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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3823-3828
Copyright © 2003 by The Endocrine Society

Effect of Long-Term Hormone Replacement Therapy on Atherosclerosis Progression in Postmenopausal Women Relates to Myeloperoxidase Promoter Polymorphism

Riikka Mäkelä, Prasun Dastidar, Hannu Jokela, Marika Saarela, Reijo Punnonen and Terho Lehtimäki

Laboratory of Atherosclerosis Genetics, Department of Clinical Chemistry, Center for Laboratory Medicine, University Hospital of Tampere (R.M., H.J., M.S., T.L.), and Department of Clinical Chemistry, Tampere University Medical School (T.L.); Departments of Diagnostic Radiology (P.D.) and Obstetrics and Gynecology (R.P.), University Hospital of Tampere, and University of Tampere (R.P., P.D.), 33521 Tampere, Finland

Address all correspondence and requests for reprints to: Dr. Riikka Mäkelä, Laboratory of Atherosclerosis, Genetics Department of Clinical Chemistry, Center for Laboratory Medicine, University Hospital of Tampere, P.O. Box 2000, 33521 Tampere, Finland. E-mail: riikka.makela{at}uta.fi.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Myeloperoxidase (MPO) is an oxidative enzyme present in phagocytes and atherosclerotic lesions. The MPO gene has a promoter polymorphism -463G/A, which leads to high (GG) and low expression (AG, AA) genotypes. We investigated the effect of long-term hormone replacement therapy (HRT) on the progression of atherosclerosis in a 5-yr follow-up study of postmenopausal women with different MPO genotypes. Eighty-seven nonsmoking postmenopausal women, aged 45–71 yr, were divided into three groups based on the use of HRT. The HRT-EVP group (n = 25) used sequential estradiol valerate plus progestin, the HRT-EV group used estradiol valerate alone (n = 32), and the control group (n = 30) used no HRT. The atherosclerosis severity score (ASC) for abdominal aorta and carotid arteries was determined by ultrasonography, and the MPO genotype was analyzed. In subjects with the GG genotype, the progression of ASC was significantly faster in the control group than in the HRT group (genotype by time interaction, P = 0.042), whereas in A allele carriers there were no significant differences in ASC progression between control and HRT. The effects of HRT on atherosclerosis progression in subjects with the GG genotype seem to be especially beneficial compared with controls with the same genotype but without HRT. These results may help us understand in greater detail the benefit and possible risk of HRT in atherosclerotic diseases.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
COMPLICATIONS OF ATHEROSCLEROSIS are the major sources of morbidity and mortality in postmenopausal women. Several observational studies have indicated that estrogens reduce the risk of atherosclerosis and coronary heart disease in these women (1, 2, 3, 4, 5, 6). In contrast, the results of randomized clinical trials show that hormone replacement therapy (HRT) has no effect or even a harmful influence on atherosclerosis (7, 8, 9). The reasons for this contradiction are unclear, but are speculated to arise from the type of HRT used, selection bias, or other unmeasured factors (1, 7). Discrepancy in the results of observational and randomized clinical trials may also have some unknown genetic background (10), and understanding of the genetic factors involved might promote a clearer understanding of the benefit and possible risks associated with HRT in atherosclerotic diseases.

Atherosclerosis is a chronic inflammatory process characterized by an oxidative activity of inflammatory cells in the vessel wall (11). Myeloperoxidase (MPO) is an antimicrobial oxidative enzyme found in phagocytes (12). Elevated blood leukocyte and MPO levels are associated with an increase in the incidence of coronary artery disease (CAD) (13). MPO is present in atherosclerotic lesions (14) and is able to form proatherosclerotic particles by its oxidative intermediates (15, 16). Persons with MPO deficiency have a reduced risk of cardiovascular damage (17). Interestingly, MPO-deficient mice have significantly increased atherosclerosis compared with wild-type controls (18). Estrogen is known to enhance MPO activity in myeloid cells (19) and also to increase the amount of MPO in plasma (20). Accordingly, the MPO concentration in plasma is significantly lowered at menopause, but is restored during HRT (21).

