Study On The Functional Differences And Mechanism In Ischemic Myocardium Repair Bv Exosomes Derived From Various Mesenchymal Stem Cells

OBJECTIVE:This study was to compare the therapeutic effects of two MSC-derived exosomes(bone marrow mesenchymal stem cells(BMSC)and umbilical cord mesenchymal stem cells(UMSC))on acute myocardial infarction treatment.The proteomics analysis was used to investigate its molecular mechanism of functional differences.This study will provide a theoretical basis for the clinical transformation of MSC-derived exosomes in the treatment of ischemic heart disease.METHODS:At first,the frozen P3 BMSC were thawed and passaged.The collagenase-trypsin digestion method was used to isolate and culture UMSC.Stem cell related identification was carried out on P5 generation cells and further experiments were carried out.EXO was extracted and identified from BMSC and UMSC cultured supernatant fluid by differential overspeed centrifugation.In the second part,we compared the function differences in myocardium repair between two exosomes through experiments in vitro and vivo(Exosomes were transplanted into the rat models of acute myocardial infarction).In the third part,the proteins involved in EXO were extracted and analyzed by liquid chromatography-mass spectrometry.According to the analysis results,differentially expressed proteins were selected for verification in vitro.RESULTS:The inoculated P3 generation human BMSC were uniform in spindle-shaped,and proliferated rapidly after resuscitation.The cells fusion reached more than 90%in about 5 days.After the umbilical cord tissue was digested and adhered the culture dishes,the adherent fibroblast-like cells were observed under the microscope.The shape was polygonal or fusiform.The cell colonies fused to each other more than 80%after 3-week cultivation.The passaging cells growth rate is significantly faster than that of the primary cells.Flow cytometry detection of MSC cell phenotypes revealed that more than 95%of human MSC express CD44,CD90 and CD 105,and do not express CD34 and HLA-DR.The growth curve showed that the proliferation rate and quantity of P5 generation UMSCs were significantly higher than that of BMSC.Under the same induction conditions,both BMSC and UMSC could be induced to differentiate into osteoblasts,chondrocytes and adipocytes.BMSC was more likely to be induced to adipocytes and chondrocytes,while UMSC was more likely to be induced to osteoblasts.Observed under transmission electron microscopy,MSC-EXOs showed a typical "cup holder-like" structure with low-density bright areas in the cavity.Particle size analysis results showed the average particle size of EXO from BMSC is 131.5nm,and the average particle size of EXO from UMSC is 137.5nm.Western Blot results showed that both BMSC and UMSC-derived EXO were positive for CD63 and TSG101.In vitro:After H2O2-induced injury,flow cytometry results showed that MSC-derived EXO-treated H9C2 cells have reduced apoptosis.MSC-derived EXO could induce HUVEC cells to accelerate migration and form tube-like structures in vitro.MSC-derived EXO could inhibit fibroblast-to-myofibroblast transdifferentiation induced by TGF-? in vitro.UMSC-EXO was superior to BMSC-EXO in the above regulation abilities.In vivo:3 days after the injection,the resident rate of DiI-labeled UMSC-EXO is high than that of BMSC-EXO.The results of HE staining,macrophage polarization response,RT-qPCR,and Western Blot showed that MSC-derived EXO can reduce the inflammatory response,reduce fibrosis,accelerate angiogenesis,and reduce the degree of apoptosis in the ischemic area after MI with the observation of decreased inflammatory cell infiltration in the myocardial infarction area,decreased expression of inflammatory factors IL-6,TNF-?,I?Ba and NF-?B p65,decreased expression of apoptosis-related Bax,cleaved-Caspase3,and increased expression of Bc12.The expression of MMP-2/MMP-9 that related to myocardial fibrosis was decreased in the myocardial infarction area while the expression of eNOS and VEGF that related to angiogenesis increased.Sirius scarlet staining,Masson 's Trichrome staining,TUNEL staining,and CD3 1 staining showed that MSC-EXO inhibited myocardial interstitial fibrosis,inhibited myocardial cell apoptosis,and promoted new blood vessel formation after infarction.Color Doppler echocardiography results showed that MSC-EXO can improve cardiac function after acute myocardial infarction.The improvement degree of above indicators in the UMSC-EXO transplantation group were more significant than those of the BMSC-EXO transplantation group.624 quantifiable proteins in BMSC-EXO and 635 quantifiable proteins in UMSC-EXO were identified by Liquid chromatography-mass spectrometry analysis.The number of same proteins expressed by the two type of EXOs was 558.139 proteins were up-regulated in BMSC-EXO and 153 proteins were up-regulated in UMSC-EXO.The results of functional enrichment of differential proteins showed that the differentially up-regulated proteins in UMSC-EXO are more involved in cell adhesion,promoting cell proliferation and survival,anti-apoptosis,and inflammation regulation and participated in related signaling pathways more than BMSC-EXO,Significant differentially expressed protein PDGFC was screened for verification.The results showed that the expression of PDGFC in UMSC-EXO transfected with shRNA targeting PDGFC was decreased.After co-culture with HUVEC cells in vitro,the expression of p-AKT in HUVEC cells was decreased while the ability of HUVEC cells to form tubular structures was decreased.CONCLUSION:Both BMSC-EXO and UMSC-EXO can improve the antiapoptotic ability of cardiomyocytes,promote the proliferation and migration of endothelial cells and the formation of tubular structures,inhibit fibroblast-to-myofibroblast transdifferentiation induced by TGF-? in vitro.In vivo,MSC-EXO can reduce the local inflammatory response after acute myocardial infarction,improve the myocardial survival rate,reduce the degree of myocardial tissue fibrosis,promote the formation of new capillaries and improve the cardiac function.The overall effect of UMSC-EXO was better than that of MSC-EXO.The proteins contained in MSC-EXO play a certain role in the myocardial repair process after myocardial infarction,and the differences in the expression levels of differential proteins(such as PDGFC)may be one of the reasons for the difference therapeutic effects.