The Effect And Mechanisms Of IGF-1 On BWSCs Transplantation In The Treatment Of Acute Myocardial Infarction

Background:Ischemic heart disease(IHD)often leads to myocardial infarction(MI)and heart failure(HF)in patients.Even after medication and early interventional treatment,the mortality of MI remains high.Many studies have confirmed that stem cell transplantation can reduce cardiomyocytes apoptosis,inhibit cardiac remodeling,and improve impaired cardiac function through paracrine mechanisms.Bone marrow mesenchymal stem cells(BMSCs)have many advantages such as easy to isolate,culture and proliferate plus immune tolerance.After transplantation,they can promote capillary regeneration,secrete a variety of cell growth factors,and inhibit apoptosis of cardiomyocytes.However,due to the unfavorable microenvironment of the ischemic area after transplantation,the retention and survival rate of stem cells are very low,and thus limit its efficacy.Insulin-like growth factor 1(IGF-1)is a cytokine that can promote the proliferation,proliferation,and differentiation of a variety of cells in vivo and in vitro,while inhibiting apoptosis and necrosis.Many studies have showed that IGF-1 plays an active role in stem cell transplantation.Therefore,this study attempts to use genetic engineering to produce BMSCs that overexpress IGF-1(IGF-1-BMSCs)in order to obtain better post-MI transplantation efficacy.The canonical Wnt signaling pathway is widely involved in various physiological activities such as gene expression,cell movement,stem cell proliferation and differentiation,and its components are expressed in mesenchymal stem cells(MSCs),which suggesting that Wnt signaling plays an important role in the biological function of MSCs.Secreted frizzled related protein 2(SFRP2)is generally considered to be an inhibitor of the canonical Wnt pathway while subsequent studies have pointed out that it may maintain or even activate the canonical Wnt signaling pathway in some cases.In the transplantation of protein kinase B(AKT/PKB)overexpressing MSCs(AKT-MSCs)for treating MI,after silencing the SFRP2 gene,the original protective effect of AKT-MSCs disappeared.IGF-1 can play an important regulatory role in the Wnt system through the PI3K/AKT pathway,thereby affecting the transcription of downstream genes.In adipose stem cells,IGF-1 can promote SFRP2 secretion through the PI3K/AKT pathway.Therefore,we tried to analyze the role of AKT,SFRP2 protein and Wnt pathway in IGF-1-BMSCs transplantation for the treatment of acute myocardial infarction.Aim:To evaluate the effect of IGF-1 on the improvement of BMSCs transplantation after MI and elucidate the possible mechanisms.Methods:We first isolated and cultured BMSCs from SD rats,using whole bone marrow adherent culture method,and detected the cell surface markers CD73?CD 105?CD90 and CD45 by flow cytometry.Then we constructed BMSCs overexpressing insulin-like growth factor-1(BMSCs-IGF-1)or empty vector(BMSCs-NC)using lentivirus.BMSCs-IGF-1 and BMSCs-NC were cultured for 48 h under hypoxia and normoxic conditions respectively,and then MTS was used to detect apoptosis.Western Blot was used to detect markers of cell proliferation,apoptosis,and expression of stem-related proteins.Transwell assay was used to detect migration ability of cells.TUNEL was used to detect cell apoptosis.The differences of cell proliferation,migration ability,stemness and apoptosis between BMSCs-IGF-1 andBMSCs-NC under hypoxia and normoxic conditions were compared.We further used Western Blot to detect the expression of AKT/SFRP2 protein.We also used AKT inhibitor LY294002 to blocking the PI3K/AKT pathway in BMSCs,and then used Western Blot to detect the expression of apoptosis-related