Identification des facteurs d'échange de guanine-nucléotides (GEF) mécanosensibles : rôle dans l'homéostasie vasculaire

Les forces hémodynamiques jouent un rôle important dans le développement et l'homéostasie du réseau vasculaire. Les cellules endothéliales (CE) et les cellules musculaires lisses (VSMC), composant la paroi vasculaire, répondent à ces forces en réorganisant leur cytosquelette. Une altération des forces hémodynamiques provoque une alteration de ce cytosquelette et conduit à la survenue de pathologies vasculaires tels que l'athérosclérose et les anévrismes. Les facteurs d'échange de nucléotides de guanine (GEFs) médient l'activation spatio-temporelle des petites GTPases (RhoA, Rac1, Cdc42), acteurs clés de la dynamique du cytosquelette. L'identification des GEFs mécano-sensibles pourrait donc fournir de nouvelles cibles thérapeutiques dans les pathologies vasculaires. Un séquençage ARN effectuée sur des CE et des VSMC soumises à différentes forces hémodynamiques a permis d’identifier ARHGEF18 et ARHGEF40 dans les CE ainsi que NET1 dans les VSMCs comme des GEFs mecano-sensibles potentiels. Dans les CE, les contraintes de cisaillement pathologiques (3,6 et 36 dynes/cm2) réduisent l'activité d'Arhgef18 en comparaison aux contraintes de cisaillement physiologiques (16 dynes/cm2). De plus, Arhegf18 interagit uniquement avec RhoA mais l’extinction d'Arhgef18 réduit à la fois l'activité de RhoA et de Rac1. Les CEs déficientes en Arhgef18 ont une adhésion et une migration réduite en comparaison aux CEs contrôle. Sous contraintes de cisaillement physiologiques, l’extinction d’Arhgef18 altère l’alignement des CE dans le sens du flux et la localisation de ZO-1 et Claudin5 aux jonctions. Enfin, l’extinction d'Arhgef18 réduit de manière significative l'expression de COX2 médiée par TNFα. Ces résultats identifient Arhgef18 comme un GEF mécano-sensible qui joue un rôle important dans la physiologie des CEs et la prévention de l'inflammation et pourrait donc prévenir la survenue de pathologies vasculaires.Hemodynamic forces play an important role in the vascular network development and homeostasis. Altered hemodynamic forces have been shown to associate with vascular disorders such as atherosclerosis and aneurysms. Cytoskeletal rearrangement is the primary response of vascular cells such as endothelial cells (ECs) and smooth muscle cells (VSMCs) to hemodynamic forces and irregular cytoskeletal arrangements has been observed in areas prone to vascular disorders in vivo. Guanine nucleotide exchange factors (GEFs) mediate spatio-temporal activation of small-GTPases (RhoA, Rac1, Cdc42), which are key players of cytoskeletal dynamics. Identifying mechanosensitive GEFs may provide new potential therapeutic targets to treat vascular disorders. RNA sequencing performed on ECs and VSMCs subjected to various shear stress and cyclic stretch levels respectively, identified ARHGEF18, ARHGEF40 in ECs and NET1 in VSMCs as potential mechanosensitive GEFs. In ECs, pathological (3.6 & 36 dynes/cm2) shear stress reduced Arhgef18 activity compared to physiological (16 dynes/cm2) shear stress. In our hands, Arhgef18 interact with RhoA only but knocking down of Arhgef18 reduces both RhoA and Rac1 activity. Moreover, ECs silenced for Arhgef18 showed reduced adhesion and migration under static conditions. Under physiological shear stress conditions, loss of Arhgef18 altered cell alignment and junctional protein localization. Furthermore, knockdown of Arhgef18 significantly reduced TNFα mediated COX2 expression. These findings Identified Arhgef18 as a potential mechanosensitive GEF that plays important role in ECs physiology and inflammation thereby vascular diseases

ARHGEF18 participates in Endothelial Cell Mechano-sensitivity in Response to Flow

