Abstract
MicroRNAs have been appreciated in various cellular functions, including the regulation of angiogenesis. Mesenchymal-stem-cells (MSCs) transplanted to the MI heart improve cardiac function through paracrine-mediated angiogenesis. However, whether microRNAs regulate MSC induced angiogenesis remains to be clarified. Using microRNA microarray analysis, we identified a microRNA expression profile in hypoxia-treated MSCs and observed that among all dysregulated microRNAs, microRNA-377 was decreased the most significantly. We also validated that vascular endothelial growth factor (VEGF) is a target of microRNA-377 using dual-luciferase reporter assay and Western-blotting. Knockdown of endogenous microRNA-377 promoted tube formation in human umbilical vein endothelial cells. We then engineered rat MSCs with lentiviral vectors to either overexpress microRNA-377 (MSCmiR-377) or knockdown microRNA-377 (MSCAnti-377) to investigate whether microRNA-377 regulated MSC-induced myocardial angiogenesis, using MSCs infected with lentiviral empty vector to serve as controls (MSCNull). Four weeks after implantation of the microRNA-engineered MSCs into the infarcted rat hearts, the vessel density was significantly increased in MSCAnti-377-hearts, and this was accompanied by reduced fibrosis and improved myocardial function as compared to controls. Adverse effects were observed in MSCmiR-377-treated hearts, including reduced vessel density, impaired myocardial function, and increased fibrosis in comparison with MSCNull-group. These findings indicate that hypoxia-responsive microRNA-377 directly targets VEGF in MSCs, and knockdown of endogenous microRNA-377 promotes MSC-induced angiogenesis in the infarcted myocardium. Thus, microRNA-377 may serve as a novel therapeutic target for stem cell-based treatment of ischemic heart disease.
Highlights
The formation of new blood vessels is critical for the repair of ischemic myocardium, and vascular endothelial growth factor (VEGF) is one of the most extensively characterized angiogenic factors [1]
It has been demonstrated that MSCs can facilitate new blood vessel growth by secretion of proangiogenic factors (e.g. VEGF, IGF-1a, HGF, etc.) that contribute to cardiac repair and enhance the reparative process [4,5,6]
When the signal density of miRs was cut off by a value of 100, a group of 13 miRs were significantly up-regulated including miR-210, -25, -450a, -130a, -3593-3p, -34c*, -214, 181a, -23b, -34a, -31, -31*, and -140*; whereas 20 miRs were significantly down-regulated in MSCs under hypoxic conditions. miR-210 was the most significantly increased miR in hypoxia-treated MSCs (Fig. 1B), a finding consistent with previous observations in tumor cells, endothelial cells, and cardiomyocytes in responsive to hypoxia [15], [30], [31]. miR-377 was the most down-regulated in MSCs upon hypoxia treatment (Fig. 1B)
Summary
The formation of new blood vessels is critical for the repair of ischemic myocardium, and VEGF is one of the most extensively characterized angiogenic factors [1]. While direct administration of VEGF into the ischemic myocardium has been used successfully to stimulate therapeutic angiogenesis in animal models, clinical trials of VEGF have been largely unsuccessful [2], [3]. These results underscore our incomplete knowledge of myocardial angiogenesis under ischemic conditions. MSCs are, highly sensitive to ischemic conditions, and the majority of injected MSCs die within several hours of delivery in vivo [7] In this regard, multiple approaches (e.g. hypoxic treatment, genetic modification, and pre-conditioning) have been applied to MSCs in an effort to improve their survival and proangiogenic capacity both in vivo and in vitro [8]. Hypoxia is well recognized to promote MSC-mediated myocardial angiogenesis by induction of VEGF expression [9], [10], the underlying mechanisms underlying these effects have not been delineated
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