Abstract 1218: Direct Evidence for the Important Role of Erythropoietin Receptor to Promote Angiogenesis in Peripheral Ischemia in Mice
Background: Therapeutic angiogenesis is a novel promising option for severe peripheral arterial disease (PAD). Although stem cell and gene therapy has been under investigation, the efficacy is not durable and stable. We have recently identified the expression of erythropoietin (Epo) receptor (EpoR) on vascular endothelial cells under chronic hypoxic conditions. In this study, we thus tested our hypothesis that the local Epo/EpoR system critically influences angiogenesis in PAD.
Methods and Results: We examined the development of angiogenesis in EpoR−/ − mice, which lack EpoR in cardiovascular system but not in hematopoietic system. After 2 weeks of hindlimb ischemia, laser Doppler analysis showed that blood flow ratio (ischemic/non-ischemic limb) was significantly lower in EpoR−/ − mice than wild-type (WT) mice (0.29±0.08 vs. 0.75±0.11; P<0.01, n=8 each), in which EpoR−/ − mice showed the significant reduction in endothelial progenitor cell (EPC) mobilization in response to ischemia (Flk-1+/ CD133+ cells; 0.31±0.21 vs. 0.04±0.02; P<0.01, n=12 and 13, respectively). Intravenous infusion of ex-vivo cultured EPCs from WT mice, but not those from EpoR−/ − mice, improved hindlimb blood flow after ischemia in WT mice. By contrast, bone marrow (BM) transplantation with WT-BM cells significantly improved EPC mobilization and limb blood flow ratio in EpoR−/ −mice with hind-limb ischemia (WT-BM vs. EpoR−/ −-BM; 0.44±0.14 vs. 0.30±0.15; P<0.01, n=12 each). Next, we transplanted WT-BM cells (green fluorescent protein, GFP+) to WT and EpoR−/ − mice, and evaluated the number of GFP+ cells in ischemic tissue. Confocal microscopy and FACS analysis revealed that the number of BM-derived cells in ischemic tissue was significantly reduced in EpoR−/ − mice compared to WT mice (%GFP+ cells; 3.3±2.3 vs. 0.82±1.2; P<0.05, n=11 and 6, respectively), indicating the crucial role of EpoR in the recruitment of BM-derived cells to ischemic tissue. The VEGF-supplemented Matrigel implantation assay also showed that the number of capillary vessels was significantly lower in EpoR−/ − mice than WT mice.
Conclusions: The present results indicate that the Epo/EpoR system in local ischemic tissue plays an important role to promote postischemic angiogenesis in vivo.