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International audience; Microvesicles are plasma membrane-derived fragments released from various cell types during activation and/or apoptosis and posses the ability to deliver biological information between cells. Microvesicles generated from T lymphocytes undergoing activation and apoptosis bear the morphogen Sonic Hedgehog, and exert a beneficial potential effect on the cardiovascular system through their dual capacity to increase nitric oxide and reduce reactive oxygen species production. This study investigated the effect of microvesicles on the apoptosis of human umbilical vein endothelial cells triggered by actinomycin D. Microvesicles prevented apoptosis induced by actinomycin D by modulating reactive oxygen species production: during the early phase of apoptosis, microvesicles might act directly as reactive oxygen species scavengers, owing to their ability to carry active antioxidant enzymes, catalase, and isoforms of the superoxide dismutase. Furthermore, their effects were associated with the ability to increase the expression of manganese-superoxide dismutase in endothelial cells, through the internalization process. Interestingly, microvesicles bearing Sonic Hedgehog induced cytoprotection in endothelial cells through the activation of the Sonic Hedgehog pathway. These findings provide additional evidence that microvesicles from T lymphocytes exert their vasculoprotective effects by promoting internalization and induction of antioxidant messages to the endothelial monolayer.

International audience; Obesity is a major public health problem, resulting from an excess of energy storage and/or a default of energy expenditure leading to the increased occurrence of cardiovascular risk factors that favour the development of vascular complications. As a consequence, many studies are interested to find novel therapeutic chemical including flavonoids that appear to be promising natural compounds to treat obesity and its complications. Several in vitro studies addressed the mechanisms involved that might explain their beneficial effects, on adipocytes and endothelial cells, two cell types that play major role in obesity and its vascular complications. Besides the well-described antioxidant properties of flavonoids, at least a part of their beneficial effects on these cell types might be explained by their action on the regulation of mitochondrial function. In this review, we will therefore focus on the pathophysiological role of mitochondria in regulating endothelial and adipocyte functions. In addition, we will present some of the more promising flavonoids, important in human diet, including flavanols, flavonols, isoflavones, anthocyanins, flavanones and flavones; and their potential effects to improve endothelial or adipocyte functions via the mitochondria.

Neovascularization represents a pivotal process consisting in the development of vascular network during embryogenesis and adult life. Postnatally, it arises mainly through angiogenesis, which has physiological and pathological roles in health and disease. Blood vessel formation results as tightly regulated multistep process which needs coordination and precise regulation of the balance of proangiogenic and antiangiogenic factors. Sonic Hedgehog (SHH), a morphogen belonging to Hedgehog (HH) family proteins, is implicated in a remarkably wide variety of process, including vessel development. Recent evidence demonstrate that, in addition to the classic factors, microvesicles (MVs), both microparticles (MPs) and exosomes, small vesicles released distinct cellular compartments, are involved in modulation of neovascularization. MPs generated from T lymphocytes undergoing both activation and apoptosis harbor at their surface SHH and play a crucial role in modulation of neovascularization. They are able to modulate the different steps implicated in angiogenesis process in vitro and to enhance postischemic neovascularization in vivo . As the consequence, we suggest that the MPs carrying SHH contribute to generation of a vascular network and may represent a new therapeutic approach to treat pathologies associated with failed angiogenesis.

