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Endocrine Care |
Division of Endocrinology, Metabolism, and Nutrition, Department of Internal Medicine (R.C.C., S.H., L.A.F.), Department of Anatomic Pathology (W.D.E.), Department of Health Sciences Research (A.L.O.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Lorraine A. Fitzpatrick, M.D., Endocrine Research Unit, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, Minnesota 55905. E-mail: . fitz{at}mayo.edu
Abstract
Background: Coronary artery calcium, a radiographic marker for atherosclerosis and a predictor of coronary heart disease (CHD), is less extensive in women than in men of the same age. The role of estrogen in the pathogenesis of coronary artery calcification is unknown. We examined the association of estrogen status with extent of calcification and atherosclerotic plaque in coronary arteries of deceased women.
Methods: Coronary arteries were obtained at autopsy from 56 white women age 18–98 yr, 46 postmenopausal and 10 premenopausal. Exclusion criteria included patients with coronary stents, coronary artery bypass surgery, and medical-legal cases. Medical records were reviewed for demographics, CHD risk factors, menstrual status, and use of estrogen replacement therapy. Contact microradiography of coronary arteries assessed true calcium content and atherosclerotic plaque area was analyzed histologically.
Results: The coronary arteries from estrogen-treated postmenopausal women had lower mean coronary calcium content (P = 0.002), mean plaque area (P < 0.0001), and calcium-to-plaque area ratio (P = 0.004) than those from untreated menopausal women. Estrogen status, age, diabetes, and hypertension predicted calcium and plaque area by univariate analysis. After controlling for these CHD risk factors, estrogen status remained an independent predictor of both calcium (P = 0.014) and plaque area (P = 0.001) in all women. Mean calcium area (P < 0.05) but not plaque area (P = 0.44) was significantly greater in women treated with estrogen replacement therapy than in premenopausal women. Coronary calcium (P < 0.007) and plaque area (P < 0.03) varied significantly with age in untreated postmenopausal women, but not in the estrogen-treated or premenopausal women (P = 0.33).
Conclusions: Estrogen status is associated with coronary calcium and plaque area independent of age and CHD risk factors. Estrogen may modulate the calcium content of atherosclerotic plaques, as well as plaque area and may slow the progression of atherosclerosis in women.
ATHEROSCLEROTIC CALCIFICATION OCCURS by an active, regulated process with features common to bone formation (1). Coronary calcium, quantified histologically or radiographically, correlates closely with the overall extent of atherosclerotic disease (2, 3, 4) and is an independent predictor of future coronary events (5, 6, 7, 8). Coronary calcium content is lower in women than in men of the same age (2). Estrogen, which has well-documented antiatherogenic effects (9), may modulate vascular calcification through interactions with the vascular smooth muscle cells, macrophages, and bone matrix proteins found in the vessel wall (10, 11, 12, 13, 14).
The incidence of coronary heart disease (CHD) in estrogen-replete premenopausal women is one-half to one-quarter that of men of similar age and increases markedly following menopause (15, 16). Observational studies suggest that postmenopausal women treated with estrogen replacement therapy (ERT) have a significantly reduced risk of CHD and less extensive coronary calcification than untreated women (17, 18, 19). However, the role of estrogen in the primary and secondary prevention of CHD remains controversial as its cardioprotective effects have not been confirmed in prospective clinical trials in women with established heart disease (20, 21, 22).
To determine whether estrogen status is correlated with the extent of calcification and atherosclerotic disease in pre- and postmenopausal women, we measured true calcium content and plaque area in intact coronary arteries obtained at autopsy.
Materials and Methods
Study groups
Intact coronary arteries were obtained from sequential autopsies performed at the Mayo Clinic (Rochester, MN) on all eligible women (n = 56) whose deaths occurred during the 3-yr period 1997–1999 under a protocol reviewed and approved by the Mayo Clinic Institutional Review Board, IRB No. 0-655-96. Eligible women were age 18 yr or greater with consent for full autopsy and for research use of autopsy specimens. Exclusion criteria included patients with coronary stents, prior coronary artery bypass grafting, use of selective estrogen receptor modulators or bisphosphonates (e.g. alendronate), and conditions associated with dystrophic vascular calcification, such as hyperparathyroidism or chronic hemodialysis. Arteries were not obtained from women whose death occurred as a direct complication of cardiac surgery or catheterization, or in whom acute myocardial infarction was suspected.
