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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-2672
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 7 2713-2720
Copyright © 2006 by The Endocrine Society

Intimal Estrogen Receptor (ER)ß, But Not ER{alpha} Expression, Is Correlated with Coronary Calcification and Atherosclerosis in Pre- and Postmenopausal Women

Rose C. Christian, Peter Y. Liu, Sean Harrington, Ming Ruan, Virginia M. Miller and Lorraine A. Fitzpatrick

Division of Endocrinology, Metabolism, Diabetes and Nutrition, and Internal Medicine (R.C.C., P.Y.L., S.H., M.R., L.A.F.) and Departments of Surgery and Physiology and Biophysics (V.M.M.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Lorraine A. Fitzpatrick, M.D., Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91361. E-mail: lfitzpat{at}amgen.com.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) {alpha} and ß are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ER{alpha} or ERß in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ER{alpha} and ERß expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status.

Methods: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ER{alpha} and ERß, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ER{alpha} and ERß with smooth muscle actin, a marker of VSMCs.

Results: ERß and ER{alpha} were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERß expression exceeded ER{alpha} expression in all wall layers (P < 0.001) and was adjacent to areas of calcium deposition. ERß expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P < 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ER{alpha} expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ER{alpha} but not ERß declined with age (P < 0.01) in HT women only. Age had no effect on ER{alpha} or ERß expression in non-HT or premenopausal women.

Conclusions: ERß is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERß expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE CARDIOVASCULAR EFFECTS of hormone therapy (HT) in postmenopausal women are highly controversial (1, 2). Observational studies (3, 4, 5) bolstered by more recent prospective studies showing that estrogen therapy improves lipid profiles (6, 7, 8, 9) initially suggested a possible cardioprotective effect of HT. Most recently, the results from the Nurses’ Health Study (10) suggested that the timing of estrogen administration may play a role in the potential cardioprotective effect. However, randomized, placebo-controlled trials have failed to confirm any cardioprotection (11, 12, 13, 14) and, to the contrary, suggested increased cardiovascular risk (11, 14). Experimental data (15) indicate that estrogens have significant effects on vascular function and that the vascular response to estrogens may vary greatly due to differences in cell targets (endothelium, vascular smooth muscle, macrophages), receptors, or signaling pathways (16). Hence, the possibility exists that novel pharmacogenetically targeted hormonal therapies may provide cardioprotection. Investigation of the seemingly controversial effects of estrogen on vascular tissue may contribute greatly to the development of effective interventions.

The ERs are ligand-activated transcription factors belonging to the nuclear receptor superfamily. ER{alpha} and ERß have been cloned, and both are expressed in macrophages and endothelial and vascular smooth muscle cells (VSMCs) of various tissues (5, 17, 18). ER{alpha} and ERß signal through similar endothelial cell pathways (17) but differ in both transcriptional activity (17) and regulation (19). Hence, tissue-specific variations in ER expression and localization may affect the vascular response (20). Because estrogens act through these receptors, either or both receptors could potentially mediate pro- or antiatherogenic processes in coronary arteries. However, little is known about the distribution and role of ER subtypes in vascular tissue.

Calcium and plaque area are key components of atherosclerosis, and calcification of plaque is now believed to be an actively regulated process (21). Coronary calcium content is strongly correlated with the extent of atherosclerotic disease determined by autopsy (22, 23, 24) or angiography (25). There are few animal models of atherosclerotic coronary calcification; therefore, studies examining human vascular tissue are critical (26, 27). Furthermore, studies of elastic arteries (e.g. carotid, brachial, and femoral), which are largely composed of elastic fibers with interspersed VSMCs, may not be applicable to the functionally distinct coronary artery, in which the medial layer is largely composed of VSMCs. We recently reported, for the first time, that calcium and plaque area in coronary arteries obtained at autopsy from postmenopausal women treated with estrogen were strongly correlated with estrogen status (28) after correction for coronary heart disease status, which suggests an antiatherosclerotic effect with estrogen use. Several observational studies in humans corroborate these findings, although to date, no prospective trials have been performed using coronary calcium as an endpoint (10, 29, 30). To investigate the mechanistic implications of this relationship, we used immunohistochemistry to localize and quantify ER{alpha} and ERß in human vascular tissue. We examined the relationship between ER expression patterns and atherosclerotic plaque and calcification in premenopausal women and postmenopausal women receiving or not receiving HT.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study groups