proteins.BMSCs-IGF-1-si-SFRP2(BMSCs-IGF-1 cells with knocked out SFRP2 gene)were constructed by genetic engineering,and the proliferation and apoptosis of BMSCs-IGF-1-si-SFRP2 and BMSCs-IGF-1-siNC were detected respectively.Finally,in animal experiments,we established a model of acute MI by ligating the coronary arteries of SD rats,and transplanted about 1 × 106 BMSCs-IGF-1,BMSCs-NC or the same volume of solvent through the tail vein one week later.Three weeks after transplantation,cardiac Doppler ultrasound was used to evaluate the cardiac function,and histological analysis was performed to evaluate the cardiac fibrosis in each group.Western Blot was used to detect various proteins in the AKT/SFRP2 pathway in cardiac tissues to evaluate the role of IGF-1 in improving BMSCs transplantation for acute MI.Results1.Overexpression of IGF-1 protects BMSCs against hypoxia1.1 BMSCs have typical characteristics:small cell shape,which is triangular or spindle-shaped,and is characteristically adhered to the surface of the culture dish.The expression ratios of surface antigens CD73,CD 105 and CD90 detected by flow cytometry were 99.8%,97.0%,and 99.8%respectively,while only 0.55%of cells expressed CD45.BMSCs overexpressing IGF-1(BMSCs-IGF-1)were constructed by lentivirus.The level of IGF-1 in the medium secreted by BMSCs-IGF-1 was measured by ELISA,which was 4 times(95%confidence interval:2.52-6.35)higher than that of BMSCs-NC(empty vector).1.2 BMSCs-IGF-1 and BMSCs-NC were exposed to hypoxic environment for 48 h,then we detected cell viability by MTS assay.Under hypoxic conditions,the proliferation ability of BMSCs was significantly inhibited,but IGF-1 significantly improved this situation.TUNEL staining was used to evaluate the apoptosis of the cells under hypoxic conditions.IGF-1 reduced the number of hypoxia-induced apoptosis by 25%(95%confidence interval:11%-3%).Transwell assay was performed to evaluate the effect of IGF-1 overexpression on cell migration capacity and the results showed that under hypoxia,the number of BMSCs-IGF-1 cells migrated through the compartment was significantly higher than that of BMSCs-NC.1.3 Western blot experiments were performed to detect the expression of apoptosis-related proteins,including cleaved caspase-3,BAX,and BCL-2.Under hypoxic conditions,cleaved caspase-3 and BAX were increased in BMSCs-NC cells,and BCL-2 was decreased.Compared with BMSCs-NC,BMSCs-IGF-1 increased the expression of BCL-2,while decreased the levels of cleaved caspase-3 and BAX(P<0.01).Under hypoxic conditions,the expression of OCT4 and NANOG in BMSCs was decreased,while IGF-1 overexpression significantly restored its expression(P<0.01).2.Mechanisms of IGF-1 for improving BMSCs proliferation,migration and anti-apoptotic ability2.1 Western blot results showed that after 48 hours of culture under hypoxia and normoxic conditions,the expression of p-AKT and SFRP2 proteins in BMSCs-IGF-1 were higher than that in BMSCs-NC.The expression of p-AKT and SFRP2 in BMSCs decreased when exposed to hypoxia,but IGF-1 overexpression could reverse this situation(P<0.05).Hypoxia reduced total cell ?-catenin levels in BMSCs-IGF-1 and BMSCs-NC.However,the total cell ?-catenin levels in BMSCs-IGF-1 were higher than that in BMSCs-NC under both hypoxia and normoxic conditions(P<0.05).Under normoxic and hypoxia conditions,the expression of ?-catenin downstream proteins(e.g.cyclin D1 and c-Myc)in BMSCs-IGF-1 were higher than that in BMSCs-NC(P<0.05).Immunofluorescence staining showed that ?-catenin level in BMSCs-IGF-1 was significantly higher than that of BMSCs-NC under hypoxia and normoxic(P<0.001).Thees results indicated that IGF-1 may promote the expression of SFPR2 through the AKT signaling pathway,and further regulate the expression of?