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 (MPs(Shh+)) 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 MPs(Shh+) induce the formation of capillary-like structures in an in vitro model using human endothelial cells, although they inhibited cell migration. Besides, MPs(Shh+) 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 MPs(Shh+), via a Rho-associated coiled-coil-containing protein kinase inhibitor-sensitive pathway. We demonstrate that MPs(Shh+) 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 MPs(Shh+) reduces angiogenesis. Interestingly, the effects induced by MPs(Shh+) 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 MPs(Shh+) 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.

BackgroundMicroparticles (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 (MPsShh+) that are able to regulate in vitro angiogenesis.Methodology/Principal FindingsHere, we investigated the ability of MPsShh+ to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPsShh+, MPsShh+ 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 MPsShh+-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. MPsShh+-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPsShh+ increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPsShh+ enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPsShh+ on eNOS pathway. Quantitative RT-PCR revealed that MPsShh+ 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/SignificanceThese findings suggest that MPsShh+ may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis.

Aims: Circulating microparticles (MPs) from metabolic syndrome patients and those generated from apoptotic T-cells induce endothelial dysfunction; however, the molecular and cellular mechanism(s) underlying in the effects of MPs remain to be elucidated. Results: Here, we show that both types of MPs increased expression of endoplasmic reticulum (ER) stress markers XBP-1, p-eIF2alpha and CHOP and nuclear translocation of ATF6 on human aortic endothelial cells. MPs decreased in vitro nitric oxide release by human aortic endothelial cells, whereas in vivo MP injection into mice impaired the endothelium-dependent relaxation induced by acetylcholine. These effects were prevented when ER stress was inhibited suggesting that ER stress is implicated in the endothelial effects induced by MPs. MPs affected mitochondrial function and evoked sequential increase of cytosolic and mitochondrial reactive oxygen species (ROS). Pharmacological inhibition of ER stress and silencing of neutral sphingomyelinase with siRNA abrogated all MP-mediated effects. Neutralization of Fas-Ligand carried by MPs abolished effects induced by both MP types, whereas neutralization of low density lipoprotein-receptor on endothelial cells prevented T-lymphocyte MP-mediated effects. Innovation and Conclusion: Collectively, endothelial dysfunction triggered by MPs involves temporal cross-talk between ER and mitochondria with respect to spatial regulation of ROS via the neutral sphingomyelinase and interaction of MPs with Fas and/or low density lipoprotein-receptor. These results provide a novel molecular insight into the manner MPs mediate vascular dysfunction and allow identification of potential therapeutic targets to treat vascular complications associated with metabolic syndrome.