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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2789
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 8 3123-3130
Copyright © 2006 by The Endocrine Society

Effects of Hormone Replacement Therapy Type and Route of Administration on Plasma Matrix Metalloproteinases and Their Tissue Inhibitors in Postmenopausal Women

K. C. Lewandowski, J. Komorowski, D. P. Mikhalidis, M. Bienkiewicz, B. K. Tan, C. J. O’Callaghan, A. Lewinski, G. Prelevic1 and H. S. Randeva1

Department of Biological Sciences (K.C.L., B.K.T., H.S.R.), University of Warwick, Coventry CV4 7AL, United Kingdom; Departments of Endocrinology and Metabolic Diseases (K.C.L., J.K., A.L.) and Quality Control and Radiation Protection (D.P.M., M.B.), The Medical University of Lodz, 90-419 Lodz, Poland; Department of Community Health and Epidemiology (C.J.O.), Queen’s University, Kingston, Ontario, Canada K7L 3N6; and Departments of Endocrinology and Clinical Biochemistry (G.P.), The Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom

Address all correspondence and requests for reprints to: Dr. Harpal S. Randeva, M.B.C.H.B., F.R.C.P., Ph.D., Molecular Medicine Group, Department of Biological Sciences, The University of Warwick, Coventry CV4 7AL, United Kingdom. E-mail: hrandeva{at}bio.warwick.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Matrix metalloproteinases (MMPs) are implicated in numerous disease states including cardiovascular disease and cancer. Because recent studies have shown a detrimental effect of hormone replacement therapy on cardiovascular disease and breast cancer, we investigated whether there are any differences in the concentrations of MMPs and their tissue inhibitors (TIMPs) in women receiving various forms of postmenopausal therapy.

Material and Methods: A total of 195 healthy postmenopausal women were assessed: 46 were taking tibolone, 47 were taking transdermal estradiol, 46 were taking conjugated equine estrogens (CEE), and 56 were not taking any menopausal therapy (CTR). Plasma levels of MMP-2 and -9 and TIMP-1 and TIMP-2 were measured by ELISA methods.

Results: MMP-9 levels were significantly higher in the CEE group in comparison with healthy women not receiving menopausal therapy (P < 0.05). In contrast, MMP-9 levels in the tibolone group were significantly lower than in any other group (P < 0.01, compared with transdermal estradiol and CTR, and P < 0.001, compared with CEE). MMP-9 to TIMP-1 ratio was also significantly higher in the CEE, compared with CTR (P < 0.05), and lower in the tibolone group (P < 0.01, compared with all groups). MMP-2 levels were higher in the CEE group, compared with healthy women not receiving any menopausal therapy, and women taking tibolone (P < 0.05).

Conclusions: Our study demonstrates differential effects of various forms of postmenopausal therapy on serum levels of MMP-9 and MMP-2. It remains to be established whether these differences might be associated with differences in risks of cardiovascular disease and cancer in these women.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
BY MID-1990 IT WAS believed that the main benefit from hormone replacement therapy (HRT) was protection against cardiovascular disease. This was based on numerous observational and epidemiological studies, which suggested that postmenopausal HRT reduced the risk of coronary heart disease (CHD) by 30–50% (1, 2, 3, 4). Some studies have also suggested some improvements in 24-h blood pressure profiles in women receiving oral estradiol valerate (5), transdermal 17-ß-estradiol (5), or oral conjugated equine estrogens (CEE) (6). However, in a recent randomized, placebo-controlled study (7), HRT failed to demonstrate any protective effects on 24-h heart rate variability and did not modify cardiac autonomic activity. Moreover, randomized, controlled trials both for secondary (8) and primary prevention of cardiovascular disease (9) failed to document any protective effect of HRT. Of note, in these trials the combination of the CEE with medroxyprogesterone acetate (MPA) was used. On the other hand, there are no randomized studies that demonstrated any detrimental effects of tibolone on cardiovascular events. If anything, the effects of tibolone on the cardiovascular system were found to be either neutral (10) or even potentially beneficial (11, 12). The precise reason for these observations remains to be determined.

