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Venous endothelial function in postmenopausal women after six months of tibolone therapy
Maturitas 39 (2001) 63 – 70 www.elsevier.com/locate/maturitas
Venous endothelial function in postmenopausal women after six months of tibolone therapy Concepcio´n Ceballos a, Constantino Ribes a, Jose´ A. Amado b, Inmaculada de Mier c, Luis Sainz de Rozas c, Jose´ R. Berrazueta c,* a b
Department of Gynecology, Hospital Uni6ersitario M. de Valdecilla, Uni6ersity of Cantabria, Santander 39008, Spain Departmant of Endocrinology, Hospital Uni6ersitario M. de Valdecilla, Uni6ersity of Cantabria, Santander 39008, Spain c Department of Cardiology, Hospital Uni6ersitario M. de Valdecilla, Uni6ersity of Cantabria, Santander 39008, Spain Received 27 June 2000; received in revised form 29 November 2000; accepted 1 December 2000
Abstract Study Objecti6e: To test venous endothelial function in long-term climateric therapy with tibolone. Design: Measurement of dorsal hand-vein diameter by venous occlusion plethysmography during infusion of norepinephrine (NE), bradykinin (BK), NG-monomethyl L-arginine (L-NMMA) and sodium nitroprusside (SNP). Setting: Plethysmography and Menopause Units. University Hospital Valdecilla. Santander. Spain. Patients: Eleven postmenopausal women having continuous treatment with oral tibolone (2.5 mg/day) for 6 months. Inter6entions: Three plethysmography studies were made: at baseline, and at three and six months of treatment. Main outcome measures: Dorsal hand-vein diameter measured by venous occlusion plethysmography during infusion of NE, BK, L-NMMA and SNP. Results: (a) Baseline study: maximum dilation with BK was 54.2 910.2%. (b) Three-month study: BK dilation of 71.5911.9%, with a significant increase of 17.3% (P= 0.019) compared with baseline. (c) Six-month study: BK dilation of 77.5 911.9%, with a significant increase 23.3% (P=0.002) compared with baseline. Maximal vasodilation was reached with SNP in the three studies and L-NMMA infusion has a similar vasoconstrictor response in the three studies. Conclusions: Long-term climateric therapy with tibolone improves vein endothelium-dependent vasodilation suggesting a positive impact of this drug on endothelial function. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tibolone; Endothelial function; Dorsal hand-vein plethysmography; Nitric oxide
1. Introduction
* Corresponding author. Fax: + 34-942-201-334. E-mail address: [email protected] (J.R. Berrazueta).
Cardiovascular disease is the leading cause of death among women in developed countries [1]. The incidence of cardiovascular disease and the associated morbidity and mortality increase dramatically after the menopause [2]. This increased
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risk cannot be explained by changes in lifestyle or in risk factors [3]. Estrogen administration to postmenopausal women is associated with a reduction in the incidence of coronary disease of about 50% [4], when given as primary prevention, and appears to be more effective in women with a greater cardiovascular risk [5]. However, it does not have the same effect on the overall rate of coronary heart disease events in postmenopausal women with established coronary disease [6]. The cardiovascular benefits due to estrogens have been attributed to a number of mechanisms, including a beneficial effect on the plasma lipid profile [7] and on endothelial response, both in experimental animals [8] and in postmenopausal women [9,10]. Apart from natural estrogens, other types of climateric therapy (CT) are used in the menopause. Tibolone has a gestagenic, estrogenic and androgenic action, and appears to be at least as efficacious as other forms of CT in the treatment of climacteric symptoms [11]. It also prevents the long-term effects of estrogen deficit, such as osteoporosis and cardiovascular disease [12]. Tibolone may improve some lipid parameters but its androgenic action may also be responsible for a reduction in HDL cholesterol, and this would reduce its beneficial effect on lipids [13]. Both tibolone and estrogen have been shown to induce peripheral vasodilation and to have a direct effect on vascular reactivity, thereby increasing peripheral blood flow [14]. Plethysmography by means of venous occlusion of the forearm with intra-arterial injections of acetylcholine (Ach) has been used to measure vessel reactivity due to nitric oxide (NO) release, but this technique requires intra-arterial puncture, which makes it unsuited to serial studies. However, Vallance et al. [15] demonstrated that the dorsal hand-veins also release endothelial NO, when precontracted with norepinephrine (NE) and then stimulated with an agonist such as Ach or bradykinin (BK). Moreover, this effect can be inhibited by using a NO synthesis inhibitor such as NG-monomethyl L-arginine (L-NMMA) [16]. The aim of this study was to examine the endothelium-dependent response in postmenopausal women on long-term replacement
therapy with tibolone. The study was carried out on dorsal hand-veins by means of venous occlusion plethysmography, a technique that has not been used in most previous studies of vasomotion in postmenopausal women.
2. Subjects and methods The study was performed in 11 postmenopausal women, who met the following inclusion criteria: menopause of over six months, as confirmed by analytical indices (estradiol B 30 pg/ml and FSH \60 mU/ml), gynecological examination with normal cervical smear, mammogram and gynecological ultrasound. The following exclusion criteria were established: cigarette smoking, presence of high arterial blood pressure, diabetes mellitus, liver disease, obesity (Quetelet index of over 30), treatment of thyroid disorders, heart disease, evidence of atherosclerosis and lipid disorders requiring treatment. After the baseline plethysmography study, the women started a continuous six-month course of treatment with oral tibolone (2.5 mg/day), and further plethysmography studies were done at three and six months. Of the 11 women, one withdrew from the study because of weight increase. Before plethysmography, whole blood samples were taken for general biochemical analysis of total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides. Compliance was monitored in the menopausal unit every three months, and the number of used pills was calculated. All the women were selected from those attending the menopausal unit at the University Hospital ‘Marques de Valdecilla’, and all gave their written informed consent. The study protocol was approved by the Hospital’s Ethics Committee.
