Role of Cx40 in the healthy and diseased vascular endothelium

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Thesis

Reference

Role of Cx40 in the healthy and diseased vascular endothelium

DENIS, Jean-François

Abstract

L'athérosclérose est une maladie chronique, progressive, immuno-inflammatoire et multifactorielle affectant des artères de moyenne ou grande taille dans des régions exposées à un flux sanguin faible oscillatoire. Ces propriétés particulières du flux sanguin induisent une signalisation intracellulaire spécifique accompagnée d'une expression distincte de gènes et provoquent des changements dans la morphologie des cellules endothéliales. La présence de jonctions gap composées de connexines entre les cellules endothéliales permet une communication directe par le passage d'ions et de petits métabolites. Trois connexines, Cx37, Cx40 et Cx43, sont exprimées dans les cellules endothéliales artérielles et forment chacune des canaux avec des perméabilités et des paramètres d'ouverture uniques. Des études sur des modèles murins ont révélé que les connexines jouent un rôle important dans la physiologie artérielle ainsi que dans l'initiation et la progression de l'athérosclérose. Cette thèse met l'accent sur la Cx40. Cette protéine est fortement exprimée dans les cellules endothéliales d'artères [...]

DENIS, Jean-François. Role of Cx40 in the healthy and diseased vascular endothelium. Thèse de doctorat : Univ. Genève, 2017, no. Sc. 5144

DOI : 10.13097/archive-ouverte/unige:101734

Available at:

http://archive-ouverte.unige.ch/unige:101734

Disclaimer: layout of this document may differ from the published version.

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Professeur Jean-Claude Martinou

Section de Médecine fondamentale FACULTÉ DE MÉDECINE Département de Pathologie et Immunologie Professeur Brenda R. Kwak

Role of Cx40 in the Healthy and Diseased Vascular Endothelium

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention Biologie

par

Jean-François DENIS de

Bassenge (Belgique)

Thèse n° 5144 Genève

2017

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DE GENÈVE

FACUTTÉ DES SCIENCES

DocroRAT Ès sctENcES, MENTIoN BtoloctE

Thèse de Monsieur Jean-François DENIS

intitulée

<Role of ^Cx40 in the Healthy and Diseased Vascular Endothelium>

La Faculté des sciences, sur le ^préavisde Madame B. R. KWAK, professeure ordinaire et directrice de thèse (Faculté

de

médecine, Département

de

pathologie

et

immunlogie), Monsieur

J.

MARTINOU, professeur ordinaire et codirecteur de thèse (Département de biologie cellulaire), Monsieur K.-H. KRAUSE, professeur ordinaire (Faculté de médecine, Département de pathologie et immunologie), Madame E. JONES, professeure (Centre for Molecular and Vascular Biology, Leuven, Belgium), Madame T. PETROVA, professeure (Department

of

Fundamental Oncology, University

of

Lausanne, Switzerland), autorise I'impression de la présente thèse, sans exprimer d'opinion sur les propositions qui y sont énoncées.

Genève, le 10 novembre2017

Thèse

-5144-

Le Doyen

N.B.

-

La thèse doit porter la déclaration précédente et remplir les conditions énumérées dans les "lnformations

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“We make our world significant by the courage of our questions and the depth of our answers”

(Carl Sagan)

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Résumé

L'athérosclérose est une maladie chronique, progressive, immuno-‐inflammatoire et multifactorielle affectant des artères de moyenne ou grande taille dans des régions exposées à un flux sanguin faible oscillatoire. Ces propriétés particulières du flux sanguin induisent une signalisation intracellulaire spécifique accompagnée d’une expression distincte de gènes et provoquent des changements dans la morphologie des cellules endothéliales. La présence de jonctions gap composées de connexines entre les cellules endothéliales permet une communication directe par le passage d'ions et de petits métabolites. Trois connexines, Cx37, Cx40 et Cx43, sont exprimées dans les cellules endothéliales artérielles et forment chacune des canaux avec des perméabilités et des paramètres d’ouverture uniques. Des études sur des modèles murins ont révélé que les connexines jouent un rôle important dans la physiologie artérielle ainsi que dans l'initiation et la progression de l'athérosclérose. Cette thèse met l’accent sur la Cx40. Cette protéine est fortement exprimée dans les cellules endothéliales d'artères saines dans des régions rectilignes exposées à un flux sanguin laminaire élevé et atheroprotecteur, mais est absente dans les cellules endothéliales à la surface de plaques d'athérosclérose. Nous avons constaté que la Cx40 est différemment exprimée en fonction du type de flux sanguin. Son niveau d'expression est élevé dans les cellules endothéliales exposées à un flux sanguin laminaire élevé sous l’influence de la régulation positive du facteur de transcription KLF4, qui se lie directement à (au moins) trois éléments CACCC dans le promoteur du gène de la Cx40. En outre, l'absence de la Cx40 amplifie l'athérosclérose induite par les contraintes de cisaillement du flux sanguin dans

