholesterol-lowering effects of berberine might be mediated, at least in part, by regulating the liver LDLR (Singh and Liu 2019). Further mechanistic studies (Abidi et al. 2005, 2006; Dong et al. 2015; Kong et al. 2006; Li et al. 2009a; Momtazi et al. 2017) indicate that berberine can increase stability of LDLR at both mRNA (Fig. 1a) and protein (Fig. 1b) levels and, thereby, upregulate the expression of hepatic LDLR. The 30 -untranslated region (30 -UTR) of human LDLR mRNA contains three AU-rich elements (AREs) responsible for rapid mRNA turnover, which mediates berberine-induced mRNA stabilization. Berberine was found to extend LDLR mRNA half-life entirely through 30 UTR in an extracellular receptor-activated kinase (ERK) cascade-dependent manner, in which inhibit interactions of cis-regulatory sequences of 30 UTR and mRNA binding proteins that are downstream effectors of this signaling pathway (Abidi et al. 2005, 2006; Kong et al. 2006). AREs are the well-known RNA cis-regulatory elements that play a

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central role in the mRNA stability (Zhang et al. 2002). Further studies highlighted that berberine can inhibit binding of heterogeneous nuclear ribonucleoprotein 1 (hnRNP I), a destabilizing ARE binding protein, to the LDLR mRNA 30 UTR, whereby enhances LDLR mRNA stability resulting in increased LDLR expression and improved plasma LDL-C clearance (Li et al. 2009b; Singh et al. 2014). Besides, berberine also can increase the stability of LDLR protein on the surface of hepatocytes through post-translational regulation via modulatory effects on PCSK9/LDLR pathway (Cameron et al. 2008; Momtazi et al. 2017; Xiao et al. 2012). PCSK9 is mainly produced by hepatocytes and secreted to the bloodstream where it binds the extracellular domain of hepatic LDLR and targets it to lysosomal degradation, leading to insufficient hepatic LDLR to trap plasma circulating LDL-C (Qian et al. 2007), causing hypercholesterolemia and atherosclerosis-related cardiovascular disease (Abifadel et al. 2003). Mechanistically, berberine can decrease expression of PCSK9 through accelerating the degradation of two cellular transactivators, hepatocytes nuclear factor 1α (HNF1α) and sterol regulatory elementbinding protein 2 (SREBP2) that are essential for PCSK9 expression (Dong et al. 2015; Li et al. 2009a; Momtazi et al. 2017). To sum up, berberine can increase the liver LDLR at both mRNA and protein levels through two distinct mechanisms including inhibition of hnRNPI activity and reduction of PCSK9 expression, respectively. Cholesterol is finally eliminated from the liver via the conversion into bile acids and/or excretion as free cholesterol into bile, resulting in more cholesterol uptake by the liver from the bloodstream. Berberine was found to increase cholesterol secretion from the liver into bile (Guo et al. 2016; Li et al. 2015) and also elevate hepatic expression of enzymes regulating the synthesis of bile acid from cholesterol, such as mitochondrial sterol 27-hydroxylase, which was associated with a significant reduction of plasma cholesterol (Wang et al. 2010). In conclusion, berberine can regulate plasma cholesterol through inhibiting cholesterol biosynthesis by the liver together with intestinal absorption of dietary cholesterol, elevating liver LDLR stability, and enhancing cholesterol excretion via increasing biosynthesis of bile acid and secretion of free cholesterol.

3 Berberine’s Effects on Atherosclerosis Lesion Progression: In Vitro Evidence 3.1

Berberine Improves Endothelial Cell Function

Vascular endothelial dysfunction is known to be a primary event promoting initiation and progression of atherosclerotic lesions, thus improving or repairing endothelium functionality that can exert beneficial effects on the atherosclerotic process (Mordi and Tzemos 2014). Endothelial dysfunction is characterized by elevated oxidative stress and decreased nitric oxide (NO) synthesis/availability, leading to

