Hypoxia treatment enhances paracrine effect of mesenchymal stem cells (MSCs). The aim of this study was to investigate whether exosomes from hypoxia-treated MSCs (ExoH) are superior to those from normoxia-treated MSCs (ExoN) for myocardial repair. Mouse bone marrow-derived MSCs were cultured under hypoxia or normoxia for 24 h, and exosomes from conditioned media were intramyocardially injected into infarcted heart of C57BL/6 mouse. ExoH resulted in significantly higher survival, smaller scar size and better cardiac functions recovery. ExoH conferred increased vascular density, lower cardiomyocytes (CMs) apoptosis, reduced fibrosis and increased recruitment of cardiac progenitor cells in the infarcted heart relative to ExoN. MicroRNA analysis revealed significantly higher levels of microRNA-210 (miR-210) in ExoH compared with ExoN. Transfection of a miR-210 mimic into endothelial cells (ECs) and CMs conferred similar biological effects as ExoH. Hypoxia treatment of MSCs increased the expression of neutral sphingomyelinase 2 (nSMase2) which is crucial for exosome secretion. Blocking the activity of nSMase2 resulted in reduced miR-210 secretion and abrogated the beneficial effects of ExoH. In conclusion, hypoxic culture augments miR-210 and nSMase2 activities in MSCs and their secreted exosomes, and this is responsible at least in part for the enhanced cardioprotective actions of exosomes derived from hypoxia-treated cells.

Background: Age and other cardiovascular risk factors have been reported to impair the activities of mesenchymal stem cells (MSCs), which will affect the efficacy of stem cell transplantation. The objective of the study is to investigate whether exosomes derived from human umbilical cord MSCs (UMSCs) could enhance the activities of bone marrow MSCs from old person (OMSCs), and improve their capacity for cardiac repair. Methods: Exosomes extracted from conditioned medium of UMSCs were used to treat OMSCs to generate OMSCs Exo. The key molecule in the exosomes that have potential to rejuvenate aged MSCs were screened, and the role of OMSC was tested in the mouse model of mycardial infarction (MI). Results: We found the activity of senescence-associated β-galactosidase and the expression of aging-related factors such as p53, p21, and p16 were significantly higher in OMSCs than those in UMSCs. After treatment with UMSC exosomes, these senescence phenotypes of OMSCs were remarkably reduced. The proliferation, migration, differentiation, and anti-apoptotic and paracrine effect were increased in OMSCs Exo. In vivo study, mice with cardiac infarction had significantly better cardiac function, less fibrosis, and more angiogenesis after they were injected with OMSCs Exo as compared with those with OMSC. There was more miR-136 expression in UMSCs and OMSCs Exo than in OMSCs. Upregulation of miR-136 by transfection of miR-136 mimic into OMSCs significantly attenuated the apoptosis and senescence of OMSCs. Apoptotic peptidase activating factor (Apaf1) was found to be the downstream gene that is negatively regulated by miR-136 via directly targeting at its 3′UTR. Conclusion: Our data suggest that exosomes from young MSCs can improve activities of aged MSCs and enhance their function for myocardial repair by transferring exosomal miR-136 and downregulating Apaf1.

Objective The efficiency of cell therapy is limited by poor cell survival and engraftment. Here we studied the effect of the growth hormone-releasing hormone agonist, JI-34, on mesenchymal stem cells (MSCs) survival and angiogenic therapy in a mouse model of critical limb ischemia. Approach and Results Mouse bone marrow-derived MSCs were incubated with or without 10−8 mol/L JI-34 for 24 hours. MSCs were then exposed to hypoxia and serum deprivation to detect the effect of preconditioning on cell apoptosis, migration and tube formation. For in vivo, critical limb ischemia was induced by femoral artery ligation. After surgery, mice were received 50μl phosphate buffer saline or with 1×106 MSCs or with 1×106 JI-34 preconditioned MSCs. Treatment of MSCs with JI-34 improved MSCs viability and mobility and markedly enhanced their capability to promote endothelial tube formation in vitro. These effects were paralleled by increased phosphorylation and nuclear translocation of STAT3. In vivo, JI-34 pre-treatment enhanced the engraftment of MSCs into ischemic hindlimb muscles and augmented reperfusion and limb salvage compared with untreated MSCs. Significantly more vasculature and proliferating CD31+ and CD34+ cells were detected in ischemic muscles that received MSCs treated with JI-34. Conclusions Our studies demonstrate a novel role for JI-34 to markedly improve therapeutic angiogenesis in hindlimb ischemia by increasing the viability and mobility of MSCs. These findings support additional studies to explore the full potential of Growth hormone-releasing hormone agonists to augment cell therapy in the management of ischemia.

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