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un modèle murin. Cela s'explique par une fonction de la Cx40 indépendante de la fonction de jonction gap et importante pour le développement de l’athérosclérose. L'interaction fonctionnelle entre IκBα et la Cx40 réduit la réponse inflammatoire endothéliale en altérant la translocation de NFκB dans le noyau. De plus, le séquençage ARN et gene set enrichment analysis (GSEA) ont permis d’identifier le cycle cellulaire comme un processus régulé par la Cx40 en condition de flux sanguin laminaire élevé. La diminution de l’expression de la Cx40 par interférence d’ARN sous flux sanguin laminaire élevé a augmenté la proportion de cellules endothéliales positives aux proliferating cell nuclear antigen (PCNA) et a diminué la proportion de cellules endothéliales dans la phase G0/G1 in vitro. Pour finir, différents poissons-‐zèbres mutés pour les orthologues de la Cx40, c'est-‐à-‐dire Cx41.8^t1/t1, Cx41.8tq270/tq270 et Cx41.8^t1/t1Cx45.6^-‐/-‐, ont été utilisés dans le laboratoire. Ces poissons-‐zèbres qui sont soit déficients pour la Cx41.8 (Cx41.8^t1/t1), soit avec une activité réduite du canal (Cx41.8tq270/tq270) ou avec une absence des deux orthologues (Cx41.8^t1/t1Cx45.6^-‐/-‐), ont été croisés avec des poissons exprimant l'eGFP dans l'endothélium (flk1: eGFP) et les protocoles de génotypage ont été optimalisés dans le laboratoire. Cela ouvre de nouvelles perspectives pour des expériences permettant de visualiser les premières étapes de l'athérogenèse ainsi que des expériences investiguant le rôle de la "Cx40" endothéliale dans la vasculogenèse et l’angiogenèse.

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Abstract

Atherosclerosis is a multifactorial chronic progressive immuno-‐inflammatory disease that forms at regions in medium-‐ to large-‐sized arteries that are exposed to low, oscillatory shear stress. Shear stress patterns prime endothelial cells (ECs) to specific intracellular signaling inducing distinct gene expression and cell morphology. Presence of gap junction channels composed of connexins between ECs allows for direct communication by enabling the passage of ions and small metabolites. Three connexins are expressed in arterial ECs, i.e. Cx37, Cx40 and Cx43, forming each channels with unique permeabilities and gating properties.

Studies on mouse models have revealed that connexins play an important role in arterial physiology and in the initiation and progression of atherosclerosis. This thesis focuses on Cx40. This protein is highly expressed in ECs in straight parts of healthy arteries exposed to atheroprotective high laminar shear stress (HLSS), but is lost in ECs overlying atherosclerotic plaques. We found that Cx40 is a shear stress response gene. Its expression level is enhanced in ECs exposed to HLSS through the upregulation of the transcription factor KLF4, which directly binds to (at least) three CACCC elements in the Cx40 promoter. Furthermore, absence of endothelial Cx40 exacerbates shear stress-‐induced atherosclerosis in a mouse model. This is explained by a novel channel-‐independent function of Cx40 relevant for atherosclerosis; the functional interaction of IκBα-‐Cx40 reduces endothelial activation by impairing NFκB translocation to the nucleus thus reducing inflammatory responses. In addition, RNAseq and gene set enrichment analysis identified cell cycle progression as an important down-‐stream target of Cx40 under HLSS. Downregulation of Cx40 by an siRNA approach under HLSS

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increased the proportion of proliferating cell nuclear antigen (PCNA)-‐positive ECs and decreased the proportion of ECs in the G0/G1 phase in vitro. Finally, zebrafish models mutated for Cx40 orthologues, i.e. absence of Cx41.8 (Cx41.8^t1/t1), reduced channel function of Cx41.8 (Cx41.8tq270/tq270)and with absence of the 2 orthologues (Cx41.8^t1/t1Cx45.6^-‐/-‐), were introduced in the laboratory. Cross-‐breeding with a zebrafish model expressing eGFP in the endothelium (flk1:eGFP) was performed and genotyping protocols have been set up. This opens new perspectives for experiments visualizing the earliest steps in atherogenesis as well as experiments examining the role of endothelial “Cx40” in vasculogenesis/angiogenesis.

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Abbreviations

AAV8 adeno-‐associated-‐virus-‐8 AF atrial fibrillation

Akt protein kinase B

ApoE^-‐/-‐ Apolipoprotein E-‐deficient ASVD Arteriosclerotic vascular disease ATF activating transcription factor

AV atrioventricular

cAMP cyclic adenosine monophosphate CETP cholesterylester transfer protein cGMP cyclic guanosine monophosphqte

ChIP chromatin immunoprecipitation

CL cytoplasmic loop CT C-‐terminal

CVDs cardiovascular diseases CXCL12 C-‐X-‐C motif chemokine 12

d radial distance of the vessel diameter

D vessel diameter

DAPI 4’,6-‐diamidino-‐2-‐fenylindool DMJ dorsal midline junction

DMNT1 DNA-‐Methyltransferase 1

DNA deoxyribonucleic acid

dynes/cm2 dynes per square centimeter

E-‐selectin endothelial-‐leukocyte adhesion molecule

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ECA external carotid artery ECs endothelial cells ELs extracellular loops eNOS nitric oxide synthase 3

