Long noncoding TSI attenuates aortic valve calcification by suppressing TGF-81-induced osteoblastic differentiation of valve interstitial cells

Introduction: Calcific aortic valve disease (CAVD) is an active and cellular-driven fibrocalcific process charac-terised by differentiation of valve interstitial cells (VICs) towards an osteogenic-like phenotype. A recently identified lncRNA, lncTSI, has been reported to inhibit fibrogenesis through transforming growth factor (TGF)-8/Smad3 pathway. Here, the present study aimed to investigate the role of lncTSI in CAVD.Methods: The effect of TGF-81 on lncTSI of VICs was measured. TGF-81, RUNX2 and collagen I expression be-tween calcified aortic valve tissue and normal samples by immunohistochemistry and western blotting. Human VICs were cultured and treated with TGF-81. SiRNA and pcDNA3.1-lncTSI plasmid transfection were used to silence and overexpress lncTSI in VICs for 48 h, Smads phosphorylation, RUNX2 and collagen I expression were then verified by western blotting. In ApoE-/-mice fed with 0.25 % high-cholesterol diet, AAV2-lncTSI were injected intravenously to observe their effect on the formation of aortic valve calcification.Results: lncTSI was highly expressed in VICs treated with TGF-81. lncTSI was transcriptionally regulated by Smad3 and reversely inhibited TGF-81-induced Smad3 phosphorylation and downregulated profibrotic gene expression. Silencing lncTSI increased TGF-81-induced Smad3 phosphorylation, and subsequently, upregulated RUNX2 and collagen I expressions in VICs. While overexpression of lncTSI reversed the production of RUNX2 and collagen I in VICs. In a mouse CAVD model of 24 week 0.25 % high-cholesterol diet feeding, overexpression of lncTSI significantly reduced calcium deposition, RUNX2, pSmad3, and collagen I expression in aortic valve leaflets, with less aortic valve stenosis. Conclusions: The novel findings of present study suggested that lncTSI alleviated aortic valve calcification through negative regulation of the TGF-8/Smad3 pathway. The results may help elucidate new diagnostic and therapeutic targets to prevent CAVD progression.