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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4594-4600
Copyright © 2004 by The Endocrine Society

Tibolone Activates Nitric Oxide Synthesis in Human Endothelial Cells

Tommaso Simoncini, Paolo Mannella, Letizia Fornari, Antonella Caruso, Gaetano Varone, Silvia Garibaldi and Andrea R. Genazzani

Molecular and Cellular Gynecological Endocrinology Laboratory, Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Pisa 56100, Italy

Address all correspondence and requests for reprints to: Tommaso Simoncini, M.D., Ph.D., Molecular and Cellular Gynecological Endocrinology Laboratory, Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa, Via Roma, 57, 56100 Pisa, Italy. E-mail: t.simoncini{at}obgyn.med.unipi.it (Web site: http//www.med.unipi.it/mcgel).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
After the unexpected findings of the Women’s Health Initiative trial, indicating that traditional cardiovascular risk markers fail to predict the effects of hormone replacement therapy, it is of interest to characterize how steroids act on vascular cells. This is particularly important for tissue-specific drugs such as tibolone, whose actions may differ from other preparations. Because nitric oxide (NO) is a key regulator of vascular tone and atherogenesis, we studied its regulation by tibolone and its metabolites on human endothelial cells. Tibolone and its estrogenic metabolites (3{alpha}- and 3ß-OH tibolone) activate NO synthesis by recruiting functional estrogen receptors, whereas the progestogenic/androgenic metabolite ({Delta}4 isomer) has no effect. During prolonged exposures, tibolone and the estrogenic compounds enhance the expression of endothelial NO synthase (eNOS). In addition, tibolone is able to induce rapid activation of eNOS, leading to rapid increases in the release of NO. Relevant for its clinical effects, the sulfated metabolites of tibolone are also effective in activating eNOS. Different from estrogen, rapid activation of eNOS does not rely on recruitment of phosphatidylinositol-3 kinase but rather on MAPK-dependent cascades. These results help to understand the mechanisms of action of tibolone on the cardiovascular system and have relevant clinical implications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR DISEASE IS the major cause of morbidity and mortality in postmenopausal women in developed countries (1). This observation, along with the wealth of evidence indicating that estrogen has positive vascular actions in vitro as well as in vivo (2), has supported the hypothesis that postmenopausal hormone therapy may protect from cardiovascular disease. However, recent randomized clinical trials (3, 4) have unexpectedly questioned this assumption.

One of the lessons coming from the Heart and Estrogen/progestin Replacement Study and the Women’s Health Initiative trials is that modifications of surrogate cardiovascular risk markers during hormone replacement therapy (HRT) do not predict cardiovascular prognosis. This missing link between lipid modifications and cardiovascular benefits fits with the notion that a major part of sex hormones effects on the cardiovascular system is exerted directly on vascular cells (2).

The studies on the cardiovascular actions of HRT have been limited to selected preparations, and one of the main tasks that stands ahead on the research for a cardiovascular-acting hormonal preparation is to define the direct vascular effects of each compound. This is particularly important after the recent report that women receiving only conjugated equine estrogens (CEE) in the Women’s Health Initiative trial did not develop excessive coronary heart disease, as opposed to women receiving CEE + medroxyprogesterone acetate (4, 5), suggesting that certain compounds may alter the vascular actions of estrogens (6).

Tibolone is a synthetic steroid used for the treatment of postmenopausal symptoms and osteoporosis. After absorption, tibolone is rapidly metabolized into two estrogenic metabolites, the 3{alpha}- and 3ß-hydroxy tibolone, as well as into a progestogenic/androgenic metabolite, the {Delta}4 isomer. Due to the complexity of its actions, tibolone has been proposed to represent the first member of a new class of compounds, the selective tissue estrogenic activity regulators (7). The majority of the metabolites circulate as sulfated, inactive forms, the 3{alpha}-S tibolone being the major form. Both the conversion to estrogenic or progestogenic/androgenic metabolites and the activation of the sulfated forms by desulfatation are to some extent different in the various body districts, and this confers to tibolone a characteristic tissue specificity of action (8).