International audience; Clusiaceae plants display high contents of xanthones and coumarins, the effects of which on endothelium, more particularly on angiogenesis, have not been assessed yet. We screened the capacity of six molecules from Clusiaceae - belonging to xanthones, coumarins and acid chromanes classes - to induce endothelium-dependent relaxation on mice aortic rings. Endothelial nitric oxide (NO) production was assessed in endothelial cell line using electron paramagnetic resonance technique. Then, the capacity of these molecules to induce capillary-like structures of endothelial cells was assessed. Cellular processes implicated in angiogenesis (adhesion, migration and proliferation) and Western blot analyses were then investigated. Among the tested molecules, isocalolongic acid (IA) and 2-deprenylrheediaxanthone (DRX) induced an endothelium-dependent relaxation of the aorta associated with an increase of NO production in endothelial cells. Using in vitro and ex vivo angiogenesis assays, it was shown that IA treatment promoted the formation of capillary-like network. In contrast, DRX prevented the ability of vascular endothelial growth factor (VEGF) to increase the formation of capillary-like network. IA increased endothelial cell proliferation while DRX decreased all cellular processes of angiogenesis. Western blot analysis showed that IA increased VEGF expression whereas DRX decreased ICAM-1 expression. Altogether, these data allowed identifying isolated molecules from Clusiaceae that exhibit a potential activity towards the modulation of endothelium-dependent relaxation involving NO release. Interestingly, they also highlighted paradoxical effects of the two compounds on cellular angiogenic processes, IA being pro-angiogenic and DRX anti-angiogenic.

Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation or apoptosis. Microparticles from monocytes have been implicated in atherosclerosis and vascular inflammation, but their direct effects on endothelial cells are not completely elucidated. The present study was designed to dissect the signaling pathways of monocytic microparticles in endothelial cells with respect to both NO pathway and reactive oxygen species. Microparticles were produced by treatment of human monocytic cell line THP-1 with the apoptotic agent VP-16. Human endothelial cells were treated with monocytic microparticles and then, we studied their effects on nitrosative and oxidative stresses. Incubation of human endothelial cells with microparticles enhanced the production of NO without affecting superoxide anions generation. Microparticles did not affect endothelial NO synthase expression and its phosphorylation. Interestingly, microparticles decreased caveolin-1 expression and increased its phosphorylation. Inhibition of PI-3-kinase or MEK1/2 reversed the effects of microparticles on caveolin-1 expression but not its phosphorylation. Moreover, microparticles increased nitration of several proteins, reflecting peroxynitrite production, which was prevented by blockade of PI-3-kinase pathway. In summary, monocyte microparticles active multiple pathways related to nitrosative stress in endothelial cells including both PI-3-kinase and ERK1/2 in the regulation of caveolin-1 expression. These data underscore the pleiotropic effect of microparticles on endothelial cells and suggest that they probably play a critical role on vascular function.

International audience; BACKGROUND: Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPs(Shh+)) that are able to regulate in vitro angiogenesis.METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated the ability of MPs(Shh+) to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPs(Shh+), MPs(Shh+) plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPs(Shh+)-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPs(Shh+)-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPs(Shh+) increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPs(Shh+) enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPs(Shh+) on eNOS pathway. Quantitative RT-PCR revealed that MPs(Shh+) treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles. CONCLUSIONS/SIGNIFICANCE: These findings suggest that MPs(Shh+) may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis.

International audience; Sonic Hedgehog (Shh) is a morphogen involved in embryonic development of nervous system. Also, it has been shown that recombinant Shh can modulate angiogenesis under ischemic conditions. However, angiogenic effects of endogenous Shh have not been completely elucidated. Using small membrane-derived vesicles expressing Shh (MPsShh+), we have shown that, although MPsShh+ decrease endothelial cell proliferation and migration, they are able to favour angiogenesis through the increase of both endothelial cell adhesion and expression of pro-angiogenenic factors. Activation of proteins implicated in cell adhesion, such as Rho A, as well as upregulation of pro-angiogenic factors were sensitive to inhibition of Shh pathway. Although whole composition of MPsShh+ needs to be characterized to understand potential effects of MPsShh+, these results highlight a new role of MPsShh+ in vascular pathophysiology and may have significant implication for therapy in pathologies associated with altered angiogenesis in order to re-address angiogenic switch.

