Abstract 18216: Epigenetic Up-regulation of α-smooth Muscle Actin Expression and Cell Aggregation in Human Aortic Valve Interstitial Cells Promotes Calcific Nodule Formation

Abstract

Older people are at risk of calcific aortic valve disease. Aortic valve interstitial cells (AVICs) play an important role in nodular calcification in aortic valve leaflets. AVICs in human aortic valves consist of fibroblasts and myofibroblasts that express α-smooth muscle actin (α-SMA). We have observed that AVICs of diseased aortic valves have greater osteogenic activities. However, molecular mechanism underlying AVIC formation of calcification nodules is not well understood. We hypothesized that an epigenetic mechanism promotes AVIC calcification nodule formation through induction of α-SMA expression and cell aggregation. Methods and Results: MiRNA profiles in AVICs from normal and diseased human aortic valves were analyzed by miRNA array and real-time qPCR. Diseased AVICs displayed higher levels of miR-486. Immunoblotting and immunofluorescence staining revealed that diseased AVICs had higher levels of α-SMA and α-SMA fibers. Inhibition of miR-486 by lentiviral-delivered miR-486 antagomir in diseased AVICs suppressed α-SMA expression and cell aggregation, resulting in reduced calcification nodule formation. Conversely, lentiviral-delivered miR-486 mimic in normal AVICs induced α-SMA expression and cell aggregation, leading to exacerbated calcification nodule formation. Stimulation of normal AVICs with pro-osteogenic mediators TGF-β1 and BMP-2 up-regulated miR-486 levels. MiR-486 antagomir reduced α-SMA expression, cell aggregation and calcification nodule formation in cells exposed to TGF-β1 or BMP-2. Further, miR-486 mimic induced AKT phosphorylation. Inhibition of AKT decreased α-SMA expression and cell aggregation induced by miR-486 mimic in normal AVICs. Knockdown of α-SMA suppressed cell aggregation and calcification nodule formation. Conclusions: The pro-osteogenic phenotype of AVICs of diseased aortic valves is associated with up-regulated levels of miR-486 and α-SMA. MiR-486 modulates the AKT pathway to up-regulate α-SMA expression and cell aggregation that are required for calcification nodule formation. These novel findings indicate that miR-486 contributes to the mechanism underlying aortic valve calcification and appears to be a therapeutic target for suppression of valvular osteogenic activity.