Abstract 1223: Conditional Knockout of CXCR4 Receptor Reveals its Role in Vascularization and the Differentiation of Endothelial Progenitor Cells (EPCs).
SDF-1/CXCR4 pathways are essential for the retention and maintenance of hematopoietic stem cells. Based on the relationship between HSCs and EPCs in its origin and functional kinetics in response to cytokine stimuli, we investigated a pivotal role of CXCR4 signals on EPC biology and kinetics for postnatal vasculognesis. Cre-mediated removal of the CXCR4 gene in mice resulted in the defect of neovascularization in hindlimb ischemia model consistent with decreased capillary density in ischemic tissue. Tissue analysis demonstrated impaired incorporation of CXCR4 null EPCs into sites of ischemia. In vitro evidences in EPC colony assay (decrease in large EPC-CFU, committed EPC status) and FACS analysis suggested the significant impairment of EPC differentiation in bone marrow structure. This finding further supported by transplantation experiment of EPC enriched cells into ischemic models. CXCR4 null EPC group did not restore the revascularization from ischemic disease with immature vascular structure (p <0.01, n=7). Thus, to investigate the mechanistic importance of SDF-1/CXCR4 signals for EPC differentiation, we explored the EPC colony status formed by ex vivo expanded cord blood AC133 (+) cells. The prolonged SDF treatment followed by precondition of hypoxia caused the dominant frequency of large EPC-CFU with very less small EPC-CFU appearance, possible the conversion from early EPC status to committed EPC with spindle like morphology. The high proliferative activity of SDP-1 treated EPCs, was also assessed by the colony size index. Blocking of SDF-1 signals using specific neutralization antibodies completely diminished morphological conversion into large EPC. Furthermore, we investigated the significance of SDF/CXCR4/eNOS signals for EPC differentiation. The abrogation of NO synthesis by L-NAME in EPC expansion culture has inhibited the onset of large EPC colonies in EPC colony assay.. Together with the results from in vovo and in vitro experiments, we concluded that SDF-1/CXCR4 signal might be a target of commitment of immature hemangioblast into functional EPC. Thus, our present data suggest that the full understanding of CXCR4 signals for EPC differentiation may provide a cue for therapeutic potential of the EPC therapy for hind limb disease.