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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 852-859
Copyright © 2004 by The Endocrine Society

Tibolone and Its Metabolites Induce Antimitogenesis in Human Coronary Artery Smooth Muscle Cells: Role of Estrogen, Progesterone, and Androgen Receptors

Raghvendra K. Dubey, Delbert G. Gillespie, Marion Grögli, Helenius J. Kloosterboer and Bruno Imthurn

Department of Medicine (R.K.D., D.G.G., B.I.), Center for Clinical Pharmacology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213; Department of Obstetrics and Gynecology (R.K.D., M.G., B.I.), Clinic for Endocrinology, University Hospital Zurich, 8091 Zurich, Switzerland; and Research and Development Laboratories (H.J.K.), NV Organon, 5340 BH Oss, The Netherlands

Address all correspondence and requests for reprints to: Dr. Raghvendra K. Dubey, Clinic for Endocrinology (D217, NORD-1), Department of Obstetrics and Gynecology, University Hospital Zurich, Frauenklinikstrasse 10, 8091 Zurich, Switzerland. E-mail: raghvendra.dubey{at}usz.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tibolone, a hormone replacement drug, protects postmenopausal women against osteoporosis and climacteric symptoms without inducing adverse effects on the endometrium and breast. Compared with other estrogens, little is known about the cardiovascular effects of tibolone. Because abnormal growth of smooth muscle cells (SMCs) is a prerequisite for coronary artery disease, here we investigated the effects of tibolone on SMC growth.

We examined the effects of tibolone and its metabolites on human arterial SMC growth (DNA synthesis, cellular proliferation, cell migration, collagen synthesis) and MAPK expression. Fetal calf serum-induced SMC growth, phosphorylated MAPK expression, and platelet-derived growth factor-induced SMC-migration were concentration-dependently inhibited by tibolone and its endogenous estrogenic and progestogenic/androgenic metabolites in the following order of potency: {Delta}4-tibolone>3ß-OH-tibolone {cong} 3{alpha}-OH-tibolone. The antimitogenic effects of tibolone were partially blocked by ER antagonist (ICI182780), progesterone receptor antagonist (RU486) but not by the androgen receptor antagonist (flutamide); moreover, RU486 was more potent than ICI182780. The antimitogenic effects of tibolone were completely blocked by RU486 plus ICI182780. In addition, the inhibitory effects of equimolar concentrations of the three tibolone metabolites summed up to the inhibitory effects of tibolone.

In conclusion, tibolone inhibits SMC growth and MAPK phosphorylation via both its estrogenic and progestogenic metabolites, and these inhibitory effects involve both progesterone and ERs. Hence, tibolone may induce antivasoocclusive actions and protect women against coronary artery disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROTECTIVE EFFECTS OF estrogen on the cardiovascular system is supported by several epidemiological and observational studies (1). However, findings of randomized hormone replacement therapy trials for the primary and secondary prevention of cardiovascular disease do not support this notion (2, 3). The lack of protective effects have been attributed to the differences in the chemical characteristic of conjugated equine estrogens used and coadministration of the synthetic progestin, medroxyprogesterone (1, 4, 5). Therefore, it is important to know whether other menopausal therapies have adverse or beneficial effects on mechanism(s) that play a key role in the vascular remodeling process associated with coronary artery disease.

Tibolone, a novel hormone replacement drug widely used in several countries, protects postmenopausal women against osteoporosis and climacteric symptoms, without inducing adverse effects on the endometrium and breast (6, 7). In vivo tibolone is rapidly metabolized to two estrogenic metabolites, 3{alpha}-OH-tibolone and 3ß-OH-tibolone, and {Delta}4-tibolone, a metabolite with both progestogenic and androgenic properties (6). Tibolone itself is biologically inactive, and its mixed hormonal activities are attributed to its estrogenic and progestogenic/androgenic metabolites (6). Moreover, because of its unique properties of mediating the protective effects on bone via estrogen receptors (ERs) and blocking the growth effects in breast and endometrium by inhibiting steroid metabolizing enzymes (8) rather than acting as a receptor antagonist, as shown for selective ER modulators (8), tibolone is also classified as a selective tissue estrogen activity regulator (8).

