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A Textbook for Vascular Specialists

Robert FitridgeEditor Third Edition

Mechanisms of Vascular Disease

Mechanisms of Vascular Disease

Robert FitridgeEditor

Mechanisms of Vascular DiseaseA Textbook for Vascular Specialists

3 rd Edition

ISBN 978-3-030-43682-7 ISBN 978-3-030-43683-4 (eBook)https://doi.org/10.1007/978-3-030-43683-4

© Springer Nature Switzerland AG 2007, 2011, 2020This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, 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 AGThe registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

EditorRobert FitridgeDiscipline of SurgeryThe University of AdelaideAdelaide, SAAustralia

https://doi.org/10.1007/978-3-030-43683-4

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Contents

1 Vascular Endothelium in Health and Disease . . . . . . . . . . . . . . . . . . . 1Ran Wei, Paul M. Kerr, Stephen L. Gust, Raymond Tam, and Frances Plane

2 Pathophysiology of Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Sanuja Fernando, Christina A. Bursill, Stephen J. Nicholls, and Peter J. Psaltis

3 Mechanisms of the Vulnerable Atherosclerotic Plaque and Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Khizar Rana, Stephen J. Nicholls, and Johan W. Verjans

4 Current and Emerging Therapies for Atherosclerosis . . . . . . . . . . . . 71Adam J. Nelson and Stephen J. Nicholls

5 Pathophysiology of Angiogenesis and Its Role in Vascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Nathan K. P. Wong, Emma L. Solly, Christina A. Bursill, Joanne T. M. Tan, and Martin K. C. Ng

6 Vascular Biology of Smooth Muscle Cells and Restenosis . . . . . . . . . 117Victoria Nankivell, Khalia Primer, Achini Vidanapathirana, Peter Psaltis, and Christina Bursill

7 Vascular Haemodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Shirley Jansen, Michael Lawrence-Brown, Siamak Mishani, Christopher Lagat, Brian Evans, Kurt Liffman, and Ilija D. Šutalo

8 Computational Fluid Dynamics in the Arterial System: Implications for Vascular Disease and Treatment . . . . . . . . . . . . . . . . 171Siamak Mishani, Shirley Jansen, Michael Lawrence-Brown, Christopher Lagat, and Brian Evans

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9 Physiological Haemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Simon McRae

10 Hypercoagulable States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215Simon J. McRae

11 Platelets in the Pathogenesis of Vascular Disease and Their Role as a Therapeutic Target . . . . . . . . . . . . . . . . . . . . . . . . 233James McFadyen and Karlheinz Peter

12 Abdominal Aortic Aneurysm Pathology and Progress Towards a Medical Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263Joseph V. Moxon, Smriti M. Krishna, Tejas P. Singh, and Jonathan Golledge

13 Pathophysiology and Principles of Management of Hereditary Aneurysmal Aortopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293Mèlanie H. A. M. Perik, Aline Verstraeten, and Bart L. Loeys

14 Pathophysiology, Classification and Principles of Management of Acute Aortic Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317Mark Hamilton

15 Biomarkers in Vascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341Ashraf Cadersa and Ian M. Nordon

16 Pathophysiology and Principles of Management of Vasculitis and Fibromuscular Dysplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361Maureen Rischmueller, Sarah Downie-Doyle, and Robert Fitridge

17 Sepsis and Septic Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395Benjamin Reddi

18 Pathophysiology of Reperfusion Injury . . . . . . . . . . . . . . . . . . . . . . . . 415Prue Cowled and Robert Fitridge

19 Abdominal Compartment Syndrome and Open Abdomen Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441Martin Björck

20 Pathophysiology and Management of Limb Compartment Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455David Lindström and Carl-Magnus Wahlgren

21 Pathophysiology of Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469Stephan A. Schug

22 Post Amputation Pain Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489Stephan A. Schug

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23 Treatment of Neuropathic Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505Stephan A. Schug

24 Pathophysiology of Varicose Veins, Chronic Venous Insufficiency and Venous Ulceration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525Manj S. Gohel

25 Pathophysiology of Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . 541Stuart J. Mills, Ben R. Hofma, and Allison J. Cowin

26 Pathophysiology and Principles of Management of the Diabetic Foot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563Guilherme Pena, David G. Armstrong, Joseph L. Mills, and Robert Fitridge

27 Lymphoedema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593Matt Waltham and Kristiana Gordon

28 Graft Materials: Present and Future . . . . . . . . . . . . . . . . . . . . . . . . . . 621Mital Desai and George Hamilton

29 Vascular Graft Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653Mauro Vicaretti

30 Radiation Physics and Biological Effects of Radiation in Vascular Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671Joseph Dawson and Stephan Haulon

31 Radiation Stewardship: Radiation Exposure, Protection and Safety in Contemporary Endovascular Practice . . . . . . . . . . . . . 695Joseph Dawson and Stephan Haulon

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731

Contents

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Abbreviations

18 F-FDG Fluorodeoxyglucose18 F-FDG PET-CT 18F-fluorodeoxyglucose positron emission tomography/

computed tomography18 F-NaF Sodium fluoride3 D-IF 3D-image fusion5 -HT 5-Hydroxytryptamine (also known as serotonin)α-SMA Alpha-smooth muscle actinAAA Abdominal aortic aneurysmAAS Acute aortic syndromeAASV Anterior accessory saphenous veinsAAV ANCA-associated vasculitidesABC Automatic brightness controlABCA1 ATP-binding cassette transporter A1ABCG1 ATP-binding cassette transporter G1ABI Ankle-brachial indexAC Abdominal complianceACE Angiotensin-converting enzymeACS Acute coronary syndromeACS Abdominal compartment syndromeACT Acceptance and commitment therapyActa2 Smooth muscle α-actinACTA2 Actin alpha 2 geneADA2 Adenosine deaminase-2ADAMTS13 Disintegrin and metalloprotease with a thrombospondin

type 1 motif, member 13ADAMTS3 Disintegrin and metalloprotease with thrombospondin

motifs-3 proteaseADP Adenosine diphosphateAGE Advanced glycated end-productsAK Above-kneeAKI Acute kidney injury

x

ALARA As low as reasonably achievableALCS Acute limb compartment syndromeAMP Adenosine monophosphateAMPA α-Amino-3 hydroxy-5-methylisoxazole receptorsANA Antinuclear antibodiesANCA Anti-neutrophil cytoplasmic antibodyAng AngiopoietinAngII Angiotensin IIAnti-GBM Anti-glomerular basement membrane diseaseAP Antero-posteriorAP-1 Activating protein-1APC Activated protein CAPLAS Antiphospholipid antibody syndromeapoA-I Apolipoprotein A-IapoB Apolipoprotein BApoE Apolipoprotein EAPP Abdominal perfusion pressureARCL1 Autosomal recessive cutis laxa type 1ARDS Acute respiratory distress syndromeASCVD Atherosclerotic cardiovascular diseaseAT AntithrombinATP Adenosine triphosphateATS Arterial tortuosity syndromeAVG Arteriovenous graftsAZA AzathioprineBAPN 3-AminopropionitrileBAV Bicuspid aortic valveBC Boundary conditionsBC Bacterial celluloseBCL-2 B-cell lymphoma-2BCRL Breast cancer-related lymphoedemaBD Behcet’s diseaseBEIR VII Biological Effects of Ionizing Radiation VII CommitteeBEVAR Branched endovascular aneurysm repairBGN Biglycan geneBK Below-kneeBKCa Large-conductance Ca2+-activated K+ channelBMI Body mass indexBMP Bone morphogenetic proteinBNC Bacterial nanocelluloseBTK Bruton tyrosine kinaseBVS Bioresorbable vascular scaffoldC-ANCA Cytoplasmic anti-neutrophil cytoplasmic antibodyCAA Coronary artery aneurysmsCAC Coronary artery calcification

Abbreviations

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CAD Coronary artery diseaseCAD Computer-aided designCAK Cumulative air kermaCAM CalmodulincAMP Cyclic adenosine monophosphateCBCT Cone-beam CTCBD CannabidiolCCA Clonal chromosomal abnormalitiesCCK CholecystokininCCL5 C-C motif chemokine ligand 5CCTA Coronary computed topographic angiographyCD36 Cluster of differentiation-36Cdc42 Cell division cycle 42CDs Clusters of differentiationCE-MRA Contrast-enhanced magnetic resonance angiographyCEA Carotid endarterectomyCEAP Clinical, etiological, anatomical, pathophysiologicalCEMRI Contrast-enhanced magnetic resonance imagingCETP Cholesteryl ester transfer proteinCFD Computational fluid dynamicscGMP Cyclic guanosine monophosphateCGRP Calcitonin gene-related peptideCHCC Chapel Hill Consensus ConferenceCKD Chronic kidney diseaseCLI Critical limb ischaemiaCMR Cardiac magnetic resonanceCNC Cardiac neural crestCNCP Chronic noncancer paincNOS Constitutive nitric oxide synthaseCNS Central nervous systemCO Carbon monoxideCOX Cyclo-oxygenaseCOX-1 Cyclooxygenase-1COX-2 Cyclo-oxygenase-2CPIs Checkpoint inhibitorsCRC Colorectal cancerCRM Crew resource management (CRM)CRP C-Reactive proteinCRPS Complex regional pain syndromesCRRETAWAC Cysteine‐arginine‐arginine‐glutamic acid‐threonine‐

alanine‐tryptophan‐cysteine peptideCSE Cystathionine γ-laseCT Computerised tomographyCTA Coronary tomography angiographyCTLA4 Cytotoxic T-lymphocyte-associated protein 4