3. Study protocol Plethysmography was performed early in the morning with the women fasting overnight and having taken no non-steroid anti-inflammatory drug in the 48 h prior to the test. The test was
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carried out at a controlled temperature between 28 and 30°C. The patients were relaxed, lying in a supine position with their hand raised and resting on a flat surface angled upward at 30° to allow spontaneous emptying of the dorsal hand-vein. Each test was designed to last no more than two hours. The diameter of a dorsal hand-vein was measured by a constant distension pressure, as described previously [17]. Briefly, the study consists of inserting a 23-gauge butterfly needle into a preselected vein on the back of the hand. A tripod supporting a linear variable differential transformer is attached with sticking plaster to the perivenous surface. The transformer has a thin central vertically sliding steel rod that rests on the summit of the vein. This small rod is located 10 mm downstream from the tip of the needle inserted in the vein under study and moves with the distension and constriction of the vein. The vertical movements allow measurement of the variations in the diameter of the vein. Measurements are made by the distension induced by intermittent venous occlusion for periods of 5 min with the inflation to 40 mm Hg of a cuff (Hockanson) placed on the upper part of the arm under study. Physiologic saline and the drugs were administered by means of constant rate infusion pump (Terufusion. Syringe pump mod STC526) at a rate of 0.25 ml/min. With the subject at rest and in a warm environment, the superficial dorsal hand-vein lacks tone. For observation of the vasodilation response, the vein was first constricted by continuous infusion of NE at doses between 40 and 640 pmol/min, until the diameter of the vein was reduced to 20% of its diameter measured during initial infusion of saline. The NE dose producing 50% constriction was chosen for continuous infusion for the test. Next the different substances were administered: first BK (at doses of 2, 4, and 8 pmol/min) and then, after a period of stabilization, BK was administered (at the maximum dose) with L-NMMA (1 mmol/min). After 10 min of infusion with saline, the 50% NE constriction dose was administered together with sodium nitroprusside (SNP) (at doses of 0.25, 0.5 and 1 mg/min). Results are presented as dose–response curves. The diameter
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of the vein during the initial saline infusion was defined as 100% relaxation. Initial distension with the constrictive dose was considered to be 0% dilation. Vasodilation induced by BK was calculated as a percentage between 100 and 0% vasodilation.
4. Drugs The drugs used for the plethysmography study were as follows: NE (L-Noradrenaline Bitartrate, Braun), BK (Clinalfa AG, Zurich, Switzerland), L-NMMA (Clinalfa AG, Zurich, Switzerland), SNP (Fides-Rottapharm, Barcelona, Spain). All drugs were diluted in physiologic saline immediately before use.
5. Statistical analysis Vein size was expressed in arbitrary units, which were converted into mm after calibration at the end of each test. Constriction is expressed as a percentage reduction in vein diameter in relation to the baseline diameter obtained with saline infusion. Dilation is expressed as a percentage reversal of NE-induced constriction. All the data were analyzed with the SPSS software package (Statistical Package for Social Sciences, Chicago, IL). The parametric distribution of the data for each variable was first checked with the Kolmogorov–Smirnov test. All the data were expressed as mean9 standard deviation, and the median and range were also obtained for each of the variables. Differences between time points were evaluated by ANOVA, since the distribution of the data was parametric. When differences were observed, a Scheffe test was used to determine where the differences occurred. To avoid observer bias, the person evaluating the study was blind to the visit number.
6. Results The clinical and biochemistry data of the patients are given in Table 1. We have found a
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66 Table 1 Clinical variablesa Clinical variables
Basal study
Three months study
Six months study
Age (yr/range) Menopause (yr/range) Menarche (yr/range) BMI (Kg.m2) Time after menopause (yr) Glucose (mg /dl) Creatinine (mg /dl) ALT (U/L) Cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg)
51.05 93 46.7 936 13.2 9 1.45 25.29 3.07 492 92 9 9 0.990.1 26 910 2349 52.1 146.69 46.6 61.89 14.9 118.089 78.1 1309 15 83 9 6
203 9 56 146 950 45 9 6 78.2 9 26 132 917 889 3
242.1 9 52.8 172.9 9 50.9b 47.2 9 9.96c 83.1 9 23.3 135 9 15 869 7
Values given as mean 9SD. HDL, high density lipoproteins; LDL, low density lipoproteins. PB0.05, indicating significant change from baseline value. c P a
b
significant increase in LDL cholesterol level and a decrease in HDL cholesterol level. The other changes were not significants.
6.1. Baseline study The maximum venous dilation, achieved with BK (8 pmol/min) was 54.29 10.2% (Fig. 1). The combination of the maximum BK dose and LNMMA induced a constriction in the dorsal hand-vein of − 85.10 95.2% (Fig. 2). The vasodilation induced with SNP was close to maximum even at the lowest dose (Fig. 3).
6.2. Three month study After three months of tibolone therapy, the maximum venous dilation with BK was 71.59 11.9%, representing a significant increase of 17.3% (P=0.019) compared with baseline (Fig. 1). The maximum dose of BK and L-NMMA combined induced a constriction in the vein of −82.75 9 8.2%. The maximum SNP-induced vasodilation was virtually 100%.
Fig. 1. Dose response curve of NO-dependent relaxation of the dorsal hand-vein induced by BK at 2, 4 and 8 pmol/min in postmenopausal women. Bars represent mean 9 SEM. Hatched bars, basal study. Open bars, second study three months later with climateric therapy with tibolone; the NO dependent relaxation improved significantly ((b) P=0.01). Closed bars, third study six months later; the NO dependent relaxation improved significantly ((c) P= 0.002) against the basal study.
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The maximum SNP-induced vasodilatation was again close to 100% (Fig. 3).
7. Discussion
Fig. 2. Dose response curve of relaxation of the dorsal handvein with L-NMMA (NO synthesis inhibitor) in postmenopausal women treated with tibolone for six months. Differences between basal and third study. The maximum relaxation with BK is suppressed by the effect of L-NMMA, indicating that Tibolone relaxation obtained by BK is through the release of endothelial NO. There is no difference between the two studies.
6.3. Six month study After six months of treatment, the maximum vasodilatation with BK was 77.5911.9%. The mean increase compared with baseline was 23.3% (P = 0.002) (Fig. 1). The combination of the maximum dose of BK and L-NMMA induced a constriction in the vein of −79.6 910.3% (Fig. 2).
Fig. 3. Exogenous NO-independent relaxation of the dorsal hand-vein induced by SNP in postmenopausal women is similar before and after six months of tibolone treatment.
