Microparticles carrying sonic hedgehog favor neovascularization through the activation of nitric oxide pathway in mice

Abstract

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 (MPsShh+) 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. © 2010 Benameur et al.