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Effect of estradiol valerate alone or in association with cyproterone acetate upon vascular function of postmenopausal women at increased risk for cardiovascular disease
Maturitas 40 (2001) 239– 245 www.elsevier.com/locate/maturitas
Effect of estradiol valerate alone or in association with cyproterone acetate upon vascular function of postmenopausal women at increased risk for cardiovascular disease Cristiana Vitale, Massimo Fini, Filippo Leonardo, Paola Rossini, Elena Cerquetani, Daniela Onorati, Giuseppe M.C. Rosano * Department of Internal Medicine, Cardio6ascular Research Unit, Via della Pisana 235, San Raffaele — Roma, TOSINVEST SANITA’, 00163 Rome, Italy Received 25 September 2000; received in revised form 22 May 2001; accepted 11 June 2001
Abstract Objecti6es: a large body of evidence has been accumulated suggesting that impairment of vascular endothelial function is an initial step in the development of atherosclerosis. Recent studies have shown that estrogen replacement therapy in postmenopausal women (PMW) improves endothelium-dependent, flow-mediated dilatation (FMD) while the cyclical adjunct of a progestin may reverse this effect. Methods: the purpose of this study was to evaluate endothelium-dependent, FMD in the brachial artery and the plasma levels of Endothelin-1 in menopausal females treated with estradiol valerate with and without cyclical cyproterone acetate in 20 PMW (mean age 64 9 6 years) with more than one risk factor for coronary artery disease. After a baseline evaluation, PMW entered a double-blinded, placebo controlled single cross-over study and were randomized to receive either estradiol valerate (2 mg) for 21 days or estradiol valerate (2 mg) for 11 days and estradiol valerate (2 mg) and cyproterone acetate (1 mg) for 10 days. Patients were crossed-over the complementary treatment 7 days after completing the first treatment phase. The study of forearm blood flow was repeated at the end of each treatment period. Results: estradiol valerate significantly increased FMD as compared with baseline (12 93 vs. 7 94%, PB0.01) the adjunct of cyproterone acetate did not affect the effect of estradiol valerate upon FMD (12 93 vs. 1194%, P= NS). Similarly reactive hyperemic flow increased after estradiol valerate alone (24%) or in association with cyproterone acetate (24%) compared with baseline. Plasma levels of Endothelin-1 were significantly reduced by estradiol valerate alone or in association with cyproterone acetate. Conclusions: in conclusion hormone replacement therapy with estradiol valerate and cyproterone acetate improves endothelial function and reduces plasma levels of Endothelin-1 in PMW at risk of coronary artery disease. These effects may be relevant for cardioprotection. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Estradiol valerate; Cyproterone acetate; Postmenopausal women
* Corresponding author. Tel.: + 390-6-660581. E-mail address: [email protected] (G.M.C. Rosano). 0378-5122/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 5 1 2 2 ( 0 1 ) 0 0 2 4 2 - 0
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1. Introduction A large body of evidence suggests that the impairment of vascular endothelial function is an initial step in the development of atherosclerosis. Ovarian hormones have anti-atherogenic properties influencing all the steps involved in the formation of the atherosclerotic plaque: accumulation of cholesterol in the arterial wall, arterial smooth muscle cell proliferation, platelet aggregation, collagen and elastin production-and act as vasoactive substances [1–6]. In fact, these hormones may modulate the blood flow in many arterial districts, such as the vagina, vulva, uterine and carotid vessels, as well as the coronary arteries, both through endothelium-dependent mechanisms and endothelium-independent mechanisms [1,2]. Animal studies have shown that estrogens reverse the acetylcholine-induced vasoconstriction of coronary arteries in ovariectomized monkeys suggesting that estrogens modulate vasomotor responses of atherosclerotic arteries through an endothelium-dependent mechanism. Similar effects have been reported in human females but not in men [6]. The cessation of ovarian function is associated with an impairment of endothelial function, which is restored, at least in part, by hormone replacement therapy [7–9]. An important component of endothelial dysfunction is the impairment of the release of endothelial-derived relaxing factor. Flow mediated vasodilatation induced by reactive hyperemia has been shown to be endothelium-dependent and can be assessed by high-resolution ultrasound in superficial arteries. Vascular function is also influenced by production of vasoactive peptides such as Endothelin-1. Improvement of endothelial function as been reported after estrogen administration in vascular districts different from the coronary arteries and recent studies have shown that estrogen replacement therapy in postmenopausal women (PMW) improves endothelium-dependent, flow-mediated dilatation (FMD) in the forearm [7]. Other studies have shown that endothelium-dependent vasodilatation varies during the menstrual cycle and that 17b-estradiol is involved in this variation [8]. Unopposed estrogen replacement therapy has cardioprotective effects but may increase the
likelihood of uterine malignancies if not associated with progestin administration. Progestins, however, may reverse the beneficial effect of estrogen replacement therapy upon cardiovascular functions [10,11]. Thus, an improved therapy for menopausal disease should have the positive effect of estrogen on the cardiovascular system and the antagonistic effect of progestins upon the uterus but not on the vascular bed. Our group has shown that different progestins and different schemes of progestin administration have different effect upon the vascular response of brachial artery [12]. The association of estradiol valerate and cyclical cyproterone acetate has been suggested for hormonal replacement of menopausal women. To date no data exist on the systemic vascular effect of this association. The purpose of this study was to evaluate endothelium-dependent, flow-mediated vasodilatation in the brachial artery and plasma levels Endothelin-1 in menopausal females treated with estradiol valerate and receiving cyclical cyproterone acetate.
