Vesicle-Associated Membrane Protein-Associated Protein B Promotes Osteogenic Differentiation in Aortic Valve Interstitial Cells via Activation of the SMAD Signaling Pathway
Calcific aortic valve disease (CAVD) is increasingly affecting the aging population and is involved in the osteogenic reprogramming of valvular interstitial cells (VICs). However, the exact underlying molecular regulatory mechanisms remain unclear. Vesicle-associated membrane-protein-associated protein B (VAPB) mediates the generation of contact sites between the endoplasmic reticulum (ER) and other cellular membranes. However, the role of VAPB in CAVD remains unclear. This study was to elucidate the relationship between vesicle-associated membrane protein-associated protein B (VAPB) and aortic valve calcification at both tissue and cellular molecular levels, exploring potential regulatory mechanisms. The expression levels of VAPB were assessed using immunohistochemical analysis. Osteogenic differentiation and calcification levels of valve interstitial cells (VICs) were assessed through alizarin red staining, calcium content quantification, and the detection of the osteogenic marker RUNX2. Gene set enrichment analysis was used, and immunoprecipitation was performed. VAPB expression was significantly upregulated in the aortic valve leaflets of patients with calcific aortic valve disease (CAVD) compared to normal aortic valves. VICs with VAPB overexpression exhibited a significant increase in calcium content (p < 0.001) and upregulation of the transcription factor RUNX2 and the osteogenic markers osteocalcin and osteopontin at both mRNA and protein levels (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. The findings indicate that VAPB promotes osteogenic differentiation in aortic valve interstitial cells by activating the SMAD signaling pathway. Overall design: RNA-seq profiling of wildtype human VICs  and their  knockdown derivatives (shVAPB) cultured in osteogenic medium for 3 days