Compared with various estrogens, very little is known with regard to the vascular effects of tibolone. A limited number of recent studies provides evidence that tibolone may positively influence mechanisms that would have antivasoocclusive actions. In this context tibolone protects against cholesterol-induced endothelial damage/dysfunction and accumulation of cholesterol and fatty streak formation within the vessel wall (9); improves endothelium-dependent smooth muscle relaxation and lowers serum cholesterol (9), lipoprotein (a), and triglycerides levels (10, 11, 12); down-regulates the cytokine-induced expression of vascular cell adhesion molecule-1, E-selectin (13), plasminogen activator inhibitor-1, and pro-matrix metalloproteinase-1 (14) in human arterial endothelial cells; inhibits endothelin-1 synthesis (14, 15); and induces nitric oxide synthesis (16) and antiischemic effects (17). However, in contrast to other clinically used estrogens, tibolone decreases high-density lipoprotein (HDL) levels (18), raising concerns about its vasoprotective actions.

Abnormal growth of smooth muscle cells (SMCs) [migration from media into the intima, proliferation, and deposition of extracellular matrix proteins such as collagen] contributes to the vascular remodeling process associated with coronary artery disease and atherosclerosis (19, 20). Moreover, the inhibitory effects of estradiol on SMC growth largely contributes to its antivasoocclusive/vasoprotective effects (1). Although tibolone mimics the effects of estradiol on several vasoprotective mechanisms, its effects on SMC growth have not been investigated. Moreover, the mechanisms by which it influences SMC growth and the relative contribution of the estrogenic and progestogenic components remains unknown. Therefore, in the present study, we investigated the effects of tibolone and its estrogenic and progestogenic metabolites on serum-induced growth and platelet-derived growth factor (PDGF)-BB-induced SMC migration. Moreover, the role of estrogen, progesterone, and androgen receptors (ARs) in mediating the effects of tibolone on SMC activity were investigated. Because the MAPK pathway plays a key role in mediating the mitogenic signal for multiple mitogens implicated in the pathogenesis of SMC growth in atherosclerosis and coronary artery disease, we also investigated the effects of tibolone and its metabolites on MAPK expression.


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

All tissue culture reagents and culture ware were purchased from GIBCO Laboratories (Grand Island, NY). Fetal calf serum (FCS) was obtained from HyClone Laboratories Inc. (Logan, UT). 17ß-Estradiol, RU486, flutamide, and PD98059 were purchased from Sigma Chemical Co. (St. Louis, MO). Tibolone, 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone were provided by N.V. Organon (Oss, The Netherlands). ER antagonist ICI 182,780 was from Tocris (Langford, Bristol, UK). 3H-thymidine (specific activity 11.8 Ci/mmol) was purchased from ICN Biomedicals (Costa Mesa, CA). L-[3H]-proline (specific activity 23 Ci/mmol) was purchased from Amersham Biosciences (Piscataway, NJ). Antibodies for ERK1/2 were procured from Calbiochem (La Jolla, CA).

Cell culture

Well-characterized female human coronary artery SMCs were obtained from Cascade Biologics (Portland, OR) and cultured as described by us previously (4). SMC purity was recharacterized by immunofluorescence staining with smooth muscle-specific anti-smooth muscle {alpha}-actin monoclonal antibodies and morphologic criteria specific for SMC as described in detail previously (4). SMCs in fourth passage were used for all the studies.

Growth studies

3H-thymidine incorporation (index of DNA synthesis) and cell number (cell proliferation) studies were conducted to investigate the effects of various test agents on cell growth. SMCs were plated at a density of 5 x 103 cells/well in 24-well tissue culture dishes and allowed to grow to subconfluence in DMEM/F12 (phenol red free) medium containing 10% FCS (steroid free and delipidated) under standard tissue culture conditions. The cells were then growth arrested by feeding DMEM (phenol red free) containing 0.4% albumin for 48 h. For DNA synthesis, growth was initiated by treating growth arrested cells for 20 h with DMEM containing 2.5% FCS and containing or lacking the test agent(s). To evaluate the effects of estrogen, progesterone, and AR antagonists, cells were pretreated for 30 min with ICI 182,780, RU486 or flutamide before the treatment with the test agents. After 20 h of incubation, the treatments were repeated with freshly prepared solutions but supplemented with 3H-thymidine (1 µCi/ml) for an additional 4 h. The experiments were terminated by washing the cells twice with Dulbecco’s PBS and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized in 500 µl of 0.3 N NaOH and 0.1% sodium dodecylsulfate after incubation at 50 C for 2 h. Aliquots from 4 wells for each treatment with 10 ml scintillation fluid were counted in a liquid scintillation counter. For cell-number experiments, SMCs were allowed to attach overnight, growth arrested for 48 h, and then treated every 24 h for 4 d, and on d 5 cells were dislodged and counted on a coulter counter.