Abbreviations

xii

CV Cryoglobulinaemic vasculitisCVD Cardiovascular diseasesCXCL1 Chemokine (C-X-C motif) ligand 1CXCL16 C-X-C motif chemokine 16CyA Cyclosporin ACYC CyclophosphamideCYP CytochromeDAG DiacylglycerolDAMPs Danger (or damage)-associated molecular patternsDAP Dose area productDAPT Dual anti-platelet therapyDEJ Dermo-epidermal junctionDFI Diabetic foot infectionDFO Diabetic foot osteomyelitisDFU Diabetic foot ulcersDICOM Digital imaging and communications in medicineDIT Diffuse intimal thickeningDL Decompression laparotomyDLL4 Delta-like ligand 4DLT Decongestive lymphatic therapyDM Diabetes mellitusDMARDs Disease modifying antirheumatic drugsDN Diabetic neuropathyDOAC Direct oral anticoagulantDOF Degrees of freedomDREZ Dorsal root entry zoneDRG Dorsal root ganglionDRLs Diagnostic reference levelsDSA Digital subtraction angiographyDSCT Dual-source CTDTS Dense tubular systemDUS Duplex ultrasonographyDVT Deep vein thrombosisEC Endothelial cellECM Extracellular matrixECMO Extracorporeal membrane oxygenationED Effective doseEDCF Endothelium-derived contracting factorEDH Endothelium-dependent hyperpolarisationEDHF Endothelium-derived hyperpolarising factorEDS Ehlers-Danlos SyndromeEDV End-diastolic velocityEETs Epoxyeicosatrienoic acidsEFEMP2 Fibulin-4 geneEFNS European Federation of Neurological Societies

Abbreviations

xiii

EGDT Early goal-directed therapyEGF Epidermal growth factorEGPA Eosinophilic granulomatosis with polyangiitisELAM-1 Endothelial-leukocyte adhesion moleculeELG Endoluminal graftsELISA Enzyme-linked immunosorbent assayEMR Electromagnetic radiationeNOS Endothelial nitric oxide synthaseEPC Endothelial progenitor cellEPCR Endothelial protein C receptorEPS Extracellular polymer substancesePTFE Expanded polytetrafluoroethyleneERK Extracellular signal-regulated kinaseESR Erythrocyte sedimentation rateET Essential thrombocytosisET-1 Endothelin-1EULAR European League Against RheumatismEVAR Endovascular aneurysm repairFBLN4/EFEMP2 Fibrillin-4FBN1 Fibrillin-1 geneFDA Food and Drug AdministrationFDPs Fibrin degradation productsFEA Finite element analysisFEVAR Fenestrated endovascular aneurysm repairFGF Fibroblast growth factorFLNA Filamin A geneFMD Fibromuscular dysplasiaFOV Field of viewsFOXE3 Forkhead box E3 geneFSI Fluid–solid interactionFSP Fluorosurfactant polymerFT Fluoroscopy timeFTAAD Familial thoracic aortic aneurysm dissectionFVL Factor V LeidenG-CSF Granulocyte colony-stimulating factorGABA γ-Aminobutyric acidGCA Giant cell arteritisGI GastrointestinalGLP-1 Glucagon-like peptide-1GLUT10 Glucose transporter 10Gly GlycineGM-CSF Granulocyte-macrophage colony- stimulating factorGP GlycoproteinGPA Granulomatosis with polyangiitisGPCR G Protein-coupled receptor

Abbreviations

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GPELF Global Programme to Eliminate Lymphatic FilariasisGPI GlycosylphosphatidylinositolGs-IVUS Greyscale IVUSGSM Greyscale medianGSV Great saphenous veinGTPase Guanosine triphosphataseGWAS Genome-wide association studiesGy GrayH2O2 Hydrogen peroxideH2S Hydrogen sulphideHARM Hyperintense acute reperfusion injury markerHBOT Hyperbaric oxygen therapyHBV Hepatitis B virusHCV Hepatitis C virusHDL High-density lipoproteinsHDL-C High-density lipoprotein cholesterolHES Hydroxyethyl starchHETE Hydroxyeicosatetraenoic acidHIF Hypoxia-inducible factorHIT Heparin-induced thrombocytopeniaHIV Human immunodeficiency virusHLA Human leucocyte antigenHMGB1 High-mobility group box 1HMW High molecular weightHMWK High-molecular-weight kininogenHO Haem oxygenaseHPETE Hydroperoxyeicosatetraenoic acidhs-CRP High-sensitivity C-reactive proteinHSPs Heat shock proteinsHU Hounsfield unitHUV Human umbilical veinHUV Hypocomplementaemic urticarial vasculitisI/R Ischaemia-reperfusionIA Intussusceptive angiogenesisIAH Intra-abdominal hypertensionIAP Intra-abdominal pressureIASP International Association for the Study of PainIB-IVUS Integrated backscatter IVUSICA Internal carotid arteryICAM-1 Intercellular adhesion molecule-1ICD-11 International Classification of DiseasesICG Indocyanine greenICRP International Commission on Radiation ProtectionICU Intensive care unitICV Immune complex vasculitides

Abbreviations

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IDL Intermediate-density lipoproteinsIFC-ACS ACS due to intact fibrous capsIFN InterferonIFN-γ Interferon gammaIFX InfliximabIgAV Immunoglobulin-A vasculitisIGF Insulin-like growth factorIgG4-RD IgG4-related diseaseIH Intimal hyperplasiaIIF Indirect immunofluorescenceIKCa Intermediate-conductance Ca2+-activated K+ channelsIL InterleukinIL-6 Interleukin-6IL-6R Interleukin-6 receptorIle IsoleucineIM Internal membraneiMAP-IVUS iMAP-intravascular ultrasoundIMH Intra-mural haematomaIMP Inosine monophosphateiNOS Inducible nitric oxide synthaseInsP3 Inositol 1,4,5-trisphosphateIP ProstanoidIP3 Inositol (1,4,5) trisphosphateIP3 Inositol trisphosphateIPH Intraplaque haemorrhageIRAD International Registry of Acute Aortic DissectionirAEs Immune-related adverse eventsIRI Ischaemia-reperfusion injuryIRP Interventional reference pointITPKC Inositol-triphosphate 3-kinaseIVC Inferior vena cavaIVIG Intravenous immunoglobulinIVUS Intravascular ultrasoundIWGDF International Working Group in Diabetic FootJAK Janus kinasesKAP Kerma area productKATP Adenosine triphosphate-sensitive K+KD Kawasaki diseaseKLF Krüppel-like factorLAO Left anterior obliqueLDL Low-density lipoproteinLDL-C Low-density lipoprotein cholesterolLDLR Low-density lipoprotein receptorLDS Loeys-Dietz syndromeLECs Lymphatic endothelial cells

Abbreviations

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LEF LeflunomideLeu LeucineLMWH Low-molecular-weight heparinLNP Localised neuropathic painLOPS Loss of protective sensationLOX Lysine 6 oxidase (lysyl oxidase)LOX-1 Lectin-like low-density lipoprotein receptor-1Lp-PLA2 Lipoprotein-associated phospholipase A2LP(a) Lipoprotein-(a)LPS LipopolysaccharideLR-NC Lipid-rich necrotic coreLTA Light transmittance aggregometryLVA Lymphatico-venous anastomosisLVV Large vessel vasculitisM-CSF Macrophage colony-stimulating factorMAC Medial arterial calcificationMAC-1 Macrophage-1 antigenMACE Major adverse cardiovascular eventsMAGP2 Microfibril-associated glycoprotein 2MAP Mean arterial pressureMAPK Mitogen-activated protein kinasesMAT2α Methionine adenosyltransferase IIαMCP-1 Monocyte chemotactic protein-1MCSV Multiple cross-sectional viewsMDCT Multidetector-row CTMEFV Mediterranean fever geneMEKK1 Mitogen-activated protein kinase 1MFAP5 Microfibrillar-associated protein 5 geneMFS Marfan syndromeMGP Matrix gla-proteinMH1/2 Mad-homology 1 and 2MHC Major histocompatibility complexMHC-II Class II major histocompatibility complexMhy11 Smooth muscle myosin heavy chainMI Myocardial infarctionMIF Macrophage inhibitory factorMIP-1α Macrophage inflammatory protein 1-alphaMIP2 Macrophage inflammatory protein 2miR MicroRNAsMLC20 Myosin light chain proteinMLCK Myosin light chain kinaseMLCP Myosin light chain phosphataseMLD Manual lymphatic drainageMLS Meester-Loeys syndromeMMF Mycophenolate mofetil

Abbreviations

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mmHg Millimetres of mercuryMMP Matrix metalloproteinasemmPb LE Lead equivalenceMOD Multi-organ dysfunctionMPa Mega-PascalMPA Microscopic polyangiitisMPN Myeloproliferative neoplasmsMPO MyeloperoxidaseMRA Magnetic resonance angiographyMRI Magnetic resonance imagingMRL Magnetic resonance lymphangiographyMRSA Methicillin-resistant S. aureusMRTA Magnetic resonance tomographic angiographyMTHFR Methylenetetrahydrofolate reductaseMTX MethotrexateMVV Medium vessel vasculitisMyD88 Myeloid differentiation primary response 88MYH11 Smooth muscle myosin heavy chainMYLK Smooth muscle myosin light chain kinaseNADPH Nicotinamide adenine dinucleotide phosphateNaV1.7, NaV1.8 and NaV1.9

Voltage-gated sodium channels

NCP Non-calcified plaquesNETs Neutrophil extracellular trapsNeuPSIG Neuropathic Pain Special Interest Group of the IASPNFκB Nuclear factor kappa-light-chain-enhancer of

activated B-cellsNGAL Neutrophil gelatinase-associated lipocalinNGF Nerve growth factorNIR Near-infrared lymphangiographyNIRAF Near-infrared autofluorescenceNIRF Intravascular near-infrared fluorescenceNIRS Near-infrared spectroscopyNK-1 NeurokininNMB Neuro-muscular blockadeNMDA N-methyl d-aspartateNNH Number needed to harmNNT Number needed to treatNO Nitric oxideNOD Nucleotide-binding and oligomerization domainNOS Nitric oxide synthaseNPWT Negative pressure wound therapyNSAIDs Non-steroidal anti-inflammatory drugsNSCLC Non-small cell lung cancerNSTEACs Non-ST elevated acute coronary syndromes

