Myocardial Protective Effect Of EcSOD Gene-Modified Bone Marrow Mesenchymal Stem Cells On Functional Enhancement In Infarcted Mice Hearts Via Akt-FoxO3a Pathway

Background：Despite continuous therapeutic advancements, myocardial infarction (MI) remains amajor cause of death world-wide. Unlike other organs, the heart has a limited ability toregenerate cardiomyocytes to repair injured tissue after an MI. Therefore，after MI, toprevent cardiomyocytes from death, reduce infarct size, has become the key forcardioprotection. Considerable evidence indicates that reactive oxygen species (ROS),such as superoxide anion (O2-) and hydrogen peroxide (H2O2), contribute importantly tomyocardial ischemic injury. Excessive O2-formation has been detected in patients withacute myocardial infarction (MI) and unstable angina pectoris. Superoxide dismutase(SOD) could provide antioxidant protection by inactivating O2-, sparing nitric oxide fromdestruction, and preventing O2-from forming more destructive reactive oxygen species,such as peroxynitrite and its reaction products. Extracellular superoxide dismutase(ecSOD), the only known isoform of SOD secreted from cells, binds to heparin sulfate proteoglycans on cellular surfaces, as a unique scavenger of superoxide anions in theextracellular space. Many studies have shown that direct gene transfer of the cDNAencoding membrane-bound human ecSOD offers transient cardioprotection effect.However, gene delivery via plasmid or virus containing ecSOD gene causes severalproblems, such as the inefficiency and instability of ecSOD expression in local tissue, andthe elicited local immune response. Bone marrow mesenchymal stem cells (BMSCs), asmulti-potent adult stem cells, hold promise as vehicles for adult stem cell–based genetherapy due to their unique advantages. Recently, Abdel et al. demonstrated for the firsttime that intravenous administration of BMSCs transduced with ecSOD could improve thesurvival of irradiated mice. We hypothesized that administration of BMSCs as a genetherapy carrier of ecSOD could offer improved cardioprotection post-MI.Objectives：1. To investigate whether ecSOD-gene modified BMSCs is involved in thecardioprotective effect after MI in vivo.2. To verify the cardioprotective effect of ecSOD-gene modified BMSCs in vitro.3. To study the potential mechanism of the cardioprotective effect underlyingecSOD-gene modified BMSCs.MethodsBMSCs, isolated from Fluc+transgenic mice（Tg〔Fluc+〕）, were transfected by aadenovirus which incorporated the cDNA for human ecSOD. ELISA was performed todetermine the concentration of ecSOD secreted from the transduced BMSCs(BMSCs-ecSOD). Female FVB mice (n=100) were randomized into4groups:1) Sham；2) MI+PBS (MI)；3) MI+BMSCs (BMSCs)；4）MI+BMSCs-ecSOD (BMSCs-ecSOD). MI wasaccomplished by ligation of the mid left anterior descending artery.2x106cells (in50uL)or same volume PBS were injected into the peri-infarct zone respectively. In vivobioluminescence imaging (BLI) was performed to monitor viable transplanted BMSCsquantitatively. Longitudinal echocardiography and histological staining revealed thatBMSCs-ecSOD significantly reduces the region at risk and infarct size. Lucigeninchemiluminescence and ELISA demonstrated that BMSCs-ecSOD increases ecSODconcentration and reduces the total ROS and apoptosis in the injury tissue. After hypoxia and serum deprivation (H/SD) injury, the apoptotic rate and formation of O2-incardiomyocytes were decreased when co-cultured with BMSCs-ecSOD in vitro. Westernblotting revealed that ecSOD supplementation increases Akt and Erk phosphorylation incardiomyocytes; and forkhead box protein O3a (FoxO3a) phosphorylation ofcardiomyocytes’ cytoplasm were found up-regulated at48-hour time point after H/SDinjury.Result1. The cardioprotective effect of ecSOD-gene modified BMSCs after MIin vivo.1.1Dose-dependent secretion of ecSOD in ecSOD-transfected BMSCsBMSCs were transfected with Ad5CMVecSOD. When the MOI were less than300, thesecretion of ecSOD was dose-dependent in24,48, and72h-experimental groups.However, the secretion of ecSOD no longer increased accordingly at MOI>300. Secretionof ecSOD from BMSCs-ecSOD at MOI=300reached its peak in48h-group compared withthose in24h and72h-groups. Therefore BMSCs were transfected at MOI=300for thefollowing experiments.1.2Increased survival of transplanted BMSCs-ecSODCompare with BMSCs,thesignal of BMSCs-ecSOD increased significantly.(p<0.05vs. BMSCs at7days after MI, p<0.001vs. BMSCs at21days after MI).1.3Transplantation of BMSCs-ecSOD improved the cardiac function significantly andreduced region at risk and infarct sizeThe improvement by echocardiography was significantly greater in BMSCs-ecSODgroup compared to the BMSCs-alone and MI group. This improvement peaked on day7post-MI, and was sustained at least through to Day28. Masson trichrome staining onDay28post-initial operation demonstrated that improvement of infarct size wasconsistent with the result in echocardiography.1.4The transplantation of BMSCs-ecSOD produces sustained increase of ecSODaccompanied with reduction of ROS in the injury tissueThe expression of ecSOD48h after MI in BMSCs-ecSOD group was significantly elevated compared with the other three groups. Meanwhile, the production of total ROSin BMSCs-ecSOD group was decreased significantly, although the expression of ecSODreduced over time, at least sustained for10days.1.5Anti-apoptotic effects of ecSOD in vivoAnti-apoptotic effect was more pronounced in BMSCs-ecSOD groupscompared withthe other three groups in vitro.2. The anti-apoptotic effect of ecSOD supplement in cardiomyocytes after H/SD invivo.2.1In vitro study showed that apoptosis in cardiomyocytes co-cultured withBMSCs-ecSOD, which were subjected to H/SD injury, was decreased by usingTUNEL staining and caspase-3activity assay.2.2Hypoxia-induced rises in O2-was attenuated in the myocardial tissue co-culturedwith BMSCs-ecSOD.3. The potential mechanism of cardioprotective effect underlying ecSOD-genemodified BMSCs.Activation of Akt, Erk1/2and FoxO3a signaling pathways may partly participate inthis cardioprotective effect.Conclusion1. In present study, we used the new stem cell-based gene therapy: stem cells were usedas “carriers” to transduce the ecSOD gene. This is the first both in vitro and in vivostudy to show that ecSOD-transfected BMSCs can efficiently attenuate myocardialdamage.2. Our data showed that the loss of BMSCs-ecSOD reduced in myocardial tissue afterMI, and meanwhile, the expression of ecSOD significantly longer in BMSCs-ecSODgroup.3. Anti-apoptotic effects of ecSOD supplement were significantly increased incardiomyocytes both in vitro and in vivo.4. Activation of Akt, Erk1/2and FoxO3a signaling pathways may partly participate inthis cardioprotective effect. 5. This method holds a great therapeutic promise in the development of novelcardioprotective strategies.