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Background Metabolic syndrome (MetS) is characterized by a cluster of interconnected risk factors -hyperglycemia, dyslipidemia, hypertension and obesity- leading to an increased risk of cardiovascular events. Small extracellular vesicles (sEVs) can be considered as new biomarkers of different pathologies, and they are involved in intercellular communication. Here, we hypothesize that sEVs are implicated in MetS-associated endothelial dysfunction. Methods Circulating sEVs of non-MetS (nMetS) subjects and MetS patients were isolated from plasma and characterized. Thereafter, sEV effects on endothelial function were analyzed by measuring nitric oxide (NO) and reactive oxygen species (ROS) production, and mitochondrial dynamic proteins on human endothelial aortic cells (HAoECs). Results Circulating levels of sEVs positively correlated with anthropometric and biochemical parameters including visceral obesity, glycaemia, insulinemia, and dyslipidemia. Treatment of HAoECs with sEVs from MetS patients decreased NO production through the inhibition of the endothelial NO-synthase activity. Injection of MetS-sEVs into mice impaired endothelium-dependent relaxation induced by acetylcholine. Furthermore, MetS-sEVs increased DHE and MitoSox-associated fluorescence in HAoECs, reflecting enhanced cytosolic and mitochondrial ROS production which was not associated with mitochondrial biogenesis or dynamic changes. MetS patients displayed elevated circulating levels of LPS in plasma, and, at least in part, it was associated to circulating sEVs. Pharmacological inhibition and down-regulation of TLR4, as well as sEV-carried LPS neutralization, results in a substantial decrease of ROS production induced by MetS-sEVs. Conclusion These results evidence sEVs from MetS patients as potential new biomarkers for this syndrome, and TLR4 pathway activation by sEVs provides a link between the endothelial dysfunction and metabolic disturbances described in MetS.

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis., The mechanisms underlying immunpathologies in dengue virus (DENV) infection are incompletely understood. Here, authors show that TLR2 recognizes DENV particles inducing cytokine expression and activating vascular endothelium cells in vitro, and that TLR2 expression on monocytes correlates with disease severity in patients.

Voir aussi le même article publié dans le livre : https://onlinelibrary.wiley.com/doi/book/10.1128/9781683670261 ; Dépôt : https://hal-pasteur.archives-ouvertes.fr/pasteur-03116237; International audience; The endothelium is the layer of endothelial cells lining the inner surface of blood vessels, which span the entire body and ensure the distribution of blood throughout the organism. It is estimated that the human body contains a staggering 100,000 km of blood vessels, more than twice the earth’s circumference. Therefore, a bacterium reaching the circulation is engaged in a network of huge proportions. Moreover, a pathogen traveling through the circulatory system does not encounter a homogeneous environment, as an important feature of the vascular network is its diversity. Although endothelial cells are present in all vessels, the organization of the vessel wall, which is formed by three layers—the tunica intima, media, and adventitia (from the vessel lumen outward)—is different among different vessel types and different organs. Vessels can be first differentiated by the complex extracellular matrix layers surrounding them. For instance, elastic arteries such as the aorta are surrounded by 50 elastic layers, providing them with unique mechanical properties. Second, the cellular content is also different according to vessel type; the walls of arteries and veins contain a layer of smooth muscle cells that gives them the capacity to relax or constrict in response to vasoactive molecules. An additional level of complexity in the network stems from the fact that larger vessels, veins or arteries, are themselves vascularized by smaller vessels, the vasa vasorum.

International audience; Mice with a global deletion of α1AMPK are characterized by endothelial dysfunction and NADPH oxidase subunit 2 (NOX-2)-mediated vascular oxidative stress. However, the underlying mechanisms are incompletely understood and may involve endothelial NOX-2 upregulation or facilitated vascular infiltration of phagocytic cells. Therefore, the current study was designed to investigate the vascular effects of chronic angiotensin II (AngII) infusion in mice with an endothelial-specific α1AMPK deletion. A mouse strain with endothelial-specific α1AMPK deletion was generated by breeding α1AMPKflox/flox mice with TekCre+ or Cadh5Cre+ mice. Chronic AngII infusion (0.5 mg/kg/day for 7day) caused mild endothelial dysfunction in wild-type mice that was significantly aggravated in endothelial α1AMPK knockout mice. Aortic NOX-2 and CD68 expression were increased, indicating that infiltrating leukocytes may significantly contribute to enhanced vascular oxidative stress. Flow cytometry revealed a higher abundance of aortic CD90.2+ T-cells, CD11b+F4/80+ macrophages and Ly6G-Ly6C+ monocytes. Vascular mRNA expression of monocyte chemoattractant protein 1, CCL5 and vascular cell adhesion molecule 1 was enhanced in AngII-infused mice lacking endothelial α1AMPK, facilitating the recruitment of inflammatory cells to the vessel wall. In addition, AngII-induced upregulation of cytoprotective heme oxygenase 1 (HO-1) was blunted in mice with endothelial α1AMPK deletion, compatible with an impaired antioxidant defense in these animals. In summary, endothelial expressed α1AMPK limits the recruitment of inflammatory cells to the vessel wall and maintains HO-1 mediated antioxidant defense. Both mechanisms reduce vascular oxidative damage and preserve endothelial function during chronic AngII treatment.

International audience; Blood flowing in arteries generates shear forces at the surface of the vascular endothelium that control its anti-atherogenic properties. However, due to the architecture of the vascular tree, these shear forces are heterogeneous and atherosclerotic plaques develop preferentially in areas where shear is low or disturbed. Here we review our recent study showing that elevated shear forces stimulate endothelial autophagic flux and that inactivating the endothelial macroautophagy/autophagy pathway promotes a proinflammatory, prosenescent and proapoptotic cell phenotype despite the presence of atheroprotective shear forces. Specific deficiency in endothelial autophagy in a murine model of atherosclerosis stimulates the development of atherosclerotic lesions exclusively in areas of the vasculature that are normally resistant to atherosclerosis. Our findings demonstrate that adequate endothelial autophagic flux limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence and inflammation.

Rationale: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. Objective: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. Methods and Results: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. Conclusions: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.

