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Gender Difference in Improvement of Endothelium-Dependent Vasodilation After Estrogen Supplementation
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VASCULAR FUNCTION
Gender Difference in Improvement of Endothelium-Dependent Vasodilation After Estrogen Supplementation HIROAKI KAWANO, MD, TAKESHI MOTOYAMA, MD, KIYOTAKA KUGIYAMA, MD, OSAMU HIRASHIMA, MD, MASAMICHI OHGUSHI, MD, HIROMI FUJII, MD, HISAO OGAWA, MD, HIROFUMI YASUE, MD Kumamoto, Japan
Objectives. We examined whether there is a gender difference in the improvement of endothelium-dependent vasodilation after estrogen supplementation. Background. Estrogen therapy reduces the risk of cardiovascular events in postmenopausal women, and the augmented release of endothelium-derived nitric oxide (NO) by estrogens has been suggested to be one of the mechanisms for the cardioprotective effects of estrogen. Methods. With ultrasound technique, we measured the diameter and blood flow of the brachial artery at rest, during reactive hyperemia after transient occlusion and after nitroglycerin administration before and after estradiol supplementation in 15 postmenopausal women (mean 63 years) and in 15 men matched for age and risk factors for atherosclerosis. Results. Estradiol supplementation augmented the flowmediated vasodilation and serum level of nitrite/nitrate (metabo-
lites of NO) in women (respectively, from a mean 6 SEM of 8.0 6 0.6% to 12.9 6 0.6% [p < 0.01 by analysis of variance (ANOVA)] and from 64.9 6 8.7 to 93.7 6 9.4 mmol/liter [p < 0.05 by ANOVA]) but not in men (respectively, from 8.1 6 0.6% to 8.3 6 0.7% and from 57.8 6 6.7 to 60.8 6 5.4 mmol/liter). The increases in blood flow during reactive hyperemia and in diameter after nitroglycerin administration were not affected by estradiol supplementation in either men or women. Conclusions. Estradiol supplementation improves endotheliumdependent vasodilation in women, probably because of augmented NO production/release, but not in men. Thus, there may be gender differences in the effects of estrogen therapy on endothelial functions and NO production/release. (J Am Coll Cardiol 1997;30:914 –9) ©1997 by the American College of Cardiology
Life expectancy is greater in women than in men, in large part because of the lower incidence of cardiovascular death in women (1). Previous studies (2– 4) have suggested that ovarian hormones, especially estrogens, can through their protective action against atherosclerosis, reduce the risk of cardiovascular events in women within the reproductive age group. This protection is lost with the onset of menopause or after surgical castration (2,3,5). Thus, estrogen therapy may have beneficial effects for postmenopausal or castrated women in reducing the risk of cardiovascular events, as reported previously (2,4 – 6), and the American Heart Association consensus panel (7) recommends estrogen replacement therapy for postmenopausal women with coronary artery disease. However, all previous studies reported the beneficial effects of estrogen
supplementation in reducing the risk of cardiovascular events only for women. Therefore, it remains unknown whether estrogen supplementation also reduces the risk of cardiovascular events in men. The precise mechanism of the cardioprotective effect of estrogens on the development of atherosclerosis is unclear at present. However, several reports (8 –10) have shown that estrogens stimulate the release of endothelium-derived nitric oxide (NO). This phenomenon may partly contribute to the cardioprotective action of estrogens with respect to cardiovascular events and the development of atherosclerosis because NO is known to induce coronary dilation and to suppress platelet aggregation and migration of monocytes and smooth muscle cells (11–15). To examine whether there is a gender difference in the improvement of flow-mediated endotheliumdependent vasodilation after estrogen supplementation, we measured the flow-mediated endothelium-dependent vasodilation of brachial arteries in men and women by using an ultrasound technique before and after estradiol (E2) supplementation. We also measured the serum levels of E2 and nitrite/nitrate, metabolites of NO, and compared these levels with the magnitude of endothelium-dependent vasodilation (15).
From the Division of Cardiology, Kumamoto University School of Medicine, Kumamoto, Japan. This study was supported in part by Grant-in-Aid for Scientific Research A 08407019 from the Ministry of Education, Science, Sports and Culture in Japan and the Smoking Research Foundation Grant for Biomedical Research, Tokyo, Japan. Manuscript received December 19, 1996; revised manuscript received May 28, 1997, accepted June 20, 1997. Address for correspondence: Dr. Hirofumi Yasue, Division of Cardiology, Kumamoto University, 1-1-1 Honjo, Kumamoto City 860, Japan. E-mail: [email protected]. ©1997 by the American College of Cardiology Published by Elsevier Science Inc.