Microparticles (MPs) are membrane vesicles harboring cell surface proteins and containing cytoplasmic components of the original cell. High levels of circulating MPs have been detected in pathological states associated with vascular dysfunction. We took advantage of the self-assembly method of tissue engineering to produce in vitro three vascular constructs from human vascular smooth muscle cells and fibroblasts to investigate the role of the adventitia in the modulation of vascular tone by MPs, comparing the contractile response of each of these constructs to histamine. The first two were composed of an adventitia (tissue-engineered vascular adventitia (TEVA)) or a media (tissue-engineered vascular media (TEVM)) solely, and the third one contained a media and an adventitia (tissue-engineered vascular media and adventitia (TEVMA)). In the three constructs, the results show that histamine induces contraction insensitive to blockade of inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) and not affected by MP treatment. MPs decreased NO production and nuclear factor (NF)-kappaB expression but did not affect superoxide anion (O(2)(-)) release in TEVA. MPs enhanced NF-kappaB expression but did not affect iNOS and COX-2 expression or NO or O(2)(-) release in TEVM. In TEVMA, MPs did not enhance NF-kappaB expression, but COX-2 expression was higher, and O(2)(-) release was lower. Thus, MPs affected NO, O(2)(-), NF-kappaB, and COX-2 in a subtle fashion to maintain the contractile response to histamine. The use of tissue-engineered vascular constructs results in a better understanding of the effect of MPs on human adventitia and media.

Date du colloque: 2009; National audience

International audience; Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation or apoptosis. We have recently shown that MPs harboring the morphogen Sonic Hedgehog (MPsShh+) correct endothelial injury by release of nitric oxide from endothelial cells [Agouni, Mostefai, Porro, Carusio, Favre, Richard, Henrion, Martínez and Andriantsitohaina (2007) FASEB J., 21, 2735–2741]. Here, we show that MPsShh+ induce the formation of capillary-like structures in an in vitro model using human endothelial cells, although they inhibited cell migration. Besides, MPsShh+ regulate cell proliferation. Both cell adhesion and expression of proteins involved in this process such as Rho A and phosphorylation of focal-activated kinase were increased by MPsShh+, via a Rho-associated coiled-coil-containing protein kinase inhibitor-sensitive pathway. We demonstrate that MPsShh+ increase messenger RNA and protein levels of proangiogenic factors as measured by quantitative reverse transcription–polymerase chain reaction and western blot. In spite of vascular endothelial growth factor expression, conditioned media from endothelial cells treated avec MPsShh+ reduces angiogenesis. Interestingly, the effects induced by MPsShh+ on the formation of capillary-like structures, expression of adhesion molecules and proangiogenic factors were reversed after silencing of the Shh receptor, using small interfering RNA or when Sonic Hedgehog (Shh) signaling was pharmacologically inhibited with cyclopamine. Taken together, we show that Shh carried by MPsShh+ regulate angiogenesis probably through both a direct and an indirect mechanisms, and we propose that MPs harboring Shh may contribute to the generation of a vascular network in pathologies associated with tumor growth.

International audience; Hedgehog (Hh) proteins belong to a class of morphogens involved in many biological processes during embryonic development; they are relatively silent during normal adult life although they may be recruited postnatally in response to tissue injury. Three secreted proteins have been identified: Sonic hedgehog (Shh), Desert hedgehog and Indian hedgehog. The interaction of Hh ligand with its receptor Patched-1 triggers the activation of smoothened and initiates transduction events that lead to the regulation of transcriptional factors belonging to the Gli family. Hh pathway orchestrates both coronary development and adult coronary neovascularisation by controlling the expression of multiple proangiogenic genes and anti-apoptotic cytokines. Shh pathway enhances the recruitment of endothelial progenitor cells in addition to the mechanisms described for other Hh and concurs to its myocardial protection. In cerebral ischemia, Hh mimicking molecules has been reported to limit damages caused by vessel occlusion. Besides, Shh carried by microparticles corrects endothelial injury through nitric oxide release. Anomalous activations of Hh pathway are implicated in various types of tumours including medulloblastoma, carcinoma of esophagus, stomach, pancreas and colon. Hh can influence angiogenesis in both positive and negative manner and they may have implication for therapeutic strategies to treat either ischemic or cancer diseases.