ER endoplasmic reticulum

ERK extracellular signal-‐regulated kinase FACS fluorescent-‐activated cell sorting FCS fetal calf serum

FDR fold discovery rate

GSEA gene set enrichment analysis HCAECs human coronary artery ECs HCD high cholesterol diet

HLSS high laminar shear stress HUVECs human umbilical vein ECs ICA internal carotid artery

ICAM-‐1 intercellular adhesion molecule-‐1

IFN interferon

IFN-‐γ interferon γ

IKK IκB kinase

IL interleukin

IP3 inositol triphosphate

JNK c-‐Jun N-‐terminal kinase

KLFs Krüppel-‐like family of transcription factors LCA left common carotid artery

LDA lateral dorsal aorta

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LDL low-‐density lipoprotein LDL LECs lymphatic endothelial cells LLSS low laminar shear stress

LPS lipopolysaccharide

MAP mitogen activated protein MCP-‐1 monocyte chemotactic protein-‐1

MKP-‐1 mitogen-‐activated protein kinase phosphatase 1 N/m2 Newton per square meter

NADPH nicotinamide adenine dinucleotide phosphatase

NES normal enrichment score

NF-‐kB nuclear factor kappa-‐light-‐chain-‐enhancer of activated B cells

NGS normal goat serum

NO nitric oxide

Nrf2 nuclear factor erythroid 2 related factor-‐2 NT N-‐terminal

OA occipital artery

ODDD oculodentodigital dysplasia OSI oscillatory shear index OSS oscillatory shear stress P2X4 P2X purinoreceptor 4

Pa Pascal

PA-‐1 plasminogen activator inhibitor PBS phosphate buffered saline

PBT phoshate buffered tween

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PCNA proliferating cell nuclear antigen PCSK9 protein subtilisin kexin type 9

PECAM-‐1 platelet endothelial cell adhesion molecule-‐1

PFA paraformaldehyde

PFB pectoral fin buds PGI2 prostaglandin 2 PI3K PI 3-‐kinase

PPARα peroxisome proliferator-‐activated receptor-‐alpha

PVDF polyvinylideenfluoride

Q, flow rate

Re Reynolds number

RelA transcription factor p65

RNA ribonucleic acid

ROS reactive oxygen species RPKM reads per kilobase per million

RT room temperature

SA sinoatrial

SDS-‐PAGE sodium dodecyl sulfate polyacryl gel electrophoresis SEM standard error of the mean

SHP-‐2 protein-‐tyronase phosphatase 1D SIDS sudden infant death syndrome siRNA short interfering RNA

SIRT1 sirtuin-‐1

SNP single nucleotide polymorphism

SSC saline-‐sodium citrate

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STA superior thyroid artery TF transcription factor

TFBS transcription factor binding sites TGF-‐β transforming growth factor

TM thrombomodulin

TNF tumor necrosis factor TNF-‐α tumor necrosis factor α

TRPV transient receptor potential cation channel TWIST1 twist related protein-‐1

UTR 3’-‐untranslated region

VCAM-‐1 vascular cell adhesion molecule-‐1 VEGFR2 vascular endothelial growth factor-‐2 VSMCs vascular smooth muscle cells

vWF von Willebrand factor

WISH whole mount in situ hybridisation WSS Wall shear stress

WT wild type

ZO-‐1 zona occludens-‐1 μ fluid viscosity

μ blood viscosity

ρ density of blood

τ shear stress

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1.1 General introduction

Cardiovascular diseases (CVDs) are the first cause of death worldwide [1-‐3].

They form a group of disorders of the heart and blood vessels that include:

coronary heart disease, cerebrovascular disease (stroke), peripheral artery disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis and pulmonary embolism [4]. In 2012, it was estimated that 17.5 million people died from CVDs, accounting for 31% of all deaths worldwide. Prediction by the World Health Organization (WHO) for 2030 show that death due to CVDs will rise to 37% of which approximately 75% will occur in low and middle income countries [1]. In line with this data the WHO launched in September 2016 the initiative “Global Hearts” to beat the global threat of CVD, including heart attack and stroke, illustrating the importance of this threat [5].

The underlying cause of acute events like heart attack and stroke is the blockade of an artery preventing oxygen rich blood to reach the heart or brain [6]. The main cause of such a blockade is the rupture of an atherosclerotic plaque, a fibrous-‐fatty deposit on the inner wall of arteries [7].

1.2 Atherosclerosis

Arteriosclerotic vascular disease (ASVD) or atherosclerosis is a multifactorial chronic progressive inflammatory disease of medium-‐ to large-‐sized arteries [8].

Risk factors for atherosclerosis such as hyperlipidemia, diabetes mellitus, hypertension and cigarette smoking are linked to damaged endothelium [9].

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accumulation of low-‐density lipoprotein (LDL) particles in the sub-‐endothelial space of the arteries. Biochemical processes and oxidative modifications lead to the formation of oxidized LDL particles that aggregate and increase LDL infiltration and subsequently trigger an innate inflammatory response [10, 11].

