Studies Of Erythropoietin With Stromal Cell-derived Factor-1 In Promoting Therapeutic Neovascularization Of Endothelial Progenitor Cells

PartⅠIsolation and characterization of endothelial progenitor cellsObjective:To establish the culturing system of EPCs according to the former established protocols of our lab,and provide cell source for following experiments.Method:Mononuclear cells(4×10~6) were isolated by density gradient centrifugation from rabbit peripheral blood(20 ml) plated on culture dishes coated with fibronectin (0.1%),and cultured in endothelial cell basal medium-2(EBM-2) supplemented with 20%fetal bovine serum(FBS) and Clonetics EGM-2 SingleQuots.The EPC were characterized by acetylated low density lipoprotein(DiI-acLDL) uptake,lectin binding(FITC-UEA-1),and immunochemical staining for CD34,CD133,CD45,von Willebrand factor(vWF),at 7 days after onset of culture.Results:Attached spindle-like cells appeared after 3 days culture and became more and larger at 7 days of culture.Cord-like structure was observed.The positive rates of CD133,CD34,andⅧfactor of ex vivo expanded EPC at 7 days of culture were 83.11±3.46%,82.87±4.66%and 77.45±6.71%,respectively.CD45 is negative.The cells had the capacity to incorporate DiI-acLDL and FITC-UEA-1(＞90%).Conclusion:EPCs have been shown to be present in circulation,and could be isolated and cultured from peripheral blood MNCs.PartⅡStromal cell-derived factor-1 effects on endothelial progenitor cell in vitroObjective:To prepare NIH 3T3 transduced with the SDF-1αretroviral vector(NIH 3T3/SDF-1) and with LacZ retroviaral vector(NIH 3T3/LacZ) and investigate the effect of stromal cell-derived factor-1(SDF-1) secreted by NIH 3T3/SDF-1 on migration and apoptosis of ex vivo expanded EPCs. Methods:Thaw the freezing NIH 3T3/SDF-1 cells and the SDF-1 concentration in the supernatant of cultured cells with or without EPO was measured with enzyme-linked immunosorbent assay(ELISA).Thaw the freezing NIH 3T3/LacZ cells and was conformed by theβ-Gal staining.Amodified Boyden chamber assay was used to determine the effect of secreted SDF-1 on EPC migration.Apoptosis of EPCs under influence of SDF-1 were investigated by stained with DAPI with or without L-NMMA.Results:The freezing cells were thawed successfully and cells viability≥90%.NIH 3T3/SDF-1 cells produced 147±6 ng of SDF-1 per 10~6 cells in 24 h and EPO can not influence the expression of SDF-1 in NIH 3T3/SDF-1 cells.NIH3T3/LacZ cells were conformed by positiveβ-Gal staining.EPCs migrated more when they were co-cultured with NIH 3T3/SDF-1 than with unmodified NIH 3T3 cells(31±9 versus 13±4 migrated cells,respectively;P＜0.05).The migrated cells were significantly reduced to 16±4 when EPCs were cocultured with NIH 3T3/SDF-1 in the presence of L-NMMA(P＜0.05).Apoptosis was found in 30±7%of nutrient deprived EPCs co-cultured with NIH 3T3 cells.EPCs co-cultured with NIH 3T3/SDF-1 demonstrated a significant reduction in apoptosis(17±4%,P＜0.05) L-NMMA did not significantly affect apoptosis in EPCs co-cultured with NIH 3T3/SDF-1 cells(18±5%,P＞0.05).Conclusion:NIH3T3/SDF-1 cells can secrete SDF-1 to induce EPC migration in vitro through NO pathway.Apoptosis of EPCs can be reduced by SDF-1 secreated by NIH3T3/SDF-1cells and NO is not involved in the inhibition of EPC apoptosis.PartⅢErythropoietin with Stromal cell-derived factor-1 in therapeutic nevascularization of endothelial progenitor cells in ischemia limbObjective:Erythropoietin(EPO) mobilizes bone marrow mononuclear cells into the peripheral circulation.Stromal cell-derived factor-1(SDF-1) enhances the homing of progenitor cells mobilized from the bone marrow and augments neovascularization in ischemic tissue.We hypothesize that SDF-1 will boost the pro-angiogenic effect of EPO.Methods:NIH 3T3/SDF-1(10~6 cells) cells were injected into the ischemic muscles immediately after resection of the left femoral artery and vein of C57BL/6J mice. EPO(1000IU/kg/day) was injected intraperitioneally daily for 3 days after surgery. Blood perfusion was examined using a laser Doppler perfusion imaging system.Mice were sacrificed 21 days after surgery.Immunostaining and Western blot assay of the tissue lysates to confonfirm the cells in ischemia tissues and expressed SDF-1. Capillary density in the ischemia tissue were assessed with alkaline phosphatase staining,and the apoptosis of muscle cells was viewed using an in situ cell death detection kit.Results:The perfusion ratio of ischemic/non-ischemic limb increased to 0.75±0.06 and 0.63±0.03 with the treatment of either SDF-1 or EPO only,respectively,3 weeks after surgery,which was significantly higher than the saline-injected control group (0.50±0.02,P＜0.05).Combined treatment with both SDF-1 and EPO resulted in an even better perfusion ratio of 0.89±0.08(P＜0.05 versus the single treatment groups). CD34~+ cells were detected with immunostaining,and Western blot assay showed that the injected NIH 3T3/SDF-1 survived and expressed SDF-1.More CD34~+ cells, increased capillary density,and less apoptotic muscle cells were found in both EPO and SDF-1 treated group(P＜0.05 versus other groups).Conclusion:Combination of EPO-mediated progenitor cell mobilization and SDF-1-mediated homing of EPCs promotes neovascularization in the ischemic limb and increases the recovery of blood perfusion without increasing the number of red blood cells and hematocrit.