2. Methods
2.1. Patient population The study population included 30 postmenopausal women (aging \45 and B 70 years). All patients have written informed consent. After a screening visit to evaluate the fulfilling of inclusion criteria, 20 PMW have been included in the study. Inclusion criteria were age between 45 and 70 years, presence of more than two risk factors for coronary artery disease causing a 10 year cardiovascular risk of 40%, intact uterus. No subject had taken any cholesterol-lowering agent, estrogen therapy, or antioxidant vitamin supplements during the preceding 2 months. Aspirin and non-steroidal anti-inflammatory agents were stopped beginning 10 days prior to study. Menopausal status was assessed by absence of menses by \6 months and by plasma levels of 17b-estradiol B 100 pmol/l, coupled with baseline FSH \ 25 mIU/ml.
C. Vitale et al. / Maturitas 40 (2001) 239–245
Patient with clinically significant findings on physical exam or presence of known clinically significant disease that would interfere with study evaluation, those with current use of medications (or products) that would have interfered with forearm blood flow have been excluded from the study as those receiving ovarian hormonal therapy during the past 3 months preceding the study. Patients with cardiac conditions such as unstable angina and/or arrhythmia (Lown class \ 3), recent acute MI (B3 months), primary valvular, congenital heart disease, myocardial, pericardial or endocardial disease, congestive heart failure were also excluded. Women presenting with contra indications to hormonal replacement therapy presence of undiagnosed vaginal bleeding, known or suspected breast or genital organs malignancy, active thrombophlebitis or thromboembolic disorders and those unable to comply with the protocol or refusing the exam related to end-point were excluded.
2.2. Study protocol The study design was a double-blinded, placebo controlled and with a single crossover. The treatment period was 21 days for each arm of the study. Patients have been studied four times, at baseline and at the end of each treatment period and before the second treatment period. After 1 week wash-out of all cardio-active drugs patients underwent baseline study of forearm blood flow and then randomized to receive either estradiol valerate (2 mg) for 21 days or estradiol valerate (2 mg) (Progynova®, Schering AG) for 11 days and estradiol valerate (2 mg) and cyproterone acetate (1 mg) for 10 days (Climen®, Schering AG). Patients were crossed-over the complementary treatment 7 days after completing the first treatment phase. During visit 1, each patient received a supply of either estradiol valerate followed by estradiol valerate+cyproterone acetate for the second sequence or estradiol valerate+ cyproterone acetate followed by estradiol valerate and were instructed regarding the proper self-administration of drug About 10 ml of blood were withdrawn at the
241
end of each test and were assayed for FSH and E2 and Endothelin-1.
2.3. E6aluation of brachial artery reacti6ity Flow-mediated vasodilatation and nitroglycerine-induced (endothelium-independent) vasodilatation of brachial artery were measured by an experienced investigator unaware of the clinical data. The same investigator performed the four studies in each patient in order to avoid inter-observer variability. Studies were conducted in quiet and temperature controlled room (22–23 °C). Patients were asked to avoid caffeine-containing drinks and to refrain from smoking for the 6 h preceding the study. Imaging studies of the left brachial artery were performed using a Hewlett-Packard SONOS 2000 ultrasound machine equipped with a 7.5 MHz linear-array transducer prior to and at the end of each of the three treatment periods. Patients were studied in the supine position after 15 min rest. The right brachial artery was scanned over a longitudinal section 3–5 cm above the right elbow, at the site where the clearest image was obtained. The focus zone was set to the depth of the anterior vessel wall. Depth and gain settings were optimized to identify the lumen-vessel wall interface. Blood pressure was monitored every 2 min from the left arm. The electrocardiogram was recorded throughout the study by means of an ECG monitor inbuilt in the ultrasound machine. When an optimal image of the right brachial artery was obtained the surface of the skin was marked for the future measurements. The changes in diameter of the right brachial artery were measured at rest, during reactive hyperemia, after 10 min recovery and after 5 min of sublingual nitroglycerine (spray). A pneumatic tourniquet was placed around the forearm distal to the target artery and have been inflated to a pressure of 250 mmHg for 5 min. Reactive hyperemia was induced by sudden cuff deflation. A scan was performed continuously for 30 s before and for 90 s after cuff deflation. About 15 min later a third scan was recorded to confirm vessel recovery. A fourth scan was performed for the 2 min proceed-
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ing and the 180 s following sublingual nitroglycerine (0.4 mg). The ultrasound images were recorded on S-VHS videotape and printed on paper. The diameter of the brachial artery was measured from the anterior to the posterior interface, the mean diameter was calculated from 4 cardiac cycles synchronized with the R wave peak on the electrocardiogram. All measurements were made at end diastole. The diameter change was expressed as the percent change compared with baseline. Pulse wave velocity profile of blood flow was also recorded at 15 s intervals. Mean flow velocity was calculated by measurement of the area under the velocity profile curve. Blood flow (ml/min) was calculated from vessel cross-sectional area and brachial artery blood flow velocity.