0735-1097/97/$17.00 PII S0735-1097(97)00234-9
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KAWANO ET AL. GENDER DIFFERENCE IN ENDOTHELIAL FUNCTIONS
Abbreviations and Acronyms ANOVA 5 analysis of variance E2 5 estradiol NO 5 nitric oxide
Methods Study subjects. The study subjects were 15 healthy postmenopausal female volunteers (mean age 63.6 6 2.6 years) and 15 healthy male volunteers matched for age and risk factors for atherosclerosis (mean age 63.1 6 0.7 years). Women were eligible if menopause had occurred at least 1 year previously (mean age at which the women reached menopause 51.2 years [range 47 to 55]). Menopause was confirmed by measuring serum follicular-stimulating hormone levels. No subject had received hormone replacement therapy for $2 months before the study. All subjects were asymptomatic, normotensive, nondiabetic and nonsmoking. None had congestive heart failure or any other serious disease. The subjects’ characteristics are shown in Table 1. All subjects were studied before and after supplementation of 17-beta estradiol (Estraderm TTS, 4 mg, Ciba, Basel, Switzerland) transdermally for 36 h. Written informed consent was obtained from all volunteers before the study. The study was in agreement with the guidelines approved by the ethics committee at our institution. Study design. In this study, flow-mediated vasodilation and nitroglycerin-induced vasodilation of the brachial artery were measured by two skillful examiners who did not know whether their study was done before or after E2 supplementation. We excluded subjects with inadequate scan quality of either brachial artery before the study. All studies were performed in a quiet temperature-controlled room (22° to 23°C). During the study, blood pressure was monitored in the right arm every 2 min by an automated blood pressure recorder. All study subjects were right-handed.
The vasodilator responses in the brachial arteries were measured by ultrasound technique. The validity of the method has been confirmed by our previous studies and others (16 – 20). Briefly, the diameter of the left brachial arteries was measured from B-mode ultrasound images with the use of a 7.5-MHz linear array transducer (SSH-160A ultrasound system, Toshiba Corp., Tokyo, Japan). Flow velocity in the brachial arteries was measured by using a pulsed Doppler signal at a 70° angle to the vessel, with the range gate (1.5 mm) in the center of the artery. The brachial arteries were scanned in the antecubital fossa in longitudinal fashion. Depth and gain settings were optimized at the beginning of the study and were kept constant throughout the recording period. When a satisfactory transducer position was found, the surface of the skin was marked, and the arm remained in the same position throughout the study. The subjects lay quietly for 10 min before the first scan. After baseline measurements of the diameter and flow velocity in the brachial arteries were obtained, a blood pressure cuff placed around the forearm was inflated with a pressure of 250 to 300 mm Hg. After 4.5 min, the cuff was released. The diameter and flow velocity were continuously measured between cuff inflation and after cuff deflation. Thereafter, the subjects lay quietly for 15 min. After confirmation that the diameter and the flow velocity returned to the baseline levels, sublingual nitroglycerin (0.3 mg) was administered, and 3 to 4 min later, the last measurements were made. Corretti et al. (19) reported that, compared with 5 mins of arterial occlusion, 1 and 3 min of arterial occlusion were inadequate to elicit statistically significant vasodilation responses. Furthermore, Celermajer et al. (16,17) reported that 4.5 mins of arterial occlusion was adequate to elicit statistically significant vasodilation responses. Thus, we used 4.5 min of arterial occlusion to evaluate the flow-mediated endotheliumdependent vasodilation. The ultrasound images were recorded on a super-VHS video cassette recorder (BR-S601M, Victor Corp., Tokyo, Japan) and arterial diameters were measured at a fixed dis-
Table 1. Clinical Characteristics of Study Subjects Before and After Estradiol Supplementation Men Before Age (yr) Heart rate (beats/min) Mean blood pressure (mm Hg) Basal arterial diameter (mm) Basal blood flow (ml/min) Increase in blood flow during reactive hyperemia (%) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglyceride (mg/dl)
915
Women After
Before
After
63.1 6 0.7 62.0 6 2.0 63.4 6 2.1 105.3 6 1.9 107.1 6 2.1 4.04 6 0.15* 4.06 6 0.16* 279.3 6 30.1* 280.5 6 28.7* 241.5 6 29.9 245.6 6 28.8
63.6 6 2.6 63.8 6 1.7 64.1 6 2.3 106.7 6 2.3 107.5 6 2.5 3.21 6 0.13 3.20 6 0.14 165.2 6 19.1 167.3 6 18.7 249.5 6 27.5 247.6 6 29.3
155.7 6 6.7 46.2 6 1.8 97.2 6 6.2 58.7 6 8.4
156.8 6 6.0 48.3 6 2.8 96.4 6 5.7 59.1 6 7.1
157.3 6 7.0 45.6 6 2.0 98.1 6 5.9 57.2 6 7.9
155.9 6 6.5 47.1 6 2.1 97.0 6 6.1 58.4 6 6.9
*p , 0.01 versus before and after estradiol supplementation in women. Data are expressed as mean value 6 SE. HDL 5 high density lipoprotein; LDL 5 low density lipoprotein.
