Therapeutic Effects And Mechanisms Of Human Amniotic Mesenchymal Stem Cells On Ventricular Remodeling In Mice

Objective:Ventricular remodeling is an adaptive response after heart injury and often leads to left ventricular dysfunction and develops end-stage heart failure,which is a pathophysiological process followed by damage repair and overall ventricular compensation,including myocardial hypertrophy,cardiomyocyte apoptosis,myocardial fibrosis,inflammatory response,and vascular remodeling.In recent years,an increased number of drugs have been used to prevent and treat ventricular remodeling,however,the morbidity and mortality rates of ventricular remodeling remain high,so there is an urgent need to find a more effective prevention and treatment method.Stem cell therapy has become a research hotspot for the treatment of a variety of diseases,human amniotic mesenchymal stem cells(hAMSCs)are the kind of stem cells with multilineage differentiation,high proliferation,non-tumorigenic,anti-inflammatory properties and low immunogenicity and other unique advantages.It has been shown that mesenchymal stem cells possess therapeutic effects for cardiovascular diseases(such as myocardial infarction,and atherosclerosis).Do hAMSCs have a preventive or therapeutic effect on ventricular remodeling?This project will explore the role and possible mechanism of hAMSCs in the treatment of ventricle remodeling,aiming to provide a feasible theoretical and experimental basis for cell therapy in patients with clinical ventricular remodeling.Methods:1.Isolation,culture and identification of hAMSCs:1)hAMSCs were isolated by enzymatic digestion,and routinely cultured to P3 generation for cell identification;2)Flow cytometry to detect surface antigens associated with hAMSCs;3)Cellular immunofluorescence to detect whether hAMSCs express embryonic stem cell markers;4)The ability of in vitro osteogenic differentiation induction experiments to detect multidirectional differentiation of hAMSCs;5)Severe immunodeficient mice(M-NSG)in vivo transplantation cell experiments and soft agar colony formation experiments to detect the tumorigenicity of hAMSCs in vivo and vitro.2.The effect of hAMSCs transplantation on ventricular remodeling:1)Preparation of ventricular remodeling model:a mouse ventricular remodeling model was constructed by subcutaneous injection of 40mg/kg isoproterenol(ISO)per day;2)Transplantation of hAMSCs:7 days after ISO injection,tail vein transplantation hAMSCs entered the ventricle remodeling mice(cell quantity:8×10~5cells/piece),transplanted once every 7 days,a total of 3 transplants;3)Mouse heart morphology and function detection:small animal echocardiograph detected the changes of cardiac function before and after molding of mice in each group(normal saline group,ISO group,ISO+hAMSCs group);Heart tissue samples are collected,cardiac weight ratio is measured,and blood cell analyzers measure blood routine;4)Cardiac histology detection:HE and Mason staining of heart tissue to detect myocardial tissue structure changes and the degree of fibrosis;5)Myocardial injury and fibrosis related index detection:Western blot and RT-q PCR experiments detect ANP,BNP and collagen III;6)Heart tissue vascular density detection:use immunohistochemistry to observe the change of the number of blood vessels in heart tissue;7)Migration of hAMSCs:tail vein transplantation of carboxylfluorescein diacetate succinimidyl ester(CFSE)-labeled hAMSCs into mice,fluorescence microscopy to observe the colonization ability of hAMSCs in heart tissue after 1 and 3 days of transplantation;Cardiac tissue paraffin sections were subjected to CD90 and MAB1281 dual immunofluorescence to detect whether hAMSCs transdifferentiated into other functionally impaired cells.3.Effects of hAMSCs on macrophages:1)Macrophage infiltration detection:tissue immunofluorescence detection of CD206 expression level in cardiac tissue;2)Macrophage polarization detection:detection of protein and m RNA expression levels of CD206,CD86,IL-10 in cardiac tissue by Western blot and RT-q PCR;3)In vivo experiment RAW264.7 macrophage polarization detection:In in vitro experiments,RAW264.7 macrophages were induced to polarize to M1 type by ISO,and then hAMSCs-CM was co-incubated with RAW264.7 to detect