tively). Peak brachial artery diameters and forearm blood flows during reactive hyperemia and the percent increase in flow during reactive hyperemia were significantly increased by both treatments but no difference was noted between treatments (Table 2). The brachial artery dilator response to nitroglycerin was similar with both therapies (P= 0.362 by ANOVA), and was not significantly changed from respective pretreatment measurements (Table 2). The plasma levels of Endothelin-1 were reduced after administration either estradiol valerate alone or in combination with cyproterone acetate compared with baseline levels (1.229 0.5 ng/l, vs. 1.429 0.3 and 1.399 0.6 ng/l, P B 0.01 for both treatments vs. baseline). No significant difference was found between treatment values.
2.4. Statistical analysis
4. Discussion
Data are presented as mean9 1 S.D. or percentages when appropriate. After testing data for normality, Student’s paired t-test or Wilcoxon Signed Rank test was used to compare values before and after each therapy and the relative changes in values in response to each therapy. The effects of the two hormone therapy regimens on vascular function and plasma levels of Endothelin-1 relative to respective pretreatment values were analyzed by one way repeated measures analysis of variance (ANOVA) or Friedman Repeated ANOVA on Ranks. A P B0.05 have been considered statistical significant.
The present study confirms the beneficial effect of estrogens upon endothelial function in PMW and shows that the adjunct of cyproterone acetate to estradiol valerate does not negatively affect the beneficial effect of estrogen therapy. The improvement of vascular function observed in the present study is in part attributable to an increased production of nitric oxide from endothelial cells and in part related to the decrease of the vasoactive peptide Endothelin-1. The cessation of ovarian function in women with menopause leads to a reduction of sex steroid hormone levels with important metabolic and pathophysiological implications affecting dif-
3. Results Clinical characteristics of study patients are shown in Table 1. About 20 patients met the inclusion criteria and entered the study. Baseline values of brachial artery diameter and flow prior to each treatment period were compared and no significant differences were noted (Table 2). Baseline brachial artery diameter and forearm blood flows were also similar at the 2 baseline point and during the two treatment periods (P= 0.530 and P = 0.694 by ANOVA, respec-
Table 1 Clinical features of study patients (n = 20) Mean age (years) Time since menopause (years)
64 9 6 12 9 4
Risk factors for coronary artery disease Cholesterol\220 mg/dl Diabetes Hypertension Cigarette smoking Family history
10 4 8 7 9
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Table 2 Effect of Estradiol valerate alone or in combination with cyproterone acetate on Endothelial function and plasma Endothelin-1 levels Estradiol valerate
Estradiol valerate plus Cyproterone acetate
Baseline
After therapy
Baseline
After therapy
Brachial artery diameter (mm) Basal-1 Hyperemia Basal-2 Nitroglycerin
4.269 0.66 4.55 9 0.64 4.279 0.68 5.039 0.52
4.279 0.62 4.76 9 0.62a 4.27 90.58 5.02 9 0.69
4.27 90.53 4.56 9 0.57 4.28 9 0.69 5.04 90.74
4.28 9 0.59 4.77 9 0.63a 4.28 90.57 5.03 9 0.55
Brachial artery flow (ml/min) Basal-1 Hyperemia Increase in flow (%) Basal-2 Nitroglycerin
1289 74 6149 243 4809 272 1199 55 1559 59
1219 85 7189 347b 594 9312b 116 9 51 1619 63
126 968 580 9192 461 9231 121 9 51 147 9 83
127 9 46 726 9263b 572 9199b 118 945 157 962
1.429 0.3
1.2190.4a
1.39 9 0.6
1.22 9 0.5a
Endothelin-1 (ng/l)
N =20. Data are expressed as means 9 S.D. a PB0.05 for comparison with the respective baseline values. b PB0.01, for comparison with the respective baseline values.
