Correlating Androgen and Estrogen Steroid Receptor Expression with

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The Journal of Clinical Endocrinology & Metabolism 90(2):1041–1046 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-1211

Correlating Androgen and Estrogen Steroid Receptor Expression with Coronary Calcification and Atherosclerosis in Men without Known Coronary Artery Disease Peter Y. Liu, Rose C. Christian, Ming Ruan, Virginia M. Miller, and Lorraine A. Fitzpatrick Division of Endocrinology, Metabolism, Diabetes and Nutrition (P.Y.L., R.C.C., M.R., L.A.F.), Department of Surgery (V.M.M.) and Department of Physiology and Biophysics (V.M.M.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905 Background: Accumulating data emphasize the gender specificity of key components of the atherosclerotic process and the importance of gonadal steroids on the human vasculature. Steroid receptors, including the androgen receptor (AR) and estrogen receptors (ERs) ␣ and ␤ are expressed in key vascular tissues, including endothelial cells and vascular smooth muscle cells. However, the relative abundance and importance of these receptors in the coronary artery are not well defined, particularly in men. We therefore examined AR, ER␣, and ER␤ expression as a function of key components of atherosclerosis, namely plaque and calcium area, in male human coronary arteries. Methods: Coronary arteries were obtained at autopsy from 24 men without known coronary artery disease. Coronary calcification was measured by contact microradiography, and atherosclerotic plaque area was quantified histologically. Coronary artery cross-sections were immunostained for AR,

T

HE MALE PREDOMINANCE in the incidence and prevalence of cardiovascular disease is well recognized (1– 5). Whether this excess is due to gender differences in vascular endothelial function, macrophage lipid loading, or other factors that predispose to atherosclerosis is not known (5). Nevertheless, these epidemiological and mechanistic observations have implicated gonadal hormones and their receptors in the pathogenesis of cardiovascular disease, leading to “estrogen protective” and “androgen harmful” hypotheses (4). Concurrently, fundamental gender differences modulating the impact of gonadal hormones on the vasculature are becoming apparent (6). For example, androgens increase monocyte binding to human umbilical endothelial cells harvested from males but not females (7). Testosterone inhibits proximal aortic arch intimal thickening in castrated male, but not ovarectomized female, rabbits (8). Physiological estrogen and progesterone exposure decreased macrophage lipid loading but only in macrophages obtained from female, not male, donors (9). For these reasons, genderFirst Published Online November 9, 2004 Abbreviations: AR, Androgen receptor; ER, estrogen receptor; GMA, glycolmethylmethacrylate; LUM, lumen; VSMC, vascular smooth muscle cell. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

ER␣, and ER␤ and then measured semiquantitatively in each arterial wall layer (intima, adventitia, and media). Results: AR, ER␤, and ER␣ were expressed in all artery wall layers but most avidly in the media (P < 0.001). ER␤ exceeded ER␣ expression (P < 0.0005). AR expression in the media correlated negatively with plaque area (P ⴝ 0.006, R ⴝ ⴚ0.55), whereas intimal ER␤ expression correlated positively with plaque area (P ⴝ 0.012, R ⴝ 0.50). Conclusions: We conclude that both AR and ER␤ are important in relatively early coronary atherosclerosis, but inversely so, because decreasing AR and increasing ER␤ expression correlate with more extensive atherosclerosis. ER␤ seems to be the predominate ER in coronary arteries harvested from men without known coronary artery disease. Interventional studies are required to assess the functional significance of these observations. (J Clin Endocrinol Metab 90: 1041–1046, 2005)

specific research examining the role of gonadal hormones in the vascular biology of cardiovascular disease is crucial. The androgen receptor (AR) and estrogen receptors (ERs) ␣ and ␤ are all expressed in endothelial and vascular smooth muscle cells (VSMCs) (4, 7, 10 –13). However, the relative extent and role of these receptors in men is not well studied, although data in women (14, 15) and rodents (16), as well as noncoronary arteries (17), are available. Nevertheless, specific data implicating androgens, estrogens, and their receptors in the pathogenesis of male cardiovascular disease are available (18). Randomized trials show that androgen therapy improves markers of vascular function, such as exercise stress testing and flow-mediated dilatation; however, longterm outcome-based studies are lacking (4). In contrast, excess cardiovascular mortality was observed in older men with prostate cancer randomly assigned to medical castration with high-dose oral estrogen, compared with those who underwent orchidectomy (19). Similarly, in postmenopausal women, estrogen therapy alone or combined with progestins does not improve, and may actually worsen, cardiovascular outcome (20 –22). Furthermore, genomic polymorphisms of the AR and ER␣ are associated with impaired vascular function and/or coronary atherosclerosis in men (23–26). The coronary artery is anatomically and functionally distinct from elastic arteries (such as the carotid, brachial, and femoral arteries) because the medial layer is largely composed of VSMCs instead of elastic fibers. Investigating ath-

