The Mechanism Of Hypoxia Preconditioning Improving The Efficacy Of Exosomes Secreted From Mesenchymal Stem Cells On Myocardial Infarction

Myocardial infarction is a major hazard disease to human health. Cell transplantation is one of the most promising treatment for myocardial tissue repair, and how to improve the repair efficacy is the key issue. The therapeutic effect of transplanted cells is thought through secretion of stimulaing factors, which is named as paracrine effect. Nowadays, a great deal of evidence suggested that exosomes secreted from stem cells may play a critical role in paracrine effects. Our preliminary study has shown that exosomes derived from Hypoxia preconditioning (HP) mesenchymal stem cells (MSCs) own superior capability of angiogenesis and cytoprotection, which was associated with significantly upregulated expression of microRNA 210. While, It is still unknow that Hypoxia preconditioning through which way to modify the profile of exosomes. It is reported that nSMase2 may play an important role in controling the release of exosomes and the microRNA secretory mechanism. Our study showed that improved expression level of nSMase2 under hypoxia, and usage of GW4869, a inhibitor of nSMase2, can impair the biological function of exosomes derived from HP-MSCs, which was in accord with reduced miR-210 level in exosome. Our study focus on the effect of HP on exosomal profile derived from MSCs, Which will enhance our understanding of stem cell biology, enable us to refine cell-based therapeutic strategies, and develop new cell-free, exosome-based therapeutic reagent for the treatment of cardiovascular diseases.Objective:We performed this study to investigate the effect of HP on exosomes derived from MSCs and to explore the related mechanism. We aim to investigate the cardio-protective effects of exosomes derived from hypoxia preconditioning mesenchymal stem cells on MI, and to explore the underlying regulatory mechanism in delivery specific microRNA-210.Methods:Exosomes secreted from MSCs are purified from conditioned medium and identified by protein marker expression, transmission electron microscopy and nanoparticle tracking analysis. In vivo study, we divided it into four groups including Sham, PBS, exosomecontro1 and exosomeHP group. After myocardial infarction, exosomes or PBS are delivered intramyocardially into adult C57BL/6J mice at the border of an ischemic region following ligation of the left anterior descending coronary artery. Echocardiography was performed to evaluate cardiac function at baseline and then 3, 7,14 and 28 days after MI. We test the biological functionality of the microRNA-210 in H9C2 and HUVEC cells transfected with microRNA-210 mimic with Iipo3000.Results:This study first compares Exo-H-MSCs and Exo-N-MSCs in quantity of exosomes and hypoxic related microRNAs. We find Hypoxia condition dose not lead to a change in the amount of exosomes released from MSCs compared with Normoxia. In a mouse myocardial infarction model, Exo-H-MSCs have superior capacity of angiogenesis, anti-apoptosis, reducing fibrosis and improving cardiac function compared with other groups. In vitro studies, Exo-H-MSCs have more ability to enhance tube formation in human umbilical vein endothelial cells, and decrease apoptosis in primary cultured neonatal mouse cardiomyocytes (CMs) under oxidative stress, which are associated with significantly upregulated expression of microRNA 210. Transfected cells with microRNA-210 mimic have similar biological function as well as Exo-H-MSCs. In addition, the requirement for exosome biogenesis in the increased expression of secreted microRNA 210 is demonstrated. Hypoxia only increased the expression of sphingomyelinase (nSMase2) rather than Rab27a, which are two proteins crucial in exosome secretion. The expression of nSMase2 is also increased by adding DMOG (a stabilizer of Hif-la). nSMase2 can promotes angiogenesis and anti-apoptosis, while blocking the activity of nSMase2 by GW4869, a inhibitor of nSMase2, can abrogate the biological function of exosomes derived from Exo-H-MSCs, and resulted in reduced miR210 secretion after hypoxia pretreating.Conclusion:Our studies demonstrate that hypoxic-MSC derived exosomes provide more cardio-protective effects after MI by means of increasing exosomal miR210 transfer to recipient cells in a nSMase2-dependent secretory machinery way.