ferent organs and system. Ovarian hormone deficiency is of particular importance for the development of cardiovascular diseases, because of the unfavorable metabolic changes associated with estrogen deficiency and because of its effects on impairment of endothelial function [13– 18]. The decrease in endothelial function was assessed in the present study by brachial artery FMD. Although the brachial artery is not a common site for the development of atherosclerosis, several studies have shown that brachial artery FMD is impaired in patients with coronary artery disease and in those at increased risk of cardiac events and have also shown a close similarity between brachial and coronary vasomotor responses [18]. Patients included in the present study had all an overall risk of future cardiac events between 20 and 40% in 10 years and, therefore, had an impaired vascular reactivity because of their menopausal status and because of the adjunctive effect of cardiovascular risk factors [19– 21]. Acute and chronic administration of estrogens to PMW restores the endothelium-dependent vasodilatation, which is impaired after the menopause [5,6,12]. Gilligan et al. showed that PMW with
cardiovascular risk factors have an impaired vasodilator response to acetylcholine and sodium nitroprusside and that physiological doses of estradiol potentiates the acetylcholine-induced increase in coronary flow. Thus suggesting an improvement of endothelial function. The increase in coronary blood flow after acetylcholine challenge was higher in subjects in whom endothelium-dependent coronary vasomotor responsiveness was more compromised. Several studies have shown that acute estrogen administration improves endothelial function in the brachial artery of postmenopausal estrogen depleted women [7,22]. The effect of estrogen administration upon coronary vasomotor tone is present also after chronic administration of estrogen [5,6,12]. In PMW with intact uterus receiving estrogen replacement therapy progestins are prescribed in order to protect the endometrium from hyperplastic and potentially carcinomatous effects of prolonged unopposed estrogen therapy. Since the administration of some progestins may reverse the beneficial effect of estrogen replacement therapy upon vascular reactivity, a replacement therapy for menopausal women with increased cardio-
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vascular risk should include those estrogen– progestin associations able to sustain the positive effect of estrogens on the cardiovascular system during the progestin phase. Progesterone receptors are present in the arterial wall and there is evidence that the arterial effects of progestins are mediated through progesterone receptors as well as through down-regulation of estradiol receptors [23]. Progestin therapy affects arterial function, since it may stabilize arteries in a state of vasomotor instability, but may also induce vasoconstriction of estrogenized vessels [24]. Therefore, a careful selection of the dose, type and schedule of administration of progestin to add to estrogens is crucial to preserve and possibly enhance the beneficial effects of estrogens. More androgenic progestins and progestins given in cyclical regimen have been shown to have unfavorable vascular effect. Our group has shown that cyclical administration of medroxyprogesterone acetate when given at high doses and on a cyclical scheme abolishes the beneficial effect of estrogens upon vascular functions and exercise-induced myocardial ischemia and that norethisterone acetate has a detrimental effect upon the endothelium-dependent vasodilatation of the forearm in PMW and increases the brachial artery resistance [25,12]. The effect seen with cyclical medroxyprogesterone is not seen with continuous combined administration of the progestin thereby suggesting that the effect may be related to the dose and scheme of administration of medroxyprogesterone. In the present study we have shown cyproterone acetate given in association to estradiol valerate does not reverse the beneficial effect of estrogen replacement therapy. The scheme of administration of cyproterone acetate tested in the present study was the continuous combined, theoretically the more detrimental to vascular function. The fact that in a cyclical scheme of administration, cyproterone acetate sustains the beneficial effect of estradiol valerate upon endothelial function and Endothelin-1 plasma levels suggests that this progestin may be safely used for the hormonal replacement therapy of menopausal women, at risk of coronary artery disease and in those with increased vascular reactivity.
5. Conclusion The present study shows that both estradiol valerate and estradiol valerate plus cyproterone acetate improves by a similar degree endothelial function in the forearm of PMW at increased cardiovascular risk. These findings suggest that this association could be safely administered in women with increased cardiovascular risk of with increased vascular reactivity.
References [1] Wagner JD, Clarkson TB, St. Clair RW, et al. Estrogen and progesterone replacement therapy reduces LDL accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest 1991;88:1995 – 2002. [2] Adams MR, Kaplan JR, Manuck SB, et al. Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. Lack of an effect of adding progesterone. Arteriosclerosis 1990;10:1051 – 7. [3] Gisclard V, Miller VM, Vanhoutte PM. Effect of 17-beta estradiol on endothelium-dependent responses in the rabbit. J Pharmacol Exp Ther 1988;244:19 – 22. [4] Williams JK, Adams MR, Klopfenstein HS. Estrogen modulates responses of atherosclerotic coronary arteries. Circulation 1990;81:1680 – 7. [5] Herrington DM, Braden GA, Williams JK, et al. Endothelial-dependent coronary vasomotor responsiveness in postmenopausal women with and with-out estrogen replacement therapy. Am J Cardiol 1994;73:951 – 2. [6] Collins P, Rosano GM, Sarrel PM, et al. 17-beta Estradiol attenuates acetylcoline-induced coronary arterial constriction in women but no men with coronary heart disease. Circulation 1995;92:24 – 30. [7] Volterrani M, Rosano GMC, Coats A, et al. Estrogen acutely increases peripheral blood flow in postmenopausal women. Am J Med 1995;99:119 – 22. [8] Sarrel PM. Ovarian hormones and the circulation. Maturitas 1990;12:287 – 98. [9] Collins P, Shay J, Jiang C, et al. Nitric oxide accounts for dose-dependent estrogen-mediated coronary relaxation following acute estrogen withdrawal. Circulation 1994;90:1964 – 8. [10] Adams MR, Register TC, Golden DL, et al. Medroxyprogesterone acetate antagonizes inhibitory effects of coniugated equine estrogens on coronary artery atherosclerosis. Arterioscler Thromb Vasc Biol 1997;17:217 – 21. [11] Sarrel PM. Ovarian hormones and the circulation. Maturitas 1990;12:287 – 98.
