Effects of tibolone on selectins in postmenopausal women

Maturitas 53 (2006) 166–170

Effects of tibolone on selectins in postmenopausal women Kathrin Sator a , Michael O. Sator a,∗ , Paul G. Sator b , Christian Egarter c , Johannes C. Huber a a

Department of Obstetrics and Gynaecology, Division of Endocrinology & Reproductive Medicine, University of Vienna, General Hospital, Waehringer Guertel 18-20, A-1090 Vienna, Austria b Department of Dermatology, Lainz, Vienna, Austria c Department of Obstetrics and Gynaecology, Division of Obstetrics and Gynaecology, University of Vienna, General Hospital, Waehringer Guertel 18-20, A-1090 Vienna, Austria Received 10 November 2004; received in revised form 22 March 2005; accepted 30 March 2005

Abstract Objective: The first step in atherosclerosis is characterized by the adherence of lymphocytes and monocytes to cell adhesion molecules expressed by endothelial cells. The precise mechanism by which steroid hormones may be exerting a protective action against atherogenesis remains unclear. Therefore, we wanted to investigate the effect of tibolone on the circulating levels of various selectins in postmenopausal women. Methods: Thirty healthy postmenopausal women were enrolled in a prospective, randomized, double blind, placebo-controlled outpatient trial. Results: Patients treated with tibolone revealed a significant decrease for the variables sE-selectin, sL-selectin, and sPECAM-1 after 8 weeks of treatment. Conclusions: By reducing leukocyte adhesion molecule expression on human endothelial cells, tibolone may have the intrinsic potential to exert additional, lipid-independent, cardiovascular protective effects that may explain the clinical benefits of cardiovascular diseases in postmenopausal women. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Cell adhesion molecules; Selectins; Tibolone; Hormone replacement therapy; Cardiovascular disease

1. Introduction Cardiovascular disease is the leading cause of mortality in developed countries, and yet the pathogenesis ∗ Corresponding author. Tel.: +43 1 40400 2816; fax: +43 1 40400 2817. E-mail address: [email protected] (M.O. Sator).

of the disease is incompletely understood [1]. It appears that premenopausal women are protected against atherosclerosis compared with men, and this gender difference initially led investigators to propose that estrogens may well be protective against cardiovascular disease [2]. However, the precise mechanism by which steroid hormones may be exerting this protective action remains unclear.

0378-5122/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2005.03.009

K. Sator et al. / Maturitas 53 (2006) 166–170

One of the most important events initiating the development of atherosclerosis is the adhesion of leucocytes to endothelium, which precedes their emigration to the tissues and is central to the processes of inflammation and immune reaction. Leucocyte adhesion to the endothelium is mediated by adhesion molecule pairs, principally the selectins E, L and P, members of the immunglobulin superfamily (ICAM-1 and VCAM1), and the integrins [3]. The selectins are type I membrane glycoproteins that mediate adhesion of leukocytes and platelets to vascular surfaces. L-selectin (sL) is expressed on most leukocytes. E-selectin (sE) is expressed on cytokineactivated endothelial cells, whereas P-selectin (sP) is rapidly redistributed from membranes of secretory granules to the surfaces of activated platelets as well as endothelial cells. sP and sE primarily bind to ligands on leukocytes, and sP also interacts with ligands on platelets and some endothelial cells. sL binds to ligands on endothelial cells of high endothelial venules in lymph nodes, on activated endothelial cells and on other leukocytes. These selectins are a group of cell adhesion receptors that have been shown to mediate cell–cell contact and support rapidly and reversibly cell adhesion under hydrodynamic flow [4]. They have thus attracted great interest recently. Selectins and their ligands play a crucial role in the adhesion of leukocytes to endothelium and leukocyte recruitment has been considered to be a sequential process of selectin-dependent rolling, followed by chemokine-induced leukocyte activation, which leads to rapid integrin-dependent arrest. The first transmembrane protein at interendothelial cell contacts that was suggested to participate in transendothelial migration of leukocytes was platelet endothelial adhesion molecule-1 (PECAM-1). Besides endothelial cells, platelets, neutrophils and monocytes express PECAM-1. Transendothelial migration of neutrophils and monocytes can be inhibited with antibodies blocking PECAM-1, and leukocyte extravasation could be blocked in vivo. PECAM-1 is certainly a very efficient signaling molecule, and the subcellular localization may argue for an early participation in migration [5]. The growth factor macrophage colony-stimulating factor (MCSF) is produced by endothelial cells, stimulates the proliferation and differentiation of macrophages and influences various macrophage functions such as the expression of scavanger receptors [6].