Abbreviations

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NSTEMI Non-ST segment elevation myocardial infarctionO2- Superoxide anionOA Open abdomenOCI Operator-controlled imagingOCT Optical coherence topographyOH• Hydroxyl radicalOM OsteomyelitisOPG OsteoprotegerinOPN OsteopontinOR Odds ratioOSL Optically stimulated luminescenceOSR Open surgical repairOxLDL Oxidised low-density lipoproteinsP-ANCA Perinuclear anti-neutrophil cytoplasmic antibodyPAD Peripheral artery diseasePAF Platelet-activating factorPAI-1 Plasminogen activator inhibitor-1PAIs Plasminogen activator inhibitorsPAMPs Pathogen-associated molecular pathogensPAN Polyarteritis nodosaPARs Protease-activated receptorsPASV Posterior accessory saphenous veinsPAU Penetrating aortic ulcerPBT Probe to bone testPC Protein CPCI Percutaneous coronary intervention (angioplasty)PCL PolycaprolactonePCSK9 Proprotein convertase subtilisin/kexin type 9PCT ProcalcitoninPD-1 Programmed cell death protein-1PD-L1 Programmed cell death ligand-1PDE-5 Phosphodiesterase type 5PDGF Platelet-derived growth factorPDGFB Platelet-derived growth factor BPDGFRβ Platelet-derived growth factor receptor βPECAM-1 Platelet endothelial cell adhesion molecule-1PEG Polyethylene glycolPET Positron emitting topographyPGA Polyglycolic acidPGI2 Prostaglandin I2/ProstacyclinPGM Prothrombin gene mutationPHD Prolyl hydroxylase domainPHN Postherpetic neuralgiaPHZ Para-anastomotic hyper-compliant zonePI3K Phosphatidylinositol 3-kinase

Abbreviations

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PIP2 Phosphatidylinositol 4,5-bisphosphatePKC Protein kinase CPLA Poly-lactic acidPLC Phospholipase CPLGF Placental growth factorPLLA Poly-l-lactic acidPLP Phantom limb painPMR Plaque-to-myocardial signal intensity ratioPMR Polymyalgia rheumaticaPNH Paroxysmal nocturnal haemoglobinuriaPPAR-γ Peroxisome proliferator-activated receptor gammaPPARα Peroxisome proliferator-activated receptor αPPI Proton-pump inhibitor (e.g. omeprazole)PRKG1 Type I cGMP-dependent protein kinase genePRP Primary Raynaud’s phenomenonPRTN3 Proteinase 3 genePRV Polycythaemia rubra veraPS Protein SPSC Posterior subcapsular cataractsPSD Peak skin dosePSGL P-Selectin glycoprotein ligand-1pSS Sjögren’s syndromePSV Peak systolic velocityPTA Percutaneous transluminal angioplastyPTFE PolytetrafluoroethylenePTS Post thrombotic syndromePU PolyurethanePVNH Periventricular nodular heterotopiaRAAA Ruptured abdominal aortic aneurysmRac-1 Ras-related C3 botulinum toxin substrate 1RAGE Receptors for advanced glycated end productsRAK Reference air kermaRANTES Regulated upon activation, normal T-cell expressed

and secretedRAO Right anterior obliqueRCC Renal cell carcinomaRCTs Randomised controlled trialsRFC-ACS ACS due to ruptured fibrous capsRGD Arginine-glycine-aspartic acidRGDS Arginine-glycine-aspartic acid-serinerHDL Reconstituted high-density lipoproteinRIAM Rap1-GTP interacting adapter moleculeRLC Integrin subunit alpha 9RM Regenerative medicineROS Reactive oxygen species

Abbreviations

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RP Raynaud’s phenomenonRPAK Reference point air kermaRSD Reflex sympathetic dystrophyRTX RituximabSAA Serum amyloid ASAM S-AdenosylmethionineSAP Stable angina plaquesSARA SMAD anchor for receptor activation (involved in TGF-β

signalling)SBTE Sheet-based tissue engineeringSC Schwann cellsSCS Spinal cord stimulationSCVs Small colony variantsSDF Stromal cell-derived factorSDF-1α Stromal-derived growth factor-1αSEP Serum elastin peptideSERPINA1 Alpha-1-antitrypsin geneSFA Superficial femoral arterySGLT2 Sodium glucose cotransporter-2SGS Shprintzen-Goldberg syndromeSIRS Systemic inflammatory response syndromeSKCa Small-conductance Ca2+-activated K+ channelsSLC2A10 Solute carrier family 2 member 10 geneSLV Single longitudinal viewSMAD2/3 Receptor-regulated mothers against decapentaplegic

homologue 2 and 3 proteinsSMCs Smooth muscle cellsSMP Sympathetically maintained painSNPs Single nucleotide polymorphismsSNRIs Serotonin/noradrenaline reuptake inhibitorsSOFA Sequential [Sepsis-related] Organ Failure AssessmentSP Substance PSPP Skin perfusion pressureSR-A1 Scavenger receptor type 1SR-A2 Scavenger receptor type 2SR-PSOX Scavenger receptor for phosphatidylserine and oxidised

low-density lipoproteinSRB1 Scavenger receptor B1SRP Secondary Raynaud’s phenomenonSSC Surviving sepsis campaignSSRIs Selective serotonin reuptake inhibitorsSSV Small saphenous veinSTEMI ST-elevation myocardial infarctionSv SievertSvO2 Venous oxygen saturation

Abbreviations

xxi

SVV Small vessel vasculitisT-regs Regulatory T-cellsTA Temporal arteryTAAD Thoracic aortic aneurysm and dissectionTAAs Thoracic aortic aneurysmsTAB Temporal artery biopsyTAC Temporary abdominal closureTAD Thoracic aortic dissectionTAFI Thrombin-activatable fibrinolysis inhibitorTAK Takayasu arteritisTAO Thromboangitis obliteransTBAD Type B aortic dissectionTBI Toe:brachial pressure indexTCA Tricyclic antidepressantsTCC Total contact castTCFA Thin-cap fibroatheromaTcPO2 Transcutaneous oxygen tensionTCZ TocilizumabTE Tissue engineeringTEE Transesophageal echocardiograpyTENS Transcutaneous electrical nerve stimulationTESA Tissue engineering by self-assemblyTEVAR Thoracic endovascular aortic repairTEVG Tissue-engineered vascular graftTF Tissue factorTfh T follicular helper cellTFPI Tissue factor pathway inhibitorTGF-α Transforming growth factor-alphaTGF-β Transforming growth factor-betaTh T-helper cellTh1 T-helper type 1 lymphocyteTHC TetrahydrocannabinolTIA Transient ischaemic attackTIE-2 Tyrosine kinase with immunoglobulin-like and epidermal

growth factor-like domain-2TIMP-3 Tissue inhibitors of matrix metalloproteinase-3TIMPs Tissue inhibitors of metalloproteinasesTIR Toll/interleukin‐1 receptorTKIs Tyrosine kinase inhibitorsTLD Thermoluminescent dosimeterTLRs Toll-like receptorsTNF Tumour necrosis factorTNF-α Tumour necrosis factor-alphaTNFi Tumour necrosis factor inhibitorsTOE Trans oesophageal echocardiography

Abbreviations

xxii

TP ThromboxanetPA Tissue plasminogen activatorTPs Toe pressuresTreg T regulatoryTRIF Toll/interleukin‐1 receptor domain‐containing adaptor‐

inducing IFN‐βTrKa Tropomyosin receptor kinase ATRLs Triglyceride-rich lipoproteinsTRP Transient receptor potential ion channelTRPV1 Transient receptor potential vanilloid type 1 ion channelTTE Transthoracic echocardiographyTXA2 Thromboxane A2uPA Urokinase-type plasminogen activatorVACM Vacuum-assisted wound closure and mesh- mediated fascial

tractionvasDCs Vascular dendritic cellsVC Vascular calcificationVCAM-1 Vascular cell adhesion molecule 1VE-cadherin Vascular endothelial cadherinvEDS Vascular EDS (EDS type IV)VEGF Vascular endothelial growth factorVEGFA Vascular endothelial growth factor AVEGFR1 Vascular endothelial growth factor receptor 1VH-IVUS Virtual histology IVUSVLA-4 Very late antigen-4VLDL Very low-density lipoproteinsVRT Venous refilling timeVSMC Vascular smooth muscle cellVTE Venous thromboembolismvWF von Willebrand factorWBC White blood cellsWHO World Health OrganisationWIfI Wound, Ischaemia and foot Infection classification systemWSS Wall shear stress

Abbreviations

1© Springer Nature Switzerland AG 2020R. Fitridge (ed.), Mechanisms of Vascular Disease, https://doi.org/10.1007/978-3-030-43683-4_1

Chapter 1Vascular Endothelium in Health and Disease

Ran Wei, Paul M. Kerr, Stephen L. Gust, Raymond Tam, and Frances Plane

Key Learning Points• All vascular endothelial cells have a common embryonic origin but show clear

bed-specific heterogeneity in morphology, function, gene and protein expres-sion, determined by both environmental stimuli and epigenetic features acquired during development.

• Endothelial heterogeneity is also demonstrated by regional differences in the release of vasoactive and inflammatory mediators in response to stimuli such as changes in shear stress and hypoxia, and in expression of pro- and anti-coagulant molecules.

• The first identified endothelium-derived relaxing factor (nitric oxide; NO) is a short-lived free radical synthesised from L-arginine by endothelial NO synthase (eNOS) and destroyed by reactive oxygen species (ROS).

• Carbon monoxide (CO) and hydrogen sulphide (H2S) are two other gaseous mediators contributing to endothelium-dependent modulation of vascular tone.

• Endothelium-dependent hyperpolarisation (EDH) is an important regulator of blood flow and blood pressure and plays a predominant role in smaller vessels.

• Arachidonic acid is released from endothelial cell membrane phospholipids and metabolised into a number of vasoactive factors by cyclooxygenase (COX), lipoxygenase and cytochrome P450 monooxygenase enzymes.

• Endothelin-1 (ET-1) is a potent vasoconstrictor which is released from the vascu-lar endothelium. NO strongly inhibits the release of ET-1 and ET-1 attenuates

R. Wei · S. L. Gust · R. Tam · F. Plane (*) Department of Pharmacology, University of Alberta, Edmonton, AB, Canadae-mail: fplane@ualberta.ca

P. M. Kerr Faculty of Nursing, Robbins Health Learning Centre, MacEwan University, Edmonton, AB, Canada

http://crossmark.crossref.org/dialog/?doi=10.1007/978-3-030-43683-4_1&domain=pdfhttps://doi.org/10.1007/978-3-030-43683-4_1#DOImailto:fplane@ualberta.ca

2

NO-mediated dilation. Therefore, ET-1 and NO are functionally interdependent and many of the cardiovascular complications associated with endothelial dys-function may be due to an imbalance in this relationship.