3H-proline incorporation studies were done to investigate the effects of various test agents on collagen synthesis. Confluent monolayers of SMCs were made quiescent by feeding DMEM containing 0.4% BSA for 48 h. SMCs growth arrested for 48 h were treated with DMEM supplemented with 2.5% FCS plus 3H-L-proline (1 µCi/ml) and containing or lacking the test agents. To evaluate the effects of estrogen, progesterone, and AR antagonists, cells were pretreated for 30 h with ICI 182,780, RU486, or flutamide, respectively, before the treatment with the test agents. The experiments were terminated by washing the cells twice with PBS and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized as described above and aliquots from 4 wells for each treatment counted in a liquid scintillation counter. Each experiment was conducted in triplicate and with three separate cultures of SMCs. To make sure that the inhibitory effects of the experimental agents on collagen synthesis were not due to changes in cell number, the experiments were conducted in confluent monolayers of cells in which changes in cell number were precluded. Additionally, cell counting was performed in cells treated in parallel to the cells used for the collagen synthesis studies, and the data were normalized to cell number.

SMC migration

Modified Boydens chamber (Neuro Probe Inc., Cabin John, MD) was used to evaluate the effects of various estrogens on PDGF-BB-induced SMC migration and as previously described (4). To evaluate the effects of estrogen, progesterone, and AR antagonists, cells were pretreated for 30 min with ICI 182,780, RU486, or flutamide before the treatment with the test agents.

MAPK (ERK1/2) expression

The effects of the test agents on MAPK expression were also assessed because MAPK is an important mediator of cell growth. SMCs grown to subconfluence in 35-mm2 culture dishes were treated for 24 or 48 h with or without various test agents in the presence of FCS (2.5%). Then the treatments the cells were washed with ice-cold PBS and solubilized by adding lysis buffer. Equal amounts of proteins (20 µg/lane) were denatured by boiling at 95 C for 5 min and resolved in 10% sodium dodecyl sulfate-polyacrylamide gel. After separation the proteins were transferred to a nitrocellulose membrane and the membranes probed with ERK1/2 antibodies and the bands visualized after staining with peroxidase-conjugated secondary antibody and enhanced chemiluminescence system and using X-OMAT LS films (Kodak, Rochester, NY) for exposure. Densitometry of both p42 and p44 bands was conducted to assess the changes in phosphorylated MAPK.

All experiments were performed in triplicate or quadruplicate with three separate cultures. Data are presented as mean ± SEM. Statistical analysis was performed using ANOVA, paired or unpaired Student’s’ t test, or Fisher’s least significant difference test as appropriate. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment with 2.5% FCS stimulated 3H-thymidine incorporation by {cong} 7-fold (P < 0.001 vs. 0.4% BSA) and 3H-proline incorporation by {cong} 5-fold (P < 0.05 vs. 0.4% BSA). Treatment with tibolone as well as 17ß-estradiol inhibited FCS-induced 3H-thymidine incorporation in a concentration-dependent manner (Fig. 1Go). At equimolar concentrations tibolone was as potent as 17ß-estradiol in inhibiting 3H-thymidine incorporation. Physiological concentrations of 17ß-estradiol (0.001 µmol/liter) and equimolar concentration of tibolone (0.001 µmol/liter) significantly inhibited FCS-induced 3H-thymidine incorporation by 10.7 ± 1.2% and 12.3 ± 3%, respectively (Fig. 1Go). A {cong} 50% decrease in FCS-induced 3H-thymidine incorporation was observed at approximately 1 µmol/liter of tibolone and 17ß-estradiol, respectively (Fig. 1Go). Similar to the effects on 3H-thymidine incorporation, tibolone and 17ß-estradiol inhibited 2.5% FCS-induced 3H-proline incorporation (Fig. 2Go). Tibolone and 17ß-estradiol decreased proline incorporation by 50% at 0.1 µmol/liter.



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  		FIG. 1. Inhibition of FCS-induced DNA synthesis (3H-thymidine incorporation) by 0.001–1 µmol/liter of tibolone, its metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone), and 17ß-estradiol in human coronary artery SMCs. Values are mean ± SEM from three experiments conducted in quadruplicate. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone.

 


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  		FIG. 2. Inhibition of FCS-induced collagen synthesis (3H-proline incorporation) by 0.001–1 µmol/liter of tibolone, its metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone), and 17ß-estradiol in human coronary artery SMCs. Values are mean ± SEM from three experiments conducted in quadruplicate. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone.

