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Background Obstructive sleep apnea is characterized by repetitive pharyngeal collapses during sleep, leading to intermittent hypoxia ( IH ), the main contributor of obstructive sleep apnea–related cardiovascular morbidity. In patients and rodents with obstructive sleep apnea exposed to IH, vascular inflammation and remodeling, endothelial dysfunction, and circulating inflammatory markers are linked with IH severity. The nonmuscle myosin light chain kinase (nm MLCK ) isoform contributes to vascular inflammation and oxidative stress in different cardiovascular and inflammatory diseases. Thus, in the present study, we hypothesized that nm MLCK plays a key role in the IH ‐induced vascular dysfunctions and inflammatory remodeling. Methods and Results Twelve‐week‐old nm MLCK +/+ or nm MLCK −/− mice were exposed to 14‐day IH or normoxia. IH was associated with functional alterations characterized by an elevation of arterial blood pressure and stiffness and perturbations of NO signaling. IH caused endothelial barrier dysfunction (ie, reduced transendothelial resistance in vitro) and induced vascular oxidative stress associated with an inflammatory remodeling, characterized by an increased intima‐media thickness and an increased expression and activity of inflammatory markers, such as interferon‐γ and nuclear factor‐κB, in the vascular wall. Interestingly, nm MLCK deletion prevented all IH ‐induced functional and structural alterations, including the restoration of NO signaling, correction of endothelial barrier integrity, and reduction of both oxidative stress and associated inflammatory response. Conclusions nm MLCK is a key mechanism in IH ‐induced vascular oxidative stress and inflammation and both functional and structural remodeling.

International audience; Normal arterial aging processes involve vascular cell dysfunction associated with wall stiffening, the latter being due to progressive elastin and elastic fiber degradation, and elastin and collagen cross-linking by advanced glycation end products (AGEs). These processes progressively lead to cardiovascular dysfunction during aging. Elastin is only synthesized during late gestation and childhood, and further degradation occurring throughout adulthood cannot be physiologically compensated by replacement of altered material. However, the ATP-dependent K(+) channel opener minoxidil has been shown to stimulate elastin expression in vitro and in vivo in the aorta of young adult rats. Therefore, we have studied the effect of a 10-week chronic oral treatment with minoxidil (120 mg/L in drinking water) on the aortic structure and function in aged 24-month-old mice. Minoxidil treatment increased tropoelastin, fibulin-5, and lysyl-oxidase messenger RNA levels, reinduced a moderate expression of elastin, and lowered the levels of AGE-related molecules. This was accompanied by the formation of newly synthesized elastic fibers, which had diverse orientations in the wall. A decrease in the glycation capacity of aortic elastin was also produced by minoxidil treatment. The ascending aorta also underwent a minoxidil-induced increase in diameter and decrease in wall thickness, which partly reversed the age-associated thickening and returned the wall thickness value and strain-stress relation closer to those of younger adult animals. In conclusion, our results suggest that minoxidil presents an interesting potential for arterial remodeling in an antiaging perspective, even when treating already aged animals.

Williams-Beuren syndrome (WBS) (OMIM# 194050) is a rare, most often sporadic, genetic disease caused by a chromosomal microdeletion at locus 7q11.23 involving 28 genes. Among these, the elastin gene codes for the essential component of the arterial extracellular matrix. Developmental disorders usually associate an atypical face, cardiovascular malformations (most often supravalvular aortic stenosis and/or pulmonary artery stenosis) and a unique neuropsychological profile. This profile is defined by moderate mental retardation, relatively well-preserved language skills, visuospatial deficits and hypersociability. Other less known or rarer features, such as neonatal hypercalcemia, nutrition problems in infancy, ophthalmological anomalies, hypothyroidism, growth retardation, joint disturbances, dental anomalies and hypertension arising in adolescence or adulthood, should be treated. The aim of this paper is to summarize the major points of WBS regarding: (i) the different genes involved in the deletion and their function, especially the elastin gene and recent reports of rare forms of partial WBS or of an opposite syndrome stemming from a microduplication of the 7q11.23 locus, (ii) the clinical features in children and adults with a focus on cardiovascular injury, and (iii) the specific neuropsychological profile of people with WBS through its characteristics, the brain structures involved, and learning.

International audience; In vascular endothelium, adherens junctions between endothelial cells are composed of VE-cadherin (vascular endothelial cadherin), an adhesive receptor that is crucial for the proper assembly of vascular structures and the maintenance of vascular integrity. As a classical cadherin, VE-cadherin links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with the actin cytoskeleton via catenins. Although, from structural crystallographic data, a dimeric structure arranged in a trans orientation has emerged as a potential mechanism of cell-cell adhesion, the cadherin organization within adherens junctions remains controversial. Concerning VE-cadherin, its extracellular part possesses the capacity to self-associate in solution as hexamers consisting of three antiparallel cadherin dimers. VE-cadherin-based adherens junctions were reconstituted in vitro by assembly of a VE-cadherin EC (extracellular repeat) 1-EC4 hexamer at the surfaces of liposomes. The artificial adherens junctions revealed by cryoelectron microscopy appear as a two-dimensional self-assembly of hexameric structures. This cadherin organization is reminiscent of that found in native desmosomal junctions. Further structural studies performed on native VE-cadherin junctions would provide a better understanding of the cadherin organization within adherens junctions. Homophilic interactions between cadherins are strengthened intracellularly by connection to the actin cytoskeleton. Recently, we have discovered that annexin 2, an actin-binding protein connects the VE-cadherin-catenin complex to the actin cytoskeleton. This novel link is labile and promotes the endothelial cell switch from a quiescent to an angiogenic state.

