| BackgroundEndothelial progenitor cells(EPCs), which can differentiate into endothelial cells, play an important role in postnatal vasculogenesis. Injured or hypoxic tissues can express several angiogenic factors, which recruit EPCs from the bone marrow into the circulation, and toward to the injured site. The coronary collateral circulation is an important factor in terms of the amount of damage to the myocardium and prognosis after myocardial infarction (MI). Well-developed collaterals may greatly limit or even completely eliminate myocardial infarction despite complete occlusion of a coronary artery. However, the ability to recover from acute MI often appeares to be inadequate. Transplanting EPCs into myocardium was proved to improve cardiac function after MI. EPCs can not only directly integrate into vascular structures, but also have paracrine effects to magnify neovascularization. However, there is little understanding of the signaling pathways in EPC transplantation. Although the EPC-based cell therapy was encouraging, previous studies showed that the survival of transplanted EPCs in vivo was less than ideal, and most of them died in a short time.objectivesTo investigate the potential signaling mechanism of the EPC-based cell therapy after MI.To investigate whether exogenous erythropoietin (EPO) delivery could improve the survival of transplanted EPCs (tEPCs) and enhance the efficiency of EPC-based cell therapy.Methods 1. Bone marrow cells were harvested from BALB/c mice, and mononuclear cells were separated. Cells were induced cultured in Endothelial Cell Growth Medium (EGM) with additional murine growth factors, cells were identified for EPC-s.2. Myocardial infarction was induced in wild-type mice by ligating the left anteri-or descending coronary artery (LAD). EGFP-EPCs (enhanced green fluorescent protein-EPCs) with or without EPO were transplanted into peri-infarct myocardi-um. ECG was recorded15min after LAD ligation by an Electrocardiograph, wortmannin was injected intraperitoneally after surgery.3. EGFP-EPCs were detected after EPCs transplantation with a fluorescence micro-scope.4. Expressions of stromal cell-derived factor-1alpha (SDF-1a), vascular endothe-lial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) in the peri-infarct myocardium were measured by ELISA. The expression of VEGFR-2was determined by immunohistochemistry. Expressions of Akt and p-Akt were assessed by Western blot.5. The amount of circulating EPCs (cEPCs) was analyzed by flow cytometry.6. Microvessel density in the peri-infarct myocardium was determined by immuno-histochemistry, fibrosis by Masson’s trichrome stain, and apoptosis by TUNEL assay.7. Cardiac function was assessed by echocardiography. Results1. Cells appeared to be clusters at day3and quite a few of them were spindle shaped at day7. After14days of culture, cells showed cobblestone appearance. EPCs were double stained with Dil-ac-LDL and UEA-1lectin. They expressed both CD34and Flk, and formed tubelike structures.2. The typical elevation of’ST’segment in I and aVL leads was recorded15min after surgery. 3. Compared to vehicle, the expression level of VEGF was elevated (P<0.01), along with elevated expression of VEGFR-2and p-Akt in the peri-infarct myocardium after EPC transplantation. Meanwhile, the expression of eNOS was elevated too (P<0.05).4. Compared to vehicle, cEPCs level was elevated after EPC transplantation (P<0.01). This effect could be inhibited by wortmannin.5. EPC transplantation increased microvessel density (P<0.01) and reduced the vol-ume of collagen (P<0.01) in the peri-infarct myocardium. The fractional short-ening (FS) was increased (P<0.05) and the left ventricular diameter was smaller after EPC treatment (P<0.05). Wortmannin inhibited the effect of EPC trans-plantation on cardiac function.6. More EGFP-EPCs were found in the hearts treated with EPCs+EPO than in those treated with EPCs alone (P<0.01).7. cEPC level was markedly elevated after EPC+EPO treatment compared with EPC application alone (P<0.01). SDF-1alpha and VEGF were increased accordingly (P<0.05).8. EPC+EPO therapy could further increase microvessel density (P<0.01), decrease apoptosis (P<0.05) and reduce fibrosis (P<0.01) in the peri-infarct myocardium compared to only EPC therapy.9. Left ventricular fractional shortening (LVFS) was higher with smaller left ventricu-lar end-diastolic dimension (LVEDd) and end-systolic dimension (LVEDs) after EPC+EPO treatment compared to EPC treatment alone (P<0.05). Conclusions1) EPC transplantation after MI could lead to a coordinated upregulation of VEGF/VEGFR2/p-Akt/eNOS, which may play important roles in autologous EPC mobilization.2) EPC transplantation could enhance neovascularization, reduce fibrosis in the peri-infarct myocardium, and improve cardiac function after MI.3) Supportive EPO could enhance the survival of transplanted EPCs and autologous EPC mobilization. 4) EPO improved the efficiency of EPC therapy in mice with MI, associated with further enhanced neovascularization, reduced fibrosis and apoptosis in the peri-infarct myocardium. |
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