International audience; A sedentary lifestyle has adverse effects on the cardiovascular system, including impaired endothelial functions. Subjecting healthy men to 7 days of dry immersion (DI) presented a unique opportunity to analyze the specific effects of enhanced inactivity on the endothelium. We investigated endothelial properties before, during, and after 7 days of DI involving eight subjects. Microcirculatory functions were assessed with laser Doppler in the skin of the calf. We studied basal blood flow and endothelium-dependent and -independent vasodilation. We also measured plasma levels of microparticles, a sign of cellular dysfunction, and soluble endothelial factors, reflecting the endothelial state. Basal flow and endothelium-dependent vasodilation were reduced by DI (22 + or - 4 vs. 15 + or - 2 arbitrary units and 29 + or - 6% vs. 12 + or - 6%, respectively, P < 0.05), and this was accompanied by an increase in circulating endothelial microparticles (EMPs), which was significant on day 3 (42 + or - 8 vs. 65 + or - 10 EMPs/microl, P < 0.05), whereas microparticles from other cell origins remained unchanged. Plasma soluble VEGF decreased significantly during DI, whereas VEGF receptor 1 and soluble CD62E were unchanged, indicating that the increase in EMPs was associated with a change in antiapoptotic tone rather than endothelial activation. Our study showed that extreme physical inactivity in humans induced by 7 days of DI causes microvascular impairment with a disturbance of endothelial functions, associated with a selective increase in EMPs. Microcirculatory endothelial dysfunction might contribute to cardiovascular deconditioning as well as to hypodynamia-associated pathologies. In conclusion, the endothelium should be the focus of special care in situations of acute limitation of physical activity.

Breaking the Barrier Being able to deliver drugs into the brain to treat degenerative diseases such as Alzheimer's or Parkinson's requires the ability to traverse the blood-brain barrier (BBB). Understanding the formation of the very specific adherent junctions (AJ) and tight junctions present at the BBB cell junctions is a prerequisite to the design of such therapeutics. However, diminishing the expression of any one component involved in the formation of these intercellular junctions destroys them. Coureuil et al. (p. 83 , published online 11 June) exploited the specific recruitment of AJ proteins by Neisseria meningitidis to dissect this process. Adhesion of the bacteria to human brain endothelial cells recruited the polarity complex Par3/Par6/PKCζ required for the establishment of eukaryotic cell polarity and the formation of intercellular junctions. The bacterial recruitment of the polarity complex depleted junctional proteins at the cell-cell interface opening the intercellular junctions at the brainendothelial interface.

International audience; Obstructive sleep apnea (OSA) is a highly prevalent sleep disorder leading to cardiovascular and metabolic complications. OSA is also a multicomponent disorder, with intermittent hypoxia (IH) as the main trigger for the associated cardiovascular and metabolic alterations. Indeed, recurrent pharyngeal collapses during sleep lead to repetitive sequences of hypoxia-reoxygenation. This IH induces several consequences such as hemodynamic, hormonometabolic, oxidative, and immuno-inflammatory alterations that may interact and aggravate each other, resulting in artery changes, from adaptive to degenerative atherosclerotic remodeling. Atherosclerosis has been found in OSA patients free of other cardiovascular risk factors and is related to the severity of nocturnal hypoxia. Early stages of artery alteration, including functional and structural changes, have been evidenced in both OSA patients and rodents experimentally exposed to IH. Impaired vasoreactivity with endothelial dysfunction and/or increased vasoconstrictive responses due to sympathetic, endothelin, and renin-angiotensin systems have been reported and also contribute to vascular remodeling and inflammation. Oxidative stress, inflammation, and vascular remodeling can be directly triggered by IH, further aggravated by the OSA-associated hormonometabolic alterations, such as insulin resistance, dyslipidemia, and adipokine imbalance. As shown in OSA patients and in the animal model, genetic susceptibility, comorbidities (obesity), and life habits (high fat diet) may aggravate atherosclerosis development or progression. The intimate molecular mechanisms are still largely unknown, and their understanding may contribute to delineate new targets for prevention strategies and/or development of new treatment of OSA-related atherosclerosis, especially in patients at risk for cardiovascular disease.

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.

International audience; BACKGROUND: Moderate physical activity enhances endothelium-dependent vasorelaxation. Whether the frequency of exercise affects endothelial function is unclear. The purpose of this study was to investigate the effects of various frequencies of training on endothelium-dependent vasorelaxation. DESIGN: Male Wistar rats were trained for 8 weeks on a treadmill at various frequencies [1 (Ex1), 3 (Ex3) or 5 days/week (Ex5)] and compared with age-matched sedentary animals (SED). A control group allowed us to assess endothelial function before the exercise protocol. Rings of thoracic aorta were precontracted with phenylephrine. RESULTS: Endothelium-independent relaxation elicited by sodium nitroprusside was similar in all groups. The maximal response elicited by acetylcholine (ACh) was not different between groups, whereas pD2 values (-logEC50, EC50 being the concentration of ACh that elicited 50% of the maximal response) significantly correlated with frequency of training. nitro-L-arginine methyl ester (L-NAME) reduced the relaxation elicited by 10(-7) mol/l ACh or higher in control and all trained groups, and by 10(-6) mol/l ACh or higher in SED group. Indomethacin inhibited the vasodilating response to 10(-7) mol/l ACh or higher in control, SED and Ex1 groups, and to 10(-8) mol/l or more in Ex3 and Ex5 animals. Tetraethylammonium attenuated the response to 10(-6) mol/l ACh or higher in control and SED groups and to 10(-7) mol/l or more in all trained animals. CONCLUSION: This data suggest that decreased ACh-induced vasorelaxation after physical inactivity may result from impairment of endothelial nitric oxide synthase, prostacyclin and endothelium-derived hyperpolarizing factor pathways. This effect is prevented by training in a frequency-dependent manner.

International audience; PURPOSE OF REVIEW: Whereas hormone therapy may increase the risk of coronary heart disease and stroke in menopausal women, epidemiological studies (protection in premenopausal women) suggest and experimental studies (prevention of fatty streak development in animals) demonstrate a major atheroprotective action of estradiol. There is also evidence for a thrombogenic effect of oral estrogens. An understanding of the deleterious and beneficial effects of estrogens is thus required. RECENT FINDINGS: The immuno-inflammatory system plays a key role in the development of fatty streak deposit as well as in the rupture of the atherosclerotic plaque. Whereas estradiol favors an anti-inflammatory effect in vitro (cultured cells), it rather elicits a pro-inflammatory response in vivo involving several subpopulations of the immuno-inflammatory system, which could contribute to plaque destabilization. Endothelium is another important target for estrogens, since estradiol potentiates endothelial nitric oxide and prostacyclin production. The respective actions of estrogens on these cell populations may be influenced by the timing of hormonal therapy initiation, hormone regimens, status of the vessel wall and expression of isoforms of estrogen receptors alpha and beta. SUMMARY: A better understanding of the balance between the deleterious and beneficial effects of estrogens is required and should help to improve hormonal therapy safety and to optimize the prevention of cardiovascular disease after menopause.