The promoter region of the MPO gene has a single G-to-A base substitution at position -463 which creates high (G) and low expression (A) alleles (22, 23). The -463A mutation creates an estrogen receptor-binding site in the MPO promoter (24). In women, the GG genotype is overrepresented among subjects with Alzheimer’s disease (25) and multiple sclerosis (26) and is a risk factor for MPO-antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (27). The low expression genotypes, AG and AA, are known to have a protective role in coronary artery disease (CAD) (28).

MPO is a potent contributor to atherosclerosis, and estrogen may affect MPO activity. However, there are no previous prospective studies concerning the effect of HRT on the development of atherosclerosis in subjects with different MPO -463G/A genotypes. We here hypothesized that the MPO genotype might modulate the effect of HRT on the development of atherosclerosis and therefore investigated the effect of long-term HRT vs. placebo control on the progression of atherosclerosis in a 5-yr follow-up study of postmenopausal women stratified by MPO high (GG) and low expression (AG, AA) genotypes.


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

In 1993, women attending a private out-patient clinic in Tampere for annual routine gynecological examinations were invited to participate. For the cross-sectional baseline study in 1993 (5), 120 nonsmoking and nondiabetic postmenopausal women, aged 45–71 yr, were enrolled. In 1998, all of these 120 women were invited by letter to participate in the 5-yr follow-up study; 87 of 120 (72.5%) consented. They had no clinically evident cardiovascular diseases or hypertension and were classified into 3 groups based on the use of HRT. The HRT-EVP group (n = 26) used estradiol valerate (EV; 2 mg/d for 11 d), followed by EV continued with progestin (P; levonorgestrel, 0.25 mg/d) for 10 d. The HRT-EV group (n = 32) used EV alone, and the control group (n = 30) had never used HRT. In the HRT-EVP and HRT-EV groups, there was a pause in therapy for 7 d after each 21-d cycle. None of these women discontinued the therapy during follow-up. HRT, when used, was started for climacteric symptoms at the time of menopause. There were no major differences in the baseline characteristics shown in Table 1Go, i.e. age, body mass index (BMI), or lipids between dropped controls or HRT-treated subjects, compared with corresponding subjects participating in the follow-up study. In addition, both dropped and participating subjects were nonsmoking, nondiabetic, postmenopausal women without clinically evident cardiovascular diseases, hypertension or chronic medication.


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  		TABLE 1. Baseline study: serum lipids and apolipoproteins in postmenopausal women by HRT and MPO genotype status

 
In the control group, the main reason for non-use of HRT was the absence of vasomotor and other climacteric symptoms and subject’s aversion to HRT. At baseline, the mean duration of EVP and of EV treatment was 10.7 ± 2.2 yr and 9.2 ± 3.7 yr, respectively. The mean time from menopause in the control group was 11.9 ± 4.1 yr. The mean ages in the HRT-EV, HRT-EVP and control groups were 60.4 ± 4.8, 59.5 ± 5.5, and 61.5 ± 5.8 yr, respectively (P = 0.377 for all groups, by ANOVA). The mean BMI values in the HRT-EV, HRT-EVP, and control groups were 25.7 ± 2.9, 25.5 ± 3.1, and 25.9 ± 3.2 kg/m2, respectively (P = 0.881 for all groups, by ANOVA). At baseline, all women were clinically healthy and used no lipid-lowering or other chronic medication. Ultrasonography and blood sampling were carried out at the University Hospital of Tampere. The ethics committee of the University Hospital of Tampere approved the study.

Blood samples

Blood samples for serum lipid and genotype analyses were taken after the overnight fasting. Sampling took place within 3 wk after the ultrasonography and, for HRT users, during the third week of the hormone regimen. After separation of serum by low speed centrifugation, the sera were divided into aliquots and stored at –70 C until analyzed.