 
FCS increased cell number in growth-arrested SMCs by {cong}8-fold (data not shown). Tibolone and 17ß-estradiol inhibited FCS-induced increases in cell number in a concentration-dependent manner (Fig. 3Go). The lowest concentrations of tibolone and 17ß-estradiol that significantly inhibited FCS-induced increases in cell number were 0.001 µmol/liter, and these concentrations inhibited cell number by 18 ± 2% and 14 ± 2%, respectively. Trypan blue exclusion tests and dimethylthiazoldiphenyltetra-zoliumbromide assay indicated no loss in viability of cells treated with tibolone and 17ß-estradiol (data not shown).



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  		FIG. 3. Inhibition of FCS-induced cell number (cell-proliferation) by 0.001–1 µmol/liter of tibolone, its metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone), and 17ß-estradiol in human coronary artery SMCs. Values are mean ± SEM from three experiments conducted in quadruplicate. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone.

 
Similar to tibolone, FCS-induced 3H-thymidine incorporation, 3H-proline incorporation, and cell number were inhibited in a concentration-dependent fashion by its metabolites 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone (Figs. 1–3GoGoGo). The inhibitory effects of {Delta}4-tibolone were comparable with the effects of tibolone. Compared with {Delta}4-tibolone and tibolone, the estrogenic metabolites of tibolone were significantly less potent in inhibiting FCS-induced increases in 3H-thymidine incorporation, 3H-proline incorporation, and cell number. Tibolone inhibited 3H-thymidine incorporation by 54%, 3H-proline incorporation by 49.8%, and cell number by 60% at a concentration of 0.1 µmol/liter. At this concentration 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone inhibited thymidine incorporation by approximately 24, 23.5, and 34%, respectively, proline incorporation by 22.6, 26.6, and 31%, and cell number by 41, 34.21, and 47.6%.

Treatment of SMCs with PDGF-BB stimulated migration of human aortic SMCs (P < 0.05 vs. cells treated with 0.4% BSA; Fig. 4Go). PDGF-BB-induced SMC migration was inhibited in a concentration-dependent manner in SMCs pretreated with tibolone (Fig. 4AGo). The inhibitory effects of tibolone on PDGF-BB-induced SMC migration were mimicked by its metabolites 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone (Fig. 4BGo). Concentrations of tibolone (0.001 µmol/liter) equivalent to the physiological concentrations of 17ß-estradiol inhibited SMC migration by 16 ± 2% (P < 0.05 vs. SMCs treated with vehicle alone). At 0.001 µmol/liter, tibolone inhibited PDGF-BB-induced SMC migration by 58%. At this concentration 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone inhibited SMC migration by 29.4, 20.6, and 35%, respectively.



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  		FIG. 4. Inhibition of PDGF-BB (25 ng/ml)-induced cell migration by 0.001–1 µmol/liter tibolone (A) and 0.1 µmol/liter of tibolone, its metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone), and 17ß-estradiol (B). *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone.

 
To investigate whether the antimitogenic effects of tibolone metabolites add up to the effects of tibolone, we studied the additive effects of 0.1 µmol/liter of 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone on SMC growth. As shown in Fig. 5Go, the inhibitory effects of 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone on 3H-thymidine incorporation, cell proliferation, 3H-proline incorporation, and SMC migration were additive in nature, with the final effect equal to that observed in presence of 0.1 µmol/liter of tibolone. Treatment of SMCs with 0.1 µmol/liter of {Delta}4-tibolone, 3{alpha}-OH-tibolone, 3ß-OH-tibolone, 3{alpha}-OH-tibolone plus 3ß-OH-tibolone, 3{alpha}-OH-tibolone plus 3ß-OH-tibolone plus {Delta}4-tibolone, and tibolone inhibited DNA synthesis by 28, 13, 18, 23, 44, and 48%, respectively. Moreover, treatment of SMCs with 0.1 µmol/liter of 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone inhibited SMC migration in an additive fashion, with the final inhibitory effect equal to that observed with 0.1 µmol/liter tibolone.



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  		FIG. 5. Bar graph demonstrating that estrogenic and progestogenic metabolites can account for the inhibitory effects of tibolone. Figure depicts a comparison of the inhibitory effects of 0.1 µmol/liter tibolone with 0.1 µmol/liter of 3{alpha}-OH-tibolone (3{alpha}-T), 3ß-OH-tibolone (3ß-T), {Delta}4-tibolone ({Delta}4-T), 3{alpha}-T plus 3ß-T, 3{alpha}-T plus 3ß-OH-T plus {Delta}4-T on FCS (2.5%)-induced SMC growth (DNA synthesis, collagen synthesis, cell number) and PDGF-BB (25 ng/ml)-induced cell migration. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone.