The TWIK related K+ channel TREK1 is an important member of the class of two-pore-domain K+ channels. It is a background K+ channel and is regulated by hormones, neurotransmitters, intracellular pH and mechanical stretch. This work shows that TREK1 is present both in mesenteric resistance arteries and in skin microvessels. It is particularly well expressed in endothelial cells. Deletion of TREK1 in mice leads to an important alteration in vasodilation of mesenteric arteries induced by acetylcholine and bradykinin. Iontophoretic delivery of acetylcholine and bradykinin in the skin of TREK1+/+ and TREK1−/− mice also shows the important role of TREK1 in cutaneous endothelium-dependent vasodilation. The vasodilator response to local pressure application is also markedly decreased in TREK1−/− mice, mimicking the decreased response to pressure observed in diabetes. Deletion of TREK1 is associated with a marked alteration in the efficacy of the G-protein-coupled receptor-associated cascade producing NO that leads to major endothelial dysfunction.

International audience; Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation. Accordingly, VEGF-induced fibrinolysis correlated with an increase in the expression of tPA and of some MMPs, such as MT2-MMP and was completely blocked by a neutralizing antibody against tPA. Overall, these results indicate that efficient proteolysis of three dimensional fibrin matrices during VEGF-mediated angiogenesis involves a complex interplay between the MMP and plasmin-mediated proteolytic systems.

International audience; The apelin receptor is a G protein-coupled receptor activated by several apelin fragments. Its tissue distribution suggests that apelin signalling is involved in a broad range of physiological functions. Endothelial cells, which express high levels of apelin receptors, respond to apelin through the phosphorylation of key intracellular effectors associated with cell proliferation and migration. In addition, apelin is a mitogen for endothelial cells and exhibits angiogenic properties in matrigel experiments. This review focuses on the therapeutic potential of apelin signalling, which is associated with pathologies that result from decreased vascularisation (ischemias) or neovascularisation (retinopathies and solid tumors).

International audience; 12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.

International audience; Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)(y1) zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.

International audience; Because angiogenesis is a key step in a number of pathologic processes, including tumor growth and atherosclerosis, many research studies have investigated the regulatory signals active at various stages of vascular invasion. The differential activities of the endothelial junction protein vascular endothelial (VE)-cadherin reflect the versatile behavior of endothelial cells between vascular quiescence and angiogenesis. VE-cadherin function and signaling are deeply modified in proliferating cells, and this conversion is accompanied by phosphorylation of the protein on tyrosine residues and enhanced transcription of its gene. Recent advances in the complex interplay between protein tyrosine kinases and phosphatases regulating VE-cadherin phosphorylation and function are discussed in this review.

We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.

International audience; The seeding of endothelial cells (ECs) on biomaterial surfaces became a major challenge, allowing to improve the non-thrombogenic properties of these surfaces. Recently, the use of polyelectrolyte films has been suggested as a new versatile technique of surface modification aimed at tissue engineering. In this study, we evaluate the adhesion properties of ECs on two types of polyelectrolyte films ending either by poly(D-lysine) (PDL), or poly(allylamine hydrochloride) (PAH), and compared them to data obtained on PDL or PAH monolayers, glass and fibronectin (Fn)-coated glass. ECs seeded on polyelectrolyte films showed a good morphology, allowing ECs to resist physiological shear stress better compared to ECs seeded on glass or Fn. The expression of beta1 integrins was slightly lower on polyelectrolyte films than on control surfaces. However, the phosphorylation of focal adhesion kinase, involved in the transduction of adhesion signal, was not modified on PAH ending films compared to control surfaces; whereas it became lower on PDL ending films. Finally, PAH ending films improve strongly ECs adhesion without disturbing the adhesion mechanism, necessary for the development of a new endothelium. These types of films or similar build-ups could thus be used in the future as a way to modify surfaces for vascular tissue engineering.

International audience; We examined the expression and function of a gene we previously cloned from its downregulation in a muscle atrophy model. The encoded protein was named myodulin because of sequence homologies with the cartilage-specific chondromodulin-I (ChM-I) protein, its restricted expression in skeletal muscle tissue, and its modulating properties on vascular endothelial cells described here. We investigated the expression of myodulin in muscle fibers and cultured muscle cells. Myodulin RNA messengers were found in muscle fibers and their tendon extensions. Overexpression of myodulin fused to a FLAG peptide showed evidence of a muscle cell surface protein. Myodulin functions were assessed from similarities with chondromodulin-I. Coculture experiments using C(2)C(12) mouse myoblasts or myotubes, which stably overexpress myodulin, with H5V mouse cardiac vascular endothelial cells revealed that myodulin had a very active role in the invasive action of endothelial cells, without any evidence of extracellular myodulin secretion. Our results suggest that myodulin may be a muscle angiogenic factor operating through direct cell-to-cell interactions. This role is consistent with the correlation between modulations in myodulin expression and modifications in muscle microvascularization associated with activity-dependent muscle mass variations.

International audience; For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical (*OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 microM FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of *OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced *OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with *OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular *OH in damage associated with intracerebral iron injection.

International audience; Previous studies have shown that aldosterone, which activates the mineralocorticoid receptor (MR), promotes thrombosis in animal models. Our objective was to determine whether MR activation/expression in the vascular endothelium could modify thrombotic risk in vivo and to examine thrombin generation at the surface of aortic endothelial cells (HAECs). MR was conditionally overexpressed in vivo in vascular endothelial cells in mice (MR-EC mice) or stimulated with aldosterone in HAECs. Thrombosis after ferric chloride injury was delayed in MR-EC mice compared with controls as well as in wild-type FVB/NRj mice treated with aldosterone (60 μg/kg/d for 21 d). Thrombin generation in platelet-poor plasma did not differ between MR-EC mice and controls. In MR-EC mice, aortic endothelial cell protein C receptor (EPCR) expression was increased. Aldosterone (10(-8) M) attenuated thrombin generation at the surface of cultured HAECs, and this effect was associated with up-regulation of expression of EPCR, which promotes formation of activated protein C. Aldosterone increases EPCR expression via a transcriptional mechanism involving interaction of MR with the specificity protein 1 site. These findings demonstrate that MR activation acts on endothelial cells to protect against thrombosis in physiological conditions and that MR-mediated EPCR overexpression drives this antithrombotic property through enhancing protein C activation.

