| Back ground:Bone marrow-derived endothelial progenitor cells (EPCs) have been isolated from peripheral blood and shown to differentiate into mature endothelial in vitro and in vivo. The EPCs in the circulation can migrate, engraft and incorporate into sites of neovascularization in response to certain cytokines and tissue ischemia. The number of homing EPCs in the ischemic tissue is crucial for the ultimate therapeutic neovascularization. Stromal cell-derived factor-1 (SDF-1, CXCL12) is a chemokine of CXC subfamily. It binds to its CXCR4 receptor to chemoattract stem cells. CXCR4 is a G-protein coupled receptor that is the only known receptor for SDF-1. Previous studies showed that CXCR4 is abundantly expressed in the cultured EPCs and, the migration of EPCs towards SDF-1 is dose-dependent. In the myocardial infarction model in rats, SDF-1 is distinctly upregulated in the damaged myocardium, suggesting that SDF-1/CXCR4 axis may play an important role in the homing of EPCs after myocardial infarction. Akt is a key downstream effector of the lipid kinase PI3K (phosphoinositide3-kinase). Experimental evidence demonstrates that the PI3K/Akt signaling pathway controls cell growth, proliferation, survival, migration and invasion. Previous studies showed that Akt is a key downstream effector molecule of VEGF and SDF-1 in endothelial cells, it involves in the survival of endothelial cells and mediated the migration of endothelial cells arosed by VEGF. Here, we aim to investigate the functional role of PI3K/Akt signaling pathway in the migration of EPCs as mediated by SDF-1/CXCR4 axis in the homing of EPCs after myocardial infarction.Objective:To investigate the homing capacity of intravenous implanted EPCs and the therapeutic effect of EPCs on neovascularization, infarct area, cardiac function in rats’acute myocardial infarction model. To investigate the functional role of PI3K/Akt signaling pathway in the SDF-1 induced EPCs migration in vitro and the homing of EPCs after myocardial infarction in vivo.Method:Bone-marrow derived mononuclear cells were isolated from the tibias and femurs of rats by density-gradient centrifugation and suspended in Endothelial Basal Medium-2. The cells were plated on six-well plates and cultured in EBM-2 supplemented with SingelQuotes supplement and 5% FBS. After 3 days in culture, nonadherent cells were removed, cells were washed by PBS, fresh medium was applied. The acetylated low-density lipoprotein uptaken and BS-1 lectin binding were tested for EPCs characterization. Immunohistochemistry staining were performed for testing the expressoion of von Willebrand factor (vWF) and endothelial NO synthase (eNOS) in EPCs.The left anterior coronary artery were ligated to produce the rats’ myocardial infarction model. E1/E3-defective adenovirus vector expressing enhanced green fluorescent reporter protein (EGFP) was used to label the EPCs. The 5×106 EPCs in 1ml medium or 1ml medium without cells were transfused into the circulation by tail vein 24 hours after the ligation. After 14 days of transfusion, vWF immunofluorescence staining was performed to measure the vessel density in the infarcted border zone. After 28 days of transfusion, the infarction area were measured by TTC staining, cardiac function was measured by echocardiography and hemodynamic measuring.Fluorescence-Activated Cell Sorting was used to detect the CXCR4 expression rate of EPCs after 7 days in culture. AMD3100 and LY294002 were used to block the CXCR4 or PI3K/Akt pathway in EPCs. A migration assay was performed using an 8-μm pore size 24-well transwell polycarbonate insert system to evaluate the migratory ability of EPCs towards SDF-1.Elisa assay was performed to evaluate the SDF-1 expression in the infarct myocardium.5×106 EPCs with or without AMD3100 (10μg/ml) or LY294002 (20μM) pretreatment were injected via tail vein after 24 hours of the coronary left anterior coronary artery ligation. After 14 days of EPCs implantation, the homing number of implanted EPCs were measured under fluorescence microscope, vWF immunofluorescence staining were performed to measure the vessel density in the infarcted border zone. After 28 days of EPCs implantation, the infarction area were measured and cardiac function was measured.Result:After 3 days in culture, some of the bone marrow-derived EPCs showed a elliptical or teardrop-shaped appearance. After 5 days in culture, the cells fomed typical clone. The red (DiI-acLDL) and green (Fitc-BS-1 lectin) fluorescence doube positive cell proportion is (90.61±3.73)%, the vWF posive proportion is (91.64±3.12)%, the eNOS positive proportion is (89.68±3.47)%.After 14 days of cell implantation, the vessel density was distinctly increased in EPCs group compare with with the control group. The labeled EPCs were detected in the infracted border zone and formed tube-like structures. vWF staining showed that the tube-like structures were new vessels.After 28 days of implantation, the cardiac function were improved in EPCs group compared with control group, the percentage of infarction area in EPCs group was (21.82±2.94)% which were lower than that in control group (30.06±2.01)%.FACS analysis showed that 52.06±2.10% of cultured EPCs in day 7 express CXCR4, whereas only 7.21±2.18% of freshly isolated bone-marrow mononuclear cells expressed CXCR4. SDF-1 treatment leads to a significant phosphorylation of Akt in EPCs, which was inhibited by AMD3100 and LY294002. SDF-1 induced a dose-dependent migration of EPCs. The migration was inhibited when the EPCs were pretreated with AMD3100 or LY294002.The SDF-1 expression was sifnificant elevated after myocardial infarction. In vivo, the homing capacity of EPCs to the peri-infarct region, neovascularization, and restoration of cardiac function were distinctly impaired in AMD3100 or LY294002 pretreated EPCs implantation group compared with unpretreated EPCs implantation group.Conclusion:The purified EPCs could be achieved by in vitro culture of bone marrow-derived mononuclear cells with EBM-2+SingelQuotes culture. Intravenous implanted EPCs can effectively homing to the infarcted border zone, promote neovascularization, reduce infarct area and improve the cardiac function. PI3K/Akt signaling pathway is crucial in the SDF-1 induced migration of EPCs in vitro and, SDF-1/CXCR4 mediated PI3K/Akt activation is important for the EPCs homing after myocardial infarction. |
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