Tibolone exerts antiatherosclerotic actions directly on the vascular wall, in which it inhibits leukocyte adhesion molecule expression in animal (9) as well as human (10) endothelial cells. Recent data also indicate that tibolone and its estrogenic metabolites inhibit vascular smooth muscle cell growth via regulation of MAPKs (11), therefore reinforcing the concept that a significant part of the cardiovascular actions of this drug is exerted at the vascular level. Indeed, tibolone induces relaxation in selected vascular districts (12, 13, 14, 15), and tibolone administration is associated with increased plasma levels of nitric oxide (NO) metabolites, suggesting a regulation of the synthesis of this potent vasodilatory molecule (16). However, no mechanism has been described to explain tibolone-induced vascular relaxation, and the proof of a link with NO metabolism is missing.

Because endothelial cell-derived NO plays a critical role in cardiovascular physiology and disease, we studied whether tibolone and its metabolites exert direct vascular effects on human endothelial cells through genomic or nongenomic regulation of the endothelial NO synthase (eNOS).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures and treatments

Human umbilical vein endothelial cells (HUVECs) were harvested enzymatically with type I A collagenase (1 mg/ml) as previously described (17) and maintained in phenol red-free DMEM (Life Technologies, Inc., Gaithersburg, MD), containing HEPES (25 mmol/liter), heparin (50 U/ml), endothelial cell growth factor (50 ng/ml), L-glutamine (2 mmol/liter), antibiotics, and 10% fetal bovine serum (FBS). Before each experiment, HUVECs were kept for at least 48 h in DMEM containing 10% steroid-deprived FBS (by activated charcoal stripping). All experiments were performed on confluent monolayers of endothelial cells. Before every experiment investigating rapid, nontranscriptional effects (up to 30-min treatments), HUVECs were serum starved in DMEM containing no FBS for 8 h before treatment to avoid the confounding effects of serum. Whenever an inhibitor was to be used (ICI 182,780, PD 98059, wortmannin, ORG 31710, or Emate), we added the compound 30 min before the treatments. Tibolone, 3{alpha}-OH tibolone, 3ß-OH tibolone, 3{alpha}-sulfated tibolone, 3{alpha}-17ß-disulfated tibolone, ORG 31710, and Emate were provided by Dr. H. J. Kloosterboer (N.V. Organon, Oss, The Netherlands).

eNOS activity assay

Endothelial cells were collected in 320 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µg/ml phenylmethylsulfonyl fluoride. eNOS activity was determined as conversion of [3H]arginine to [3H]citrulline, incubating cell extracts for 10 min at 30 C in 50 mM potassium phosphate, 1.2 mM L-citrulline, 1.2 mM CaCl2, 120 µM nicotinamide adenine dinucleotide phosphate reduced, 24 µM L-[3H]arginine. Converted citrulline was separated by unconverted arginine using the acidic ion-exchange resin Dowex 50 W, 200–400 mesh (Sigma, St. Louis, MO), as described (18). Extracts incubated with the eNOS inhibitor, N-{omega}-nitro-L-arginine methyl ester (1 mM), served as blank. Converted eNOS activity was obtained subtracting the blank to the samples.

Nitrite assay

NO production was determined by a modified nitrite assay using 2, 3 diaminonaphtalene as described (19). Fluorescence of 1-(H)-naphtotriazole was measured with excitation and emission wavelengths of 365 and 450 nm. Standard curves were constructed with sodium nitrite. Nonspecific fluorescence was determined in the presence of NG-monomethyl-L-arginine (3 mM).

Immunoblottings

Cell lysates were separated by SDS-PAGE. Antibodies used were: eNOS (Transduction Laboratories, Lexington, KY), wild-type or Tyr204-P-ERK 1/2 (Calbiochem, San Diego, CA). Primary antibodies were incubated with the membranes overnight at 4 C. The blots were hybridized with a secondary antibody coupled to horseradish peroxidase, as described (20). Immunodetection was accomplished using enhanced chemiluminescence.

Statistical analysis

All values are expressed as the mean ± SD. Statistical differences between mean values were determined by ANOVA, followed by Fisher’s protected least significance difference test for comparison of mean values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tibolone triggers eNOS activity and NO synthesis in a concentration-dependent manner

To compare the effects of tibolone and of its metabolites (3{alpha}-OH tibolone, 3ß-OH tibolone, {Delta}4 isomer, 3{alpha}-sulfate tibolone, 3{alpha}-17ß-disulfate tibolone) on eNOS enzymatic activity and NO synthesis, we exposed steroid-deprived HUVECs to increasing concentrations of the investigated compounds for 30 min or 48 h.