International audience; Maturation of newly formed vessels is a multistep phenomenon during which functional endothelial barriers are established. Disruption of vessel integrity is an important feature in many physiological and pathological processes. We previously reported that lymphoblastic leukemia-derived sequence 1 (LYL1) is required for the late stages of postnatal angiogenesis to limit the formation of new blood vessels, notably by regulating the activity of the small GTPase Rap1. In this study, we show that LYL1 is also required during the formation of the mature endothelial barrier in the lungs of adult mice. Specifically, LYL1 knockdown in human endothelial cells downregulated the expression of ARHGAP21 and ARHGAP24, which encode two Rho GTPase-activating proteins, and this was correlated with increased RhoA activity and reorganization of the actin cytoskeleton into stress fibers. Importantly, in lungs of Lyl1-deficient mice, both vascular endothelial (VE)-cadherin and p120-catenin were poorly recruited to endothelial adherens junctions, indicative of defective cell-cell junctions. Consistent with this, higher Evans blue dye extravasation, edema, and leukocyte infiltration in the lung parenchyma of Lyl1-/- mice than in wild-type littermates confirmed that lung vascular permeability is constitutively elevated in Lyl1-/- adult mice. Our data show that LYL1 acts as a stabilizing signal for adherens junction formation by operating upstream of VE-cadherin and of the two GTPases Rap1 and RhoA. As increased vascular permeability is a key feature and a major mechanism of acute respiratory distress syndrome, molecules that regulate LYL1 activity could represent additional tools to modify the endothelial barrier permeability.

OBJECTIVE: In endothelial cells, nitric oxide (NO) is produced by the endothelial isoform of nitric oxide synthase (eNOS), which is localized in the cholesterol-rich plasmalemmal microdomains involved in signal transduction, known as caveolae. The present study was undertaken to evaluate the effect of hypercholesterolemia and fatty streak formation on the endothelial caveolae and on endothelial function, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production. METHODS AND RESULTS: We first studied the effect of atheroma on endothelial NO production. Fatty streak infiltrated aorta of cholesterol-fed New Zealand White rabbits demonstrated an impairment of acetylcholine-induced relaxation and nearly normal calcium ionophore A23187-induced maximal relaxation. The abundance of caveolae in the endothelium covering the fatty streak, as well as their 'grape-like' clustering, appeared to be decreased. We therefore investigated the effect, on endothelial NO production, of the cholesterol-binding agents 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CD) and filipin, known to alter caveolae structure and/or function. Treatment with either hp-beta-CD (2%) or filipin (4 microg/ml) did not affect contraction to phenylephrine or relaxant responses to A23187 or to the NO donor sodium nitroprusside. In contrast, both treatments impaired acetylcholine-induced relaxation. Cultured bovine aortic endothelial cells (BAEC) similarly treated with hp-beta-CD demonstrated a 50% decrease of total cellular cholesterol and a decreased abundance of caveolae as well as their 'grape-like' clustering. Cholesterol depletion decreased the bradykinin-induced transient peak of free intracellular calcium and subsequent receptor-stimulated NO production (assessed using reporter cells rich in soluble guanylyl cyclase), whereas that elicited by A23187 remained unaltered. CONCLUSION: Fatty streak deposit is associated with a decrease in caveolae 'transductosomes' abundance which appears to represent a novel mechanism of endothelial dysfunction.

Vascular endothelial growth factor (VEGF) is mitogenic for endothelial cells and has been shown to induce angiogenesis and endothelial cell migration through stimulation of endothelial tyrosine-kinase receptors. Here, using confocal microscopy and the patch-clamp technique on endothelial cells, membrane permeability to calcium as well as cytoplasmic and nuclear free calcium levels have been investigated in the first stages of tyrosine-kinase receptor activation by VEGF. VEGF (0.5nM) as well as inositol trisphosphate (IP3) induced an activation of membrane calcium-permeable channels exhibiting a similar low conductance in the range of 10 pS. The VEGF-triggered activation of these calcium channels, mediated by IP3 and involving the intracellular calcium stores, results in an increase in both cytoplasmic and nuclear calcium levels in endothelial cells, potentially modulating gene expression. Finally, the effect of Ni2+, a calcium channel blocker, on endothelial cell proliferation has been studied. The results show that inhibition of extracellular calcium influx significantly inhibits VEGF-induced cell proliferation. In the process of cell stimulation by VEGF, and possibly by other growth factors, activation of calcium channels could then be a key step in calcium-regulated gene expression and cell activation. These results suggest that the use of calcium channel blockers could be a novel way of prevention or reversion of VEGF-induced tumoral angiogenesis.

International audience; Extracellular matrix (ECM) receptors of the integrin family initiate changes in cell shape and motility by recruiting signaling components that coordinate these events. Integrin-linked kinase (ILK) is one such partner of beta1 integrins that participates in dynamic rearrangement of cell-matrix adhesions and cell spreading by mechanisms that are not well understood. To further elucidate the role of ILK in these events, we engineered a chimeric molecule comprising ILK fused to a membrane-targeted green fluorescent protein (ILK-GFP-F). ILK-GFP-F is highly enriched in cell-matrix adhesions, and its expression in fibroblasts leads to an accumulation of focal adhesions (2-5 microm) and elongated adhesions (>5 microm). ILK-GFP-F enhances cell spreading on fibronectin and induces a constitutive increase in the levels of GTP-bound Rac-1. Conversely, ILK knock-down by siRNA transfection decreases active Rac-1. Endogenous ILK was found to associate with PKL (paxillin kinase linker) and the Rac/Cdc42 guanine nucleotide exchange factor betaPIX. Further, expression of a dominant negative betaPIX mutant reversed the increase in active Rac-1 levels of ILK-GFP-F-expressing cells, thus placing betaPIX in the pathway leading from ILK to Rac-1 activation. However, expression of constitutively active Rac only partially restores the spreading defects of ILK-depleted cells, suggesting that an additional ILK-dependent signal is required for cell spreading.