Matrix metalloproteinases (MMPs) are a family of zinc-binding proteolytic enzymes that normally remodel the extracellular matrix. Increased activity of MMPs has been reported in numerous disease processes including tumor growth, arthritis, and cardiovascular disease. Increased matrix degradation by MMPs within the atherosclerotic plaque has been implicated as one of the key factors that lead to plaque instability (13) and consequently to cardiovascular events. The major MMP species in the myocardium and vasculature are the gelatinases (MMP-2 and -9), MMP-1 (interstitial collagenase), and membrane type 1-MMP. MMP-2 and MMP-9 specifically attack type IV collagen, laminin, and fibronectin, major components of the basal lamina around blood vessels (14). MMP-2 and MMP-9 play a major role in acute myocardial ischemia, reperfusion injury, and vascular matrix remodeling (15, 16). Increased expression and activation of MMP-2 and MMP-9 have been noted in vulnerable regions of human atherosclerotic plaques, cerebral ischemia, and acute coronary syndromes (17, 18, 19). The regulation of MMP activity is complex and occurs at multiple levels, including interactions of secreted MMPs with tissue inhibitors of metalloproteinases (TIMPs) (20). Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein, plays an important role in vasculogenesis, atherogenesis, and vascular remodeling in response to injury (21, 22). VEGF, stimulated by hypoxia (21) or reactive oxygen intermediates (22), is also known to up-regulate the expression of MMPs (23).

Because MMPs are implicated in the pathogenesis of CHD and stroke and also neoplastic disease, we decided to investigate whether there are any differences in the plasma concentrations of MMP-2, MMP-9, TIMP-1, and TIMP-2 in women receiving medium or long-term HRT in comparison with healthy controls. In view of the aforementioned and previous findings of the effects of HRT on traditional parameters of cardiovascular disease risk (24), we assessed, whether there were any differences in the concentrations of MMPs and TIMPs in women receiving various forms of menopausal therapy (CEE, transdermal estradiol, and tibolone).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
From a database of more than 2000 postmenopausal women who have been followed up in the Menopause Clinic of the Royal Free and University College Hospitals (London, UK), we identified more than 500 women who had been menopausal for at least 10 yr and had taken uninterruptedly for at least 5 yr any of the three most commonly prescribed preparations for the control of menopausal symptoms in our clinic or who had not taken any menopausal therapy during the same period of time. Women fulfilling these criteria were contacted through a questionnaire and invited to participate in our cross-sectional study. More than 80% replied positively and agreed to participate. The Royal Free and University College Hospital Ethics Committee approved the study. Written informed consent was obtained from each subject entering the study.

All women were assessed clinically, and details of their past medical history, family history, and current medication were recorded. Because diabetes, hypertension, obesity, and cardiovascular disease are known cardiovascular disease risk markers, we excluded women with diabetes and fasting hyperglycemia (fasting glucose above 6 mmol/liter, or hemoglobin A1c > 7.0%), women on statin therapy, those with hypertension (systolic >140 mm Hg, diastolic > 90mm Hg, and those on antihypertensive therapy) and those with a history of angina and all those with a body mass index (BMI) more than 30 kg/m2. This analysisis therefore limited to 195 healthy postmenopausal women who, apart from the study medications, were taking no other medications. Women attending our clinic were invited and recruited consecutively to take part in our study. They were offered the different preparations of HRT, and there were those who refused HRT (controls); however, like those on HRT, these women agreed to be followed up in our menopause clinic. The decision as to the kind of menopausal therapy selected by each woman was based on individual choice and preference. Women who preferred to retain their menses opted for CEE or transdermal estrogens, whereas women who preferred a nonbleed regimen preferred to use tibolone.