 
To investigate whether the inhibitory effects of tibolone on SMC growth were mediated via estrogen, progesterone, or ARs, the effects of tibolone in the presence and absence of ICI 182,780 (a potent ER antagonist), RU486 [a specific progesterone receptor (PR) antagonist], and flutamide (a potent AR antagonist) were examined. The inhibitory effects of tibolone on FCS-induced 3H-thymidine incorporation, 3H-proline incorporation, cell migration, and cell number were partially, but significantly, reversed (Fig. 6Go) in SMCs pretreated with ICI 182,780 (10 µmol/liter) and RU486 (1 µmol/liter) but not in cells pretreated with flutamide (10 µmol/liter). Moreover, when combined, the antagonistic effects of ICI 182,780 and RU486 were additive in nature and blocked the inhibitory effects of tibolone completely (Fig. 6Go). The inhibitory effects of 3{alpha}-OH-tibolone and 3ß-OH-tibolone were completely reversed in SMCs pretreated with ICI182,780 but not with RU486 (Fig. 7Go). Similarly, the inhibitory effects of {Delta}4-tibolone were completely reversed in SMCs pretreated with RU486 but not in cells pretreated with ICI182,780 or flutamide (Fig. 8Go). In the presence of ICI182780, the inhibitory effects of 0.1 µmol/liter of 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and 3{alpha}-OH-tibolone plus 3ß-OH-tibolone on cell proliferation were reversed from 26 to 8%, 17 to 6.5%, and 31 to 9%, respectively. Similarly, RU486 reversed the inhibitory effects of 0.1 µmol/liter of {Delta}4-tibolone of SMC proliferation from 43% to 12%.



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  		FIG. 6. Modulatory effects of estradiol receptor antagonist [ICI 182,780 (ICI); 10 µmol/liter], PR antagonist [RU486 (RU); 1 µmol/liter] and AR antagonist [flutamide (FLU); 10 µmol/liter] on the inhibitory effects of tibolone (TIB; 100 nmol/liter) on FCS (2.5%)-induced thymidine incorporation, collagen synthesis, and PDGF-BB (25 ng/ml)-induced migration of human coronary artery SMCs. Results are expressed as percent of control and represent mean ± SEM from n = 3 experiments each in triplicate. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone (significant reversal of the inhibitory effects of tibolone).

 


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  		FIG. 7. Modulatory effects of estradiol receptor antagonist [ICI 182,780 (ICI); 10 µmol/liter], PR antagonist [RU486 (RU); 1 µmol/liter] and AR antagonist [flutamide (FLU); 10 µmol/liter] on the inhibitory effects of 3-{alpha}-OH-tibolone (3ß-OH-T; 100 nmol/liter) and 3{alpha}-OH-tibolone (3{alpha}-OH-T; 100 nmol/liter) on FCS (2.5%)-induced thymidine incorporation, collagen synthesis, cell number, and PDGF-BB (25 ng/ml)-induced migration of human coronary artery SMCs. Results are expressed as percent of control and represent mean ± SEM from n = 3 experiments each in triplicate. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. 3{alpha}-OH-T or 3ß-OH-T alone (significant reversal of the inhibitory effects of 3{alpha}-OH-T or 3ß-OH-T).

 


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  		FIG. 8. Modulatory effects of estradiol receptor antagonist [ICI 182,780 (ICI); 10 µmol/liter], PR antagonist [RU486 (RU); 1 µmol/liter], and AR antagonist [flutamide (FLU); 10 µmol/liter] on the inhibitory effects of {Delta}4-tibolone ({Delta}4-T; 100 nmol/liter) on FCS (2.5%)-induced thymidine incorporation, collagen synthesis, and PDGF-BB (25 ng/ml)-induced migration of human coronary artery SMCs. Results are expressed as percent of control and represent mean ± SEM from n = 3 experiments each in triplicate. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. {Delta}4-tibolone alone (significant reversal of the inhibitory effects of {Delta}4-tibolone).

