Wang Yueheng, He Shengping, Lan Lan, Yu Hongjiao, Zhao Huan, Xie Yuchen, Zhong Guoli, Yuan Liang, Li Kun, Hu Xiao, Macrae Vicky E, Fu Xiaodong, Chen Guojun, Zhu Dongxing
Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, Guangdong 510260, China.
Department of Cardiovascular Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, Guangdong 510515, China.
Cardiovasc Res. 2025 Apr 15;121(1):190-204. doi: 10.1093/cvr/cvae253.
Calcific aortic valve disease (CAVD) is a common heart valve disease with significant clinical consequences. The mechanisms that drive the pathogenesis of CAVD remain to be fully elucidated. N6-methyladenosine (m6A), the most prevalent RNA epigenetic regulator, has recently been implicated in cardiovascular disease, but its role in CAVD has yet to be investigated. In this study, we investigated the potential function of m6A modification in CAVD.
Using clinical samples from CAVD patients in combination with human valve interstitial cell (hVIC) calcification model, we screened the expression of m6A modulators and discovered that alkB homolog 5, RNA demethylase (ALKBH5), a key m6A demethylase, was significantly down-regulated in calcified hVICs and human aortic valves. Consistently, increased m6A levels were seen in calcified hVICs, and treatment with 3-deazaadenosine (DAA), an inhibitor of m6A modification, significantly reduced hVIC osteogenic differentiation and calcification. In addition, we showed that silencing of ALKBH5 expression increased global m6A levels and accelerated hVIC osteogenic differentiation and calcification, whereas overexpression of ALKBH5 resulted in the opposite effect. We demonstrated that ALKBH5 directly modulate m6A levels of TGFBR2 and its mRNA stability, leading to altered TGFBR2 expression and SMAD2 signalling in hVICs. We further showed that inhibition of TGFBR2 or knockdown of SMAD2 attenuated ALKBH5 knockdown-induced hVIC osteogenic differentiation and calcification. The expression of the m6A reader protein YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) was up-regulated during the process of hVIC calcification. Intriguingly, we revealed that the ALKBH5 silencing-induced increased hVIC osteogenic differentiation and calcification were abolished after knockdown of YTHDF1. These data suggest a potential role YTHDF1 in aortic valve calcification.
This study showed that ALKBH5 attenuated aortic valve calcification through the TGFBR2/SMAD2 signalling pathway via direct m6A modification of TGFBR2.
钙化性主动脉瓣疾病（CAVD）是一种常见的心脏瓣膜疾病，具有严重的临床后果。驱动CAVD发病机制仍有待充分阐明。N6-甲基腺苷（m6A）是最普遍的RNA表观遗传调节剂，最近已被证明与心血管疾病有关，但其在CAVD中的作用尚未得到研究。在本研究中，我们调查了m6A修饰在CAVD中的潜在功能。
使用CAVD患者的临床样本并结合人瓣膜间质细胞（hVIC）钙化模型，我们筛选了m6A调节剂的表达，发现关键的m6A去甲基化酶alkB同源物5、RNA去甲基化酶（ALKBH5）在钙化的hVIC和人主动脉瓣中显著下调。一致地，在钙化的hVIC中观察到m6A水平升高，用m6A修饰抑制剂3-脱氮腺苷（DAA）处理显著降低了hVIC的成骨分化和钙化。此外，我们表明沉默ALKBH5表达会增加整体m6A水平并加速hVIC的成骨分化和钙化，而ALKBH5的过表达则产生相反的效果。我们证明ALKBH5直接调节TGFBR2的m6A水平及其mRNA稳定性，导致hVIC中TGFBR2表达和SMAD2信号通路改变。我们进一步表明，抑制TGFBR2或敲低SMAD2可减弱ALKBH5敲低诱导的hVIC成骨分化和钙化。m6A阅读蛋白YTH N6-甲基腺苷RNA结合蛋白F1（YTHDF1）的表达在hVIC钙化过程中上调。有趣的是，我们发现敲低YTHDF1后，ALKBH5沉默诱导的hVIC成骨分化和钙化增加被消除。这些数据表明YTHDF1在主动脉瓣钙化中具有潜在作用。
本研究表明，ALKBH5通过对TGFBR2进行直接m6A修饰，经由TGFBR2/SMAD2信号通路减轻主动脉瓣钙化。
