Abstract Body (Do not enter title and authors here): Background and Objective:
Calcific aortic valve disease (CAVD) is a debilitating condition characterized by excessive oxidative stress and inflammation. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been implicated in CAVD pathogenesis. Lysine-specific demethylase 2B (KDM2B) possesses antioxidant properties. This study aimed to investigate the protective role of KDM2B against TMAO-mediated CAVD and explores the underlying mechanisms. We hypothesized that KDM2B mitigates TMAO-induced inflammatory and osteogenic response of valve interstitial cells (AVICs) by reconstructing intracellular oxidative balance.
Methods and Results:
KDM2B expression was diminished in calcified aortic valves and TMAO-treated valve interstitial cells (AVICs) compared to healthy controls, as confirmed by western blot, qPCR, immunohistochemistry (IHC), and immunofluorescence. KDM2B silencing via siRNA exacerbated TMAO-induced inflammation and osteogenic response in AVICs, while KDM2B overexpression (Ad-KDM2B) mitigated these effects. RT-qPCR array identified NADPH Oxidase 4 (Nox4) as a key KDM2B-regulated oxidizing gene. Ad-KDM2B significantly downregulated Nox4 mRNA expression in AVICs. Chromatin immunoprecipitation with qPCR (Chip-qPCR) revealed enhanced JunD binding to the Nox4 promoter upon KDM2B overexpression. KDM2B promoted JunD expression by demethylating H3K4me3, thereby suppressing Nox4 transcription. Western blot, along with alkaline phosphatase and Alizarin Red staining, demonstrated that Nox4 overexpression abolished the protective effects of KDM2B overexpression. In vivo, adeno-associated virus-mediated KDM2B overexpression significantly attenuated aortic valve lesions in mice fed a high-fat, choline diet (HFCD).
Conclusion:
KDM2B protects against TMAO-induced CAVD by regulating oxidative stress. It achieves this by decreasing H3K4me3 on JunD, thereby preventing Nox4 transcription. KDM2B represents a potential therapeutic target for CAVD.