Microparticles (MPs) are small vesicles released from the plasma membrane after cell stimulation or apoptosis. Recent studies show that MPs can be implicated in modulation of angiogenesis, an essential cellular process from cardiovascular system during physiological and pathological conditions. Moreover, engineered MPs generated from apoptotic/stimulated human lymphocytes harbour at their surface the morphogen Sonic Hedgehog (Shh) which also play a critical role in vessel formation. Accordingly, the present study was aimed to examine how engineered human MPs bearing Shh (MPsShh+) are able to modulate the different steps implicated in angiogenesis process. In human endothelial cells (ECs), MPsShh+ induce the formation of capillary-like structures. Moreover, MPsShh+ are able to modulate EC proliferation, adhesion and mRNA levels of proangiogenic factors. Silencing of Shh receptor, by siRNA or Shh signalling by pharmacological inhibitor cyclopamine, strongly reverses tubulogenesis, and concomitantly decreases mRNA levels of pro-angiogenic factors. Furthermore, we investigate the effects of MPsShh+ on post-ischemic neovascularization using mice model of femoral artery ligation. The recovery of ischemic/normal blood flow ratio is greater in MPsShh+-treated mice than in control. In muscle, MPsShh+ enhance the expression of eNOS, decrease expression of caveolin-1 and modify expression of several angiogenic genes. Pharmacological inhibition of Shh pathway completely reverses the improvement of blood flow and the effects on eNOS expression. Besides, in light of our results, we proposed the hypothesis that MPsShh+ may represent a new therapeutic approach to treat pathologies associated with failed angiogenesis and a new target in pathologies associated with exaggeration of angiogenesis, such as cancer., Les microparticules (MPs) sont des fragments de la membrane plasmique libérés suite à une stimulation cellulaire ou apoptotique. Des études récentes ont montré que les MPs peuvent être impliquées dans la modulation de l'angiogenèse, un processus essentiel du système cardio-vasculaire pendant les états physiologiques et pathologiques. De plus, les MPs dérivées des cellules T humaines activées et apoptotiques sont porteuses du morphogène Sonic Hedgehog (Shh), lequel a aussi un rôle dans la formation des vaisseaux. En conséquence, l'objectif de cette étude est d'élucider l'implication des MPs porteuses de Shh (MPsShh+) sur les différentes étapes associées à l'angiogenèse. Dans les cellules endothéliales, les MPsShh+ provoquent la formation des structures capillaires. Également, les MPsShh+ sont capables de moduler la prolifération, l'adhésion et le niveau d'expression des ARNm des facteurs pro-angiogéniques dans les cellules endothéliales. De plus, l'inhibition de l'expression du récepteur de Shh, ou l'utilisation de la cyclopamine, un inhibiteur pharmacologique de Shh, restitue la capacité des MPsShh+ de former des capillaires et aussi diminue l'expression des facteurs pro-angiogéniques. Ensuite, nous avons évalué les effets des MPsShh+ sur la neovascularization post-ischémique en utilisant le modèle in vivo de la ligature de l'artère fémorale chez la souris. Une meilleure récupération du flux sanguin est observée chez les souris traitées avec les MPsShh+ par rapport aux témoins. Dans le muscle squelettique, les MPsShh+ augmentent l'expression de la eNOS et diminuent celle de la caveoline-1. L'inhibition pharmacologique de la voie Shh prévient l'amélioration du flux sanguin et les effets induits sur l'expression de la eNOS. L'ensemble des résultats suggère que les MPsShh+ pourraient être utilisées comme de nouveaux outils thérapeutiques dans certaines pathologies caractérisées par une angiogenèse défaillante et elles pourraient représenter une nouvelle cible dans les pathologies associées à une angiogenèse exagérée, comme le cancer.