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tance from an anatomic marker with ultrasound calipers by two independent observers who did not know whether the study subject was male or female or whether the study was done before or after E2 supplementation. Measurements were taken from the anterior to the posterior interface between the media and adventitia (“m” line) at end-diastole, incident with the R wave on a continuously recorded electrocardiogram (16,17,19 – 21). The diameter at four cardiac cycles was analyzed for each scan, and the measurements were averaged. The diameter measurements for reactive hyperemia were taken 45 to 90 s after cuff deflation to measure peak diameter (19). The responses of the vessel diameters to reactive hyperemia and nitroglycerin were expressed as a percent increase of the baseline value of the diameter. Blood flow was calculated by multiplying the velocity-time integral of the Doppler flow signal by heart rate and the vessel cross-sectional area. The percent of increase in brachial blood flow observed immediately after cuff deflation was calculated as the maximal flow recorded within the 1st 15 s after cuff deflation divided by the flow at baseline (16,17,19,20). In our studies, the repeated measurements of rest arterial diameter had an interobserver variability of 0.05 6 0.02 mm, and an intraobserver variability of 0.02 6 0.02 mm. When these studies were performed at the same time on 2 separate days in 20 control subjects, the between-occasions within-patient difference for the measurements of arterial diameter during reactive hyperemia was 0.05 6 0.04 mm. Blood sampling and assays. Blood samples were obtained from all study subjects on the study day while the subjects were in the fasting state. Serum E2 concentrations were measured by a specific immunoradiometric assay for E2 (Diagnostic Products Corp.) (20,22). Serum total cholesterol and triglyceride concentrations were measured enzymatically, and serum high density lipoprotein (HDL)-cholesterol concentration was measured by heparin-Ca21/Ni21 precipitation (23). Nitrite/ nitrate, stable metabolites of NO, were measured with an autoanalyzer (flow injection analyzer, TCI-NOX1000, Tokyo Kasei Kogyo, Tokyo, Japan) that used a method based on the Griess reaction (15,20,24). Briefly, the samples were passed through a column containing copper-coated cadmium, which reduced all nitrate to nitrite. The nitrite then was detected when it reacted with Griess reagent (24). Absorbance was measured at 540 nm by spectrophotometer. Statistical analysis. The changes in variables were assessed by two-way analysis of variance with repeated measures followed by post hoc testing with the Scheffe´ test. Statistical significance was defined as p , 0.05.
Results All study subjects tolerated the studies well. E2 supplementation did not make any difference in heart rate, mean blood pressure, lipid profile or percent increase in blood flow during reactive hyperemia in either the men or the women (Table 1).
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Figure 1. Bar graph showing the percent increase in arterial diameter during reactive hyperemia in men and women before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE. *Significant effect of estradiol supplementation compared with values in male subjects (p , 0.01 by ANOVA).
The percent increase in arterial diameter during reactive hyperemia was comparable in the men and the women before E2 supplementation (8.1 6 0.6% vs. 8.0 6 0.6%, p 5 0.97). After E2 supplementation, the percent increase in arterial diameter during reactive hyperemia was augmented in the women (from 8.0 6 0.6% to 12.9 6 0.6%, p , 0.01) but not in the men (from 8.1 6 0.6% to 8.3 6 0.7%, p 5 0.85) (Fig. 1). The effect of E2 supplementation on arterial dilation during reactive hyperemia in the women was significantly different from that in the men (p , 0.01). The percent increase in arterial diameter after nitroglycerin administration was comparable in the men and the women before and after E2 supplementation (before E2, 19.7 6 2.0% vs. 19.7 6 2.8%, p 5 0.97; after E2, 20.3 6 1.6% vs. 21.6 6 2.6%, p 5 0.60). E2 supplementation did not alter the increase in diameter after nitroglycerin administration in either the men or the women (men, from 19.7 6 2.0% to 20.3 6 1.6%, p 5 0.46; women, from 19.7 6 2.8% to 21.6 6 2.6%, p 5 0.25) (Fig. 2). The serum level of nitrite/nitrate was comparable between the men and the women before E2 supplementation (64.9 6 8.7 vs. 57.8 6 6.7 mmol/liter, p 5 0.61). After E2 supplementation, the serum level of nitrite/nitrate was increased in the women (from 64.9 6 8.7 to 93.7 6 9.4 mmol/liter, p , 0.05) but not in the men (from 57.8 6 6.7 to 60.8 6 5.4 mmol/liter, p 5 0.57) (Fig. 3). The effect of E2 supplementation on serum nitrite/nitrate levels in the women was significantly different (p , 0.05) from that in the men. There was no statistical difference in the serum E2 level before E2 supplementation between the men and the women (29.7 6 7.0 vs. 19.7 6 2.8 pg/ml, p 5 0.21). The serum E2 level increased after E2 supplementation in both groups (men, from 29.7 6 7.0 to 88.3 6 6.6 pg/ml, p , 0.01; women, from 19.7 6 2.8 to 94.6 6 9.6 pg/ml, p , 0.01), and there was no difference between the men and the women (p 5 0.59) in the serum level of E2 after E2 supplementation (Fig. 4).
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KAWANO ET AL. GENDER DIFFERENCE IN ENDOTHELIAL FUNCTIONS
Figure 2. Bar graph showing the percent increase in arterial diameter after sublingual nitroglycerin administration in men and women before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE.
Discussion The present study demonstrated that short-term E2 supplementation augmented dilation of the brachial artery during reactive hyperemia after transient occlusion and increased the serum level of nitrite/nitrate in the women but not in the men. The response of arterial diameter to nitroglycerin (an endothelium-independent vasodilator) did not change after E2 supplementation in either the men or the women. Furthermore, the response of arterial diameter to nitrovasodilators has been reported (20,22,25) to be unaffected by the physiologic level of serum estrogens. Coronary risk factors, such as serum lipid profiles and blood pressure, are known to affect flow-mediated arterial dilation (16,17). However, they are unlikely to have modified the present results, because they were not changed, regardless of E2 supplementation, in either
Figure 3. Bar graph showing the level of serum nitrite/nitrate in men and women before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE. *Significant effect of estradiol supplementation compared with values in male subjects (p , 0.05 by ANOVA).