• Angiogenesis is the growth of new blood vessels formed by endothelial cell tubes sprouting from existing vessels. This process involves the following sequence of events:

– Activation of endothelial cells – Degradation of extracellular matrix by matrix metalloproteinases – Proliferation and directional migration of endothelial cells – Formation of endothelial tubes – Maturation of new vessels by recruitment of pericytes and smooth muscle

cells to stabilise endothelial sprouts and secrete extracellular matrix mole-cules to form the vascular basement membrane

• Angiogenesis in response to ischaemia is largely controlled by hypoxia- inducible factor-1 (HIF-1). Recruitment and proliferation of bone marrow-derived endo-thelial progenitor cells to form new vessels (vasculogenesis) is a separate but complimentary process which occurs simultaneously in ischaemic and wound tissue to augment perfusion.

• The endothelium is one of the few surfaces that can maintain blood in a liquid state during prolonged contact. The endothelium also prevents thrombin forma-tion by expressing tissue factor pathway inhibitor that binds to clotting factor Xa and thrombomoduli, which in turn bind to and inactivate thrombin, thus blocking its pro-coagulant activity. Several other haemostatic factors are also expressed by the endothelium, in particular von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1).

• Vascular endothelium has potent anti-platelet aggregation properties which are mediated by the synthesis of prostacyclin (PGI2) and NO.

• The endothelial response to inflammation and infection involves the production of inflammatory cytokines (such as interleukin-8) and the expression of surface adhesion molecules (selectins), which facilitate leukocyte adhesion and migra-tion to the site of inflammation/infection.

1.1 Introduction

The endothelium, first described over 100 years ago as an inert anatomical barrier between the blood and cells of the vessel wall, is now recognized as a dynamic organ with secretory, synthetic, metabolic, and immunologic functions. Endothelial cells play an obligatory role in modulating vascular tone and permeability, angio-genesis, and in mediating haemostatic, inflammatory and reparative responses to local injury. To fulfil these roles, the endothelium is continuously responding to spatial and temporal changes in mechanical and biochemical stimuli. Such respon-siveness is affected through receptors for growth factors, lipoproteins, platelet

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products, and circulating hormones, which regulate changes in RNA and protein expression, cell proliferation, and migration or the release of vasoactive and inflam-matory mediators [1].

All vascular endothelial cells have a common embryonic origin but show clear bed-specific heterogeneity in morphology, function, and gene and protein expres-sion, determined by both environmental stimuli and epigenetic features acquired during development. Thus, the endothelium should not be regarded as an homoge-nous tissue but rather a conglomerate of distinct populations of cells sharing many common functions but also adapted to meet regional demands [2]. In most blood vessels, continuous endothelium provides an uninterrupted barrier between the blood and tissues and ensures tight control of permeability at the blood-brain bar-rier. In regions of increased trans-endothelial transport such as capillaries of endo-crine glands and the kidney, the presence of fenestrae, transcellular pores approximately 70 nm in diameter with a thin fenestral diaphragm across their open-ing, facilitate the selective permeability required for efficient absorption, secretion, and filtering. In hepatic sinuses, the presence of a discontinuous endothelium with large fenestrations (0.1–1 mm in diameter) lacking a fenestral diaphragm, provides a highly permeable and poorly selective sieve essential for transfer of lipoproteins from blood to hepatocytes [1, 3].

Beyond these structural variations, endothelial heterogeneity is also manifest in regional differences in the release of vasoactive and inflammatory mediators, in responsiveness to stimuli such as changes in shear stress and hypoxia, and in expres-sion of pro- and anti-coagulant molecules. For example, the contribution of nitric oxide (NO) to endothelium-dependent vasodilation appears to be greater in large conduit arteries compared to small resistance vessels [4]. Expression of von Willebrand factor (vWF), a circulating glycoprotein that mediates platelet adhesion to the subendothelial surface of injured blood vessels, displays a mosaic pattern in the aorta and in selected capillary beds [5]. These regional differences between endothelial cells extend to their susceptibility to injury in the face of cardiovascular risk factors such as hypercholesterolemia, diabetes and smoking, and thus impact the function of the vasculature both in health and disease.

This chapter provides an overview of how the endothelium regulates four key aspects of cardiovascular homeostasis; vascular tone, angiogenesis, haemostasis and inflammation.

1.2 Endothelium-Dependent Regulation of Vascular Tone

Since the first report of endothelium-dependent modulation of the contractile state of smooth muscle cells in the artery wall [6], it has become apparent that endothelial cells release a plethora of vasoactive factors in response to a wide range of mechani-cal and chemical stimuli. Many of these factors also modulate processes such as inflammation, cell adhesion, and coagulation, highlighting the crucial physiological role of the endothelium, and why endothelial dysfunction is pivotal in the

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development of cardiovascular diseases such as atherosclerosis and hypertension. Here we will focus on the four major pathways underlying endothelium-dependent modulation of vascular tone; the gaseous mediators NO, carbon monoxide (CO) and hydrogen sulfide (H2S), endothelium-dependent hyperpolarisation (EDH), metabo-lites of arachidonic acid, and endothelin.

1.2.1 Gaseous Mediators

The first endothelium-derived relaxing factor described by Furchgott and Zawadski was subsequently identified as NO, a short-lived free radical synthesized from L-arginine by endothelial NO synthase (eNOS) and destroyed by reactive oxygen species (ROS). Once released from the endothelium, NO activates the haem- dependent enzyme soluble guanylyl cyclase in surrounding smooth muscle cells, leading to formation of cyclic guanosine monophosphate (cGMP). Subsequent acti-vation of cGMP-dependent kinase leads to phosphorylation of a diverse range of target proteins such as large conductance Ca2+-activated K+ (BKCa) channels, Rho kinase, myosin light chain phosphatase and phospholamban, that mediate smooth muscle cell relaxation and hence vasodilation [7]. This signalling pathway is termi-nated by phosphodiesterase enzymes that degrade cGMP. These enzymes are inhib-ited by the drug sildenafil used for treatment of erectile dysfunction [8]. NO can also act in a cGMP-independent manner (e.g. nitrosylation of proteins), which will not be discussed here but has been reviewed by Lima et al. [9].

eNOS is a bidomain enzyme; an N-terminal oxygenase domain with binding sites for haem, tetrahydrobiopterin, oxygen and the substrate L-arginine which sup-ports catalytic activity, and also a C-terminal reductase domain which binds the co-factors nicotinamide adenine dinucleotide phosphate (NADPH), flavin mono-nucleotide and flavin adenine dinucleotide. Transfer of electrons from NADPH to flavins in the reductase domain and then to the haem in the oxygenase domain is required so that the haem iron can bind oxygen and catalyze the synthesis of NO from L-arginine. Binding of the ubiquitous Ca2+ regulatory protein calmodulin (CAM) facilitates transfer of electrons from the reductase to the oxygenase domain and is critical for activation of the enzyme [10].

eNOS is constitutively expressed in all endothelial cells but regulation of enzyme activity by physiological and pathophysiological stimuli occurs via a complex pat-tern of transcriptional and post-translational modifications. Both eNOS mRNA and protein levels are increased by fluid shear stress via activation of a pathway involv-ing c-Src-tyrosine kinase and transcription factor nuclear factor κ-light-chain- enhancer of activated B cells (NFκB). At the post-translational level, eNOS activity is highly regulated by substrate and cofactor availability as well as by endogenous inhibitors, lipid modification, direct protein-protein interactions, phosphorylation, O-linked glycosylation, and S-nitrosylation.

Agonists at endothelial G-protein coupled receptors (GPCRs) such as bradykinin and acetylcholine elicit Ca2+-CAM-dependent NO production via phospholipase

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C-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and subsequent release of Ca2+ from intracellular stores. Activation of tyrosine kinase linked recep-tors such as the vascular endothelial growth factor (VEGF) receptor, and mechani-cal stimulation of the endothelium by shear stress, lead to phosphorylation of eNOS at Ser1177 to increase the Ca2+ sensitivity of the enzyme so that it can be activated at resting Ca2+ levels. Distinct kinase pathways can mediate eNOS phosphorylation; shear stress elicits phosphorylation of Ser1177 via protein kinase A, whereas insulin and VEGF cause phosphorylation of the same residue via the serine/threonine pro-tein kinase Akt. Conversely, phosphorylation of the enzyme at Tyr657 within the flavin mononucleotide domain, or Thr495 within the CAM-binding domain, inhib-its enzyme activity [11].

Within endothelial cells, eNOS is targeted to invaginations of the cell membrane called caveolae, membrane microdomains enriched in cholesterol and sphingolip-ids, and defined by the presence of the scaffolding protein caveolin-1. Caveolae sequester diverse receptors and signalling proteins including GPCRs, growth factor receptors, and Ca2+ regulatory proteins such as CAM. Thus, targeting of eNOS to this region facilitates communication with upstream and downstream pathways. Within caveolae, caveolin-1 tonically inhibits eNOS activity, thereby limiting the production of NO. The binding of Ca2+-CAM leads to disruption of the caveolin-1/eNOS interaction and increases eNOS activity [12]. Other associated proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1) modulate eNOS activ-ity by virtue of their function as scaffolds for the binding of signalling molecules such as tyrosine kinases and phosphatases [13].

The release of NO by stimuli such as shear stress, circulating hormones (e.g. catecholamines, vasopressin), plasma constituents (e.g. thrombin), platelet prod-ucts (e.g. serotonin), and locally-produced chemical mediators (e.g. bradykinin) plays a critical role in mediating acute changes in  local blood flow and tissue perfusion. Shear stress-stimulated NO production is central to exercise-induced increases in blood flow in skeletal muscle [14]. Production of NO in response to serotonin released from aggregating platelets, dilates coronary arteries to prevent clots from occluding vessels [15]. Mice lacking eNOS are hypertensive and infu-sion of L-arginine analogues, competitive inhibitors of eNOS, cause alterations in  local blood flow and in systemic blood pressure, demonstrating the impor-tance of endothelium- derived NO in long-term cardiovascular control in  vivo [11]. In humans, elevated levels of an endogenous inhibitor of eNOS, asymmet-ric dimethylarginine, are associated with hypertension and increased cardiovas-cular risk [16].

