| Purpose:Myocardial remodeling is the structural basis for the progression of various cardiovascular diseases to heart failure,and its subsequent fatality rate is still high.Myocardial remodeling is usually caused by changes in structure and function caused by myocardial cell necrosis,functional decline and decrease,and a large amount of collagen fiber deposition in the intercellular matrix.It is urgent to find an effective prevention and treatment method.In recent years,scholars have found that stem cell therapy has a positive effect on the prevention and treatment of myocardial remodeling.Human amniotic epithelial cells(hAECs)are characterized by differentiating ability as embryonic stem cells and immunomodulatory properties as adult stem cells.The hAECs have been considered as ideal seeding cells in regenerative medicine since they have been widely used in preclinical studies of various diseases.However,the role of hAECs in myocardial remodeling remains unclear.The purpose of this study was to explore the protective effect of hAECs on myocardial remodeling and its mechanism.Method:1.Isolation,culture and identification of hAECsThe amniotic layer was removed from the fresh placenta,and digested by trypsin.The hAECs were isolated and cultured in a serum-free medium until passage 3.The properties of hAECs were identified by flow cytometry,immunofluorescence,intracellular and extracellular tumorigenicity,and osteogenic differentiation.2.Establishment of myocardial remodeling model by TAC and transplantation of hAECsC57BL/6 male mouse at 8-10 weeks were divided into a control group(sham operation group),a TAC model group,and an AEC treatment group,with 8-10 mice in each group.The myocardial remodeling model was constructed by aortic arch constriction(TAC).One week later,the ultrasound imaging system was used to detect the peak velocity of blood flow of the aorta at the constriction site,and the sharp increase in the peak velocity of the aorta was regarded as a sign of successful surgery.Two weeks after modeling,hAECs were injected into mice through the caudal vein at8×10~5 cells/200μL and followed by once a week for 5 weeks.3.Cardiac function was detected by the ultrasound imaging systemAfter 8 weeks of modeling,the images of the long and short axis section of the mouse heart and the aortic arch were collected using the small animal ultrasound imaging system,and the cardiac function of the mouse was evaluated by analyzing the ultrasonic data.4.HE and Masson stainingEight weeks after modeling,mouse heart tissue was collected for fixation,dehydration,embedding,and sectioning.The degree of myocardial fibrosis was evaluated by HE and Masson staining.5.Measurement of cell apoptosis and vascular distribution in cardiac tissueThe expressions of Bcl2 and Bax in heart tissue were detected by immun-ohistochemistry;blood vessel distribution was detected by tissue immunofluorescence.6.Measurements of cell viability,apoptosis,migration,and tube formation of human umbilical vein endothelial cellsThe injury models of myocardial cells(H9C2)and human umbilical vein endothelial cells(HUVEC)were constructed under hypoxia and serum deprivation(H/SD).The cell viability of H9C2 and HUVEC cells was detected by CCK8;a mitochondrial membrane potential kit was used to detect the apoptosis of H9C2 and HUVEC cells;the migration and tube formation of HUVEC were detected by scratch and matrigel assay.Results:1.Properties of hAECsThe hAECs were isolated from amniotic tissue,and after subculture,hAECs were in typical cobblestone shapes under the inverted microscope;differentiating ability of hAECs was confirmed by osteogenic differentiation;hAECs were expressing CD29,CD90,and CD73 was,and CD105,but did not express CD34,CD45,and HLA-DR with flow cytometry;cell immunofluorescence assay hAECs were expressing OCT4and E-cadherin via cellular immunofluorescence assay;no tumorigenicity of hAECs was proved by tumorigenicity tests in vitro and in vivo.2.The cardiac remodeling was successfully constructed in mice via TACThe peak velocity of blood flow was increased dramatically at the constricted part of the cardiac aortic arch by the ultrasound imaging system one week after the modeling,which was evidence of successful cardiac remodeling via surgeries.3.hAECs attenuated myocardial remodeling and improved cardiac functionAfter 8 weeks of modeling,cardiac tissue was collected from mice,the results showed that the AEC group could effectively reduce the degree of cardiac hypertrophy compared with the TAC model group.The ultrasonographic results showed that the left ventricular dilatation in the AEC group was significantly decreased compared with that in the TAC group under M mode,and LVEF and LVFS in the AEC group were significantly higher than those in the TAC group;LV mass,LVAWd,LVPWs,LVPWd,LVIDs and LVIDd were decreased significantly in AEC group at end of 8 weeks of remodeling.The results of HE staining showed that the degrees of hypertrophy and disorder of myocardial cells was significantly decreased in the AEC group compared with those in the TAC group.The results of Masson staining showed that the AEC group could effectively reduce the area of myocardial fibrosis compared with the TAC group.In addition,the results of Western blot and Q-PCR showed that the expressions of Vimentin and ANP proteins in the AEC group were significantly decreased compared with those in the TAC group,and the expressions of COL1,COL3,Vimentin,BNP,and ANP genes were also significantly decreased.4.hAECs alleviated apoptosis of cardiomyocytes in remodeled myocardiumImmunohistochemical analysis showed that the AEC group could effectively reduce myocardial cell apoptosis compared with the TAC group.In vitro H/SD conditions,the results of the mitochondrial membrane potential kit and CCK8 kit showed that the AEC cell secretions(H/SD-CM)could significantly improve the activity of H9C2 cells and reduce the H/SD-induced apoptosis.5.hAECs promote the formation of blood vessels in myocardial remodeled tissueThe results of tissue immunofluorescence showed that the number of blood vessel formations in the AEC group was significantly higher than that in the TAC group.In vitro H/SD conditions,the results of the mitochondrial membrane potential kit and CCK8 kit showed that CM from hAECs significantly increased the activity of HUVEC cells and reduced the apoptosis of HUVEC cells,and the matrigel assay results showed that the number of tube formation was less in the H/SD group,but increased significantly in the H/SD-CM group;scratch test results showed that CM from hAECs significantly increased the migration rate of HUVEC cells under H/SD condition.Conclusion:hAECs have a protective effect on myocardial remodeling in mouse,that is,hAECs can significantly improve cardiac function and reduce the formation of myocardial fibrosis via reducing the apoptosis of myocardial cells and promoting angiogenesis. |
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