Background Lymphomatoid papulosis (LyP) is classified as an indolent cutaneous lymphoma, but outcome dramatically worsens if LyP is associated with lymphoma. The frequency of this association remains unclear in the literature. Here, we assess the frequency and risk factors of association between LyP and another lymphoma in an 11-year retrospective study conducted in 8 dermatology departments belonging to the French Study Group on Cutaneous Lymphoma (FSGCL). Patients and methods Patients with LyP were identified and data extracted from the FSGCL registry between 1991 and 2006. Patients were followed up to January 2014. Age, sex, number of skin lesions, histologic subtype, and genotype were recorded at baseline. Risk factors were determined using univariate and multivariate analysis. Cumulative probability of association was calculated using the Kaplan-Meier method. Results We observed 52 cases of lymphomas (cutaneous, n = 38; systemic, n = 14) in 44 of 106 patients (41%). Lymphoma diagnosis was concomitant with or prior to LyP diagnosis in 31 cases and occurred during the course of LyP in 21 cases (cutaneous, n = 14; systemic, n = 7; median delay: 5 years; interquartile range: 1.5-7 years). In multivariate analysis, main prognostic factors for association between LyP and another lymphoma were older age (odds ratio [OR]: 1.05 per year; 95% confidence interval [CI]: 1.01-1.08; p = .011) and presence of a T-cell clone in LyP lesions (OR: 7.55; 95% CI: 2.18-26.18; p = .001). Conclusion Older age and presence of a T-cell clone in LyP lesions are risk factors for associated lymphomas in patients with LyP. These findings should help to identify patients who require close management in clinical practice. Implications for practice The management of lymphomatoid papulosis (LyP) is that of an indolent cutaneous lymphoma, based on its excellent prognosis. However, this good prognosis is altered if LyP is associated with lymphoma. Furthermore, risk factors for and frequency of this association remain unclear in the literature. The results presented here demonstrate a high rate of association between LyP and other lymphomas (41%) as well as a long median delay of occurrence (5 years), which emphasizes the need for prolonged follow-up of patients with LyP. Moreover, two main risk factors (i.e., older age and presence of a T-cell clone in LyP lesions) are highlighted, which should help clinical practitioners to identify patients who require close management.

International audience; OBJECTIVE:Hypocomplementemic urticarial vasculitis (HUV) is an uncommon vasculitis of unknown etiology that is rarely described in the literature. We undertook this study to analyze the clinical spectrum and the therapeutic management of patients with HUV.METHODS:We conducted a French nationwide retrospective study that included 57 patients with chronic urticaria, histologic leukocytoclastic vasculitis, and hypocomplementemia. We assessed clinical and laboratory data and evaluated the patients' cutaneous and immunologic responses to therapy. We evaluated treatment efficacy by measuring the time to treatment failure.RESULTS:Urticarial lesions were typically more pruritic than painful and were associated with angioedema in 51% of patients, purpura in 35%, and livedo reticularis in 14%. Extracutaneous manifestations included constitutional symptoms (in 56% of patients) as well as musculoskeletal involvement (in 82%), ocular involvement (in 56%), pulmonary involvement (in 19%), gastrointestinal involvement (in 18%), and kidney involvement (in 14%). Patients with HUV typically presented with low C1q levels and normal C1 inhibitor levels, in association with anti-C1q antibodies in 55% of patients. Hydroxychloroquine or colchicine seemed to be as effective as corticosteroids as first-line therapy. In patients with relapsing and/or refractory disease, rates of cutaneous and immunologic response to therapy seemed to be higher with conventional immunosuppressive agents, in particular, azathioprine, mycophenolate mofetil, or cyclophosphamide, while a rituximab-based regimen tended to have higher efficacy. Finally, a cutaneous response to therapy was strongly associated with an immunologic response to therapy.CONCLUSION:HUV represents an uncommon systemic and relapsing vasculitis with various manifestations, mainly, musculoskeletal and ocular involvement associated with anti-C1q antibodies, which were found in approximately half of the patients. The best strategy for treating HUV has yet to be defined.

International audience

We previously reported that vascular endothelial growth factor induced vascular endothelial (VE)-cadherin tyrosine phosphorylation at Y685 in a Src-dependent manner in vitro. Here, we studied the occurrence of Y685 phosphorylation in vivo in the female reproductive tract because it is a unique model of physiological vascular remodeling dependent on vascular endothelial growth factor. We first developed and characterized an anti-phospho-specific antibody against the site Y685 of VE-cadherin to monitor VE-cadherin phosphorylation along the four phases of mouse estrous cycle, termed proestrus, estrus, metestrus, and diestrus. A dynamic profile of tyrosine phosphorylated proteins was observed in both uterus and ovary throughout mouse estrous cycle, including kinase Src, which was found highly active at the estrus phase. The extent of tyrosine phosphorylated VE-cadherin was low at proestrus but strongly increased at estrus and returned to baseline at metestrus and diestrus, suggesting a potent hormonal regulation of this specific process. Indeed, C57Bl/6 female mice treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin confirmed a significant increase in phosphoY685-VE-cadherin compared with that in untreated mice. These results demonstrate that VE-cadherin tyrosine phosphorylation at Y685 is a physiological and hormonally regulated process in female reproductive organs. In addition, this process was concomitant with the early steps of vascular remodeling taking place at estrus stage, suggesting that phosphoY685-VE-cadherin is a biomarker of endothelial cell activation in vivo.