In addition to its vasodilator actions, NO is now recognized as playing other protective roles in the vasculature as a regulator of inflammation and vessel repair. Loss of NO-mediated vasodilation, due to reduced expression or activity of eNOS and/or oxidative stress-mediated reductions in NO bioavailability, is a hallmark of endothelial dysfunction associated with cardiovascular risk factors such as hypercholesterolemia, smoking, diabetes and obesity. Loss of endothe-lium-derived NO tips the homeostatic balance in favour of vasoconstriction, proliferation, activation of platelets and blood clot formation, and inflammation.

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Therefore loss of NO contributes to clinical manifestations such as high blood pressure, atherosclerosis, and thrombosis, which are associated with significant morbidity and mortality [17].

Although they have received much less attention than NO, two other gaseous mediators, CO and H2S, also contribute to endothelium-dependent modulation of vascular tone. CO is generated within endothelial cells during catabolism of heme by the enzyme heme oxygenase (HO). Of the two known isoforms, HO1 is an inducible form associated with increased oxidative stress while HO2 is constitu-tively expressed in endothelial cells and, like eNOS, activated by Ca2+-CAM. The strongest evidence for a physiological role of endothelium-derived CO in regulation of vascular tone has come from studies of the cerebral circulation in which GPCR agonists and hypoxia cause Ca2+-CAM- and HO-dependent vasodilation. CO-mediated smooth muscle relaxation is due to activation of soluble guanylyl cyclase which increases cGMP levels, leading to cGMP-dependent kinase-mediated activation of BKCa channels. In addition, CO can bind directly to the heme moiety bound to BKCa channels to directly elevate Ca2+ sensitivity of the channels [18]. To date, little is known about the mechanisms regulating CO production or alterations in HO/CO signalling in disease states.

Release of endothelium-derived H2S, synthesized from cysteine by cystathio-nine γ-lase (CSE), is stimulated by many factors including acetylcholine, increases in shear stress, oestrogens, and plant flavonoids. Evidence from mice lacking CSE suggests an important role for H2S in the physiological maintenance of blood pressure [19]. Like eNOS, CSE activity is regulated by a complex integration of transcriptional, post-transcriptional, and post-translational mechanisms. The rapid metabolism of H2S via an oxygen-dependent pathway within mitochondria suggests that it may play a greater role in hypoxic rather than normoxic tis-sues [20].

Several signalling mechanisms have been described for H2S such as reaction with heme-containing proteins, protein S-sulfhydration, reaction with ROS, and reduction of protein disulfide bonds to thiols [21]. The vasodilator action of H2S has largely been attributed to hyperpolarization of the smooth muscle membrane poten-tial mediated by opening adenosine triphosphate-sensitive K+ (KATP) channels; H2S sulfhydrates KATP channels at Cys43 to facilitate the binding of phosphatidylinosi-tol(4,5)bisphosphate, a physiological activator of these channels [22]. However, the sensitivity of H2S-evoked vasodilation to blockers of small (SKCa) and intermediate (IKCa) conductance Ca2+-activated K+ channels has also raised the possibility that H2S may evoke relaxation via EDH [23].

Interactions between NO and H2S signalling pathways can occur at a number of points, making for a complex relationship between the two mediators; NO inhibits CSE activity via S-nitrosation but can increase CSE expression and cel-lular uptake of its substrate cystine, and conversely, H2S potentiates cGMP accu-mulation via the inhibition of phosphodiesterase. Alterations in H2S production and/or signalling have been observed in animal models of endothelial dysfunction associated with pathological conditions such as diabetes and obesity, leading to H2S donors being considered as potential therapeutic agents for the treatment of these diseases [24].

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1.2.2 Endothelium-Dependent Hyperpolarisation

Observations of agonist-induced endothelium-dependent vasorelaxation which per-sisted in the presence of inhibitors of COX and eNOS and was accompanied by hyperpolarisation  of the vascular smooth muscle cell membrane potential led to identification of a third endothelium-derived relaxing factor, endothelium-derived hyperpolarising factor (EDHF). Hyperpolarisation of the smooth muscle cells reduces the probability of opening the voltage-dependent Ca2+ channels, thus reduc-ing Ca2+ influx to cause relaxation. A range of agents have been proposed to account for the actions of EDHF including K+, epoxyeicosatrienoic acids (EETs) and C-type natriuretic peptide. However, it is now widely accepted that, rather than a diffusible factor, endothelium-dependent hyperpolarisation (EDH) of vascular smooth muscle is mediated by direct electrical coupling between endothelial and smooth muscle cells via myoendothelial gap junctions [25].

The initiating step in EDH-mediated vasorelaxation is activation of endothelial SKCa and IKCa channels. These channels, activated by increases in intracellular Ca2+ via CAM which is constitutively associated with the channels, are voltage- independent and thus can operate at negative membrane potentials close to the K+ equilibrium potential. Inhibition of endothelium-dependent relaxation by a combination of SKCa/IKCa channel blockers is now regarded as the hallmark of EDH- mediated vasodilation, but the relative contribution of the two channels varies between different stimuli. This ability of endothelial cells to generate stimulus- specific responses to diverse inputs is facilitated by organization of SKCa and IKCa channels into spatially distinct microdo-mains that allow for differential activation by localized increases in Ca2+. SKCa chan-nels are located within caveolae at inter- endothelial junctions on the luminal surface where together with eNOS, they can respond to local Ca2+ increases elicited by shear stress-induced activation of transient receptor potential vanilloid type 4 (TRPV4) channels. In contrast, IKCa channels are located on the abluminal surface close to myoendothelial contact points where they are activated by localized, InsP3-mediated increases in Ca2+ evoked by GPCR agonists (Fig. 1.1) [26].

Fig. 1.1 Schematic showing differential localization and activation of endothelial SKCa and IKCa channels. SKCa channels are located on the luminal surface and respond to local Ca2+ increases elicited by shear stress-induced activation of TRPV4 channels. IKCa channels are located on the abluminal surface where they are activated by localized, InsP3-mediated increases in Ca2+ evoked by GPCR agonists

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The importance of EDH as a regulator of blood flow and blood pressure in vivo is demonstrated by enhanced resistance artery tone and elevated systemic blood pressure seen in mice lacking endothelial SKCa and/or IKCa channels. Loss of EDH, due to changes in expression or activity of SKCa/IKCa channels, contributes to exper-imental hypertension and diabetes-related erectile dysfunction. In contrast, resis-tance of the EDH pathway to the deleterious actions of ROS may allow EDH-mediated vasodilation to be maintained in the face of reduced bioavailability of NO in athero-sclerosis and heart failure. Thus, selective activation of endothelial SKCa and IKCa channels is a potential therapeutic avenue for the future [27].

1.2.3 Metabolites of Arachidonic Acid

Within endothelial cells, arachidonic acid, released from cell membrane phospholip-ids by phospholipases, is metabolized by COX, lipoxygenase (LO), and cytochrome P450 monooxygenase (CYP) enzymes to yield an array of vasoactive factors.

COX enzymes metabolise arachidonic acid to endoperoxide intermediates which are then converted to a range of eicosanoids (e.g. prostacyclin (PGI2), thromboxane A2 (TXA2)) through the actions of various synthases. Two isoforms of COX are found in the endothelium. The constitutively expressed COX-1 has long been regarded as vas-culoprotective, the predominant product being PGI2 which acts on prostanoid (IP) receptors to cause vasodilation and inhibition of platelet aggregation via activation of adenylyl cyclase to increase levels of cyclic-adenosine monophosphate (cAMP). PGI2 also inhibits platelet and lymphocyte adhesion to endothelium, limits vascular smooth muscle cell proliferation and migration, and counteracts the production of pro-inflam-matory growth factors [17]. However, evidence is now emerging that GPCR-mediated activation of endothelial COX-1 can generate other products such as TXA2 which activates thromboxane (TP) receptors on smooth muscle cells and so functions as an endothelium-derived contracting factor (EDCF). Stimulation of TP receptors elicits not only vasoconstriction but also proliferation of vascular smooth muscle cells, plate-let adhesion and aggregation, and expression of adhesion molecules on endothelial cells. A shift from production of endothelium- derived relaxing factors to COX-dependent EDCFs is implicated in endothelial dysfunction associated with ageing, diabetes, and hypertension in both animal models and humans. Since activation of TP receptors is the common downstream effector, selective antagonists of this receptor may have therapeutic potential in the treatment of cardiovascular diseases [28].

COX-2 was first identified as an inducible form of the enzyme regulated at the level of gene expression and associated with inflammation. However, it is expressed in some blood vessels in the absence of overt signs of inflammation, and may be a major source of vasculoprotective PGI2; hence the deleterious cardiovascular conse-quences seen in some patients treated with selective COX-2 inhibitors [29].

LO enzymes deoxygenate polyunsaturated fatty acids to hydroperoxyl metabolites. The three LO isoforms expressed in endothelial cells are 5-LO, 12-LO, and 15-LO, which correspond to the carbon position of arachidonic acid oxygenation. Each LO oxygenates arachidonic acid to form a stereospecific hydroperoxyeicosatetraenoic

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acid (HPETE) which are unstable and rapidly reduced to the corresponding hydroxye-icosatetraenoic acid (HETE). 5-LO is the initial enzyme in the synthesis of pro-inflammatory leukotrienes but 5-LO products do not seem to be involved in regulation of vascular tone. In contrast, products from the 12-LO and 15-LO pathways are vaso-active but show species and vessel variation in the responses they elicit. 12-HETE causes relaxation of a number of peripheral arteries including human coronary arteries but causes vasoconstriction in dog renal arteries. In the same vessels, 15-HPETE and 15-HETE cause slight vasorelaxation at lower concentrations but contractions at higher concentrations mediated by activation of TP receptors.