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Fig. 2 Improved functions of berberine-treated endothelial cells

proinflammatory and proliferative processes that assist all steps of atherogenesis progression (Yuyun et al. 2018). Berberine was found to protect against endothelial dysfunction in both the cultured human umbilical vein endothelial cells (HUVECs) and blood vessels isolated from rat aorta. This protective effect of berberine is through activating AMPK signaling that upregulates the expression of endothelial nitric oxide synthase (eNOS) and downregulates the expression of NADPH oxidase (NOX4) resulting in decreased generation of reactive oxygen species (ROS) and increased production of NO (Wang et al. 2009; Zhang et al. 2013). The activated endothelial cells express adhesion molecules, such as E- and P-selectins, intercellular adhesion molecules (ICAM) like ICAM-1 and vascular cell adhesion molecules (VCAM) like VCAM-1, which act as a receptor for recruitment of the inflammatory monocytes (Collins et al. 2000; Dong et al. 1998; Shih et al. 1999). The improved functionality of injured endothelial cells by berberine treatment can be further supported by other studies showing berberine suppressed the production of ICAM-1 and VCAM-1, leading to a decrease adhesion of monocytes to endothelial cells (Chen et al. 2014; Ko et al. 2007; Wu et al. 2012). To sum up, berberine can improve endothelial cell function through balancing NO and ROS production via modulating AMPK/eNOS/NOX4 signaling pathway together with reducing the adhesion capacity to monocytes (Fig. 2).

3.2

Berberine Modulates Atherogenic Activities of Macrophages

Macrophages, derived from recruited monocytes, play a central role in atherosclerotic plaque formation and progression. After adhesion, monocytes subsequently infiltrate into the subendothelial layer and differentiate into macrophages. When these cells transmigrate across the endothelial monolayer into the intima, they uptake ox-LDL-C and form the so-called foam cells (Moore et al. 2013; Moore and Tabas 2011). Berberine was shown to decrease the expression of the infiltration marker CD68, revealing decreased macrophage infiltration into aortic plaques (Chen et al. 2014). Mechanistically, berberine can suppress transendothelial migration through reducing the toll-like receptor (TLR)-mediated macrophage migration ability via inhibiting

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the enzymatic activity of Src that is an inducible tyrosine kinase having a pivotal role in cell movement (Cheng et al. 2015). It is further approved by the other study that showed berberine treatment could significantly and highly reduce macrophages contents in mice’s atherosclerotic plaques (Yang et al. 2020). The impaired cholesterol efflux and the excessive internalization of LDL-C lead to cholesterol accumulation in the infiltrated macrophages, causing foam cell formation and atherosclerosis lesion progression (Khera et al. 2011). Reverse cholesterol transporters, such as class B scavenger receptor type I (SR-BI) and ATP-binding cassette transporter (ABC)A1 and G1 (ABCG1), play crucial roles in the efflux of intracellular cholesterol in foam cells (Ohashi et al. 2005). Upon macrophage cholesterol loading, autophagy enhances the lysosomal hydrolysis of stored cholesterol droplets and generates free cholesterol mainly for ABCA1mediated efflux, whereby it facilitates cholesterol efflux (Ouimet et al. 2011; Razani et al. 2012; Wang et al. 2016a). During atherogenic states, the autophagy process is imparted in macrophages, and berberine treatment was found to enhance autophagy in macrophages, prevent autophagy resistance in the foam cells, and consequently induce cholesterol efflux in both treated cells. Berberine could promote autophagydependent cholesterol efflux through suppression of the PI3K/AKT/mTOR pathway resulting in ABCA1-dependent cholesterol efflux in macrophage-derived foam cells (Kou et al. 2017). The positive effect of berberine on cholesterol efflux is further confirmed by other studies that show berberine can increase expression of SR-BI (Chi et al. 2014) and ABCA1/G1 via activating the liver X receptor α (LXRα) transcription factor (Lee et al. 2010; Yang et al. 2020). Meanwhile, autophagy dysfunction and consequent intracellular lipid accumulation induces macrophage-mediated inflammation and thus exacerbates atherosclerotic development (Razani et al. 2012). Berberine was shown to inhibit oxLDLinduced inflammatory factors, such as macrophage inflammatory protein 1 alpha (MIP-1α) and RANTES in macrophages, through inducing autophagy that was mediated by activation of the AMPK/mTOR signaling pathway (Fan et al. 2015). Berberine was also found to decrease macrophage inflammation through reducing the expression and secretion of tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and Interleukin 6 (IL-6), in vitro (Chen et al. 2008). Potential inhibitory effect of berberine on atherosclerotic plaque progression can be supported by other studies that show therapeutic approaches enhancing autophagy in macrophages potentially prevent or treat atherosclerosis progression (Maiuri et al. 2013; Schrijvers et al. 2011). Besides, berberine was also demonstrated to inhibit cholesterol accumulation and foam cell formation promoted by the influx of either native or oxLDL-C in macrophages. These cells can internalize native LDL-C through a receptor-independent route called macropinocytosis that has been demonstrated to promote the formation of foam cells and suggests a new therapeutic target to decrease cholesterol accumulation in atherosclerotic plaques (Kruth 2013). Of note, berberine was found to inhibit macropinocytosis and, thereby, decrease cholesterol accumulation and corresponding negative feedbacks promoted by foam cell formation (Zimetti et al. 2015). Macrophages also internalize oxLDL-C using scavenger receptors class A SR