The results of this study show that long-term tibolone therapy (6 months) improves endothelium-dependent dilation through mechanisms mediated by NO release, and that analysis of endothelial function in the dorsal hand-vein with venous occlusion plethysmography may be a useful model to study comparative changes occurring in vascular endothelium. In this model, the most striking variations were observed in the endothelium-dependent response to administration of BK, a vasodilator that induces NO release from the venous bed, when these have been pre-contracted with NE [15]. Since the discovery that NO is an endotheliumderived relaxation factor [18], many in vitro [19] and in vivo studies have found that in different diseases there is an impaired endothelium-dependent relaxation in arterial [20] and venous [15] beds in response to stimulation with substances, such as Ach and BK, that mediate NO release, whereas there is normal relaxation due to nitrovasodilators, such as nitroglycerine or SNP, that are transformed into NO, and this has led to the concept of endothelial dysfunction [21]. Endothelial function can be studied in arterial or venous beds. Venous response may be affected by biochemical but not mechanical factors (shear stress, pulsatile flow) that induce endothelial dysfunction. So we chose to study venous instead of arterial beds. To our knowledge, there are not studies comparing venous endothelial function with brachial or coronary artery responses, but Lehmann et al. showed that tibolone had not effect on aortic compliance [22]. The incidence of cardiovascular disease increases in women after the menopause because of a decrease in estrogen levels [2], but is reduced to premenopausal levels in postmenopausal women, who receive estrogen therapy [4,23]. The beneficial effect of estrogens results from the reduction in lipid levels but this only occurs in some patients [24,25]. Other mechanisms resulting in a lower
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risk of thrombosis are due to a reduction in factors such as fibrinogen or plasminogen [26,27]. Other possible protective mechanisms, such as changes in contractility and hemodynamic status, have also been studied [28], but the most recent studies suggest that the protective role of estrogens derives from a direct action on the vessel wall [29]; in particular, the mechanism responsible for the cardiovascular benefit of estrogen is its action on the endothelium [30]. The physiologic levels of 17 b-estradiol enhance endothelium-dependent relaxation of the coronary arteries through a mechanism involving greater NO release [31,32]. The effect of estrogen on endothelial function has been demonstrated mainly in studies of its acute effects and of short-term treatment [33,34], which have shown improved endothelium dependent relaxation. Less attention has been given to the effects of prolonged administration but a positive response has been demonstrated in this setting [35]. Tibolone has proved effective as CT to control climacteric symptoms [11] and to prevent the long-term effects of estrogen deficit, such as osteoporosis and cardiovascular disease [12] through mechanisms similar to those of CT with estrogens. However, its androgenic action may be responsible for a reduction in HDL cholesterol, as we have shown in our study, in contrast to what occurs with combined estrogen– progestin treatment, which increases HDL cholesterol levels [36]. We have also found an increase in LDL cholesterol that has not been shown in other studies that describe a beneficial effect of tibolone on lipids [37,38]. Tibolone, like estrogen, has been shown to induce peripheral vasodilation and also has a direct effect on vascular reactivity, increasing peripheral blood flow [14,39] with no changes in blood pressure or cardiac output. Studies of endothelial function in the coronary arteries entail a degree of risk. The model of venous endothelial function used in our study has a very low risk, and it is the easiest way to study both the endothelial dysfunction in a generalized hormonal or metabolic disorder, as occurs in the menopause, and the beneficial effect of treatment with drugs to modify action on the endothelium.
In addition to testing endothelial function, studies performed on the veins are not affected by the shear stress from variations in blood flow and arterial pressure, which modulates arterial NO release [40]. Our results show that CT with tibolone produces improvement in endothelium-dependent relaxation and this effect appears after a prolonged period of treatment. Moreover, relaxation with SNP, an endothelium independent vasodilator, is normal indicating that the relaxing function of the smooth muscle, in this case of the veins, is normal along all the study. It has been suggested that both estrogens and tibolone may reduce synthesis of vasoconstrictive substances such as angiotensin II or endothelin [41,42]. Other possible mechanisms for estrogens involve increased synthesis of endothelial NO, as suggested by some in vitro [31] and in vivo [33] studies. However, this mechanism has so far not been proposed to explain the mode of action of tibolone although tibolone has the intrinsic potential to protect the arterial vessel wall against atherosclerotic processes [43]. Our results suggest that a possible mechanism of tibolone could be an increase in the synthesis and release of NO from the endothelium. One of the fundamental elements required to study a metabolic pathway is the possibility of using substances to block its metabolism. LNMMA, an L-arginine analogue and a competitive inhibitor of NO synthesis [16] does not affect the vasoconstrictive action of noradrenaline and does not have a direct vasoconstrictive effect in human veins; it inhibits vasodilation induced by Ach and BK, but not that induced by endothelium-independent vasodilators [15]. In our study, there were no differences between the tests when the constriction response obtained with L-NMMA was compared with maximum BK relaxation. However, as is well known, this shows that maximum BK relaxation is mediated by NO, and that tibolone exerts its effect on the venous endothelium, and that a background of continuous endothelial NO synthesis is required to produce this relaxation effect.
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7.1. Study limitation Since we did not introduce a placebo control group, we can not exclude some kind of bias, but to avoid observer bias, we assigned a visit number to each study, and the person evaluating the study was blind to the visit number. Six months is a short follow-up treatment, and longer-term follow-up will be needed to determine whether tibolone continues to have beneficial effects on endothelium and other vascular effects. ln conclusion, measurement of the dorsal handvein is a simple method to study endothelial dysfunction and this plethysmographic model is suitable for serial studies. This study has demonstrated that CT with tibolone improves long-term venous endothelium-dependent vasodilation by a mechanism that requires background NO release.
[8]
[9]
[10]
[11] [12]
[13]
Acknowledgements Concepcio´ n Ceballos was a fellow of the Fundacio´ n Marque´ s de Valdecilla (Cantabria, Spain) and this study was supported by a grant from this foundation.
[14]
[15]
[16]
References [1] Hennekens CH. Risk factors for coronary heart disease in women. Cardiol Clin 1998;16:1 –8. [2] Lerner DJ, Kannel WB. Patterns of coronary heart disease in women: perspective from the Framingham study. Am Heart J 1986;113:383 –90. [3] Kannel WB, Hjortland MC, McNamara PM, Gordon T. Menopause and risk of cardiovascular disease: the Framinghan study. Ann Intern Med 1976;85:447 – 52. [4] Stampler MJ, Golditz GA. Estrogen replacement theraphy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med 1991;20:47 – 63. [5] Wenger NK. Postmenopausal hormone therapy. Its useful for coronary prevention? Cardiol Clin 1998;16:17 –25. [6] Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/ progestin replacement study (HERS) Research group. JAMA 1998;280:605 –13. [7] Bush TL, Barret-Connor E, Cowan LD, Criqui MH, Wallace RB, Suchindran CM, Tyroler HA, Rifkind BM.