The presence of serum anti-Ro and anti-La autoantibodies directed against the ribonucleoproteins Ro and La has been associated with Sjögren's syndrome (SS), an autoimmune rheumatic disease that targets salivary and lachrymal glands. There is increasing evidence of the direct involvement of autoantibodies in the pathogenesis of tissue injury and correlation of their presence with clinical manifestations in SS. The focus of this work was to explore the cellular apoptotic pathway triggered by binding and penetration of anti-Ro and anti-La autoantibodies in human salivary gland cell line A-253 and to identify the membrane receptors through which anti-Ro and anti-La could exert their effect.Anti-Ro and anti-La autoantibodies were purified from IgG fractions, obtained from eleven healthy volunteers and patients with primary Sjögren's syndrome, using Sepharose 4B-Ro and Sepharose 4B-La affinity columns. Flow cytometry, RT-PCR, western blot and confocal microscopy analysis were used to visualize the FCgammaRI, FCgammaRII and FCgammaRIII receptors on the A-253 cell membrane. DNA laddering and western blot analysis of caspases activation were studied to evaluate in A-253 cells treated with anti-Ro and anti-La autoantibodies.The results yeilded the evidence of the presence of members of the Fcgamma receptors (FcgammaRs) family on the cell membrane of the human salivary gland cell line A-253. Furthermore, we demonstrated that, in the A-253 cell line, anti-Ro and anti-La autoantibodies can access the cells probably through Fcgamma receptors, and trigger apoptotis.We conclude that anti-Ro and anti-La autoantibodies have pathogenic effects that could depend on binding to Fcgamma receptors.

Recent studies have demonstrated that microparticles (MPs) generated from T lymphocytes undergoing both activation and apoptosis correct endothelial injury, and regulate angiogenesis. These effects are mediated by an increase of NO release and a decrease of reactive oxygen species (ROS) production. Apoptosis is an important mechanism that controls endothelial cell number and regeneration. We studied the effects of these MPs on human umbilical vein endothelial cell (HUVEC) apoptosis induced by actinomycin D (actD). Engineered MPs were obtained by activation of human lymphocyte CEM-T cell line with phytohemagglutinin and then, by stimulation with phorbol-12-myristate-13-acetate and actD. HUVECs were grown for 24 h in absence or presence of the proapoptotic agent actD (1 μg/ml), and/or 10 μg protein/ml of MPs. In another set of experiments, endothelial cells were pre-incubated either with nonselective caspases inhibitor, z-vad.fmk (50 μm), PI3-kinase inhibitor, LY294002 (10 μm), ERK inhibitor, U0126 (10 μm), NOS inhibitor, L-NA (100 μm), or the superoxide dismutase (SOD) mimetic, manganese(III)tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride (MnTMPyP, 100 μm). The proportion of nuclei with hypodiploid DNA (sub-G1 peak) corresponding to apoptotic cells was determined by flow cytometry after staining with propidium iodide, and by microscopy using TUNEL labeling. Our results clearly indicate that activated/apoptotic T lymphocytes MP treatment significantly reduces HUVEC apoptosis evoked by actD. Moreover, pancaspases inhibitor reduces the degree of cell death either in presence or in absence of MPs, indicating the implication of caspases in actD-induced apoptosis. Although, the PI3-kinase inhibitor induces apoptosis by itself, it does not blunt the capacity of MPs in reducing apoptosis induced by actD. In contrast, the inhibitory effect of MPs is prevented in the presence either of the ERK inhibitor or the NOS inhibitor. Interestingly, MnTMPyP reduces actD-evoked apoptosis and the protective effects of MPs. Thus, these MPs may act as an SOD mimetic under these experimental conditions. Altogether, T lymphocyte MPs evoked cell protection against apoptosis at the level of caspases inhibition, MAP kinases, NO and ROS formations. They underscore the potential therapeutic of such MPs against vascular pathologies in close association with apoptosis. Supported by ANR-07-PHYSIO-010-01.