International audience; Prostate cancer can usually be treated at a clinically localized stage by radical prostatectomy. Unfortunately, within 10 years following surgery, 30% of patients experience local or distant relapse. Few data exist on the association of markers of angiogenesis and distant relapse after radical prostatectomy. By immunohistochemistry in tissue microarray, we compared the expression pattern of hypoxia inducible factor 1, alpha subunit (HIF1α) and vascular endothelial growth factor (VEGF) and its receptors in 45 patients with distant relapse and 68 patients without relapse after radical prostatectomy. Expressions of HIF1α and VEGF were assessed in prostate tumor cells and those of VEGFR1, VEGFR2 and neuropilin 1 in tumor and endothelial cells. The five molecules studied were expressed by all tumors, with the exception of neuropilin 1 in endothelial cells for one tumor. Strong endothelial expression of VEGFR1 appeared to be an independent predictor of distant relapse. A moderate to strong endothelial expression of neuropilin 1 was in turn an independent predictor of absence of distant relapse. No significant difference was found for HIF1α, VEGF, VEGFR1, VEGFR2 and neuropilin 1 expression in tumor cells, nor for VEGFR2 in endothelial cells, between the two groups. To our knowledge, this is the first study to evaluate the prognostic value of VEGFR1, VEGFR2 and neuropilin 1 in endothelial cells in prostate cancer after radical prostatectomy. The evaluation by immunohistochemistry of endothelial expression of neuropilin 1 and VEGFR1 could be an additional tool in the assessment of tumor aggressiveness of clinically localized prostate cancer to better identify patients at high risk of distant relapse.

International audience; The α4β1 integrin facilitates both monocyte rolling and adhesion to the vascular endothelium and is physiologically activated by monocyte chemoattractant protein (MCP-1). The current study investigated the initial events in the adhesion of THP-1 cells to immobilized Vascular Cell Adhesion Molecule 1 (VCAM-1). Using AFM force measurements, cell adhesion was shown to be mediated by two populations of α4β1/VCAM-1 complexes. A low affinity form of α4β1 was anchored to the elastic elements of the cytoskeleton, while a higher affinity conformer was coupled to the viscous elements of the cell membrane. Within 100 ms of contact, THP-1 cells, stimulated by co-immobilized MCP-1, exhibited a tremendous increase in adhesion to VCAM-1. Enhanced cell adhesion was accompanied by a local decoupling of the cell membrane from the cytoskeleton and the formation of long membrane tethers. The tethers were individually anchored by multiple α4β1/VCAM-1 complexes that prolonged the extension of the viscous tethers. In vivo, the formation of these membrane tethers may provide the quantal structural units for the arrest of rolling monocytes within the blood vessels.

P. Menua,∗, J.F. Stoltzb and H. Kerdjoudjc aFaculte de medecine, Groupe Ingenierie Cellulaire et Tissulaire, Universite de Lorraine, UMR CNRS 7561, Vandœuvre, France bDepartement Unite Therapeutique Cellulaire et Tissulaire, Universite de Lorraine, Centre Hospitalier Universitaire de Nancy, Vandœuvre, France cEA 4691, Laboratoire Biomateriaux et inflammation en site osseux, UFR Odontologie, SFR CAP-Sante, Universite de Reims, Champagne Ardenne (URCA), France

International audience; Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.

Rationale: Positive signals, such as vascular endothelial growth factor, direct endothelial cells (ECs) to specific locations during blood vessel formation. Less is known about repulsive signal contribution to shaping vessels. Recently, “neuronal guidance cues” have been shown to influence EC behavior, particularly in directing sprouting angiogenesis by repelling ECs. However, their role during de novo blood vessel formation remains unexplored. Objective: To identify signals that guide and pattern the first mammalian blood vessels. Methods and Results: Using genetic mouse models, we show that blood vessels are sculpted through the generation of stereotyped avascular zones by EC-repulsive cues. We demonstrate that Semaphorin3E (Sema3E) is a key factor that shapes the paired dorsal aortae in mouse, as sema3E −/− embryos develop an abnormally branched aortic plexus with a markedly narrowed avascular midline. In vitro cultures and avian grafting experiments show strong repulsion of ECs by Sema3E-expressing cells. We further identify the mouse notochord as a rich source of multiple redundant neuronal guidance cues. Mouse embryos that lack notochords fail to form cohesive aortic vessels because of loss of the avascular midline, yet maintain lateral avascular zones. We demonstrate that lateral avascular zones are directly generated by the lateral plate mesoderm, a critical source of Sema3E. Conclusions: These findings demonstrate that Sema3E-generated avascular zones are critical regulators of mammalian cardiovascular patterning and are the first to identify a repulsive role for the lateral plate mesoderm. Integration of multiple, and in some cases redundant, repulsive cues from various tissues is critical to patterning the first embryonic blood vessels.

OBJECTIVE Major depression is an independent risk factor for the development of cardiovascular diseases. However, the exact mechanism by which depression may induce cardiovascular events is unclear. Endothelial dysfunction has been reported as a possible link between depression and subsequent cardiovascular events as described in depressed subjects. The purpose of this study was to investigate endothelial dysfunction and atherosclerosis formation in the aorta of mice exposed to the unpredictable chronic mild stress (UCMS) procedure. METHODS BALB/c mice were exposed to two 7-week UCMS procedures separated by 6 weeks. Treatments (fluoxetine 10 mg/kg; NaCl 0.9%) started at the third week until the end of the seventh week of each procedure. Endothelial function was evaluated by in vitro assessment of acetylcholine-induced vasorelaxation in aortic rings. By using specific inhibitors for nitric oxide (NO)- and prostacyclin-dependent relaxation, we assessed the part played by NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF)-like mediators in endothelium-dependent relaxation. Atherosclerosis was evaluated by histological examination. RESULTS Depression-like behavior was increased in the UCMS versus unstressed group and was reversed by chronic fluoxetine treatment. Vascular reactivity study indicated that UCMS induced a decrease in the NO-dependent relaxation that was partially compensated by an EDHF-like dependent relaxation. Because fluoxetine per se increased the NO-dependent relaxation, fluoxetine was able to reverse UCMS effect on the NO component and abolished the EDHF-like component. Atherosclerotic lesion was found in aorta of UCMS and nonstressed animals. CONCLUSIONS As an independent risk factor, UCMS reproduced the endothelial alterations observed in depression but was not sufficient to provoke morphological alterations.