Intact coronary arteries were obtained sequentially from women requiring full autopsy at the Mayo Clinic (Rochester, MN) during the 3-yr period from 1997–1999 under a protocol reviewed and approved by the Mayo Clinic Institutional Review Board (IRB no. 0-655-96). Eligible women were at least 18 yr of age and provided written consent for research use of autopsy specimens. Patients with a history of coronary stents, prior coronary artery bypass grafting, angioplasty, or other interventions were excluded because our study design required intact coronary arteries. Patients who died of unexpected coronary events were not included as autopsy, and extensive dissection of the coronary arteries was required. Patients with a history of use of selective ER modulators, bisphosphonates, or conditions associated with dystrophic vascular calcification, such as hyperparathyroidism or chronic hemodialysis, were excluded (28).

Inpatient and outpatient medical records were reviewed for demographics, menstrual status, and estrogen use. Women whose last menstrual period or surgical menopause was at least 1 yr before death were considered postmenopausal (n = 46). Current estrogen use was confirmed if documented in the outpatient record and in the admission history of the patient’s final hospitalization. HT-treated women were current users of oral or transdermal estrogen with or without a progestogen for 6 months or greater at the time of death (n = 13). Postmenopausal women were considered untreated (non-HT) if they were never users or had been prescribed estrogen less than 6 months and were nonusers at the time of death (n = 33).

Specimen preparation

The three major coronary arteries, left anterior descending, right coronary, and left circumflex were dissected intact from the epicardial surface to preserve the integrity of the vessel lumen (LUM). Four sequential segments were cut from the proximal 3 cm of each artery. Specimens were not decalcified during sample preparation to preserve true calcium content; this approach, used in all of our studies, is essential to accurate quantification of calcium content (23, 24). The first and third arterial segments were cut to 1 cm, dehydrated in ascending alcohols, and embedded in glycolmethylmethacrylate (GMA) using a temperature-controlled method, as previously reported (28, 31). GMA is a hard, clear plastic monomer that allows excellent preservation of vessel morphology and calcium content during preparation of sections. The second and fourth arterial sections were cut to 0.5 cm, placed in formalin, and embedded in paraffin for immunohistochemistry.

Contact microradiography and histological measurements

Cross sections (200 µm) were cut from the proximal end of each GMA-embedded arterial segment and imaged by contact microradiography by a method previously described and known to be highly precise (28, 32). Sections were stained with aldehyde fuchsin and eosin counterstain to optimally delineate the elastin laminae to distinguish the intimal, medial, and advential components of the arterial wall. Images of stained sections and contact microradiographs were captured digitally as previously described (28). Calcium area and the areas circumscribed by the LUM, internal elastic lamina, and external elastic lamina were determined from these images. Plaque area (square millimeters) was calculated by subtracting LUM area from the internal elastic lamina area. Each section was radiographed, and the calcium content was determined by pixel count as in computerized tomography. Calcium area (square millimeters) was calculated by dividing the pixel count by the pixel size calibration factor ({kappa} = pixels/millimeters squared). The calibration factor was calculated on a standardized microscope slide bearing a scored 1-mm square as the number of pixel counts within 1 mm2. The calcium-to-plaque ratio reflects the percentage of atheromatous plaque that is calcified and was calculated by dividing calcium area by plaque area.

The mean calcium and plaque areas were calculated for each subject from all measurable proximal and distal sections of intact coronary arteries, a maximum of six sections per subject. Sections in which the arterial circumference was interrupted or LUM area not well preserved were excluded from analysis. Two hundred twenty-three sections were analyzed for calcium area (n = 71 for HT and n = 152 for non-HT), and 226 sections (n = 69 for HT and n = 157 for non-HT) were analyzed for plaque area.

Immunohistochemistry

One arterial segment, randomly selected from among the three coronary arteries collected, was analyzed for each subject [n = 9 premenopausal (PRE) women, n = 13 HT, n = 33 non-HT]. Each paraffin-embedded arterial segment was cut 5 µm thick and immunostained for ER{alpha} and ERß using the ABC Elite Vectastain Kit and avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). ER{alpha} immunostaining was performed with rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA); the rabbit IgG for ERß was specific for ß1, ß2, ß1d3, and ß2d3 isoforms (Alpha Diagnostics International, San Antonio, TX). Due to formalin fixation of the arteries, steam antigen retrieval was performed on paraffin-embedded sections with citric acid (pH 6.0) to digest cross-links. For negative control sections, antibody was preadsorbed with peptide overnight at a temperature of 4 C. Also, sections were incubated without secondary antibody to determine auto fluorescence.