Administration of tibolone, 3{alpha}-OH tibolone, and 3ß-OH tibolone was associated with significant, concentration- dependent, rapid increases in eNOS activity (Fig. 1AGo) and NO release (not shown). At each concentration tested, the two estrogenic 3{alpha}-OH and 3ß-OH metabolites were more potent than the parent compound. The 3{alpha}-sulfated and 3{alpha}-17ß disulfated tibolone also displayed some stimulatory activity on eNOS (Fig. 1AGo) and NO synthesis (not shown), although it became significant only at the highest concentrations. On the contrary, the administration of the {Delta}4 isomer did not alter eNOS activity (Fig. 1AGo) or NO release (not shown).



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  		FIG. 1. Tibolone and its estrogenic metabolites activate eNOS. Steroid-deprived HUVECs were exposed for 30 min (A) or 48 h (B) to increasing concentrations (0.1, 1, 10, 100 nM) of tibolone (TIB), 3{alpha}-OH tibolone (3{alpha} TIB), 3ß-OH tibolone (3ß TIB), {Delta}4 isomer ({Delta}4 ISO), 3{alpha}-sulfate tibolone (3{alpha} S TIB), or 3{alpha}–17ß-disulfate tibolone (3{alpha}–17ß S TIB), and eNOS enzymatic activity was assayed in whole-cell lysates as conversion of [3H]arginine to [3H]citrulline. *, P ≤ 0.05 vs. control (CON). The experiments were repeated three times in triplicates, with equal results.

 
When endothelial cells were treated with tibolone and its metabolites for 48 h, eNOS activity (Fig. 1BGo) and NO release (not shown) further increased during the administration of tibolone and the estrogenic metabolites in a concentration-dependent manner. Again, the sulfated compounds were weak stimulators of eNOS (Fig. 1BGo) and NO synthesis (not shown), and only the highest concentration of the 3{alpha}-sulfated tibolone induced statistically significant effects. As seen during rapid exposures and long-term treatment, the {Delta}4 isomer did not modify the activity of eNOS (Fig. 1BGo) or the synthesis of NO (not shown).

Tibolone estrogenic metabolites induce rapid NO synthesis and eNOS activation through estrogen receptor (ER)-dependent nongenomic mechanisms

Because rapid activation of eNOS and NO synthesis are strongly induced only by the compounds with higher affinity for ER (21), we checked whether, by blocking ER, these actions could be impaired. Indeed, cotreatment with the pure ER antagonist, ICI 182,780, completely prevented the rapid activation of NO synthesis and eNOS by 3{alpha}-OH and 3ß-OH tibolone (Fig. 2AGo). Rapid NO synthesis induction and activation of eNOS are independent from protein synthesis because no increase in eNOS protein amount could be detected in whole-cell lysates (Fig. 2AGo), therefore suggesting the recruitment of nongenomic signaling mechanisms.



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  		FIG. 2. Tibolone and its estrogenic metabolites activate eNOS through genomic and nongenomic, ER-dependent mechanisms. Steroid-deprived HUVECs were exposed for 30 min (A) or 48 h (B) to the estrogenic metabolites 3{alpha}-OH tibolone (3{alpha} TIB, 10 nM) or 3ß-OH tibolone (3ß TIB, 10 nM) in the presence or absence of the pure ER antagonist, ICI 182,780 (ICI, 100 nM), and NO release in the cell culture medium (white bars) and eNOS enzymatic activity (black bars) as well as eNOS protein amount in whole-cell lysates were assayed. *, P ≤ 0.05 vs. control (CON). The experiments were repeated three times in triplicates, with equal results.

 
Tibolone estrogenic metabolites induce NO synthesis via ER-dependent eNOS gene induction

In addition to recruiting rapid signaling, the two estrogenic tibolone metabolites are also able to activate eNOS gene expression during longer (48 h) exposures, as shown by increased levels of eNOS protein in HUVEC extracts (Fig. 2BGo). The induction of eNOS by 3{alpha}-OH and 3ß-OH tibolone relies on the activation of functional ER because the addition of ICI 182,780 abolished the increase in protein expression (Fig. 2BGo). Moreover, enhanced eNOS expression explains the additional increase in eNOS activity seen during prolonged administration of the compounds (Fig. 2BGo).