International audience; Confining cells on adhesive patterns allows performing robust, weakly dispersed, statistical analysis. A priori, adhesive patterns could be efficient tools to analyze intracellular cell stress fields, in particular when patterns are used to force the geometry of the cytoskeleton. This tool could then be very helpful in deciphering the relationship between the internal architecture of the cells and the mechanical, intracellular stresses. However, the quantification of the intracellular stresses is still something delicate to perform. Here we first propose a new, very simple and original method to quantify the intracellular stresses, which directly relates the strain the cells impose on the extracellular matrix to the intracellular stress field. This method is used to analyze how confinement influences the intracellular stress field. As a result, we show that the more confined the cells are, the more stressed they will be. The influence of the geometry of the adhesive patterns on the stress patterns is also discussed.

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action., Author Summary Pseudomonas aeruginosa is a leading cause of nosocomial infections and is a particular threat for cystic fibrosis and immunodepressed patients. One of the most aggressive toxins in its arsenal is ExoU, injected directly into target cells by a needle-like complex located on the surface of the bacterium, the type III secretion system. P. aeruginosa strains that express ExoU cause rapid cell death as a consequence of the membrane-destruction (phospholipase) potential of the toxin. In this work, we report the three-dimensional structure of ExoU in complex with a partner molecule, SpcU. ExoU contains three distinct regions, and the fold suggests how ExoU binds to the membrane or other molecules within the target cell and becomes activated. In addition, we also show that once it is translocated into the cell, ExoU co-localizes with intracellular organelles of the endosomal pathway, potentially in an attempt of the target cell to destroy the toxin. This work provides new insight into the cellular destruction mechanism of this aggressive toxin and could be a basis for the development of new inhibitors of P. aeruginosa pathogenesis.

International audience; Adherens junctions are required for vascular endothelium integrity. These structures are formed by the clustering of the homophilic adhesive protein VE-cadherin, which recruits intracellular partners, such as β- and α-catenins, vinculin, and actin filaments. The dogma according to which α-catenin bridges cadherin*β-catenin complexes to the actin cytoskeleton has been challenged during the past few years, and the link between the VE-cadherin*catenin complex and the actin cytoskeleton remains unclear. Recently, epithelial protein lost in neoplasm (EPLIN) has been proposed as a possible bond between the E-cadherin*catenin complex and actin in epithelial cells. Herein, we show that EPLIN is expressed at similar levels in endothelial and epithelial cells and is located at interendothelial junctions in confluent cells. Co-immunoprecipitation and GST pulldown experiments provided evidence that EPLIN interacts directly with α-catenin and tethers the VE-cadherin*catenin complex to the actin cytoskeleton. In the absence of EPLIN, vinculin was delocalized from the junctions. Furthermore, suppression of actomyosin tension using blebbistatin triggered a similar vinculin delocalization from the junctions. In a Matrigel assay, EPLIN-depleted endothelial cells exhibited a reduced capacity to form pseudocapillary networks because of numerous breakage events. In conclusion, we propose a model in which EPLIN establishes a link between the cadherin*catenin complex and actin that is independent of actomyosin tension. This link acts as a mechanotransmitter, allowing vinculin binding to α-catenin and formation of a secondary molecular bond between the adherens complex and the cytoskeleton through vinculin. In addition, we provide evidence that the EPLIN clutch is necessary for stabilization of capillary structures in an angiogenesis model.

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International audience; Microfibrils are macromolecular complexes associated with elastin to form elastic fibers that endow extensible tissues, such as arteries, lungs, and skin, with elasticity property. Fibrillin-1, the main component of microfibrils, is a 350-kDa glycoprotein for which genetic haploinsufficiency in humans can lead to Marfan syndrome, a severe polyfeatured pathology including aortic aneurysms and dissections. Microfibrils and fibrillin-1 fragments mediate adhesion of several cell types, including endothelial cells, while fibrillin-1 additionally triggers lung and mesangial cell migration. However, fibrillin-1-induced intracellular signaling is unknown. We have studied the signaling events induced in human umbilical venous endothelial cells (HUVECs) by aortic microfibrils as well as recombinant fibrillin-1 Arg-Gly-Asp (RGD)-containing fragments PF9 and PF14. Aortic microfibrils and PF14, not PF9, substantially and dose dependently increased HUVEC cytoplasmic and nuclear calcium levels measured using the fluorescent dye Fluo-3. This effect of PF14 was confirmed in bovine aortic endothelial cells. PF14 action in HUVECs was mediated by αvβ3 and α5β1 integrins, phospholipase-C, inosital 1,4,5-trisphosphate, and mobilization of intracellular calcium stores, whereas membrane calcium channels were not or only slightly implicated, as shown in patch-clamp experiments. Finally, PF14 enhanced endothelial cell proliferation and migration. Hence, fibrillin-1 sequences may physiologically activate endothelial cells. Genetic fibrillin-1 deficiency could alter normal endothelial signaling and, since endothelium dysfunction is an important contributor to Marfan syndrome, participate in the arterial anomalies associated with this developmental disease.

