Bone Marrow Mesenchymal Stem Cell-derived Exosomes Attenuates Pressure Overload Induced Cardiac Remodeling In Vivo And Vitro

Background: There is an increasing prevalence of heart failure worldwide,causing enormous economic cost and loss of lives.One of the leading causes of heart failure is pathological cardiac remodeling.The molecular basis of cardiac remodeling includes the process of cardiac hypertrophy,myocyte loss and fibrosis,which have several detrimental overlapping effects affecting cardiac structure and function at multiple levels.Over the last two decades,stem cell therapy has emerged as a new strategy for heart failure.Mesenchymal stem cells(MSC)are among the most promising cell types due to their multiple biological functions such as pluripotent differentiation,anti-inflammation,immune regulation,neuroprotection and so on.Accumulating evidences suggest that MSC exert their therapeutic effects mainly through paracrine mechanisms like secreting exosomes,rather than a direct manner.Exosomes are extracellular vesicles of endosomal origin which have emerged as key mediators of intercellular communication.They are classified to be originating from endosomal compartment or the microvesicles of cellular membrane,which are 30–100 nm in size and enriched in proteins,messenger RNAs,and micro RNAs.It has been reported that MSC-derived exosomes can enhance myocardial viability and prevent adverse remodeling both in myocardial ischemia and ischemic/reperfusion models.However,studies on the precise role and therapeutic potential of MSC-derived exosomes in cardiac hypertrophy are still lacking.Here,we hypothesized that MSC-derived exosomes would regulate pathological hypertrophy,protect cardiomyocytes from apoptosis and retard the progression of fibrosis as well.Methods: Exosomes collected from the conditioned media of murine bone marrow mesenchymal stem cell were isolated using ultracentrifugation method.Wild-type C57BL/6 male mice were subjected to transverse aortic constriction to induce cardiomyocyte hypertrophy,and myocardium was injected with either phosphatebuffered saline(PBS,control),or MSC-derived exosomes.After 6 weeks,the hearts were harvested and performed for histopathological analysis,cardiac fibrosis and cell apoptosis measurements.Neonatal rat ventricular cardiomyocytes and myofibroblasts were exposed by angiotensin Ⅱ or co-treatment with MSC-derived exosomes in vitro.Three groups were designed:(1)the control group: Dulbecco’s modified Eagle’s medium(DMEM)treatment for 48 hours;(2)the Angiotensin Ⅱ(AngⅡ)group: 10μmol/LAngⅡ treatment for 48 hours;(3)the AngⅡ+ exosomes group:10 μmol/LAngⅡ and 10μg/L exosomes treatment for 48 hours.Results: Most of the exosomes derived from MSC conditioned media presented membrane vesicles,with typical cup-shaped morphology and within 100 nm in diameter,which demonstrated that MSC-derived exosomes were successfully purified in our experiments.The mice received MSC exosomes exhibited increased LVEF,FS and decreased septal and LV wall thickness,as well as decreased LV dimension at6 week compared with PBS injection.In addition,MSC-derived exosomes significantly reduce myocardial fibrosis and apoptosis,attenuate cardiac hypertrophy after transverse aortic constriction.Similar results were obtained in neonatal rat ventricular cardiomyocytes stimulated with angiotensin Ⅱ.AngⅡ increased cell surface area,whereas co-treatment with exosomes blocked this effect.Exosomes also lowered the expression of hypertrophic markers such as ANP and β-MHC,which are respectively up-regulated by AngⅡ,suggesting that MSC exosomes restored cardiomyocyte phenotype under hypertrophic stimuli.We also found that when exposed to angiotensin Ⅱ in vitro,co-treatment with MSC-derived exosomes may induce premature cellular senescence of cardiofibroblasts,suppress the activities and reduce fibrosis,which can be a potential target for antifibrotic therapies.Conclusions: In this study,we firstly found that mesenchymal stem cells derived exosomes could preserve myocardial structure,reduce myocardial fibrosis and apoptosis,attenuate cardiac hypertrophy and improve cardiac function in a mouse pressure overload model.The results obtained from the in vivo experiments were further validated in vitro.Exosomes protected cardiomyocytes from AngⅡ-induced pathological hypertrophy,and increased premature cellular senescence in myofibroblasts when exposed to AngⅡ with MSC exosomes in vitro.We revealed that mice bone marrow MSC-derived exosomes played a critical role in suppressing cardiac hypertrophy,apoptosis and fibrosis in pressure overload induced remodeling,which may provide a new therapeutic approach for pressure overload heart failure.