Bone Marrow Mesenchymal Stem Cells Overexpress ACE2 To Protect Cardiomyocytes And Endothelial Cells

Part 1 Mesenchymal stem cells with overexpression of angiotensin-converting enzyme 2 regulate RAS in cardiomyocytesBackground:Myocardial infarction is a multifactorial disease which has become a major cause for deaths worldwide.Mesenchymal stem cells(MSCs)have many characteristics,such as high expansion potential,ease of isolation,and the ability to differentiate into endothelial cells,cardiomyocytes and vascular smooth muscle cells,which makes them a suitable candidate for regenerative therapy for ischemic heart disease.Several studies have reported that MSCs can significantly attenuate ventricular remodeling and improve recovery of cardiac function after myocardial infarction.Recently,genetic modification and therapeutic agent incorporation techniques have been used to improve MSCs' innate properties to increase the therapeutic efficacy.The renin-angiotensin system(RAS),which involves the association of renin,angiotensin-converting enzyme(ACE),angiotensin II(Ang II)and AT1 receptor,has been demonstrated playing an essential role in the pathogenesis of myocardial infarction.Accumulating evidence suggests that Ang II plays a key role in cardiovascular pathology and remodeling.ACE2,the first homologue of ACE(a counter-regulatory enzyme of ACE),degrades Ang II into Ang 1-7 and mitigates the detrimental effects of Ang II.Therefore,the combination of MSCs and the protective gene ACE2 may be a potential strategy for the treatment of myocardial infarction.Objective: To test the hypothesis that MSCs overexpressing ACE2 may have further benefits in rescuing myocardial infarction,and investigate the mechanisms of it in vitro.Methods: MSCs were isolated and cultured,and then examined by Flow cytometry(FCM)analysis.MSCs were infected by lentivirus encoding recombinant enhanced green fluorescent protein(GFP)gene and ACE2 gene(Lv-GFP and Lv-GFP-ACE2).The transfection efficiency was determined by inverted fluorescence microscope,and then measured the cell viability by CCK-8 kits.The changes of RAS in ACE2-MSCs were determined by Real-time PCR;Specific ACE2 activity was examined by ACE2 fluorescence assay;Real-time PCR was used to detect the expression of RAS and NOS in H9c2 after co-culture with ACE2-MSCs.Ang ? concentration in the medium was tested by radioimmunoassay,and Ang-(1-7)level was determined by ELISA.We also tested the paracrine factors in the medium by proteome profiler array.Results: The efficiency of lentiviral vector transduction of MSCs was as high as 95% and was well maintained after passage.MSCs modified with ACE2 showed a sustained high expression of ACE2 m RNA and activity.The expression of AT1 R was down-regulated and Mas R was up-regulated.In vitro,the expression of ACE and AT1 R in H9c2 were decreased,while the expression of Mas R was increased after co-culture with ACE2-MSCs.Moreover,the expression of n NOS,e NOS in H9c2 were up-regulated.The modified MSCs reduced the concentration of Ang II and increased the production of Ang-(1-7).Proteome profiler array found that Angiopoietin-like3?HGF?IGFBP-1?IGFBP-6?Leptin?M-CSF have obvious changes.Conclusion: These data demonstrate that MSCs modified to overexpress the ACE2 gene can produce biologically active ACE2 protein and have an enhanced ability to rescue myocardial infarction.These benefits could be due to the inhibition of RAS,the degradation of Ang II and up-regulation of Ang-(1-7).In addition,ACE2-MSCs could regulate the NOS in cardiomyocytes.Part2 MSCs modified with ACE2 protect LPS-induced endothelial cell injuriesBackground: Although the effectiveness of MSCs transplantation on myocardial infarction has been confirmed,the effects on vascular restenosis after PCI remains controversial while the improvement of the cardiac function after myocardial infarction.Inflammation and leukocyte recruitment play a key role in the pathogenesis of restenosis.Therefore,we have hypothesized MSCs overexpressing ACE2 may possess anti-inflammatory activities and protect the function of endothelial cell,thereby preventing restenosis.It could provide meaningful cell-based experiments for clinically prevention and treatment of vascular restenosis after PCI.Objective: To investigate the effects and mechanisms of MSCs modified by ACE2 gene on LPS-induced endothelial cell injuries.Methods: The Real-time PCR was used to detect the expression of TNF-?,IL-6,IL-1? in ECs after treatment with LPS for different concentration,and the changes of that after co-culture with ACE2-MSCs.The effect of ACE2-MSCs on the viability of endothelial cells was measured using CCK-8 assay.Nitric oxide(NO)in the culture medium was measured using Griess method.The reactive oxygen species(ROS)were measured by DCFH-DA.The expression of NOS and Mas R in EC after co-culture with ACE2-MSCs was determined by Real-time PCR.We also tested the paracrine factors in the medium by proteome profiler array.Results: Treatment of ECs with 1000ng/ml of LPS for 24 hours increased the expression of TNF-?,IL-6,IL-1?.LPS(1000ng/ml)significantly decreased cell viability,increased NO secreation and ROS production.MSCs modified by ACE2 resulted in inhibition of such effects induced by LPS,while GFP-MSCs have no that protective effects.In addition,the activation of i NOS induced by LPS in ECs was inhibited by ACE2-MSCs,and the expression of e NOS,n NOS,Mas R was increased after co-culture with ACE2-MSCs.Proteome profiler array found that HGF ?ICAM-1?IGFBP-1?IGFBP-5?IL-6?IL-10?IL-11?Resistin?TIMP-1?TNF-??VEGF have obvious changes.Conclusion: MSCs overexpressing ACE2 inhibited LPS-induced inflammatory response.The mechanism underlying this protective effect might be related to the regulation of NOS and the improvement of Mas receptor.