Endothelium integrity is maintained by junctional adhesion structures, in particular adherens junctions. Classical cadherins are the main molecule belonging to these structures. Two classical cadherins are expressed in the endothelium. First, the vascular endothelial cadherin or VE-cadherin is only expressed in endothelial cells and located to adherens junctions. Several studies have shown that this molecule is strictly necessary during vascular development. The second classical endothelial cadherin is represented by the neural cadherin or N-cadherin. Contrary to classical cadherins, the endothelial N-cadherin shows diffuse location on the entirely cell surface and interacts with mural cells for vessel stabilization. Moreover, its role in angiogenic process is still debated. The purpose of our study was to decipher the differential role of N- and VE-cadherin in endothelial cells and during angiogenesis. We have shown that VE-cadherin was able to organize and displace N-cadherin location on the cell surface. Moreover, VE-cadherin binding to p120 catenin is required for N-cadherin junctional exclusion. Furthermore, endothelial lipid rafts, known to organize classical cadherin at the membrane, contain exclusively VE-cadherin, where it specifically binds p120 catenin. So, this protein complex could form a platform that stabilizes cadherin at the cell membrane and in the same way, strengthens intercellular interactions. Also, we have found that three endothelial genes : PECAM, Flk-1 and Tie-2, could be transcriptionally controlled by VE-cadherin in a ES cell-derived endothelial differential model. We didn't found this molecular mechanism but, a cross talk between the VE-cadherin dependant PECAM regulation and adherens molecule to extracellular matrix is possible. In the end, we characterized a new role of VE-cadherin. This adherent molecule might be crucial to endothelium biology, especially in the control of important endothelial proteins., L'intégrité de l'endothelium vasculaire est maintenue par des structures d'adhérence jonctionelle, notamment les jonctions adhérentes, dont la VE-cadhérine est le constituant majeur. Cette cadhérine est exclusivement exprimée dans les cellules endothéliales et joue un rôle clé au cours de la formation du plexus vasculaire. Les cellules endothéliales expriment également une autre cadhérine : la N-cadhérine. Bien que possédant une forte homologie structurale avec la VE-cadhérine, celle-ci possède une localisation subcellulaire originale pour une cadhérine classique puisqu'elle est localisée de manière diffuse au niveau de la membrane. Dans le développement vasculaire, elle pourrait ainsi permettre le recrutement des cellules murales par l'endothelium conduisant à la stabilisation du vaisseau. Mon travail de thèse a été d'analyser les propriétés différentielles de ces deux cadhérines dans la cellule endothéliale ainsi que le mécanisme d'exclusion jonctionelle de la N-cadhérine. Nous avons montré que la VE- et la N-cadhérine interagissent similairement à l'- et à la -caténine mais à l'inverse, p120 caténine lie préférentiellement la VE-cadhérine. Cette propriété confère à la VE-cadhérine la capacité d'exclure la N-cadhérine de la jonction interendothéliale. D'autre part, nous avons montré qu'une petite proportion de la VE-cadhérine est enchâssée dans des microdomaines riches en cholestérol : les radeaux lipidiques, à laquelle p120 est très fortement associée. Cette fraction pourrait représenter un pool de VE-cadhérine assurant la stabilité de la jonction et le maintien de la force cohésive entre cellules endothéliales. Nous avons également montré dans un modèle de différenciation cellulaire dérivé des cellules ES murines (embryonic stem cell), que l'expression de la VE-cadhérine était nécessaire à l'expression d'autres gènes endothéliaux : PECAM, Flk-1 et Tie-1. La VE-cadhérine exercerait un contrôle transcriptionel de l'expression de ces gènes mais le mécanisme exact de régulation n'a pas pu être décrypté. L'ensemble de ce travail permet donc de montrer un autre visage de la VE-cadhérine dans la biologie de l'endothelium qui serait de réguler des protéines-clé de l'endothelium.

International audience; Vascular endothelium (VE), the monolayer of endothelial cells that lines the vascular tree, undergoes damage at the basis of some vascular diseases. Its integrity is maintained by VE-cadherin, an adhesive receptor localized at cell-cell junctions. Here, we show that VE-cadherin is also located at the tip and along filopodia in sparse or subconfluent endothelial cells. We observed that VE-cadherin navigates along intrafilopodial actin filaments. We found that the actin motor protein myosin-X is colocalized and moves synchronously with filopodial VE-cadherin. Immunoprecipitation and pulldown assays confirmed that myosin-X is directly associated with the VE-cadherin complex. Furthermore, expression of a dominant-negative mutant of myosin-X revealed that myosin-X is required for VE-cadherin export to cell edges and filopodia. These features indicate that myosin-X establishes a link between the actin cytoskeleton and VE-cadherin, thereby allowing VEcadherin transportation along intrafilopodial actin cables. In conclusion, we propose that VE-cadherin trafficking along filopodia using myosin-X motor protein is a prerequisite for cell-cell junction formation. This mechanism may have functional consequences for endothelium repair in pathological settings.

