Opposing Effects of Reactive Oxygen Species and Cholesterol on Endothelial Nitric Oxide Synthase and Endothelial Cell Caveolae

Abstract

Abstract-Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular homeostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invaginations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifugation. Treatment of BAECs with 30 g/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 mol/L LY83583, a superoxide-generating napthoquinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine plus xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions. (Circ Res. 1999;85:29-37.)

Transactivation of Vascular Endothelial Growth Factor (VEGF) Receptor Flk-1/KDR Is Involved in Sphingosine 1-Phosphate-stimulated Phosphorylation of Akt and Endothelial Nitric-oxide Synthase (eNOS)

“…Cell Culture-Bovine aortic endothelial cells (BAEC) were purchased from Clonetics (San Diego, CA) and were cultured in medium 199 supplemented with 10% fetal bovine serum (Invitrogen), basal MEM vitamins, and amino acids (Invitrogen) as described before (29). Confluent cells cultured in 60-mm dishes were serum-starved for 18 - Immunoblot Analysis-Western blot analyses were performed as described previously (24,31).…”

TNFα and oxLDL Reduce Protein S-Nitrosylation in Endothelial Cells

“…In line with these findings, the data of the present study revealed that the stimulation of endothelial cells with pro-inflammatory or pro-atherogenic stimuli reduced the S-nitrosylation of distinct proteins in endothelial cells and specifically reduced the Snitrosylation of the p17 subunit of caspase-3. The decline of S-nitrosylation could be due to an inhibition of eNOS expression and/or NO bioavailability induced by TNF␣-mediated eNOS mRNA destabilization (27) or increased oxidative stress (30,31). However, pharmacological inhibition of NO synthesis for 18 h with L-arginine-L-NAME or L-NMMA did not decrease the S-NO levels, although control experiments confirmed the inhibition of eNOS (data not shown).…”

Implications of Early Structural-Functional Changes in the Endothelium for Vascular Disease

“…58 Evidence exists that ROS induce endothelial dysfunction by affecting eNOS expression or by inactivation of NO through the formation of lipid peroxidation products and peroxynitrite radicals that disturb directly the EC membrane. [61][62][63] In ECs, the enzymatic systems that contribute to the increase production of ROS are xanthine oxidase, NADH/ NAD(P)H oxidase, and eNOS. Within the atherosclerotic plaque, the inflammatory cells and SMCs are a source of superoxide possibly via angiotensin II-activated NAD(P)H oxidase.…”