Abstract Hemodynamic forces play an important role in vascular network development and homeostasis. In physiological condition, shear stress generated by laminar flow promotes endothelial cells (EC) health and induces their alignment in the direction of flow. In contrast, altered hemodynamic forces induce endothelial dysfunction and lead to the development of vascular disorders such as atherosclerosis and aneurysms. Following mechano-sensor activation, Rho protein-mediated cytoskeletal rearrangement is one of the first steps in transforming flow-induced forces into intracellular signals in EC via guanine nucleotide exchange factors (RhoGEFs) that mediate the spatio-temporal activation of these Rho proteins. Here we identified ARHGEF18 as a flow-sensitive RhoGEF specifically activating RhoA. Both ARHGEF18 expression and activity were controlled by shear stress level. ARHGEF18 promotes EC adhesion, focal adhesion formation and migration. ARHGEF18 localized to the tight junction by interacting with ZO-1 and participated to shear stress-induced EC elongation and alignment via its nucleotide exchange activity and the activation of p38 MAPK. Our study therefore characterized ARHGEF18 as the first flow-sensitive RhoA GEF in ECs, whose activity is essential for the maintenance of intercellular junctions and a properly organized endothelial monolayer under physiological flow conditions

Rare coding variants in CTSO , a potential new actor of arterial remodeling, are associated to familial intracranial aneurysm

Background Intracranial aneurysm (IA) is a common cerebrovascular abnormality characterized by localized dilation and wall thinning in intracranial arteries, that frequently leads to fatal vascular rupture. The mechanisms underlying IA formation, growth and rupture are mostly unknown, and while increasing evidence suggest a genetic component of IA, identification of specific genes or causal molecular pathways remains largely inconclusive and only a small fraction of the risk attributable to genetics for IA in the general population. Methods: Here, we combined whole exome sequencing and identity-by -descent analyses with functional investigations to identify rare IA predisposing variants in familial forms of IA and understand their contribution to the pathophysiology of IA. Results We identified two rare missense variants in the CTSO gene shared by all the affected relatives in two large pedigrees with multiple IA-affected relative. CTSO encodes for the cysteine-type papain-like cathepsin CTSO. Functional analyses revealed that CTSO acts as an extracellular protease controlling vascular smooth muscle cell migration and adhesion to the extracellular matrix. CTSO depletion, as well as expression of the two CTSO variants, which were poorly secreted, led to increase the amount of fibronectin. This effect is associated with a marked increase in VSMC stiffness which was rescued by exogenous CTSO. Conclusions This report identifies rare CTSO variants in familial IA patients and suggests that the increased susceptibility to IA induced by these variants is likely related to their primary effects on the vascular tissue, and more particularly on the media layer of the wall of cerebral arteries

SH2 domain protein E and ABL signaling regulate blood vessel size.

Blood vessels in different vascular beds vary in size, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vessel size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow, eventually leading to the DA collapse. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA size in she mutants correlated with an increased endothelial expression of claudin 5a (cldn5a), which encodes a protein enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates vessel and lumen size during vascular tubulogenesis

Hybridization chain reaction (HCR) analysis of <i>cldn5b</i> mRNA expression at 24 hpf.

(A,B) cldn5b (purple) and kdrl:GFP fluorescence in the trunk region of she mutant and wild-type sibling embryos. DA, dorsal aorta; PCV, posterior cardinal vein. cldn5b fluorescence is shown in A’,B’. (C) Quantification of cldn5b fluorescence in the DA. p = 0.17, Student’s t-test. Error bars show SEM. Data show combined results from two independent experiments. (TIF)</p

Blood flow in the tail region of wild-type zebrafish embryos.

Wild-type siblings obtained from an incross of sheci26 heterozygous adults in kdrl:GFP; gata1:dsRed background were imaged at 4 dpf using confocal microscopy. (MP4)</p

Expression of <i>abl1</i> and <i>abl2</i> in different cell types during zebrafish embryogenesis based on single-cell RNA seq expression atlas.

Data were generated by Daniocell atlas https://daniocell.nichd.nih.gov/ [31]. (TIF)</p

Numerical original data used for graphs and summary statistics.

Tab numbers in the Excel table match to the figure numbers of corresponding graphs. (XLSX)</p