 
The inhibitory effects of 0.1 µmol/liter tibolone on SMC migration were reduced from 57% to 45, 32, and 5.6%, in presence of ICI182780, RU486, and ICI182780 plus RU486, respectively. The inhibitory effects of 3{alpha}-OH-tibolone and 3ß-OH-tibolone on SMC migration were blocked by ICI182780 but not RU486 (Fig. 7Go), whereas the inhibitory effects of {Delta}4-tibolone on SMC migration were blocked by RU486 (Fig. 8Go). In presence of ICI182780, the inhibitory effects of 0.1 µmol/liter of 3{alpha}-OH-tibolone and 3ß-OH-tibolone were reduced from 17% to 4% and from 14% to 3.3%, respectively. Similarly, in presence of RU486, the inhibitory effects of 0.1 µmol/liter of {Delta}4-tibolone were reduced from 35% to 13%. We also observed that ICI182780 blocked the inhibitory effects of estradiol on SMC migration, whereas RU486 blocked the inhibitory effects of progesterone on SMC migration (data not shown). Together these findings suggest that 3{alpha}-OH-tibolone and 3ß-OH-tibolone inhibit SMC migration via ERs, and {Delta}4-tibolone inhibits SMC migration via PRs. Indeed, in presence of both ICI182780 and RU486, the inhibitory effects of tibolone as well as a combination of 3{alpha}-OH-tibolone and 3ß-OH-tibolone plus {Delta}4-tibolone on SMC migration were completely reversed (Fig. 6Go). In contrast to ICI182780 and RU486, flutamide failed to block the inhibitory effects of tibolone on SMC migration (Fig. 6Go); moreover, flutamide did not abrogate the effects of 3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone on SMC migration (Figs. 7Go and 8Go).

Phosphorylated MAPK (ERK1/2) expression was observed in SMCs treated with 2.5% FCS. In SMCs pretreated for 24–48 h with tibolone (1 µmol/liter), the stimulatory effects of FCS on ERK 1/2 phosphorylation were inhibited in a time-dependent manner (Fig. 9Go). The inhibitory effects of tibolone on ERK1/2 phosphorylation were also mimicked in SMCs treated for 48 h with 1 µmol/liter of 3{alpha}-OH-tibolone, 3ß-OH-tibolone, or {Delta}4-tibolone (Fig. 9Go). To investigate whether there is a causal relationship between inhibition of SMC growth and MAPK activity, we assessed the inhibitory effects of tibolone and its metabolites in presence and absence of the MAPK inhibitor, PD98059. As shown in Fig. 10Go, treatment of cells with PD98059 (1–30 µmol/liter) inhibited 2.5% serum-induced DNA synthesis in SMCs; moreover, the inhibitory effects of 0.1 µmol/liter of tibolone, {Delta}4-tibolone, 3{alpha}-OH-tibolone, and 3ß-OH-tibolone were enhanced in presence of 10 µmol/liter PD98059 (Fig. 10AGo). Tibolone, PD98059, and tibolone plus PD98059 inhibited DNA synthesis by 43, 40, and 71%, respectively (Fig. 10BGo). That the inhibitory effects were additive in nature suggests that the actions were mediated via inhibition of a common (MAPK) pathway; however, participation of other mechanism(s) cannot be ruled out.



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  		FIG. 9. Representative Western blots showing the inhibition of FCS-induced expression of phosphorylated MAPK (ERK1/2) by 1 µmol/liter of tibolone and its metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone) in human coronary artery SMCs treated for 24 or 48 h. The bar graph shows the densitometric analysis of the changes observed in both p42 and p44 bands and normalized to the internal standard, nonphosphorylated MAPK (ERK1/2). Values for the bar graph are mean ± SEM from three to four experiments conducted in triplicates. T, Treated with tibolone or its metabolites in presence of 2.5% FCS; C, control cells treated with 2.5% FCS alone. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone.

 


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  		FIG. 10. A, Inhibition of FCS-induced DNA synthesis (3H-thymidine incorporation) by 1–30 µmol/liter of MAPK inhibitor, PD98059. B, Inhibitory effects of 0.1 µmol/liter of tibolone, its metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone, and {Delta}4-tibolone) on human coronary artery SMCs in presence and absence of 10 µmol/liter of PD98059. Values are mean ± SEM from three experiments conducted in quadruplicate. *, P < 0.05 vs. control (SMCs treated with FCS alone); §, P < 0.05 vs. tibolone or its metabolites alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that, similar to estradiol, tibolone inhibits FCS-induced DNA synthesis, collagen synthesis, and cell proliferation as well as PDGF-BB-induced SMC migration. Our findings provide the first evidence that tibolone is capable of inducing vasoprotective effects by directly interacting with SMCs in the vessel wall and inhibiting SMC growth. This contention is further supported by the finding that tibolone inhibits cholesterol-induced atherosclerotic as well as injury-induced lesions in animals fed a hypercholesterolemic diet (9). Moreover, in postmenopausal women, tibolone treatment decreases both the intima-media thickness as well as resistive indices of the common carotid artery (21).