[17] [18]
[19]
[20]
[21]
[22]
69
Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation 1987;75:1102 – 9. Williams JK, Adams MR, Klopfenstein HS. Estrogen modulates response of atherosclerotic coronary arteries. Circulation 1990;881:1680 – 7. Stevenson JC, Crook D, Godsland IF, Collins P, Whitehead MI. Hormone replacement therapy and the cardiovascular system: nonlipid effects. Drugs 1994;47(Suppl. 1):35 – 41. Ceballos C, Ribes C, Amado JA, Pe´ rez J, Poveda JJ, Garcı´a Unzueta MT, Berrazueta JR. Venous endothelial function in postmenopausal women on long-term estrogen and progestagen therapy. Fertility and Sterility, in press. Albertazzi P, Di Micco R, Zanardi E. Tibolone: a review. Maturitas 1998;30:295 – 305. Bjarnason NH, Bjarnason K, Haarbo J, Bennink HJ, Christiansen C. Tibolone: influence on markers of cardiovascular disease. J Clin Endocrinol Metab 1997;82:1752 – 6. Farish E, Barnes JF, Rolton HA, Spowart K, Fletcher CD, Hart DM. Effects of Tibolone on lipoprotein(a) and HDL subfractions. Maturitas 1994;20:215 – 9. Hardiman P, Nihoyannopoulous P, Kicovic P, Kicovic P, Ginsburg J. Cardiovascular effects of Org OD14-a new steroidal therapy for climateric symptoms. Maturitas 1991;13:235 – 42. Vallance P, Collier J, Moncada S. Nitric oxide synthesed from L-arginine mediates endothelium dependent dilatation in human veins in vivo. Cardiovasc Res 1989;23:1053 – 7. Moncada S, Palmer RMJ, Higgs A. The discovery and biological significance of the L-arginine: nitric oxide pathway.In: Moncada S, Higgs A, Berrazueta JR, editors. En Clinical relevance of nitric oxide in the cardiovascular system. Madrid. Edicomplet. 1991;3 – 28. Collier JG. Veins on the back of the hand. In: Arteries and veins. London: Churchill Livingstone, 1975:136 – 42. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature 1987;327:524 – 6. Salas E, Berrazueta JR, Fleitas M, Poveda J, Ochoteco A, Amado J. Reduced endothelium-mediated relaxing responses in peripheral atherosclerotic arteries from patients with and witout diabetes mellitus. Diabet Med 1992;9:875 – 6. Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothelium-dependent relaxation in patients with essential hypertension. N Eng J Med 1990;323:22 – 7. Ludmer PL, Selwyn AP, Shooh TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstricion induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med 1986;315:1046 – 51. Lehmann ED, Hopkins KD, Parker JR, Turay RC, Rymer J, Fogelman I, Gosling RG. Aortic distensibility
70
[23]
[24]
[25]
[26]
[27]
[28]
[29] [30]
[31] [32]
[33]
C. Ceballos et al. / Maturitas 39 (2001) 63–70 in post menopausal women receiving tibolone. Br J Radiol 1994;67:701 – 5. Bush TL. Evidence for primary and secondary prevention of coronary artery disease in women taking oestrogen replacement therapy. Eur Heart J 1996;17(Suppl. DP):9 – 14. Stevenson JC, Crook D, Godsland IF. Influence of age and menopause on serum lipids and lipoproteins in healthy women. Atherosclerosis 1993;98:83 – 90. Bhathena RK, Anklesaria BS, Ganatra AM, Pinto R. The influence of transdermal oestradiol replacement therapy and medroxyprogesterone acetate on serum lipids and lipoproteins. Br J Clin Pharmacol 1998;45:170 –2. Falkeborn M, Persson I, Terent A, Adami HO, Lihell H, Bergstrom R. Hormone replacement therapy and the risk of stroke. Follow-up of a population-based cohort in Sweden. Arch Int Med 1993;153:1201 – 9. Koh KK, Mincemoyer R, Bui MN, Csako G, Pucino F, Guetta V, Waclawiw M, Cannon RO III. Effects of hormone replacement therapy on fibrinolysis in postmenopausal women. N Engl J Med 1997;336:683 –90. Rozenberg S, Liebens I, Vandrome J, Hotimsky A, Van Rijsselberge M. Cardiovascular protection by estrogen: a hemodynamic mechanism? Int J Fertil Menopausal Stud 1994;39(Suppl 1P):36 –42. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen. FASEB J 1996;10:615 –24. Gerhard M, Walsh BW, Tawakol A, Haley EA, Creager SJ, Seely EW, Ganz P, Creager MA. Estradiol therapy combined with progesterone and endothelium-dependent vasodilation in postmenopausal women. Circulation 1998;98:1158 – 63. Nathan I, Chaudhuri G. Estrogens and atherosclerosis. Annu Rev Pharmacol Toxicol 1997;37:477 –515. Collins P, Shay Y, Jiang C, Moss J. Nitric oxide accounts for dose-dependent estrogen-mediated coronary relaxation after acute estrogen withdrawal. Circulation 1994;90:1964 – 8. Tagawa H, Shimokawa H, Tagawa T, Kuroiwa-Matsumoto M, Hirooka Y, Takeshita A. Short term estrogen augments both nitric oxide-mediated and non-nitric oxide-mediated endothelium-dependent forearm vasodilation in postmenopausal women. J Cardiovasc Pharmacol 1997;30:481 – 8.
.
[34] Roque´ M, Heras M, Roig E, Masotti M, Rigol M, Betriu A, Balasch J, Sanz G. Short-term effects of transdermal estrogen replacement therapy on coronary vascular reactivity in postmenopausal women with angina pectoris and normal results on coronary angiograms. J Am Coll Cardiol 1998;31:139 – 43. [35] Gilligan DM, Badar DM, Panza JA, Quyymi AA, Cannon RO III. Effects of estrogen replacement therapy on peripheral vasomotor function in postmenopausal women. Am J Cardiol 1995;75:264 – 8. [36] The writing group for the PEPI trial. Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. JAMA 1995;273:199 – 208. [37] Farish E, Barnes JF, Fletcher CD, Ekevall K, Calder A, Hart DM. Effects of tibolone on serum lipoprotein and apolipoprotein levels compared with a cyclical estrogen/ progestogen regimen. Menopause 1999;6:98 – 104. [38] Castelo-Branco C, Casals E, Figueras F, Sanjuan A, Vicente JJ, Balasch J, Vanrell JA. Two-year prospective and comparative study on the effects of tibolone on lipid pattern, behavior of apolipoproteins AI and B. Menopause 1999;6:92 – 7. [39] Haenggi W, Linder HR, Birkhauser MH, Schneider H. Microscopic findings of the nail-fold capillaries-dependence on menopausal status and HRT. Maturitas 1995;22:37 – 46. [40] Perez Gonza´ lez J, Ceballos C, Vazquez L, Mier I, Amado JA, Ochoteco A, Poveda JJ, Berrazueta JR. Modification of endothelial response with changes of the pulsatile flow in patients treated by aortic valve replacement. Second International Congress on NO in Cardiovascular Regulation and Intensive Care. Stockholm, Sweden. 1997; p. 46. [41] Schunkert H, Danser AHJ, Hense HW, Derks FHM, Kurzinger S, Riegger GAJ. Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women. Circulation 1997;95:39 – 45. [42] Haenggi W, Bersinger NA, Mueller MD, Birkhaeuser MH. Decrease of serum endothelin levels with postmenopausal hormone replacement therapy or tibolone. Gynecol Endocrinol 1999;13:202 – 5. [43] Zandberg P, Peters JL, Demacker PN, Smit MJ, de Reeder EG, Meuleman DG. Tibolone prevents atherosclerotic lesion formation in cholesterol-fed, ovariectomized rabbits. Arterioscler Thromb Vasc Biol 1998;18:1844 – 54.