Kinetics of eNOS activation by tibolone

To further characterize the process of eNOS recruitment by tibolone, we performed a time-course analysis looking at the kinetics of enzymatic activation in endothelial cells using the active estrogenic metabolite 3{alpha}-OH tibolone. 3{alpha}-OH tibolone induced eNOS activation as early as 2 min after administration (Fig. 3Go), and eNOS activity progressively continued to increase thereafter, reaching maximal levels between 12 and 48 h (Fig. 3Go).



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  		FIG. 3. Kinetics of eNOS activation by tibolone. Steroid-deprived HUVECs were exposed for different times (2 min to 48 h) to the estrogenic metabolite 3{alpha}-OH tibolone (3{alpha} TIB, 10 nM), and eNOS enzymatic activity in whole-cell lysates were assayed. *, P ≤ 0.05 vs. control. The experiments were repeated three times in triplicates, with equal results.

 
Effects on eNOS of tibolone sulfated derivatives

Around 80% of tibolone metabolites are found in their sulfated form in the circulation, and these are considered to represent a relatively inactive pool that provides continuous availability of active estrogenic metabolites through tissue-specific desulfatation (22). To understand whether human endothelial cells are also capable of desulfating these compounds, we performed experiments with the monosulfated (3{alpha}-S) and the disulfated (3{alpha}-17ß-S) metabolites in the presence or absence of the specific sulfatase inhibitor, estrone-3-O-sulfamate (Emate). We found that, in the presence of Emate, both the sulfated metabolites lose their ability to recruit eNOS during short- (Fig. 4AGo) or longer-term (Fig. 4BGo) exposures. When used alone, instead, some induction of eNOS was found, with an inverse correlation with the number of sulfoxy groups present in the molecule (Fig. 4Go, A and B).



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  		FIG. 4. Effect of sulfatase inhibition on eNOS regulation by tibolone-sulfated metabolites. Steroid-deprived HUVECs were exposed for 30 min (A) or 48 h (B) to 3{alpha}-sulfate tibolone (3{alpha} S TIB, 10 nM) or 3{alpha}-17ß-disulfate tibolone (3{alpha}–17ß S TIB, 10 nM) in the presence or absence of the sulfatase inhibitor, estrone-3-O-sulfamate (Emate, 1 µM), and eNOS enzymatic activity in whole-cell lysates was assayed. *, P ≤ 0.05 vs. control (CON). The experiments were repeated three times in triplicates, with equal results.

 
Tibolone activates rapid nongenomic signaling to eNOS via an ER/MAPK cascade

To further characterize the nongenomic activation of eNOS induced by tibolone and its metabolites, we explored the effect of the selective inhibitors of the estrogen receptor (ICI 182,780), progesterone receptor (PR) (ORG 31710), MAPK kinase (MEK) 1/2 (PD98059), or phosphatidylinositol- 3 kinase (PI3K) (wortmannin) pathways.

3{alpha}-OH tibolone-dependent induction of eNOS activity was not affected by blocking PR or interfering with PI3K (Fig. 5Go). On the other hand, complete abolition of eNOS activation was obtained by blocking ER or MAPK (Fig. 5Go). None of the inhibitors was able to alter eNOS activity when given alone (Fig. 5Go). To confirm the role of MAPK in tibolone signaling to eNOS, we measured the degree of ERK 1/2 activation by using phospho-specific antibodies toward the wild-type or Tyr204-phosphorylated (active) ERK 1/2 after exposure to 3{alpha}-OH tibolone. In agreement with the previous data, we found marked phosphorylation of ERK 1/2 after a brief exposure to 3{alpha}-OH tibolone (Fig. 5Go). ERK phosphorylation was prevented by blocking ER signaling (with ICI 182,780) and inhibition of the upstream kinase, MEK 1/2 (with PD98059), but not by the inhibitors of PR or of PI3K (Fig. 5Go).