Cytochrome P450 (CYP) enzymes add oxygen across the double bonds of ara-chidonic acid to produce four cisepoxides, 14,15-, 11,12-, 8,9-, and 5,6- epoxyeicosatrienoic acids (EETs). Two CYP enzymes have been cloned from human endothelium, CYP2C8/9 and CYP2J2, both of which produce mainly 14,15- EET with lesser amounts of 11,12-EET. The latter are also the major EETs released from endothelial cells in response to GPCR agonists (e.g. acetylcholine, bradykinin) and physical stimuli such as cyclic stretch and shear stress. EETs are rapidly metab-olized by esterification into phospholipids or hydration to dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. EETs can cause vasodilation via a number of different pathways. They can stimulate endothelial TRPV4 channels, which are non-selective cation channels that mediate Ca2+ influx, to activate eNOS or IKCa/SKCa channels to cause EDH. In contrast, endothelium-dependent flow-induced dilation is linked to release of 5,6-EET to activate smooth muscle TRPV4 channels which form a complex with BKCa channels, thus coupling local increases in Ca2+ to membrane hyperpolarisation and vasorelaxation [30]. Development of 14,15-EET analogues such as 14,15-epoxyeicosa-5Z-enoic acid revealed strict structural and stereoiso-meric requirements for relaxations suggesting a specific binding site or receptor mediating EETs actions. G protein coupled receptor 40 (GPR40), a member of a family of GPCRs that have fatty acids as ligands, was recently detected in endothe-lial and smooth muscle cells, and suggested to be a low-affinity EET receptor. 11,12-EET stimulation of GPR40 increased expression of COX-2 and connexin 43, a key component of myoendothelial gap junctions, but a role for this receptor in EET-mediated changes in vascular tone remains to be established [31].

In some models of endothelial dysfunction reduced bioavailability of NO is counteracted by increased production of EETs which can maintain endothelium- dependent vasodilator responses. Thus, strategies aimed at enhancing production of endothelium-derived EETs or inhibiting their degradation, may represent a new therapeutic approach to endothelial dysfunction [32].

1.2.4 Endothelins

Endothelins are a family of 21 amino acid peptides of which there are three mem-bers (ET-1, ET-2, ET-3) with a high level of homology and similar structure [33]. Endothelial cells produce only ET-1; endothelin ET-2 is produced in the kidney and

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intestine, while ET-3 has been detected in the brain, gastrointestinal tract, lung and kidney. ET-1 is a potent vasoconstrictor inducing long-lasting vasoconstriction at a half maximum effective concentration in the nano-molar range, at least one order of magnitude lower than values reported for other vasoconstrictor peptides such as angiotensin II [34].

ET-1 is not stored by endothelial cells. Production is regulated at the level of gene expression with the rate of transcription being responsive to stimulants and inhibitors to allow rapid changes in the amounts released. Pro-inflammatory factors such as transforming growth factor-β and tumour necrosis factor–α (TNFα), insulin, and angiotensin II up-regulate ET-1 mRNA whereas NO, PGI2 and shear stress cause down-regulation. ET-1 is synthesized as a larger protein, the pre-proET-1 (203 amino acids) that is cleaved to pro-ET-1 (38 amino acids) and then to ET-1 (21 amino acids) by endothelin-converting enzymes. The half-life of ET-1 protein and mRNA is 4–7 min and 15–20 min, respectively, and most plasma ET-1 (90%) is cleared by the lungs during first passage.

The biological effects of ET-1 are mediated by two GPCR subtypes, ETA and ETB which have opposing effects on vascular tone. ETA receptors on vascular smooth muscle cells are responsible for the majority of ET-1 induced vasoconstric-tion; activation of phospholipase C increases formation of InsP3 and diacylglycerol, and the resultant increase in intracellular Ca2+ and activation of protein kinase C cause vasoconstriction. ETB receptors are mainly present on endothelial cells and play an important role in clearing ET-1 from the plasma by internalising the recep-tor complex once ET-1 has bound. Activation of endothelial ETB receptors induces vasodilatation by stimulating the release of PGI2 and NO.  Inhibition of ETB increases circulating ET-1 levels and blood pressure in healthy subjects demonstrat-ing that although ET-1 is regarded as primarily a vasoconstrictor, ETB-mediated vasodilation is also physiologically important [34].

ET-1 is not only a vasoactive factor. Acting via ETB receptors, ET-1 modulates the formation and degradation of extracellular matrix (ECM) and thus plays a role in vascular remodelling. Acting via ETA, ET-1 promotes smooth muscle prolifera-tion contributing to neointima formation following vascular injury and to thickening of the arterial wall in pathological conditions such as pulmonary arterial hyperten-sion, atherosclerosis and venous graft occlusion. As NO strongly inhibits the release of ET-1 from the endothelium, and ET-1 attenuates NO-mediated dilation, ET-1 and NO are functionally interdependent and many of the cardiovascular complications associated with endothelial dysfunction may be due to an imbalance in this relation-ship [35].

1.3 Angiogenesis

Angiogenesis is the growth of new blood vessels formed by endothelial cells sprout-ing from existing vessels. In adults it is a protective mechanism initiated in response to tissue hypoxia, ischemia or injury. It is also a key process in pathological

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conditions such as proliferative diabetic retinopathy and neovascularization of tumours and as such, inhibitors of angiogenesis have received considerable interest as a potential therapeutic strategy. The angiogenic process depends on a complex transcriptional network coordinating production and release of numerous cytokines and growth factors [36].

Angiogenesis requires a sequence of individual processes:

1. Activation of endothelial cells, 2. Degradation of ECM by metalloproteinase enzymes, 3. Proliferation and directional migration of endothelial cells, 4. Formation of endothelial tubes, 5. Maturation of new vessels by recruitment of pericytes and smooth muscle cells

to stabilize endothelial sprouts and secrete ECM molecules to form the vascular basement membrane (Fig. 1.2).

The endothelial cells that sprout from the parent vessel (tip cells) possess long and motile filopodia that extend towards the source of pro-angiogenic growth factors and respond to other guidance cues to enable directional vessel growth [37].

Endothelial cell migration requires the dynamic regulation of interactions between integrins and the surrounding ECM.  Integrins are cell surface receptors which provide adhesive and signalling functions and link the actin cytoskeleton of the cell to the ECM at areas called focal adhesions. Phosphorylation of focal adhe-sion kinase, a cytoplasmic non-receptor tyrosine kinase, in response to pro- angiogenic signal molecules stimulates cell contraction thus allowing cell movement

Fig. 1.2 Schematic of process of angiogenesis. Angiogenesis involves the following complex sequence of events: (1) Activation of endothelial cells. (2) Degradation of extracellular matrix by matrix metalloproteinases. (3) Proliferation and directional migration of endothelial cells. (4) Formation of endothelial tubes. (5) Maturation of new vessels by recruitment of pericytes and smooth muscle cells to stabilise endothelial sprouts and secrete extracellular matrix molecules to form the vascular basement membrane

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on adhesive contacts. Subsequent integrin inactivation destroys the adhesive com-plex and allows detachment of the cell in its new location [38].

Cell-cell contacts between endothelial cells, essential for development of patent vessels, are mediated by cell surface receptors such as PECAM-1, a 130 kDa mem-ber of the immunoglobulin superfamily, which acts like a docking molecule to allow other proteins to provide further strength to vascular structures. Cadherins such as vascular endothelial cadherin are transmembrane proteins which provide weak adhesive cell-cell forces, further stabilized by catenins, intracellular proteins linking the cadherin cell surface molecule to the actin cytoskeleton.

Angiogenesis in response to hypoxia and ischaemia is largely controlled by the transcription factor hypoxia-inducible factor-1 [39]. HIF-1 has multiple subunits; HIF-1α which is produced continuously but rapidly degraded in the presence of oxygen, and HIF-1β which is constitutively expressed. Under hypoxic conditions, HIF-1α degradation is inhibited, and the stabilized protein translocates to the nucleus, where it dimerizes with HIF-1β. The dimer binds to hypoxia response ele-ments on more than 60 HIF–responsive genes that function to enhance oxygen delivery and increase metabolism. Central angiogenic signals driven by increased HIF-1 activity include VEGF, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and angiopoietins [40].

FGF, VEGF and PDGF stimulate endothelial cell proliferation and migration. Their high affinity for heparan sulfate glycosaminoglycans on the endothelial cell surface facilitates binding to receptors and provides a reservoir of these factors in the ECM, which can be released during wounding or inflammation. FGF binds to the receptor tyrosine kinase FGFR-1 to increase endothelial migration and promote capillary formation. FGF also enhances PDGF expression via a VEGF-dependent mechanism illustrating the cross talk and synergism that occurs within these growth factor pathways. In addition, FGF-mediated proteolysis of ECM components and induction of the synthesis of collagen, fibronectin, and proteoglycans by endothelial cells contribute to ECM remodelling.

VEGF stimulates endothelial replication and migration and increases vessel permeability, facilitating extravasation of plasma proteins to form a provisional ECM to support cell migration. mRNA for VEGF and VEGF-receptors has been detected in the tips of invasive angiogenic sprouts, and antibody blockade of VEGF signalling significantly decreases microvessel outgrowth. PDGF produced by angiogenic endothelial cells is required for the recruitment, proliferation, and survival of pericytes for vessel stabilization and maturation. PDGF acts on two transmembrane receptor tyrosine kinases, PDGF-α and -β. PDGF-β expressed on pericytes is critical to their recruitment. Disruption of signalling at these kinases is associated with vascular abnormalities in physiological and pathological angiogenesis.

Angiopoietins are ligands of endothelial-specific Tie receptors that have multiple effects on the angiogenic process, particularly interactions between endothelial cells, pericytes, and the basement membrane. For example, angiopoietin-1 acts on Tie-2 to stimulate secretion of growth factors from endothelial cells, which in turn stimulate differentiation of surrounding pericytes into smooth muscle cells.

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Conversely, angiopoietin-2 is an antagonist of the actions of angiopoietin-1 and so acts as a naturally occurring inhibitor of angiogenesis [40].

Recruitment and proliferation of bone marrow–derived endothelial progenitor cells (EPCs) to form new vessels (vasculogenesis) is a distinct but complimentary process which occurs simultaneously in ischaemic and wounded tissue to augment perfusion [41]. First described in 1997, classification of EPCs is still controversial, although the most well-accepted definition is that they are circulating endothelial cells expressing CD45, CD34 and CD133 surface antigens [42]. EPCs express pro-teins such as L-selectin and mucosal vascular cell adhesion molecule 1 (VCAM-1) which facilitate adhesion to mature endothelial cells. Incorporation of EPCs into the endothelial cell layer induces the release of proangiogenic factors such as VEGF, resulting in further recruitment of pro-angiogenic cells and enhanced angiogenesis. EPCs have been proposed as a potential cell-therapy to promote neovascularization in ischaemic tissues. This idea is supported by many studies demonstrating the role of EPCs in improved tissue perfusion in animal models of ischaemia, and clinical data showing that administration of EPCs to patients with myocardial infarction or chronic angina is associated with positive trends in perfusion [43].

