SMS2 Inhibition Alleviates Endothelial Dysfunction and Atherosclerosis via Promoting NFE2L1 expression to Suppress Ferroptosis.

Sphingomyelin synthase 2 (SMS2) is a key enzyme in sphingomyelin synthesis and is implicated in atherosclerosis (AS). Both endothelial dysfunction (ED) and ferroptosis contribute to AS, but their relationship with SMS2 remains unclear. To explore this, we inhibited SMS2 with Ly93 and then performed RNA sequencing to assess its relationship with ferroptosis, as well as the expression, cholesterol homeostasis, and alternative splicing of NFE2L1 in endothelial cells. We also validated the relationship between SMS2, ferroptosis, and NFE2L1 in ApoE-/- mice and clinical atherosclerotic plaque samples. Results showed that SMS2 inhibition by Ly93 reduced ferroptosis and ED. SMS2 inhibition also decreased cholesterol accumulation, enhanced skipping-exon (SE) alternative splicing of NFE2L1, and increased L-NFE2L1 levels; knockdown of L-NFE2L1 reversed these effects. In ApoE-/- mice, endothelial-specific SMS2 overexpression worsened ED and plaque formation while reducing NFE2L1/GPX4 expression. Conversely, the ferroptosis inhibitor Fer-1 attenuated both ED and plaque burden. In unstable human plaques, SMS2 was elevated, whereas NFE2L1, GPX4, and SLC7A11 were reduced. Collectively, these findings suggest that SMS2 inhibition promotes L-NFE2L1 expression, and this effect is mediated, at least in part, by the suppression of cholesterol accumulation-driven alternative splicing of the NFE2L1 splicing event, a process that ultimately reduces oxidative stress. This depress ED via ferroptosis and ultimately decrease AS, identifying SMS2 as a potential therapeutic target.