Effet de la thrombine sur la vésiculation endothéliale (apport d'une approche génomique à l'étude des mécanismes de génération des microparticules endothéliales)

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocSudocFranceF

Mécanismes de génération des microparticules endothélialesmicroparticules endotheliales (influence du territoire vasculaire sur la capacité des cellules endothéliales à libérer les microparticules)

En réponse à l'activation et/ou une apoptose, les cellules endothéliales (CE) libèrent dans le milieu environnant des microparticules endothéliales (MPE). Les microparticules sont des vésicules de petite taille résultant d'évènements membranaires liés à une perte de l'asymétrie des phospholipides. Peu de travaux concernent l'étude des mécanismes de génération des MPE et l'influence du territoire vasculaire sur la capacité de vésiculation des CE n'est pas connue. Nous avons montré que la thrombine induit la génération de MPE par un mécanisme complexe impliquant un lien entre coagulation et inflammation sur les CE macro et microvasculaires. A partir de l'étude du transcriptome, nous avons mis en évidence l'implication de TRAIL dans la génération des MPE procoagulantes par la thrombine. A l'état basal, les CE issues de différents territoires vasculaires présentent des capacités différentes de vésiculation, les CE microvasculaires présentant une plus grande capacité à libérer des MPE. L'étude du transcriptome a permis de déterminer des signatures moléculaires représentatives des territoires macro et microvasculaires. Nos travaux mettent en évidence une génération différentielle de MPE en fonction du calibre des vaisseaux et les signatures spécifiques pourraient permettre une meilleure caractérisation du territoire endothélial lésé.In response to activation and/or apoptosis, endothelial cells (EC) release endothelial microparticles (EMP) in the extracellular space. Microparticles are small-sized vesicles resulting from membrane events leading to a loss of phospholipid asymmetry. Few works have documented the mechanisms underlying EMP generation and the influence of vascular bed on EMP release remains largely unknown. Our works showed that thrombin induced EMP generation through a complex mechanism involving a cross-talk between coagulation and inflammation in macro and microvascular EC. Using a global gene expression study, we have evidenced the involvement of TRAIL in the procoagulant EMP generation induced by thrombin. In a basal state, EC from various vascular beds showed different capacities of EMP release, with microvascular EC displaying the highest capacity. Gene expression study identified molecular signatures representative of EC from macro and microvascular beds. Our works highlight the role of the vascular beds in the capacity of EC to generate EMP according to the vessel caliber. The specific gene signatures could improve our knowledge the endothelial dysfunction and a better characterization of the damaged vascular beds.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Analyse du transcriptome (application au modèle de l'activation de l'endothélium par la thrombine et les anticorps antiphospholipides)

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Système d'étude du comportement cellulaire en condition de flux

International audienc

Altered angiogenesis in low birth weight individuals: a role for anti-angiogenic circulating factors

International audienceObjective: Low birth weight (LBW) is a risk factor for hypertension at adulthood. Endothelial progenitor cells (EPCs) dysfunction has been characterized in LBW neonates. We hypothesized that changes in soluble, plasma pro-or anti-angiogenic factors are associated with EPCs dysfunction and impaired angiogenesis in LBW neonates. Method: Venous umbilical cord blood was collected from 42 normal, term neonates and 75 LBW neonates. Cord blood endothelial colony forming cells (ECFC) from control patients were cultured in the presence of 10% of serum obtained from both groups. Results: The proliferation and the migration of ECFC were significantly reduced when cultured with 10% of serum of LBW neonates compared to serum of control neonates. Matrigel invasion assay was not significantly altered. Umbilical vein plasma VEGF concentration was significantly reduced in LBW neonates while that of sVEGFR and PF4 were significantly higher. Addition of VEGF corrected the inhibitory effect of LBW serum on normal ECFC proliferation. Conclusions: Serum obtained from LBW babies contains factors that exhibit an antiangiogenic effect on ECFC proliferation and migration. VEGF/sVEGF/PF4 pathway seems to be involved in the EPCs dysfunction in LBW neonates

TRAIL/Apo2L mediates the release of procoagulant endothelial microparticles induced by thrombin in vitro: a potential mechanism linking inflammation and coagulation.