Up-‐regulation of adhesion molecules (e.g. vascular cell adhesion molecule-‐1 (VCAM-‐1) and intercellular adhesion molecule-‐1 (ICAM-‐1)) and the secretion of chemokines by ECs triggers the recruitment of monocytes, lymphocytes and neutrophils that adhere on ECs and transmigrate into the lesion [6, 10, 12, 13].

(Figure 1a) Subsequently, monocytes differentiate in macrophages and oxidized LDL is taken up by these macrophages becoming foam cells to form early plaques (fatty streaks) [14, 15]. (Figure 1b) These early plaques progress to more mature atherosclerotic plaques due to the accumulation of additional inflammatory cells and extracellular lipids forming a core region [15]. This core region consisting of apoptotic cells, necrotic cells, cholesterol crystals and extracellular debris, known as the necrotic core is surrounded by a collagen matrix and vascular smooth muscle cells (VSMCs). Indeed, activated VSMCs, proliferate, dedifferentiate and migrate from the media to the intima due to the release of chemokines and growth factors by inflammatory cells in the plaque [16-‐19].

Furthermore, VSMCs have been found to trans-‐differentiate into macrophage-‐

like cells increasing the role of VSMC plasticity in atherogenesis [18, 20-‐22]. The proliferation of VSMCs in atherosclerosis is mainly reparative (e.g. formation of fibrous cap). Transforming growth factor (TGF)-‐β promotes collagen synthesis by VSMCs providing mechanical strength to the fibrous cap [23]. In contrast, VSMC senescence and death promote atherogenesis and multiple features of plaque instability [24].

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As years pass and the plaque grows it becomes more complex. Intraplaque neovascularization results in a growing network of micro-‐vessels inside the plaque leading to increased entry of inflammatory cells and this is associated with increased plaque vulnerability [25, 26]. Progression and growth of the plaque lead to reduction of the vessel lumen with subsequent reduced ability to augment blood flow during exercise for instance [27]. (Figure 1c) Finally, atherosclerotic plaques can rupture and the following thrombosis are the main cause of acute coronary syndromes and sudden coronary death [28]. (Figure 1d) Plaque rupture is induced by a complex multifactorial process that includes inflammation and biomechanical factors [29, 30]. Intraplaque lipid-‐related inflammation leads to degradation and weakening of the plaque tissue. On the other hand, collagen synthesis and smooth muscle cell proliferation exert stabilizing and reparative effects. The interplay between these two biological features mostly determines the vulnerability of the plaque [31]. Secondly, if the local wall stress due to the blood pressure and pulsatile forces exceeds the fracture stress of the fibrous cap the plaque may break and expose the highly thrombogenic plaque core to the blood [30, 32]. Usually, the plaque ruptures at the shoulder to the cap, a plaque site exposed to maximal biomechanical stresses [33, 34].

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Figure 1: Different stages in the development of atherosclerotic plaque. a) Dysfunction and activation of the endothelium under pro-‐inflammatory conditions lead to platelet and leukocyte adhesion and increased permeability of the endothelium. b) Monocytes in the intima start to accumulate lipids becoming macrophages or foam cells, which make up fatty streaks. Continued deposition of matrix components, mononuclear-‐cell influx and the recruitment of VSMCs give rise to fibro-‐proliferative progression of the plaque. c) Apoptosis of macrophages and other plaque cells give rise to the necrotic core. Fibrous cap formation consisting of matrix and a VSMC layer. Possible neovascularization within the plaque from the adventitia. d) Fibrous cap thinning and erosion in unstable plaques resulting in plaque rupture leading to arterial occlusion and myocardial infarction of stroke.[17]

Atherosclerotic plaques are categorized in function to their vulnerability to rupture. Stable plaques are characterized by a thick fibrous cap, few inflammatory cells and are rich in VSMCs and collagen that protects against disruption. In contrast, vulnerable plaques are characterized by an active inflammatory state (high inflammatory cell content), low VSMC content, a thin fibrous cap, a large necrotic core and sometimes intra-‐plaque hemorrhage [35-‐

38].

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Mechanical stimuli have been shown to affect EC and VSMC phenotype contributing to initialization and progression of the atherosclerotic disease [32, 39]. Indeed, ECs have been found to sense these mechanical signals and in consequence activate intracellular signaling pathways that regulate gene expression in ECs themselves, sometimes also leading to secretion of factors that regulate VSMCs function [40, 41]. In the next paragraph, plaque localization and EC phenotype in function of shear stress will be discussed.

1.3 Blood Flow and EC biology

ECs regulate vascular permeability, vascular tone, thrombosis and hemostasis [42]. As previously described, they have also been found to be highly implicated in the onset of atherosclerotic lesion development. Although being a multifactorial disease, atherosclerotic plaques have been observed in specific regions of the vasculature. ECs are subjected to hemodynamic shear stress, the frictional force that the flowing blood exerts on the endothelium. Interestingly, atherosclerotic plaques have typically been observed in curved regions and at bifurcations of the arteries exposed to disturbed blood flow. Indeed, these predilection regions experience repetitive phases of flow reversal resulting in steep multidirectional temporal and spatial gradients of wall shear stress (WSS) [43-‐46].

