Circulating Estradiol Is an Independent Predictor of Progression of ...

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The Journal of Clinical Endocrinology & Metabolism 91(11):4433– 4437 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2006-0932

Circulating Estradiol Is an Independent Predictor of Progression of Carotid Artery Intima-Media Thickness in Middle-Aged Men Åsa Tivesten, Johannes Hulthe, Karin Wallenfeldt, John Wikstrand, Claes Ohlsson, and Bjo¨rn Fagerberg Institute of Internal Medicine (Å.T., K.W., C.O., B.F.), The Wallenberg Laboratory for Cardiovascular Research (Å.T., J.H., K.W., J.W., B.F.), and Department of Clinical Pharmacology (C.O.), Sahlgrenska University Hospital, Go¨teborg University, S-413 45 Go¨teborg, Sweden; and Medical Advisors at AstraZeneca (J.H., J.W.), S-431 83 Mo¨lndal, Sweden Context: Estrogen treatment of men with prostate cancer is associated with increased cardiovascular morbidity and mortality; however, the role of endogenous estrogen levels for atherosclerotic disease in men is unknown. Objective: The objective of the study was to determine whether endogenous serum estradiol (E2) levels predict the progression of carotid artery intima-media thickness in men. Design, Setting and Participants: This was a population-based, prospective cohort study (the Atherosclerosis and Insulin Resistance study) conducted in Go¨teborg, Sweden, among 313 Caucasian men without cardiovascular or other clinically overt diseases. Carotid artery intima-media thickness, an index of preclinical atherosclerosis, was measured by ultrasound at baseline (58 yr of age) and after 3 yr of follow-up. Serum sex hormone levels and cardiovascular risk factors (body mass index, waist to hip ratio, systolic blood pressure, serum triglycerides, plasma c-peptide, and smoking status) were assessed at study entry.

T

HE MALE PREPONDERANCE in cardiovascular disease has often been ascribed to the protective effects of endogenous estrogens in premenopausal women (1–3). Support for this assumption derives from numerous studies reporting beneficial effects of estrogens on serum lipids, vascular function, and experimental atherosclerosis in both males and females (2, 4, 5). Recently the estrogen protection hypothesis was challenged by results from the Women’s Health Initiative, which showed that hormone replacement therapy may increase the risk for myocardial infarction and stroke in postmenopausal women (6). However, already in the late 1960s, it was recognized that estrogen treatment of men with prostate cancer is associated with increased cardiovascular morbidity and mortality (7). Moreover, the Coronary Drug Project in the 1960s showed an excess of deaths and recurrent infarction in men treated with estrogen after myocardial infarction (8). It is believed that the major mechanism underlying the adverse cardiovascular effects of oral estrogen treatment in men is the first passage effect in the liver, leading to increased First Published Online August 29, 2006 Abbreviations: BMI, Body mass index; CV, coefficient of variation; E2, estradiol; HDL, high-density lipoprotein; LDL, low-density lipoprotein. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Intervention: There was no intervention. Main Outcome Measures: Association between baseline total and free E2 levels and progression of carotid intima-media thickness over 3 yr with adjustments for cardiovascular risk factors was measured. Results: In univariate analyses, both total and free E2 levels at baseline were positively associated with the annual change in intimamedia thickness. In linear regression models including E2 and cardiovascular risk factors, low-density lipoprotein and high-density lipoprotein cholesterol and E2 were identified as independent predictors of progression of carotid artery intima-media thickness (total E2 beta ⫽ 0.187, P ⫽ 0.001; and free E2 beta ⫽ 0.183, P ⫽ 0.003). Conclusions: Circulating E2 is a predictor of progression of carotid artery intima-media thickness in middle-aged men. Further studies are needed to investigate the role of endogenous E2 for incident cardiovascular disease events. (J Clin Endocrinol Metab 91: 4433– 4437, 2006)