Inpatient and outpatient medical records of the Mayo Clinic were reviewed to obtain demographics, menstrual status, estrogen use, and cardiovascular risk factors for each subject. Premenopausal women (n = 10) had a documented menstrual period within one year of death. Women whose last menstrual period or surgical menopause was at least 1 yr before death were considered postmenopausal (n = 46). Estrogen use was documented in the outpatient record and in the admission history and physical exam note of the patient’s final hospitalization to be considered valid. ERT-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-ERT) if they were never users or had been prescribed estrogen <6 months before death.
The coronary heart disease (CHD) risk factors documented for each subject included smoking, hypertension, diabetes mellitus, obesity, and family history of heart disease. These CHD risk factors were defined by these criteria: 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 clinician as a medical diagnosis or noted on autopsy, and 5) family history of CHD if one or more first degree family members were affected. Hyperlipidemia was not included as a risk factor because lipid profiles were collected during subject’s final hospitalization(s) and therefore not reflective of usual health status.
Specimen preparation
The three major coronary arteries, left anterior descending, right coronary, and left circumflex were dissected intact from the heart at autopsy. Specimens were not decalcified during sample preparation to preserve true calcium content. Two sequential one-centimeter segments were cut from the proximal 3 centimeters of each artery, dehydrated in ascending alcohols and embedded in glycolmethylmethacrylate using a temperature-controlled method previously reported (10, 23). Glycolmethylmethacrylate is a hard, clear plastic monomer which allows excellent preservation of vessel morphology and calcium content during preparation of sections.
Contact microradiography
Cross-sections (200 µm) were cut from the proximal end of each arterial segment on an Isomet low speed saw (Buehler, Ltd., Lake Bluff, IL). Each section was placed on a high-resolution emulsion coated glass slide (Microtome Technology, Inc., San Jose, CA) and exposed to radiation for 5 min at 20KV. The section was removed from the slide after exposure and the microradiographs were developed, fixed, washed and dried to the manufacturers’ specifications (Eastman Kodak Co., Rochester, NY) as previously described (10, 23).
Histologic measurements
After contact microradiography, sections were stained with aldehyde fuchsin and eosin counterstain for optimal delineation of the elastin laminae. Images of stained sections and contact microradiographs were captured under 10x to 20x power using a Nikon microphot FXA microscope equipped with a Progress 3012 camera. All images were captured using Photoshop (Adobe Systems, Inc., San Jose, CA) and software macros were calibrated using images of a stage micrometer at a resolution of 1996 x 1450 pixels. KS 400 analysis software macros were written to calculate the calcium area and the areas circumscribed by the lumen, internal elastic lamina, and external elastic lamina. Lumen area was subtracted from internal elastic lamina area to give plaque area in square millimeters.
The calcium content of each radiographed section was analyzed by pixel count as in computerized tomography. The pixel count was divided by the pixel size calibration factor (
= pixels/mm2) to obtain calcium area in square millimeters.
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 6 sections per subject. Sections in which the arterial circumference was interrupted or lumen area not well preserved were excluded from analysis. A total of 319 sections were analyzed for calcium area [n = 57 for premenopausal women (PRE), n = 77 for ERT and n = 185 for non-ERT] and 297 sections (n = 46 for PRE, n = 76 for ERT and n = 175 for non-ERT) were analyzed for plaque area.
Figure 1
demonstrates the methodology used to assess true calcium content and plaque area. Figure 1A represents a methylmethacrylate-embedded section of a right coronary artery stained with aldehyde fuchsin and eosin to delineate the internal elastic lamina. The plaque area fills the space between the internal elastic lamina (arrow) and the lumen (white). The black stained areas are calcified, and the yellow and red stained areas are uncalcified plaque. Figure 1B is the contact microradiograph of the same artery indicating the calcified area in light green. The dotted outline traced from the stained artery is superimposed on the microradiograph. Digital imaging measured the total calcium content.
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Average calcium and plaque areas were calculated for each woman from values on all analyzable sections, a maximum of two sections from each artery, yielding up to six sections per subject. Square roots of the calcium and plaque areas were used for all analyses to stabilize the variance and skewness of the data.
