Therapeutic angiogenesis by ex vivo expanded erythroid progenitor cells

Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Here, the impact of erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. During the in vitro differentiation, vascular endothelial growth factor (VEGF) secretion by ECFCs was observed from day 3 (burst-forming unit erythroid cells) to day 10 (erythroblasts). ECFCs from day 5 to day 7 (colony-forming unit erythroid cells) showed the highest VEGF productivity, and day 6 ECFCs were used for the experiments. ECFCs contained larger amounts of VEGF and fibroblast growth factor-2 (FGF-2) than peripheral blood mononuclear cells (PBMNCs). In tubule formation assays with human umbilical vein endothelial cells, ECFCs stimulated 1.5-fold more capillary growth than PBMNCs, and this effect was suppressed by antibodies against VEGF and FGF-2. Using an immunodeficient hindlimb ischemia model and laser-Doppler imaging, we evaluated the limb salvage rate and blood perfusion after intramuscular implantation of ECFCs. ECFC implantation increased both the salvage rate (38% vs. 0%, P < 0.05) and the blood perfusion (82.8% vs. 65.6%, P < 0.01). In addition, ECFCs implantation also significantly increased capillaries with recruitment of vascular smooth muscle cells and the capillary density was 1.6-fold higher than in the control group. Continuous production of human VEGF from ECFCs in the skeletal muscle was confirmed at least 7 days after the implantation. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis.