S-nitrosylation contributes to ER stress and aggresome formation in secosterol-B-mediated endothelial dysfunction.

The involvement and the in vivo relevance of endoplasmic reticulum (ER) stress in the atherosclerotic process are well established, but the mechanisms have been only partly elucidated. Emerging evidence indicates that ER protein folding pathways are sensitive to nitric oxide (NO) fluctuations and therefore heavily vulnerable under conditions of nitrosative stress. Recent research indicates that protein S-nitrosylation (S-NO), a key redox-mediated modification involved in several disorders, affects neuronal function by altering ER stress sensor proteins. However, the mechanisms by which ER protein S-NO impacts vascular diseases remain unclear. Here, we provide evidence that secosterol-B (SEC-B), an oxysterol found in atherosclerotic plaques, induces nitrosative stress and protein S-NO in vascular endothelium, leading to ER stress. In detail, our findings demonstrate that SEC-B triggers activation of the inositol-requiring enzyme-X-box binding protein 1 signaling pathway and causes ER-membrane expansion and the accumulation of misfolded proteins in human umbilical vein endothelial cells. In parallel, increased NO levels with upregulation of inducible nitric oxide synthase protein expression and alterations in the nitrosylation levels of various proteins, including protein disulfide isomerase and glucose regulatory protein 78, were observed. Interestingly, pretreatment with NG-nitro-L-arginine methyl ester strongly reduced ER swelling and aggresome formation. Collectively, our findings demonstrate that NO and protein S-NO play a critical role in SEC-B-induced ER dysfunction, providing new insights into the mechanisms underlying vascular dysfunction observed in atherosclerosis and highlighting potential therapeutic targets to preserve endothelial integrity.