Ultrasonography

Ultrasonography at baseline and follow-up were performed with Toshiba Sonolayer V SSA 100 equipment (Toshiba Corp., Tokyo, Japan), as reported in detail previously (5). Briefly, all ultrasonographies were performed in a blinded manner by one experienced ultrasonographer and radiologist (P.D.). Transverse and longitudinal scans of extracranial carotid arteries were made bilaterally at four different segments of the carotid (5). Only fibrous and calcified atherosclerotic lesions were considered and were defined as plaques when distinct areas of mineralization or/and focal protrusion into the lumen were identified. An intimal-media far wall thickness equal to or more than 1.3 mm at any segment in the carotid arteries was defined as an atherosclerotic plaque (29), and the total number of plaques (NAP) was calculated. The carotid intima-media far wall thickness at each of the four different segments in ultrasonography examination was the average of five different individual measurements. All carotid artery examinations were made with a 5.0-MHz convex transducer probe.

Longitudinal ultrasonographs of the abdominal aorta were obtained at 1-cm intervals, and transverse scans were obtained at 2-cm intervals, in an area of three aortic segments. Significant aortic plaques were defined as an intima-media far wall thickness equal to or more than 3.0 mm (29). The reproducibility of our ultrasonographic protocol for significant aortic and carotid plaques was also examined: 1 month after the first assessment, 20 randomly selected subjects were invited to attend for a repeat examination. For each patient, the depth of field, gain, and other monitor settings were kept similar at both baseline and follow-up ultrasonographic examinations to reduce measurement variability. In all 20 patients for each single plaque the intima-media far wall thickness in the aorta remained the same between the initial and the follow-up ultrasonography examination. However, in carotid artery measurement in two patients there were differences of 0.5 and 0.7 mm in a previously measured plaque, the measurement being less in the follow-up examination. The reason for this null/minimal variability was the very short interval of 1 month between the two examinations. The repeatability of NAP between the first and second examinations was 90% for the carotid artery segment areas and 100% for the aortic segments.

The atherosclerosis severity score (ASC) was constructed by dividing the atherosclerosis in the abdominal aorta and carotid arteries into three severity classes, i.e. 1 = minor (1.3–2 mm), 2 = moderate (2–3 mm), and 3 = severe (more than 3 mm). The ASC was then calculated as the sum of the severity classes in aorta and carotid artery. The total NAP was calculated (in the baseline study only, as 5 yr data were not available) according to NAP and the severity of plaques (criteria for plaques given above). Scoring was performed in a blinded manner, without knowledge of HRT and MPO genotype status, by one person (P.D.).

MPO genotyping

DNA was isolated from white blood cells using a commercial kit (QIAGEN, Valencia, CA). The DNA fragment of the MPO gene promoter area was first amplified and then digested with AciI restriction endonuclease (New England Biolabs, Inc., Beverly, MA) as previously described (30).

Other laboratory analyses

Lipid measurements were made at baseline and after 5-yr follow-up. Serum total cholesterol and triglycerides were determined by a commercial method (Kodak Echtachem 700XR, Eastman Kodak Co., Rochester, NY). Serum high density lipoprotein cholesterol and its subfractions (HDL2 and HDL3) were separated with a dextran-sulfate- magnesium precipitation procedure (31), and the cholesterol content was analyzed with a Monarch 2000 Analyzer (Instrumentation Laboratory, Lexington, KY), using the cholesterinoxidase-peroxidase/antiperoxidase cholesterol reagent (catalog no. 237574, Roche, Mannheim, Germany) and a primary cholesterol standard (catalog no. 530, Orion Diagnostics, Helsinki, Finland). The low density lipoprotein cholesterol content was calculated according to the Friedewald formula (32). Apolipoproteins A1 and B were determined on a Monarch Analyzer by an immunoturbidimetric method (33) (catalog no. 67265 and 67249, Orion Diagnostics). In the follow-up study all lipid analyses were performed with a Cobas Integra 700 automatic analyzer with the reagents and calibrators recommended by the manufacturer (Roche Diagnostics, Basel, Switzerland).