Venous endothelial function in postmenopausal women after six months of tibolone therapy Concepcio´n Ceballos a, Constantino Ribes a, Jose´ A. Amado b, Inmaculada de Mier c, Luis Sainz de Rozas c, Jose´ R. Berrazueta c,* a b
Department of Gynecology, Hospital Uni6ersitario M. de Valdecilla, Uni6ersity of Cantabria, Santander 39008, Spain Departmant of Endocrinology, Hospital Uni6ersitario M. de Valdecilla, Uni6ersity of Cantabria, Santander 39008, Spain c Department of Cardiology, Hospital Uni6ersitario M. de Valdecilla, Uni6ersity of Cantabria, Santander 39008, Spain Received 27 June 2000; received in revised form 29 November 2000; accepted 1 December 2000
Abstract Study Objecti6e: To test venous endothelial function in long-term climateric therapy with tibolone. Design: Measurement of dorsal hand-vein diameter by venous occlusion plethysmography during infusion of norepinephrine (NE), bradykinin (BK), NG-monomethyl L-arginine (L-NMMA) and sodium nitroprusside (SNP). Setting: Plethysmography and Menopause Units. University Hospital Valdecilla. Santander. Spain. Patients: Eleven postmenopausal women having continuous treatment with oral tibolone (2.5 mg/day) for 6 months. Inter6entions: Three plethysmography studies were made: at baseline, and at three and six months of treatment. Main outcome measures: Dorsal hand-vein diameter measured by venous occlusion plethysmography during infusion of NE, BK, L-NMMA and SNP. Results: (a) Baseline study: maximum dilation with BK was 54.2 910.2%. (b) Three-month study: BK dilation of 71.5911.9%, with a significant increase of 17.3% (P= 0.019) compared with baseline. (c) Six-month study: BK dilation of 77.5 911.9%, with a significant increase 23.3% (P=0.002) compared with baseline. Maximal vasodilation was reached with SNP in the three studies and L-NMMA infusion has a similar vasoconstrictor response in the three studies. Conclusions: Long-term climateric therapy with tibolone improves vein endothelium-dependent vasodilation suggesting a positive impact of this drug on endothelial function. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tibolone; Endothelial function; Dorsal hand-vein plethysmography; Nitric oxide
1. Introduction
* Corresponding author. Fax: + 34-942-201-334. E-mail address: [email protected] (J.R. Berrazueta).
Cardiovascular disease is the leading cause of death among women in developed countries [1]. The incidence of cardiovascular disease and the associated morbidity and mortality increase dramatically after the menopause [2]. This increased
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risk cannot be explained by changes in lifestyle or in risk factors [3]. Estrogen administration to postmenopausal women is associated with a reduction in the incidence of coronary disease of about 50% [4], when given as primary prevention, and appears to be more effective in women with a greater cardiovascular risk [5]. However, it does not have the same effect on the overall rate of coronary heart disease events in postmenopausal women with established coronary disease [6]. The cardiovascular benefits due to estrogens have been attributed to a number of mechanisms, including a beneficial effect on the plasma lipid profile [7] and on endothelial response, both in experimental animals [8] and in postmenopausal women [9,10]. Apart from natural estrogens, other types of climateric therapy (CT) are used in the menopause. Tibolone has a gestagenic, estrogenic and androgenic action, and appears to be at least as efficacious as other forms of CT in the treatment of climacteric symptoms [11]. It also prevents the long-term effects of estrogen deficit, such as osteoporosis and cardiovascular disease [12]. Tibolone may improve some lipid parameters but its androgenic action may also be responsible for a reduction in HDL cholesterol, and this would reduce its beneficial effect on lipids [13]. Both tibolone and estrogen have been shown to induce peripheral vasodilation and to have a direct effect on vascular reactivity, thereby increasing peripheral blood flow [14]. Plethysmography by means of venous occlusion of the forearm with intra-arterial injections of acetylcholine (Ach) has been used to measure vessel reactivity due to nitric oxide (NO) release, but this technique requires intra-arterial puncture, which makes it unsuited to serial studies. However, Vallance et al. [15] demonstrated that the dorsal hand-veins also release endothelial NO, when precontracted with norepinephrine (NE) and then stimulated with an agonist such as Ach or bradykinin (BK). Moreover, this effect can be inhibited by using a NO synthesis inhibitor such as NG-monomethyl L-arginine (L-NMMA) [16]. The aim of this study was to examine the endothelium-dependent response in postmenopausal women on long-term replacement
therapy with tibolone. The study was carried out on dorsal hand-veins by means of venous occlusion plethysmography, a technique that has not been used in most previous studies of vasomotion in postmenopausal women.
2. Subjects and methods The study was performed in 11 postmenopausal women, who met the following inclusion criteria: menopause of over six months, as confirmed by analytical indices (estradiol B 30 pg/ml and FSH \60 mU/ml), gynecological examination with normal cervical smear, mammogram and gynecological ultrasound. The following exclusion criteria were established: cigarette smoking, presence of high arterial blood pressure, diabetes mellitus, liver disease, obesity (Quetelet index of over 30), treatment of thyroid disorders, heart disease, evidence of atherosclerosis and lipid disorders requiring treatment. After the baseline plethysmography study, the women started a continuous six-month course of treatment with oral tibolone (2.5 mg/day), and further plethysmography studies were done at three and six months. Of the 11 women, one withdrew from the study because of weight increase. Before plethysmography, whole blood samples were taken for general biochemical analysis of total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides. Compliance was monitored in the menopausal unit every three months, and the number of used pills was calculated. All the women were selected from those attending the menopausal unit at the University Hospital ‘Marques de Valdecilla’, and all gave their written informed consent. The study protocol was approved by the Hospital’s Ethics Committee.
