Abstract 464: Membrane Microparticles From Ischemic Muscle Induce In Vitro Progenitor Cells Differentiation And In Vivo Neovascularization.
Postischemic neovascularization is mediated, at least in part, by the homing of progenitor cells to sites of injury and their differentiation into endothelial cells (EC). However, the mechanisms of in situ progenitor differentiation remain for the most part unknown. We hypothesized that miproparticles (MPs) released following ischemia-induced cell activation or apoptosis are the endogenous signal of postischemic vasculogenesis. MPs were detected by electron microscopy as vesicles of 0.1 to 1 μm in diameter in mice ischemic hindlimb muscle, 48hrs after unilateral femoral artery ligation. After isolation by sequential centrifugations, flow cytometry analyses showed that AnnexinV+ MPs concentration in ischemic calf was 3.5-fold higher than in control muscle (1392±406 vs. 394±180 AnnV+MPs/mg, p<0.005) and mainly originated from endothelial cells (47% of MPs are CD144+). MPs isolated from ischemic muscles induced a more potent in vitro bone marrow-mononuclear cells (BM-MNC) differentiation into EPC, than those isolated from control muscle (6.1±1.0% vs. 3.5±0.7% Dil-LDL/BS1lectin+ cells/field, p<0.05). Moreover, MPs isolated from GFP+ ischemic muscles colocalized with differentiated BM-MNC. MPs isolated from atherosclerotic plaque were uneffective whereas those isolated from apoptotic or IL-1 activated EC also promoted BM-MNC differentiation. Interestingly, MPs from ischemic muscles produced more reactive oxygen species and expressed significantly higher levels of NADPH oxidase p47 (6 fold ; p<0.05) and p67 subunits (16 fold ; p<0.005) than those from control, whereas gp91 subunit expression was unchanged. The MPs-induced BM-MNC differentiation was reduced by two fold with MPs isolated from gp91-deficient mice (p<0.05). MPs effect on post-ischemic vasculogenesis and revascularization was examined in the ischemic hindlimb model. MPs isolated from ischemic muscles were injected into ischemic legs in parallel with venous injection of BM-MNC. MPs increased the pro-angiogenic effect of BM-MNC transplantation and this effect was blunted with MPs from gp91−/− mice. These results demonstrate that MPs produced after tissue ischemia stimulate progenitor cell differentiation and subsequently promote postnatal neovascularization.