17beta-estradiol prevents early-stage atherosclerosis in estrogen receptor-alpha deficient female mice

Abstract

Estrogen is atheroprotective and a high-affinity ligand for both known estrogen receptors, ERalpha and ERbeta. However, the role of the ERalpha in early-stage atherosclerosis has not been directly investigated and is incompletely understood. ERalpha-deficient (ERalpha-/-) and wild-type (ERalpha+/+) female mice consuming an atherogenic diet were studied concurrent with estrogen replacement to distinguish the actions of 17beta-estradiol (E(2)) from those of ERalpha on the development of early atherosclerotic lesions. Mice were ovariectomized and implanted with subcutaneous slow-release pellets designed to deliver 6 or 8 mug/day of exogenous 17beta-estradiol (E(2)) for a period of up to 4 months. Ovariectomized mice (OVX) with placebo pellets (E(2)-deficient controls) were compared to mice with endogenous E(2) (intact ovaries) and exogenous E(2). Aortas were analyzed for lesion area, number, and distribution. Lipid and hormone levels were also determined. Compared to OVX, early lesion development was significantly (p < 0.001) attenuated by E(2) with 55-64% reduction in lesion area by endogenous E(2) and >90% reduction by exogenous E(2). Compared to OVX, a decline in lesion number (2- to 4-fold) and lesser predilection (~4-fold) of lesion formation in the proximal aorta also occurred with E(2). Lesion size, development, number, and distribution inversely correlated with circulating plasma E(2) levels. However, atheroprotection was independent of ERalpha status, and E(2) athero-protection in both genotypes was not explained by changes in plasma lipid levels (total cholesterol, triglyceride, and high-density lipoprotein cholesterol). The ERalpha is not essential for endogenous/exogenous E(2)-mediated protection against early-stage atherosclerosis. These observations have potentially significant implications for understanding the molecular and cellular mechanisms and timing of estrogen action in different estrogen receptor (ER) deletion murine models of atherosclerosis, as well as implications to human studies of ER polymorphisms and lipid metabolism. Our findings may contribute to future improved clinical decision-making concerning the use of hormone therapy.

Fig. 1

a Plasma estradiol levels in ERα−/− and ERα+/+ female mice. Levels of plasma 17β-estradiol (pg/ml; mean ± SEM) in ERα−/− and ERα+/+ female study mice are shown for ovariectomized (OVX, n = 14–15), intact (n = 6–10), and ovariectomized replaced with estradiol pellets designed to deliver 6 μg/day (E₂-6; n = 10–12) and 8 μg/day (E₂-8; n = 8–13). Statistically significant (p < 0.05) and non-significant (ns) comparisons are shown for each genotype. There were no significant differences for treatment group comparisons between genotypes. b Uterine weights in ERα−/− and ERα+/+ female mice. Uterine weights (g; mean ± SEM) in ERα−/− and ERα+/+ female study mice are shown for ovariectomized (OVX, n = 4–6), intact (n = 5–7), and ovariectomized replaced with estradiol pellets designed to deliver 6 μg/day (E₂-6; n = 13–14) and 8 μg/day (E₂-8; n = 6–14). Statistically significant (p < 0.01) and non-significant (ns) comparisons are shown for each genotype. In addition, there were significant differences (p < 0.01) for uterine weight comparisons between ERα+/+ E₂-6 and E₂-8 and all ERα−/− treatment groups

Fig. 2

Plasma lipid levels in ERα−/− and ERα+/+ female mice. Fasting lipid levels (mg/dl; mean ± SEM) for total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL) were determined in non-pooled plasma of ERα−/− (n = 50 samples for each lipid) and ERα+/+ (n = 49 samples for each lipid) female mice on the atherogenic diet for animals ovariectomized (OVX), intact, and ovariectomized replaced with estradiol pellets designed to deliver 6 μg/day (E₂-6) and 8 μg/day (E₂-8). *p < 0.05 versus lipid baseline values (not shown, see text) prior to cholesterol feeding for each genotype; **p < 0.05 versus TC in other treatment groups for a given genotype

Fig. 3

Early lesion formation in ERα+/+ and ERα−/− female mice. Lesion area (μm² ± SEM) of early atherosclerotic fatty streaks in the aortas of ERα−/− (n = 80) and ERα+/+ female mice (n = 77) was measured at baseline and 2 and 4 months of cholesterol feeding (n = 5–7 mice each per genotype, treatment group, and time point). Lesion area is shown for mice ovariectomized (OVX), intact, and ovariectomized replaced with estradiol pellets designed to deliver 6 μg/day (E₂-6) and 8 μg/day (E₂-8). Data are mean ± SEM lesion area (in μm²). *p < 0.001 compared to the indicated treatment groups for a given genotype

Fig. 4

Representative cross sections of lesions from the aortas of ERα−/− And ERα+/+ mice. Histochemistry of examples of representative cross sections (10 μm) of lesions from the proximal aorta of cholesterol fed (4 months) ERα−/− (right panel) and ERα+/+ (left panel) female mice for the following experimental groups: ovariectomized (OVX), ovaries intact, and ovariectomized replaced with estradiol pellets designed to deliver 6 μg/day (E₂-6) and 8 μg/day (E₂-8). The vessels have been stained for lipid with Oil-red-O and counterstained with Gill’s Hematoxylin. A normal vessel prior to the initiation of cholesterol feeding, baseline, is shown for comparison. The representative images (×20 magnification) demonstrate red-staining subintimal lipid deposits and fatty streaks. Cross sections are oriented with the lumen to the right and the adventitial surface to the left of each image. Size markers = 20 μm

Fig. 5

Lesion distribution in aortic segments of ERα−/− and ERα+/+ female mice. Lesion area (μm2; mean ± SEM) was determined in the proximal, mid, and distal aortic segments of female ERα−/− and ERα+/+ mice ovariectomized (OVX, n = 29), intact (n = 23), and ovariectomized replaced with estradiol pellets designed to deliver 6 μg/day (E₂-6; n = 25) and 8 μg/day (E₂-8; n = 24). See text for details. *p < 0.01 versus other treatment groups of the same genotype

Fig. 6

Reduction in lesion area as a function of plasma estradiol. Lesion area (μm² ± SEM) as a function of plasma 17β-estradiol (E₂; pg/ml; mean) in ERα−/− and ERα+/+ female study mice. Estradiol values (mean, pg/ml) are indicated for mice ovariectomized, OVX (E₂ = 13 pg/ml), intact (E₂ = 58 pg/ml), and ovariectomized and replaced with E₂ pellets designed to deliver 6 μg/day (E₂-6 = 247 pg/ml) and 8 μg/day (E₂-8 = 284 pg/ml)