3. Study protocol Plethysmography was performed early in the morning with the women fasting overnight and having taken no non-steroid anti-inflammatory drug in the 48 h prior to the test. The test was
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carried out at a controlled temperature between 28 and 30°C. The patients were relaxed, lying in a supine position with their hand raised and resting on a flat surface angled upward at 30° to allow spontaneous emptying of the dorsal hand-vein. Each test was designed to last no more than two hours. The diameter of a dorsal hand-vein was measured by a constant distension pressure, as described previously [17]. Briefly, the study consists of inserting a 23-gauge butterfly needle into a preselected vein on the back of the hand. A tripod supporting a linear variable differential transformer is attached with sticking plaster to the perivenous surface. The transformer has a thin central vertically sliding steel rod that rests on the summit of the vein. This small rod is located 10 mm downstream from the tip of the needle inserted in the vein under study and moves with the distension and constriction of the vein. The vertical movements allow measurement of the variations in the diameter of the vein. Measurements are made by the distension induced by intermittent venous occlusion for periods of 5 min with the inflation to 40 mm Hg of a cuff (Hockanson) placed on the upper part of the arm under study. Physiologic saline and the drugs were administered by means of constant rate infusion pump (Terufusion. Syringe pump mod STC526) at a rate of 0.25 ml/min. With the subject at rest and in a warm environment, the superficial dorsal hand-vein lacks tone. For observation of the vasodilation response, the vein was first constricted by continuous infusion of NE at doses between 40 and 640 pmol/min, until the diameter of the vein was reduced to 20% of its diameter measured during initial infusion of saline. The NE dose producing 50% constriction was chosen for continuous infusion for the test. Next the different substances were administered: first BK (at doses of 2, 4, and 8 pmol/min) and then, after a period of stabilization, BK was administered (at the maximum dose) with L-NMMA (1 mmol/min). After 10 min of infusion with saline, the 50% NE constriction dose was administered together with sodium nitroprusside (SNP) (at doses of 0.25, 0.5 and 1 mg/min). Results are presented as dose–response curves. The diameter
65
of the vein during the initial saline infusion was defined as 100% relaxation. Initial distension with the constrictive dose was considered to be 0% dilation. Vasodilation induced by BK was calculated as a percentage between 100 and 0% vasodilation.
4. Drugs The drugs used for the plethysmography study were as follows: NE (L-Noradrenaline Bitartrate, Braun), BK (Clinalfa AG, Zurich, Switzerland), L-NMMA (Clinalfa AG, Zurich, Switzerland), SNP (Fides-Rottapharm, Barcelona, Spain). All drugs were diluted in physiologic saline immediately before use.
5. Statistical analysis Vein size was expressed in arbitrary units, which were converted into mm after calibration at the end of each test. Constriction is expressed as a percentage reduction in vein diameter in relation to the baseline diameter obtained with saline infusion. Dilation is expressed as a percentage reversal of NE-induced constriction. All the data were analyzed with the SPSS software package (Statistical Package for Social Sciences, Chicago, IL). The parametric distribution of the data for each variable was first checked with the Kolmogorov–Smirnov test. All the data were expressed as mean9 standard deviation, and the median and range were also obtained for each of the variables. Differences between time points were evaluated by ANOVA, since the distribution of the data was parametric. When differences were observed, a Scheffe test was used to determine where the differences occurred. To avoid observer bias, the person evaluating the study was blind to the visit number.
6. Results The clinical and biochemistry data of the patients are given in Table 1. We have found a
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66 Table 1 Clinical variablesa Clinical variables
Basal study
Three months study
Six months study
Age (yr/range) Menopause (yr/range) Menarche (yr/range) BMI (Kg.m2) Time after menopause (yr) Glucose (mg /dl) Creatinine (mg /dl) ALT (U/L) Cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg)
51.05 93 46.7 936 13.2 9 1.45 25.29 3.07 492 92 9 9 0.990.1 26 910 2349 52.1 146.69 46.6 61.89 14.9 118.089 78.1 1309 15 83 9 6
203 9 56 146 950 45 9 6 78.2 9 26 132 917 889 3
242.1 9 52.8 172.9 9 50.9b 47.2 9 9.96c 83.1 9 23.3 135 9 15 869 7
Values given as mean 9SD. HDL, high density lipoproteins; LDL, low density lipoproteins. PB0.05, indicating significant change from baseline value. c P a
b
significant increase in LDL cholesterol level and a decrease in HDL cholesterol level. The other changes were not significants.
6.1. Baseline study The maximum venous dilation, achieved with BK (8 pmol/min) was 54.29 10.2% (Fig. 1). The combination of the maximum BK dose and LNMMA induced a constriction in the dorsal hand-vein of − 85.10 95.2% (Fig. 2). The vasodilation induced with SNP was close to maximum even at the lowest dose (Fig. 3).
6.2. Three month study After three months of tibolone therapy, the maximum venous dilation with BK was 71.59 11.9%, representing a significant increase of 17.3% (P=0.019) compared with baseline (Fig. 1). The maximum dose of BK and L-NMMA combined induced a constriction in the vein of −82.75 9 8.2%. The maximum SNP-induced vasodilation was virtually 100%.
Fig. 1. Dose response curve of NO-dependent relaxation of the dorsal hand-vein induced by BK at 2, 4 and 8 pmol/min in postmenopausal women. Bars represent mean 9 SEM. Hatched bars, basal study. Open bars, second study three months later with climateric therapy with tibolone; the NO dependent relaxation improved significantly ((b) P=0.01). Closed bars, third study six months later; the NO dependent relaxation improved significantly ((c) P= 0.002) against the basal study.
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The maximum SNP-induced vasodilatation was again close to 100% (Fig. 3).
7. Discussion
Fig. 2. Dose response curve of relaxation of the dorsal handvein with L-NMMA (NO synthesis inhibitor) in postmenopausal women treated with tibolone for six months. Differences between basal and third study. The maximum relaxation with BK is suppressed by the effect of L-NMMA, indicating that Tibolone relaxation obtained by BK is through the release of endothelial NO. There is no difference between the two studies.
6.3. Six month study After six months of treatment, the maximum vasodilatation with BK was 77.5911.9%. The mean increase compared with baseline was 23.3% (P = 0.002) (Fig. 1). The combination of the maximum dose of BK and L-NMMA induced a constriction in the vein of −79.6 910.3% (Fig. 2).
Fig. 3. Exogenous NO-independent relaxation of the dorsal hand-vein induced by SNP in postmenopausal women is similar before and after six months of tibolone treatment.