All participants except the comparison group were being treated at the time of study and therefore sample analysis. All subjects received treatment for an uninterrupted period of at least 5 yr, and the duration of treatment was: tibolone (6.5 ± 1.0 yr), transdermal estradiol (TRD) (7.5 ± 2.5 yr), and CEE (9.8 ± 5.5 yr). For analysis, women were separated into four groups. Group 1 (CTR, n = 56) consisted of women who had never received any menopausal therapy, or who took it for less than 2 yr and did not receive any therapy for at least 5 yr before assessment. Those women, who chose not to take HRT, were followed up in our menopause clinic at which they received advice on prevention of osteoporosis and assessment of their bone mineral density. Twelve of those women had had previous hysterectomy. Group 2 (TRD, n = 47) consisted of women on transdermal estradiol alone (Estraderm TTS; Novartis, Surrey, UK), taken by 29 women who had had previous hysterectomy, or transdermal estradiol with sequential norethisterone acetate (Estrapack or Estracombi, Novartis) taken by the remaining 18 women with intact uterus. Group 3 (tibolone, n = 46, of which 36 had intact uterus) consisted of women on tibolone (Livial; Organon, Cambridge, UK) 2.5 mg daily. Group 4 (CEE, n = 46) consisted of women on CEE 0.625 mg/d (Premarin; Wyeth, Maidenhead, UK): 19 women who had had previous hysterectomy or CEE with sequential norgestrel (Prempack C, Wyeth) taken by the remaining 27 women with intact uterus.

After an overnight fast, blood samples were taken for total cholesterol, triglycerides, insulin, MMP-2, MMP-9, TIMP-1, TIMP-2, and VEGF. The samples were taken on ice and stored within 1 h at –70 C until analyzed. Plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by ELISAs [MMP-2: human, Biotrak ELISA system; Amersham Biosciences, Buckinghamshire, UK; intraassay precision coefficient of variation (CV) 6.3%; MMP-9 (total): R & D Systems, Minneapolis, MN, CV 2.9%; TIMP-1 and TIMP-2: Amersham Pharmacia Biotech U.K. Ltd., Little Chalfont, UK; CVs 11.4 and 5.4%, respectively]. VEGF was measured using enzyme immunoassays (Cytokit Red enzyme immunoassay kits; Peninsula Labs, College Park, MD; CV 6.4%). All measurements, done in duplicates, were performed in the central laboratory of the Medical University of Lodz (Lodz, Poland).

Statistical analysis

Overall significance of difference between groups was assessed by both parametric test (ANOVA) and a nonparametric test (Wilcoxon rank-sum test). If the difference of the measured parameter was found to be statistically significant with one of these tests, then further detailed analysis of comparisons between the subgroups was performed by means of simple descriptive statistics and nonparametric test of significance (the Mann-Whitney U test). In that way we tried to diminish the chance of finding a false-positive difference of a measured parameter while comparing MMP and TIMP levels between multiple subgroups. Associations between parameters of MMPs and covariates of interest (demographic and clinical) were qualified by both Pearson’s and Spearman rank correlations. Associations between qualitative variables were tested by the {chi}2 test of independence for contingency tables. In all analyses, statistical significance was considered achieved for a value of P ≤ 0.05. All the calculations were derived by means of Statistica (Tulsa, OK) software (version 6.0).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical data on patients are presented on Table 1Go. There were no statistically significant differences in age or BMI between the control group and groups receiving hormone replacement (menopausal) therapy. The only exception was the tibolone group, in which the mean age (65.5 ± 7.0 yr) was higher than in other groups (P < 0.02). However, we did not note any significant correlations between MMPs/TIMPs and age (MMP-2: r = 0.10, P = 0.16; MMP-9: r = –0.05, P = 0.31; TIMP-1: r = 0.12, P = 0.13; TIMP-2: r = 0.13, P = 0.10).


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  		TABLE 1. Descriptive demographics and univariate contrasts of covariates between groups