synthesis of clotting factors and a hypercoagulable state (7). Because estrogen therapy is suggested to be an efficient treatment of prostate cancer, clinical trials with parenteral administration of a slow-release preparation of estradiol (E2) have been initiated (9, 10). However, the results indicate that even this treatment regimen increases the risk of ischemic heart disease events in men (9, 10). E2 circulates in low, but measurable, levels in men; however, few studies have investigated the role of endogenous E2 for male cardiovascular health. The aim of the present study was to examine whether endogenous serum E2 levels predict the progression of atherosclerosis in men. We analyzed the association between circulating baseline levels of E2 and progression of carotid intima-media thickness, a wellestablished index of atherosclerosis (11–13), in a cohort of apparently healthy, middle-aged men. Patients and Methods Study design As described previously (14), this is a longitudinal study based on a stratified sampling of randomly selected and screened men (n ⫽ 818), with the underlying aim to include men with different degrees of obesity and insulin sensitivity (n ⫽ 391) to examine whether insulin resistance is associated with subclinical atherosclerosis. The subjects were randomly selected from among 58-yr-old men listed in the county council register of Gothenburg, Sweden. To keep age and the sociocultural components within a limited range, inclusion criteria were 58 yr of age,

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J Clin Endocrinol Metab, November 2006, 91(11):4433– 4437

Swedish ancestry, and living in Go¨teborg, Sweden. Exclusion criteria were cardiovascular or other clinically overt diseases (e.g. malignancy or psychiatric disease) and continuous medication with cardiovascular drugs. In connection with the screening examination, the subjects were divided into quintiles of a body mass index (BMI)/blood glucose score (14), which allowed immediate stratification and final inclusion in the study. All subjects in quintiles 1 and 5 and every fifth subject in quintiles 2– 4 of the BMI/blood glucose score were invited to participate in future cross-sectional and prospective studies (n ⫽ 391). This procedure resulted in a final population sample that was very similar to the screened men in mean characteristics. All men received both written and oral information before giving written consent to participate in the study. The study was approved by the ethics committee at the Sahlgrenska University Hospital. The mean follow-up time was 3.2 ⫾ 0.2 yr. We were able to reexamine 342 subjects, of whom 313 had complete data on sex hormone levels and intima-media thickness. Exclusion from present analyses were due to death (n ⫽ 4), having moved or refusal to participate (n ⫽ 45), or incomplete intima-media thickness data (n ⫽ 27; see below) or sex hormone data (n ⫽ 2). A comparison of men with (n ⫽ 313) and without (n ⫽ 27) available follow-up data on intima-media thickness yielded no statistically significant differences in total (89.8 ⫾ 32.9 vs. 94.1 ⫾ 31.8 pmol/liter; P ⫽ 0.52) or free (1.61 ⫾ 0.66 vs. 1.70 ⫾ 0.61 pmol/liter; P ⫽ 0.54) E2 levels at baseline. In a subgroup of patients (n ⫽ 118), dualenergy x-ray absorptiometry was performed at baseline (see Refs. 15 and 16).

Measurements and procedures All measurements were performed in the morning. Information on general health and smoking habits were obtained by a self-administered questionnaire. Alcohol intake was evaluated by adding up the current average daily intakes of wine, beer, and spirits. Blood pressure was measured twice (14). Venous blood samples were drawn after a fasting period of 10 –12 h; serum was separated and frozen within 4 h at ⫺70 C. Cholesterol and triglyceride levels were determined by fully enzymatic techniques (14). High-density lipoprotein (HDL) cholesterol was determined after precipitation of apolipoprotein B-containing lipoproteins with Mn-chloride and dextran sulfate. Low-density lipoprotein (LDL) cholesterol was calculated as described (14). Plasma levels of c-peptide, which reflect the amount of insulin secretion better than serum insulin levels (17), were assessed by a RIA (Guildhay, Guildford, UK).