Two observers who were blinded to the source of the arteries scored arteries independently, and the scores were averaged. Intensity of staining was graded semiquantitatively in each of the arterial layers (intima, media, adventitia) on a scale of 0–4, with 0, no staining; 1, faint; 2, minimal; 3, moderate; and 4, intense immunostaining. The ER{alpha}-to-ERß ratio was determined by the formula (ER{alpha} + 1)/(ERß + 1). The addition of one to each score converted the scale from a 0–4 range to a 1–5 range to avoid division by zero.

Immunofluorescent staining

Sections of paraffin-embedded arteries were stained for smooth muscle actin, ER{alpha}, and ERß. The sections were dewaxed in Histoclear (National Diagnostics, Atlanta, GA), hydrated in graded alcohol solutions, and steamed in citric acid (pH 6.0). The samples were blocked for 30 min in 4% normal donkey serum in PBS containing 0.1% Triton X-100. The samples were incubated overnight at 4 C in PBS containing 1:50 dilutions of fluorescein isothiocyanate-conjugated antibody to polyclonal rabbit anti-ER{alpha} (Santa Cruz Biotechnology), monoclonal mouse anti-ERß (Sigma Chemicals, St. Louis, MO), and muscle actin (Sigma Chemicals) antibodies. The sections were washed in PBS containing cy3-conjugated antirabbit and cy5-conjugated antimouse antibodies (Jackson ImmunoResearch, Avondale, PA) for 2 h. The sections were washed in PBS and mounted in aqueous mounting medium. The images were obtained by an Olympus FluoView 200 laser-scanning confocal microscope system (Olympus America Inc., Melville, NY) equipped with argon and krypton laser (488, 568, and 647 nm), mounted on an Olympus BX50WI microscope. An Olympus 20x oil immersion objective lens was used for imaging.

Statistical analysis

Nonparametric one-way ANOVA (Kruskal-Wallis test) was used to detect differences according to hormone status (premenopausal, postmenopausal on HT; and postmenopausal not on HT) in ranked layer (intima, media, and adventitia) scores for ER{alpha} and ERß staining. Analogous comparisons were performed according to HT status (HT vs. non-HT) and menopausal status (premenopausal vs. postmenopausal). Where appropriate, paired comparisons within individuals were assessed by Wilcoxon signed rank test. Correlation between ER immunostaining and calcium area, plaque area, or calcium-to-plaque ratio was assessed by nonparametric Spearman test using values obtained in the same arterial section wherever possible. Spearman correlation between ER immunostaining and age was also calculated. Correlations were performed examining all women (n = 55), as well as according to hormone status. All tests were two-tailed, with P < 0.05 considered significant. Bonferroni adjustment was not required as each was an a priori analysis. Analyses were performed using SAS version 8.02 (SAS Institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Subjects

The demographics and cardiovascular risk factors of these 55 human subjects have been published previously (28). As expected, premenopausal women were younger than postmenopausal women (premenopausal mean age, 40 ± 10 yr). There were no statistically significant differences in cardiovascular risk factors or clinical characteristics among groups. Non-HT women tended to be older [74 ± 14 vs. 66 ± 13 yr, P = not significant (NS)] and were more likely to have hypertension (61 vs. 46%, P = NS) and diabetes mellitus type 2 (24 vs. 0%, P = 0.05) than HT women, although differences were not statistically significant. HT women were more likely to have undergone hysterectomy (77 vs. 36%, P = NS), have osteoporosis (39 vs. 30%, P = NS), and to be smokers (23 vs. 12%, P = NS) compared with non-HT women. No differences in years of education, an indicator of socioeconomic status, were noted among the three groups.

Coronary calcification

We have previously reported the association of coronary calcium with estrogen status in this cohort (28). The results are summarized briefly to understand the interactions among the variables reported. In premenopausal women, mean coronary artery calcium area was 0.001 + 0.003 mm2, and plaque area was 1.12 + 0.53 mm2. In estrogen-treated postmenopausal women, mean coronary artery calcium area was 0.129 + 0.23 mm2, and mean plaque area was 1.55 + 0.69 mm2. In contrast, postmenopausal women who were not on estrogen therapy had a mean coronary artery area of 0.970 + 1.00 mm2 and a mean plaque area of 3.28 + 1.31 mm2. Coronary arteries from estrogen-treated postmenopausal women had lower mean coronary calcium content and calcium-to-plaque ratio (P < 0.001 and P = 0.004, respectively) than those from untreated postmenopausal women. After controlling for risk factors such as diabetes and hypertension, estrogen status remained an independent predictor of both calcium and plaque area (P = 0.014 and P = 0.001, respectively) in all women. Estrogen status was associated with coronary calcium and plaque area independent of age and coronary heart disease risk factors, suggesting that estrogen may modulate the calcium content of atherosclerotic plaques as well as plaque area.

