Experimental Study Of Transplantation Of Akt Overexpressing Amniotic Fluid-derived Mesenchymal Stem Cells In A Rabbit Model Of Myocardial Ischemia Reperfusion Injury

For patients presenting with acute myocardial infarction(STEMI),timely myocardial reperfusion treatment has become a primary strategy.However,the following ischemia reperfusion injury is a major problem to be solved in the field of cardiovascular disease now.Recently the breakthrough in molecular biological technology and stem cell study has brought new hope.The improvement of easily available,long survival,absence of teratoma formation,self-renewal capability and the enhancement of survival rate of transplanted stem cells in ischemia anoxic environment is the focus on stem cell therapy.AFMSCs represent a promising alternative in cell–based therapy,given their superior qualities,such as ease of acquisition,pluripotency,self-renewal capability,few ethical concerns,and absence of teratoma formation.Furthermore,some studies have shown that genetic modification of stem cell will enhance transplantation efficiency.In our previous studies,AFMSCs were successfully isolated and cultured from amniotic fluid,transformed into cardiomyocyte-like cells after combined induction in vitro,and were transfected by using lentivirus with Akt gene.In the present study,we continued to investigate the effect and the further mechanism of transplantation of Akt overexpressing amniotic fluid-derived mesenchymal stem cells in myocardial ischemia reperfusion injury in vitro and vivo.Part ?: The effect of hypoxia on Akt overexpressing amniotic fluid-derived mesenchymal stem cells in vitro Objective To investigate the effect of hypoxia on Akt overexpressing amniotic fluid-derived mesenchymal stem cells in vitro.Methods Null-AFMSCs and Akt-AFMSCs were cultured in vitro and divided into normoxia group and hypoxia group respectively.The survival rate of null-AFMSCs and Akt-AFMSCs was measured by WST-1 after 12 hours of hypoxia.The expression of P-Akt was analyzed by Western blot.The activity of caspase-3 in null-AFMSCs and Akt-AFMSCs was determined by caspase 3 activity assay kit.The expression of some trophic cytokines in cell-conditioned media such as VEGF,HGF,bFGF and IGF-1 was evaluated by real-time PCR.Results WST-1 analysis showed that the survival rate of Akt-AFMSCs sigfinicantly increased under hypoxia compared to null-AFMSCs(P<0.05).Western blot showed that P-Akt increased in both groups under hypoxia,and P-Akt in Akt-AFMSCs was further higher after 12 h of hypoxia than null-AFMSCs.Coincidently,the increased activity of caspase-3 was sigfinicantly decreased in Akt-AFMSCs under hypoxia compared to null-AFMSCs(P<0.05).Real-time PCR showed that the genes of a myriad of cytokines secreted by stem cells including VEGF?HGF?bFGF ? IGF-1 were significantly up-regulated in Akt-AFMSCs under hypoxia compared to null-AFMSCs(P<0.05).Conclusion Hypoxia could up-regulate the expression of P-Akt in AFMSCs.Akt gene modification could further increase the expression of P-Akt,enhance the hypoxia tolerance of AFMSCs,and promote secreting more cytokines,which contributed to cell proliferation and differentiation,promotion of neoangiogenesis and anti-apoptosis.Part ?: The effect of transplantation of Akt overexpressing amniotic fluid-derived mesenchymal stem cells in a rabbit model of myocardial ischemia reperfusion injuryObjective To investigate the effect and the further mechanism of transplantation of Akt overexpressing amniotic fluid-derived mesenchymal stem cells in myocardial ischemia reperfusion injury in vivo.Methods Null-AFMSCs and Akt-AFMSCs were cultured in vitro.New Zealand rabbits were randomly divided into 4 groups: sham group,control group(equally volume of DMEM,Low Glucose),null-AFMSCs group(intramyocardial injection of AFMSCs transfected with empty gene),Akt-AFMSCs group(intramyocardial injection of AFMSCs transfected with Akt gene).Before reperfusion in a rabbit model of myocardial ischemia reperfusion injury,Brdu-labelled null-AFMSCs and Akt-AFMSCs were respectively transplanted into infarct myocardium.Left ventricular function was assessed by echocardiography after 21 days of transplantation.Myocardial tissue sections were observed under optical and electron microscope respectively.The area of infarcted myocardium and apoptotic cardiomyocyte were evaluated by TTC staining and TUNEL,respectively.The distribution of transplanted AFMSCs was observed by immunofluorescence technique.The expression of GATA-4 was meassured by RT-PCR.The expression of cTNT and capillary were analyzed by immunohistochemistry techonology.The expression of connexin-43,VEGF,Akt,P-Akt,caspase-3 and bcl-2 were analyzed by Western blot.Results Echocardiography analysis showed that FS and EF in Akt-AFMSCs group and null-AFMSCs group increased significantly compared with those in control group after 21 days of transplantation(P<0.05).Furthermore,the improvement in cardiac function was more in Akt-AFMSCs group than null-AFMSCs group(P<0.05).The area of myocardial infarction was significantly reduced in Akt-AFMSCs group compared with that in control group and null-AFMSCs group [(10.4±2.6)% vs.(26.3±2.5)% and(17.5±3.3)%,P<0.05].HE staining showed that pathological changes in Akt-AFMSCs group were significantly alleviated.Observations with the transmission electron microscope demonstrated that loss of cytoplasmic vacuoles and myofibers was alleviated compared to null-AFMSCs group.There were more Brdu-labeled AFMSCs retained in Akt-AFMSCs group compared to null-AFMSCs group(P<0.05).The number of apoptotic nuclei was markedly reduced in the Akt-AFMSCs group compared to null-AFMSCs group and control group(P<0.05).Immunohistological analysis showed that the number of capillary density and the expression of cTNT in Akt-AFMSCs group increased significantly compared with null-AFMSCs group and control group(P<0.05).Western blot showed that the expression of P-Akt,bcl-2,VEGF and connexin-43 were significantly higher in Akt-AFMSCs group than those in null-AFMSCs group and control group(P<0.05).Conclusion The transplantation of AFMSCs could alleviate pathological myocardial ischemia-reperfusion injury,decrease the area of myocardial infarction and improve cardiac fuction in a rabbit model of myocardial ischemia reperfusion injury.Furthermore,Akt-AFMSCs transplantation could benefit more than null-AFMSCs.The protective effect of Akt-AFMSCs may be due to promotion of neoangiogenesis,prevention of cardiomyocyte apoptosis,paracrine mechanisms and promotion of AFMSCs viability,which was assisted by Akt gene modification.