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erosclerosis in human coronary arteries, by directly assessing key components such as calcium and plaque area, is therefore preferable. Increased coronary calcium content independently predicts future coronary events (27) and is positively correlated with the extent of atherosclerotic disease determined at autopsy (28, 29) or angiography (30). For these reasons, we directly assessed calcium and plaque in human coronary arteries obtained at autopsy from men without known coronary artery disease. We characterized, for the first time, the expression of AR, ER␣, and ER␤ in these vessels using a semiquantitative technique to explore the relative contribution of these receptors in the early pathogenesis of coronary artery disease. Materials and Methods Specimens Intact coronary arteries were obtained sequentially from 24 men requiring full autopsy at the Mayo Clinic, Rochester, MN, under a protocol reviewed and approved by the Mayo Clinic Institutional Review Board. Eligible men were at least 18 yr of age and had provided written consent for research use of autopsy specimens. Patients with known coronary artery disease, including a history of coronary stents, prior coronary artery bypass grafting, or angioplasty were excluded for legal reasons. Men using androgens, antiandrogens, bisphosphonates or with conditions associated with dystrophic vascular calcification, such as hyperparathyroidism or chronic hemodialysis, were excluded. Demographic details were obtained from inpatient and outpatient medical records.

Liu et al. • Sex Steroid Receptors and Coronary Atherosclerosis

Immunohistochemistry One arterial segment was analyzed for each subject. Each paraffinembedded arterial segment was cut 5-␮m thick and immunostained for AR, ER␣, and ER␤ using the ABC Elite Vectastain Kit and avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). Immunostaining was performed using IgG antibodies to AR (mouse antibody; DACO, Carpinteria, CA), ER␣ (rabbit antibody; Santa Cruz Biotechnology, Santa Cruz, CA), and ER␤ (rabbit antibody; Alpha Diagnostics, San Antonio, TX). The ER␤ antibody was specific for ␤1, ␤2, ␤1d3, and ␤2d3 isoforms. 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 controls, antibody was preadsorbed with peptide, overnight, at a refrigerator temperature of 4 C.

Statistical analysis Arteries were scored independently by three observers, who were blinded to the source of the arteries. Intensity of staining was graded semiquantitatively in each of the arterial layers (intima, media, and adventitia), on a scale of 0 – 4, with 0 ⫽ no staining, 1 ⫽ faint, 2 ⫽ minimal, 3 ⫽ moderate, and 4 ⫽ intense immunostaining. Nonparametric one-way ANOVA (Kruskal-Wallis test) was used to detect differences in ranked layer (intima, media, and adventia) scores for androgen and ER staining. Correlation between steroid receptor immunostaining and calcium and plaque area were assessed initially by Pearson’s test, and then confirmed by nonparametric Spearman test, using values obtained in the same arterial section, where possible, and also against mean calcium and mean plaque area. The Spearman test uses ranks, and hence is robust to the influence of outliers or extreme values. All tests were two-tailed, with P ⬍ 0.05 considered significant. A Bonferroni correction for multiple comparisons was made, as appropriate, for nonindependent post hoc tests. Analyses were performed using SAS software, version 8.02 (SAS Institute Inc., Cary, NC).

Specimen preparation The three major coronary arteries (left anterior descending, right coronary, and left circumflex) were dissected intact, and 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. The first and third segments were cut to 1 cm, dehydrated in ascending alcohol concentrations, and embedded in glycolmethylmethacrylate (GMA) using a temperature-controlled method previously reported (31, 32). 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 sections were cut to 0.5 cm, placed in formalin, and then embedded in paraffin for immunohistochemistry.