Vascular endothelial (VE) cadherin, a specific adhesive protein located at sites of endothelial intercellular contact, is required for junction integrity, vascular morphogenesis, and angiogenesis. We recently demonstrated that VE-cadherin was tyrosine phosphorylated in vivo in angiogenic conditions and not in quiescent endothelium. Tyrosine 685 is the only target for Src kinase in the VEGF pathway. Thus, phospho-VE-cadherin is characteristic for activated endothelium. The present study was designed to examine whether VE-cadherin was phosphorylated in tumoral angiogenesis in vivo, using two models of murine tumors: hepatocellular carcinoma model of mice with previous described hyperangiogenesis and glioblastoma model of rat brain with increased vascular permeability. I found the same tyrosine phosphorylation of VE-cadherin in both models of tumors and the site identified was Y658. To further investigate the mechanism of VE-cadherin phosphorylation, we used cultured cells stimulated with TNF because of the described role of Y658 in vascular permeability and extravasation of leukocytes and the related presence of inflammation in both models. I found that VE-cadherin is phosphorylated on Y658 with TNF and the kinase responsible for this phosphorylation was SYK (SiRNA of Syk, mutagenesis of VE-cadherin Y658 cytoplamsic domain). Because VE-cadherin is susceptible for extra-cellular domain proteolysis in response to TNFα, we studied in parallel the presence in cancer and inflammatory pathologies patients sera the presence of circulating VE-cadherin. VE-cadherin could be a feature of activated endothelium both by phosphorylation and proteolysis., L'endothélium vasculaire est la première couche de cellules en contact avec le flux sanguin. Au cours de processus pathologiques, les cellules endothéliales subissent des remaniements notoires dus à la présence de nombreux facteurs. Dans ce cas, l'intégrité de l'endothélium, assurée principalement par les jonctions adhérentes endothéliales dont la V(ascular)E(ndothelial)-cadhérine est la protéine clé, est perturbée. La phospho-VE-cadhérine est une caractéristique des cellules endothéliales activées in vitro. Notre laboratoire a montré l'existence d'une forme phosphorylée de VE-cadhérine in vivo au cours de l'angiogenèse physiologique. Des tyrosines importantes ont été identifiées : la tyrosine 685, qui est la cible directe de la tyrosine kinase Src en réponse au VEGF ainsi que les tyrosines 658 et 731. De plus, la VE-cadhérine peut être sensible à la protéolyse. Dans ce travail, la phosphorylation de la VE-cadhérine a été étudiée dans un contexte pathologique d'inflammation et de cancer à l'aide de deux modèles murins de tumeurs hépatiques ou cérébrales. J'ai démontré l'existence in vivo d'une forme phosphorylée de VE-cadhérine sur le site Y658. L'analyse d'une tyrosine kinase particulière, Syk, par si RNA ainsi que par des expériences de phosphorylation in vitro suggèrent fortement l'implication de SYK dans cette phosphorylation. En parallèle, dans le cadre d'une étude sur plusieurs pathologies, nous avons mis en évidence la présence d'une forme circulante de VE-cadhérine. La VE-cadhérine, tant par sa phosphorylation que par sa présence dans les fluides biologiques, pourrait être une signature de l'endothélium activé (phosphorylation) et/ou agressé (protéolyse).

Fibrillin-1, the main component of microfibrils and second main component of elastic fibers, is a glycoprotein whose gene mutation in human is responsible for Marfan syndrome, characterized at the vascular level by aortic aneurysm and dissections. In this work, we investigated on the one hand, the role of fibrillin-1 in human endothelial cell physiology and, on the other hand, the effect of fibrillin-1 quantitative deficiency on the structure and function of the cardiovascular system during aging in mice. Fibrillin-1 fragment "PF14" induced calcium signaling through α5β1 and αvβ3 integrins, phospholipase C activation and intracellular calcium store mobilization, as well as endothelial cell proliferation and migration. Fibrillin-1 severe or full deficiency leads to premature lethality (within two weeks postnatal) in mice, while partial deficiency, through fibrillin-1 heterozygous hypomorphic deletion of exons 19 to 24 (mgΔ deletion), does not affect mice longevity. Fbn-1+/mgΔ mice were hypotensive with cardiac hypertrophy and presented aortic aneurysm becoming more frequent in aged animals. The aortic wall of Fbn-1+/mgΔ mice presented structural (elastic fibers fragmentations), biomechanical (increased aortic stiffness) and vasomotor alterations. This work suggests that fibrillin- 1 quantitative deficiency may alter the signaling events normally triggered by this protein in endothelial cells during vascular morphogenesis, and subsequently induce aneurysms in the aorta wall during ageing. The opposite effects of fibrillin-1 and elastin deficiencies suggest opposite roles for these two major components of elastic fibers. Moreover, this work validates Fbn-1+/mgΔ mice as a structural and functional model for Marfan syndrome., La fibrilline-1, composant majeur des microfibrilles et second composant majeur des fibres élastiques, est une glycoprotéine dont la mutation du gène chez l'humain est responsable du syndrome de Marfan, caractérisé par des anévrismes et des dissections aortiques. Dans ce travail de thèse, nous avons étudié d'une part le rôle de la fibrilline-1 dans la physiologie des cellules endothéliales vasculaires humaines et d'autre part la conséquence du déficit quantitatif de la fibrilline-1 sur la structure et la fonction du système cardiovasculaire au cours du vieillissement chez la souris. Le fragment de fibrilline-1 "PF14" induit une signalisation calcique via les intégrines α5β1 et αvβ3, l'activation de la phospholipase C et la mobilisation des réserves intracellulaires de calcium, ainsi que la prolifération et la migration des cellules endothéliales humaines. La déficience quantitative sévère ou totale de la fibrilline-1 entraîne une létalité chez la souris dans les deux semaines après la naissance alors que celle, partielle, due à la délétion hétérozygote hypomorphique des exons 19 à 24 du gène de la fibrilline-1 (délétion mgΔ) n'affecte pas la longévité des souris. Les souris Fbn-1+/mgΔ présentent une hypertrophie cardiaque et une hypotension ainsi que des anévrismes aortiques qui deviennent plus fréquents avec l'âge. La paroi aortique de ces souris présente des altérations structurales (fragmentations des lames élastiques), biomécaniques (l'augmentation de la rigidité) et de la vasomotricité, qui s'accentuent avec l'âge. Nos résultats suggèrent que la déficience quantitative de la fibrilline-1 pourrait altérer la signalisation que cette protéine déclenche normalement dans les cellules endothéliales dès la phase de morphogénèse artérielle, et induire en conséquence une pathologie anévrismale de la paroi aortique au cours du vieillissement. Les effets opposés des déficiences quantitatives en fibrilline-1 et en élastine sur la fonction vasculaire suggèrent des rôles opposés pour ces deux composants majeurs des fibres élastiques. Ce travail permet par ailleurs de valider les souris Fbn-1+/mgΔ, tant sur le plan structural que fonctionnel, comme modèle du syndrome de Marfan.