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(SR-A), CD36, and lectin-like oxidized LDL receptor-1 (LOX-1), and berberine treatment could reduce the elevated expression of the mentioned receptors and, thereby, decrease cholesterol influx and lipid content in oxLDL-exposed macrophages/foam cells (Chi et al. 2014; Guan et al. 2010; Yang et al. 2020). Importantly, a plaque with a large lipid core and covered by a thin fibrous cap is at a higher risk for rupture (Schaar et al. 2004). Since the atherosclerotic plaque rupture followed by thrombus formation causes myocardial infarction, stroke, and death (Carr et al. 1996), suppressing the rupture of unstable plaques are crucial to prevent emergency medical conditions. The ruptured fibrous cap is found to be rich in macrophage and foam cells generating matrix metalloproteinases (MMPs) and extracellular MMP inducer (EMMPRIN), which digest extracellular matrix proteins and weaken the fibrous cap, resulting in the vulnerability of atherosclerotic plaques (Gough et al. 2006; Ha et al. 2020; Perrucci et al. 2020; Schmidt et al. 2006). Berberine treatment was indicted to decrease elevated expression of MMPs and EMMPRIN in oxLDL-exposed macrophages (Chen et al. 2014; Huang et al. 2012; Huang et al. 2011), suggesting that berberine can stabilize vulnerable plaques. To sum up, berberine has potential to prevent or treat atherosclerotic plaque formation and progression, at least in part, through suppressing macrophage migration into the intima, inhibiting foam cell formation and inflammation via suppressing intracellular cholesterol accumulation through enhancing autophagy and cholesterol efflux and reducing cholesterol influx, as well as increasing plaque stability (Fig. 3).

3.3

Berberine Blocks Proliferation and Migration of Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) are a major cell type contributing to atherosclerotic lesion progression. Proliferation and migration of VSMCs from media to intima layers of vascular wall and formation of muscle-origin foam cells after lipid ingestion promote the intimal thickening and, thereby, exert an important role in atherosclerosis pathology (Basatemur et al. 2019; Bennett et al. 2016). Therefore, inhibiting VSMCs proliferation and migration can beneficially affect atherosclerotic plaque initiation and progression. Arterial injury is known to promote cell cycle re-entry in VSMCs, following a wave of immediate early gene expression. After the injury, activation of extracellular signal-regulated kinase (ERK) is among the earliest of biochemical changes and is strongly associated with subsequent expression of early response transcription factors and growth factors (Kim et al. 1998; Koyama et al. 1998; Roostalu and Wong 2018). ERK is a key transducer of extracellular signals that induce cell growth and movement, which are essential for the promotion and development of vascular lesions (Lu et al. 2020). ERK transmits mitogenic signals by translocation to the nucleus where activates many of its substrates, such as early growth response factor

Fig. 3 Alleviating effects of berberine on atherogenic function of macrophages. Berberine can inhibit intracellular cholesterol accumulation and foam cell formation through inducing autophagy via suppressing AMPK/PI3K/AKT/mTOR signaling pathway. The autophagy activation leads to the lysosomal

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hydrolysis of cholesterol droplets and the increased ATP-binding cassette transporter A1 (ABCA1) and ABCG1 activity, resulting in the enhanced cholesterol efflux activity. Activated autophagy can also suppress oxLDL-induced inflammatory factors, such as macrophage inflammatory Protein 1 alpha (MIP-1α) and RANTES in macrophages. Berberine can also increase expression of ABCA1 and class B scavenger receptor type I (SR-BI) through inducing the activity of LXR-α transcription factor. Besides, berberine can also reduce intracellular cholesterol accumulation through reducing the expression of cholesterol influx receptors, including class A scavenger receptor (SR-A), CD38, and LOX-1. Berberine mediates stabilization of atherosclerotic plaques through reducing the macrophage-produced matrix metalloproteinase-9 (MMP-9) via inhibiting activity of extracellular MMP inducer (EMMPRIN)

