Microparticles from ischemic muscle promotes postnatal vasculogenesis.

International audienceBACKGROUND: We hypothesized that microparticles (MPs) released after ischemia are endogenous signals leading to postischemic vasculogenesis. METHODS AND RESULTS: MPs from mice ischemic hind-limb muscle were detected by electron microscopy 48 hours after unilateral femoral artery ligation as vesicles of 0.1- to 1-microm diameter. After isolation by sequential centrifugation, flow cytometry analyses showed that the annexin V(+) MP concentration was 3.5-fold higher in ischemic calves than control muscles (1392+/-406 versus 394+/-180 annexin V(+) MPs per 1 mg; P<0.001) and came mainly from endothelial cells (71% of MPs are CD(144+)). MPs isolated from ischemic muscles induced more potent in vitro bone marrow-mononuclear cell (BM-MNC) differentiation into cells with endothelial phenotype than those isolated from control muscles. MPs isolated from atherosclerotic plaques were ineffective, whereas those isolated from apoptotic or interleukin-1beta-activated endothelial cells 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<0.001) than controls, whereas gp91 subunit expression was unchanged. BM-MNC differentiation was reduced by 2-fold with MPs isolated from gp91-deficient animals compared with wild-type mice (P<0.05). MP effects on postischemic revascularization were then examined in an ischemic hind-limb model. MPs isolated from ischemic muscles were injected into ischemic legs in parallel with venous injection of BM-MNCs. MPs increased the proangiogenic effect of BM-MNC transplantation, and this effect was blunted by gp91 deficiency. In parallel, BM-MNC proangiogenic potential also was reduced in ABCA1 knockout mice with impaired vesiculation. CONCLUSIONS: MPs produced during tissue ischemia stimulate progenitor cell differentiation and subsequently promote postnatal neovascularization

NADPH Oxidase-Derived Overproduction of Reactive Oxygen Species Impairs Postischemic Neovascularization in Mice with Type 1 Diabetes

We hypothesized that diabetes-induced oxidative stress may affect postischemic neovascularization. The response to unilateral femoral artery ligation was studied in wild-type or gp91(phox)-deficient control or type 1 diabetic mice or in animals treated with the anti-oxidant N-acetyl-l-cysteine (NAC) or with in vivo electrotransfer of a plasmid encoding dominant-negative Rac1 (50 μg) for 21 days. Postischemic neovascularization was reduced in diabetic mice in association with down-regulated vascular endothelial growth factor-A protein levels. In diabetic animals vascular endothelial growth factor levels and postischemic neovascularization were restored to nondiabetic levels by the scavenging of reactive oxygen species (ROS) by NAC administration or the inhibition of ROS generation by gp91(phox) deficiency or by administration of dominant-negative Rac1. Finally, diabetes reduced the ability of adherent bone marrow-derived mononuclear cells (BM-MNCs) to differentiate into endothelial progenitor cells. Treatment with NAC (3 mmol/L), apocynin (200 μmol/L), or the p38MAPK inhibitor LY333351 (10 μmol/L) up-regulated the number of endothelial progenitor cell colonies derived from diabetic BM-MNCs by 1.5-, 1.6-, and 1.5-fold, respectively (P < 0.05). In the ischemic hindlimb model, injection of diabetic BM-MNCs isolated from NAC-treated or gp91(phox)-deficient diabetic mice increased neovascularization by ∼1.5-fold greater than untreated diabetic BM-MNCs (P < 0.05). Thus, inhibition of NADPH oxidase-derived ROS overproduction improves the angiogenic and vasculogenic processes and restores postischemic neovascularization in type 1 diabetic mice

PSGL-1–mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy