Combined Transplantation Of Endothelial Progenitor Cells And Mesenchymal Stem Cells For Isoproterenol Injuried Cardiomyopathy In Rats

Objective Stem cell transplantation is emerging as a potential therapeutic approach to treating cardiac diseases, and providing a new method for various cardiomyopathies. Endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) are important components of stem cells derived from bone marrow. Some studies showed they had the capacities to either repair damaged endothelium and generate new blood vessels, or regenerate new cardiocytes in the ischemic area of myocardium. While EPCs and MSCs have different biologic properties, their potential synergetic role in treating degenerative disease or damage myocardium might have better results. In this study, we aimed to investigate whether simultaneous implantation of MSCs and EPCs into the myocardium in rats with isoproterenol injured cardiomyopathy could enhance regional myocardium blood flow (RMBF) and improve cardiac function.Methods Bone marrow mononuclear cells (BMNCs) of 50 male rats were separated by Ficoll density gradient centrifugation, and EPCs and MSCs were cultured in EGM-2 and DMEM, respectively. The primary passage of EPCs and the third passage of MSCs were used for cells transplantation. 90 female rats (150-220g) received inguinal subcutaneous injections of ISO (250mg/kg) for two consecutive days. Before injection of ISO and 4 weeks after injection of ISO, heart function was assessed by transthoracic echocardiography to confirm the successful models (EF decreasing by 20% with the dilation of left ventricular). These successful models were then randomly divided into five groups: sham group, control group, MSCs group, EPCs group and EPCs+MSCs group. 2×106 EPCs, 2×106 MSCs and mixture of 1×106 EPCs and 1×106 MSCs resolved in 200μL EBM-2 were given evenly to left ventricular free wall for EPCs group, MSCs group and EPCs+MSCs group, respectively. Another vehicle group only received 200μL EBM-2 to test the effect of EBM-2 medium. And the sham control group was given saline. Echocardiography examination was repeated at 3 months after cell therapy for left ventricular end diastolic diameter (LVEDD), left ventricular anterior wall thickness (LVAWT), left ventricular posterior wall thickness (LVPWT) and interventricular septum thickness (IVST). Left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), ejection factors (EF), fractional shortening ratio (FS) were calculated by an independent investigator. The left ventricular end-diastolic pressure (LVEDP), the maximal rate of pressure rise (+dp/dtmax), the maximal rate of pressure fall (-dp/dtmax) were analyzed a right carotid artery catheter. RMBF was measured by colored micro spheres (CM) on the last day of the study. Fluorescence in situ hybridization (FISH) was performed to detect male cells in the female rat hearts. Immunofluorescence staining was performed with monoclonal mouse anticardiac troponin T, and polyclonal rabbit anti–von Willebrand factor (vWF) was used to assay the differentiation of transplanted cells. To detect fibrosis in myocardium, Masson's trichrome staining was performed on the left ventricle tissue. To detect capillary density in the myocardium, immunohistochemical staining of endothelial cells was performed using an antibody against vWF. The ratio of apoptosis cell in myocardium was analyzed by Tunel staining. The protein expression of vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (b-FGF) and angiopoietin 2 (Ang-2) were measured by ELISA assay. Quantitative real time polymerase chain reaction(QRT-PCR), using SYBR? Green I as a dye, was performed to analyze the mRNA expression of VEGF, b-FGF and Ang-2.Results 71 of 90 female rats survived from the ISO injection intervention. Whereas, the 6 female rats didn't manifest the significant decrease of cardiac function, and were excluded from the study. These 65 rats were then randomly divided into five groups (13 rats in each group). At the completion of 3-month cell therapy treatment, there were 11 rats in sham group, 11 rats in control group, 10 rats in MSCs group, 9 rats in EPCs group and 11 rats in MSCs+EPCs group to survive. 4 weeks after ISO injection, compared with baseline, the cardiac function showed decrease in EF and FS (P<0.05). However, LVEDD, LVESV and LVEDV were increased significantly (P<0.05). 3 months after cell transplantation, MSCs group, EPCs group and EPCs+MSCs group were improved in EF, FS, LVESV, LVEDV, +dp/dt, -dp/dt and LVEDP (P<0.05). There were no difference between sham group and the control group (P>0.05). However, there showed no statistical difference in LVEDD between EPCs+MSCs group and single stem cells treatment groups (P>0.05). Besides LVEDD, EPCs+MSCs group demonstrated even better cardiac function than either MSCs group or EPCs group (P<0.05). The combination of MSCs and EPCs induced greater RMBF comparing as the other three groups (sham group, control group and MSCs group, P<0.05), but was not significantly greater than EPCs group (P>0.05). Total capillary density in the myocardial tissue had the similar results with RMBF. The Sry positive cells detected in cardiac tissue in EPCs+MSCs group and MSCs group, however, in EPCs group they only were found in blood vessels. No Sry positive cell was presented in the sham group and control group. Immunofluorescence staining demonstrated that some of Sry positive MSCs were positive for the cardiac markers (cardiac troponin T) and some of EPCs were positive for vWF. EPCs+MSCs group had lest collagen deposition compared with the other four groups (P<0.05). There are less the ratio of apoptosis cell in EPCs+MSCs group than the other four groups (P<0.05). VEGF, b-FGF and Ang-2 protein levels slightly increased in MSCs group compared with sham group and control group (P<0.05). Both EPCs+MSCs group and EPCs group demonstrated further increase in protein expression level than MSCs group (P<0.05). Interestingly, EPCs group had higher protein expressions of b-FGF than the EPCs+MSCs groups (P<0.05). QRT-PCR analysis demonstrated that higher mRNA expressions of were found in the EPCs+MSCs group and EPCs group than either MSCs group or sham group or control group (P<0.05). But, mRNA expression of b-FGF was not different between the EPCs group and the EPCs+MSCs group (P>0.05).Conclusion The transplantation of EPCs combined with MSCs can significantly improve the cardiac function and myocardium flow for the rats with isoproterenol injured cardiomyopathy, as comparing with MSCs or EPCs transplantation. The effects are related to increase neovascularization, decrease fibrosis and apoptosis cardiocytes in cardiac tissue. The transplanted EPCs and MSCs can differentiate into cardiocytes and endothelial cells, and promote the expression of various growth factors such as VEGF, b-FGF and Ang-2. This study provided a novel treatment method for isoproterenol injured cardiomyopathy and presented experimental data for the application of combined transplantation of two kinds of stem cell.