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1 (Egr-1) (Gille et al. 1995; Goetze et al. 1999). Egr-1 is a zinc finger transcription factor that is lowly expressed in the normal vessel wall but is rapidly and transiently expressed in VSMCs in response to arterial injury (Khachigian et al. 1996; Kim et al. 1995). The activated Egr-1 regulates the expression of several genes participating in cell growth and differentiation, such as platelet-derived growth factor (PDGF) (Havis and Duprez 2020; Khachigian et al. 1996; Khachigian et al. 1995; Silverman et al. 1997) and Cyclins (Guillemot et al. 2001; Havis and Duprez 2020; Yan et al. 1997), which are known to be implicated in the pathogenesis of atherosclerosis (Guillemot et al. 2001; Havis and Duprez 2020; Khachigian and Collins 1997; Yan et al. 1997). Berberine has been found to abolish injury-induced VSMC regrowth through the inactivation of ERK and inhibiting the expression of Egr-1 and downstream production of growth mediators Cyclin D1 and PDGF-A, thereby preventing early signaling related with cell cycle re-entry induced by injury. Of note, the inhibitory effect of berberine was identified to be by suppressing the activity of mitogen-activated protein kinase 1/2 (MEK1/2) that mediates activation of ERK/ Egr-1 signaling by injury (Liang et al. 2006). Moreover, PDGF is an important growth factor released after vascular injury and is related to VSMC proliferation and migration. Berberine was shown to inhibit PDGF-stimulated VSMC proliferation through activating AMPK/p53/p21Cip1 signaling and inactivation the Rac1/Cyclin D/Cyclin-dependent kinase (Cdk) leading to G1 arrest (Liang et al. 2008). AMPK is a serine/threonine protein kinase that is known to suppress PDGF-induced proliferation in human aortic VSMC (Igata et al. 2005). The activated AMPK can induce a cell cycle G1 arrest via AMPK-dependent phosphorylation of p53 in human VSMCs (Igata et al. 2005). Inhibition of PDGFinduced VSMC proliferation by berberine was indicated to be mediated in part through increasing the activity of AMPK, which led to the activation of p53 phosphorylation and up-regulation of the Cdk inhibitor p21Cip1 (Liang et al. 2008). Furthermore, Rac1 is a signaling GTPase that regulates a wide variety of cellular activities, including cell proliferation, migration, and apoptosis. Rac1 is known to mediate PDGF-induced VSMC proliferation through activating the production of cell cycle regulatory molecules Cyclins and Cdks that are responsible for G1/S cell cycle transition (Liang et al. 2008). Of note, berberine was shown to inhibit PDGF-induced Rac1 activation and upregulation of Cyclin D1, Cyclin D3, Cdk2, and Cdk4, whereby it induces G1-phase arrest in VSMCs (Liang et al. 2008). In addition to promoting cell proliferation, PDGF can also stimulate migration in VSMCs, as PDGF is the most potent reported chemoattractant for VSMCs (Zhang et al. 2018). Many reports show PDGF increases both Rac1 activity and cell migration (Al-Koussa et al. 2020). Herewith, berberine was also found to PDGFinduced VSMCs migration through inhibiting activation of Rac1 and another GTPase, Cdc42, that mediate the migratory function of PDGF through VSMCs injury (Liang et al. 2008). Regarding the mechanism of berberine on the inhibition of Rac1 and Cdc42, there has been evidence that AMPK activation could lead to suppression of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis (Liang et al. 2008). Suppression of HMG-CoA reductase decreases cholesterol synthesis as well as

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some critical isoprenoids downstream of mevalonate such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are essential for membrane translocation and activation of Rac1 and Cdc42 (Liang et al. 2008). Therefore, AMPK activation by berberine can drive suppression of HMG-CoA reductase and reduce downstream isoprenoids that are required for Rac1 and Cdc42. Hence, berberine can indirectly inhibit activation of Rac1 and Cdc42 and prevent cell proliferation and migration induced by PDGF in VSMCs. In conclusion, berberine-elicited anti-proliferative and anti-migratory effects in the injured VSMCs are related to a multifaceted attack on multiple signaling targets that critically participated in growth suppression. Given that abnormal proliferation and migration of VSMCs is a hallmark event in atherosclerotic lesions development, such findings can support the preventive effect of berberine on initiation and progression of atherosclerosis lesions.