Statistical analyses

In view of the small number of low expression allele A homozygous (n = 2) subjects, the A allele carriers were combined into one group (AG or AA), as previously done in other studies (27, 28, 34). One-way analysis of covariance (ANCOVA), using age and BMI as covariates, was used to assess statistical differences in lipid and apolipoprotein values between the MPO genotypes within the HRT and control groups (Table 1Go). Longitudinal follow-up data were analyzed by ANOVA for repeated measures (RANOVA) to find interactions between treatment groups (controls vs. HRT) and time points. RANOVA was performed separately within the high expression allele G homozygous and low expression allele A carrier subject groups. ANCOVA (adjusted for age and BMI), with the least significant difference post hoc test, was used to test the significance of differences between treatment groups in ASC and total NAP at baseline. A similar analysis with the same covariates was made of follow-up data. Serum triglycerides were analyzed after logarithmic transformations by reason of the skewed distribution (the untransformed values are shown). The Statistica for Windows version 5.1 software package (Statsoft, Inc., Tulsa, OK) was used for statistical analyses. PS program version 2.1.15 was used for the power calculations of the test procedures. Data are presented as the mean ± SD unless otherwise stated. P <= 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The baseline distribution of known CAD risk factors among postmenopausal women with respect to MPO allele status is presented in Table 1Go. In the baseline study there were statistically significant differences in age and serum triglycerides between the carriers of high (GG) and low expression genotypes (AG, AA) in the HRT-EV group.

At baseline, subjects with the high expression genotype (GG) on HRT-EV tended to have an average of 32.2% smaller ASC (1.23 vs. 1.80 in controls), and subjects on HRT-EVP had 20.6% smaller ASC (1.44 vs. 1.80 in controls) than the controls (ANCOVA for trend, P = 0.056, adjusted by age and BMI; Fig. 1AGo). After 5 yr of follow-up, the corresponding differences between the HRT-EV and HRT-EVP groups and the controls were 31.5% (3.00 vs. 4.38, P = 0.010) and 27.2% (3.19 vs. 4.38, P = 0.040; ANCOVA for trend, P = 0.035, adjusted by age and BMI; Fig. 1CGo). At baseline, subjects with the GG genotype on HRT-EV had a mean of 49.9% (1.95 vs. 3.90 in controls, P = 0.001) lower total NAP, and subjects on HRT-EVP had a 37.6% (2.44 vs. 3.90 in controls, P = 0.020) smaller total NAP than the controls (ANCOVA for trend, P = 0.003, adjusted by age and BMI).



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  		FIG. 1. A–D, ASC in postmenopausal women by HRT group and MPO genotype status [high expression allele G homozygotes (GG) and low expression allele A carriers (AG, AA)]. A and B, Results from the baseline study; C and D, results from the cross-sectional study after a 5-yr follow-up. The P values for the mean (±SD, whiskers) differences between the HRT groups and controls shown in the figure are obtained by ANCOVA, with least significance test as a post hoc test. Results are adjusted for age and BMI.

 
At baseline, the subjects with low expression genotypes (AG or AA) on HRT-EV had an average 49.4% (0.90 vs. 1.78 in controls, P = 0.032) smaller ASC, and subjects on HRT-EVP had 43.7% (1.00 vs. 1.78 in controls, P < 0.10) smaller ASC than the controls (ANCOVA for trend, P = 0.041, adjusted by age and BMI; Fig 1BGo). After 5 yr of follow-up, the corresponding differences between the HRT-EV and HRT-EVP groups and controls were 34.3% (2.70 vs. 4.11, P = 0.018) and 24.3% (3.11 vs. 4.11, P < 0.10; ANCOVA for trend, P = 0.027, adjusted by age and BMI; Fig. 1DGo). At baseline, A allele carriers on HRT-EV had a mean of 61.4% (1.50 vs. 3.89 in controls, P = 0.003) lower total NAP, and A allele carriers on HRT-EVP had a mean of 34.3% (2.56 vs. 3.89 in controls, P < 0.10) smaller total NAP than the controls (ANCOVA for trend, P = 0.006, adjusted by baseline age and BMI).