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Figure 4. Bar graph showing the variation in serum E2 concentrations before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE.
the men or the women. Thus, the present results suggest that short-term E2 supplementation may possibly augment flowmediated endothelium-dependent dilation of the brachial arteries in women but not in men. Endothelium-dependent vasodilation has been shown to play a crucial role in the regulation of vascular tone (26 –29). Furthermore, blood flow and shear stress cause production and release of endotheliumderived NO, which plays an important role in flow-mediated endothelium-dependent arterial dilation (28 –31). Estrogens increase calcium-dependent NO synthase activity in vascular endothelium (9). In the present study, the serum level of nitrite/nitrate, stable metabolites of NO (15), was increased after E2 supplementation in the women. This finding is in agreement with a previous study (10) showing that basal release of NO from endothelium-preserved aortic rings of the female rabbits is regulated by ovarian hormones. Furthermore, we (20) previously reported that the flow-mediated endotheliumdependent vasodilation and the serum nitrite/nitrate level varied in parallel with serum estrogen levels during the menstrual cycle in premenopausal healthy women. Both endotheliumdependent vasodilation and serum nitrite/nitrate levels in postmenopausal women are lower than those in the follicular phase in premenopausal women. In the present study, the depressed arterial reactivity was normalized with increasing serum nitrite/nitrate levels when serum E2 levels were increased to follicular levels after E2 supplementation in the postmenopausal women but not in the men. Thus, our present findings strongly suggests that short-term estrogen supplementation causes induction of NO synthase and improves flowmediated endothelium-dependent dilation of the brachial arteries in women. Mechanisms of the gender differences in the improvement of endothelial functions after E2 supplementation. The precise mechanism for the gender differences in the improvement of flow-mediated endothelium-dependent vasodilation after E2 supplementation remains undetermined in the present study. Treatment with various antioxidant agents restores
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endothelium-dependent vasodilation, and estrogens have shown antioxidant activity in experimental studies (32,33). However, in the present study, flow-mediated endotheliumdependent vasodilation of the brachial arteries was increased after 36 h of E2 supplementation in the postmenopausal women but not in the men. Collins et al. (34) reported that short-term administration of E2 modulates coronary artery responses induced by acetylcholine (an endothelium-dependent vasodilator) in women but not in men. These findings of a gender-specific benefit of estrogens suggest that the rapid improvement in endothelial function cannot be accounted for by the proposed receptor-independent actions of estrogens, such as the antioxidant activity reported in experimental animal studies (32,33). Weiner et al. (9) reported that the calcium-dependent NO synthase activity was increased in female pigs but not in male pigs after 5 days of estrogen supplementation. They also reported that, although calciumdependent NO synthase activity was not increased in male pigs after 5 days of estrogen supplementation, it was increased after 10 days of estrogen supplementation. Thus, one possible explanation for these observations is that the number or availability of estrogen receptors is initially too low in most tissues of the male pigs and requires a period of estrogen priming (35). The effect of long-term E2 supplementation on the modulation of arterial tone in men remains unclear from the present study. We need further study to clarify the effect of the long-term E2 supplementation on the modulation of arterial tone in humans. Reactive hyperemia after temporary interruption of the blood flow may result from an interplay between physical (myogenic) and local metabolic factors, including prostaglandins and adenosine (36,37). Increased wall shear stress due to the increase in blood flow results in the production of the endothelium-derived vasodilators, including NO (30,38). We and others (39,40) have shown that after transient arterial occlusion, NO plays a significant role in the duration of hyperemia or flow debt repayment but not in the peak reactive hyperemia. That may be why the increase in peak blood flow before and after E2 supplementation was comparable in the men and the women despite the gender differences in the flow-mediated endothelium-dependent vasodilation. Clinical implications. Endothelium-derived NO inhibits platelet aggregation and development of atherosclerosis (11– 15). Impairment of endothelium-dependent vasodilation has been shown (41– 43) to be related to the pathophysiology of vascular diseases. A previous report (18) showed a close relation in the magnitude of endothelium-dependent vasodilation between the coronary and brachial arteries. Thus, the present study suggests that, in women, E2 supplementation may prevent various cardiovascular diseases such as coronary artery spasm, which has been shown (44,45) to be partly mediated by the dysfunction of endothelium-regulated arterial tone. Differences in endothelial functions between premenopausal and postmenopausal women. The postmenopausal women in this study demonstrated an 8% increase in arterial
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diameter during reactive hyperemia before E2 supplementation, in contrast, despite similar serum E2 levels, the premenopausal women at the menstrual phase in our previous study (20) demonstrated only a 4.9% increase. Because progesterone and other hormones in addition to estrogens vary during the menstrual cycle in premenopausal women, we cannot deny the possibility of effects of the other hormones on the variation of endothelial functions in premenopausal women. In fact, Miller and Vanhoutte (46) reported that progesterone antagonized the stimulatory action of estrogens on the release of endothelium-derived NO in a canine model. Nevertheless, it is not clear from the present study why similar levels of estrogens in women did not result in similar increases in arterial diameter. Conclusions. The present study showed that E2 supplementation may increase NO production/release and improve flow-mediated endothelium-dependent vasodilation in women but not in men. Thus, there may be gender differences in the effects of estrogen therapy on endothelial functions and NO production/release in humans. We thank all the volunteers who participated in this study. We also thank the staff in the Division of Cardiology, Kumamoto University for their help.
