Epicardial Transplantation Of Hypoxic Preconditioning Human Adipose-derived Stem Cells Treats Acute Myocardial Infarction In Swines

Objective:As a new seed cells used to treat acute myocardial infarction (AMI), the characteristic of adipose-derived stem cells (ADSCs) is easy acquirement, great quantity and multiple differentiation. Although the transplantation of ADSCs in AMI has achieved great success, the harsh and complicated microenvironment of AMI affects the survival of ADSCs and limits the therapeutic effect of MI. Hypoxic preconditioning could enhance the anti-apoptosis and paracrine ability of ADSCs. Therefore, in this study, we apply the method of hypoxic preconditioning to culture human ADSCs (hADSCs) and observe the change of anti-apoptosis and cytokine secretion. Meanwhile, we eject the hADSCs and hypoxic preconditioning human adipose-derived stem cells (HP-hADSCs) to the Chinese minipig model of AMI epicardially, compare the difference between hADSCs and HP-hADSCs transplantation and discuss the mechanisms of cell therapy in MI.Methods:The hADSCs obtained by liposuction were cultured and expanded to passage3to5. According to the different oxygen concentration, hADSCs were cultured and divide into5groups:Group A (1%O2), Group B (3%O2), Group C (5%O2), Group D (10%O2) and Group E (21%O2). After24hours, FCSA, RT-qPCR, ELISA, Western Blot and Annexin V-FITC/PI were used to analysis the phenotype, gene expression and protein secretion of cytokine and the capacity of anti-apoptosis in hADSCs, the hADSCs cultured in the optimum hypoxic environment were named as HP-hADSCs. The mid-third of LAD was ligated surgically to establish minipig AMI model. The minipigs were randomly divided into three groups (n=8in each): Group A (0.9%NaCl10ml injection), Group B (hADSCs transplantation) and Group C (HP-hADSCs transplantation). A total of1x108hADSCs or HP-hADSCs were injected into the peri-MI tissue with8-10different points. Before operation, after operation immediately and six weeks later, cardiac perfusion and function were evaluated by SPECT and echocardiography in each group. The animals were euthanized and tissue in peri-MI area were analyzed for the engraftment, proliferation of stem cells, angiogensis and myocardium apoptosis by HE&Masson, immunofluorescence and TUNEL staining. Results:(1) The phenotype of hADSCs in CD71, CD73, CD90and CD105were positive and CD34, CD45, CD54and HLA-DR were negative. RT-qPCR indicated that the mRNA expression of HGF and VEGF increased with the decreasement of oxygen concentration and it was statistically significant in Group A (P<0.05), however, there was no significant change in NGF and KGF (P>0.05). ELISA shown that hypoxic preconditioning could enhance the protein VEGF and HGF secration of hADSCs significantly. In Group B, the HGF secration was2613.33±180.37ng/L and the VEGF was2856.08±114.63ng/L, there was no significant in KGF and NGF (P>0.05).(2) Comparison with the other groups, the mRNA expression of Bax and BNip3increased in Group A, so did the mRNA expression of Bcl-2in Group B. Western Blot demonstrated that the HIF-la, Bax and BNip3protein secration were highest in Group A, so was the Bcl-2proterin in Group B. hADSCs in each group were cultured in an anaerobic and serum-deprivation condition, after12hours, Annexin V FITC/PI indicated that the survival rate of hADSCs in Group A, Group B and Group C was high, although there was no statistically siginificant among them, it was highest in Group B. Combined with the results of cytokine protein and mRNA in each group, we considered that3%O2hypoxic condition was the optimum for hADSCs culture and the Group B hADSCs was named as HP-hADSCs.(3) Six weeks later, the changes in MDP were significantly improved in hADSCs and HP-hADSCs transplantation groups and it was the most obvious in HP-hADSCs group (P<0.01). The echocardiography indicated that there was no significant difference abour LVEF and IWT among these groups (P<0.05), but the LVEDV and LVESVwere improved in HP-hADSCs groups (P<0.05). HE&Masson staining shown that there was less fibrosis in hADSCs and HP-hADSCs transplantation groups and it was obviously reduction in later group.(4) Six weeks later, Ki67and β-microglobin were positive in HP-hADSCs group, it indicated that HP-hADSCs engrafted and prolifered in the peri-MI tissue, however, there were stem cells in hADSCs group only. As a marker of vascular endothelial cells, Isolactin B4was positive in HP-hADSCs and hADSCs groups and the vascular tissue shape was clearly visible in HP-hADSCs group. TUNEL immunofluorescence staining shown that the apoptosis of cardiomyocytes in the HP-hADSCs and hADSCs transplantation groups were reduced and it was more significant in the HP-hADSCs group (P<0.01).Conclusions:(1) Hypoxic preconditioning is an effective method to enhance the ability of anti-apoptosis and paracrine for hADSCs, however, immoderate oxygen concentration (<3%O2) was not suitable to survive for hADSCs.(2) hADSCs cultured under3%O224hours, the cytokine protein secration and anti-apoptosis capacity of hADSCs could be enhanced.(3) Compared with hADSCs transplantation group, the method of intramyocardial injection of HP-hADSCs could reduce the size of MI, attenuate myocardial fibrosis, maintain the cardiac function and inhibit ventricular remodeling effectively.(4) HP-hADSCs could survive and proliferate in peri-MI tissue, reduce the cell apoptosis and improve angiogenesis by paracrine effect.