Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

BACKGROUND: Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. METHODOLOGY/PRINCIPAL FINDINGS: EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34(+)/CD45(−) cells/1×10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. CONCLUSIONS/SIGNIFICANCE: Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.