Contact microradiography and histologic measurements Cross-sections (200 ␮m) 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 (31, 33). Sections were stained with aldehyde fuchsin and eosin counterstain to optimally delineate the elastin laminae to distinguish the intimal, medial, and adventitial components of the arterial wall. Images of stained sections and contact microradiographs were captured digitally by a computer-assisted histomorphometric analysis as previously described (31). Calcium area and the areas circumscribed by the lumen (LUM), internal elastic lamina and external elastic lamina were determined from these images. Plaque area (square millimeters) was calculated by subtracting the LUM area from internal elastic lamina area. The calcium content of each radiographed section 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 (␬ ⫽ pixels/mm2). The calibration factor was calculated, on a standardized microscope slide bearing a scored 1-mm square, as the number of pixel counts with an area of 1 mm2. 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.

Results Subjects

The demographics and risk factors for coronary artery disease for these men are shown in Table 1. Although ages ranged from 18 – 86 (median, 68.5), only four men were less than 60 yr old. One or more of the following cardiovascular risk factors were present in a large proportion (80% of evaluable subjects): diabetes mellitus, hypertension, smoking, obesity, and family history of heart disease. These are defined as follows: 1) diabetes mellitus treated with an oral hypoglycemic agent and/or insulin at the time of death; 2) hypertension requiring treatment; 3) smoking if cigarette consumption occurred within 6 months of death; 4) obesity if listed by the clinician as a medical diagnosis or noted on autopsy; and 5) family history of coronary heart disease if one or more first-degree family members were affected. Hyperlipidemia was not included as a risk factor because lipid TABLE 1. Clinical characteristics Variable

Men (n ⫽ 24) mean ⫾ SEM

Age (yr) Diabetes mellitus Hypertension Smoking Obesity Family history At least one risk factor (including obesity) At least one risk factor (not including obesity) Cancer

67 ⫾ 3 4/20 (20%) 10/19 (53%) 2/18 (11%) 9/20 (45%) 6/17 (35%) 15/19 (79%) 14/19 (74%) 15/24 (63%)

For definitions of cardiovascular risk factors, see text.

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profiles were generally collected only during the terminating admission and did not reflect usual health status. The immediate cause of death was respiratory failure (n ⫽ 8), sepsis (n ⫽ 5), pneumonia (n ⫽ 4), or other causes (n ⫽ 7). The remote cause of death was malignancy in the majority of cases (n ⫽ 15). Arteries

AR, ER␣, and ER␤ expression in the media was consistently higher than either the adventitia or intima (P ⬍ 0.001 for both media vs. adventitia and media vs. intima, Fig. 1). As can be inferred from this figure, ER␤ was more avidly expressed than ER␣ within each and every vascular layer, although significantly so only for the adventitial and intimal layers (P ⬍ 0.0005, for both). ER␤ therefore appears to be the dominant ER expressed in male coronary arteries. Intimal expression of AR, ER␣, and ER␤ significantly differed, with ER␣ expressed least, AR expressed intermediately, and ER␤ expressed most (P ⬍ 0.04 for each pairwise comparison). In contrast, medial expression of AR, ER␣, and ER␤ did not significantly differ. Adventitial expression of AR and ER␣ were equivalent, although ER␤ was significantly higher (P ⬍ 0.0005 for both ER␣ vs. ER␤ and ER␤ vs. AR). Medial AR expression correlated negatively with plaque area (P ⫽ 0.006, R ⫽ ⫺0.55; Fig. 2). To exclude the potential impact of extreme values, Spearman’s test was also performed, and yielded comparable results (P ⫽ 0.002, R ⫽ ⫺0.60). However, significant correlations were not observed for AR expression in the other layers (media and adventitia) or with calcium area. In contrast, intimal ER␤ expression correlated significantly, but positively, with plaque area (P ⫽ 0.012, R ⫽ 0.50; Fig. 3). Comparable results were obtained with Spearman’s test (P ⫽ 0.04, R ⫽ 0.44). No other significant correlations were observed. ER␣ expression did not correlate with plaque or calcium area (data not shown). As illustrated in Figs. 2 and 3, calcium content ranged from 0 –7.4 mm2 but was zero or almost zero in most men, except for five subjects in whom calcium content exceeded 0.4 mm2. This is most likely explained by the exclusion, due to legal reasons, of men with known coronary artery disease. Our data therefore shows that sex steroid receptor expression is not related to coronary calcium content in men with low calcium content, because the full range of expression (from 0 – 4) was disclosed in such men. However, potential rela-