International audience; Chronic exposure to simulated urban CO pollution is reported to be associated with cardiac dysfunction. Despite the potential implication of myocardial perfusion alteration in the pathophysiology of CO pollution, the underlying mechanisms remain today still unknown. Therefore, the aim of this work was to evaluate the effects of prolonged exposure to simulated urban CO pollution on the regulation of myocardial perfusion. Cardiac hemodynamics and myocardial perfusion were assessed under basal conditions and during the infusion of a β-Adrenergic agonist. The effects of CO exposure on capillary density, coronary endothelium-dependent vasodilatation, eNOS expression and eNOS uncoupling were also evaluated. Our main results were that prolonged CO exposure was associated with a blunted myocardial perfusion response to a physiological stress responsible for an altered contractile reserve. The impairment of myocardial perfusion reserve was not accounted for a reduced capillary density but rather by an alteration in coronary endothelium-dependent vasorelaxation (-45% of maximal relaxation to ACh). In addition, though chronic CO exposure did not change eNOS expression, it significantly increased eNOS uncoupling. Therefore, the present work underlines the fact that chronic CO exposure, at levels found in urban air pollution, is associated with reduced myocardial perfusion reserve. This phenomenon is explained at the coronary-vessel level by deleterious effects of CO exposure on the endothelium NO-dependent vasorelaxation via eNOS uncoupling.

International audience; Little is known about the vascular function and expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) in Duchenne muscular dystrophy (DMD). Bradykinin is involved in the regulation of eNOS expression induced by angiotensin-converting enzyme inhibitors. We characterized the vascular function and eNOS and nNOS expression in a canine model of DMD and evaluated the effects of chronic bradykinin treatment. Vascular function was examined in conscious golden retriever muscular dystrophy (GRMD) dogs with left ventricular dysfunction (measured by echocardiography) and in isolated coronary arteries. eNOS and nNOS proteins in carotid arteries were measured by western blot and cyclic guanosine monophosphate (cGMP) content was analyzed by radioimmunoassay. Compared with controls, GRMD dogs had an impaired vasodilator response to acetylcholine. In isolated coronary artery, acetylcholine-elicited relaxation was nearly absent in placebo-treated GRMD dogs. This was explained by reduced nNOS and eNOS proteins and cGMP content in arterial tissues. Chronic bradykinin infusion (1 μg/min, 4 weeks) restored in vivo and in vitro vascular response to acetylcholine to the level of control dogs. This effect was NO-mediated through upregulation of eNOS and nNOS expression. In conclusion, this study is the first to demonstrate that DMD is associated with NO-mediated vascular endothelial dysfunction linked to an altered expression of eNOS and nNOS, which can be overcome by bradykinin.

International audience; For more than two decades, methods for the non-invasive exploration of cutaneous microcirculation have been mainly based on optical microscopy and laser Doppler techniques. In this review, we discuss the advantages and drawbacks of these techniques. Although optical microscopy-derived techniques, such as nailfold videocapillaroscopy, have found clinical applications, they mainly provide morphological information about the microvessels. Laser Doppler techniques coupled with reactivity tests are widespread in the field of microvascular function research, but many technical issues need to be taken into account when performing these tests. Post-occlusive reactive hyperemia and local thermal hyperemia have been shown to be reliable tests, although their underlying mechanisms are not yet fully understood. Acetylcholine and sodium nitroprusside iontophoresis, despite their wide use as specific tests of endothelium-dependent and -independent function, respectively, show limitations. The influence of the skin site, recording conditions, and the way of expressing data are also reviewed. Finally, we focus on promising tools such as laser speckle contrast imaging.

PURPOSE. Vascular degeneration and the ensuing abnormal vascular proliferation are central to proliferative retinopathies. Given the metabolic discordance associated with these diseases, the authors explored the role of ghrelin and its growth hormone secretagogue receptor 1a (GHSR-1a) in proliferative retinopathy. METHODS. In a rat model of oxygen-induced retinopathy (OIR), the contribution of ghrelin and GHSR-1a was investigated using the stable ghrelin analogs [Dap3]-ghrelin and GHRP6 and the GSHR-1a antagonists JMV-2959 and [D-Lys3]-GHRP-6. Plasma and retinal levels of ghrelin were analyzed by ELISA, whereas retinal expression and localization of GHSR-1a were examined by immunohistochemistry and Western blot analysis. The angiogenic and vasoprotective properties of ghrelin and its receptor were further confirmed in aortic explants and in models of vaso-obliteration. RESULTS. Ghrelin is produced locally in the retina, whereas GHSR-1a is abundantly expressed in retinal endothelial cells. Ghrelin levels decrease during the vaso-obliterative phase and rise during the proliferative phase of OIR. Intravitreal delivery of [Dap3]-ghrelin during OIR significantly reduces retinal vessel loss when administered during the hyperoxic phase. Conversely, during the neovascular phase, ghrelin promotes pathologic angiogenesis through the activation of GHSR-1a. These angiogenic effects were confirmed ex vivo in aortic explants. CONCLUSIONS. New roles were disclosed for the ghrelin-GHSR-1a pathway in the preservation of retinal vasculature during the vaso-obliterative phase of OIR and during the angiogenic phase of OIR. These findings suggest that the ghrelin-GHSR-1a pathway can exert opposing effects on retinal vasculature, depending on the phase of retinopathy, and thus holds therapeutic potential for proliferative retinopathies. (Invest Ophthalmol Vis Sci. 2011;52:5376‐5386) DOI:10.1167/iovs.10-7152

CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17– and IL-22–secreting population of CD4+ T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR–CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human β-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.

International audience; BACKGROUND: Endothelial von Willebrand factor (VWF) mediates platelet adhesion and acts as a protective chaperone to clotting factor VIII. Rapid release of highly multimerized VWF is particularly effective in promoting hemostasis. To produce this protein, an elaborate biogenesis is required, culminating at the trans-Golgi network (TGN) in storage within secretory granules called Weibel-Palade bodies (WPB). Failure to correctly form these organelles can lead to uncontrolled secretion of low-molecular-weight multimers of VWF. The TGN-associated adaptor AP-1 and its interactors clathrin, aftiphilin and γ-synergin are essential to initial WPB formation at the Golgi apparatus, and thus to VWF storage and secretion. OBJECTIVES: To identify new proteins implicated in VWF storage and/or secretion. METHODS: A genomewide RNA interference (RNAi) screen was performed in the Nematode C. elegans to identify new AP-1 genetic interactors. RESULTS: The small GTPase Rab10 was found to genetically interact with a partial loss of function of AP-1 in C. elegans. We investigated Rab10 in human primary umbilical vein endothelial cells (HUVECs). We report that Rab10 is enriched at the Golgi apparatus, where WPB are formed, and that in cells where Rab10 expression has been suppressed by siRNA, VWF secretion is altered: the amount of rapidly released VWF was significantly reduced. We also found that Rab8A has a similar function. CONCLUSION: Rab10 and Rab8A are new cytoplasmic factors implicated in WPB biogenesis that play a role in generating granules that can rapidly respond to secretagogue.