 
The concentrations of MMP-2 were found to be significantly higher in women on CEE, compared with controls [951 ± 317 ng/ml (mean ± SD) vs. 797 ± 357 ng/ml P < 0.05] and the tibolone group (844 ± 254 ng/ml, P < 0.05; Fig. 1Go). Likewise, the concentrations of MMP-9 were also significantly higher in CEE in comparison with controls (68.4 ± 62 vs. 33.6 ± 28.5 ng/ml, P < 0.05) and the tibolone group (21.3 ± 21.7 ng/ml, P < 0.001; Fig. 1Go). Furthermore, concentrations of MMP-9 in the tibolone group were also significantly lower, compared with controls (21.3 ± 21.7 vs. 33.6 ± 28.5 ng/ml, P < 0.01) and the TRD therapy group (38 ± 32 ng/ml, P < 0.01; Fig. 1Go). There were no significant differences in the concentrations of TIMP-1 among the different groups (104.2 ± 23.5 ng/ml, controls; 118 ± 53.8 ng/ml, CEE; 99.1 ± 18.7 ng/ml, TRD; and 103.7 ± 38.7 ng/ml, tibolone group: P = NS). TIMP-2 concentrations were lower in the TRD group (81.4 ± 16.4 ng/ml) in comparison with controls (93.2 ± 26.8 ng/ml; P < 0.01) and the tibolone group (96.8 ± 32 ng/ml; P < 0.05). We found no significant correlation between duration of treatment of HRT and MMPs/TIMPs (MMP-2: r = 0.13, P = 0.24; MMP-9: r = 0.10, P = 0.28; TIMP-1: r = 0.14, P = 0.20; TIMP-2: r = 0.15, P = 0.17). Given that women in the tibolone group were older, the aforementioned differences remained significant, even after accounting for age (MMP-2, P < 0.05; MMP-9, P < 0.01; TIMP-2, P < 0.05; TIMP-1, P = NS).


Figure 1
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  		FIG. 1. Mean ± SEM concentrations of MMP-2, MMP-9, and VEGF in healthy women on no HRT (Gp-0) and those on HRT: TRD (Gp-1), tibolone (Gp-2), and CEE (Gp-3), and the ratios of MMP-2 (M2) to TIMP-2 (T2) and MMP-9 (M9) to TIMP-1 (T1). a, P < 0.05 vs. Gp-0 and Gp-2; b, P < 0.01 vs. Gp-0 and Gp-1; c, P < 0.05 vs. Gp-0; d, P < 0.001 vs. Gp-2; e, P < 0.01 vs. Gp-0; f, P < 0.001 vs. Gp-1 and Gp-3; g, P < 0.01 vs. Gp-1; h, P < 0.01 vs. Gp-0 and Gp-1; i, P < 0.001 vs. Gp-3; j, P < 0.05 vs. Gp-0; k, P < 0.001 vs. Gp-2.

 
Interestingly, neither the TRD group nor the CEE group had any significant change in VEGF levels, compared with the control group (Table 1Go and Fig. 1Go). However, those women receiving tibolone had significantly lower VEGF levels, compared with controls (3.29 ± 2.11 vs. 5.0 ± 2.35 ng/ml, respectively, P < 0.01; Fig. 1Go) and those women taking TRD therapy (5.17 ± 2.66 ng/ml, P < 0.001; Fig. 1Go) or CEE therapy (4.90 ± 2.18 ng/ml, P < 0.001; Fig. 1Go).

Although the effect of hysterectomy on the concentrations of MMP-2, MMP-9, TIMP-1, and TIMP-2 was not the main subject of our study, in view of the fact that women who had intact uterus were also taking norethisterone acetate (in the TRD group) or sequential norgestrel (in the CEE group) in addition to estrogen, in each subgroup we also compared MMP-2, MMP-9, TIMP-1, and TIMP-2 concentrations between women who had prior hysterectomy and those with intact uterus. This analysis demonstrated that within each subgroup there were no significant differences in mean age and BMI as well as MMP-2, MMP-9, TIMP-1, and TIMP-2 concentrations between women with intact uterus and those who had had previous hysterectomy as shown in Table 2Go.


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  		TABLE 2. Descriptive statistics (mean ± SD) for controls and different routes of HRT administration, depending on whether the subjects had previous hysterectomy

 
Given that in its inactive form, MMP-9 is complexed to TIMP-1, whereas Pro-MMP-2/MMP-2 is complexed to TIMP-2 and the balance between MMPs and TIMPs determines the degree of matrix degradation, we determined the respective MMP to TIMP ratio. The MMP-9 to TIMP-1 ratio was significantly higher in the CEE group in comparison with both untreated controls and the tibolone group (P < 0.05 and P < 0.001, respectively; Table 1Go and Fig. 1Go). The MMP-9 to TIMP-1 ratio in the tibolone group was significantly lower in comparison with all other groups, including TRD (P < 0.01; Table 1Go and Fig. 1Go). In contrast, with the exception of lower MMP-2 to TIMP-2 ratio in the tibolone group in comparison with TRD (P < 0.01), there were no differences in MMP-2 to TIMP-2 ratio between other groups (Table 1Go and Fig. 1Go).