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Decreased estrogen levels in menopause lead to an increase in cytokine concentrations, which, in turn, increases selectin expression [7]. Conversely, 17␤estradiol (E2 ) has been shown in endothelial cell cultures to inhibit the expression of selectins [8]. The cytokine-induced expression of endothelial selectins requires transcriptional activation [9], and E2 is known to have important gene-regulatory effects [10]. Furthermore, androgens have also been shown to suppress the production of epithelial selectins in an animal model [11]. Tibolone, a substance widely used in hormone replacement therapy (HRT), is characterized by a combination of estrogenic, progesteronic, and androgenic properties [12]. As a result of the androgenic property, it effects a decrease in high-density lipoprotein cholesterol (HDL), but also reduces very low-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, and lipoprotein (a) [13]. A number of other effects of tibolone on the cardiovascular system have also been investigated and controversially discussed. While some studies showed a negative influence on independent risk factors for the development of cardiovascular diseases, like serum C-reactive protein (CRP) [14], Williams et al. found a beneficial effect of tibolone on myocardial function in ovariectomized atherosclerotic monkeys [15]. But the influence on the initial steps in atherosclerosis, especially on increased selectin levels in postmenopausal women, has not yet been studied [16,17]. Because plasma concentrations of the soluble forms of selectins are thought to reflect the level of cellular expression, the aim of the present study was to investigate the effects of tibolone on different soluble selectins in healthy postmenopausal women and to compare these effects with a placebo group.

2. Patients and methods 2.1. Study design In the present, prospective, randomized, double blind, placebo-controlled study 30 healthy, postmenopausal women between 50 and 80 years, with an E2 -level ≤30 pg/ml, no HRT within the last 3 months or implants within the last 6 months were included in a group receiving 1 × 1 tablet of tibolon 2.5 mg (Liviel® ,

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Organon, Austria) daily or 1 × 1 placebo tablet with lactose. The treatment period lasted at least 8 weeks. Exclusion criteria were uterine bleeding disorder, a history of endometrial hyperplasia, Diabetes mellitus, cardiovascular disease (including venous thrombembolism), hypertonia (RR > 170/105), Multiple sclerosis, clinically relevant liver or renal disease and a therapy with cholesterol-lowering agents or antioxidant vitamin supplement within the last 3 months. Ethical approval of the regional committee was received, and women participated voluntarily with informed consent, in accordance with the Declaration of Helsinki. 2.2. Data collection Before the beginning of treatment, participant’s age, duration of menopause, body weight, blood pressure, and E2 levels were determined. At the follow-up examination at 2 months blood pressure, body weight, and E2 levels were documented again. Assessments of the soluble forms of sE, sL, sP, MCSF and PECAM-1 were performed before and after 8 weeks of treatment. Venous blood samples were taken between 8 and 10 a.m. after at least 10 h of fasting, in a supine position after 20 min of rest and placed in tubes containing trisodium citrate. The subjects had to refrain from consuming alcohol in the 24 h before

sampling. The blood samples were placed on ice immediately and centrifuged at 3000 × g and 4 ◦ C for 30 min within 1 h of collection. Plasma was divided into aliquots and frozen at −70 ◦ C until analysis. We used commercially available ELISA tests to measure soluble selectins (R&D Systems Inc., Minneapolis, MN, USA). 2.3. Statistical analysis The statistical analysis was based on the intention to treat principle. The differences between the verum and placebo group were compared by Mann–Whitney’s Utest. Temporal changes were examined by Wilcoxon’s rank test and the comparison of temporal changes in both groups by the Mann–Whitney U-test. P-value <0.05 was considered statistically significant.