References 1. British Heart Foundation Statistics Database. Coronary Heart Disease Statistics, 1992. London: The Prevention Group, 1992. 2. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. N Engl J Med 1987;316:1105–10. 3. Witteman JCM, Grobbee DE, Kok FJ, Hofman A, Valkenburg HA. Increased risk of atherosclerosis in women after the menopause. Br Med J 1989;298:642– 4. 4. Bush TL, Barrett-Connor E, Cowan LD, et al. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation 1987;75:1102–9. 5. Samaan SA, Crawford MH. Estrogen and cardiovascular function after menopause. J Am Coll Cardiol 1995;26:1403–10. 6. Stampfer MJ, Colditz GA, Willett WC, et al. Postmenopausal estrogen therapy and cardiovascular disease. N Engl J Med 1991;325:756 – 62. 7. Smith SC, Blair SN, Criqui MH, et al. Preventing heart attack and death in patients with coronary disease. Circulation 1995;92:2– 4. 8. Kauser K, Rubanyi GM. Gender difference in bioassayable endotheliumderived nitric oxide from isolated rat aortae. Am J Physiol 1994;267: H2311–7. 9. Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci U S A 1994;91:5212– 6. 10. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci U S A 1992;89:11259 – 63. 11. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987;2:1057– 8. 12. Durante W, Kroll MH, Vanhoutte PM, Schafer AI. Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C. Blood 1992;79:110 – 6. 13. Dubey RK, Overbeck HW. Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth. Cell Tissue Res 1994;275:133– 41. 14. Bath PMW, Hassall DG, Gladwin AM, Palmer RMJ, Martin JF. Nitric oxide and prostacyclin. Arterioscler Thromb 1991;11:254 – 60.
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15. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109 – 42. 16. Celermajer DS, Sorensen KE, Gooch VM, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340:1111–5. 17. Celermajer DS, Sorensen KE, Bull C, Robinson J, Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol 1994;24:1468 –74. 18. Anderson TJ, Uehata A, Gerhard MD, et al. Close relation of endothelial function in human coronary and peripheral circulations. J Am Coll Cardiol 1995;26:1235– 41. 19. Corretti MC, Plotnick GD, Vogel RA. Technical aspects of evaluating brachial artery endothelium-dependent vasodilatation using high frequency ultrasound. Am J Physiol 1995;268:H1397– 404. 20. Kawano H, Motoyama T, Kugiyama K, et al. Menstrual cyclic variation of endothelium-dependent vasodilation of the brachial artery: possible role of estrogen and nitric oxide. Proc Assoc Am Physicians 1996;108:473– 80. 21. Pignoli P, Tremoli E, Poli A, Oreste P, Paoletti R. Intimal plus medial thickness of the arterial wall: a direct measurement with ultrasound imaging. Circulation 1986;74:1399 – 406. 22. Gilligan DM, Badar DM, Panza JA, Quyyumi AA, Cannon RO. Acute vascular effects of estrogen in postmenopausal women. Circulation 1994;90: 786 –91. 23. Noma A, Okabe H, Netsu-Nakayama K, Ueno Y, Shinohara H. Improved method for simultaneous determination of cholesterol in high- and lowdensity lipoproteins. Clin Chem 1979;25:1480 –1. 24. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and 15N nitrate in biological fluids. Anal Biochem 1982;126:131– 8. 25. Gilligan DM, Quyyumi AA, Cannon RO, Johnson GB, Schenke WH. Effects of physiological levels of estrogen on coronary vasomotor function in postmenopausal women. Circulation 1994;89:2545–51. 26. Vallance P, Collier J, Moncada S. Effect of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989;28:997–1000. 27. Sinoway LI, Hendrickson C, Davidson WR, Prophet S, Zelis R. Characteristics of flow-mediated brachial artery vasodilation in human subjects. Circ Res 1989;64:32– 42. 28. Nabel EG, Selwyn AP, Ganz P. Large coronary arteries in humans are responsive to changing blood flow: an endothelium-dependent mechanism that fails in patients with atherosclerosis. J Am Coll Cardiol 1990;16:349 –56. 29. Drexler H, Zeiher AM, Wollschla¨ger H, Meinertz T, Just H, Bonzel T. Flow-dependent coronary artery dilatation in humans. Circulation 1989;80: 466 –74. 30. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol 1986;250:H1145–9.