Among the high expression genotype (GG) subjects, the progression rate of ASC differed significantly between HRT users and controls (treatment group by time point interaction in RANOVA, P = 0.042; Fig. 2AGo). The power of the test was 83%. Among low expression allele A carriers, the progression rates of ASC in HRT users and nonusers did not differ statistically (treatment group by time point interaction in RANOVA, P = 0.274; Fig. 2BGo). The power of the test was 58%, whereas a power of 80% would be obtained with 34 subjects.



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  		FIG. 2. A and B, The effect of HRT on the progression of atherosclerosis, as measured by ASC in postmenopausal women with high expression genotype GG (A) and low expression genotype AG or AA (B) compared with the progression of controls with the same MPO genotype and time elapsed from menopause, but without HRT. The P values shown in the figure are from two-way ANOVA (grouping HRT/control) for repeated measures.

 
The corresponding changes in serum lipids during follow-up, according to MPO genotype status, are shown in Table 2Go. There was a suggestively significant difference between the MPO genotypes in the response of serum triglyceride levels (i.e. MPO genotype group by time point interaction, P = 0.051 for serum triglycerides). There were no statistically significant differences between the MPO genotypes in the response to other lipid values.


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  		TABLE 2. Changes in lipid profile during the follow-up

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the current study long-term HRT seemed to have beneficial effects on atherosclerosis progression in subjects with the high expression (GG) genotype compared with those with the same genotype but without HRT. No positive effect of HRT was seen in the low expression genotype (AG or AA) group, where the progression of atherosclerosis tended to be similar among HRT users and controls during the 5-yr follow-up. There were no significant differences in atherosclerosis score between the genotypes (data not shown). All subjects were nonsmokers, were not diabetic, had normal blood pressure, and were otherwise clinically healthy. Most of the important cardiovascular risk factors were equally distributed among the study groups, which diminishes the possibility of common background characteristics affecting our study.

Reports of the beneficial effects of HRT on atherosclerosis have concerned the early stages of the disease, whereas the use of HRT in advanced stages of the disease seems to increase CAD events (35). The risk of cardiovascular events appears to be highest in the first year of HRT use, decreasing with the years (7). HRT is known to raise plasma levels of C-reactive protein (36), which is a strong independent cardiovascular risk factor (37). It is not known whether the effects of HRT on inflammation and blood coagulation predispose to plaque instability and thrombosis (35).

The plaques detected by ultrasonography here were mostly uncalcified, constituting less advanced atherosclerotic lesions. As individuals with more advanced atherosclerotic disease, characterized by stenotic or occlusive lesions, were not included, the association of the MPO genotype with atherosclerosis severity status may have been modified. Ultrasonographic methods permit evaluation of atherosclerosis in asymptomatic subjects (38, 39), such as those attending to our study. It is therefore not clear whether the atherosclerosis progression evaluated in our study would result in clinical manifestations. Nevertheless, it has been reported that atherosclerosis affects coronary arteries in parallel to extracoronary arteries (40). An elevation in carotid intima-media thickness is associated with increased CAD events, and by preventing the disease progression in carotid arteries, an equal reduction in cardiovascular risk is achieved (39).

