A Role for Bromodomain Containing Protein 4 in Aortic Valvular Interstitial Cells

Background: Calcific aortic valve disease (CAVD) is a common valvular heart condition globally. Bromodomain-containing protein 4 (BRD4) is strongly associated with the development of various cardiovascular pathologies, most notably atherosclerosis (AS). Since CAVD and AS exhibit striking similarities in risk determinants, it is increasingly hypothesized that early-stage pathological changes in both diseases may converge on shared molecular mechanisms. To date, however, no functional or mechanistic data have been reported regarding BRD4's contribution to CAVD. This study aimed to elucidate the functional role of BRD4 in osteogenic differentiation of aortic valvular interstitial cells (AVICs). Methods: We first detected BRD4 and associated protein levels in aortic valve tissues of patients with CAVD or aortic regurgitation (AR) who underwent aortic valve replacement using immunohistochemistry (IHC) and Western blot (WB) analyses. Then AVICs were isolated from AR patient valves and cultured with complete medium (CM) and osteogenic medium (OM) for in vitro validation. To determine whether BRD4 inhibition alleviates osteogenic differentiation and calcification of AVICs, we knocked down BRD4 expression or applied the BRD4 inhibitor JQ1. The impact of BRD4 on oxidative stress, inflammatory response, and apoptosis in AVICs was assessed using WB, reactive oxygen species (ROS) assays, and Enzyme-linked immunosorbent assay (ELISA). To elucidate the mechanism of BRD4 action, we employed inhibitors targeting relevant signaling pathways. Results: BRD4 was significantly upregulated in calcific aortic valves using IHC and WB experiments (p < 0.001). BRD4 inhibition attenuated calcification of AVICs in osteogenic medium, accompanied by a decrease in runt-related transcription factor 2 (Runx2) (p < 0.001) and osteopontin (OPN) (p < 0.001). Further investigations demonstrated that BRD4 suppression reduced ROS generation (p < 0.01), lowered secretion of inflammatory cytokines (IL-6, IL-8, and MCP-1) (p < 0.001), and diminished expression of the pro-apoptotic protein Bax (p < 0.05) in AVICs. Mechanistically, in OM-treated AVICs, the down-regulation of BRD4 increased PI3K (p < 0.05) and p-AKT (p < 0.01) levels and reduced the expression of Runx2 (p < 0.01), OPN (p < 0.01), and Bax (p < 0.001), while the AKT inhibitor reversed this scenario. Conclusions: BRD4 promotes the calcification of AVICs in vitro via the PI3K/AKT signaling pathway. This research represents the initial investigation into the function of BRD4 in valvular calcification, indicating its potential as a therapeutic target for CAVD.