Tables 3Go and 4Go demonstrate correlations between MMPs and other measured parameters, as assessed by Pearson’s correlation coefficients. There were no significant correlations between age and plasma concentrations of MMP-2 and MMP-9 or TIMP-1 and TIMP-2 concentration in controls and groups of TIB and CEE therapy. Only in the TRD group were positive correlations found between age and TIMP-1 (r = 0.4, P = 0.01) and TIMP-2 (r = 0.3, P = 0.05). In all subgroups there was a significant correlation between plasma MMP-2 and MMP-9 concentrations as well as between MMP-2 and TIMP-2 concentrations. Consistent correlation between VEGF and both MMP-2 and MMP-9 was found only in women treated with tibolone. Because overall assessment of significance of the observed differences was performed both by parametric (ANOVA) and nonparametric test (Wilcoxon rank-sum test), assessment of correlation coefficients was also performed by the means of the nonparametric Spearman correlation coefficient. Table 5Go demonstrates significant correlations in each subgroup as assessed by Spearman rank correlation method.


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  		TABLE 3. Bivariate assessments of association between covariates of interest for each study group (control and TRD) expressed as Pearson correlation coefficients and noting sample size (n) in each group

 

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  		TABLE 4. Bivariate assessments of association between covariates of interest for each study group (tibolone and CEE) expressed as Pearson correlation coefficients and noting sample size (n) in each group

 

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  		TABLE 5. Bivariate assessments of significant associations between covariates of interest for each study group expressed as Spearman correlation coefficients and noting sample size (n) in each group

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The most important finding of our study is demonstration of significantly higher levels of MMP-2 and MMP-9 in healthy postmenopausal women receiving CEE, compared with healthy women not receiving menopausal therapy or those treated with tibolone. In addition, women treated with tibolone had significantly lower MMP-9 in comparison with all other groups. Furthermore, the significance of lower VEGF concentrations in women receiving tibolone is yet unclear. However, VEGF plays an important role in angiogenesis, vasculogenesis, atherogenesis, and vascular remodeling (21, 22) and is known to up-regulate the expression of MMPs (23). There are conflicting data on the effects of tibolone on VEGF, with some studies showing a decline in VEGF in rabbit carotid artery (25) but an increase in Ishikawa cells (immortalized from a well-differentiated human adenocarcinoma cell line) (26). In the only prospective study, of 12 months’ duration, administration of tibolone in 37 women caused about 15% increase in VEGF concentrations (significant only in those with low baseline VEGF), the levels of which were, however, still lower than in women who took 0.625 mg CEE plus 5 mg MPA (n = 34) for 12 months (274.8 ± 183.1 vs. 321.1 ± 181.8 pg/ml) (27). Interestingly, in our study a positive association between VEGF and both MMP-2 and MMP-9 was noted only in the tibolone-treated group.

The significance of our findings is further supported by the differences in the MMP to TIMP ratio. TIMP-1 predominantly binds to the zymogen of MMP-9 and TIMP-2 binds to the zymogen of Pro-MMP-2/MMP-2 (20), and this balance between MMPs and TIMPs determines the degree of matrix degradation. The MMP-9 to TIMP-1 ratio was significantly higher in the CEE group and significantly lower in the tibolone group, which may suggest lower plasma concentrations of active MMP-9 in the tibolone group, the opposite being true for women receiving CEE. In contrast to the CEE and tibolone groups, there were no significant differences in MMP-2, MMP-9, and TIMP-1 concentrations in the transdermal group in comparison with untreated healthy controls. The significance of decreased TIMP-2 level in transdermal group is not clear because no significant difference in the MMP-2 to TIMP-2 ratio was detected between women on transdermal estradiol and the healthy postmenopausal women not receiving therapy. Likewise, in the CEE group, there was no significant difference in the MMP-2 to TIMP-2 ratio; however, again tibolone-treated women had significantly lower MMP-2 to TIMP-2 ratio.