C. Vitale et al. / Maturitas 40 (2001) 239–245 [12] Rosano GMC, Leonardo F, Cerquetani E, et al. Comparative effects of continuos combined hormone replacement therapyregimens on brachial artery blood flow. J Am Coll Cardiol (Supplement A) 1998;2:459 Abstract. [13] Barret-Connor E, Bush TL. Estrogen and coronary heart disease in women. J Am Med Assoc 1991;265:1861 – 7. [14] Sullivan JM, VanderZwaag R, Lemp GF, et al. Postmenopausal estrogen use and coronary atherosclerosis. Ann Intern Med 1988;108:358 –63. [15] Haarbo J, Leth-Espensen P, Stender S, et al. Estrogen monotherapy and combined estrogen-progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. J Clin Invest 1991;87:1274 –9. [16] Celermajer DS, Sorensen KE, Spiegelhalter DJ, Georgakopoulos D, Robinson J, Deanfield JE. Aging is associated with endothelial dysfunction in healty men years before the age-related decline in women. JACC 1994;24:471 – 6. [17] Mendelsohn ME, Karas RH. Estrogen and the blood vessel wall, Curr. Opin. Cardiol. 1994;8:619 – 26. [18] David EB, Calvin EJ, Katherine MB, et al. Estrogen replacement reverses endothelial dysfunction in postmenopausal women. Am J Med 1998;104:552 –8. [19] Akahoshi M, Soda M, Nakashima E, Shimaoka K, Seto S, Yano K. Effect of menopause on trends of
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serum cholesterol, blood pressure, and body mass index. Circulation 1996;94:61 – 6. Anastos K, Charney P, Charon RA, Cohen E, Jones CY, Marte C, Swiderski DM, Wheat ME, Williams S. Hypertension in women: what is really known? The Women’s Caucus, Working Group on Women’s Health of the Society of General Internal Medicine. Ann Intern Med 1991;115:287 – 93. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. New Engl J Med 1987;316:1105 – 10. Gilligan DM, Badar DM, Panza JA, et al. Acute vascular effects of estrogen in postmenopausal women. Circulation 1994;90:786 – 91. Ingegno MD, Money SR, Thelmo W, et al. Progesterone receptors in the human heart and great vessels. Lab Invest 1988;59:353 – 6. Sarrel PM. Clinical findings related to arterial effects of ovarian steroids. In: Forte TM, editor. Hormonal, Metabolic, and Cellular Influences on Cardiovascular Disease in Women. New York: Futura Publishing Company, 1997. Rosano GMC, Chierchia SL, Morgagni GL, et al. Effect of the association of different progestogens to estradiol 17-beta therapy upon effort-induced myocardial ischemia in female patients with coronary artery disease. J Am Coll Cardiol 2000;36(7):2154 – 9.
Effect of estradiol valerate alone or in association with cyproterone acetate upon vascular function of postmenopausal women at increased risk for cardiovascular disease Cristiana Vitale, Massimo Fini, Filippo Leonardo, Paola Rossini, Elena Cerquetani, Daniela Onorati, Giuseppe M.C. Rosano * Department of Internal Medicine, Cardio6ascular Research Unit, Via della Pisana 235, San Raffaele — Roma, TOSINVEST SANITA’, 00163 Rome, Italy Received 25 September 2000; received in revised form 22 May 2001; accepted 11 June 2001
Abstract Objecti6es: a large body of evidence has been accumulated suggesting that impairment of vascular endothelial function is an initial step in the development of atherosclerosis. Recent studies have shown that estrogen replacement therapy in postmenopausal women (PMW) improves endothelium-dependent, flow-mediated dilatation (FMD) while the cyclical adjunct of a progestin may reverse this effect. Methods: the purpose of this study was to evaluate endothelium-dependent, FMD in the brachial artery and the plasma levels of Endothelin-1 in menopausal females treated with estradiol valerate with and without cyclical cyproterone acetate in 20 PMW (mean age 64 9 6 years) with more than one risk factor for coronary artery disease. After a baseline evaluation, PMW entered a double-blinded, placebo controlled single cross-over study and were randomized to receive either estradiol valerate (2 mg) for 21 days or estradiol valerate (2 mg) for 11 days and estradiol valerate (2 mg) and cyproterone acetate (1 mg) for 10 days. Patients were crossed-over the complementary treatment 7 days after completing the first treatment phase. The study of forearm blood flow was repeated at the end of each treatment period. Results: estradiol valerate significantly increased FMD as compared with baseline (12 93 vs. 7 94%, PB0.01) the adjunct of cyproterone acetate did not affect the effect of estradiol valerate upon FMD (12 93 vs. 1194%, P= NS). Similarly reactive hyperemic flow increased after estradiol valerate alone (24%) or in association with cyproterone acetate (24%) compared with baseline. Plasma levels of Endothelin-1 were significantly reduced by estradiol valerate alone or in association with cyproterone acetate. Conclusions: in conclusion hormone replacement therapy with estradiol valerate and cyproterone acetate improves endothelial function and reduces plasma levels of Endothelin-1 in PMW at risk of coronary artery disease. These effects may be relevant for cardioprotection. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Estradiol valerate; Cyproterone acetate; Postmenopausal women
* Corresponding author. Tel.: + 390-6-660581. E-mail address: [email protected] (G.M.C. Rosano). 0378-5122/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 5 1 2 2 ( 0 1 ) 0 0 2 4 2 - 0
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C. Vitale et al. / Maturitas 40 (2001) 239–245