Assessment of sex hormones Total E2 was measured using an ultrasensitive RIA [Spectria estradiol sensitive RIA; Orion Diagnostics, Espoo, Finland; limit of detection 5 pmol/liter, functional sensitivity 9.3 pmol/liter, intraassay coefficient of variation (CV) 3%, interassay CV 6%]. The cross-reactivity of the E2 assay with other steroid hormones is as follows: estrone, 0.97%, 16-oxoestradiol, 0.86%, E2 3-glucuronide, 0.61%, estriol, 0.44%, 16-hydroxyestrone, 0.26%, progesterone, less than 0.05%, corticosterone, 0.02%, testosterone, less than 0.001%. Total testosterone was measured using a RIA (Orion Diagnostics; limit of detection 0.1 nmol/liter, intraassay CV 6%, interassay CV 5%), and SHBG was measured using an immunoradiometric assay (Orion Diagnostics; limit of detection 1.3 nmol/liter, intraassay CV 2%, interassay CV 4%). Free testosterone and free E2 were calculated according to the method described by Vermeulen et al. (18) and Van den Beld et al. (19), taking the concentrations of total testosterone, total E2, and SHBG into account and assuming a fixed albumin concentration of 43 g/liter. All samples were analyzed in duplicate in one laboratory.

Assessment of carotid intima-media thickness Examination was performed with an ultrasound scanner (Acuson 128; Acuson, Mountain View, CA) with a 7-Mhz linear transducer aperture of 38 mm. A 10-mm segment of the left and right carotid arteries was scanned at the level of bifurcation, and images for intima-media thickness measurements were recorded from the far wall in the common carotid artery and the carotid artery bulb. The software program gives the average thickness of the intima-media complex on the left and right side for the common carotid artery and the carotid artery bulb, respec-

Tivesten et al. • Estradiol Predicts IMT Progression in Men

tively. The intima-media thickness was defined as the distance from the leading edge of the lumen-intima interface to the leading edge of the media-adventitia interface of the far wall. Lumen diameter of the carotid artery was defined by the distance between the leading edges of the intima-lumen interfaces of the near wall and lumen-intima of the far wall. Images were measured in an automated analyzing system (20). The interobserver variability for measurement of intima-media thickness in the common carotid artery and the carotid artery bulb has been shown to be 5.3 and 6.0%, respectively (21). A composite measure of intimamedia thickness in the carotid artery was calculated as the mean intimamedia thickness in the common carotid and the carotid artery bulb on the left and right side.

Statistical analysis Values are given as mean ⫾ sd. Skewed variables were log transformed before statistical testing. The annual change in intima-media thickness and lumen diameter was tested against 0 in a one-sample t test, and 95% confidence intervals were calculated. Univariate associations were examined with Pearson’s correlation analysis. Partial correlations were performed between serum E2 levels and the annual change in intima-media thickness, controlling for the BMI/blood glucose score used for stratification. Multiple linear regression was used to explore the relation between serum E2 levels at baseline and the annual increase in carotid intima-media thickness with adjustment for cardiovascular risk factors (BMI, waist-to-hip ratio, systolic blood pressure, serum LDL cholesterol, HDL cholesterol and triglycerides, plasma c-peptide, and smoking status). Multiple regression using spline functions was applied to estimate the relationship between serum free E2 levels and the annual change in intima-media thickness, with the BMI/blood glucose score included as an independent variable. The regression function was comprised by linear pieces at the ends and quadratic functions in the intermediate intervals and the knots were chosen at the serum-free E2 values 1.3, 1.8, and 2.4 pmol/liter. The location of the knots were based on tertiles of the cohort with respect to free E2 levels, but because of the wide range of free E2 levels within the third tertile, the third knot was introduced dividing the upper tertile in two parts with the relationship 2:1. P ⬍ 0.05 (two tailed) was considered statistically significant. Statistical analyses were performed using SPSS for Windows (version 13.0; SPSS, Chicago, IL).