4 Berberine’s Effects on Atherosclerosis Lesion Progression: In Vivo Evidence There are several in vivo studies on mouse models of atherosclerosis that support the abovementioned in vitro studies showing potential anti-atherogenic effects of berberine. ApoE/ mouse fed with the atherogenic diet is the most common model of human atherosclerosis that is widely used to evaluate the preventive or therapeutic effects of drugs on atherosclerosis lesion progression. It was found that oral gavage of berberine at a low dose (50 mg/kg/day) and a high dose (100 mg/kg/day) after 13 weeks could significantly decrease atherosclerotic plaque area and lesion size in ApoE/ mice bearing atherosclerosis, in which the high dose was found to be more effective (Wu et al. 2020). Further study reveals that berberine treatment (8 weeks) could decrease vascular inflammation and oxidative stress via an AMPK-dependent mechanism, leading to a significant reduction of atherosclerotic plaque progression in mouse aorta (Wang et al. 2011). However, another in vivo study indicated that berberine treatment at a lower dose (orally 10 mg/kg/day, for 16 weeks) failed to exert a significant effect on atherosclerosis plaque progression in hypercholesterolemic ApoE/ mice, while the same dose of its derivatives, dihydroberberine and 8,8-dimethyldi-hydroberberine possessing higher bioavailability, were found to at the same dose effectively decrease atherosclerotic plaque size and improve plaque stability (Chen et al. 2014). This finding was suggested to be due to poor intestinal absorption of berberine (Chen et al. 2014). Thus, oral administrating of berberine derivatives with improved intestinal absorption or its administration via alternative routes can enhance therapeutic potential. It can be further supported by a recent in vivo study that showed berberine (10 mg/kg/ day, for 14 weeks) via intraperitoneal injection in hypercholesterolemic ApoE/ mice could decrease the size of atherosclerotic plaque in aortic sinus by 45% (Yang et al. 2020).

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Cellular and molecular examination of atherosclerosis lesions isolated from berberine-treated mice reveals that berberine can suppress atherosclerosis plaque progression through mechanisms consistent with those found by in vitro studies discussed in previous sections. Of note, berberine has been indicated to reduce macrophage content in mice’s atherosclerotic plaques through reducing macrophage adhesion and infiltration to the vascular endothelium via inhibiting the expression of two critical adhesion molecules ICAM-1 and VCAM-1 (Chen et al. 2014; Yang et al. 2020). Moreover, berberine treatment was shown to reduce lipids contents in aortic macrophages through increasing expression of ABCA1/G1-mediated cholesterol efflux and reducing expression of SR-A/CD36-mediated cholesterol influx in hypercholesterolemic ApoE/ mice (Yang et al. 2020). Likewise, immunohistochemical assays of mice’s aortic plaque showed that berberine treatment decreased protein expression of MMP-9 and EMMPRIN and whereby increased collagen content and, consequently, improved fibrous cap thickness and plaque stability (Chen et al. 2014). Overall, these results indicate that berberine inherently has the potential to protect and ameliorate the progression of atherosclerotic plaques. Since there is no clinically approved lipid-lowering agent that can target atherosclerotic plaques and locally inhibit the function of cellular and molecular mediators in lesion progression, these findings underscore the therapeutic importance of berberine for preventing and treating atherosclerosis. However, low bioavailability of berberine can influence such beneficial effects, and, therefore, further preclinical studies are required to determine its effectual derivatives or administration routes, such as intravenous injection, for providing an efficient anti-atherosclerotic therapeutic tool.

5 Conclusion Berberine is found to be a promising anti-atherogenic agent protecting against atherosclerosis progression, as it can modulate cholesterol metabolism and hemostasis and the other important atherogenic factors directly involved in atherosclerotic lesion formation. The majority of findings consistently demonstrate that the lipidlowering effect of berberine is mainly in term of reducing TC and LDL-C through several LDLR-dependent and LDLR -independent mechanisms. Berberine decreases plasma levels of LDL-C through upregulating the mRNA and protein expression of the hepatic LDLR via two distinct mechanisms, including evaluation of LDLR mRNA stability and inhibition of PCSK9, a main negative regulator of LDLR protein. Besides the LDLR-mediated mechanism, berberine also can impact cholesterol hemostasis through regulating the other important factors, including liver biosynthesis of cholesterol, intestinal absorption of dietary cholesterol, as well as bile acid synthesis and secretion, and secretion of free cholesterol. Berberine has been also found to have anti-inflammatory and anti-oxidant abilities, whereby it can modulate function and proliferation of inflammatory macrophages, VSMC, and endothelial cells that directly collaborate in atherosclerotic lesion formation.

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Among the signaling pathways through which berberine regulates intracellular processes, AMPK has a central and critical role. Interestingly, activated AMPK contributes to various mechanisms involved in the atheroprotective effect of berberine, including suppressing cholesterol biosynthesis, improving endothelial dysfunction via upregulating the expression of eNOS and downregulating the expression of NOX4, as well as blocking injury-stimulated VSMCs regrowth and proliferation. Acknowledgments The authors appreciate the cooperation of Departments of Medical Immunology and Medical Biotechnology of Mashhad University of Medical Sciences. Conflicts of Interest The authors declare that there are no conflicts of interest and financial support for the present review article.

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