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. 2003 May;111(10):1579-87.
doi: 10.1172/JCI16777.

HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI-dependent manner

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HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI-dependent manner

Ming Gong et al. J Clin Invest. 2003 May.

Abstract

Cardiovascular diseases remain the leading cause of death in the United States. Two factors associated with a decreased risk of developing cardiovascular disease are elevated HDL levels and sex - specifically, a decreased risk is found in premenopausal women. HDL and estrogen stimulate eNOS and the production of nitric oxide, which has numerous protective effects in the vascular system including vasodilation, antiadhesion, and anti-inflammatory effects. We tested the hypothesis that HDL binds to its receptor, scavenger receptor class B type I (SR-BI), and delivers estrogen to eNOS, thereby stimulating the enzyme. HDL isolated from women stimulated eNOS, whereas HDL isolated from men had minimal activity. Studies with ovariectomized and ovariectomized/estrogen replacement mouse models demonstrated that HDL-associated estradiol stimulation of eNOS is SR-BI dependent. Furthermore, female HDL, but not male HDL, promoted the relaxation of muscle strips isolated from C57BL/6 mice but not SR-BI null mice. Finally, HDL isolated from premenopausal women or postmenopausal women receiving estradiol replacement therapy stimulated eNOS, whereas HDL isolated from postmenopausal women did not stimulate eNOS. We conclude that HDL-associated estrodial is capable of the stimulating eNOS. These studies establish a new paradigm for examining the cardiovascular effects of HDL and estrogen.

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Figures

Figure 1
Figure 1
HDL isolated from female subjects stimulates the production of nitric oxide. HDL, LDL, and VLDL were isolated from young, reproductively competent female humans (a) and mice (b) and age-matched male humans (a) and mice (b) (31). The freshly isolated lipoproteins (10 μg/ml) were incubated with human microvascular endothelial cells that had been prelabeled with 0.75 μCi/ml of [3H]arginine for 15 minutes at 37°C (21). An additional set of cells was treated with 1 μg/ml of ionomycin to determine the maximal eNOS stimulation. The cells were then washed, lysed, and extracted, and radiolabeled arginine was separated from radiolabeled citrulline using an ion-exchange column. Each experiment included controls, using 1 mM L-NNA to demonstrate that over 99% of the generated citrulline was due to eNOS activity (data not shown). In (c), a concentration curve of the effect of female HDL on eNOS activity is shown. The data are from six to eight independent experiments, with triplicate measurements in each experiment (mean ± SE).
Figure 2
Figure 2
The ability of female HDL to stimulate eNOS requires binding to SR-BI. (a) Human microvascular endothelial cells were pretreated with buffer only, 50 μg/ml of blocking SR-BI IgG, or 50 μg/ml isotype-matched nonspecific IgG for 15 minutes at 37°C. The cells were then incubated with 0.75 μCi/ml of [3H]arginine and 10 μg/ml of female HDL or 1 μg/ml of ionomycin for 15 minutes at 37°C. The cells were processed to quantify the amount of citrulline generated. Each experiment included controls, using 1 mM L-NNA to demonstrate that over 99% of the generated citrulline was due to eNOS activity (data not shown). The data are from six independent experiments, with triplicate measurements in each experiment (mean ± SE). (b) Human microvascular endothelial cells were pretreated with 0.75 μCi/ml of [3H]arginine and then incubated with 10 μg/ml of female HDL or increasing concentrations of male HDL for 15 minutes. The amount of citrulline generated was then quantified. Each experiment included controls, using 1 mM L-NNA to demonstrate that over 99% of the generated citrulline was due to eNOS activity (data not shown). The data are from six independent experiments, with triplicate measurements in each experiment (mean ± SE).
Figure 3
Figure 3
HDL-associated estradiol is responsible for eNOS stimulation. (a) HDL was isolated from female C57BL/6 mice that had intact ovaries (Intact), had the ovaries removed (OVX), or had the ovaries removed and a 17-β-estradiol pellet implanted (OVX + E2). HDL was also isolated from male age-matched C57BL/6 mice that had a 17-β-estradiol pellet implanted (Male + E2). In addition, HDL from control male mice was isolated and enriched with 17-β-estradiol in vitro (Male HDL + E2). LDL from female mice (LDL + E2) and BSA (BSA + E2) were also enriched with 17-β-estradiol in vitro. The in vitro–modified HDL, LDL, and BSA were reisolated, and the amount of estradiol associated was quantified before use (see Methods). Human microvascular endothelial cells were pretreated with 0.75 μCi/ml of [3H]arginine, followed by treatment with 10 μg/ml of each sample or 1 μg/ml of ionomycin for 15 minutes at 37°C. The cells were then processed to quantify the amount of citrulline generated. Each experiment included controls, using 1 mM L-NNA to demonstrate that over 99% of the generated citrulline was due to eNOS activity (data not shown). The data are from eight independent experiments, with triplicate measurements in each experiment (mean ± SE). (b) The same assay as described above was used, with the exception that 10 μM of ICI 182,780 was added to each of the reactions. The data are from four independent experiments, with triplicate measurements in each experiment (mean ± SE).
Figure 4
Figure 4
Female HDL but not male HDL potently relaxes arterial contraction. Femoral artery strips from wild-type C57BL/6 mice were precontracted with 30 nM 5-hydroxytryptamine (5-HT), and cumulative concentrations of HDL, isolated from female (a) or male (b) mice, were added (indicated by arrow). In each single strip, HDL purified from both female and male mice was tested. The order of application of the female HDL versus the male HDL did not affect the results. Note that addition of 1 μM acetylcholine (ACh) caused normal relaxation in the same muscle strip in which the HDL purified from male mice did not cause detectable relaxation. (c) Quantification of the extent of relaxation caused by HDL purified from female or male mice. Six muscle strips from three mice were used.
Figure 5
Figure 5
HDL-induced relaxation is SR-BI and endothelium dependent. (a) Endothelium-intact femoral artery strips isolated from C57BL/6 or SR-BI null mice were precontracted with 30 nM 5-hydroxytryptamine. Acetylcholine (1 μM) or 10 μg/ml HDL purified from female mice was then added to the tissue bath, and the extent of relaxation was measured. Six muscle strips were used for each group. Black bars represent C57BL/6 mice and white bars SR-BI null mice. (b) Endothelium-intact or endothelium-denuded femoral artery strips isolated from control C57BL/6 mice were precontracted with 30 nM 5-hydroxytryptamine. Acetylcholine (1 μM) or 10 μg/ml HDL purified from female mice was then added to the tissue bath, and the extent of relaxation was measured. Three to six muscle strips were used for each group. Black bars represent endothelium intact and white bars endothelium denuded.
Figure 6
Figure 6
HDL isolated from women receiving estrogen replacement therapy stimulates nitric oxide generation. HDL was isolated from five premenopausal women, five postmenopausal women, and five postmenopausal women on estradiol replacement therapy. Human microvascular endothelial cells were pretreated with 0.75 μCi/ml of [3H]arginine and then incubated with 10 μg/ml of the above HDL for 15 minutes. The amount of citrulline generated was then quantified. Each experiment included controls, using 1 mM L-NNA to demonstrate that over 99% of the generated citrulline was due to eNOS activity (data not shown). The data for each HDL are shown, and each value is the average of six measurements.

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