KAWANO ET AL. GENDER DIFFERENCE IN ENDOTHELIAL FUNCTIONS
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31. Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res 1994;74:349 –53. 32. Sugioka JM, Shimosegawa Y, Nakano MM. Estrogens as natural antioxidants of membrane lipid peroxidation. FEBS Lett 1987;210:37–9. 33. Keaney JF, Gaziano JM, Xu A, et al. 17-beta estradiol preserves endothelial vasodilator function and limits low-density lipoprotein oxidation in hypercholesterolemic swine. Circulation 1994;89:2251–9. 34. Collins P, Rosano GMC, Sarrel PM, et al. 17b-Estradiol attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease. Circulation 1995;92:24 –30. 35. Rosser M, Chorich L, Howard E, Zamorano P, Mahesh VB. Changes in rat uterine estrogen receptor messenger ribonucleic acid levels during estrogenand progesterone-induced estrogen receptor depletion and subsequent replenishment. Biol Reprod 1993;48:89 –98. 36. Sparkes HV, Belloni FL. The peripheral circulation: local regulation. Annu Rev Physiol 1978;40:67–92. 37. Carlsson I, Sollevi I, Wennmalm A. The role of myogenic relaxation, adenosine and prostaglandines in human forearm reactive hyperemia. J Physiol (Paris) 1987;389:147– 61. 38. Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 1986;8:37– 44. 39. Yamabe H, Okumura K, Ishizaka H, Tsuchiya T, Yasue H. Role of endothelium-derived nitric oxide in myocardial reactive hyperemia. Am J Physiol 1992;263:H8 –14. 40. Tagawa T, Imaizumi T, Endo T, Shiramoto M, Harasawa Y, Takeshita A. Role of nitric oxide in reactive hyperemia in human forearm vessels. Circulation 1994;90:2285–90. 41. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:27–36. 42. Dzau VJ, Gibbons GH, Cooke JP, Omoigui N. Vascular biology and medicine in the 1990s: scope, concepts, potentials, and perspectives. Circulation 1993;87:705–19. 43. Zeiher AM, Drexler H, Wollschla¨ger H, Just H. Modulation of coronary vasomotor tone in humans: progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 1991;83:391– 401. 44. Kugiyama K, Yasue H, Okumura K, et al. Nitric oxide production is deficient in spasm arteries of patients with coronary spastic angina. Circulation 1996;94:266 –72. 45. Yasue H, Horio Y, Nakamura N, et al. Induction of coronary spasm by acetylcholine in patients with variant angina: possible role of the parasympathetic nervous system in the pathogenesis of coronary artery spasm. Circulation 1986;74:955– 63. 46. Miller VM, Vanhoutte PM. Progesterone and modulation of endotheliumdependent responses in canine coronary arteries. Am J Physiol 1991;261: R1022–7.
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VASCULAR FUNCTION
Gender Difference in Improvement of Endothelium-Dependent Vasodilation After Estrogen Supplementation HIROAKI KAWANO, MD, TAKESHI MOTOYAMA, MD, KIYOTAKA KUGIYAMA, MD, OSAMU HIRASHIMA, MD, MASAMICHI OHGUSHI, MD, HIROMI FUJII, MD, HISAO OGAWA, MD, HIROFUMI YASUE, MD Kumamoto, Japan
Objectives. We examined whether there is a gender difference in the improvement of endothelium-dependent vasodilation after estrogen supplementation. Background. Estrogen therapy reduces the risk of cardiovascular events in postmenopausal women, and the augmented release of endothelium-derived nitric oxide (NO) by estrogens has been suggested to be one of the mechanisms for the cardioprotective effects of estrogen. Methods. With ultrasound technique, we measured the diameter and blood flow of the brachial artery at rest, during reactive hyperemia after transient occlusion and after nitroglycerin administration before and after estradiol supplementation in 15 postmenopausal women (mean 63 years) and in 15 men matched for age and risk factors for atherosclerosis. Results. Estradiol supplementation augmented the flowmediated vasodilation and serum level of nitrite/nitrate (metabo-
lites of NO) in women (respectively, from a mean 6 SEM of 8.0 6 0.6% to 12.9 6 0.6% [p < 0.01 by analysis of variance (ANOVA)] and from 64.9 6 8.7 to 93.7 6 9.4 mmol/liter [p < 0.05 by ANOVA]) but not in men (respectively, from 8.1 6 0.6% to 8.3 6 0.7% and from 57.8 6 6.7 to 60.8 6 5.4 mmol/liter). The increases in blood flow during reactive hyperemia and in diameter after nitroglycerin administration were not affected by estradiol supplementation in either men or women. Conclusions. Estradiol supplementation improves endotheliumdependent vasodilation in women, probably because of augmented NO production/release, but not in men. Thus, there may be gender differences in the effects of estrogen therapy on endothelial functions and NO production/release. (J Am Coll Cardiol 1997;30:914 –9) ©1997 by the American College of Cardiology
Life expectancy is greater in women than in men, in large part because of the lower incidence of cardiovascular death in women (1). Previous studies (2– 4) have suggested that ovarian hormones, especially estrogens, can through their protective action against atherosclerosis, reduce the risk of cardiovascular events in women within the reproductive age group. This protection is lost with the onset of menopause or after surgical castration (2,3,5). Thus, estrogen therapy may have beneficial effects for postmenopausal or castrated women in reducing the risk of cardiovascular events, as reported previously (2,4 – 6), and the American Heart Association consensus panel (7) recommends estrogen replacement therapy for postmenopausal women with coronary artery disease. However, all previous studies reported the beneficial effects of estrogen
supplementation in reducing the risk of cardiovascular events only for women. Therefore, it remains unknown whether estrogen supplementation also reduces the risk of cardiovascular events in men. The precise mechanism of the cardioprotective effect of estrogens on the development of atherosclerosis is unclear at present. However, several reports (8 –10) have shown that estrogens stimulate the release of endothelium-derived nitric oxide (NO). This phenomenon may partly contribute to the cardioprotective action of estrogens with respect to cardiovascular events and the development of atherosclerosis because NO is known to induce coronary dilation and to suppress platelet aggregation and migration of monocytes and smooth muscle cells (11–15). To examine whether there is a gender difference in the improvement of flow-mediated endotheliumdependent vasodilation after estrogen supplementation, we measured the flow-mediated endothelium-dependent vasodilation of brachial arteries in men and women by using an ultrasound technique before and after estradiol (E2) supplementation. We also measured the serum levels of E2 and nitrite/nitrate, metabolites of NO, and compared these levels with the magnitude of endothelium-dependent vasodilation (15).