International audience; The endothelium, lining the inner side of all vessel types, is constituted of a monolayer of endothelial cells with cobblestone morphology. Endothelial cell-cell contacts contain numerous transmembrane adhesive proteins that are either clustered in junctional structures or located along the intercellular cleft. These proteins promote cell-cell adhesion and control vascular permeability to fluids and molecules, as well as transmigration of various types of leukocytes. In addition, recent findings showed that constituents of the junctions might be part of the vascular invasion machinery by activating cell protrusions. Such activities may thus be considered as markers of pathological angiogenesis or targets of antiangiogenic therapy. L'endothélium, localisé sur la face interne de tous les types de vaisseaux, est constitué d'une monocouche pavimenteuse de cellules endothéliales. La zone de contact intercellulaire endothéliale contient plusieurs protéines transmembranaires à activité adhésive qui sont soit incluses dans des structures jonctionnelles spécifiques, soit localisées le long de la zone de contact. Ces protéines sont essentielles pour l'adhérence intercellulaire et le contrôle de la perméabilité vasculaire aux fluides, aux molécules et à la transmigration de plusieurs types de globules blancs. Récemment, il a été montré que certains constituants des jonctions pouvaient également être impliqués dans l'activité protrusive des cellules permettant l'invasion cellulaire. Ces propriétés peuvent donc être considérées comme des cibles pour des thérapies antiangiogéniques.

Fibrillin-1, the main component of microfibrils and second main component of elastic fibers, is a glycoprotein whose gene mutation in human is responsible for Marfan syndrome, characterized at the vascular level by aortic aneurysm and dissections. In this work, we investigated on the one hand, the role of fibrillin-1 in human endothelial cell physiology and, on the other hand, the effect of fibrillin-1 quantitative deficiency on the structure and function of the cardiovascular system during aging in mice. Fibrillin-1 fragment "PF14" induced calcium signaling through α5β1 and αvβ3 integrins, phospholipase C activation and intracellular calcium store mobilization, as well as endothelial cell proliferation and migration. Fibrillin-1 severe or full deficiency leads to premature lethality (within two weeks postnatal) in mice, while partial deficiency, through fibrillin-1 heterozygous hypomorphic deletion of exons 19 to 24 (mgΔ deletion), does not affect mice longevity. Fbn-1+/mgΔ mice were hypotensive with cardiac hypertrophy and presented aortic aneurysm becoming more frequent in aged animals. The aortic wall of Fbn-1+/mgΔ mice presented structural (elastic fibers fragmentations), biomechanical (increased aortic stiffness) and vasomotor alterations. This work suggests that fibrillin- 1 quantitative deficiency may alter the signaling events normally triggered by this protein in endothelial cells during vascular morphogenesis, and subsequently induce aneurysms in the aorta wall during ageing. The opposite effects of fibrillin-1 and elastin deficiencies suggest opposite roles for these two major components of elastic fibers. Moreover, this work validates Fbn-1+/mgΔ mice as a structural and functional model for Marfan syndrome., La fibrilline-1, composant majeur des microfibrilles et second composant majeur des fibres élastiques, est une glycoprotéine dont la mutation du gène chez l'humain est responsable du syndrome de Marfan, caractérisé par des anévrismes et des dissections aortiques. Dans ce travail de thèse, nous avons étudié d'une part le rôle de la fibrilline-1 dans la physiologie des cellules endothéliales vasculaires humaines et d'autre part la conséquence du déficit quantitatif de la fibrilline-1 sur la structure et la fonction du système cardiovasculaire au cours du vieillissement chez la souris. Le fragment de fibrilline-1 "PF14" induit une signalisation calcique via les intégrines α5β1 et αvβ3, l'activation de la phospholipase C et la mobilisation des réserves intracellulaires de calcium, ainsi que la prolifération et la migration des cellules endothéliales humaines. La déficience quantitative sévère ou totale de la fibrilline-1 entraîne une létalité chez la souris dans les deux semaines après la naissance alors que celle, partielle, due à la délétion hétérozygote hypomorphique des exons 19 à 24 du gène de la fibrilline-1 (délétion mgΔ) n'affecte pas la longévité des souris. Les souris Fbn-1+/mgΔ présentent une hypertrophie cardiaque et une hypotension ainsi que des anévrismes aortiques qui deviennent plus fréquents avec l'âge. La paroi aortique de ces souris présente des altérations structurales (fragmentations des lames élastiques), biomécaniques (l'augmentation de la rigidité) et de la vasomotricité, qui s'accentuent avec l'âge. Nos résultats suggèrent que la déficience quantitative de la fibrilline-1 pourrait altérer la signalisation que cette protéine déclenche normalement dans les cellules endothéliales dès la phase de morphogénèse artérielle, et induire en conséquence une pathologie anévrismale de la paroi aortique au cours du vieillissement. Les effets opposés des déficiences quantitatives en fibrilline-1 et en élastine sur la fonction vasculaire suggèrent des rôles opposés pour ces deux composants majeurs des fibres élastiques. Ce travail permet par ailleurs de valider les souris Fbn-1+/mgΔ, tant sur le plan structural que fonctionnel, comme modèle du syndrome de Marfan.