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  		FIG. 5. Tibolone activates eNOS via an ER-dependent, MAPK-dependent nongenomic pathway. Steroid-deprived HUVECs were exposed for 30 min to 3{alpha}-OH tibolone (3{alpha} TIB, 10 nM) in the presence or absence of the pure ER antagonist ICI 182,780 (ICI, 100 nM), the selective PR antagonist ORG 31170 (ORG, 100 nM), the MEK 1/2 inhibitor PD 98059 (PD, 5 µM), or the specific PI3K inhibitor wortmannin (WM, 30 nM). eNOS enzymatic activity (A) and wild-type or Tyr204-phosphorylated-ERK 1/2 protein amount (B) were assayed in whole-cell lysates. *, P ≤ 0.05 vs. control (CON). The experiments were repeated three times in triplicates, with equal results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HRT is effective for the treatment of menopausal symptoms and of some of the long-term complications of ovarian hormone depletion, but the side effects may be significant, and the fear of developing uterine and breast cancer is an important reason for noncompliance. The development of compounds that act as estrogens on the desired tissues and behave as estrogen antagonists on the mammary gland and endometrium has thus great clinical relevance.

Cardiovascular disease is a major concern for postmenopausal women, and estrogen deficiency is an important concurrent factor in the pathophysiology of vascular degeneration (2, 23). Recent trials (3, 4) have heightened the attention on the effects of hormonal preparations on the cardiovascular system, and newer approaches are required to correctly understand the action of each compound. Indeed, because the modifications of traditional surrogate cardiovascular risk markers have not been found to reflect the clinical effects of postmenopausal hormone therapies, the direct effects on vascular cells should be specifically investigated. In addition, the recent news of the absence of any excess of coronary heart disease in women participating in the CEE-only arm of the Women’s Health Initiative (and actually of a trend toward protection in younger women), as opposed to the women who received CEE plus medroxyprogesterone acetate (4, 5), reinforced the hypothesis that specific medications may have detrimental effects on cardiovascular disease, independently from the amelioration of the lipid profile (6).

Tibolone is a synthetic steroid acting as a selective tissue estrogenic activity regulator (7), whose action depends on its metabolism in the target tissues. The cardiovascular action of tibolone has been particularly debated due to the fact that this compound induces a modification of the lipid profile that is markedly different with respect to standard estrogen/progestin HRTs. Indeed, significant decreases of high- density lipoprotein (HDL) cholesterol are found in postmenopausal women receiving tibolone (8), which have raised serious concerns about the safety of this drug in the past. However, this drug has been widely used in Europe, and no apparent increase in cardiovascular disease incidence has been observed (8). Moreover, animal models investigating the effect of a long-term treatment with tibolone after castration do not show enhanced atherosclerosis (24, 25, 26). In agreement, several reports indicate that, notwithstanding the decreased HDL cholesterol levels, tibolone is neutral (27) or actually decreases the extent of carotid atherosclerosis in humans (14) and prevents the formation of cholesterol- induced atherosclerosis in rabbits (28). However, the results obtained in animal models may not translate directly to humans due to significant differences in the modifications of HDL cholesterol and triglyceride levels induced by tibolone in these species (24, 25, 26, 28).

Recent evidence of preserved reverse cholesterol transport notwithstanding the decreased HDL cholesterol concentrations has been provided (29, 30), and this may explain the absence of an excess of atherosclerotic degeneration in women as well as in animal models during prolonged tibolone administration. However, the presence of protective actions on vascular cells may also explain this apparent paradox, and indeed tibolone and its estrogenic metabolites have been shown to exert direct vascular antiinflammatory actions, inhibiting leukocyte adhesion molecules expression in human endothelial cells (10).

NO synthesis by endothelial cells is of paramount importance for the regulation of vascular tone and blood flow and control of the hemostatic process (31). Furthermore, endothelium-derived NO is a potent antiinflammatory and antiatherogenic factor, being able to prevent endothelial cell dysfunction and vascular degenerative processes (31). Moreover, NO is a primary vascular target of estrogens. In fact, estrogens activate the synthesis and release of NO through the stimulation of eNOS gene expression (32) as well as through nontranscriptional enhancement of eNOS enzymatic activity (20, 33, 34).