From the Division of Cardiology, Kumamoto University School of Medicine, Kumamoto, Japan. This study was supported in part by Grant-in-Aid for Scientific Research A 08407019 from the Ministry of Education, Science, Sports and Culture in Japan and the Smoking Research Foundation Grant for Biomedical Research, Tokyo, Japan. Manuscript received December 19, 1996; revised manuscript received May 28, 1997, accepted June 20, 1997. Address for correspondence: Dr. Hirofumi Yasue, Division of Cardiology, Kumamoto University, 1-1-1 Honjo, Kumamoto City 860, Japan. E-mail: [email protected]. ©1997 by the American College of Cardiology Published by Elsevier Science Inc.
0735-1097/97/$17.00 PII S0735-1097(97)00234-9
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Abbreviations and Acronyms ANOVA 5 analysis of variance E2 5 estradiol NO 5 nitric oxide
Methods Study subjects. The study subjects were 15 healthy postmenopausal female volunteers (mean age 63.6 6 2.6 years) and 15 healthy male volunteers matched for age and risk factors for atherosclerosis (mean age 63.1 6 0.7 years). Women were eligible if menopause had occurred at least 1 year previously (mean age at which the women reached menopause 51.2 years [range 47 to 55]). Menopause was confirmed by measuring serum follicular-stimulating hormone levels. No subject had received hormone replacement therapy for $2 months before the study. All subjects were asymptomatic, normotensive, nondiabetic and nonsmoking. None had congestive heart failure or any other serious disease. The subjects’ characteristics are shown in Table 1. All subjects were studied before and after supplementation of 17-beta estradiol (Estraderm TTS, 4 mg, Ciba, Basel, Switzerland) transdermally for 36 h. Written informed consent was obtained from all volunteers before the study. The study was in agreement with the guidelines approved by the ethics committee at our institution. Study design. In this study, flow-mediated vasodilation and nitroglycerin-induced vasodilation of the brachial artery were measured by two skillful examiners who did not know whether their study was done before or after E2 supplementation. We excluded subjects with inadequate scan quality of either brachial artery before the study. All studies were performed in a quiet temperature-controlled room (22° to 23°C). During the study, blood pressure was monitored in the right arm every 2 min by an automated blood pressure recorder. All study subjects were right-handed.
The vasodilator responses in the brachial arteries were measured by ultrasound technique. The validity of the method has been confirmed by our previous studies and others (16 – 20). Briefly, the diameter of the left brachial arteries was measured from B-mode ultrasound images with the use of a 7.5-MHz linear array transducer (SSH-160A ultrasound system, Toshiba Corp., Tokyo, Japan). Flow velocity in the brachial arteries was measured by using a pulsed Doppler signal at a 70° angle to the vessel, with the range gate (1.5 mm) in the center of the artery. The brachial arteries were scanned in the antecubital fossa in longitudinal fashion. Depth and gain settings were optimized at the beginning of the study and were kept constant throughout the recording period. When a satisfactory transducer position was found, the surface of the skin was marked, and the arm remained in the same position throughout the study. The subjects lay quietly for 10 min before the first scan. After baseline measurements of the diameter and flow velocity in the brachial arteries were obtained, a blood pressure cuff placed around the forearm was inflated with a pressure of 250 to 300 mm Hg. After 4.5 min, the cuff was released. The diameter and flow velocity were continuously measured between cuff inflation and after cuff deflation. Thereafter, the subjects lay quietly for 15 min. After confirmation that the diameter and the flow velocity returned to the baseline levels, sublingual nitroglycerin (0.3 mg) was administered, and 3 to 4 min later, the last measurements were made. Corretti et al. (19) reported that, compared with 5 mins of arterial occlusion, 1 and 3 min of arterial occlusion were inadequate to elicit statistically significant vasodilation responses. Furthermore, Celermajer et al. (16,17) reported that 4.5 mins of arterial occlusion was adequate to elicit statistically significant vasodilation responses. Thus, we used 4.5 min of arterial occlusion to evaluate the flow-mediated endotheliumdependent vasodilation. The ultrasound images were recorded on a super-VHS video cassette recorder (BR-S601M, Victor Corp., Tokyo, Japan) and arterial diameters were measured at a fixed dis-
Table 1. Clinical Characteristics of Study Subjects Before and After Estradiol Supplementation Men Before Age (yr) Heart rate (beats/min) Mean blood pressure (mm Hg) Basal arterial diameter (mm) Basal blood flow (ml/min) Increase in blood flow during reactive hyperemia (%) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglyceride (mg/dl)
915
Women After
Before
After
63.1 6 0.7 62.0 6 2.0 63.4 6 2.1 105.3 6 1.9 107.1 6 2.1 4.04 6 0.15* 4.06 6 0.16* 279.3 6 30.1* 280.5 6 28.7* 241.5 6 29.9 245.6 6 28.8
63.6 6 2.6 63.8 6 1.7 64.1 6 2.3 106.7 6 2.3 107.5 6 2.5 3.21 6 0.13 3.20 6 0.14 165.2 6 19.1 167.3 6 18.7 249.5 6 27.5 247.6 6 29.3
155.7 6 6.7 46.2 6 1.8 97.2 6 6.2 58.7 6 8.4
156.8 6 6.0 48.3 6 2.8 96.4 6 5.7 59.1 6 7.1
157.3 6 7.0 45.6 6 2.0 98.1 6 5.9 57.2 6 7.9
155.9 6 6.5 47.1 6 2.1 97.0 6 6.1 58.4 6 6.9
*p , 0.01 versus before and after estradiol supplementation in women. Data are expressed as mean value 6 SE. HDL 5 high density lipoprotein; LDL 5 low density lipoprotein.
