Mitochondrial regulation of exosomal transported miRNA play a central role in vascular smooth muscle cell proliferation and migration associated with atherogenesis and re-stenosis

Abstract

Vascular smooth muscle (VSM) cell proliferation and migration are the hallmark of atherosclerosis and re-stenosis. In this study, we have investigated the role of mitochondrial bioactivity in determining the content of exosomes released by vascular smooth muscle cells and the biological effect these exosomes have on vascular smooth muscle (VSM) cell proliferation and migration. VSM cells were isolated from 12 week old male Sprague-Dawley rats. Experiments were undertaken using day zero isolated (contractile phenotype), 21 day cultured (synthetic phenotype) and 21 day mitochondrial incompetent (synthetic phenotype- Rho cell). The effect of balloon angioplasty on rat aorta structural remodelling was also studied. Inhibition of mitochondrial network formation with the DRP1 inhibitor MDivi (10 uM) inhibited angioplasty-dependent remodelling. This observation was confirmed in VSM cell cultures where MDivi significantly reduced proliferation and migration. Total exosomal release was significantly greater in the 21 day cultured cells when compared with quiesced non-proliferating cells and 21 day cultured Rho cells (480±20 ng, 520±10 ng and 420±10 ng per ml respectively) and total exosomal RNA yield was 70.2±10.2 ng/ul, 118.7±2.4 ng/ul and 70.8±4.7 ng/ul respectively. Mitochondrial function significantly influenced miRNA and mRNA measured in exosomes. When compared with the hyperproliferative synthetic VSM cell miR-21 expression was reduced by 88±12.1% and miR-145 expression increased by 73±19.8%. We also measured a 7-fold decrease and 6.6-fold decrease mTOR, PI3K and 4EBP1 respectively. A significant increase expression of P53, cdkn2a and ROS scavenging proteins including SOD1 and SOD2 were measured in 21 day cultured Rho cells vs. 21 day hyperproliferative VSM cells. In this study we have further correlated VSM cell hyperproliferative phenotype with mitochondrial function. Moreover, further demonstrated mitochondrial function/VSM cell phenotype with exosomal release and cargo that potentially drives the hyperproliferative/migratory phenotype central to atherosclerosis and re-stenosis.