MPO activity is higher in normal women than in men (41). Accordingly, estrogen is known to enhance MPO activity in myeloid cells (19) and increase the amount of MPO in plasma (20). At the time of menopause, MPO activity is significantly decreased, but may be restored by HRT (21, 42). This is in accordance with the other findings concerning estrogen actions on immunity (43). Oxidative intermediates produced by MPO have been related to low density lipoprotein oxidation and atherosclerosis progression (44, 45). However, estrogen-induced MPO activity may diminish superoxide anion generation and thereby reduce the overall production of free radicals (21, 46). Interestingly, if the first stage metabolism in the liver is passed by transdermal administration of estradiol, plasma levels of MPO are reduced (47).

MPO is present in atherosclerotic plaques (14), and in patients with CAD, the leukocyte and blood MPO levels are higher than in controls (13). MPO activity is increased in patients suffering cardioembolic stroke (48), and, accordingly, MPO deficiency may be assumed to provide protection against cardiovascular damage (17). However, in a murine model of atherosclerosis, MPO-deficient mice developed 50% larger atherosclerotic lesions than controls, indicating an unexpected protective role of MPO in atherosclerosis (18). In the present study the positive effect of HRT on atherosclerosis progression was evident only in the high expression genotype GG carriers. A previous study in subjects with or without angiographically proved CAD has reported a protective role of the low expression A allele in atherosclerotic disease (28). In that study, however, women and men were unequally distributed among controls and patients with CAD, and according to recent reports, the MPO -463G/A polymorphism may be a gender-dependent risk factor for Alzheimer’s disease, multiple sclerosis, and MPO-ANCA-associated vasculitis. In women, the high expression genotype GG is associated with a greater morbidity risk of these disease states, whereas in men the A allele is overrepresented (24, 26, 27). The possible association of the GG genotype with female sex may partly explain our findings, although the exact mechanism remains to be established.

There were some limitations to our study. The subjects had already used HRT for almost a decade at the beginning of the follow-up study, and the early progression of atherosclerotic lesions could not be examined. The cardiovascular end processes were not studied, and we cannot estimate the risk of cardiovascular events. The study was exposed to selection bias. Firstly, it was based on self-selected HRT treatment, and the possible association between climacteric symptoms and CAD or MPO genotype cannot be excluded. Secondly, 33 subjects dropped out. There were no major differences between the background characteristics of the drop-outs and the participants or the HRT users and control subjects. However, other factors, such as socioeconomic status, may differ between HRT users and nonusers and therefore bias our results. The number of A allele carriers was low, and the statistical power remained insignificant (58%). For a power of 80%, the estimated number of subjects would have been 34. We cannot therefore exclude the possibility of type II error in the study.

The data obtained provide evidence of a possible role of genetic factors in the response of HRT. The follow-up study indicated that the effect of long-term HRT on atherosclerosis progression in postmenopausal women differs according to MPO genotype. Due to the observational nature of our study, this finding needs to be confirmed in larger and randomized clinical trials. However, if our findings are repeated, they may be of clinical importance when hormonal treatment of atherosclerotic diseases is planned for postmenopausal women.


   Acknowledgments
 
We thank Miss Nina Peltonen for her skillful technical assistance.


   Footnotes
 
This work was supported by grants from the Finnish Foundation of Cardiovascular Research (Helsinki, Finland), the Elli and Elvi Oksanen Fund of the Pirkanmaa Fund under the auspices of the Finnish Cultural Foundation, the Emil Aaltonen Foundation, the Juho Vainio Foundation, the Research Foundation of Orion Corp., and the Medical Research Fund of Tampere University Hospital (Tampere, Finland).

Abbreviations: ANCA, Antineutrophil cytoplasmic antibodies; ANCOVA, analysis of covariance; ASC, atherosclerosis severity score; BMI, body mass index; CAD, coronary artery disease; EV, estradiol valerate; HRT, hormone replacement therapy; MPO, myeloperoxidase; NAP, number of plaques; P, progestin; RANOVA, ANOVA for repeated measures.

Received November 11, 2002.

Accepted April 24, 2003.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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