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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Release of eNOS from the caveolin immune complex by Ca2+–CaM. Endothelia...
Release of eNOS from the caveolin immune complex by Ca2+–CaM. Endothelial cells exposed for 48 h to 50% normocholesterolemic (NC) or hypercholesterolemic (HC) human serum were collected, lysed, and solubilized as described in the text. (a) A bar graph (mean ± SEM, n = 3) illustrating the maximal eNOS enzyme activity, measured by the conversion of [3H]arginine in [3H]citrulline in the corresponding cell extracts immunoprecipitated with anti–caveolin-1 antibody. The data are expressed as percent of total eNOS activity in the immunoprecipitate obtained from cells exposed to NC serum. (b) Immunoblot with an anti-eNOS antibody of the same extracts immunoprecipitated with anti–caveolin-1 antibody and exposed, in presence of Ca2+, to increasing concentrations of exogenous CaM: 0, 0.1, 1, 10, 100 μg/ml. The immune complexes bound to protein G–Sepharose beads were extensively washed in OG buffer, and the beads were then equally distributed in five separate aliquots. After 1 h incubation at 4°C in the presence of the indicated amounts of CaM, the beads were repelleted, the supernatant discarded, and the immune complex processed for SDS-PAGE and immunoblot analysis. These experiments were performed three times with similar results. CaM, calmodulin; OG, octylglucoside.