1. Introduction A large body of evidence suggests that the impairment of vascular endothelial function is an initial step in the development of atherosclerosis. Ovarian hormones have anti-atherogenic properties influencing all the steps involved in the formation of the atherosclerotic plaque: accumulation of cholesterol in the arterial wall, arterial smooth muscle cell proliferation, platelet aggregation, collagen and elastin production-and act as vasoactive substances [1–6]. In fact, these hormones may modulate the blood flow in many arterial districts, such as the vagina, vulva, uterine and carotid vessels, as well as the coronary arteries, both through endothelium-dependent mechanisms and endothelium-independent mechanisms [1,2]. Animal studies have shown that estrogens reverse the acetylcholine-induced vasoconstriction of coronary arteries in ovariectomized monkeys suggesting that estrogens modulate vasomotor responses of atherosclerotic arteries through an endothelium-dependent mechanism. Similar effects have been reported in human females but not in men [6]. The cessation of ovarian function is associated with an impairment of endothelial function, which is restored, at least in part, by hormone replacement therapy [7–9]. An important component of endothelial dysfunction is the impairment of the release of endothelial-derived relaxing factor. Flow mediated vasodilatation induced by reactive hyperemia has been shown to be endothelium-dependent and can be assessed by high-resolution ultrasound in superficial arteries. Vascular function is also influenced by production of vasoactive peptides such as Endothelin-1. Improvement of endothelial function as been reported after estrogen administration in vascular districts different from the coronary arteries and recent studies have shown that estrogen replacement therapy in postmenopausal women (PMW) improves endothelium-dependent, flow-mediated dilatation (FMD) in the forearm [7]. Other studies have shown that endothelium-dependent vasodilatation varies during the menstrual cycle and that 17b-estradiol is involved in this variation [8]. Unopposed estrogen replacement therapy has cardioprotective effects but may increase the
likelihood of uterine malignancies if not associated with progestin administration. Progestins, however, may reverse the beneficial effect of estrogen replacement therapy upon cardiovascular functions [10,11]. Thus, an improved therapy for menopausal disease should have the positive effect of estrogen on the cardiovascular system and the antagonistic effect of progestins upon the uterus but not on the vascular bed. Our group has shown that different progestins and different schemes of progestin administration have different effect upon the vascular response of brachial artery [12]. The association of estradiol valerate and cyclical cyproterone acetate has been suggested for hormonal replacement of menopausal women. To date no data exist on the systemic vascular effect of this association. The purpose of this study was to evaluate endothelium-dependent, flow-mediated vasodilatation in the brachial artery and plasma levels Endothelin-1 in menopausal females treated with estradiol valerate and receiving cyclical cyproterone acetate.
2. Methods
2.1. Patient population The study population included 30 postmenopausal women (aging \45 and B 70 years). All patients have written informed consent. After a screening visit to evaluate the fulfilling of inclusion criteria, 20 PMW have been included in the study. Inclusion criteria were age between 45 and 70 years, presence of more than two risk factors for coronary artery disease causing a 10 year cardiovascular risk of 40%, intact uterus. No subject had taken any cholesterol-lowering agent, estrogen therapy, or antioxidant vitamin supplements during the preceding 2 months. Aspirin and non-steroidal anti-inflammatory agents were stopped beginning 10 days prior to study. Menopausal status was assessed by absence of menses by \6 months and by plasma levels of 17b-estradiol B 100 pmol/l, coupled with baseline FSH \ 25 mIU/ml.
C. Vitale et al. / Maturitas 40 (2001) 239–245
Patient with clinically significant findings on physical exam or presence of known clinically significant disease that would interfere with study evaluation, those with current use of medications (or products) that would have interfered with forearm blood flow have been excluded from the study as those receiving ovarian hormonal therapy during the past 3 months preceding the study. Patients with cardiac conditions such as unstable angina and/or arrhythmia (Lown class \ 3), recent acute MI (B3 months), primary valvular, congenital heart disease, myocardial, pericardial or endocardial disease, congestive heart failure were also excluded. Women presenting with contra indications to hormonal replacement therapy presence of undiagnosed vaginal bleeding, known or suspected breast or genital organs malignancy, active thrombophlebitis or thromboembolic disorders and those unable to comply with the protocol or refusing the exam related to end-point were excluded.
2.2. Study protocol The study design was a double-blinded, placebo controlled and with a single crossover. The treatment period was 21 days for each arm of the study. Patients have been studied four times, at baseline and at the end of each treatment period and before the second treatment period. After 1 week wash-out of all cardio-active drugs patients underwent baseline study of forearm blood flow and then randomized to receive either estradiol valerate (2 mg) for 21 days or estradiol valerate (2 mg) (Progynova®, Schering AG) for 11 days and estradiol valerate (2 mg) and cyproterone acetate (1 mg) for 10 days (Climen®, Schering AG). Patients were crossed-over the complementary treatment 7 days after completing the first treatment phase. During visit 1, each patient received a supply of either estradiol valerate followed by estradiol valerate+cyproterone acetate for the second sequence or estradiol valerate+ cyproterone acetate followed by estradiol valerate and were instructed regarding the proper self-administration of drug About 10 ml of blood were withdrawn at the
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end of each test and were assayed for FSH and E2 and Endothelin-1.