National audience; Oxidative stress plays an important role in the initial occurrence of cardiovascular diseases. Previous studies demonstrated that microparticles (MPs) released from activated and apoptotic lymphocytes improve endothelial function due to their ability to decrease reactive oxygen species (ROS). Here, we evaluated the capacity of these MPs to modulate ROS production during actinomicyn D (ActD)-induced apoptosis in human umbilical vein endothelial cells (HUVECs).MPs were generated from T lymphocytes undergoing activation with phytohemagglutinin and then, by stimulation with phorbol-12 myristate-13 acetate and ActD. HUVECs were grown for 24h in absence or presence of ActD (1—g/ml), and/or 10Pg protein/ml of MPs.Once evaluated that MPs are able to prevent ActD-evoked apoptosis, we investigated the time course of modifcations in ROS production (i.e. 1, 2, 4, 6, 8, 10 and 24h) by electron paramagnetic resonance. ActD enhances ROS production after 2h and 10h of treatment that was prevented by MPs after 2h but not at 10h, indicating that they act during the early phase of apoptosis. Interestingly at 24h, superoxide dismutase (SOD) mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP) decreases cell death and inhibition of apoptosis induced by MPs, suggesting that MPs may behave as an SOD mimetic. Although these MPs carry functionally active extracellular SOD and catalase, as demonstrated by western blot analysis, treatment of HUVECs with these MPs does not modify expression of these enzymes.Under the same experimental conditions, MPs increase the expressions of other antioxidant enzymes such as Cu/Zn SOD and Mn SOD in HUVECs that may explain the protective effects induced by MPs. Thus, the present study illustrates new mechanisms by which MPs from activated/apoptotic lymphocytes may confer endothelial protection under pathological conditions in which oxidative stress is enhanced by a subtle modulation of antioxidant defense mechanism.

National audience

International society for extracellular vesicles; International audience

Date du colloque: 04/2010; National audience

During cell activation or apoptosis, cells release vesicles, also called microparticles (MPs) from the plasma membrane. Since composition of MPs is dependent on cell origin and the type of stimulation, we compared the effects of MPs generated from both apoptotic T lymphocytes and apoptotic monocytes on endothelial function with respect to both nitric oxide (NO) pathway and reactive oxygen species (ROS). MPs were produced by treatment of either human T lymphocytes with the apoptotic agent actinomycin D or human monocytic cell line THP-1 with the apoptotic agent, the etoposide VP-16. Human Eahy 926 endothelial cells were incubated with 10 μg/ml MPs for 24 h. Apoptotic MPs from human T lymphocytes decreased NO production that was associated with overexpression and phosphorylation of endothelial NOsynthase (eNOS). Also, T lymphocytes MPs enhanced expression of caveolin-1 and decreased its phosphorylation. T lymphocytes MPs enhanced ROS by a mechanism sensitive to xanthine oxidase and P-IkappaBalpha inhibitors. PI3-kinase inhibition reduced the effects of T lymphocytes MPs on eNOS, but not on caveolin-1, whereas it enhanced the effects of MPs on ROS production. Inhibition of MEK reversed eNOS phosphorylation but it had no effect on ROS production induced by T lymphocytes MPs. By contrast, apoptotic MPs from human monocytes increased both NO production and in much less extent ROS. These effects were associated with a decrease of caveolin-1 expression and increased its phosphorylation, without affecting eNOS expression and phosphorylation. The inhibitor of the PI-3kinase, LY294002, reversed the effects of monocyte MPs on caveolin-1 expression but not on its phosphorylation. The MEK1/2 inhibitor, U0126, reversed the decrease of caveolin-1 expression induced by MPs from monocytes. Interestingly, U0126 potentiated ROS production induced by monocyte MPs. Whereas in vivo injection of T lymphocytes MPs in mice impaired endothelial function, apoptotic MPs from human monocytes did not affect endothelium-dependent relaxation. In addition, monocyte MPs were able to promote in vitro angiogenesis. In summary, these results highlight differential effects of apoptotic MPs from different origins by activating diverse multiple pathways related to NO and ROS productions.