-catenin in BMSCs to play a biological role.2.2 In order to explore the role of the AKT pathway in the biological characteristics of BMSCs-IGF-1,we further used the AKT inhibitor LY294002 to block the PI3K/AKT pathway in BMSCs.We found that the proliferation and migration capabilities of BMSCs-IGF-1 were significantly suppressed by LY294002.The expression of SFRP2,?-catenin,c-Myc,and BCL-2 caused by overexpression of IGF-1,and the down regulation of BAX expression was attenuated by LY294002(all P<0.01).2.3 We further constructed the BMSCs-IGF-1-si-SFRP2 and BMSCs-IGF-1-siNC groups to explore the role of SFRP2 in improving the biological characteristics of BMSCs.The following five groups were set:normoxic+BMSCs-NC group;hypoxia+BMSCs-NC;hypoxia+BMSCs-IGF-1;hypoxia+BMSCs-IGF-1-si-SFRP2;hypoxia+BMSCs-IGF-1-siNC group.MTS assay showed that the cell viability of the BMSCs-IGF-1-si-SFRP2 group was significantly lower than that of the BMSCs-IGF-1-siNC group under hypoxic conditions(P<0.01).At the same time,the migration ability of SFRP2 group was significantly weaker than that of BMSCs-IGF-1-siNC group(P<0.01).Western blot showed that the levels of ?-catenin,cyclin D1,and c-Myc were lower in BMSCs-IGF-1-si-SFRP2 group than the BMSCs-IGF-1-siNC group.In addition,compared with the BMSCs-NC group,the BMSCs-IGF-1 group had higher BCL-2 levels and lower BAX levels,while BMSCs-IGF-1-si-SFRP2 significantly reversed this effect.3.BMSCs-IGF-1 protects H9C2 rat cardiomyocytes against hypoxia3.1 MTS detection of cell proliferation:compared with normoxic conditions,hypoxia significantly inhibited the proliferation of H9C2 cells(P<0.01).Co-culture with BMSCs-IGF-1 obviously restored the proliferation ability of H9C2 cells.3.2 Annexin V-FITC staining found that when H9C2 cells co-cultured with BMSCs-IGF-1,the number of hypoxia-induced apoptotic cells(14.52 ± 1.58%)was significantly less than that of the culture control group(35.2 ± 5.02%)(P<0.01);while co-cultured with BMSCs-NC,the number of apoptotic cells induced by hypoxia in H9C2 cells did not change significantly compare with BMSCs-IGF-1(22.33±2.29%).3.3 Western blot detection found that under hypoxic conditions,the expression of BCL-2 in H9C2 cells was decreased,while the expression of BAX and cleaved caspase-3 increased(P<0.01).When BMSCs-IGF-1 was co-cultured with H9C2 cells,the expression levels of cleaved caspase-3,BAX,and BCL-2 in H9C2 cells under hypoxia were similar to those in H9C2 cells under normoxia.In contrast,when BMSCs-NC and H9C2 cells were co-cultured under hypoxic conditions,the expression of BCL-2,BAX and cleaved caspase-3 in H9C2 were only slightly up-regulated.4.BMSCs-IGF-1 transplantation reduces infarct volume4.1 Compared with the control group,BMSCs-NC transplantation reduced the area of fibrosis after MI,and BMSCs-IGF-1 transplantation further reduced the area of myocardial fibrosis.4.2 TUNEL and IHC(immunohistochemistry)detection showed that the number of apoptotic cells in the myocardium of the BMSCs-IGF-1 transplantation group was lower than that of the BMSCs-NC group and the control group.At the same time,Western blot analysis revealed that the levels of cleaved caspase-3 and BAX were decreased(P<0.01)while the levels of BCL-2 increased in the infarcted myocardium(P<0.001).Conclusions:BMSCs-IGF-1 exhibited higher cell proliferation and migration ability,stronger stemness and resistance to apoptosis,and stronger protection of cardiomyocytes under hypoxia compared with BMSCs-NC.These effects are mediated by the AKT/SFRP2/?-catenin axis,and SFRP2 acts as an agonist rather than an antagonist of the canonical Wnt/?-catenin signaling pathway in this process.