Vegf-Vegf Receptor 1 (Flt-1) Interactions Significantly Modulate Endothelial Progenitor Cell (Epc) Migration But Have Only Minor Effects On The Migration Of Differentiated Endothelial Cells

Abstract There is a growing body of data demonstrating that vasculogenesis, whereby bone marrow -derived circulating progenitor cells (EPCs) home to sites of neovascularization, results in significant contribution to blood vessel formation during tumor growth, ischemic injury and wound healing. Vascular Endothelial Growth Factor (VEGF) has recently been shown to augment vasculogenesis. In the current study, we examined if VEGF/VEGF Receptor (VEGFR) interactions are important for EPC recruitment. Both VEGFR1 (flt-1) and VEGFR2 (flk-1) are strongly expressed, as detected by immunofluorescent and FACS analysis, on EPCs obtained by ex vivo expansion of human peripheral blood. In a modified Boyden chamber migration assay, EPCs showed dose dependent migration to VEGF. To examine receptor specificity, EPCs were preincubated with receptor blocking anti-VEGFR1 or -VEGFR2 prior to the migration assay. Level of inhibition by VEGFR1 blocking antibody was commensurate with blocking VEGFR2. Furthermore, migration in response to a VEGFR1-specific agonist, PlGF, was comparable to that induced by VEGF and was completely ablated by preincubation with VEGFR1 blocking antibody. By contrast, differentiated endothelial cells had diminished migration in response to PlGF as compared to VEGF. Furthermore, blocking VEGFR1 only mildly disrupted VEGF-induced migration of differentiated endothelial cells in vitro. Hence, unlike differentiated endothelial cells, EPC migration in vitro was mediated by both VEGF receptors. By quantitative RT-PCR, we examined the level of VEGFR1 and VEGFR2 mRNA transcripts in EPCs versus differentiated endothelial cells. VEGFR1 transcripts in EPCs were expressed 3-fold higher than in differentiated endothelial cells. VEGFR2 and neuropilin transcript levels in EPCs, however, were lower than in differentiated endothelial cells. These results suggest that VEGF/VEGFR1 interactions are important in EPC migration in vitro. We have subcloned VEGFR1 cDNA into a retrovirus vector and have shown by western blot that we can direct increased cellular expression of VEGFR1. In further experiments, we will examine the role of VEGFR1 in human EPC recruitment using murine xenotransplant models of hindlimb ischemia and wound healing. These studies will provide valuable insight towards developing EPCs as cellular therapy to augment blood vessel formation.