| In this study,we investigated the effectiveness of cytokines on post-ischemic heart failure and the mechanisms underlying the effect.This study was divided into 3 parts.Part 1:In vivo tracking of green fluorescent protein labeled mesenchymal stem cells in hearts of rats with myocardial infarctionObjective:There are rapid advances in the use of mesenchymal stem cells for tissue regeneration and repair in the heart in recent years.However,there is limited ability to identify and "track" transplanted or migrating stem cells in vivo.In this study,we transplanted bone marrow derived mesenchymal stem cells infected with the recombinant replication-defective adenovirus vector carrying green fluorescent protein into rat heats,to evaluate the effectiveness of green fluorescent protein identifying and tracking implanted cells in vivo in order to provide a effective way to track the delivered mesenchymal stem cells in vivo.Methods:Mesenchymal stem cells isolated from rat femur marrow were cultured and expanded,then infected by recombinant retrovirus containing green fluorescent protein.Transplant the green fluorescent protein labeled mesenchymal stem cells into rat heart infarct zone.The survival,distribution and migration of labeled mesenchymal stem cells were analyzed at 3 days,7 days,14 days,21 days,28 days after transplantation.Results:Green fluorescent protein was successfully expressed by mesenchymal stem cells in vitro and in vivo.The adenoviral vectors' infection of mesenchymal stem cells had high efficiency(98%) and low toxicity,green fluorescent protein positive cells were detected by confocal microscope during the initial 28 days after transplantation.Green fluorescent protein labeled mesenchymal stem cells were gradually retained into host tissue and migrated.Conclusions:Green fluorescent protein can be used to track mesenchymal stem cell location and fate after delivery in the rat heats with myocardial infarction. Part 2:Locally Overexpressing Hepatocyte Growth Factor Prevents Post-ischemic Heart Failure by Inhibition of Apoptosis via Calcineurin-Mediated Pathway and AngiogenesisObjective:Myocardial infarction is a significant cause of heart failure.Currently, therapies are limited,and novel revascularization methods might have a role.We investigated the effects of hepatocyte growth factor expressing by bone marrow-derived mesenchymal stem cells(MSCs) on post-ischemic heart failure.Methods:4 weeks after MI,SD rats(F≤30%) were randomly divided into 1.control group:injection of saline into the infarct zone,n=6;2.MSC group:transplantation of bone marrow derived MSCs transfected with Ad-GFP into the infarct zone,n=11;3, HGF group:transplantation of MSCs transfected with Ad-HGF into the infarct zone, n=6;4.HGF+CsA group:transplantation of MSCs transfected with Ad-HGF into the infarct zone and intraperitoneal injection of cyclosporine A,25mg/kg daily for 14 days,n=6.After another 4 weeks,hearts were analyzed for ventficular geometry, myocardial function,angiogenesis and endothelial cell density,apoptosis and the expression of HGF,calcineurin,Akt and Bcl-2 protein.Results:we detected the expression of HGF protein at 2days,7days of transplantation in HGF group.We found a significant improvement in left ventricular function in the HGF-MSC group compared with GFP-MSC group.The difference in LVFS,LVEDP, LVSPand dp/dtmax in the MSC-HGF group compared to the saline control group and MSC-GFP group(p＜0.05).Overexpression of HGF was associated with a greater number of CD34and von vWF positive cells proliferated in the infarct zone (27.00±4.66 vs.13.00±2.72,30.33±2.65 vs.14.17±1.74 per mm2,respectively). There were no difference in the number of cardiac myosin heavy chain positive cells between HGF and MSC group.TUNEL assays results showed that about 0.90±0.46‰nuclei were TUNEL-positive in HGF group,whereas 2.26±0.45‰nuclei were TUNEL-positive staining,in MSC group(p＜0.05).However,the antiapoptotic effect of HGF decreased when HGF and cyclosporine A were given to rats simultaneously and 2.06±0.66‰in CsA group(p＜0.05).The effects of HGF on apoptosis were associated with the expression level of calcineurin Akt and Bcl-2 protein.Conclusion:Our findings suggest that overexpression of HGF improved ischemic cardiac function through angiogenesis and reduction of apoptosis partly mediated by up-regulation of calcineurin.Part 3:Hepatoeyte growth factor and granulocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathyObjective:Myocardial infarction(MI) is a significant cause of heart failure. Currently,therapies are limited,and novel revascularization methods might have a role.The present study was to investigated whether hepatocyte growth factor(HGF) expressed by genetically modified mesenchymal stem cells(MSCs) combined with granulocyte colony-stimulating factor(G-CSF) 8 weeks from myocardial infarction can offer synergistic therapeutic benefit and the mechanisms underlying the synergistic effect.Methods:4 weeks after MI,SD(FS＜30%) rats were randomly divided into control group(n=11);HGF group(transplantation MSCs transfected with Ad-HGF into the infarct zone,n=11);G-CSF group(intraperitoneal injection G-CSF,n=11); HGF+G-CSF group(transplantation MSCs transfected with Ad-HGF into the infarct zone and intraperitoneal injection G-CSF,n=11).After another 4 weeks,hearts were analyzed for endothelial cell density and angiogenesis,ventricular geometry, myocardial function and the expression of VCAM-1 and MMP-9 protein.Results:The rats in HGF+G-CSF group exhibited better LV systolic and diastolic function(p＜0.05),experienced less adverse ventricular remodeling,as manifested by less left ventricular dilatation(4.7±0.34mm vs.6.86±0.36mm in control group, 6.78±0.36mm in G-CSF group,5.72±0.3mm in HGF group,p＜0.05) and increased border-zone wall thickness(2.22±0.13mm vs.1.46±0.13mm in control group, 1.48±0.14mm in G-CSF group,1.85±0.09mm in HGF group p＜0.05) after 8 weeks of MI.Angiogenesis was significantly enhanced in HGF+G-CSF group by inducing proliferation of CD34 positive(20.67±1.82 per mm~2 vs.8.83±1.30 per mm~2 in control group,6.83±1.13 per mm~2 in G-CSF group,14.00±1.83,per mm~2 in HGF group, p＜0.05) and vWF positive(33.00±2.87 per mm~2 vs.11.00±1.82 per mm~2 in control group,14.00±1.63 per mm~2 in G-CSF group,26.01±2.44 per mm~2 in HGF group,p＜0.05) endothelial cells.HGF induced the expression of VCAM-1 and HGF together with G-CSF treatment synergistically stimulated MMP-9 expression in ischemic heart.Conclusion:The combination of G-CSF and HGF had a significant synergistic effect and enhanced myocardial endothelial density,angiogenesis,geometric preservation, and heart function in the model of ischemic cardiomyopathy.
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