Results

The general characteristics of the subjects at baseline have been previously described elsewhere (22). Briefly, mean ⫾ sd BMI was 26.2 ⫾ 4.2 kg/m2, waist to hip ratio was 0.94 ⫾ 0.06, systolic blood pressure was 134 ⫾ 18 mm Hg, plasma cpeptide was 600 ⫾ 276 pmol/liter, serum LDL cholesterol was 4.06 ⫾ 0.97 mmol/liter, serum HDL cholesterol was 1.28 ⫾ 0.36 mmol/liter, and serum triglycerides was 1.48 ⫾ 0.91 mmol/liter. Alcohol consumption was 12.9 ⫾ 14.8 g/d and 20% were current smokers. The composite intima-media thickness was 888 ⫾ 154 ␮m at study entry and 899 ⫾ 164 ␮m after 3 yr. During follow-up there was a significant increase in the composite intima-media thickness measure after adjustment for follow-up time (mean 3.32 ␮m/yr, 95% confidence interval 0.28 – 6.36 ␮m/yr, P ⫽ 0.03). There was also a small but significant increase in carotid lumen diameter during follow-up (data not shown). Sex hormone levels at baseline are presented in Table 1. Both total and free E2 levels were positively correlated to the annual change in intima-media thickness (Table 2). Furthermore, baseline total and free E2 levels were negatively correlated with the change in lumen diameter over 3 yr (total E2 r ⫽ ⫺0.138, P ⫽ 0.01 and free E2 r ⫽ ⫺0.121, P ⫽ 0.03). There was no association between SHBG or total or free testosterone levels at baseline and change in intima-media thickness (Table 2). To eliminate the possible influence of the stratified



TABLE 1. Serum sex steroid levels at baseline (n ⫽ 313) Baseline levels

89.8 ⫾ 32.9 1.61 ⫾ 0.66 18.3 ⫾ 6.2 0.34 ⫾ 0.11 40.7 ⫾ 19.0

Total E2 (pmol/liter) Free E2 (pmol/liter) Total testosterone (nmol/liter) Free testosterone (nmol/liter) SHBG (nmol/liter) Values are given as means ⫾

SD.

sampling of subjects, partial correlations were performed, showing that the correlations between total and free E2 levels and the annual change in intima-media thickness were still present when controlling for the variable used for the stratification, i.e. a BMI/blood glucose score (total E2 r ⫽ 0.176, P ⫽ 0.002; and free E2 r ⫽ 0.168, P ⫽ 0.003). In regression models, including total or free E2 and cardiovascular risk factors, E2 and LDL cholesterol were identified as independent positive predictors and HDL cholesterol as a negative predictor of progression of carotid artery intima-media thickness. The nominally highest predictive value was seen for total and free E2, respectively (Table 3). BMI, waist to hip ratio, systolic blood pressure, serum triglycerides, plasma c-peptide and smoking status did not contribute to the variability in intima-media thickness during follow-up (data not shown). Adjustment for baseline intimamedia thickness or annual change in lumen diameter in the multivariate model did not affect the results (data not shown). Inclusion of the square of total or free E2 in the model did not improve the explained variance. The association between baseline E2 and the annual change in intima-media thickness was also studied in a subset of randomly selected subjects (n ⫽ 118) in whom measurements of fat mass by dual-energy x-ray absorptiometry had been performed at baseline. Substituting total body fat, as assessed by dual-energy x-ray absorptiometry and expressed as percentage of total body weight, for BMI and waist to hip ratio in the model did not affect the predictive value of total and free E2 for the annual increase in intima-media thickness (data not shown). Adjustment for alcohol intake did not change the relation between total or free E2 and intima-media thickening (data not shown). Multiple regression using spline functions was applied to further explore the relationship between serum free E2 and the annual increase in intima-media thickness (Fig. 1). The spline analysis suggests that there is a positive association between free E2 and annual increase in intima-media thickness at higher, but not lower, free E2 levels. TABLE 2. Univariate correlation between serum sex steroid levels at baseline and annual change in carotid artery intimamedia thickness

Total E2 Free E2 Total testosterone Free testosterone SHBG

r

P value

0.164 0.145 0.037 0.024 0.025

0.004 0.010 0.51 0.67 0.66

Pearson’s correlation analysis, n ⫽ 313. r, Pearson’s correlation coefficient.

