Mesenchymal stem cells modified with miR-126 release angiogenic factors and activate Notch ligand Delta-like-4, enhancing ischemic angiogenesis and cell survival

Abstract

The endothelial cell-specific microRNA (miRNA), miR-126, is considered a master regulator of physiological angiogenesis. Transplanted mesenchymal stem cells (MSCs) release soluble factors contributing to neoangiogenesis and cardiac repair. Therefore, we hypothesized that the over-expression of miR-126 may prolong MSC survival and enhance the cell secretome, thereby improving post-infarction angiogenesis and cardiac function. In this study, MSCs harvested from male C57BL/6 mouse bone marrow were infected in vitro with miR-126 (MSC(miR-126)) by using recombinant lentiviral vectors; the control cells were either non-transfected or transduced with mock vectors (MSC(null)). The results showed an increased secretion of angiogenic factors and a higher resistance against hypoxia in MSC(miR-126) compared with the control cells. The expression of the Notch ligand Delta-like (Dll)-4 in the MSC(miR-126) group was also increased. For in vivo experiments, MSC(miR-126) cultures were intramyocardially injected into the infarct region of the hearts of female C57BL/6 mice (an acute myocardial infarction model) who had undergone ligation of the left anterior descending coronary artery. The survival of MSC(miR-126) cultures, determined by Sry expression, was increased at 7 days after transplantation. MSC(miR-126)-treated animals showed significantly improved cardiac function as assessed by echocardiography 2 weeks later. The expression levels of angiogenic factors and Dll-4 in the infarcted myocardium were further increased by MSC(miR-126) compared with MSCs or MSC(null) cultures. Furthermore, fluorescent microsphere and histological studies revealed that myocardial blood flow and microvessel density were significantly increased in the MSC(miR-126)-transplanted animals. In addition, we found increased immature vessel proliferation following the transplantation of MSC(miR-126) cultures in which the expression of Dll-4 had been knocked down. However, blood vessels with lumen were barely detected, which indicated that Dll-4 plays a key role in tubulogenesis. We conclude that the transplantation of MSCs overexpressing miR-126 can further enhance functional angiogenesis in the ischemic myocardium possibly by the secretion of angiogenic factors and the activation of Dll-4, thus increasing MSC survival. Therefore, MSCs modified with miR-126 may represent a novel and efficient therapeutic approach for ischemic angiogenesis and the improvement of cardiac function.