FIG. 2. Scatter-plot relating AR expression in the adventitia (top), media (middle), and intima (bottom) with coronary plaque (left) and calcium (right) area. The solid line indicates significant correlation.

tionships in men with higher calcium content cannot be excluded. Although it is inappropriate to perform formal statistical analysis in this post hoc subgroup of five men, a negative regression between medial AR and calcium content and a positive regression between intimal ER␤ and calcium content are suggested from Figs. 2 and 3, respectively. Age did not correlate with steroid receptor expression, calcium area, or plaque area, because most men were in the narrow range between 60 and 86 yr old (data not shown). Discussion

FIG. 1. AR (left), ER␣ (middle), and ER␤ (right) expression of the intimal (I, single-hatched), medial (M, cross-hatched), and adventitial (A, gray) layers of the male coronary artery. Data are shown as the mean ⫾ SEM. The asterisk indicates the arterial layer in which the significantly highest receptor expression occurred (P ⬍ 0.001).

This study is the first to examine the relative protein expression of AR, ER␣, and ER␤ in male coronary arteries, and the relationships among receptor expression and directly measured calcium and plaque content in these same vessels. Importantly, this study adds to the sparse human data and the accumulating experimental data showing the inverse relationship between androgenic and estrogenic action, and the importance of ER␤, as well as ER␣, in coronary artery disease in men. Our finding, that ER␤ exceeds ER␣ mRNA, extends previous observations in a study that included three male arteries obtained during coronary artery bypass surgery (34). Correctly identifying the relative role of these ste-

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FIG. 3. Scatter-plot relating ER␤ expression in the adventitia (top), media (middle), and intima (bottom) with coronary plaque (left) and calcium (right) area. The solid line indicates significant correlation.

roid receptors will allow rational, pharmacogenetically appropriate drug development (35, 36). Although estrogen protective and androgen harmful hypotheses have long been touted, these hypotheses are not mutually exclusive, and the actions of androgens and estrogens are likely to be much more complicated than a simple dichotomy (4). Nevertheless, our data clearly show inverse, but equally strong (R ⬃ 0.5, for each), relationships between AR or ER␤ expression with plaque size. Furthermore, the media and intima differentially mediate these inverse relationships. The lack of relationship between medial ER␤ expression and plaque size emphasizes the importance of intimal ER␤ in atherosclerosis, which may possibly be mediated by altered endothelial function. However, function was not directly assessed in this study, which was strictly observational. Because our subjects did not have known coronary artery disease, we speculate that these relationships may reflect compensatory mechanisms to limit relatively early atherosclerosis. For this reason, plaque calcification was not prominent in our cohort, because this occurs later and is an actively regulated and potentially crucial process (37, 38). Our data suggests that sex steroid receptor expression is not related to low-level coronary calcification, al-

Liu et al. • Sex Steroid Receptors and Coronary Atherosclerosis

though relationships with more extensive coronary calcification cannot be excluded. Hence, increasing intimal ER␤ expression probably reflects a protective response to intimal plaque development. Experimental data are consistent with the concept that ER␤ mediates the antiproliferative effect of estrogens on VSMCs, which is widely believed to be an early developmental event in the atherosclerotic process. Transfection with ER␤, but not ER␣, further inhibits estradiolstimulated proliferation of VSMCs harvested from men (39). Studies examining the arterial response to injury, rather than de novo atherosclerosis, are also available, although these two processes may not be directly comparable. In female rats, selective ER␤ agonists inhibit the in vivo intimal proliferation of VSMCs after carotid denudation injury (40). Finally, in male animal models, ER␤, but not ER␣, mRNA (16, 41, 42) and protein (42) expression increases after traumatic balloon injury to the endothelium (41, 42) or during transplant rejection (16). However, in most mouse models of atherosclerosis, female, rather than male, gender imparts greater atherosclerotic risk, emphasizing that species-specific factors may modulate hormonal effects. Hence, studies of de novo male human coronary artery disease are required. Our data does not exclude a role for ER␣ in coronary artery disease in men. However, it suggests that ER␣-mediated effects may occur later in the atherosclerotic process. This hypothesis is consistent with epidemiological studies, which largely relate various ER␣ polymorphisms with atherosclerosis in men with presumed late and severe disease, typified by actual myocardial infarction, sudden death, or unstable angina (24 –26). Whether these relationships with ER␣ remain in men with early cardiovascular disease is not known. Furthermore, our finding that ER␤, not ER␣, is significantly correlated with atherosclerosis in men without clinically apparent coronary artery disease, and that ER␤ exceeds ER␣ immunostaining in all layers of the coronary artery, provides circumstantial evidence that ER␤ is also important during earlier stages of atherosclerosis. Whether this is true after progression of atherosclerosis cannot be answered by this study. Nevertheless, it suggests that therapeutic interventions targeting ER␤ may be more useful in the earlier developing stages of atherosclerosis. Such preventative therapy may be feasible in men. For example, in a young man with an inactivating mutation of aromatase, carotid ultrasound detected lipid plaques, which regressed and eventually disappeared with estradiol therapy (43). From our study, research to develop selective estrogen receptor modulators that target vascular ER␤ and their application in men with early coronary disease is warranted. Such studies may compliment similar pilot studies already performed in men with established coronary disease (44). Decreasing CAG repeat length in exon 1 of the AR results in increased transactivation and is associated with reduced endothelial response to ischemia and lower high-density lipoprotein cholesterol (both of which are risk factors for coronary artery disease) (23). These data suggest that increased AR activity is associated with greater cardiovascular risk. Hence, decreasing medial AR expression may reflect a protective response to accumulating plaque. Our data are therefore consistent with the androgen harmful hypothesis and