2.3. E6aluation of brachial artery reacti6ity Flow-mediated vasodilatation and nitroglycerine-induced (endothelium-independent) vasodilatation of brachial artery were measured by an experienced investigator unaware of the clinical data. The same investigator performed the four studies in each patient in order to avoid inter-observer variability. Studies were conducted in quiet and temperature controlled room (22–23 °C). Patients were asked to avoid caffeine-containing drinks and to refrain from smoking for the 6 h preceding the study. Imaging studies of the left brachial artery were performed using a Hewlett-Packard SONOS 2000 ultrasound machine equipped with a 7.5 MHz linear-array transducer prior to and at the end of each of the three treatment periods. Patients were studied in the supine position after 15 min rest. The right brachial artery was scanned over a longitudinal section 3–5 cm above the right elbow, at the site where the clearest image was obtained. The focus zone was set to the depth of the anterior vessel wall. Depth and gain settings were optimized to identify the lumen-vessel wall interface. Blood pressure was monitored every 2 min from the left arm. The electrocardiogram was recorded throughout the study by means of an ECG monitor inbuilt in the ultrasound machine. When an optimal image of the right brachial artery was obtained the surface of the skin was marked for the future measurements. The changes in diameter of the right brachial artery were measured at rest, during reactive hyperemia, after 10 min recovery and after 5 min of sublingual nitroglycerine (spray). A pneumatic tourniquet was placed around the forearm distal to the target artery and have been inflated to a pressure of 250 mmHg for 5 min. Reactive hyperemia was induced by sudden cuff deflation. A scan was performed continuously for 30 s before and for 90 s after cuff deflation. About 15 min later a third scan was recorded to confirm vessel recovery. A fourth scan was performed for the 2 min proceed-
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ing and the 180 s following sublingual nitroglycerine (0.4 mg). The ultrasound images were recorded on S-VHS videotape and printed on paper. The diameter of the brachial artery was measured from the anterior to the posterior interface, the mean diameter was calculated from 4 cardiac cycles synchronized with the R wave peak on the electrocardiogram. All measurements were made at end diastole. The diameter change was expressed as the percent change compared with baseline. Pulse wave velocity profile of blood flow was also recorded at 15 s intervals. Mean flow velocity was calculated by measurement of the area under the velocity profile curve. Blood flow (ml/min) was calculated from vessel cross-sectional area and brachial artery blood flow velocity.
tively). Peak brachial artery diameters and forearm blood flows during reactive hyperemia and the percent increase in flow during reactive hyperemia were significantly increased by both treatments but no difference was noted between treatments (Table 2). The brachial artery dilator response to nitroglycerin was similar with both therapies (P= 0.362 by ANOVA), and was not significantly changed from respective pretreatment measurements (Table 2). The plasma levels of Endothelin-1 were reduced after administration either estradiol valerate alone or in combination with cyproterone acetate compared with baseline levels (1.229 0.5 ng/l, vs. 1.429 0.3 and 1.399 0.6 ng/l, P B 0.01 for both treatments vs. baseline). No significant difference was found between treatment values.
2.4. Statistical analysis
4. Discussion
Data are presented as mean9 1 S.D. or percentages when appropriate. After testing data for normality, Student’s paired t-test or Wilcoxon Signed Rank test was used to compare values before and after each therapy and the relative changes in values in response to each therapy. The effects of the two hormone therapy regimens on vascular function and plasma levels of Endothelin-1 relative to respective pretreatment values were analyzed by one way repeated measures analysis of variance (ANOVA) or Friedman Repeated ANOVA on Ranks. A P B0.05 have been considered statistical significant.
The present study confirms the beneficial effect of estrogens upon endothelial function in PMW and shows that the adjunct of cyproterone acetate to estradiol valerate does not negatively affect the beneficial effect of estrogen therapy. The improvement of vascular function observed in the present study is in part attributable to an increased production of nitric oxide from endothelial cells and in part related to the decrease of the vasoactive peptide Endothelin-1. The cessation of ovarian function in women with menopause leads to a reduction of sex steroid hormone levels with important metabolic and pathophysiological implications affecting dif-
3. Results Clinical characteristics of study patients are shown in Table 1. About 20 patients met the inclusion criteria and entered the study. Baseline values of brachial artery diameter and flow prior to each treatment period were compared and no significant differences were noted (Table 2). Baseline brachial artery diameter and forearm blood flows were also similar at the 2 baseline point and during the two treatment periods (P= 0.530 and P = 0.694 by ANOVA, respec-
Table 1 Clinical features of study patients (n = 20) Mean age (years) Time since menopause (years)
64 9 6 12 9 4
Risk factors for coronary artery disease Cholesterol\220 mg/dl Diabetes Hypertension Cigarette smoking Family history
10 4 8 7 9
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Table 2 Effect of Estradiol valerate alone or in combination with cyproterone acetate on Endothelial function and plasma Endothelin-1 levels Estradiol valerate
Estradiol valerate plus Cyproterone acetate
Baseline
After therapy
Baseline
After therapy
Brachial artery diameter (mm) Basal-1 Hyperemia Basal-2 Nitroglycerin
4.269 0.66 4.55 9 0.64 4.279 0.68 5.039 0.52
4.279 0.62 4.76 9 0.62a 4.27 90.58 5.02 9 0.69
4.27 90.53 4.56 9 0.57 4.28 9 0.69 5.04 90.74
4.28 9 0.59 4.77 9 0.63a 4.28 90.57 5.03 9 0.55
Brachial artery flow (ml/min) Basal-1 Hyperemia Increase in flow (%) Basal-2 Nitroglycerin
1289 74 6149 243 4809 272 1199 55 1559 59
1219 85 7189 347b 594 9312b 116 9 51 1619 63
126 968 580 9192 461 9231 121 9 51 147 9 83
127 9 46 726 9263b 572 9199b 118 945 157 962
1.429 0.3
1.2190.4a
1.39 9 0.6
1.22 9 0.5a
Endothelin-1 (ng/l)
N =20. Data are expressed as means 9 S.D. a PB0.05 for comparison with the respective baseline values. b PB0.01, for comparison with the respective baseline values.