We observed that among the tibolone metabolites, the inhibitory effects of tibolone on SMC biology are partially mimicked by both its estrogenic metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone) and progestogenic metabolite ({Delta}4-tibolone). Moreover, the inhibitory effects of tibolone were partially reversed in presence of both PR and ER antagonists, implying that it inhibits SMC activity via both ERs and PRs. A major role of the progestogenic component of tibolone in mediating its antimitogenic effects is evident from the observation that, compared with the ER antagonist, ICI182780, the PR antagonist, RU486, was more efficacious in blocking the antimitogenic effects of tibolone. This notion is further supported by the finding that {Delta}4-tibolone, a progestogenic metabolite of tibolone, was more potent than the estrogenic metabolites of tibolone (3{alpha}-OH-tibolone, 3ß-OH-tibolone) in inhibiting SMC activity. Similar to the inhibitory effects on SMC activity, tibolone inhibited serum-induced expression of phosphorylated ERK1/2; moreover, these effects were also mimicked by its metabolites. Taken together, our findings provide evidence that tibolone and its metabolites can inhibit SMC growth; moreover, they mediate their inhibitory effects on SMCs by inhibiting mitogen-induced MAPK phosphorylation and that the inhibitory effects of tibolone are mediated via both ERs and PRs. Finally, tibolone is as potent as estradiol in inhibiting SMC growth, suggesting that it may be as effective preventing against vasoocclusive disorders in postmenopausal women receiving estradiol treatment.

The tibolone metabolite {Delta}4-tibolone binds to both androgen and progestogen receptors and has both progestogenic and androgenic activities (6). This suggests that the growth-inhibitory effects of tibolone may, in part, be mediated via PR or AR. Because androgens have also been suggested to protect the vasculature, the role of ARs in mediating the effects of tibolone and its metabolites was also investigated. The observation that the inhibitory effects of tibolone as well as {Delta}4-tibolone were blocked by the PR antagonist, RU486, and not by the AR antagonist, flutamide, provides direct evidence that ARs are not involved in mediating the inhibitory effects of tibolone and {Delta}4-tibolone. In this context it is important to note that there is very little evidence to support the notion that androgens protect against cardiovascular disease (22).

In addition to the {Delta}4-tibolone, the estrogenic metabolites of tibolone also inhibited SMC growth, and these effects were completely blocked by the ER antagonist, ICI182780. This suggests that the inhibitory effects of 3{alpha}-OH-tibolone and 3ß-OH-tibolone are solely ER mediated. In contrast to the estrogenic metabolites, the inhibitory effects of tibolone were only partially blocked by ICI182780, implying that ERs, only in part, contribute to the overall inhibitory effects of tibolone. This contention is strongly supported by the finding that the PR antagonist, RU486, was more efficacious in blocking the inhibitory effects of tibolone on SMC growth. Moreover, at equimolar concentrations, {Delta}4-tibolone was more potent than 3{alpha}-OH-tibolone and 3ß-OH-tibolone in inhibiting SMC growth. Our data suggest that the antimitogenic effects of tibolone is mediated collectively via both the estrogenic and progestogenic metabolites and involve both ERs and PRs. This contention is also supported by our observation that the antimitogenic effects of tibolone were almost completely blocked in presence of both ICI182780 (ER antagonist) and RU486 (PR antagonist).

Although the antimitogenic effects of tibolone are mediated via both the estrogenic and progestogenic metabolites, the relative contribution of these metabolites in mediating the antivaoocclusive actions remains unclear and can only be speculated. Based on the pharmacokinetics of tibolone metabolism, the 3{alpha}-OH-tibolone and 3ß-OH-tibolone are the major metabolites (23), whereas due to the rapid clearance, the progestogenic metabolite, {Delta}4-tibolone, is a minor metabolite (23, 24). Taken together, these findings imply that even though the estrogenic metabolites were less efficacious in inhibiting SMC growth due to their long half-life and higher circulating levels, they may play a dominant role in inducing antivasoocclusive actions of tibolone. Indeed, administration of tibolone has been shown to prevent neointima formation in animal models (8). Finally, the estrogenic metabolites of tibolone can accumulate in sulfated form and released slowly to induce local effects (23), including inhibition of SMC growth. This notion is supported by our recent observations that sulfated estrogenic metabolites of tibolone are as potent as estradiol sulfate in inhibiting FCS-induced SMC growth (our unpublished personal observations).