1.3.1 Shear Stress

Shear stress is expressed in Newton per square meter (N/m²), Pascal (Pa) or dynes per square centimeter (dynes/cm²), and is defined by the parallel stress produced by the movement of blood compared to the static ECs of the vessel wall [32]. Depending on their anatomical localization, ECs experience different levels

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of shear stress and shear gradients (pulsatile and directional changes) [44]. In humans shear stress values in straight large conduit arteries vary between 5 and 20 dynes/cm² (1dyne = 0.1N/m²) and between 19 and 60 dynes/cm²for small arterioles [47-‐49]. If we consider blood as a Newtonian fluid and the vessel diameter of large conduit arteries relatively constant, the profile of blood flow in straight parts of arteries is assumed to be parabolic and shear stress laminar.

Shear stress can in this condition be determined with the Haagen-‐Poisseuille equation τ=4μQ/πd³ (shear stress (τ), flow rate (Q), fluid viscosity (μ) and radial distance of the vessel diameter (d)) [44, 48, 50]. WSS in straight parts of arteries can thus be determined by calculating the gradient of local blood flow velocity close to the vessel wall multiplied by the blood viscosity [51]. In bending arterial segments or at bifurcations, physical laws determine that blood flow velocities are spatially distributed (due to a balance of pressure, centrifugal and viscous forces) such that skewed and asymmetric velocity profiles can be observed at these locations. In addition, the direction and the velocity of blood flow in arteries vary throughout the cardiac cycle. As a result, arterial locations that experience low blood flow fluctuating in direction with both forward and reverse velocities during the cardiac cycle are considered as “disturbed shear stress regions” or as “exposed to low, oscillatory shear stress” (atheroprone flow) [44, 48, 50]. (Figure 2) In contrast, unbranched and straight parts of arteries that are exposed to high unidirectional laminar shear stress (atheroprotective flow) do not develop atherosclerotic lesions [52, 53]. (Figure 2)

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Figure 2: Atherosclerotic plaques develop preferentially in areas (Atherosclerosis-‐susceptible region) at arterial bifurcations exposed to atheroprone flow. In contrast, ECs exposed to high unidirectional laminar shear stress (atheroprotective flow) are athero-‐resistant (Atherosclerosis-‐

resistant region). Adapted from [52]

These findings point to both the magnitude and the direction of shear stress that ECs experience and these cells adjust their phenotype in consequence. Various mechanoreceptors have been identified on the surface of ECs, which convert mechanical signals into a chemical response inside the cell.

1.3.2 Shear stress sensors/mechanotransduction

ECs have been found to act as shear stress sensors through mechanotransduction. I.e. they transform the physical stress of the exerted shear stress into intracellular biochemical signals to change cell function, cell morphology and gene expression. The signal transduction in ECs has been proposed to follow several sequential steps [44, 54]. Firstly, physical deformation of the cell surface; secondly, intracellular stress transmission;

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feedback signaling [44]. Although the exact mechanism by which signal transduction occurs and which mechanotransducers can sense which type of shear stress in ECs is not yet fully understood, several mechanosensing and mechanotransduction systems have been put forward [52, 55]. The first shear stress sensing mechanism has been described in 1988. Olesen and colleagues described a K⁺-‐channel acting as shear stress sensor [56]. Since then, other ion channels like transient receptor potential cation channel (TRPV) [57], P2X purinoreceptor 4 (P2X4) [58] and Piezo1 [59] have been linked to shear stress sensing [60]. In addition, membrane-‐bound molecules like Integrins [61], the junctional complex of platelet endothelial cell adhesion molecule-‐1(PECAM-‐1), vascular endothelial (VE)-‐cadherin and vascular endothelial growth factor-‐2 (VEGFR2) [62, 63], the tyrosine receptors Tie1 and Tie2 [64], G protein-‐coupled S1P1 [65] and the transmembrane heparin sulfate proteoglycan syndecan 4 [66]

have been proposed to be implicated in mechanosensing. Last but not least, the glycocalyx and specific membrane microdomains such as primary cilia and calveolae have been described as mechanosensor [44, 54, 55, 67, 68]. (Figure 3) At this moment, it is not yet fully understood which mechanosensor is used for laminar and oscillatory shear stress, for instance, and whether these mechanotransduction systems can act individually or act synergistically, this way amplifying each other.

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Figure 3: ECs Mechanotransduction. The shear stress on the ECs is sensed on the luminal surface by different receptors and various ion channels that activate downstream effects.[32]

1.3.3 Shear stress models

In order to study the effect of shear stress on ECs different in vivo, ex vivo and in vitro models have been developed. In vivo mouse models can be used for their naturally flow disturbed regions such as curved and branched arterial regions, however this will give at best “comparative results” between different arterial regions but will not “provide causative insight” onto the relation between shear stress and the expression of certain genes [69]. In consequence, surgical models have been used to investigate shear stress effects on the ECs, like arteriovenous fistulas, constrictive perivascular cuffs and partial ligation of arteries.