peer reviewedMicroparticles are small vesicles playing a crucial role in cell communication by promoting prothrombotic and proinflammatory responses. However, the molecular mechanisms underlying their release are still elusive. We previously established that thrombin promoted the generation of endothelial microparticles (EMPs). In the present study, gene profiling identified TRAIL/Apo2L, a cytokine belonging to the tumor necrosis factor-alpha superfamily, as a target of thrombin. Thrombin increased the expression of cell-associated and soluble forms of TRAIL (sTRAIL) in HMEC-1 cells and human umbilical vein endothelial cells (HUVECs). Blocking TRAIL by specific antibodies or by small interfering RNA reduced both the number and the procoagulant activity of EMPs released by thrombin. Consistent with an involvement of sTRAIL in thrombin-induced EMP release, we showed that (1) exogenously added sTRAIL generated procoagulant EMPs; (2) supernatants from thrombin-stimulated endothelial cells induced EMP release by HMEC-1 cells and HUVECs, whereas those recovered from TRAIL knockdown endothelial cells displayed no effect. TRAIL/TRAIL-R2 complex mediated EMP release by initiating the recruitment of adaptor proteins and the activation of nuclear factor kappaB. Moreover, sTRAIL modulated intercellular adhesion molecule-1 and interleukin-8 expression induced by thrombin by a downstream pathway involving nuclear factor kappaB activation. Our data reveal a novel mechanism controlling EMP release and identify TRAIL as a key partner in the pathway linking coagulation and inflammation elicited by thrombin

Endothelial-derived microparticles: Biological conveyors at the crossroad of inflammation, thrombosis and angiogenesis

peer reviewedEndothelial microparticles (EMP) are complex vesicular structures that can be shed by activated or apoptotic endothelial cells. EMP are composed of a phospholipid bilayer that exposes transmembrane proteins and receptors and encloses cytosolic components such as enzymes, transcription factors and mRNA derived from their parent cells. Thus, EMP behave as biological conveyors playing a key role in the tuning of vascular homeostasis. This review focuses on the multifaceted roles of EMP, notably in coagulation, inflammation and angiogenesis and also on the mechanisms that trigger their formation. In this context, EMP could compromise vascular homeostasis and then represent key players in the pathogenesis of several inflammatory and thrombotic diseases. Consequently, elucidating their role and their mechanisms of formation will bring new insights into the understanding of endothelial-associated diseases. Moreover, in the future, it can open novel therapeutic perspectives based on the inhibition of EMP release

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis.: Fibrinolytic microparticles

International audienceBACKGROUND: We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. DESIGN AND METHODS: Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. RESULTS: Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. CONCLUSIONS: Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium

Accelerated senescence of cord blood endothelial progenitor cells in premature neonates is driven by SIRT1 decreased expression

International audienceKey Points • We demonstrate that PT promotes ECFCs dysfunction by inducing stress-induced premature senescence. • Our data reveal that SIRT1 deficiency drives PT-ECFC senescence, and acts as a critical determinant of the PT-ECFC angiogenic defect. Epidemiological and experimental studies indicate that early vascular dysfunction occurs in low-birth-weight subjects, especially preterm (PT) infants. We recently reported impaired angiogenic activity of endothelial colony-forming cells (ECFCs) in this condition. We hypothesized that ECFC dysfunction in PT might result from premature senescence and investigated the underlying mechanisms. Compared with ECFCs from term neonates (n 5 18), ECFCs isolated from PT (n 5 29) display an accelerated senescence sustained by growth arrest and increased senescence-associated b-galactosidase activity. Increased p16 INK4a expression, in the absence of telomere shortening, indicates that premature PT-ECFC aging results from stress-induced senescence. SIRT1 level, a nicotinamide adenine dinucleotide-dependent deacetylase with anti-aging activities, is dramatically decreased in PT-ECFCs and correlated with gestational age. SIRT1 deficiency is subsequent to epigenetic silencing of its promoter. Transient SIRT1 overexpression or chemical induction by resveratrol treatment reverses senescence phenotype, and rescues in vitro PT-ECFC angiogenic defect in a SIRT1-dependent manner. SIRT1 overexpression also restores PT-ECFC capacity for neovessel formation in vivo. We thus demonstrate that decreased expression of SIRT1 drives accelerated senescence of PT-ECFCs, and acts as a critical determinant of the PT-ECFC angiogenic defect. These findings lay new grounds for understanding the increased cardiovascular risk in individuals born prematurely and open perspectives for therapeutic strategy