International audience; Estrogens, and in particular 17β-estradiol (E2), play a pivotal role in sexual development and reproduction and are also implicated in a large number of physiological processes including the cardiovascular system. Although epidemiological studies and Nurses' Health Study suggested, and all animal models of early atheroma clearly demonstrated a vasculoprotective action of both endogenous and exogenous estrogens, the Women's Health Initiative did not confirm the preventive action of estrogens against coronary heart disease (CHD). However, women who initiated hormone therapy closer to menopause tended to have reduced CHD risk compared with increased CHD risk among women more distant from menopause. Thus, it is now mandatory to try to understand the mechanisms that could have influenced the actions of estrogens at various stages of atherosclerosis and/or of life. In this current review, we will summarize our understanding of the potential cellular targets and mechanisms of the vasculoprotective actions of estrogens, as well as of the lack of action of estrogens when administered after a period of hormonal deprivation. The mechanisms of the aggravating role of progestogens such as medroxyprogesterone acetate will be considered. Finally, we will analyze the possibilities to uncouple some beneficial from other undesirable actions following the partial/selective activation of estrogen receptors.

International audience; Purpose: Retinopathy of prematurity (ROP) is a major cause of visual handicap in the pediatric population. To date, this disorder is thought to stem from deficient retinal vascularization. Intriguingly, functional electrophysiological studies in patients with mild or moderate ROP and in the oxygen-induced retinopathy (OIR) model in rats reveal central photoreceptor disruption that overlies modest retinal vessel loss; a paucity of retinal vasculature occurs predominantly at the periphery. Given that choroidal circulation is the major source of oxygen and nutrients to the photoreceptors, the authors set out to investigate whether the choroidal vasculature system may be affected in OIR.Methods: Rat models of OIR treating newborn animals with 80% or 50/10% alternated oxygen level for the first two postnatal weeks were used to mimic ROP in humans. Immunohistology staining and vascular corrosion casts were used to investigate the vessel layout of the eye. To investigate the effect of 15-deoxy-Δ12,14-PGJ(2) (15d-PGJ(2); a nonenzymatic product of prostaglandin D(2)) on endothelial cells, in vitro cell culture and ex vivo choroid explants were employed and intravitreal injections were performed in animals.Results: The authors herein demonstrate that deficient vascularity occurs not only in the retinal plexus but also in the choroid. This sustained, marked choroidal degeneration is specifically confined to central regions of the retina that present persistent photoreceptor loss and corresponding functional deficits. Moreover, the authors show that 15d-PGJ(2) is a prominent contributor to this choroidal decay.Conclusions: The authors demonstrate for the first time pronounced, sustained choroidal vascular involution during the development of ROP. Findings also suggest that effective therapeutic strategies to counter ROP should consider choroidal preservation.

28 pages; International audience; AMP-activated protein kinase AMP-activated protein kinase (AMPK AMPK ), a phylogenetically conserved serine/threonine protein kinase, is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. It is now recognized that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile, and blood pressure in insulin-resistant rodents. Indeed, AMPK activation mimics the beneficial effects of physical activity or those of calorie restriction calorie restriction by acting on multiple cellular targets. In addition, it is now demonstrated that AMPK is one of the probable (albeit indirect) targets of major antidiabetic drugs drugs including the biguanides (metformin metformin ) and thiazolidinedione thiazolidinedione s, as well as of insulin-sensitizing adipokines (e.g., adiponectin adiponectin ). Taken together, such findings highlight the logic underlying the concept of targeting the AMPK pathway for the treatment of metabolic syndrome and type 2 diabetes.

The progression of viral infections is notoriously difficult to follow in whole organisms. The small, transparent zebrafish larva constitutes a valuable system to study how pathogens spread. We describe here the course of infection of zebrafish early larvae with a heat-adapted variant of the Infectious Hematopoietic Necrosis Virus (IHNV), a rhabdovirus that represents an important threat to the salmonid culture industry. When incubated at 24°C, a permissive temperature for virus replication, larvae infected by intravenous injection died within three to four days. Macroscopic signs of infection followed a highly predictable course, with a slowdown then arrest of blood flow despite continuing heartbeat, followed by a loss of reactivity to touch and ultimately by death. Using whole-mount in situ hybridization, patterns of infection were imaged in whole larvae. The first infected cells were detectable as early as 6 hours post infection, and a steady increase in infected cell number and staining intensity occurred with time. Venous endothelium appeared as a primary target of infection, as could be confirmed in fli1:GFP transgenic larvae by live imaging and immunohistochemistry. Disruption of the first vessels took place before arrest of blood circulation, and hemorrhages could be observed in various places. Our data suggest that infection spread from the damaged vessels to underlying tissue. By shifting infected fish to a temperature of 28°C that is non-permissive for viral propagation, it was possible to establish when virus-generated damage became irreversible. This stage was reached many hours before any detectable induction of the host response. Zebrafish larvae infected with IHNV constitute a vertebrate model of an hemorrhagic viral disease. This tractable system will allow the in vivo dissection of host-virus interactions at the whole organism scale, a feature unrivalled by other vertebrate models., Author Summary The zebrafish larva is uniquely amenable to imaging among vertebrate models because of its small size, transparency, and ease of anesthesia, making it a useful model to understand host-pathogen interactions. We have performed the first detailed analysis of a viral infection in zebrafish. Infection of zebrafish larvae with a salmonid rhabdovirus adapted to growth at the appropriate temperatures resulted in a predictable succession of pathological signs before death. Detection of infected cells in whole larvae revealed that blood vessels were a major target of the virus, providing an explanation to hemorrhages and subsequent loss of blood flow observed in infected larvae. Destruction of vascular cells caused by the viral infection was readily observed in transgenic larvae with fluorescent endothelium. We could identify the critical moments of the infection with simple temperature shift experiments. This work provides a basis to dissect the role of host factors in controlling the propagation of viral infections.