Data are presented as mean ± SD unless otherwise indicated. Group characteristics were compared using nonparametric Wilcoxon rank-sum or 
-square tests, as appropriate. Linear regression was used to assess relationships among calcium or plaque areas and clinical variables such as estrogen status, age, diabetes, and other CHD risk factors. Significant univarate predictors were evaluated simultaneously via multiple regression analyses. Dummy variables were used to represent estrogen status, with the premenopausal women being the reference group. No difference was found in square root calcium or plaque between the PRE and ERT groups when controlling for age (P = 0.56 and P = 0.84, respectively). Hence, for multiple regression models, the PRE and ERT groups are combined.
Results
Premarin (conjugated equine estrogen) alone (n = 5) or in combination with medroxyprogesterone (n = 4) was the predominant oral estrogen used. Three subjects were on transdermal E2, and one subject was taking ethinyl E2.
Immediate causes of death included pneumonia (n = 20), respiratory failure (n = 16), infection (n = 4), gastrointestinal bleeding (n = 4), pulmonary embolism (n = 6), sudden death/arrhythmia (n = 3) or other (n = 3). A history of clinical CHD (myocardial infarction, arrhythmia, or congestive heart failure) was documented in 13 of 46 (28%) postmenopausal women and in none of the 10 premenopausal women.
The CHD risk factors and demographic and clinical characteristics of the PRE, ERT, and non-ERT groups are listed in Table 1. As expected, the PRE women (n = 10) were younger than the postmenopausal women. No significant difference in age was noted between ERT and non-ERT women. Diabetes mellitus type II (DM II) was more prevalent in the non-ERT than ERT women (24.2% vs. 0%, P = 0.05). The prevalence of other CHD risk factors did not vary significantly between the ERT and non-ERT groups. There were no significant differences in mean years of education, an indicator of socioeconomic status, among the three groups. Women in the ERT group were more than twice as likely to have undergone a total abdominal hysterectomy-bilateral salpingo-oophorectomy (9/13, 70%) than women in the non-ERT group (12/33, 36.4%, P = 0.055). The clinical differences we report between the treated and untreated postmenopausal women are consistent with national surveys. In NHANES III, only 6% of current hormone replacement therapy (HRT) users had DM type I or II. The greater prevalence of hysterectomy in the ERT women is also consistent with NHANES III, which found that 75% of women with surgical menopause had ever used HRT, vs. 30% of women with natural menopause (24).
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). In a multivariate analysis, estrogen status, age and diabetes, but not hypertension, were significant independent predictors of plaque and calcium area (Table 3).
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). Calcium content was 90% lower in women on ERT than in nonusers (non-ERT) (P = 0.002).
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Figure 2 indicates the amount of plaque (SQRT plaque) in each group (non-ERT, ERT, and PRE). There was significantly more plaque in the non-ERT group compared with the ERT or PRE group (P = 0.0001 and P = 0.0003, respectively). Similar findings for calcification are demonstrated in Fig. 3. The mean SQRT calcium is greater in the non-ERT group compared with ERT-treated women (P = 0.0003) and was greater in non-ERT vs. PRE (P < 0.0001). We assessed the mean amount of CAC and plaque in each group as a function of age. Coronary calcium varied significantly with age in untreated postmenopausal women (non-ERT, P < 0.007), but not in the ERT or PRE women (P = 0.4) (Fig. 4). Plaque area varied significantly with age only in non-ERT women (P < 0.03) (Fig. 5).
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This is the first study to examine the association of estrogen status with the true calcium content of atheromatous plaques. Estrogen status independently correlated with coronary calcium, plaque burden, and the proportion of calcium to plaque in both postmenopausal and premenopausal women. These associations were not explained by differences in the distribution of known CHD risk factors (age, diabetes, hypertension), osteoporosis, or socioeconomic status. We found that estrogen use in postmenopausal women was associated with a significantly lower calcium-to-plaque ratio (Table 4
). Differences in these ratios suggest that calcification and plaque formation may progress at different rates rather than in parallel, and that unique mechanisms may be involved in these two pathologic processes.
