The Apoptosis Of Myocardial Cells Is Regulated By Bone Marrow Mesenchymal Stem Cells With Overexpressing Integrin-linked Kinase

Myocardial infarction（MI）is one of the most serious cardiac diseases which are harmful to human health in the world.Traditional treatment methods mainly contain drug therapy,thrombolytic therapy,percutaneous coronary intervenstion and coronary artery bypass grafting.In recent years,it has been found that stem cell transplantation can protect myocardial cells in the infarcted area and promote angiogenesis to improve cardiac function,thus it is becoming one of the treatment methods with great potential for development.Bone marrow mesenchymal stem cell（BMSC）is a commonly used type of stem cell transplantation in the cardiovascular field.Although BMSC transplantation can improve cardiac function to a certain extent,problems such as low survival rate,poor cell activity and cell senescence will occur within the ischemic and hypoxic micro-environment in the infarct area,which seriously reduces its efficacy.Therefore,it is necessary to find the relevant solutions.It have been found that overexpressing some genes in stem cells is a good idea.Integrin linked kinase（ILK）is a highly expressed serine/threonine protein kinase in the cardiovascular system which is involved in a variety of cardiac functions,such as myocardial contraction,compensatory hypertrophy,myocardial survival,angiogenesis,the controling of endothelial progenitor cell migration to ischemic tissue and so on.Our early experiments have confirmed that ILK modified c-kit⁺stem cells can improve cardiac function after myocardial infarction.So we consider ILK-related further study on the influence of BMSCs,and ILK-modified BMSCs effect on myocardial cell function and molecular mechanism,and it is also highly hoped to determine the active site of ILK,in order to clarify modification principle of ILK regulating BMSCs and relevant mechanism of myocardial cell function which is regulated and controlled by the ILK-modified BMSCs,and provide more effective methods to improve the efficacy of stem cell transplantation in improving cardiac function after myocardial infarction.Objective:To investigate the effects of different ILK activities on the survival,proliferation,migration and apoptosis of BMSCs,to further explore the effects of ILK-overexpressed BMSCs on apoptosis and the relative molecular mechanisms of cardiomyocytes（H9c2）,and to determi ne the active sites of ILK effects.Methods:1.Lentiviral expression vectors containing different ILKs were constructed to transfect with BMSCs.Lentiviral expression vectors containing wild-type ILK（WT-ILK）,activity-silent kinase ILK（ILK-S343A）,high-activity-kinase ILK（ILK-S343D）and dominant mutant ILK（ILK-E359K）were constructed to respectively infected with BMSCs.GFP fluorescence and Western blotting（WB）were used to detect the expression of ILK protein in the BMSCs to identify the infection efficiency,and purinomycin was used to screen the positive cells to obtain stable infected BMSCs.BMSCs were divided into control group,null group,WT-ILK group,ILK-E359K group,ILK-S343A group and ILK-S343D group.2.Effects of ILK on survival,proliferation,migration and apoptosis of BMSCsBMSCs survival and proliferation in each group were detected by live cell count test and CCK8（Cell Counting Kit-8）kit,and proliferation-related genes PCNA and Cyclin D₁ were detected by WB.The migration of BMSCs in each group was detected by scarification test and Transwell transmembranemigration test.Cell apoptosis induced by hydrogen peroxide（H₂O₂）was identified by DAPI staining and TUNEL staining,the protein expression of apoptosis-related gene Caspase3 was detected by western blotting.3.The effect of BMSCs with overexpressed ILK on apoptosis of myocardial cellsBMSCs in each group were co-cultured with myocardial cells（H9c2）in a non-contact manner.The apoptosis of H9c2 was detected by DAPI staining and TUNEL staining.Exosomes from BMSCs in null group,WT-ILK group,ILK-S343A group and ILK-S343D group were extracted and identified.After the exosomes were co-cultured with H9c2,apoptosis of cardiomyocytes was detected by the above method,and Caspase3 protein expression.Results:1.Lentiviral vectors expressing WT-ILK,ILK-E359K,ILK-S343A andILK-S343D were successfully constructed and stably expressed in BMSCs.2.Overexpression of ILK regulates BMSCs survival and proliferationThe results of live cell count and CCK8 assay showed that the survival and proliferation of cells in WT-ILK group and ILK-S343D group weresignificantly increased compared with control group and null group,and the increase was more obvious in the latter group.The results of ILK-S343Dgroup were（1.58±0.39）times（P<0.05）and（1.60±0.03）times（P<0.01）,respectively,than those in WT-ILK group.In contrast,ILK-E359K andILK-S343A groups showed decreased proliferation.Furthermore,WB results showed that the expressions of proliferation-related genes PCNA andCyclin D1 in the ILK-S343D group were higher than those in the WT-ILK group,suggesting that overexpression of ILK regulates the survival andproliferation of BSMCs,and is related to the kinase activity at site 343 and the function at site 359.3.Overexpression of ILK modulates the plane migration and transmembrane migration of BMSCsThe results of scarification test and transwell transmembrane migration assay showed that cell migrations in WT-ILK group and ILK-S343D group were significantly increased,because the relative residual area of WT-ILK group was（0.64±0.06）times than that of null group（P<0.05）and the number of transmembrane migratory cells was（1.67±0.03）times than that of null group（P<0.05）.The ILK-S343D group was（0.64±0.03）times（the relative residual area）（P<0.05）and（1.32±0.05）times（the number of transmembrane migratory cells）than that of WT-ILK group（P<0.05）,while cell migrations in ILK-E359K group and ILK-S343A group were decreased.This suggests that ILK overexpression modulates BMSCs migration,and this effect is related to the activity at sites 343 and 359.4.Overexpression of ILK modulates the apoptosis of BMSCsThere are similar results of DAPI and TUNEL experiments,showing that compared with null group,apoptosis of cells in WT-ILK group and ILK-S343D group were decreased,and ILK-S343D group was more obvious,while the apoptosis of cells in ILK-E359K group and ILK-S343A group was slightly increased,the expression of Caspase3 protein was consistent with the trend of the above groups,indicating that ILK regulates the apoptosis of BMSCs through kinase activity.5.BMSCs with overexpressed ILK affect myocardial apoptosis through the exosome-pathwayAfter the BMSCs in each group were co-cultured with H9c2,the apoptosis of cardiomyocytes was detected by DAPI and TUNEL test,which showed that the apoptosis of H9c2 was decreased in BMSCs that were co-cultured with WT-ILK group and ILK-S343D group,and the ILK-S343D group was（60.23±0.12）%（P<0.05）and（65.45±1.02）%（P<0.05）of that in WT-ILK group.In exosome co-culturing experiments,we confirmed that exosomes derived from BMSCs in the ILK-S343D group（SD-BSMC-Exo）were more effective against H₂O₂-induced myocardial apoptosis than those in the null group（Null-BMSC-Exo）,and the expression of Caspase3 protein was also decreased,indicating that BMSCs with high kinase activity can regulate myocardial apoptosis through exosomes through the paracrine mechanism.Conclusions:1.Overexpression of ILK can regulate the survival,proliferation,migrati on and apoptosis of BMSCs,which is related to the kinase activity at 343 site and the functional activity at 359 site on ILK gene sequence.2.BMSCs modified by overexpressed ILK,especially ILK S343D decrea sed H9c2 apoptosis induced by H₂O₂.3.ILK-modified BMSCs regulate H₂O₂-induced apoptosis of cardiomyocy tes through the exosome-pathway.