Protocadherins are transmembrane proteins exhibiting homophilic adhesive activities through their extracellular domain. Protocadherin 12 ( Pcdh12) is expressed in angiogenic endothelial cells, mesangial cells of kidney glomeruli, and glycogen cells of the mouse placenta. To get insight into the role of this protein in vivo, we analyzed PCDH12-deficient mice and investigated their placental phenotype. The mice were alive and fertile; however, placental and embryonic sizes were reduced compared with wild-type mice. We observed defects in placental layer segregation and a decreased vascularization of the labyrinth associated with a reduction in cell density in this layer. To understand the molecular events responsible for the phenotypic alterations observed in Pcdh12−/− placentas, we analyzed the expression profile of embryonic day 12.5 mutant placentas compared with wild-type placentas, using pangenomic chips: 2,289 genes exhibited statistically significant changes in expressed levels due to loss of PCDH12. Functional grouping of modified genes was obtained by GoMiner software. Gene clusters that contained most of the differentially expressed genes were those involved in tissue morphogenesis and development, angiogenesis, cell-matrix adhesion and migration, immune response, and chromatin remodeling. Our data show that loss of PCDH12 leads to morphological alterations of the placenta and to notable changes in its gene expression profile. Specific genes emerging from the microarray screen support the biological modifications observed in PCDH12-deficient placentas.

International audience; The vascular endothelial cadherin (VE-cad)-based complex is involved in the maintenance of vascular endothelium integrity. Using immunoprecipitation experiments, we have demonstrated that, in confluent human umbilical vein endothelial cells, the VE-cad-based complex interacts with annexin 2 and that annexin 2 translocates from the cytoplasm to the cell-cell contact sites as cell confluence is established. Annexin 2, located in cholesterol rafts, binds to both the actin cytoskeleton and the VE-cad-based complex so the complex is docked to cholesterol rafts. These multiple connections prevent the lateral diffusion of the VE-cad-based complex, thus strengthening adherens junctions in the ultimate steps of maturation. Moreover, we observed that the down-regulation of annexin 2 by small interfering RNA induces a delocalization of VE-cad from adherens junctions and consequently a destabilization of these junctions. Furthermore, our data indicate that the decoupling of the annexin 2/p11 complex from the VE-cad-based junction, triggered by vascular endothelial growth factor treatment, facilitates the switch from a quiescent to an immature state.

International audience; The bacterial lacZ gene encoding for beta-galactosidase (beta-gal) is a common reporter gene used in transgenic mice. Nonetheless, the absence of fluorigenic substrates usable in live animals greatly hampered the non-invasive follow-up of this reporter gene expression. We used far-red fluorescence for imaging beta-Gal expression in live cells in vitro or in vivo. The 9H-(1,3-dichloro-9,9-dimethylacridin- 2-one-7-yl) beta-D-galactopyranoside substrate was used to monitor beta-Gal expression as a reporter of tumor growth, or of the physiological levels of an endogenous gene or of gene transfer in lung. A quantitative evaluation of this method as well as a comparison of its sensitivity with Firefly Luciferase-based bioluminescence was also performed. In vivo measurements showed that 10(3) beta-Gal tumor cells located under the skin were detectable. In deeper organs like lung, as little as 5 ng of beta-Gal or Luciferase enzymes per mg of proteins were measured, confirming that both techniques reached similar sensibilities. Nonetheless, quantitative comparison of beta-Gal levels measured with far-red imaging or with a standardized enzymatic evaluation after killing revealed that the 2D-fluorescent reflectance imaging method is submitted to a color-dependent disparity of the organs and cannot supply quantitative measurements but that a simple correction can be applied.

Tyrosine phosphorylation of VE-cadherin in response to VEGF : molecular mechanisms and physiopathologic implications. Vascular integrity deeply depends on endothelial cell-cell junction stability. VE- (Vascular Endothelial)-cadherin is a key component of this junctions, and its tyrosine phosphorylation is thought to be a potent mechanism that controls vascular permeability. In this report, we demonstrated the existence of a tyrosine phosphorylated form of VE-cadherin in vivo. Remarkably, VE-cadherin tyrosine phosphorylation was strongly increased in ovary and uterus during hormone-induced angiogenesis, but not in other adult quiescent tissues. For the first time, we demonstrated that Src had a permanent association with VE-cadherin, in vivo and in vitro. Moreover, in agreement with other in vitro data, we found a transient association between VEGFR-2 and VE-cadherin in vivo in angiogenic tissues. Using several approaches, we demonstrated that VE-cadherin is a direct substrate for Src kinase in VEGF signaling pathway, and that tyrosine 685 is the unique phosphorylated site. In vitro wound healing test suggested that phosphorylation of Y685 in response to VEGF may be a key event in the intracellular signaling involved in migration process of endothelial cells. Finally, studying human breast carcinoma we also demonstrated the existence of a tyrosine phosphorylated form of VE-cadherin in tumor angiogenesis and its association with a PI3K and Src family kinase activity. Overall, this data concluded that Src kinase directly phosphorylates VE-cadherin on tyrosine 685 in response to VEGF, and this phosphorylation may be in vivo a sign of an angiogenic endothelium., L'intégrité de l'endothélium vasculaire est dépendante de l'organisation des jonctions adhérentes entre les cellules endothéliales. La VE (Vascular Endothelial)-cadhérine un constituant essentiel de ces jonctions et sa phosphorylation sur tyrosine pourrait être un moyen de régulation de la perméabilité endothéliale. Ce travail de thèse a permis de valider l'existence in vivo de la phosphorylation sur tyrosine de la VE-cadhérine. Nous montrons en effet la très forte induction de cette phosphorylation au cours de l'angiogenèse physiologique dans l'ovaire et l'utérus, par rapport aux tissus quiescents. Pour la première fois, nous rapportons l'association constante de la tyrosine kinase Src à la VE-cadhérine, à la fois in vivo et in vitro. De plus, en accord avec les données de la littérature in vitro, nous confirmons in vivo l'association transitoire du VEGFR2 à la VE-cadhérine dans un contexte angiogénique. Par différentes approches, nous apportons la preuve que la VE-cadhérine est un substrat direct de Src, et que la tyrosine 685 du domaine cytoplasmique est la cible unique de la kinase en réponse au VEGF. Grâce à un test de blessure d'HUVECs, nous montrons l'importance de la phosphorylation par Src de la tyrosine 685 dans la migration des cellules endothéliales induite par le VEGF. Enfin, l'étude de carcinomes mammaires nous as permis de démontrer l'existence d'une forme phosphorylée sur tyrosine de la VE-cadhérine également dans l'angiogenèse tumorale, et son association avec une activité kinase de type Src et PI3K. L'ensemble de ce travail permet de conclure à la phosphorylation par Src de la VEcadhérine sur la Y685 en réponse au VEGF, et suggère que cette phosphorylation représente in vivo la signature d'un endothélium activé.