In contrast to the various beneficial effects, tibolone has also been shown to reduce the levels of HDL (18), a counterindication for cardiovascular health. Because growth of SMCs in response to autocrine/paracrine factors is the ultimate process leading to vasoocclusion (19), our finding that tibolone inhibits SMC growth suggests that tibolone would induce antivasoocclusive effects even in the presence of high HDL. Indeed, in a rabbit model, Zandberg et al. (9) demonstrated that tibolone prevents high-cholesterol, diet-induced atherosclerosis (cholesterol accumulation in the aortic arch, fatty streak, and foam cell formation) and neointimal thickening both in the presence and absence of endothelial lesions. Similarly, Clarkson et al. (25) demonstrated that decreases in HDL levels in response to tibolone did not increase coronary atherosclerosis.

One common signaling pathway that is activated by multiple growth factors and implicated in the vascular remodeling process is the MAPK pathway (19, 26). The MAPK pathway is activated at sites of balloon injury-induced neointima formation (19, 26). Moreover, MAPK activation plays a key role in mediating mitogen (bovine fibroblast growth factor, PDGF, and angiotensin II)-induced migration and proliferation of vascular SMCs (26). Our observation that tibolone inhibited FCS-induced MAPK activity and these effects were mimicked by its estrogenic and progestogenic metabolites suggests that inhibition of the MAPK pathway via both estrogen and PRs contributes to the inhibitory effects of tibolone on DNA synthesis, proliferation, collagen synthesis, and cell migration. This notion is further supported by the fact that inhibition of MAPK with PD98059-abrogated SMC growth; moreover, the inhibitory effects of both tibolone and its metabolites were enhanced in an additive fashion in presence of PD98059.

The metabolism of tibolone to its estrogenic and progestogenic/androgenic metabolites has been shown to be tissue specific (27, 28). The progestogenic effects of tibolone on the endometrium are largely due to its selective metabolism to {Delta}4-tibolone (29). However, in T-47D ATCC and T-47D Sutherland breast cancer cells and HOS TE-85 and MG-63 osteoblast-like cells, the ratio of {Delta}4-tibolone and the estrogenic metabolite (3ß-OH-tibolone) is approximately 3:1 (30). In the present study, we found that the RU486 (PR antagonist) was 2.5 times more potent than ICI182780 (ER antagonist) in reversing the inhibitory effects of tibolone on SMC growth, suggesting that in SMCs, tibolone is largely converted to {Delta}4-tibolone and to a lesser extent to the estrogenic metabolites (3{alpha}-OH-tibolone, 3ß-OH-tibolone). However, additional studies are required to accurately quantify the conversion of tibolone to {Delta}4-tibolone, 3{alpha}-OH-tibolone, and 3ß-OH-tibolone by vascular SMCs.

What are the clinical implication of our finding that the effects of tibolone on SMC biology are ER and PR mediated? Tibolone is as effective as other estrogens/hormone replacement therapy treatments in relieving climacteric symptoms (6, 7). Moreover, in contrast to other clinically used estrogens for postmenopausal hormone therapy, tibolone does not induce deleterious effects on the breast (induces antiproliferative and proapoptotic effects in breast cancer cells) and the endometrium (6, 7, 27). Having both estrogenic and progestogenic properties, unlike other estrogens tibolone, does not require coadministration of synthetic progestins (medroxyprogesterone acetate, cyproterone acetate, norethiesterone acetate) known to abrogate various cardiovascular protective effects of estradiol, including inhibitory effects on neoimtima formation and induction of nitric oxide synthesis (1, 5, 31, 32, 33, 34). Our finding that tibolone is as potent as estradiol in inhibiting SMC growth suggests that it may be as effective as 17ß-estradiol alone in preventing against vasooccluisve disorders. Taken together the findings suggest that tibolone may have an advantage over other pharmacological regimens used for postmenopausal hormone therapy.

In conclusion, we provide evidence that tibolone, a hormone therapy compound classified as selective tissue estrogen activity regulator, is as potent as estradiol in inhibiting SMC growth; moreover, these effects are mediated via its endogenous estrogenic and progestogenic metabolites. Tibolone alters SMC biology in part by inhibiting MAPK activity, and these effects of tibolone on SMC biology are mediated via both ERs and PRs. Finally, our study indicates that tibolone may induce antivasoocclusive effects by inhibiting the SMC activity during vascular remodeling process associated with coronary artery disease in postmenopausal women.


   Footnotes
 
This work was supported by the Swiss National Science Foundation Grant 32-64040.00 and in part from an educational grant from N.V. Organon (Oss, The Netherlands).

Abbreviations: AR, Androgen receptor; ER, estrogen receptor; FCS, fetal calf serum; HDL, high-density lipoprotein; PDGF, platelet-derived growth factor; PR, progesterone receptor; SMC, smooth muscle cell.

Received July 22, 2003.

Accepted November 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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