VAPB Promotes Osteogenic Differentiation in Aortic Valve Interstitial Cells via Activation of the SMAD Signaling Pathway

OBJECTIVE: Prior studies have associated vesicle-associated membrane protein- associated protein B (VAPB) with various cardiovascular diseases, yet its role in calcific Aortic Valve Disease (CAVD) is not fully understood. This study aims to elucidate the relationship between VAPB and aortic valve calcification at both tissue and cellular molecular levels, exploring potential regulatory mechanisms. METHODS: The expression levels of VAPB in calcified valve tissues were assessed using immunohistochemical analysis. Osteogenic differentiation and calcification levels of valve interstitial cells (VICs) were evaluated by alizarin red staining, calcium content quantification, and the detection of the osteogenic markers. Gene set enrichment analysis (GSEA) was used to identify downstream pathways regulated by VAPB, immunoblotting was used to detect the expression of key molecules of SMAD signaling, and immunoprecipitation was performed to detect interactions between VAPB and SMAD1/5/9. RESULTS: VAPB expression was significantly upregulated in the aortic valve leaflets of patients with CAVD. VICs with VAPB overexpression exhibited a significant increase in calcium content (p < 0.001) and upregulation of osteogenic markers RUNX2, osteocalcin, and osteopontin (p <0. 01). Conversely, VAPB knockdown reduced osteogenic differentiation in VICs. Furthermore, VAPB overexpression led to the enhanced expression of p-SMAD1/5/9 through activation of the SMAD signaling pathway (p < 0.05), while inhibition of the SMAD pathway abrogated the pro-calcification effects of VAPB. Dual immunofluorescence staining demonstrated colocalization of VAPB with SMAD1/5/9, and immunoprecipitation confirmed an interaction between VAPB and SMAD1/5/9. CONCLUSION: The findings indicate that VAPB promotes osteogenic differentiation in aortic valve interstitial cells by activating the SMAD signaling pathway.