The results of this study show that long-term tibolone therapy (6 months) improves endothelium-dependent dilation through mechanisms mediated by NO release, and that analysis of endothelial function in the dorsal hand-vein with venous occlusion plethysmography may be a useful model to study comparative changes occurring in vascular endothelium. In this model, the most striking variations were observed in the endothelium-dependent response to administration of BK, a vasodilator that induces NO release from the venous bed, when these have been pre-contracted with NE [15]. Since the discovery that NO is an endotheliumderived relaxation factor [18], many in vitro [19] and in vivo studies have found that in different diseases there is an impaired endothelium-dependent relaxation in arterial [20] and venous [15] beds in response to stimulation with substances, such as Ach and BK, that mediate NO release, whereas there is normal relaxation due to nitrovasodilators, such as nitroglycerine or SNP, that are transformed into NO, and this has led to the concept of endothelial dysfunction [21]. Endothelial function can be studied in arterial or venous beds. Venous response may be affected by biochemical but not mechanical factors (shear stress, pulsatile flow) that induce endothelial dysfunction. So we chose to study venous instead of arterial beds. To our knowledge, there are not studies comparing venous endothelial function with brachial or coronary artery responses, but Lehmann et al. showed that tibolone had not effect on aortic compliance [22]. The incidence of cardiovascular disease increases in women after the menopause because of a decrease in estrogen levels [2], but is reduced to premenopausal levels in postmenopausal women, who receive estrogen therapy [4,23]. The beneficial effect of estrogens results from the reduction in lipid levels but this only occurs in some patients [24,25]. Other mechanisms resulting in a lower
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risk of thrombosis are due to a reduction in factors such as fibrinogen or plasminogen [26,27]. Other possible protective mechanisms, such as changes in contractility and hemodynamic status, have also been studied [28], but the most recent studies suggest that the protective role of estrogens derives from a direct action on the vessel wall [29]; in particular, the mechanism responsible for the cardiovascular benefit of estrogen is its action on the endothelium [30]. The physiologic levels of 17 b-estradiol enhance endothelium-dependent relaxation of the coronary arteries through a mechanism involving greater NO release [31,32]. The effect of estrogen on endothelial function has been demonstrated mainly in studies of its acute effects and of short-term treatment [33,34], which have shown improved endothelium dependent relaxation. Less attention has been given to the effects of prolonged administration but a positive response has been demonstrated in this setting [35]. Tibolone has proved effective as CT to control climacteric symptoms [11] and to prevent the long-term effects of estrogen deficit, such as osteoporosis and cardiovascular disease [12] through mechanisms similar to those of CT with estrogens. However, its androgenic action may be responsible for a reduction in HDL cholesterol, as we have shown in our study, in contrast to what occurs with combined estrogen– progestin treatment, which increases HDL cholesterol levels [36]. We have also found an increase in LDL cholesterol that has not been shown in other studies that describe a beneficial effect of tibolone on lipids [37,38]. Tibolone, like estrogen, has been shown to induce peripheral vasodilation and also has a direct effect on vascular reactivity, increasing peripheral blood flow [14,39] with no changes in blood pressure or cardiac output. Studies of endothelial function in the coronary arteries entail a degree of risk. The model of venous endothelial function used in our study has a very low risk, and it is the easiest way to study both the endothelial dysfunction in a generalized hormonal or metabolic disorder, as occurs in the menopause, and the beneficial effect of treatment with drugs to modify action on the endothelium.
In addition to testing endothelial function, studies performed on the veins are not affected by the shear stress from variations in blood flow and arterial pressure, which modulates arterial NO release [40]. Our results show that CT with tibolone produces improvement in endothelium-dependent relaxation and this effect appears after a prolonged period of treatment. Moreover, relaxation with SNP, an endothelium independent vasodilator, is normal indicating that the relaxing function of the smooth muscle, in this case of the veins, is normal along all the study. It has been suggested that both estrogens and tibolone may reduce synthesis of vasoconstrictive substances such as angiotensin II or endothelin [41,42]. Other possible mechanisms for estrogens involve increased synthesis of endothelial NO, as suggested by some in vitro [31] and in vivo [33] studies. However, this mechanism has so far not been proposed to explain the mode of action of tibolone although tibolone has the intrinsic potential to protect the arterial vessel wall against atherosclerotic processes [43]. Our results suggest that a possible mechanism of tibolone could be an increase in the synthesis and release of NO from the endothelium. One of the fundamental elements required to study a metabolic pathway is the possibility of using substances to block its metabolism. LNMMA, an L-arginine analogue and a competitive inhibitor of NO synthesis [16] does not affect the vasoconstrictive action of noradrenaline and does not have a direct vasoconstrictive effect in human veins; it inhibits vasodilation induced by Ach and BK, but not that induced by endothelium-independent vasodilators [15]. In our study, there were no differences between the tests when the constriction response obtained with L-NMMA was compared with maximum BK relaxation. However, as is well known, this shows that maximum BK relaxation is mediated by NO, and that tibolone exerts its effect on the venous endothelium, and that a background of continuous endothelial NO synthesis is required to produce this relaxation effect.
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7.1. Study limitation Since we did not introduce a placebo control group, we can not exclude some kind of bias, but to avoid observer bias, we assigned a visit number to each study, and the person evaluating the study was blind to the visit number. Six months is a short follow-up treatment, and longer-term follow-up will be needed to determine whether tibolone continues to have beneficial effects on endothelium and other vascular effects. ln conclusion, measurement of the dorsal handvein is a simple method to study endothelial dysfunction and this plethysmographic model is suitable for serial studies. This study has demonstrated that CT with tibolone improves long-term venous endothelium-dependent vasodilation by a mechanism that requires background NO release.
[8]
[9]
[10]
[11] [12]
[13]
Acknowledgements Concepcio´ n Ceballos was a fellow of the Fundacio´ n Marque´ s de Valdecilla (Cantabria, Spain) and this study was supported by a grant from this foundation.
[14]
[15]
[16]
References [1] Hennekens CH. Risk factors for coronary heart disease in women. Cardiol Clin 1998;16:1 –8. [2] Lerner DJ, Kannel WB. Patterns of coronary heart disease in women: perspective from the Framingham study. Am Heart J 1986;113:383 –90. [3] Kannel WB, Hjortland MC, McNamara PM, Gordon T. Menopause and risk of cardiovascular disease: the Framinghan study. Ann Intern Med 1976;85:447 – 52. [4] Stampler MJ, Golditz GA. Estrogen replacement theraphy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med 1991;20:47 – 63. [5] Wenger NK. Postmenopausal hormone therapy. Its useful for coronary prevention? Cardiol Clin 1998;16:17 –25. [6] Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/ progestin replacement study (HERS) Research group. JAMA 1998;280:605 –13. [7] Bush TL, Barret-Connor E, Cowan LD, Criqui MH, Wallace RB, Suchindran CM, Tyroler HA, Rifkind BM.