Estrogen may modulate arterial calcification, which has many features of bone formation and repair, by interactions with the bone matrix proteins and smooth muscle cells in the arterial wall. The extracellular matrix of atheromas, like bone osteoid, contains the components required for mineralization: collagen type I, bone matrix proteins, such as GLA, osteopontin, and osteonectin, osteoblast-like cells and matrix vesicles (1). Estrogen inhibits the proliferation of vascular smooth muscle cells (VSMC), which may differentiate into osteoblast-like cells in atheromas, and are capable of calcifying in vitro (25). Estrogen down-regulates TNF-
, a cytokine found in bone and in atheromas; TNF- enhances VSMC calcification in vitro (26). Thus, inhibition of TNF- in VSMCs by estrogen may prevent the accumulation of matrix and calcification in vascular tissue. Both estrogen and TNF- regulate the OPG/RANK/RANKL cytokine signaling pathway, which plays a major role in both dystrophic and physiologic calcification (27). An animal model demonstrating the role of cytokines in calcification is the OPG-deficient mouse, which develops both osteoporosis and premature arterial calcification (1).
Neither coronary artery calcium content nor plaque area was associated with age in the estrogen-replete women (premenopausal or postmenopausal) (Figs. 4 and 5). In contrast, plaque area and coronary calcium increased significantly as a function of age in postmenopausal, estrogen-deplete women. These data suggest that estrogen, endogenous or exogenous, may inhibit progression of atherosclerosis and associated calcification in aging women.
In our study, users of ERT had only 10% of the calcium area and 50% of the plaque area of untreated postmenopausal women. Our findings are consistent with those of many observational studies, which suggested that estrogen is cardioprotective (15, 16, 17, 28, 29, 30, 31, 32), although secondary prevention trials have not confirmed these observations to date (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). A causal relationship between ERT use and atherosclerosis would be supported by demonstrating a greater effect with a specific regimen and/or longer duration of use. Our sample size was not adequate to determine how different hormone replacement regimens correlated with plaque and calcium content. A large prospective clinical trial of significant duration is needed to determine the effect of ERT on EBCT-quantified coronary calcium and coronary events.
We can only speculate whether the lower calcium-to-plaque ratio observed in the estrogen-replete women is protective against coronary events, as the effect of calcification on plaque behavior remains controversial. Although the majority of lesions that rupture and cause sudden death are calcified, it is unclear whether calcium makes plaques vulnerable to rupture (34, 35, 36, 37, 38). Regardless, it is well documented that calcified arterial plaques are significantly more likely to dissect or experience restenosis after angioplasty (39).
A strength of our study is the use of human coronary arteries, rather than surgical specimens of the carotid or femoral arteries. We were able to investigate plaque morphology and calcium content in the coronary circulation rather than in surrogate vessels or animal models. A limitation inherent to autopsy studies is the retrospective collection of clinical data. In our study, this limitation was offset by using arteries collected within hours of death from patients at our own institution; this gave us immediate access to medical records from the hospitalizations and outpatient visits that directly preceded death.
Another strength of our study is the application of methods unique to the analysis of calcified tissue (such as bone) to determine the true calcium content of even minimally calcified coronary arteries. First, we did not decalcify specimens during preservation. Decalcification is performed in most histopathology labs to facilitate cutting sections, and results in underestimation of calcium content. Contact microradiography of arterial sections allowed us to accurately measure calcium deposits which were small, scattered, and multifocal.
In conclusion, our data indicate a strong negative association between estrogen status and both coronary calcium and plaque, and suggest that coronary calcification and plaque formation may progress at different rates in the presence of estrogen. In light of the recent controversies related to findings of prospective ERT trials, such as Heart and Estrogen/Progestin Replacement Study (20) and Estrogen Replacement and Atherosclerosis Study (21), understanding the role of estrogen in the pathogenesis of the calcified, atherosclerotic plaque has become increasingly relevant.
Acknowledgments
We acknowledge the assistance of Ms. Ruth Kiefer for excellent editorial assistance, and Ms. Nancy Diehl for data entry. We are grateful to the Bone Histomorphometry Laboratory (Ms. Janis Donovan, Ms. Julie Burgess, and Ms. Donna Jewison) for their expert advice and resources.
Footnotes
This work has been supported, in part, by NIH Grant RO1-HL-51736-5 and unrestricted grants from American Home Products and the Mayo Foundation.
Abbreviations: CHD, Coronary heart disease; DM II, type 2 diabetes mellitus; ERT, estrogen replacement therapy; HRT, hormone replacement therapy; non-ERT, postmenopausal women not treated by ERT; PRE, premenopausal women; VSMC, vascular smooth muscle cells.
Received August 6, 2001.
Accepted December 6, 2001.
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