MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE^−/−) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases.

MALT1（粘膜相关淋巴组织淋巴瘤易位蛋白 1）是一种人副半胱天冬酶蛋白，通过其半胱天冬酶样结构域具有蛋白水解活性。 MI-2（一种特定的化学抑制剂）对 MALT1 的药理抑制作用会减弱内皮细胞对炎症刺激的反应。然而，MALT1 如何调节血管平滑肌细胞 (SMC) 的功能尚不清楚。本研究旨在探讨 MI-2 抑制 MALT1 对体外和体内血管 SMC 功能的影响。 MI-2 治疗导致培养的主动脉 SMC 出现浓度和时间依赖性细胞死亡，这种死亡可以通过铁螯合剂去铁胺 (DFO) 或铁他汀-1 (Fer-1)（一种铁死亡的特异性抑制剂）来挽救，但不能通过抑制剂来挽救细胞凋亡 (Z-VAD-fmk)、焦亡 (Z-YVAD-fmk) 或坏死 (Necrostatin-1、Nec-1)。 MI-2 处理下调了谷胱甘肽过氧化物酶 4 (GPX4) 和铁蛋白重多肽 1 (FTH1) 的表达，而用 DFO 或 Fer-1 预处理可以阻止这种情况。 MI-2 治疗还激活了自噬，而 Atg7 缺陷或巴弗洛霉素 A1 会抑制自噬，从而防止 MI-2 诱导的铁死亡。 MI-2 治疗减少了柱状瘤病 (CYLD) 的裂解，CYLD 是 MALT1 的特异性底物。值得注意的是，MI-2 治疗导致小鼠主动脉收缩力迅速丧失，而与 Fer-1 共孵育可以防止这种情况发生。此外，局部应用 MI-2 分别显着减少了 C57BL/6J 小鼠和载脂蛋白 E 敲除 (ApoE ^−/− ) 小鼠的颈动脉新内膜病变和动脉粥样硬化，而与 Fer-1 联合治疗均得到改善。 总之，本研究表明，MALT1 抑制可诱导血管 SMC 的铁死亡，可能有助于其改善增殖性血管疾病。