[17] [18]
[19]
[20]
[21]
[22]
69
Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation 1987;75:1102 – 9. Williams JK, Adams MR, Klopfenstein HS. Estrogen modulates response of atherosclerotic coronary arteries. Circulation 1990;881:1680 – 7. Stevenson JC, Crook D, Godsland IF, Collins P, Whitehead MI. Hormone replacement therapy and the cardiovascular system: nonlipid effects. Drugs 1994;47(Suppl. 1):35 – 41. Ceballos C, Ribes C, Amado JA, Pe´ rez J, Poveda JJ, Garcı´a Unzueta MT, Berrazueta JR. Venous endothelial function in postmenopausal women on long-term estrogen and progestagen therapy. Fertility and Sterility, in press. Albertazzi P, Di Micco R, Zanardi E. Tibolone: a review. Maturitas 1998;30:295 – 305. Bjarnason NH, Bjarnason K, Haarbo J, Bennink HJ, Christiansen C. Tibolone: influence on markers of cardiovascular disease. J Clin Endocrinol Metab 1997;82:1752 – 6. Farish E, Barnes JF, Rolton HA, Spowart K, Fletcher CD, Hart DM. Effects of Tibolone on lipoprotein(a) and HDL subfractions. Maturitas 1994;20:215 – 9. Hardiman P, Nihoyannopoulous P, Kicovic P, Kicovic P, Ginsburg J. Cardiovascular effects of Org OD14-a new steroidal therapy for climateric symptoms. Maturitas 1991;13:235 – 42. Vallance P, Collier J, Moncada S. Nitric oxide synthesed from L-arginine mediates endothelium dependent dilatation in human veins in vivo. Cardiovasc Res 1989;23:1053 – 7. Moncada S, Palmer RMJ, Higgs A. The discovery and biological significance of the L-arginine: nitric oxide pathway.In: Moncada S, Higgs A, Berrazueta JR, editors. En Clinical relevance of nitric oxide in the cardiovascular system. Madrid. Edicomplet. 1991;3 – 28. Collier JG. Veins on the back of the hand. In: Arteries and veins. London: Churchill Livingstone, 1975:136 – 42. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature 1987;327:524 – 6. Salas E, Berrazueta JR, Fleitas M, Poveda J, Ochoteco A, Amado J. Reduced endothelium-mediated relaxing responses in peripheral atherosclerotic arteries from patients with and witout diabetes mellitus. Diabet Med 1992;9:875 – 6. Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothelium-dependent relaxation in patients with essential hypertension. N Eng J Med 1990;323:22 – 7. Ludmer PL, Selwyn AP, Shooh TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstricion induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med 1986;315:1046 – 51. Lehmann ED, Hopkins KD, Parker JR, Turay RC, Rymer J, Fogelman I, Gosling RG. Aortic distensibility
70
[23]
[24]
[25]
[26]
[27]
[28]
[29] [30]
[31] [32]
[33]
C. Ceballos et al. / Maturitas 39 (2001) 63–70 in post menopausal women receiving tibolone. Br J Radiol 1994;67:701 – 5. Bush TL. Evidence for primary and secondary prevention of coronary artery disease in women taking oestrogen replacement therapy. Eur Heart J 1996;17(Suppl. DP):9 – 14. Stevenson JC, Crook D, Godsland IF. Influence of age and menopause on serum lipids and lipoproteins in healthy women. Atherosclerosis 1993;98:83 – 90. Bhathena RK, Anklesaria BS, Ganatra AM, Pinto R. The influence of transdermal oestradiol replacement therapy and medroxyprogesterone acetate on serum lipids and lipoproteins. Br J Clin Pharmacol 1998;45:170 –2. Falkeborn M, Persson I, Terent A, Adami HO, Lihell H, Bergstrom R. Hormone replacement therapy and the risk of stroke. Follow-up of a population-based cohort in Sweden. Arch Int Med 1993;153:1201 – 9. Koh KK, Mincemoyer R, Bui MN, Csako G, Pucino F, Guetta V, Waclawiw M, Cannon RO III. Effects of hormone replacement therapy on fibrinolysis in postmenopausal women. N Engl J Med 1997;336:683 –90. Rozenberg S, Liebens I, Vandrome J, Hotimsky A, Van Rijsselberge M. Cardiovascular protection by estrogen: a hemodynamic mechanism? Int J Fertil Menopausal Stud 1994;39(Suppl 1P):36 –42. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen. FASEB J 1996;10:615 –24. Gerhard M, Walsh BW, Tawakol A, Haley EA, Creager SJ, Seely EW, Ganz P, Creager MA. Estradiol therapy combined with progesterone and endothelium-dependent vasodilation in postmenopausal women. Circulation 1998;98:1158 – 63. Nathan I, Chaudhuri G. Estrogens and atherosclerosis. Annu Rev Pharmacol Toxicol 1997;37:477 –515. Collins P, Shay Y, Jiang C, Moss J. Nitric oxide accounts for dose-dependent estrogen-mediated coronary relaxation after acute estrogen withdrawal. Circulation 1994;90:1964 – 8. Tagawa H, Shimokawa H, Tagawa T, Kuroiwa-Matsumoto M, Hirooka Y, Takeshita A. Short term estrogen augments both nitric oxide-mediated and non-nitric oxide-mediated endothelium-dependent forearm vasodilation in postmenopausal women. J Cardiovasc Pharmacol 1997;30:481 – 8.
.
[34] Roque´ M, Heras M, Roig E, Masotti M, Rigol M, Betriu A, Balasch J, Sanz G. Short-term effects of transdermal estrogen replacement therapy on coronary vascular reactivity in postmenopausal women with angina pectoris and normal results on coronary angiograms. J Am Coll Cardiol 1998;31:139 – 43. [35] Gilligan DM, Badar DM, Panza JA, Quyymi AA, Cannon RO III. Effects of estrogen replacement therapy on peripheral vasomotor function in postmenopausal women. Am J Cardiol 1995;75:264 – 8. [36] The writing group for the PEPI trial. Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. JAMA 1995;273:199 – 208. [37] Farish E, Barnes JF, Fletcher CD, Ekevall K, Calder A, Hart DM. Effects of tibolone on serum lipoprotein and apolipoprotein levels compared with a cyclical estrogen/ progestogen regimen. Menopause 1999;6:98 – 104. [38] Castelo-Branco C, Casals E, Figueras F, Sanjuan A, Vicente JJ, Balasch J, Vanrell JA. Two-year prospective and comparative study on the effects of tibolone on lipid pattern, behavior of apolipoproteins AI and B. Menopause 1999;6:92 – 7. [39] Haenggi W, Linder HR, Birkhauser MH, Schneider H. Microscopic findings of the nail-fold capillaries-dependence on menopausal status and HRT. Maturitas 1995;22:37 – 46. [40] Perez Gonza´ lez J, Ceballos C, Vazquez L, Mier I, Amado JA, Ochoteco A, Poveda JJ, Berrazueta JR. Modification of endothelial response with changes of the pulsatile flow in patients treated by aortic valve replacement. Second International Congress on NO in Cardiovascular Regulation and Intensive Care. Stockholm, Sweden. 1997; p. 46. [41] Schunkert H, Danser AHJ, Hense HW, Derks FHM, Kurzinger S, Riegger GAJ. Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women. Circulation 1997;95:39 – 45. [42] Haenggi W, Bersinger NA, Mueller MD, Birkhaeuser MH. Decrease of serum endothelin levels with postmenopausal hormone replacement therapy or tibolone. Gynecol Endocrinol 1999;13:202 – 5. [43] Zandberg P, Peters JL, Demacker PN, Smit MJ, de Reeder EG, Meuleman DG. Tibolone prevents atherosclerotic lesion formation in cholesterol-fed, ovariectomized rabbits. Arterioscler Thromb Vasc Biol 1998;18:1844 – 54.