Liu et al. • Sex Steroid Receptors and Coronary Atherosclerosis

the extensive molecular and clinical data in support thereof (4, 5). Several caveats are important. First, a correlational study cannot definitively determine causality or the direction of the relationships. This is an observational study only, and interventional studies are required to assess the functional significance of these observations, which may be modulated by other factors, such as cofactor expression. Second, the low rates of coronary calcification observed are largely explained by selection bias, which is present here predominately because men with known coronary artery disease were excluded due to legal reasons. Hence, our data are most applicable to men with early coronary artery disease and may not even be generalizable to other groups of patients, particularly those with higher degrees of coronary calcification or more advanced disease. For this reason, the relationships disclosed herein relate either to the initiation of or to very early atherosclerosis. Third, our antibodies may not have detected membrane-bound ER␣, which has been recently described in rat vascular tissues (45), and hence this study did not examine membrane receptor-mediated effects. Fourth, the presence of aromatase in the vascular wall and the possible local production of estradiol from circulating testosterone mean that some of the actions of ER␤ could potentially be mediated by serum testosterone (18). Although this study does not exclude the possibility that other factors, such as macrophages, cell adhesion molecules, or inflammatory mediators, may contribute to the development of coronary artery disease, randomized placebo-controlled studies examining aromatizable and nonaromatizable androgenic compounds in young or older men do not support this hypothesis (46 – 49). Further studies focusing on male and female difference and the effects of menopause, with or without hormone replacement therapy, on steroid receptor expression in coronary and noncoronary arteries (particularly those that appear resistant to atherosclerosis) are desirable. Nevertheless, we have assessed androgen and ER expression in functionally distinct layers of the coronary artery wall and correlated receptor expression with the extent of atherosclerosis. ER␤ is the predominant ER subtype in all coronary artery wall layers, at least in men without clinically evident coronary artery disease. We conclude that AR and ER␤ are both important, although inversely so, in the development of relatively early coronary atherosclerosis. This information may provide therapeutic targets to decrease the male preponderance of ischemic heart disease. Acknowledgments We thank Sean Harrington and Carmen Wick for technical assistance. Received June 24, 2004. Accepted November 3, 2004. Address all correspondence and requests for reprints to: Peter Y. Liu, M.B.B.S., Ph.D., Division of Endocrinology, Harbor-UCLA Medial Center, 1000 West Carson Street, Box 446, Torrance, California 90502. E-mail: [email protected]. This work was supported, in part, by Public Health Service Grants NHLBI RO1HL 51736 – 8 (to V.M.M.) and National Center for Research Resources K24RR 17593–1 (to L.A.F.). P.Y.L. was supported by fellowships from the National Health and Medical Research Council of Aus-

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tralia (Grant ID 262025) and the Royal Australasian College of Physicians (Vincent Fairfax). Current address for R.C.C.: Section of Endocrinology, Diabetes and Metabolism, University of Wisconsin Medical School, Madison, Wisconsin 53717. Current address for L.A.F.: Amgen Inc., One Amgen Center Drive, Mailstop 38 –2-B, Thousand Oaks, California 91320.

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