The mechanism that might explain higher levels of MMP-9 and MMP-2 in women treated with CEE is not entirely clear, although it seems likely to be direct enhancement of enzyme synthesis by oral but not the transdermal route because no significant differences, in comparison with controls, were seen in the TRD group. Indeed, there is evidence that 17ß-estradiol increases synthesis of MMP-2 in human vascular smooth muscle cells (28) and MMP-9 in endothelial cells (our unpublished data). Furthermore, although classical estrogen response elements have not been identified in the promoter regions of MMPs, several MMPs, including MMP-9, have consensus sequences for activator protein-1 sites (29, 30). Estrogen bound to its receptor (especially estrogen receptor-{alpha}) activates transcription target genes under the control of activator protein-1 response elements (31). In contrast, tibolone, which is a tissue-specific steroid, does not seem to induce MMPs synthesis. The mechanism of this difference is not known, although it might be the result of the inhibition of local effects of estrogens (32). Tibolone has been found to inhibit the conversion of estrone sulfate to more potent estradiol (33), mostly through inhibition of sulfatase, the enzyme that forms estrone from estrone sulfate, the former subsequently being converted to estradiol by 17ß-hydroxysteroid dehydrogenase type I (34, 35). To some extent, tibolone, by its {Delta}4-metabolite, also induces the activity of 17ß-hydroxysteroid dehydrogenase type II (that converts estradiol back to estrone) (36). In such context estrogen might potentially increase MMP-2 and MMP-9 levels, whereas the opposite might be true for tibolone.

So far, the data on MMP and TIMP concentrations in women receiving HRT have been very few and inconclusive. Zanger et al. (37), reported significantly raised MMP-9 levels in 10 postmenopausal women receiving oral HRT (equine estrogens 0.625 mg ± 2.5 mg MPA), who incidentally also had a history of established coronary artery disease. In contrast, in our study we observed raised concentrations of both MMP-9 and MMP-2 in a larger group of subjects receiving conjugated oral estrogens ± 2.5 mg MPA. Apart from the size difference, we note that our study assessed MMP and TIMP concentrations in healthy subjects, rather than in those with established coronary artery disease. The presence of a significant correlation between plasma MMP-2 and MMP-9 concentrations in all subgroups might suggest, however, that administration of oral conjugated estrogens or tibolone is more likely to affect concentrations of both metalloproteinases.

In another study, Wakatsuki et al. (38) found no difference in MMP-9 levels in 20 women receiving daily 0.625 mg CEE in comparison with the control (n = 15) and transdermal groups (n = 19), although on treatment, they observed a significant decline in TIMP-1 in the group receiving oral equine estrogen. MMP-2 and TIMP-2 concentrations were not measured in that study. Although the authors did not formally assess the MMP-9 to TIMP-1 ratio, there is a possibility that a decrease in TIMP-1 might lead to an increase in the concentration of active MMP-9, despite the lack of a significant increase in total MMP-9 levels.

Another difference between our study and the study by Wakatsuki et. al. (38) pertains to the fact that their subjects received CEE or transdermal estrogen only, i.e. without a progestin component. We note in our study, however, that previous hysterectomy (women not taking a progestin) had no effect on mean MMP-2, MMP-9, TIMP-1, and TIMP-2 levels. Moreover, if anything, physiological concentrations of progesterone may reduce the protein expression and activity of certain MMPs, including MMP-2 and MMP-9 (39). Furthermore, while comparing our results with the studies by Zanger et al. (37) or Wakatsuki et al. (38), we should note that in those studies treatment was administered for a short period, 1 month and 3 months, respectively. It is therefore possible that the effects of treatment may differ during short-term vs. long-term administration of any given treatment modality. We note, however, an increase in total MMP-9 (37), with possible increase of free MMP-9 (38), was noted after a relatively short treatment period. In our study, all women were on HRT for at least 5 yr, with those on CEE taking treatment the longest on average; however, we failed to note any correlation between duration of HRT treatment and circulating MMP to TIMP levels, as described earlier.

Finally, another possible explanation for differences between our results and those of Wakatsuki et al. (38) may be related to interracial differences in MMP concentrations. Our subjects were Caucasians, whereas subjects assessed by Wakatsuki et al. (38) were Japanese. Interestingly, a recent study by Tayebjee et al. (40) demonstrated lower MMP-9 concentrations in individuals of Far Eastern/Chinese origin, regardless of age or gender.