Our present data show that tibolone and its metabolites act like estrogens on endothelial cells, triggering NO synthesis. We also demonstrate that tibolone metabolites have a double mechanism of action, inducing a rapid nongenomic stimulation of eNOS activity as well as an up-regulation of eNOS protein during longer exposures. These results are in agreement with recent work indicating increased amounts of NO metabolites in postmenopausal women receiving tibolone (16), as well as NO-dependent vasorelaxation of animal (35) and human vessels (36) on challenge with tibolone. However, it has to be mentioned that the functional regulation of peripheral arterial vessels in postmenopausal women receiving tibolone is not yet established because two well- conducted prospective, randomized trials looking at brachial artery flow-mediated vasodilation reported contrasting results, either showing improved dilation in women administered daily tibolone (12) or showing neutral effects (37).

Our experiments indicate that the activation of endothelial NO synthesis by tibolone depends on the recruitment of functional ERs, as shown by the experiments with ER inhibitors as well as by the approximately 1000-fold stronger effect of 3{alpha}-OH and 3ß-OH tibolone with respect to the parent steroid, which fits well with the relative affinities for ER{alpha} (21). This is also consistent with the previous evidence that only the estrogenic 3{alpha}-OH and 3ß-OH tibolone are effective in inhibiting cytokine-induced leukocyte adhesion molecules expression in human endothelial cells (10). In addition, the progestogenic/androgenic metabolite, {Delta}4 isomer, was completely neutral on NO synthesis, therefore reinforcing the concept that estrogen receptors are the major effectors of tibolone effects on NO synthesis in endothelial cells.

Like estrogen, tibolone is found to exert both genomic and nongenomic actions which influence NO synthesis. The transcriptional induction of eNOS is likely to be important in the vessel because NO has been found to be a potent antiinflammatory and antiatherogenic mediator. However, nongenomic signaling through ERs is also of primary importance in endothelial cells. Indeed, we have recently shown that through rapid activation of NO, steroid hormones induce in vivo relevant protective actions such as the prevention of vascular injury during ischemia-reperfusion (20) and myocardial ischemia (38).

Rapid eNOS activation on exposure to estrogen relies on nongenomic recruitment of MAPK and PI3K pathways (20, 33, 34). Tibolone is instead found to act exclusively through MAPK but not through PI3K. This difference could be explained by the inability of ER{alpha} to physically interact with the regulatory subunit of PI3K, p85{alpha}, when engaged by tibolone or its metabolites because different ER{alpha} agonists have been shown to systematically induce distinct receptor conformations (39). To our knowledge, this is the first molecular characterization of a difference between ER-dependent intracellular signaling induced by estradiol or tibolone and may help to understand some of the differential effects of this steroid with respect to estrogens seen in clinical practice.

Both the genomic induction of eNOS and the rapid activation of the enzyme coordinately leading to enhanced NO production by endothelial cells could be relevant in the clinical setting because the concentrations used in our experiments are in the range associated with effective transactivation of ERs (21).

This is further supported by the evidence that endothelial cells are able to convert the inactive sulfated tibolone metabolites to their active forms trough desulfatation, thus suggesting that the endothelial lining may well be reached by a biologically effective concentration of active metabolites due to the fact that the 3{alpha}-sulfated tibolone, and the 3{alpha}-17ß-sulfated tibolone are present in high concentration in the bloodstream after absorption and thus provide an abundant source of active compounds.

In conclusion, we here show that tibolone is able to directly modulate human endothelial cells, triggering NO synthesis through both ER-dependent transcriptional and nontranscriptional signaling. By enhancing endothelial-derived NO production, tibolone and its estrogenic metabolites induce potentially important vascular antiinflammatory and antiatherogenic effects; however, the clinical effects of tibolone on heart disease have still to be tested in clinical trials.


   Footnotes
 
Abbreviations: CEE, Conjugated equine estrogen; eNOS, endothelial NO synthase; ER, estrogen receptor; FBS, fetal bovine serum; HDL, high-density lipoprotein; HRT, hormone replacement therapy; HUVEC, human umbilical vein endothelial cell; MEK, MAPK kinase; NO, nitric oxide; PI3K, phosphatidylinositol-3 kinase; PR, progesterone receptor.

Received December 22, 2003.

Accepted May 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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