Tumoral cells as leucocytes get through the vascular endothelium across adherens junctions, mainly composed of VE-cadherin. This transmigration process disturbs transiently the junctions, impairing so the endothelium integrity.To understand the mechanisms leading to the opening of adherens junctions, we first determined the proteic composition of the VE-cadherin-based complex of primary endothelial cell (HUVEC) mature adherens junctions. To do so, we developed a method coupling immunoprecipitation and mass spectrometry-based proteomic analysis (LC-nanoESI-MS/MS). Partners of VE-cadherin, so far unknown such as annexin 2 and moesin, were so identified.Our results demonstrated that, in confluent HUVECs, the VE-cadherin-based complex interacts with annexin 2 and that annexin 2 translocates from the cytoplasm to the plasma membrane as cell reach confluence. Annexin 2, located in cholesterol rafts, binds both to the actin cytoskeleton and the VE-cadherin-based complex so the complex is docked to cholesterol rafts. These multiple connections prevent the lateral diffusion of the VE-cadherin-based complex thus strengthening adherens junctions in the ultimate steps of maturation. Moreover, we observed that the down-regulation of annexin 2 by siRNA induces a delocalization of VE-cadherin from adherens junctions and consequently a destabilization of these junctions. Furthermore, our data suggest that the decoupling of the annexin 2/p11 complex from the VE-cadherin-based junction, triggered by Vascular Endothelial Growth Factor facilitates the switch from a quiescent to an immature state.Moesin seems to interact with the VE-cadherin-based complex in immature junctions of sub-confluent HUVECs. Moesin might be involved in production of early cell-cell contact rather than in adherens junction maturation., Les cellules tumorales, comme les leucocytes, franchissent l'endothélium vasculaire au niveau des jonctions intercellulaires à l'endroit même où s'exprime la VE-cadhérine, la protéine majeure des jonctions adhérentes. Cette transmigration déstabilise transitoirement les jonctions, ce qui affecte l'intégrité de ce tissu.Afin de comprendre les mécanismes sous-jacents à l'ouverture des jonctions, nous avons établi la composition protéique du complexe à base de VE-cadhérine des jonctions adhérentes matures de cellules endothéliales primaires confluentes de type HUVEC. Pour cela, nous avons couplé immunoprécipitation et analyse protéomique par spectrométrie de masse (LC-nanoESI-MS/MS). Nous avons ainsi identifié de nouveaux partenaires du complexe à base de VE-cadhérine jamais identifiés auparavant au niveau de la jonction adhérente endothéliale. Parmi ceux-ci se trouvent des protéines liant l'actine telles l'annexine 2 et la moésine.Nos résultats indiquent que l'annexine 2, qui s'accumule à la membrane plasmique au niveau des radeaux de cholestérol, entre en interaction directe avec le complexe à base de VE-cadhérine. Ainsi, l'annexine 2 connecte le complexe jonctionnel à base de VE-cadhérine au cytosquelette d'actine dans les HUVEC confluentes. L'utilisation de siRNA nous a permis d'établir que l'expression de l'annexine 2 est absolument nécessaire au maintien de la VE-cadhérine à la membrane plasmique. Nos résultats suggèrent que l'annexine 2, connectée à l'actine, arrime le complexe à base de VE-cadhérine au niveau des radeaux de cholestérol. En limitant la diffusion membranaire du complexe jonctionnel, ces interactions aboutissent à une consolidation des jonctions adhérentes ce qui contribue à maintenir l'intégrité de l'endothélium vasculaire. Lorsque les HUVEC sont soumises à des molecules destabilisant les jonctions adhérentes, une délocalisation de l'annexine 2 de la membrane vers le cytosol et une perturbation de la localisation de la VE-cadhérine sont observés. Ceci suggère que le prérequis à l'ouverture des jonctions adhérentes nécessite une rupture de l'interaction existant entre le complexe à base de VE-cadhérine et l'annexine 2.La moésine, quant à elle, semble interagir avec le complexe à base de VE-cadhérine dans les jonctions immatures formées entre cellules sub-confluentes. La moésine serait donc impliquée dans l'établissement des contacts intercellulaires précoces plutôt que dans la maturation des jonctions endothéliales.