title: Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells.
abstract: Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.
authors: Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada.
authors: Ruchi
authors: R
authors: Chaube
authors: Vasantha Madhuri
authors: VM
authors: Kallakunta
authors: Michael Graham
authors: MG
authors: Espey
authors: Ryan
authors: R
authors: McLarty
authors: Adam
authors: A
authors: Faccenda
authors: Sirinart
authors: S
authors: Ananvoranich
authors: Bulent
authors: B
authors: Mutus
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D055785:Gene Knockdown Techniques; D006360:Heat-Shock Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D026361:Reactive Nitrogen Species / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D002784:Cholesterol; D052250:Nitric Oxide Synthase Type III; D013108:Sphingomyelin Phosphodiesterase