Ex Vivo Expanded Erythroid Progenitor Cells Augment Therapeutic Angiogenesis by Extensive Cytokine Production.

Alternative therapies for clinical limb ischemia have become very important for patients with peripheral ischemic disease (PAD), who cannot undergo surgical or percutaneous revascularization. Delivery of angiogenic growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF), stromal cell-derived factor-1α (SDF-1α), and placental growth factor (PlGF), using recombinant proteins or gene transfer have been considered for alternative treatment of PAD and their efficacies have been demonstrated. Recent reports have demonstrated that erythroid progenitor cells contain and secrete various angiogenic cytokines. Although this suggest an important role for erythroid progenitor cells in angiogenesis, no studies have reported in vivo evidence of angiogenesis induction by erythroid progenitor cells. Here, the impact of purified human erythroid colony-forming cell (ECFC) implantation on therapeutic angiogenesis was investigated in murine models of hindlimb ischemia. ECFCs were purified from human peripheral blood and expanded ex vivo. ECFCs from day 5 to 7, those are considered as erythroid progenitors of colony-forming unit erythroid(CFU-E) level of maturation, showed the highest vascular endothelial growth factor (VEGF) productivity. THerefore, day-6 ECFCs were used for the following experiments. Day-6 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). The capillary density was 1.6-fold higher in the ECFC group than in the control group. Implantation of ECFCs promoted angiogenesis in ischemic limbs by supplying angiogenic cytokines (VEGF and FGF-2), suggesting a possible novel strategy for therapeutic angiogenesis for the patients with PDA.