Wen Liming, Lin Xiangjie, Hu Dongtu, Li Juncong, Xie Kaiji, Li Shunyi, Su Shuwen, Duan Xiaolin, Zhong Guoheng, Lin Yingwen, Chen Yangchao, Xu Tianyu, Zeng Qingchun
State Key Laboratory of Organ Failure Research, Department of Cardiology, Nanfang Hospital, Southern Medical University, 510515, Guangzhou, China; Guangdong Provincial Key Laboratory of Cardiac Function and Microcirculation, Southern Medical University, 510515, Guangzhou, China; Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), 510005, Guangzhou, China.
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin NT, Hong Kong, China.
Atherosclerosis. 2025 Mar;402:119109. doi: 10.1016/j.atherosclerosis.2025.119109. Epub 2025 Jan 30.
Trimethylamine N-oxide (TMAO) is a gut microbial metabolite that promotes calcified aortic valve disease (CAVD), but the underlying mechanism remains obscure. Herein, we aim to test the hypothesis that TMAO regulated the inflammatory process in aortic valves via N6-methyladenosine (m6A) RNA methylation-mediated macrophage polarization.
In vitro, we stimulated macrophages (Phorbol-12-Myristate-13-Acetate-induced THP-1) with TMAO and assessed the expression of methyltransferase-like 3 (Mettl3), IL-1 receptor associated kinase M (IRAK-M) and polarization markers. The interaction between YTH domain family protein 2 (YTHDF2) and IRAK-M mRNA was explored by RNA-IP and RNA decay assay. Functionally, the effects of macrophages on human aortic valve interstitial cells (AVICs) were measured via macrophage adhesion assay and co-culture system. In vivo, the influence of IRAK-M on CAVD development was verified using Irak-m mice.
Mettl3 was highly expressed while IRAK-M was decreased in human calcified aortic valves. In vitro, TMAO upregulated the expression of Mettl3, while the expression of IRAK-M, an important negative regulator of the NF-κB pathway, was remarkably decreased in macrophages. TMAO also induced classical macrophage activation (M1 polarization). Mechanistically, IRAK-M was identified as a target of Mettl3-mediated m6A modification, indicating the involvement of m6A methylation in the regulation of NF-κB activation. Moreover, RIP assay revealed the direct interaction between YTHDF2 and IRAK-M mRNA and this process was dependent on Mettl3. TMAO-treated macrophage conditioned medium induced inflammatory responses in human aortic valve interstitial cells (AVICs). In vivo experiments showed that the deletion of IRAK-M significantly accelerated the progression of aortic valve lesion in mice administrated with high-fat and choline diet (HFCD).
TMAO induces the expression of Mettl3 in macrophages. Mettl3 promotes M1 polarization of macrophages by inhibiting IRAK-M through a m6A/YTHDF2 pathway. TMAO-treated macrophages aggravate the inflammation of human AVICs.
氧化三甲胺（TMAO）是一种肠道微生物代谢产物，可促进钙化性主动脉瓣疾病（CAVD），但其潜在机制仍不清楚。在此，我们旨在验证以下假设：TMAO通过N6-甲基腺苷（m6A）RNA甲基化介导的巨噬细胞极化调节主动脉瓣中的炎症过程。
在体外，我们用TMAO刺激巨噬细胞（佛波醇-12-肉豆蔻酸酯-13-乙酸酯诱导的THP-1），并评估甲基转移酶样3（Mettl3）、白细胞介素-1受体相关激酶M（IRAK-M）和极化标志物的表达。通过RNA免疫沉淀（RNA-IP）和RNA降解试验探索YTH结构域家族蛋白2（YTHDF2）与IRAK-M mRNA之间的相互作用。在功能上，通过巨噬细胞黏附试验和共培养系统测量巨噬细胞对人主动脉瓣间质细胞（AVICs）的影响。在体内，使用Irak-m小鼠验证IRAK-M对CAVD发展的影响。
在人钙化主动脉瓣中，Mettl3高表达而IRAK-M降低。在体外，TMAO上调Mettl3的表达，而NF-κB途径的重要负调节因子IRAK-M在巨噬细胞中的表达显著降低。TMAO还诱导经典巨噬细胞活化（M1极化）。机制上，IRAK-M被确定为Mettl3介导的m6A修饰的靶点，表明m6A甲基化参与NF-κB活化的调节。此外，RNA免疫沉淀试验揭示了YTHDF2与IRAK-M mRNA之间的直接相互作用，且该过程依赖于Mettl3。TMAO处理的巨噬细胞条件培养基诱导人主动脉瓣间质细胞（AVICs）产生炎症反应。体内实验表明，在给予高脂胆碱饮食（HFCD）的小鼠中，敲除IRAK-M可显著加速主动脉瓣病变的进展。
TMAO诱导巨噬细胞中Mettl3的表达。Mettl3通过m6A/YTHDF2途径抑制IRAK-M，促进巨噬细胞的M1极化。TMAO处理的巨噬细胞加剧人AVICs的炎症。
