Abstract 18239: Microrna-204 and -486 Regulate the Expression of Osteogenic Transcription Factors in Human Aortic Valve Cells: Evidence for Epigenetic Modulation of Valvular Cell Osteogenic Reprogramming

Abstract

Calcific aortic valve disease affects a large number of elderly people. Aortic valve interstitial cells (AVIC) play an important role in valvular calcification. However, pharmacological prevention of the progression of this disease is unavailable due to limited knowledge of underlying molecular mechanisms. Transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) are capable of inducing AVIC reprogramming (expression of osteogenic transcription factors) and have been implicated in the pathogenesis of calcific aortic valve disease. We hypothesized that microRNAs modulate the responses of human AVIC to TGF-β1 and BMP-2 stimulation. Methods and results: Stimulation of AVIC isolated from normal human aortic valves with TGF-β1 or BMP-2 up-regulated the expression of osteogenic transcription factors Runx2 and Osx. Prolonged stimulation with TGF-β1 or BMP-2 induced calcium deposit formation. MicroRNA array and real-time qRT-PCR analysis revealed that TGF-β1 and BMP-2 each down-regulated microRNA-204 and up-regulated microRNA-486. MicroRNA-204 mimic suppressed the expression of Runx2 and Osx, and microRNA-204 inhibitor increased Runx2 and Osx levels in cells exposed to TGF-β1 or BMP-2. Conversely, microRNA-486 mimic increased Runx2 and Osx levels, and microRNA-486 inhibitor reduced Runx2 and Osx expression in cells exposed to TGF-β1 or BMP-2. More importantly, treatment of human AVIC with a combination of microRNA-204 inhibitor and microRNA-486 mimic up-regulated the expression of Runx2 and Osx, and induced calcium deposit formation in the absence of TGF-β1 or BMP-2. Conclusions: TGF-β1 and BMP-2 induce the expression of Runx2 and Osx in human AVIC that is associated with down-regulation of microRNA-204 and up-regulation of microRNA-486. MicroRNA-204 negatively, but microRNA-486 positively modulates Runx2 and Osx expression in human AVIC, and microRNA-204 inhibitor and microRNA-486 mimic synergistically promote human AVIC osteogenic reprogramming. These findings provide new insight into the mechanism by which TGF-β1 and BMP-2 promote aortic valve calcification and indicate that microRNAs are potential therapeutic targets for suppression of the progression of calcific aortic valve disease.