1.4 Haemostasis

Endothelial cells play a pivotal role in regulating blood flow by exerting effects on the coagulation system, platelets, and fibrinolysis. Under normal physiological con-ditions, the endothelium provides one of the few surfaces which can maintain blood in a liquid state during prolonged contact [3]. A key factor in blood clot formation is activation of the serine protease thrombin which cleaves fibrinogen, producing fragments that polymerise to form strands of fibrin. Thrombin also activates factor XIII, a fibrinoligase, which strengthens fibrin-to-fibrin links, thereby stabilising the clot and stimulating platelet aggregation. Heparan sulfate proteoglycan molecules provide an anti-thrombotic endothelial cell surface by serving as co-factors for anti-thrombin III, causing a conformational change that allows this inhibitor to bind to, and inactivate, thrombin and other serine proteases involved in the clotting cascade. The endothelium also prevents thrombin formation by expressing tissue factor path-way inhibitor (TFPI) which binds to clotting factor Xa. TFPI and antithrombin III both contribute to physiological haemostasis, and both show impairment in acquired thrombotic states. A third endothelial anti-coagulation mechanism is expression of thrombomodulin. Binding of thrombin to cell surface thrombomodulin removes its pro-coagulant activity, and the thrombin-thrombomodulin complex activates pro-tein C, a vitamin K-dependent anticoagulant. Activated protein C, helped by its cofactor protein S, inactivates clotting factors Va and VIIa [44].

The anti-platelet aggregation properties of the endothelium are largely mediated by release of PGI2 and NO. As with smooth muscle relaxation, PGI2 inhibits platelet aggregation through the activation of IP receptors and activation of adenylyl cyclase, whereas NO inhibits platelet adhesion, activation, secretion, and aggregation

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through a cGMP-dependent mechanism. NO inhibits agonist-dependent increases in intra-platelet Ca2+ to suppress the Ca2+-sensitive conformational change in the heterodimeric integrin glycoprotein IIb–IIIa required for fibrinogen binding. NO also promotes platelet disaggregation by impairing the activity of phosphoinositide 3-kinase, which normally supports conformational changes in glycoprotein IIb–IIIa, rendering its association with fibrinogen irreversible. Should a blood clot form, fibrinolysis depends primarily on the action of plasmin, an active protease formed from its precursor, plasminogen, upon stimulation by tissue-type plasminogen acti-vator [44].

Under physiological conditions there is a haemostatic balance, and in addition to these anti-thrombotic mechanisms, the endothelium also synthesises several key haemostatic components, with vWF and plasminogen activator inhibitor-1 (PAI-1) being particularly important. PAI-1 is secreted in response to angiotensin IV, pro-viding a link between the renin-angiotensin system and thrombosis. In addition to anti- coagulant activity, binding of thrombin to thrombomodulin accelerates its capacity to activate thrombin-activatable fibrinolysis inhibitor which cleaves fibrin and other proteins, resulting in the loss of plasminogen/plasmin and tissue plas-minogen activator binding sites and thus retarding fibrinolysis. Perturbations such as those that may occur at sites of injury, inflammation, or high shear stress tip this haemostatic balance in favour of a pro-thrombotic and anti-fibrinolytic microenvi-ronment. Critical steps include loss of cell surface heparan proteoglycan molecules and increased expression of the transmembrane glycoprotein tissue factor which initiates coagulation by stimulating the activation of clotting factors IX and X, and pro- thrombinase, with subsequent fibrin formation. Tissue factor accumulates in experimentally injured vessels and accumulation in some atherosclerotic plaques is likely to account for their high thrombogenicity [45].

1.5 Inflammation

The development of inflammatory reactions by the endothelium in response to injury or infection is critical for the maintenance and/or repair of the normal struc-ture and function of the vessel wall. However, excessive inflammation can lead to severe tissue damage and contribute to the development of atherosclerosis. The interaction between endothelial cells and inflammatory cells such as leukocytes depends on the production of inflammatory cytokines (e.g. interleukin 8; IL-8) to attract leukocytes, and expression of adhesion molecules (e.g. selectins) to facili-tate their adhesion and migration towards the site of infection. Loosely tethered leukocytes first roll over the endothelial surface, then arrest, spread, and finally migrate between endothelial cells to attach onto underlying ECM components [46] (Fig. 1.3).

Leukocyte rolling involves endothelial adhesion molecules which transiently bind to carbohydrate ligands on leukocytes to slow passage through the blood ves-sel. E- and P-selectin are expressed only on the surface of activated endothelial cells

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whereas L-selectin is constitutively expressed on leukocytes and binds to ligands induced on the endothelium at sites of inflammation or on other leukocytes. The role of individual types of selectins in leukocyte rolling shows stimulus- and time- dependent variation. Immediate stimulation of leukocyte rolling induced by hista-mine or thrombin depends on rapid expression of P-selectin. Surface levels of this adhesion molecule decline after only 30 min. In contrast, TNFα stimulates delayed leukocyte rolling and adhesion to endothelial cells through the induction of E-selectin, surface levels of which peak after 12 h and decline after 24 h. Both E- and P-selectin are expressed on the surface of endothelial cells overlying atheroscle-rotic plaques, affirming the importance of these molecules in the development of atherosclerosis.

Firm adhesion of leukocytes is promoted by binding of cytokines to leukocyte GPCRs resulting in rapid activation of β1 and β2 integrins to increase their affinity for adhesion molecules of the immunoglobulin superfamily, intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1). ICAM-1 is constitutively expressed on endothelial cells, but levels are increased by stimuli such as TNFα peaking at 6 h and remaining elevated for 72 h. ICAM-1 mediates firm adhesion of blood cells by acting as a ligand for leucocyte β2 integrins. VCAM, a ligand for integrins α4β1 and α4β7, principally mediates the adhesion of mono-cytes, lymphocytes, eosinophils, and basophils to the endothelial surface. Expression of VCAM-1 is induced by cytokines, oxidized low-density lipoproteins, and ROS acting, as with induction of ICAM-1, primarily via NF-κB.

The migration of leukocytes through the endothelium requires the transient dis-assembly of endothelial cell junctions. Firm adhesion of leukocytes to the endothe-lium induces clustering of adhesion molecules like ICAM-1 and VCAM-1, triggering activation of intracellular signalling pathways which induce endothelial cell actin cytoskeleton and cell junction remodelling. The remodelling process

Fig. 1.3 Schematic of adhesion and migration of leukocytes. Inflammatory cytokines attract leu-kocytes and increase expression of adhesion molecules (e.g. selectins) to facilitate their adhesion and migration towards the site of infection. Loosely tethered leukocytes first roll over the endothe-lial surface, then arrest and migrate between endothelial cells to attach onto underlying ECM

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involves numerous pathways including Rho GTPase signalling, protein phosphory-lation, and ROS generation, but a key event is reorganization of PECAM-1 dimers. PECAM-1 localizes to intercellular junctions of endothelial cells, forming homodi-mers linking two cells. Leukocytes also express PECAM-1 and the dissociation of PECAM-1 dimers between endothelial cells to form dimers between emigrating leukocytes and endothelial cells is critical for leukocyte migration [47].

1.6 Conclusions

The endothelium, once viewed as an inert physical barrier, is a dynamic secretory organ fulfilling numerous roles in maintenance of cardiovascular homeostasis. Endothelial cells from different parts of the vasculature show highly differentiated functions. Advances in defining endothelial functions at the molecular level may lead to targeted therapies to alleviate chronic endothelial dysfunction associated with the progression of cardiovascular disease.

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7. Gao Y. The multiple actions of NO. Pflugers Arch. 2010;459(6):829–39. 8. Keravis T, Lugnier C. Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the

intracellular signalling network: benefits of PDE inhibitors in various diseases and perspec-tives for future therapeutic developments. Br J Pharmacol. 2012;165:1288–305.

9. Lima B, Forrester MT, Hess DT, Stamler JS. S-nitrosylation in cardiovascular signaling. Circ Res. 2010;106:633–46.

10. Mayer B, Hemmens B. Biosynthesis and action of nitric oxide in mammalian cells. Trends Biochem Sci. 1997;22:477–81.

11. Förstermann U, Sessa WC.  Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33:829–37.

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13. Park S, Sorenson CM, Sheibani N. PECAM-1 isoforms, eNOS and endoglin axis in regulation of angiogenesis. Clin Sci. 2015;129:217–34.

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14. Green DJ, O’Driscoll G, Blanksby BA, Taylor RR.  Control of skeletal muscle blood flow during dynamic exercise: contribution of endothelium-derived nitric oxide. Sports Med. 1996;21:119–46.

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16. Willeit P, Freitag DF, Laukkanen JA, Chowdhury S, Gobin R, Mayr M, et  al. Asymmetric dimethylarginine and cardiovascular risk: systematic review and meta-analysis of 22 prospec-tive studies. J Am Heart Assoc. 2015;4:e001833.

17. Vanhoutte PM, Shimokawa H, Feletou M, Tang EHC. Endothelial dysfunction and vascular disease—a 30th anniversary update. Acta Physiol. 2017;219:22–96.

18. Leffler CW, Parfenova H, Jaggar JH, Wang R. Carbon monoxide and hydrogen sulfide: gas-eous messengers in cerebrovascular circulation. J Appl Physiol. 2006;100:1065–76.

19. Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, et al. H2S as a physiologic vasorelaxant: hyper-tension in mice with deletion of cystathionine γ-lyase. Science. 2008;322:587–90.

20. Olson KR. Hydrogen sulfide as an oxygen sensor. Clin Chem Lab Med. 2013;51:623–32. 21. Ono K, Akaike T, Sawa T, Kumagai Y, Wink DA, Tantillo DJ, et  al. Redox chemistry and

chemical biology of H2S, hydropersulfides, and derived species: implications of their possible biological activity and utility. Free Radic Biol Med. 2014;77:82–94.

