Stromal cell derived factor 1 (SDF-1/CXCL 12) and C-X-C chemokine receptor type 4 (CXCR4) axis plays a critical role in vascular injury recovery and angiogenesis.However, the multi-functional characteristics of native SDF-1 limit its potential application.We developed a novel strategy to block CXCR4 and promote mobilization of endothelial progenitor cells (EPCs) necessary for vascular injury recovery and angiogenesis.To this end, we generated a CXCR4 antagonist namely, SDF-1βP2G (P2G), which was derived from human SDF-1p by replacing the second amino acid proline with glycine in the N-terminus.P2G was expressed in bacteria and purified to homogeneity by nickel solid phase affinity chromatography and reversed phase-high-performance liquid chromatography.As opposed to native SDF-1, P2G exerted antagonistic effects against CXCR4 by promoting CXCR4 internalization and competitively inhibiting downstream signaling events in vitro, and doesand time-dependently stimulating the mobilization of EPCs (CD31+/c-Kit+) from bone marrow into peripheral circulation in vivo.Moreover, intravenous administration of P2G significantly stimulated angiogenesis and skeletal muscle regeneration in a mouse model with acute hindlimb ischemia as early as 7 days after surgical operation (p<0.001,vs PBS).Compared to AMD3100, another chemical antagonist of CXCR4, P2G stimulated a rapid revascularization (p<0.01).Mechanistic study showed that P2G significantly stimulated EPCs mobilization in the peripheral blood and promoted their infiltration into ischemic skeletal muscle tissues.In addition, P2G enhanced the activation and/or expression of angiogenesis and progenitor cell migration-related genes including Akt, ERK, mTOR, MMP9, SDF-1/CXCR4 and vascular endothelial growth factor (VEGF).Furthermore, neutralization of VEGF with its specific antibody abolished P2G-induced blood reperfusion and angiogenesis.More importantly, no obvious inflammatory and apoptotic effects were observed in multiple organs after P2G administration.These data suggest that the new antagonist of CXCR4, P2G, can be successfully utilized to stimulate angiogenesis and muscle regeneration through mobilization of EPCs in a VEGF dependent manner.Our work has demonstrated for the first time that P2G is non-toxic, specific CXCR4 antagonist with a great potential for clinical application for ischemic vascular diseases.