3. Results In this study, 30 postmenopausal women were recruited, 15 women in the placebo group and 15 in the tibolone group. All women fulfilled inclusion and exclusion criteria and gave informed consent. One woman in the placebo group stopped treatment after 3 weeks because of disturbing hot flushes and two women in the tibolone group were lost to follow up at 8 weeks.

Table 1 Values before and after therapy in the tibolone group are compared to the values of the placebo group Date of measurement in respect of therapy

P-valueb

Tibolone group (n = 13) Pvaluea

Median (ng/ml)

Minimum (ng/ml)

Maximum (ng/ml)

Placebo group (n = 14) Median (ng/ml)

Minimum (ng/ml)

Maximum (ng/ml)

Pvaluea

sE-selectin

Before After

0.019

39.6 27.8

20.4 16.1

85.8 68.6

n.s. 0.033

41.9 44.4

22.9 21.9

65.6 71.3

n.s.

sL-selectin

Before After

0.003

863.0 746.2

442.0 514.1

1204.4 1057.7

n.s. 0.017

944.5 892.8

717.7 654.8

1213.0 1225.1

n.s.

sP-selectin

Before After

n.s.

103.7 131.9

35.5 49.0

305.2 235.9

n.s. n.s.

138.1 129.9

31.4 37.0

454.0 189.2

n.s.

MCSF

Before After

n.s.

352.8 422.6

224.7 238.5

1565.7 731.6

n.s. n.s.

453.4 453.7

233.3 299.4

1169.4 647.0

n.s.

sPECAM-1

Before After

0.016

48.2 45.2

35.1 29.7

74.0 65.1

n.s. n.s.

44.4 46.5

39.1 37.8

63.9 57.1

n.s.

N.S.: NOT SIGNIFICANT a P-values of the Wilcoxon test, which compared temporal differences. b P-values of the Mann–Whitney test, which compared inter-group differences.

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Table 2 Changes from values at baseline to values after therapy in the tibolone group are compared to respective changes in the placebo group by Mann–Whitney’s U-test P-valuea

Tibolone group (n = 13)

sE-selectin sL-selectin sP-selectin MCSF sPECAM-1

Median

Minimum

Maximum

−8.2 −136.8 14.4 38.3 −5.0

−17.0 −348.0 −173.0 −1055.0 −22.0

5.0 72.0 83.0 426.0 4.0

0.006 0.001 n.s. n.s. 0.029

Placebo group (n = 14) Median

Minimum

Maximum

1.3 8.6 4.4 −2.4 0.6

−7.0 −100.0 −317.0 −757.0 −9.0

10.0 71.0 30.0 222.0 7.0

The negative medians represent decreases of values after therapy. a P-values were calculated by Mann–Whitney’s U-test.

Patients treated with tibolone revealed a significant decrease for the variables sE, sL, and PECAM-1 after 8 weeks compared to baseline, whereas placebo patients did not exhibit any significant changes between the dates of measurements. The variables did not differ significantly before therapy in both groups. At 8 weeks, tibolone patients revealed significantly lower values than controls in above mentioned variables (Table 1). The inter-group comparison of changes between the date before and during tibolone therapy also yielded significant results for the variables sE, sL, and PECAM1. The values of these variables were strongly decreased after 8 weeks (Table 2). The levels of sP and the substance MCSF did not change significantly in both groups.