International audience; Pulmonary vascular remodeling is key to the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH). We recently reported that fibroblast growth factor (FGF)2 is markedly overproduced by pulmonary endothelial cells (P-ECs) in IPAH and contributes significantly to smooth muscle hyperplasia and disease progression. Excessive FGF2 expression in malignancy exerts pathologic effects on tumor cells by paracrine and autocrine mechanisms.We hypothesized that FGF2 overproduction contributes in an autocrine manner to the abnormal phenotype of P-ECs, characteristic of IPAH. In distal pulmonary arteries (PAs) of patients with IPAH, we found increased numbers of proliferating ECs and decreased numbers of apoptotic ECs, accompanied with stronger immunoreactivity for the antiapoptotic molecules, B-cell lymphoma (BCL)2, and BCL extra long (BCL-xL) compared with PAs from control patients. These in situ observations were replicated in vitro, with cultured P-ECs from patients IPAH exhibiting increased proliferation and diminished sensitivity to apoptotic induction with marked increases in the antiapoptotic factors BCL2 and BCL-xL and levels of phosphorylated extracellular signal-regulated (ERK)1/2 compared with control P-ECs. IPAH P-ECs also exhibited increased FGF2 expression and an accentuated proliferative and survival response to conditioned P-EC media or exogenous FGF2 treatment. Decreasing FGF2 signaling by RNA interference normalized sensitivity to apoptosis and proliferative potential in the IPAH P-ECs. Our findings suggest that excessive autocrine release of endothelial-derived FGF2 in IPAH contributes to the acquisition and maintenance of an abnormal EC phenotype, enhancing proliferation through constitutive activation of ERK1/2 and decreasing apoptosis by increasing BCL2 and BCL-xL.

International audience; MELK (maternal embryonic leucine zipper kinase) is a cell cycle dependent protein kinase involved in diverse cell processes including cell proliferation, apoptosis, cell cycle and mRNA processing. Noticeably, MELK expression is increased in cancerous tissues, upon cell transformation and in mitotically-blocked cells. The question of how MELK protein level is controlled is therefore important. Here, we show that MELK protein is restricted to proliferating cells derived from either cancer or normal tissues and that MELK protein level is severely decreased concomitantly with other cell cycle proteins in cells which exit the cell cycle. Moreover, we demonstrate in human HeLa cells and Xenopus embryos that approximately half of MELK protein is degraded upon mitotic exit whereas another half remains stable during interphase. We show that the stability of MELK protein in M-phase is dependent on its phosphorylation state.

International audience; Giant cell arteritis (GCA) is characterized by intimal hyperplasia and luminal obstruction leading to ischemic manifestations involving extra-cranial branches of carotid arteries and aorta. Histopathological lesions involve all layers of the arterial wall and are associated with multinucleated giant cells, fragmented internal elastic lamina and polymorphic cellular infiltrates, including T lymphocytes and macrophages. The pathophysiology of GCA is still poorly understood. After dendritic cell activation, CD4(+) T lymphocytes, T helper 1 (Th1) cells, produce interferon gamma and modulate macrophage activation and functions, and Th17 cells produce interleukin 17 (IL-17), which can induce cytokine production by macrophages and fibroblasts. Macrophages in the adventitia produce pro-inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor alpha. These cytokines promote arterial wall and systemic inflammation. Questions remain regarding the nature of the antigen(s) triggering dendritic cell activation and the mechanisms underlying vascular remodeling. Here we review recent advances in the pathogenesis of GCA, with emphasis on the interactions between cells of the immune system and components of the vessel wall, including vascular smooth muscle cells and endothelial cells, leading to vascular remodeling. Finally, we propose new areas of investigation that could help understand the triggering factors and key pathogenic events in GCA.

International audience; Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1) in a dose-dependent manner without being cytotoxic. Using Matrigel and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of alphav beta3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration.

International audience; BACKGROUND: A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERalpha) could be involved in the transduction of the vascular benefits of polyphenols. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used ERalpha deficient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, Provinols, or delphinidin, an anthocyanin that possesses similar pharmacological profile, is mediated by ERalpha. Indeed, Provinols, delphinidin and ERalpha agonists, 17-beta-estradiol and PPT, are able to induce endothelial vasodilatation in aorta from ERalpha Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing the effects of ERalpha completely prevented the effects of Provinols and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between delphinidin and ERalpha activator site is demonstrated using both binding assay and docking. Most interestingly, the ability of short term oral administration of Provinols to decrease response to serotonin and to enhance sensitivity of the endothelium-dependent relaxation to acetylcholine, associated with concomitant increased NO production and decreased superoxide anions, was completely blunted in ERalpha deficient mice. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benefits via ERalpha activation. It is a major breakthrough bringing new insights of the potential therapeutic of polyphenols against cardiovascular pathologies.

Background— The prevention of early atheroma by estrogens has been clearly demonstrated in all animal models and appears to be mediated through a direct action on the arterial wall rather than through an effect on the lipoprotein profile. The goal of the present study was to evaluate which cellular target is crucial in this beneficial action of estradiol. Methods and Results— We first confirmed the key role of estrogen receptor-α (ERα) in the atheroprotective effect of estradiol, because this action was completely abolished in mice deficient in both the low-density lipoprotein receptor (LDLr) and ERα. Second, using chimeric mice with an ERα deficiency in the hematopoietic lineage, we showed the persistence of the protective action of estradiol, which suggests the involvement of extrahematopoietic ERα. Third, we showed that loxP-flanked ERα mice (ERα flox/flox ) bred with Tie2-Cre + mice on an LDLr −/− background had complete inactivation of ERα in most hematopoietic and all endothelial cells. Remarkably, in this mouse model, the atheroprotective effect of estradiol was completely abolished. Fourth, the atheroprotective effect of estradiol remained abolished in Tie2-Cre + ERα flox/flox LDLr −/− mice transplanted with either Tie2-Cre + ERα flox/flox or ERα −/− bone marrow, whereas it was present in analogous chimeric Tie2-Cre − ERα flox/flox LDLr −/− receivers expressing endothelial ERα. Conclusions— We demonstrate directly and for the first time that endothelial ERα represents a key target of the atheroprotective effect of estradiol, whereas hematopoietic ERα is dispensable. Selective estrogen receptor modulators that mimic the endothelial action of estradiol should now be considered in atheroprotection.

International audience; BACKGROUND: Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. DESIGN AND METHODS: Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. RESULTS: Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. CONCLUSIONS: Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target.