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TABLE 3. The independent predictive values of LDL cholesterol, HDL cholesterol, and total or free E2 for the annual change in carotid artery intima-media thickness

Model including total E2 Total E2 LDL cholesterol HDL cholesterol Model including free E2 Free E2 LDL cholesterol HDL cholesterol

Standardized beta

P value

0.187 0.159 ⫺0.165

0.001 0.008 0.01

0.183 0.165 ⫺0.161

0.003 0.006 0.02

The multiple regression model used the annual change in carotid artery intima-media thickness as the dependent variable and serum total or free E2 and major cardiovascular risk factors (BMI, waist to hip ratio, systolic blood pressure, serum LDL cholesterol, HDL cholesterol and triglycerides, plasma c-peptide, and smoking status) as independent variables. The table shows the significant independent predictors in the model.

Discussion

There is abundant evidence supporting a role of sex steroids for cardiovascular health; however, little attention has been given to the role of endogenous estrogen levels for atherogenesis in males. Our study demonstrates that circulating E2 is a strong predictor of progression of carotid artery atherosclerosis, as estimated by carotid intima-media thickness, in a cohort of apparently healthy, middle-aged men. Only a few studies have addressed the relation between endogenous E2 levels and atherosclerosis in men (23, 24). In a recent 4-yr longitudinal study on the relation between baseline E2 levels and progression of carotid artery intimamedia thickness in men, Muller et al. (23) found a tendency toward a positive association between serum E2 and intimamedia thickening, but the data did not reach statistical significance. In comparison, our results showed that the predictive value of E2 was nominally equal to those of LDL and HDL cholesterol. The differences in results of the two studies may be explained by the fact that our study cohort was larger (n ⫽ 313) than that of Muller et al. (n ⫽ 195); our subjects were middle-aged with an age span of only 1 yr (58 yr vs. 77–96 yr); subjects with a medical history at baseline were excluded from our study, whereas Muller et al. included subjects both with and without prevalent cardiovascular disease and medication; and 80% of our baseline population participated in the follow-up examination vs. 48% in the study by Muller et al. The dominant belief has long been that endogenous estrogens are protective from cardiovascular disease in females, and this hypothesis has been extended to include males as well (4, 25). Support for this hypothesis derives from the finding of premature atherosclerosis in two men with a disruptive mutation in the estrogen receptor-␣ gene and aromatase deficiency, respectively (4). However, there is accumulating evidence suggesting a detrimental effect of endogenous estrogens in males. For example, male estrogen receptor-␣ knockout mice surprisingly exhibited decreased susceptibility to atherosclerosis (26), and the CC genotype of the estrogen receptor-␣ c.454 –397T⬎C polymorphism, possibly associated with enhanced receptor function (27), has been coupled to increased incidence of myocardial infarction in men (28, 29). Furthermore, in a case-control study of men

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FIG. 1. Description of the relationship between serum free E2 at baseline and the annual increase in intima-media thickness by a spline curve generated by a multiple regression using spline functions. The variable used for stratification of subjects (a BMI/blood glucose score) was included as an independent variable in the regression analysis. For the curve shown in the figure, the BMI/blood glucose score was put equal to the mean 8.82.

in the Framingham cohort, serum E2 levels were significantly increased in subjects with coronary heart disease (30). Our data support the notion of a positive association between endogenous E2 and atherosclerotic disease. At present, there are still not enough data to allow any firm conclusions about the relation between endogenous E2 levels and male cardiovascular disease events (31). Thus, there is a need for wellpowered longitudinal studies investigating the predictive value of serum E2 levels for incident cardiovascular events. Atherosclerosis is the principal mechanism underlying coronary heart disease, but most cases of myocardial infarction are due to the formation of an occluding thrombus on the surface of an atherosclerotic plaque. The adverse cardiovascular effects of high-dose oral estrogen to men have often been ascribed to the prothrombotic effects of estrogen (7, 8). Phillips et al. (32) suggested that high serum E2 levels may predispose to coronary thrombosis but not to atherosclerosis. However, both the area of coronary atherosclerotic lesions and the presence of coronary thrombosis have been shown to be associated with the CC genotype of the estrogen receptor-␣ c.454 –397T⬎C polymorphism, suggesting the presence of a link between endogenous estrogen action and atherosclerosis in men (28). In accordance, a recent study showed a positive correlation between coronary atherosclerosis and intimal expression of estrogen receptor-␤ in men without known coronary artery disease (33). Our findings of a positive association between serum E2 levels and intimamedia thickening supports the notion that estrogens, besides possibly increasing the risk for thrombosis and thereby cardiovascular events, also have an important impact on atherogenesis in men. In the present study, we found a positive association between free E2 and the annual increase in intima-media thickness at higher, but not lower, free E2 levels. This might suggest that high doses of E2 (and possibly other estrogens) should be avoided in clinical trials. Indeed, high-dose par-