Arteriovenous fistulas originally created for dialysis patients were the first interventions to cause acute changes in shear stress [70]. The blood flow is increased in the artery from where the fistula is created. Here, it was shown that the artery dilates (increased lumen) in such a manner that the final experienced shear stress was not significantly changed compared to the original shear stress

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[71]. The mouse aorto-‐caval fistula has been used as a model for human arteriovenous fistula [72].

The constructive perivascular cuff model is used in hyperlipidemic animals to accelerate atherogenesis. (Figure 4) Cheng et al. showed with a flow-‐modifying cuff around the carotid artery of Apolipoprotein E-‐deficient mice (ApoE^-‐/-‐ mice) that local changes in hemodynamic conditions initiate atherosclerosis.

Interestingly, they also described that plaque vulnerability was associated with low unidirectional (laminar) shear stress rather than with oscillatory shear stress. Indeed, the regions of low laminar shear stress displayed an atherosclerotic lesion with large lipid core, many macrophages, low collagen content and few VSMCs [73, 74].

Figure 4: Constrictive perivascular cuff model [75]. A: The conical shaped cast creates three regions of shear stress: a low laminar shear stress (LLSS) upstream of the cast, a region of increasingly high laminar shear stress (HLSS) inside the cast, and a region with oscillatory shear stress (OSS) downstream of the cast. B: Wall shear stress (WSS, left) and oscillatory shear index (OSI, right) determined by micro computer tomography (μCT) in carotid artery after 9weeks of cast placement.

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Several partial carotid artery ligation models exist to elicit different degrees of flow alterations and arterial remodeling in mice. Ligation of three of the four caudal branches of the left carotid artery (the internal carotid, occipital and the external carotid) after the branching of the superior thyroid artery induced significantly reduced flow but also flow reversal patterns during diastole characteristic for areas of disturbed flow [76]. (Figure 5)

Figure 5: Schematic representation of partial ligation of the left common carotid artery (LCA). Three branches of the LCA (external carotid artery (ECA), internal carotid artery (ICA), and occipital artery (OA) are ligated leaving the superior thyroid artery (STA) open. Adapted from [76] .

Ligation was shown to reduce flow in the surgically ligated artery and was resulting in shear stress-‐dependent vascular remodeling [77-‐79]. Atheroma develops here in the untouched left common carotid artery (LCA) that is not manipulated during the procedure and can be compared with the right common carotid artery (RCA). Furthermore, surgery did not affect shear rate in the right common carotid artery [80, 81]. A recent study showed that partial carotid ligation in combination with adeno-‐associated-‐virus-‐8 (AAV8)-‐mediated overexpression of proprotein convertase subtilisin/kexin type 9 (PCSK9) (AAV8-‐

PCSK9) induced within 3 weeks hyperlipidemia and atherosclerosis [82].

Inhibitors of PCSK9 are a promising new class of cholesterol lowering drug

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because of their interference with cholesterol metabolism by the means of LDL receptor recycling in hepatocytes [83, 84]. PCSK9 is involved in the degradation of the low density lipoprotein receptor (LDLR) and is found primarily in the liver, intestine, and kidney [85]. Evidence shows that PCSK9 binds to the LDLR and redirect the LDLR to the lysosome. Decreasing the available LDLRs on the cell surface and thus resulting in increase LDL in the serum. Indeed, clinical studies using monoclonal antibodies (alirocumab and evolocumab) that inhibit PCSK9 showed a reduction of approximately 50% in blood plasma LDL cholesterol levels [86-‐88].

In transgenic mice the overexpression of the PCSK9 protein leads to hypercholesterolemia and atherosclerosis [89-‐91]. Instead of using transgenic animals, Bjorklund et al. developed a gain of function mutant of PCSK9 in a recombinant AAV8. One injection of AAV8-‐PCSK9 into wild type C57BL6 mice resulted in significant hypercholesterolemia and atherosclerotic plaque formation within 3 months. Making it a good alternative for germline knockout ApoE or LDLR mice models [92]. Finally, ligation of the left external carotid artery branch in another model was shown to reduce significantly the arterial flow through the left common carotid artery and resulted in flow-‐mediated reduction of the lumen diameter and medial wall mass followed by decreased VSMC proliferation and elastin content compared with the right common carotid

artery [79, 93, 94].

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Tabel 1: Advantages and disadvantages of in vivo shear modifying models in EC physiology

IN VIVO MODEL ADVANTAGES DISADVENTAGE

Naturally flow

disturbed areas • Real chronic in vivo

situation • Chronic model

• Small sample area

• Mice do not develop spontaneous

atherosclerotic lesions[95]

• “comparative” not

“causative” results Arterio-‐venous

fistulas • Flow-‐induced arterial

remodeling

• Extensive tearing and fragmentation of the internal elastic lamina [96]

• Venous stenosis[97]

• Surgical intervention Constrictive

perivascular cuff • Distinct flow patterns in neighboring regions

• Fast atherosclerotic plaque formation[74, 98]

• Direct manipulation of the vessel

• Small sample area

• Loss of circumferential cyclic stretch region within the cast.