ferent organs and system. Ovarian hormone deficiency is of particular importance for the development of cardiovascular diseases, because of the unfavorable metabolic changes associated with estrogen deficiency and because of its effects on impairment of endothelial function [13– 18]. The decrease in endothelial function was assessed in the present study by brachial artery FMD. Although the brachial artery is not a common site for the development of atherosclerosis, several studies have shown that brachial artery FMD is impaired in patients with coronary artery disease and in those at increased risk of cardiac events and have also shown a close similarity between brachial and coronary vasomotor responses [18]. Patients included in the present study had all an overall risk of future cardiac events between 20 and 40% in 10 years and, therefore, had an impaired vascular reactivity because of their menopausal status and because of the adjunctive effect of cardiovascular risk factors [19– 21]. Acute and chronic administration of estrogens to PMW restores the endothelium-dependent vasodilatation, which is impaired after the menopause [5,6,12]. Gilligan et al. showed that PMW with
cardiovascular risk factors have an impaired vasodilator response to acetylcholine and sodium nitroprusside and that physiological doses of estradiol potentiates the acetylcholine-induced increase in coronary flow. Thus suggesting an improvement of endothelial function. The increase in coronary blood flow after acetylcholine challenge was higher in subjects in whom endothelium-dependent coronary vasomotor responsiveness was more compromised. Several studies have shown that acute estrogen administration improves endothelial function in the brachial artery of postmenopausal estrogen depleted women [7,22]. The effect of estrogen administration upon coronary vasomotor tone is present also after chronic administration of estrogen [5,6,12]. In PMW with intact uterus receiving estrogen replacement therapy progestins are prescribed in order to protect the endometrium from hyperplastic and potentially carcinomatous effects of prolonged unopposed estrogen therapy. Since the administration of some progestins may reverse the beneficial effect of estrogen replacement therapy upon vascular reactivity, a replacement therapy for menopausal women with increased cardio-
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vascular risk should include those estrogen– progestin associations able to sustain the positive effect of estrogens on the cardiovascular system during the progestin phase. Progesterone receptors are present in the arterial wall and there is evidence that the arterial effects of progestins are mediated through progesterone receptors as well as through down-regulation of estradiol receptors [23]. Progestin therapy affects arterial function, since it may stabilize arteries in a state of vasomotor instability, but may also induce vasoconstriction of estrogenized vessels [24]. Therefore, a careful selection of the dose, type and schedule of administration of progestin to add to estrogens is crucial to preserve and possibly enhance the beneficial effects of estrogens. More androgenic progestins and progestins given in cyclical regimen have been shown to have unfavorable vascular effect. Our group has shown that cyclical administration of medroxyprogesterone acetate when given at high doses and on a cyclical scheme abolishes the beneficial effect of estrogens upon vascular functions and exercise-induced myocardial ischemia and that norethisterone acetate has a detrimental effect upon the endothelium-dependent vasodilatation of the forearm in PMW and increases the brachial artery resistance [25,12]. The effect seen with cyclical medroxyprogesterone is not seen with continuous combined administration of the progestin thereby suggesting that the effect may be related to the dose and scheme of administration of medroxyprogesterone. In the present study we have shown cyproterone acetate given in association to estradiol valerate does not reverse the beneficial effect of estrogen replacement therapy. The scheme of administration of cyproterone acetate tested in the present study was the continuous combined, theoretically the more detrimental to vascular function. The fact that in a cyclical scheme of administration, cyproterone acetate sustains the beneficial effect of estradiol valerate upon endothelial function and Endothelin-1 plasma levels suggests that this progestin may be safely used for the hormonal replacement therapy of menopausal women, at risk of coronary artery disease and in those with increased vascular reactivity.
5. Conclusion The present study shows that both estradiol valerate and estradiol valerate plus cyproterone acetate improves by a similar degree endothelial function in the forearm of PMW at increased cardiovascular risk. These findings suggest that this association could be safely administered in women with increased cardiovascular risk of with increased vascular reactivity.
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