Immunohistochemistry

Differences in ER{alpha} and ERß expression were apparent as shown in typical sections illustrated in Fig. 1Go. Coronary artery sections from a non-HT subject were immunostained for ER{alpha} (Fig. 1Go, A–C) and ERß (Fig. 1Go, D–F). ERß immunostaining in the intimal layer was most intense in areas within or adjacent to calcium deposits within atheromatous plaques (Fig. 2Go). In Fig. 2Go, the photomicrograph shows an area of ERß immunostaining corresponding to an area of dense calcification in the contact microradiograph of a contiguous arterial section. This coronary artery section was from a postmenopausal woman not on HT. In contrast, ER{alpha} immunostaining did not correspond with calcification in any contact microradiograph.


Figure 1
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  		FIG. 1. Immunohistochemistry for ER{alpha} and ERß. Coronary arteries from non-HT women were stained for ER{alpha} and ERß as described in Subjects and Methods. A to C, Photomicrographs of a coronary artery stained for ER{alpha} at varying magnifications (x40, x100, and x200). D to F, Photomicrographs of an artery stained for ERß. Inset, Incubation of a section of coronary artery with nonimmune serum used as a control.

 

Figure 2
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  		FIG. 2. Contact microradiography and immunohistochemistry for ERß. Using nondecalcified technology, contact microradiography was performed on a section of coronary artery (right). A contiguous section was stained for ERß (left). Note the intense ERß staining in the area of calcification.

 
Double-labeled immunofluorescence was used to determine which cell type was expressing the ER{alpha} and ERß (Fig. 3Go). ER{alpha} and ERß were colocalized with {alpha}-actin, a marker of VSMCs.


Figure 3
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  		FIG. 3. Immunofluorescence of ER{alpha} and ERß in coronary arteries. Triple label immunofluorescence was performed on a coronary artery for actin, ER{alpha}, and ERß, and the images were merged. Actin fluoresced green, ER{alpha} fluoresced red, and ERß fluoresced blue-purple. Control section indicates the lack of autofluorescence, magnification x16. L, Artery LUM; I, intima; M, media; A, adventitia.

 
Semiquantitative correlational and subgroup analysis

ERß immunostaining scores were significantly greater than ER{alpha} scores in each coronary artery wall layer (intima, media, or adventitia), whether analyzed in all women (P < 0.001 for ER{alpha} vs. ERß, Fig. 4Go) or within each subgroup (HT, non-HT, or PRE, P < 0.01). Both ER{alpha} and ERß expression were greater in the media than in either the adventitia (P < 0.001) or intima (P < 0.001), whereas there was no difference between intimal and adventitial layers.


Figure 4
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  		FIG. 4. Immunostaining for ER{alpha} and ERß was performed in coronary arteries from premenopausal women and postmenopausal women on or off HT. Each artery layer was scored independently in a semiquantitative fashion. In all women combined, ERß was much more intense in each artery layer (intima, media, or adventitia) (*, P < 0.001 for ER{alpha} vs. ERß in the intima, media, or adventitia). When comparing the various artery layers, there was significantly more staining in the media than in the intima or adventitia for either Er{alpha} (P < 0.001) or ERß (*, P < 0.001).

 
ERß expression in the intimal layer correlated significantly with mean calcium area (r = 0.36, P = 0.008, Fig. 5AGo), mean plaque area (r = 0.34, P = 0.01, Fig. 5BGo), and calcium-to-plaque ratio (r = 0.37, P < 0.008, Fig. 5CGo) in all women. However, this correlation was not observed with ERß expression in the other layers (media r = –0.22, P = 0.11; adventitia r = –0.16, P = 0.28). Similarly, ER{alpha} expression and ER{alpha}-to-ERß ratio in any of the artery wall layers was not correlated with plaque area, calcium area or calcium-to-plaque ratio (data not shown).


