Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase
Olivier Feron, Chantal Dessy, Stephane Moniotte, Jean-Pierre Desager, Jean-Luc Balligand
Olivier Feron, Chantal Dessy, Stephane Moniotte, Jean-Pierre Desager, Jean-Luc Balligand
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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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LDL, but not HDL or VLDL, fractions account for the caveolin-mediated at...
LDL, but not HDL or VLDL, fractions account for the caveolin-mediated attenuation of NO production in endothelial cells exposed to hypercholesterolemic human serum. Endothelial cells were exposed for 48 h to 50% hypercholesterolemic human serum or to 50% of LPDS supplemented with the indicated isolated lipoprotein fraction (cholesterol content: VLDL, 25 mg/dl; LDL, 210 mg/dl; HDL2, 15 mg/dl; HDL3, 25 mg/dl). After a 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) Cells’ extracts were immunoprecipitated with anti–caveolin-1 antibody, and both the immunoprecipitate (IP) and supernatant (S) fractions were then separated on SDS-PAGE and immunoblotted with anti-eNOS antibody. Top: Immunoblot of IP and S fractions from nonstimulated cells. Bottom: Immunoblot of IP and S fractions from cells stimulated with A23187. Note that to obtain the S fractions, the supernatants from the IP were immunoprecipitated with anti-eNOS antibody in order to detect quantitatively the amount of eNOS left behind the initial caveolin IP. (b) Shown is 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 exposed to hypercholesterolemic (HC) human serum or to specific lipoprotein fractions as indicated; the data are expressed as percent of NO production in nonstimulated cells exposed to NC serum. *P < 0.05 vs. basal conditions with NC, VLDL, HDL2, and HDL3. #P < 0.01 vs. stimulated conditions with NC, VLDL, HDL2, and HDL3. HDL, high-density lipoprotein; LPDS, lipoprotein-deficient human serum; VLDL, very-low-density lipoprotein.