ER stress mediated effects on plasma membrane cholesterol and nitric oxide bioavailability

Endothelial dysfunction is due in part to the decrease in the biosynthesis and the bioavailability of nitric oxide (NO) in the endothelial cell layer. The chronic exposure of blood vessels to cardiovascular risk factors such as LDL-cholesterol, free fatty acids, homocysteine and conditions of hyperglycemia can give rise to endothelial dysfunction. Many of these same risk factors have also been shown to induce endoplasmic reticulum (ER) stress, in endothelial cells. However, the potential links between endothelial dysfunction and ER-stress have not been clearly established. The present study was undertaken with two objectives. Objective 1 was to determine how increased plasma membrane cholesterol content can affect NO diffusion, dynamics and signalling. Objective 2 was to determine whether the unfolded protein response mediated by ER stress can lead to increase in plasma membrane cholesterol in endothelial cells, how ER stress leads to increase in plasma membrane cholesterol and whether increase in plasma membrane cholesterol can influence eNOS activity and localization in the endothelial cells. Objective 1 was carried out by taking Normal Human Fibroblasts (NHF1) and sterol transport-defective Niemann Pick type C1(NPC1) fibroblasts which exhibit increase plasma membrane cholesterol content. NPC1 fibroblats showed decreased activation of both intracellular sGC and VASP (Ser239) phosphorylation induced by exposure to exogenous NO exposure relative to their normal human fibroblasts (NHF) counterparts. Objective 2 was to understand the underlying Unfolded Protein Response (UPR)/ER stress-mediated mechanisms responsible for the induction of endothelial dysfunction. We observed that ER stress and oxidative stress collectively cause elevations in plasma membrane cholesterol in endothelial cells and aortic cross-sections of ER stressed C57BL6 mice. The rise in plasma membrane cholesterol was associated with decreased eNOS activity, eNOS phosphorylation and increased eNOS localization to the Golgi. Interestingly, we observed that neutral sphingomyelinase 2 (NSMase2) becomes dysfunctional during ER stress and by S-nitrosation of cysteine and nitration of tyrosine residues. This study is the first in its kind which links the attenuation of endothelial NO production to ER stress-mediated increases in plasma membrane cholesterol and implicates a key role for NSMase2 in this process