22. Mustafa AK, Sikka G, Gazi SK, Steppan J, Jung SM, Bhunia AK, et  al. Hydrogen sulfide as endothelium-derived hyperpolarizing factor sulfhydrates potassium channels. Circ Res. 2011;109:1259–68.

23. Tang G, Yang G, Jiang B, Ju Y, Wu L, Wang R. H2S is an endothelium-derived hyperpolarizing factor. Antioxid Redox Signal. 2013;19:1634–46.

24. Wang R, Szabo C, Ichinose F, Ahmed A, Whiteman M, Papapetropoulos A. The role of H2S bioavailability in endothelial dysfunction. Trends Pharmacol Sci. 2015;36:568–78.

25. Edwards G, Félétou M, Weston AH. Endothelium-derived hyperpolarising factors and associ-ated pathways: a synopsis. Pflügers Arch. 2010;459:863–79.

26. Kerr PM, Wei R, Tam R, Sandow SL, Murphy TV, Ondrusova K, et al. Activation of endo-thelial IKCa channels underlies NO-dependent myoendothelial feedback. Vasc Pharmacol. 2015;74:130–8.

27. Kerr PM, Tam R, Narang D, Potts K, McMillan D, McMillan K, et al. Endothelial calcium- activated potassium channels as therapeutic targets to enhance availability of nitric oxide. Can J Physiol Pharmacol. 2012;90:739–52.

28. Félétou M, Huang Y, Vanhoutte PM. Endothelium-mediated control of vascular tone: COX-1 and COX-2 products. Br J Pharmacol. 2011;164:894–912.

29. Patrono C. Cardiovascular effects of cyclooxygenase-2 inhibitors: a mechanistic and clinical perspective. Br J Clin Pharmacol. 2016;82:957–64.

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31. Park SK, Herrnreiter A, Pfister SL, Gauthier KM, Falck BA, Falck JR, et al. GPR40 is a low- affinity epoxyeicosatrienoic acid receptor in vascular cells. J Biol Chem. 2018;293:10675–91.

32. Campbell WB, Imig JD, Schmitz JM, Falck JR. Orally active epoxyeicosatrienoic acid ana-logs. J Cardiovasc Pharmacol. 2017;70:211–24.

33. Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, et al. The human endo-thelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A. 1989;86:2863–7.

34. Maguire JJ, Davenport AP. Endothelin@25—new agonists, antagonists, inhibitors and emerg-ing research frontiers: IUPHAR review 12. Br J Pharmacol. 2014;171:5555–72.

35. Félétou M, Köhler R, Vanhoutte PM.  Nitric oxide: orchestrator of endothelium-dependent responses. Ann Med. 2012;44:694–716.

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37. Ucuzian AA, Gassman AA, East AT, Greisler HP. Molecular mediators of angiogenesis. J Burn Care Res. 2010;31:158–75.

38. Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007;100:782–94.

39. Rey S, Semenza GL. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling. Cardiovasc Res. 2010;86:236–42.

40. Zimna A, Kurpisz M.  Hypoxia-Inducible factor-1  in physiological and pathophysiological angiogenesis: applications and therapies. Biomed Res Int. 2015;2015:549412.

41. Pearson JD. Endothelial progenitor cells—an evolving story. Microvasc Res. 2010;79:162–8. 42. Asahara T, Murohara T, Sullivan A, Sliver M, van der Zee R, Li T, et al. Isolation of putative

progenitor endothelial cells for angiogenesis. Science. 1997;275:964–7. 43. Tongers J, Roncalli JG, Losordo DW. Role of endothelial progenitor cells during ischemia-

induced vasculogenesis and collateral formation. Microvasc Res. 2010;79:200–6. 44. van Hinsbergh VWM.  Endothelium—role in regulation of coagulation and inflammation.

Semin Immunopathol. 2012;34:93–106. 45. Kazmi SSH, Jørgensen JJ, Sundhagen JO, Krog AH, Flørenes TL, Kollerøs D, et al. A com-

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46. Muller WA.  Mechanisms of leukocyte transendothelial migration. Annu Rev Pathol. 2011;6:323–44.

47. Chistiakov DA, Bobryshev YV, Kozarov E, Sobenin IA, Orekhov AN.  Role of gut micro-biota in the modulation of atherosclerosis-associated immune response. Front Microbiol. 2015;6:671.

Further Reading

Aird WC. Endothelium and haemostasis. Hamostaseologie. 2015;35:11–6.Chatterjee S.  Endothelial mechanotransduction, redox signaling and the regulation of vascular

inflammatory pathways. Front Physiol. 2018;7:524.Maruhashi T, Kihara Y, Higashi Y.  Assessment of endothelium-independent vasodilation: from

methodology to clinical perspectives. J Hypertens. 2018;36:1460–7.Vanhoutte PM. Nitric oxide: from good to bad. Ann Vasc Dis. 2018;11:41–51.

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19© Springer Nature Switzerland AG 2020R. Fitridge (ed.), Mechanisms of Vascular Disease, https://doi.org/10.1007/978-3-030-43683-4_2

Chapter 2Pathophysiology of Atherosclerosis

Sanuja Fernando, Christina A. Bursill, Stephen J. Nicholls, and Peter J. Psaltis

Key Learning Points• Atherosclerotic plaques are formed by progressive accumulation of lipids and

inflammatory cells, and extracellular matrix deposition in arterial intima.• Atherosclerotic lesions progress through different stages from early fatty streaks

to the formation of thin cap fibroatheromas that are vulnerable to plaque rupture and athero-thrombosis.

• Plaque erosion, which is distinct from rupture, is another increasingly recognised mechanism by which plaque thrombosis occurs to cause vessel occlusion.

• Atherosclerosis is mediated by different types of innate and adaptive immune cells as well as vascular endothelial and smooth muscle cells.

• The recruitment, activation, accumulation and cross-talk of these immune cells in response to modified lipoproteins, cholesterol crystals and other stimuli, cre-ates an inflammatory cycle that aggravates plaque progression and instability.

• Plaque inflammation is countered by similarly complex healing mechanisms, involving production of anti-inflammatory cytokines, deposition of collagen and clearance of apoptotic cells.

S. Fernando · C. A. Bursill · P. J. Psaltis (*) Vascular Research Centre, Lifelong Health Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia

Adelaide Medical School, The University of Adelaide, Adelaide, SA, Australiae-mail: Sanuja.Fernando@sahmri.com; Christina.Bursill@sahmri.com; Peter.Psaltis@sahmri.com

S. J. Nicholls Monash Heart, Monash Medical Centre and Department of Medicine, Monash University, Melbourne, VIC, Australiae-mail: stephen.nicholls@monash.edu

http://crossmark.crossref.org/dialog/?doi=10.1007/978-3-030-43683-4_2&domain=pdfhttps://doi.org/10.1007/978-3-030-43683-4_2#DOImailto:Sanuja.Fernando@sahmri.commailto:Christina.Bursill@sahmri.commailto:Peter.Psaltis@sahmri.commailto:Peter.Psaltis@sahmri.commailto:stephen.nicholls@monash.edu

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2.1 Introduction

In 1904 German pathologist Felix Marchand was the first to describe the term “ath-erosclerosis” from the Greek roots “athere-” meaning “gruel”, and “-sklerosis”, meaning “hardness”. Atherosclerosis has become the leading cause of mortality and morbidity worldwide, causing clinical disease via vascular luminal narrowing which can ultimately lead to thrombus formation that obstructs blood flow to tis-sues, such as the heart (coronary heart disease), brain (ischaemic stroke) and lower extremities (peripheral vascular disease) [1]. It was formerly recognised as an ordi-nary lipid storage disease. However, major advances in basic, experimental and clinical sciences have illuminated the role of inflammation and the underlying com-plex cellular and molecular mechanisms that contribute to atherosclerosis [2]. It is now defined as a chronic inflammatory disease of the vasculature which is initiated early in life and develops over a number of decades. The intensity of the disease relies on many genetic, environmental and behavioural factors. The best-known risk factors include hypercholesterolaemia (specifically elevated low-density lipoprotein- cholesterol [LDL-C] levels), hypertension, diabetes mellitus, cigarette smoking, family history, obesity, male gender, age (male: >45 years and female: >55 years), sedentary lifestyle and diets with high saturated and trans-fatty acids.

Atherosclerosis is a multifocal disease that especially occurs at sites with low or oscillatory shear stress located near branch points in the arterial tree [1]. Thus, the most affected areas are the carotid bifurcations, coronary arteries, aortic and femo-ral artery bifurcations [3]. This lipid-driven inflammatory disease leads to subinti-mal plaque formation at specific sites due to maladaptive inflammation, fibrosis, necrosis and calcification.

2.2 Normal Vessel Wall

An understanding of the pathophysiology of atherosclerosis first requires knowledge of the normal artery wall structure (Fig. 2.1), its biology and function. Normal arteries have a trilaminar structure. The innermost layer, the tunica intima is the closest to the arterial lumen. This layer contains a monolayer of endothelial cells which resides on a basement membrane containing non-fibrillar collagen, such as type IV collagen, laminin, fibronectin, and other extracellular molecules. The middle layer, known as the tunica media is the thickest layer and lies under the intima separated by the inter-nal elastic lamina. The media contains concentric layers of smooth muscle cells (SMCs), interleaved with layers of elastin-rich extracellular matrix and serves con-tractile and elastic functions of the vessel. The external elastic lamina bounds the tunica media and the outermost layer of the artery, the tunica adventitia which ini-tially received little attention, although appreciation of its potential roles in arterial pathology has recently increased. The adventitia consists of a relatively loose array of collagen fibrils and contains blood vessels (vasa vasorum), nerve endings and lym-phatics which nourish the cellular components of the arterial wall. It is also enriched

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with fibroblasts, different subsets of leukocytes (e.g. mast cells, macrophages, lym-phocytes) and a diverse array of progenitor/stem cells [4, 5]. The adventitia provides a dynamic interface between the intima/media and the perivascular adipose and con-nective tissue, and is increasingly recognised as a complex biological processing cen-tre which serves as an injury sensor for the rest of the vessel wall [6, 7].

Atherosclerosis is heralded by the build-up of lipid-rich plaques in the subintimal compartment of the vessel wall where maladaptive remodelling responses are involved in all three vessel wall layers discussed above.

2.3 Stages of Atherosclerosis

There are major histological and molecular changes which take place in the progres-sion of