NAT10-Mediated ac4C-Modification Exacerbates Ferroptosis by Stabilizing HMOX1 in Deep Vein Thrombosis.

BACKGROUND: Deep vein thrombosis (DVT) is a prevalent peripheral vascular disorder associated with abnormal epigenetic processes and altered gene expression in endothelial cells. Accumulating evidence has demonstrated that NAT10 (N-acetyltransferase 10)-mediated N4-acetylcytidine modification exerts unique roles in ferroptosis, but its roles are still elusive in DVT. METHODS: To explore the potential mechanism of NAT10 and ferroptosis on thrombogenesis, we used NAT10 and GPX4 (glutathione peroxidase 4) knockout mice as an in vivo model, and utilized techniques, such as RNA immunoprecipitation, acRIP-qPCR (acetylated RNA immunoprecipitation-quantitative PCR), N4-acetylcytidine Dot Blotting assay, and Western blotting, for detailed molecular analysis. RESULTS: GPX4 is a pivotal gene that suppresses ferroptosis. Utilizing endothelial cell-specific GPX4 conditional knockout mice (GPX4(fl/fl)Cdh5-Cre(+)), we proved that ferroptosis in endothelial cells promotes the formation of thrombosis. Previous evidence indicates that NAT10 overexpression induces ferroptosis and downregulates GPX4 expression. Here, we found that NAT10 expression was elevated in DVT mice, and silencing of NAT10 markedly attenuated ferroptosis both in vitro and in vivo. Furthermore, endothelial cell-specific knockout of NAT10 (NAT10(fl/fl)Cdh5-Cre(+)) demonstrated a reduction in endothelial ferroptosis, thereby inhibiting both the formation and progression of DVT. Mechanistic studies indicated that NAT10 facilitated the N4-acetylcytidine modification of HMOX1 (heme oxygenase 1), which enhanced its mRNA stability, leading to the accumulation of ferrous ions, and exacerbating endothelial dysfunction in DVT. CONCLUSIONS: Collectively, our data elucidate that downregulation of NAT10 mitigates endothelial ferroptosis and prevents DVT formation and progression by modulating HMOX1 expression, which offers a potential novel strategy for the prevention and treatment of thrombosis in DVT.