4. Discussion In recent years, increasing evidence has been produced showing that the cardiovascular system represents a non-classical target of female sex steroid hormones, as well as of other steroids like tibolone [18]. The results of several studies help to understand the mechanism of action of tibolone on the cardiovascular system and its negative and positive effects on independent risk factors for the development of cardiovascular diseases. Although it is known that tibolone use is associated with an increase of CRP and decrease of HDL serum concentration, no exacerbation of atherosclerosis was found in postmenopausal monkeys treated with tibolone [19]. In fact, sex steroid hormones have been found to actively regulate important biological functions of dif-

ferent cellular components of the vascular wall, such as the endothelial cell lining, the smooth muscle cells and the vascular stroma cells, as well as the different leukocytes species [20]. Clinical observational studies suggest that tibolone may have a dilator influence on the coronary vessels [21]. Moreover, tibolone causes significant reductions in endothelin-1, sICAM-1, sICAM-3, and sVCAM-1 concentrations in postmenopausal women, suggesting that this molecule may have direct effects on endothelial cells [1]. A decrease in lipopolysaccharide-induced expression of VCAM-1 was also showed in an in vitro study by Simoncini et al. with tibolone and its two estrogenic 3␣-OH an 3␤-OH metabolites [22]. In a recent study of our group we found a highly beneficial effect of tibolone on the expression of both cell adhesion molecules, sVCAM-1 and sICAM-1 [23]. In this study tibolone and its estrogenic metabolites were able to decrease endothelial expression of sE, sL, and PECAM-1. This may be important, since these molecules participate in two different steps of the process of leukocyte adhesion to the endothelium. In fact, sE, together with these other molecules, is responsible for the initial weak attachment of circulating leukocyte to endothelial cells, which is followed by a stabilization of the production of chemotactic factors by endothelial cells [24]. Thus, tibolone may produce modifications of the adhesion molecule expression profile on endothelial cells that may act in a synergistic way to reduce leukocyte accumulation in the intimal space [25]. In agreement, rabbit arterial balloon-injury models of atherosclerosis show that treatment with tibolone is associated with a significant reduction of the post-injury intimal thickening, mainly due to decreased accumulation of lipid-laden macrophages. The mechanism for

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this reduction of intimal monocyte-derived cells may be represented by the decrease of endothelial-leukocyte adhesion molecules induced by tibolone [15]. In conclusion, our paper provides novel evidence supporting the concept of the biological importance of the direct regulation of the vascular wall by steroid receptor activating molecules regarding the prevention of atherosclerotic lesion formation. By reducing leukocyte adhesion molecule expression on human endothelial cells, tibolone may have the intrinsic potential to exert additional, lipid-independent, cardiovascular protective effects. The decrease in the expression of soluble selectins demonstrated in this study may be relevant to the observed effects of tibolone on possible clinical relief of cardiovascular diseases in postmenopausal women. However, further studies are required to specifically investigate whether treatment with tibolone increases or decreases the risk of cardiovascular events in women. References [1] Ross R. Cell biology of atherosclerosis. Annu Rev Physiol 1995;57:791–804. [2] Bush TL. The epidemiology of cardiovascular disease in postmenopausal women. Ann NY Acad Sci 1990;592:263–71. [3] Rosenfeld ME. Cellular mechanisms in the development of atherosclerosis. Diabetes Clin Pract 1996;30:1–11. [4] Adams DH, Shaw S. Leucocyte-endothelial interactions and regulation of leucocyte migration. Lancet 1994;343:832–6. [5] Cybulsky MI, Gimbrone MA. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 1991;251:788–91. [6] Clinton SK, Underwood R, Hayes L, Sherman ML, Kufe DW, Libby P. M-CSF gene expression in vascular cells and in experimental and human atherosclerosis. Am J Pathol 1992;140:301–16. [7] Barks JL, Mc Quillan JJ, Lademarco MF. TNF-␣ and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells. J Immunol 1997;159:4532–8. [8] Caulin-Glaser T, Watson CA, Pardi R, Bender JR. Effects of 17-␤-estradiol on cytokine induced endothelial cell adhesion molecule expression. J Clin Invest 1996;98:36–42. [9] Shyamala G, Guiot MC. Activation of ␬B-specific proteins by estradiol. Proc Natl Acad Sci USA 1992;89:10628–32. [10] Lademarco MF, Mc Quillan JJ, Rosen GD, Dean DC. Characterization of the promotor for vascular cell adhesion molecule-1 (VCAM-1). J Biol Chem 1992;267:16323–9.

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