The clinical significance of our findings, particularly the differential effects of CEE and tibolone on MMPs, remains to be established. We note, however, that both MMP-2 and MMP-9 remodel cardiac and vascular extracellular matrix under physiological and pathological conditions, with peripheral concentrations of MMP-2 and MMP-9 being elevated in acute coronary syndromes and cerebral ischemia (18, 19, 41, 42, 43). Moreover, MMPs are implicated not only in the pathogenesis of CHD and stroke but also neoplastic disease (44). Our observations and the aforementioned may be particularly pertinent given the findings from randomized control trials on HRT, in which women receiving CEE and MPA had a 1.26 hazard ratio for invasive breast cancer and 1.41 for stroke (9, 45). Interestingly, in the recently published estrogen-only arm of the Women’s Health Initiative study (46), there was an excess of strokes but no excess of myocardial infarction and/or breast cancer was observed. To the best of our knowledge, there are no data on the effects of tibolone treatment on blood levels of MMPs or TIMPs. There are, however, data suggesting that tibolone reduces synthesis of endothelin, E-selectin, plasminogen activator inhibitor-1, and pro-MMP-1 in endothelial cell culture in postmenopausal human female coronary artery, i.e. markers that modulate vascular tone and play a role in atherosclerosis (47). Given our findings, in contrast to women receiving CEE, those receiving tibolone might have a decreased risk of cardiovascular events, whereas in case of transdermal E2, the net effect might be close to neutral. To date, there are, however, no randomized clinical trial data on cardiovascular end points in patients treated with either tibolone or transdermal estradiol.

Limitations of the study

The limitations of our study are related to its observational and longitudinal nature. However, the differences in MMPs/TIMPs, implicated in cardiovascular disease and cancer, were statistically significant and dependent on the route/type of HRT used. However, caution must be exercised in that our preliminary observations do not explain the findings of the Women’s Health Initiative/Heart and Estrogen/progestin Replacement Study, and further studies are required to establish a possible cause-effect relationship. Moreover, the precise clinical significance of our findings needs to be confirmed in well-designed randomized control trials in women receiving various forms of postmenopausal therapy, including tibolone and the incidence of cardiovascular disease events and cancer related to their plasma concentrations of MMPs and TIMPs. This is of particular importance, given that despite the evidence of increased stroke events in women receiving oral CEE, there are also data suggesting slower progression of an increase in the intima-media thickness in women receiving 17ß-estradiol (48). Finally, although we noted no significant correlation between MMPs/TIMPs and duration of HRT (subjects had taken HRT for > 5 yr), future studies should address the potential effects of HRT in relation to short-term vs. long-term administration of any given treatment modality.

In conclusion, the results of our study clearly show a difference in the effects of various forms of menopausal therapy on plasma MMP levels, depending on the mode of administration (i.e. oral vs. transdermal) and the type of preparation used. In our observational study, there is a possibility that the apparent differences observed in plasma MMP-2 and MMP-9 concentration in women receiving different types of menopausal therapy may contribute to different cardiovascular and potentially also cancer risk in women receiving HRT.


   Footnotes
 
This work was partially supported by the European Union Center of Excellence in Molecular Medicine Grant QLK3-CT-2002-30326.

First Published Online May 16, 2006

1 G.P. and H.S.R. are joint senior last authors. Back

Abbreviations: BMI, Body mass index; CEE, conjugated equine estrogen; CHD, coronary heart disease; CV, coefficient of variation; HRT, hormone replacement therapy; MMP, matrix metalloproteinase; MPA, medroxyprogesterone acetate; TIMP, tissue inhibitor of metalloproteinase; TRD, transdermal estradiol; VEGF, vascular endothelial growth factor.

Received December 20, 2005.

Accepted May 9, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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D. Xing, S. Nozell, Y.-F. Chen, F. Hage, and S. Oparil
Estrogen and Mechanisms of Vascular Protection
Arterioscler Thromb Vasc Biol, March 1, 2009; 29(3): 289 - 295.
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