Figure 5
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  		FIG. 5. ERß score in the intima of the coronary arteries was analyzed as a function of calcium area, plaque area, and calcium-to-plaque ratio by linear regression. ERß expression in the intima layer correlated significantly with the mean calcium area (r = 0.36, P = 0.008; A), mean plaque area (r = 0.34, P = 0.01; B), and calcium-to-plaque ratio (r = 0.37, P < 0.008; C) in all women.

 
Age was associated with a significant decrease in ER{alpha} expression in the intima (r = –0.78, P < 0.002, Fig. 6AGo) and media (r = –0.69, P < 0.01, Fig. 6BGo), but not in the adventitia (r = –0.24, P = 0.43, Fig. 6CGo) of postmenopausal women receiving HT only. Similar relationships were not observed in premenopausal women or postmenopausal women not receiving HT. Age was not associated with ERß expression (r = –0.22, P = –0.10) or ER{alpha}-to-ERß ratio (r = –0.08, P = 0.57).


Figure 6
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  		FIG. 6. Linear regressions were performed with age and ER{alpha} expression. In the HT women only, there was a significant decrease in ER{alpha} expression in the HT women in the intima (r = – 0.78, P = 0.0016; A) and media (r = –0.69, P = 0.009; B). There was no significant correlation of ER{alpha} expression with the adventitia (r = –0.24, P = 0.43; C).

 
ER{alpha} and ERß expression and the ER{alpha}-to-ERß ratio did not vary among premenopausal women, postmenopausal women receiving HT, and postmenopausal women not receiving HT. ERß expression in the intima tended to be greater in non-HT women than in HT women but not significantly (P = 0.06). Furthermore, this trend was not observed in either the adventitial or medial layers. Also, neither ER{alpha} expression nor ER{alpha}-to-ERß ratio varied significantly among arteries from HT, non-HT, or PRE women in any of the wall layers (P > 0.05, data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The role of HT in cardiovascular disease in women is highly debated. Many studies implicate HT as advantageous, whereas other studies impugn its use in the postmenopausal women. Little is understood about the subtypes of ERs in coronary arteries from premenopausal women and in postmenopausal women who elected to take or not to take HT. This study is the first to examine the relationships among ER{alpha} and ERß expression, calcification, and atheromatous plaques in human coronary arteries from female subjects. Our findings add to the limited but accumulating experimental data that suggest a role for ERß in coronary atherosclerosis. A better understanding of ER expression and regulation in atherosclerosis may lead to the development of pharmacogenetically appropriate and targeted therapies (1, 33).

Our finding that semiquantitatively, ERß exceeds ER{alpha} immunostaining in all layers of the coronary artery is novel and suggests that vascular ERß may be expressed to a greater extent than ER{alpha} in advanced atherosclerosis. Our immunostaining findings are consistent with transcriptional data showing that ERß exceeds ER{alpha} mRNA in human VSMCs obtained from aorta, coronary, and iliac arteries (33, 34). Increased ERß protein expression has been observed in studies of both muscular and elastic arteries, including endomyocardial arteries from human cardiac transplant recipients (35) and carotid arteries of nonhuman primates (36) and rodents (37). However, unlike previous studies, we examined the distribution and expression of both ER subtypes by arterial wall layer, each of which is functionally and histologically distinct. A recent autopsy study of ER expression in atherosclerotic plaques of premenopausal women examined only the expression of ER{alpha}, not ERß, in the vascular wall (27).

Expression of ERß in the intimal layer correlated significantly with quantifiable indices of atherosclerotic disease severity, namely, calcium area, plaque area, and calcium-to-plaque ratio. ERß expression in the intima colocalized with calcium deposits and VSMCs as determined by immunofluorescent staining, suggesting a histological basis for this relationship, as described in other models (5). In contrast, no mathematical or histological correlation with ER{alpha} expression was observed. This provides convincing evidence that ERß plays a role in atherosclerotic plaque development and calcification and supports the thesis that atherosclerotic calcification is an actively regulated process (21) modulated by estrogen status (28, 38). The association of ERß and atherosclerotic severity does not appear to be an effect of aging because age did not correlate with ERß expression or ER{alpha}-to-ERß ratio in any of our subjects, regardless of hormonal status.

An important caveat to any observational study is that it cannot determine directionality, that is, cause and effect. Our data, although observational, suggest that ERß is not incidentally expressed but significantly involved in the process of coronary atherosclerosis and calcification. In addition, after rat carotid balloon injury, ERß mRNA and protein are up-regulated in the vascular wall. In this study, colocalization of ERß with VSMC in the neointima and media suggested a vasculoprotective effect (37). Because atherosclerosis is an inflammatory disease (21, 39), the increase in ERß expression in advanced plaque areas may reflect a compensatory reaction to chronic vascular inflammation.

