Tissue plasminogen activator enhances mobilization of endothelial progenitor cells and angiogenesis in murine limb ischemia

Background: It has been reported that plasmin induces migration of endothelial cells. We hypothesized that tissue plasminogen activator (tPA) enhanced endothelial progenitor cell (EPC) mobilization from bone marrow (BM) into the circulation and angiogenesis in murine critical limb ischemia (CLI). Methods: Forty male B6 mice were randomly divided into group 1 (control), group 2 [control + recombinant tPA (intra-venous 4 mg/kg)], group 3 (CLI) and group 4 (CLI + tPA). Results: The results demonstrated higher MMP-9 activity in BM and circulatory SDF-1α level in groups 2 and 3 than in group 1, and highest in group 4 (all p < 0.01) at 18 h after CLI. Compared with circulation, SDF-1α level in BM showed an opposite trend (all p < 0.03). The circulating EPC (C-kit/CD31, Sca-1/KDR, CXCR4/CD34) levels at 18 h after CLI were higher in group 2 than in groups 1 and 3, and highest in group 4 (all p < 0.03). EPC (C-kit/CD31, Sca-1/KDR) levels in BM were lower in group 1 than in groups 2 to 4 (all p < 0.03), whereas number of CXCR4/CD34-positive EPCs in BM did not differ among the four groups at 18 h after CLI. Protein expressions of SDF-1α, CXCR4, eNOS, and VEGF, numbers of CD31+, CXCR4+, SDF-1α+, and vWF + cells through immunofluorescent staining, numbers of small vessels (< 15 μm) and blood flow measured by Laser Doppler in an ischemic area were significantly higher in group 4 than in group 3 (all p < 0.005) at day 14 after CLI. Conclusion: tPA treatment enhanced number of circulating EPCs, angiogenesis, and blood flow to ischemic tissue in a murine model of limb ischemia.