Vascular smooth muscle cell (SMC) de-differentiation with subsequent migration and proliferation into the subendothelial space is central to the progression of cardiovascular diseases. The Nox4 NADPH oxidase (Nox4) is implicated in maintaining the differentiated phenotype of SMC in part through myocardin, a master regulator of SMC gene expression. However, this process is poorly understood. We hypothesized that microRNAs (miR)-mediate changes in Nox4 expression and regulate SMC differentiation. Treatment of human SMCs with a miR-9 or miR-25 mimic silenced Nox4 mRNA through binding to the Nox4 3’UTR. However, only miR-25 was sufficient to downregulate Nox4 protein levels. We found that miR-25 induced the expression of miR-9 through a novel mechanism involving demethylation of the miR-9 promoter by Tet methylcytosine dioxygenase 2 (TET2). Inhibition of miR-9 induction by miR-25 with a miR-9 inhibitor restored Nox4 protein expression to basal levels. Furthermore, the miR-25-mediated decrease in Nox4 protein was ameliorated by inhibiting the proteasome with MG132. These data suggest a novel mechanism wherein miR-9 and miR-25 regulate Nox4 through both translational suppression and proteosomal degradation. Overexpression of miR-9 or miR-25 in human SMCs (1) suppressed myocardin mRNA and protein expression; (2) decreased expression of multiple SMC differentiation genes; and (3) was sufficient to induce cell migration. Thrombin and tumor necrosis factor increased the expression of miR-9 and miR-25 in human SMCs and inhibition of miR-9 prevented thrombin-mediated decrease in myocardin and SMC migration. Mir-9 and miR-25 levels were increased in SMCs derived from balloon injured rat aorta as compared to medial SMCs and in murine carotid artery ten days post carotid injury. A miR-9 inhibitor decreased neointimal formation by more than 50% in following partial carotid ligation in mice. These findings identify miR-9/Nox4 as a novel regulatory pathway of SMC differentiation and a potential therapeutic target in vascular disease.
Interstitial cells from rat middle cerebral artery belong to smooth muscle cell type