enteral administration of a slow-release preparation of E2 was recently shown to increase the risk of nonfatal ischemic heart disease events in men (9). Our data suggest that these adverse cardiovascular effects of high-dose E2 treatment might, at least in part, be attributed to accelerated atherosclerotic disease. In the present paper, we found that the association between serum E2 and progression of carotid intima-media thickening was independent of traditional risk factors for atherosclerosis but other possible mechanisms for this association have not been examined. Presently we have no plausible biological explanation for this relationship and this is an important area for future research. In contrast to some previous studies reporting a negative association between serum testosterone levels and carotid intima-media thickness (23, 24, 34), we found no such association. However, with one exception (23), these have been cross-sectional studies. Our data are consistent with convincing results of a lack of relationship between circulating testosterone and incident coronary artery disease in men (25). Study limitations

A limitation of the present study is the use of RIA for E2 measurements, as it has been described that the RIA technique might result in artifacts at low E2 levels (35). However, total E2 also remained an independent predictor of intimamedia thickening (standardized beta ⫽ 0.179, P ⫽ 0.004) after removal of subjects with the 10% lowest values (⬍53 pmol/ liter) of total E2 from the analyses, indicating that the main finding in this study was not flawed by artifacts in the lower range of E2 levels. Anyway, it cannot be excluded that the findings of the spline analysis (displayed in Fig. 1) might be confounded by this limitation of the RIA technique. Furthermore, it should be noted that our results are limited to middle-aged Caucasian men without overt cardiovascular disease. The stratified sampling of the study subjects results in a study cohort that is not truly representative for the studied population, and it is not obvious that the findings of the study can be generalized across a wider population. However, the analyses have been adjusted for the stratified sampling by adjusting for the stratification variable. Lastly, our follow-up time is too short to meaningfully analyze the associations between hormone levels and cardiovascular disease events. Although carotid intima-media thickness is a well-established index of atherosclerosis that correlates with prevalent and incident heart disease and stroke (11–13), it is still a surrogate marker for the clinical manifestations of atherosclerosis. Summary and conclusions

In summary, we have shown that circulating E2 is a strong predictor of progression of carotid artery intima-media thickness in apparently healthy, middle-aged men. The predictive value of E2 was nominally equal to those of LDL and HDL cholesterol and it was independent of these and other major cardiovascular risk factors. Our data support the concept of endogenous E2 as a regulator of atherogenesis and suggest that high levels of circulating E2 may be an overlooked risk factor for atherosclerotic disease in men.


Acknowledgments We thank Dr. Lena Bokemark and Maud Peterson, Marie-Louise Ekholm, Carita Fagerlund, and Caroline Schmidt (the Wallenberg Laboratory) for excellent research assistance. We also thank Helena Johansson and Anders Ode´n, professor in biostatistics, for statistical advice and calculation of the multiple regression using spline functions. Received May 2, 2006. Accepted August 17, 2006. Address all correspondence and requests for reprints to: Åsa Tivesten, Wallenberg Laboratory for Cardiovascular Research, Bruna Stråket 16, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden. E-mail: [email protected]. This work was supported by the Swedish Heart and Lung Foundation, the Swedish Research Council, AstraZeneca (Mo¨lndal, Sweden), the Novo Nordisk Foundation, the Tore Nilson Foundation, the Emelle Foundation, the Go¨teborg Medical Society, and the Swedish Medical Society. Disclosure statement: Å.T., K.W., and C.O. have nothing to declare. J.H. and J.W. are employed by AstraZeneca R&D, Mo¨lndal, Sweden. J.H. has equity interests in AstraZeneca R&D. J.W. and B.F. have received grant support from AstraZeneca R&D.

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