• Surgical intervention Partial ligation

technique of 3 branches of the LCA

• Large portion of the carotid artery affected

• No direct manipulation of the vessel

• Large sample area

• Fast atherosclerotic plaque formation[76, 82, 99]

• Surgical intervention

• Multiple sutures

Partial ligation technique of left external carotid artery branch

• Fast vascular

remodeling [79, 94]

• Reduced blood flow to levels of internal carotid artery [100]

• One suture

• Surgical intervention

Secondly, ex vivo models using entire vessels, where ECs are surrounded by their native cell-‐cell and cell-‐matrix interactions can be used. Thus, entire vessel segments are cannulated and connected to a perfusion unit where flow patterns

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relationship between shear stress and oxidative stress used ex vivo porcine carotid arteries exposed to LLSS and OSS. Here, they showed that these explants reduced nitric oxide synthase 3 (eNOS) expression in low and oscillatory shear stress regions [103]. These ex vivo shear models have in time been refined to study the effects of other mechanical forces in the vascular environment in addition to shear stress. Here, parallel to the shear forces circumferential cyclic stretch can be controlled in addition to flow dynamics [104, 105]. The reduction of arterial compliance was shown to increase the risk of arterial disease through the interruption of the eNOS activation pathway and increasing vascular levels of oxidative stress [105]. Together, this ex vivo model makes it possible to dissect complex interactions of mechanical stresses in the vascular environment between shear and cyclic stretch [105].

The pressure myograph can be used to measure physiological functions and properties of small arteries, veins and other vessels with a maximal diameter of 6mm [106]. Here, a small segment of a vessel is mounted onto small glass cannula where they can be pressurized to a specific transmural pressure [107, 108]. In contrast to wire myograph where the constriction and dilation of the vessel is measured through a force transducer in high sensitivity isometric conditions, the pressure myograph uses a digital video edge-‐detection under isobaric conditions [107, 109, 110]. Therefore, the natural vessel diameter can be studied at a wide range of shear stresses and pressures applied to the lumen of the vessel [111]. The pressure myograph is primarily used for small vessels that have substantial vasoreactivity [112].

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Tabel 2: Advantages and disadvantages of ex vivo models in EC physiology

EX VIVO MODEL ADVANTAGES DISADVENTAGE

Vessel cannulation &

Pressure myograph • Control of shear and pressure

• Inside and outside of the vessel are

separated

• Record outer and luminal diameter of vessel

• Analysis of sample after primary experiment

• Record cross sectional area, wall thickness and media-‐lumen ratio

• Native tissue environment

• Possible occlusion of the vessel

• Quantity of animals to be used (lower

sensitivity of pressure myograph)

Finally, in vitro models have been developed using isolated cells or cell lines under different flow conditions. Maybe the easiest and cheapest method to subject ECs to shear stress is the orbital shaker method. (Figure 6D) ECs cultured in petri-‐dishes are placed on an orbital shaker platform inside a cell culture incubator. Computational fluid dynamics showed that when using a defined experimental setup (radius of orbital shaker and rotation rate) a good approximation of shear stress values could be achieved. It was shown that in the center of the plate a low shear stress with rapid variations in direction could be found and in the periphery high shear stress with uniform direction was present [113, 114]. This method makes it possible to harvest relatively high amount of cells in specific regions by using a standardized template to identify high and low shear stress regions [115].

The cone-‐and-‐plate viscometer was the first well-‐characterized in vitro shear stress device introduced by Forbes Dewey, Peter Davies and Michael Gimbrone [116, 117]. (Figure 6B) Here, the shear stress is created through the rotation of a

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cone above a stationary place containing ECs cultured on cover slips. This device was subsequently modified by other groups to integrate an optical system, which allowed the direct observation of EC in response to shear stress [118]. Next, Blackman et al. developed a shearing device based on the cone-‐and-‐plate using a micro-‐stepper motor technology to independently control the dynamics and steady components of the shear stress environment. Furthermore, this system was also fitted with a fluorescence microscope [119]. Finally, Tarbell and colleagues introduced the parallel disk viscometer [120]. (Figure 6C) Here, following the model of the cone-‐and-‐plate device the cone was replaced by a disk that was linked to a drive motor to produce a defined shear stress on the ECs and used to assess the effect of shear stress [121, 122].

Figure 6: Shear stress devices: A) parallel-‐plate flow chamber; B) cone-‐and-‐plate viscometer; C) parallel disk viscometer; D) orbital shaker; E) capillary tube. Adapted from [123]

Parallel-‐plate flow chamber systems have been used to analyze changes in the EC metabolism and morphology in response to shear stress [124-‐126]. Originally, Frangos, McIntire, and colleagues developed a flow chamber consisting of a polycarbonate plate, a rectangular silastic gasket and a glass slide with the EC monolayer [127, 128]. (Figure 7) The different parts of the device were held together by a vacuum at the periphery of the slide, forming a channel. At the time

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flow was applied to the channel by a hydrostatic pressure head between the two media reservoirs to produce steady flow or via cam-‐driven clamps upstream of the chamber to achieve pulsatile flow.