the expression level of i NOS and Arg-1 in cells.4.Effects of macrophage secretion factors on hAMSCs:1)hAMSCs anti-inflammatory gene detection:The experiment was divided into two groups,ISO+RAW264.7-treated hAMSCs,and untreated hAMSCs,the total RNA and conditioned medium were collected,and RT-q PCR experiments were performed to detect the levels of hAMSCs expressing IDO,IL-10,and COX2;2)Construction of in vitro co-incubation system:The conditioned medium was co-incubated with 3T3fibroblasts to detect the effect of hAMSCs’secretions on 3T3 cell activation.The experiment was divided into 4 groups including the 3T3 control group;ISO+RAW→3T3 model group;ISO+RAW→3T3+hAMSCs-CM treatment group 1;ISO+RAW→3T3+hAMSCs-CM←ISO+RAW treatment group 2;3)Fibroblast activation detection:RT-q PCR experiments observed the expression ofαSMA and COLIII.in each group.Results:1.Identification of hAMSCs:1)hAMSCs were successfully isolated by enzymatic digestion with typical fibroblastic form;2)Flow cytometry experiments showed that hAMSCs were highly expressed mesenchymal stem cell markers:CD90,CD73,CD44,do not express markers of hematopoietic stem cell and histo-compatibility class I antigens:CD34,CD45,and HLA-ABC;3)Cellular immunofluorescence detection showed that hAMSCs express embryonic stem cell markers:OCT-4,SSEA-4;4)osteogenic differentiation experiments showed that hAMSCs could induce differentiation into osteocytes in vitro;1)oncogenic experiments found that hAMSCs were not tumorigenic in vitro and vivo.2.hAMSCs transplantation attenuated ventricular remodeling in mice and improved cardiac function:1)Myocardial remodeling including hypertrophy,and myocardial fibrosis was induced by subcutaneous injection of ISO,and it decreased cardiac function in mice;2)Echocardiography showed that hAMSCs could reduce ventricular dilation and enhance cardiac function;hAMSCs transplantation reduced the ratio of heart and body weight and decrease the number of white blood cells,and neutrophils in the blood;3)The results of HE and Masson staining showed that transplantation of hAMSCs improved myocardial tissue arrangement and reduced the degree of fibrosis;4)Western blot and RT-q PCR results found that hAMSCs reduced the expression levels of ANP,BNP and COLIII in ventricular remodeling tissues;5)Tissue immunohistochemical detection showed that the CD31-positive cells were increased after hAMSCs were transplanted,which suggested the increased blood vessels in the remodeling heart;6)Tissue immunofluorescence detection showed that hAMSCs were migrated in small amounts in the hearts of ventricle remodeled mice,and hAMSCs were migrated in the heart and did not transdifferentiate into other damaged cells.3.hAMSCs and their secretions(hAMSCs-CM)promoted M2 macrophage polarization:1)Tissue immunofluorescence detection found that hAMSCs increased the expression level of CD206 in cardiac tissues and promoted polarization to M2-type macrophages;2)hAMSCs reduced CD86 expressions but increased the expressions of CD206 and IL-10 in the remodeling heart tissues;3)in vitro experiments,hAMSCs-CM co-incubated with RAW264.7 reduced i NOS and increased the expression level of Arg-1 of the macrophages.4.The secretions from macrophage enhanced the paracrine effect of hAMSCs and inhibit fibroblast activation:1)hAMSCs induced by the supernatants of ISO+RAW264.7 express higher levels of anti-inflammatory genes IDO,IL-10,and COX2 than untreated hAMSCs;2)ISO+RAW264.7 cell supernatant-induced hAMSCs-CM inhibited activation of 3T3 cells with fewer amount expressions ofαSMA and COLIII than that of the uninduced hAMSCs-CM group.The results showed that macrophage secretion factors significantly increased the paracrine effect of hAMSCs,which in turn inhibited fibroblast activation.Conclusion:Transplantation hAMSCs reduce ISO-induced myocardial hypertrophy,and cardiac fibrosis,improve cardiac function,increase the number of blood vessels,and prevent the occurrence and development of ventricular remodeling in mice.The protective role of hAMSCs in ventricular remodeling might be through its paracrine effects,via increasing macrophage M2 polarization and its anti-inflammatory effect.In addition,macrophages might enhance the paracrine effect of hAMSCs via their secreted cytokines.