Author + information
Stem cell-derived exosomes have been shown to have therapeutic effects on cardiac regeneration. Since hypoxia could modulate mesenchymal stem cells to have better paracrine effect, we investigated whether exosomes derived from hypoxia-treated MSCs (ExoH) have superior properties than those from normoxia-treated MSCs (ExoN) for myocardial repair.
Mouse bone marrow-derived MSCs were cultured under hypoxia or normoxia for 24 hours, and the conditioned media were collected for exosome purification by ultracentrifugation. Exosomes were identified by protein marker expression, transmission electron microscopy and nanoparticle tracking analysis. In vivo study, we divided mice into four groups including Sham, PBS, ExoH, and ExoN group. Exosomes and 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 3,7,14 and 28 days after myocardial infarction (MI). Heart sections were stained with picrosirius red staining for fibrosis quantification. Immunofluorescence staining with endothelial marker (CD31) and artery marker (aSMA) reflected angiogenesis in the peri-infarct areas of hearts at day 28 after MI. We also evaluated proliferation of endogenous Sca-1+cardiac progenitor cells (CPCs) after MI 7days with marker Ki67. For in vitro, we took tube formation in human umbilical vein endothelial cells (HUVEC) for detection angiogenesis and done TUNEL and AnnexinV-PI for apoptosis in neonatal mouse cardiomyocytes. In addition, miRNA array was done to detect the hypoxia-specific microRNA in the exosomes. We test the biological functionality of the microRNA-210 in H9C2 and HUVEC cells through transfected with microRNA-210 mimic or siRNA210.
ExoH had significantly better effects on promoting endothelial cells for tube formation and protecting cardiomyocytes from apoptosis under oxidative stress in vitro than ExoN. Intramyocardial injection of ExoH into the infarcted heart of C57BL/6 mouse resulted in significantly higher mice survival rate, smaller scar size and better recovery of cardiac functions (n=24 per group). In comparison with ExoN treatment, ExoH had superior capability to increase vascular density, decrease cardiomyocyte apoptosis, reduce fibrosis, recruit cardiac progenitor cells, and enhance cell proliferation in the infarcted heart. MicroRNA array of exosomes showed significantly higher level of microRNA-210 (miR-210) in ExoH. Transfection of endothelial cells and cardiomyocytes with miR-210 mimic also resulted in similar biological effects as ExoH did, whereas inhibiting miR-210 in MSCs diminished the superior pro-angiogenesis and anti-apoptosis effects of thereafter derived ExoH. Hypoxia treatment of MSCs increased the cellular expression of neutral sphingomyelinase 2 (nSMase2) which is crucial for exosome secretion. The expression of nSMase2 is also increased by adding DMOG (a stabilizer of Hif-1a) to MSCs under hypoxia condition. Blocking the activity of nSMase2 by inhibitor GW4869 resulted in reduced miR-210 secretion after hypoxia treatment and abrogated the beneficial effects of ExoH.
Hypoxic culture augments miR-210 and nSMase2 activities in recipient 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.