Figure 7: The parallel plate flow chamber. Cover slips were covered with confluent ECs. A silastic gasket was applied to separate the cover slip from the deck of the flow chamber. Vacuum was applied to hold the device together. Adapted from [129]

Several modified designs have been used to date. Firstly, to assess the EC monolayer permeability the flow chamber was attached to a circulating luminal loop and basal non-‐circulating loop [130]. Next, using a flow chamber with at the center a series of arrow shaped channels allowed for variable shear stresses within the same flow chamber. Thus, by changing the geometry of the center channels changes in shear stress were introduced without altering the gap width or overall flow rate [131]. With this device the effect of shear rates on platelet adhesion onto immobilized fibrinogen and von Willebrand factor (vWF) matrices was studied [132]. The sudden-‐expansion flow chamber and the backward-‐

facing step flow chamber were designed to mimic the spatial and temporal gradients in shear stress that overlap in atherosclerosis prone regions [133, 134]. The sudden-‐expansion flow chamber leads to a flow separation due to the asymmetric expansion of the flow path. Here, the fluid flows from a narrow channel directly to a wider channel. At the location of the step the flow recirculates with the direction against the main flow to finally reattach to the

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main unidirectional parabolic flow [134]. Finally, to study the effect of upstroke slopes of pulsatile flow (shear stress slew rates) the inlet and outlet of a parallel plate flow device was connected to symmetrical contractions and diffusers. Here, through precisely monitoring and controlling the frequency, amplitude and time-‐

average shear stress of pulsatile flow allowed the independent study of slew rates from other factors [135].

Finally, to study the effect of shear stress in combination with circumferential strain driven by the pulsating wall motion ECs were cultured inside elastic, silicon rubber capillary tubes applied to a pulsatile flow loop. (Figure 6E) Here, the combined effect of shear stress and strain were used to study the EC biological responses [136-‐138].

Tabel 3: Advantages and disadvantages of in vivo shear models in EC physiology

IN VITRO MODEL ADVANTAGES DISADVENTAGE

Orbital shaker • Large number of cells affected

• Conventional laboratory material

• Standardized templates to identify high and low shear regions[115]

• Shear Gradient from center to outside[114]

• Creation of secondary flows [139]

Cone & plate and parallel disk viscometer

• Re-‐usable

• Easy to clean

• Better for disturbed flow conditions [140]

• Possible creation of secondary flows [141]

Parallel flow

chambers • Ideal for laminar flow experiments [140]

• Different geometries possible

• Often sealed units (e.g.

dye injections not possible)

Silicon elastic tubes • Simultaneously shear

and strain forces • Difficult homogeneous seeding of cells inside the tubes

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1.3.4 Shear stress response

ECs respond to changes in local hemodynamic stimuli through synthesizing and metabolizing products that maintain or change vascular homeostasis [52].

Furthermore, distinct patterns of gene expression resulting in differentially activated mechanosensitive signaling pathways have been identified [142]. The resulting changes in EC phenotype to a dysfunctional state constitute a risk factor for the development of vascular diseases [52]. As noted before, atherosclerosis develops in a non-‐uniform manner in atheroprone areas.[43, 44, 143] These areas are predominantly located at arterial branch points and bifurcations or in curved arteries. Interestingly, in these areas the flow is disturbed and changes direction through the cardiac cycle resulting in low and/or oscillatory shear stress (atheroprone flow) [143]. Here, the ECs display a highly pro-‐inflammatory, pro-‐thrombotic, impaired barrier function phenotype that promotes a pathological outcome [43, 52, 53]. In contrary, unbranched and straight parts of arteries that are subjected to uniform high laminar shear stress develop no atherosclerotic lesions (atheroprotective flow) [143]. Indeed, ECs at these locations have a high expression of anti-‐inflammatory and anti-‐thrombotic genes [52, 53]. In the next paragraph intracellular signals assigned to atheroprotective and atheroprone shear stress will be discussed.

1.3.4.1 Atheroprotective shear stress

ECs exposed to unidirectional high laminar shear stress display an elongated morphology with an alignment in the direction of the flow [52]. Furthermore, ECs in these areas have been shown to form a thick glycocalyx [144]. At molecular level the transcriptional activation of the eNOS gene is one of key

Role of Cx40 in the healthy and diseased vascular endothelium

Thesis

Reference

Role of Cx40 in the healthy and diseased vascular endothelium

Role of Cx40 in the Healthy and Diseased Vascular Endothelium

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention Biologie

par

Jean-François DENIS de

Bassenge (Belgique)

Thèse n° 5144 Genève

2017

DE GENÈVE

DocroRAT Ès sctENcES, MENTIoN BtoloctE

Thèse de Monsieur Jean-François DENIS

<Role of Cx40 in the Healthy and Diseased Vascular Endothelium>

de

de

et

J.

of

of

-5144-

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Résumé

Abstract

Table of contents

Abbreviations

<Role of ^Cx40 in the Healthy and Diseased Vascular Endothelium>