We interpreted the observed increase in ERß expression as a compensatory response to coronary calcification and atherosclerosis, although we cannot exclude the possibility that ERß is a contributing factor. Regardless of directionality, our data suggest that ERß may be the predominant ER isoform involved in human atherosclerotic plaque development, although interventional studies that selectively target ERß are required to confirm these data. The use of genetic animal models lacking a specific ER or in animals that have high atherogenic potential are logical next steps to understand that mechanism of the ER on arterial plaque and calcification.

Our findings do not negate a role for ER{alpha} in human cardiovascular disease. Polymorphisms of ER{alpha} may modulate the effect of estrogen replacement on HDL cholesterol in postmenopausal women (40). Atherosclerosis in a hyperlipidemic population was associated with a specific ER{alpha} polymorphism (41). Detectable ER{alpha} protein expression correlated with autopsy-proven coronary atherosclerosis in premenopausal women, although because ERß was not measured, a stronger and potentially more important relationship with ERß cannot be excluded (26, 27). Knockout mice models of ER{alpha} and/or ERß suggest that ER{alpha} is largely responsible for the protective effects of estrogen after vascular injury (16, 42) and that ERß is important for normotension and vascular function (43). In ovariectomized mice, 17-ß estradiol accelerated reendothelialization in female ERß knockout mice and had no effect in female ER{alpha} knockout mice, suggesting that ER{alpha} mediates the beneficial effect of 17ß-estradiol (44). In a different transgenic mouse model that lacked both apoE and ER{alpha}, 17ß-estradiol treatment of ovariectomized female animals caused minimal reduction in the size of atherosclerotic lesions. The authors suggest that ER{alpha} independently mediates the atheroprotective effort of 17-ßE2 (45). In male ER{alpha} knockout mice, there was a marked reduction in atherosclerotic lesion area, number, and distribution. However, these effects on ER{alpha} were mediated by testosterone (46).

The interpretation and differences in transgenic mouse models and humans is not unusual. Iafrati et al. (47) found that estradiol was equally vasculoprotective in ER{alpha} knockout and wild-type mice, contrary to the observations described above. The controversy in the prospective clinical trials compared with observational studies in humans has also highlighted the multiple interpretations of the available data.

One must bear in mind that the response to acute endothelial injury observed in animal models reflects but one stage of a chronic and progressive disease process. These data highlight the controversy regarding the relative importance of ER{alpha} and ERß in cardiovascular disease, which is likely to differ depending on the specific vascular response and the model under investigation.

In summary, we have described ER expression in functionally distinct layers of the coronary artery wall and correlated ERß expression with the extent of atherosclerosis. ERß was the predominant ER subtype in all coronary artery wall layers. Both ER{alpha} and ERß expression were greatest in the media, a layer replete with VSMCs, and colocalized with VSMCs on immunofluorescence imaging. ER{alpha} expression did not correlate with coronary calcium, plaque area, or calcium-to-plaque ratio, despite evidence that ER{alpha} mediates the protective effects of estrogen in animal models of endothelial injury. We conclude that ERß plays an integral role in atherosclerotic plaque progression and intimal calcification in human coronary arteries.


   Acknowledgments
 
We thank Ms. K. Shogren for expertise in immunocytochemistry and Ms. R. Kiefer for expert editorial assistance.


   Footnotes
 
This work was supported, in part, by an unrestricted educational grant from Solvay Pharmaceuticals, Inc. (to R.C.C.). P.Y.L. was supported by the Neil Hamilton Fairley research fellowship from the National Health and Medical Research Council of Australia (Grant ID 262025). This work was supported, in part, by Public Health Service Grant NHLBI RO1HL 51736-8 and National Center for Research Resources Grant K24RR 17593-1.

Present address for R.C.C.: UHC-5 Endocrinology, 1 South Prospect Street, Burlington, Vermont 05401.

First Published Online April 11, 2006

Abbreviations: ER, Estrogen receptor; GMA, glycolmethylmethacrylate; HT, hormone therapy; LUM, lumen; non-HT, untreated postmenopausal; NS, not significant; PRE, premenopausal; VSMC, vascular smooth muscle cell.

Received December 9, 2005.

Accepted March 31, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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