Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase
Olivier Feron, … , Jean-Pierre Desager, Jean-Luc Balligand
Olivier Feron, … , Jean-Pierre Desager, Jean-Luc Balligand
Published March 15, 1999
Citation Information: J Clin Invest. 1999;103(6):897-905. https://doi.org/10.1172/JCI4829.
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Article

Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase

Abstract

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+–calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin–eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin–eNOS heterocomplex from HC serum–treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.

Authors

Olivier Feron, Chantal Dessy, Stephane Moniotte, Jean-Pierre Desager, Jean-Luc Balligand

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Dose-dependent increase in caveolin expression and stabilization of the ...
Dose-dependent increase in caveolin expression and stabilization of the caveolin–eNOS interaction by LDL fractions. Endothelial cells were exposed for 48 h to 50% LPDS with the indicated concentrations of LDL-C. After 5-min incubation in the presence or absence of the calcium ionophore A23187 (5 μM), cells were either tested for NO production or collected, lysed, and solubilized as described in the text. (a) Top: Extracts from A23187-treated and untreated cells were immunoprecipitated with anti–caveolin-1 antibody, and the IP fraction was separated on SDS-PAGE and immunoblotted with an anti-eNOS antibody. The extracts from the corresponding cells were also separated on SDS-PAGE and immunoblotted with either an anti-caveolin (middle) or anti-eNOS antibody (bottom). Note that longer exposure of the same blot also reveals detectable caveolin at 100 mg/dl LDL-C. These experiments were performed two times with similar results. (b) A bar graph (mean ± SEM, n = 3) illustrating the NO production (measured with a microsensor) at the basal level (open bars) and after exposure to A23187 (filled bars) in cultures of endothelial cells previously incubated in presence of the indicated amounts of LDL; the data are expressed as percent of NO production in nonstimulated cells exposed to 100 mg/dl LDL-C. #P < 0.01 vs. basal 100 mg/dl LDL and 125 mg/dl LDL conditions. *P < 0.05, **P < 0.01 vs. stimulated 100 mg/dl LDL and 125 mg/dl LDL conditions. (c) Relationship between cholesterol uptake and caveolin abundance. Individual values were obtained from endothelial cells exposed to 100, 125, 150, 175, and 200 mg/dl LDL-C. (d) Top: Extracts from cells incubated with 100 or 200 mg/dl LDL-C in presence of an antioxidant, DTPA (50 μM), and acutely treated with 5 μM A23187 were separated on SDS-PAGE and immunoblotted with anti-caveolin antibody (two lanes per condition). Bottom: The corresponding extracts were also immunoprecipitated with anti-eNOS (lanes 1 and 3) or anti-caveolin antibody (lanes 2 and 4), and the IP fraction was separated on SDS-PAGE and immunoblotted with an anti-eNOS antibody. These experiments were performed two times with similar results. DTPA, diethylenetriamine pentaacetic acid.