Effect Of Local RAS On Myocardial Ischemia Protection Of Bone Marrow Mesenchymal Stem Cells

Part ?:The influence of Ang ? pretreatment on MSC-based therapy for ischemic heart diseaseBackground: Pretreatment of mesenchymal stem cells(MSCs)with growth factors is reported to be an effective route for improving cell-based therapy of myocardial infarction(MI).Angiotensin ?(Ang ?)triggers vascular endothelial growth factor(VEGF)synthesis in MSCs.This study aimed to investigate the effects and mechanisms of Ang ? pretreatment in enhancing the therapeutic efficacy of MSCsin MI.Methods: MSCs and endothelial cells(ECs)were isolated from Sprague–Dawley(SD)rats.After pretreated with or without 100 n M Ang ? for 24 h,the MSCs were directly injected into the border zones of ischemic heart.Cardiac function,fibrosis,infarct size,VEGFexpression,angiogenesis,and cell differentiation in infarcted myocardiumwere determined after 30 d.The cell apoptosis of MSCs post hypoxia was assessed using flow cytometry.The angiogenic activity of MSCs was analyzed using tube formation assay.Thegap junction protein connexin-43(Cx43)expression was detected.Results: Compared withMSC group,pretreatment of MSCs with Ang ? resulted in better cardiac function,less cardiac fibrosis,smaller infarct size,and higher expression of VEGF and Von Willebrand Factor in ischemic myocardium,but no promotion of cardiomyocyte-like differentiation of MSCs.Ang ? pretreatment enhanced the survival of MSCs and the H9c2 cells surrounding MSCs,andaugmented the tube formation of ECs and MSCs.Ang ? pretreatment up-regulated Cx43 expression.Conclusions:The pretreatment of MSCs with Ang ? improved the outcome of MSC-based therapy for MI via the mechanisms of enhancing the paracrine production of VEGF,angiogenesis,and gap junction formation.Part ?:The influence of ACE2-overexpression on MSC-based therapy for ischemic heart diseaseBackground: MSC transplantation for the treatment of myocardial infarction is a viable therapeutic strategy,but its therapeutic effect does not last long.The bad local environment of the infarcted myocardiaum hindered the performance of MSCs.ACE2 is a new member of the RAS system discoved in the recent years,who can reverse ACE-activation indued heart injury.This research was aimed to study whether the transplantation of ACE2-overexpressed MSCs(LV-ACE2-MSCs)could improve myocardial microenvironment,and exert a long-term protective effect.Methods: MSCs and ECs were extracted from SD rats.The lentivirus infection of MSCs in a pettern of MOI=20 can achievean ideal tranfection effect.The expressions of ACE2 in MSCs were detected using RT-PCR and Western blot.The expressions of ACE2 in heart tissues were tested using immunohistochemical detection.Fluorescent activity assay kit was used for the detection of ACE2 activity in MSCs and in tissues.CCK-8 was used for MSC proliferation assay.Ang ? concentration was analyzed using radioimmunoassay kit,and Ang-(1-7)concentration was dected using ELISA kit.Rat heart function was analyzed using echocardiography.TTC staining was used to detect the myocardial infarction area.Sirius red staining was used to detect myocardial collagen deposition.Hoechst staining kit was used to detect the apoptosis of H9c2 cells.Tube formation assay was performed to test the angiogenesis activity of MSCs.The expression of RAS response component andits associated key proteins in MSCs were detected using Western blot.Cytokine array kit was used for detecting MSC paracrine growth factors.Swiftedgene expression information was obtained from high-throughput RNA sequencing.Results: the therapeutic effect of LV-ACE2-MSCs was better then LV-GFP-MSCsin the treatment of myocardial infarction,for the cardiac function was significantly improved,myocardial infarct size reduced,and local myocardial collagen deposition decreased.In vitro,MSCs itself has almost no expression of ace2 gene,and after lentivirus transfection,the expression of ACE2 was significantly increased and the activity was enhanced.Moreover,the synthesis of Ang-(1-7)increased significantly.The overexpression of ACE2 had weak inhibition effect on the proliferation of MSCs;however,enhancedthe tolerance to hypoxia of the co-cultured H9c2 cells.Among the RAS and the related key molecules in NO system of MSCs,ACE expression did not change significantly,the expression of Angiotensin ? type 1-receptor(AT1)decreased markedly;the expression of nitric oxide synthase(NOS)1 did not change,whereas the expression of NOS3 increased;STAT3 expression did not change significantly,the expression of VEGF was significantly decreased.Conclusions: MSCs is a kind of good cell gene vector.The LV-ACE2-MSCs played a protective role via converting Ang ? to the Ang-(1-7);at the same time,the overexpression of ACE2 also led to expression changes of many RAS componets and related key proteins in NO system,which had an impact on the biological functions of MSCs and the regional myocardial microenvironment.In summary,the innovation of this research includes that we reported,for the first time,the influence of Ang ? preconditioning on MSC-based therapy for MI;and for the first time,comfirmed that ace2-overexpressed MSCs could improve cardiac miocroenvironment and the therapeutic efficacy of MI.This research provides adequate evidence for clinical research and practice in MSC-based therapy for MI.