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tance from an anatomic marker with ultrasound calipers by two independent observers who did not know whether the study subject was male or female or whether the study was done before or after E2 supplementation. Measurements were taken from the anterior to the posterior interface between the media and adventitia (“m” line) at end-diastole, incident with the R wave on a continuously recorded electrocardiogram (16,17,19 – 21). The diameter at four cardiac cycles was analyzed for each scan, and the measurements were averaged. The diameter measurements for reactive hyperemia were taken 45 to 90 s after cuff deflation to measure peak diameter (19). The responses of the vessel diameters to reactive hyperemia and nitroglycerin were expressed as a percent increase of the baseline value of the diameter. Blood flow was calculated by multiplying the velocity-time integral of the Doppler flow signal by heart rate and the vessel cross-sectional area. The percent of increase in brachial blood flow observed immediately after cuff deflation was calculated as the maximal flow recorded within the 1st 15 s after cuff deflation divided by the flow at baseline (16,17,19,20). In our studies, the repeated measurements of rest arterial diameter had an interobserver variability of 0.05 6 0.02 mm, and an intraobserver variability of 0.02 6 0.02 mm. When these studies were performed at the same time on 2 separate days in 20 control subjects, the between-occasions within-patient difference for the measurements of arterial diameter during reactive hyperemia was 0.05 6 0.04 mm. Blood sampling and assays. Blood samples were obtained from all study subjects on the study day while the subjects were in the fasting state. Serum E2 concentrations were measured by a specific immunoradiometric assay for E2 (Diagnostic Products Corp.) (20,22). Serum total cholesterol and triglyceride concentrations were measured enzymatically, and serum high density lipoprotein (HDL)-cholesterol concentration was measured by heparin-Ca21/Ni21 precipitation (23). Nitrite/ nitrate, stable metabolites of NO, were measured with an autoanalyzer (flow injection analyzer, TCI-NOX1000, Tokyo Kasei Kogyo, Tokyo, Japan) that used a method based on the Griess reaction (15,20,24). Briefly, the samples were passed through a column containing copper-coated cadmium, which reduced all nitrate to nitrite. The nitrite then was detected when it reacted with Griess reagent (24). Absorbance was measured at 540 nm by spectrophotometer. Statistical analysis. The changes in variables were assessed by two-way analysis of variance with repeated measures followed by post hoc testing with the Scheffe´ test. Statistical significance was defined as p , 0.05.
Results All study subjects tolerated the studies well. E2 supplementation did not make any difference in heart rate, mean blood pressure, lipid profile or percent increase in blood flow during reactive hyperemia in either the men or the women (Table 1).
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Figure 1. Bar graph showing the percent increase in arterial diameter during reactive hyperemia in men and women before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE. *Significant effect of estradiol supplementation compared with values in male subjects (p , 0.01 by ANOVA).
The percent increase in arterial diameter during reactive hyperemia was comparable in the men and the women before E2 supplementation (8.1 6 0.6% vs. 8.0 6 0.6%, p 5 0.97). After E2 supplementation, the percent increase in arterial diameter during reactive hyperemia was augmented in the women (from 8.0 6 0.6% to 12.9 6 0.6%, p , 0.01) but not in the men (from 8.1 6 0.6% to 8.3 6 0.7%, p 5 0.85) (Fig. 1). The effect of E2 supplementation on arterial dilation during reactive hyperemia in the women was significantly different from that in the men (p , 0.01). The percent increase in arterial diameter after nitroglycerin administration was comparable in the men and the women before and after E2 supplementation (before E2, 19.7 6 2.0% vs. 19.7 6 2.8%, p 5 0.97; after E2, 20.3 6 1.6% vs. 21.6 6 2.6%, p 5 0.60). E2 supplementation did not alter the increase in diameter after nitroglycerin administration in either the men or the women (men, from 19.7 6 2.0% to 20.3 6 1.6%, p 5 0.46; women, from 19.7 6 2.8% to 21.6 6 2.6%, p 5 0.25) (Fig. 2). The serum level of nitrite/nitrate was comparable between the men and the women before E2 supplementation (64.9 6 8.7 vs. 57.8 6 6.7 mmol/liter, p 5 0.61). After E2 supplementation, the serum level of nitrite/nitrate was increased in the women (from 64.9 6 8.7 to 93.7 6 9.4 mmol/liter, p , 0.05) but not in the men (from 57.8 6 6.7 to 60.8 6 5.4 mmol/liter, p 5 0.57) (Fig. 3). The effect of E2 supplementation on serum nitrite/nitrate levels in the women was significantly different (p , 0.05) from that in the men. There was no statistical difference in the serum E2 level before E2 supplementation between the men and the women (29.7 6 7.0 vs. 19.7 6 2.8 pg/ml, p 5 0.21). The serum E2 level increased after E2 supplementation in both groups (men, from 29.7 6 7.0 to 88.3 6 6.6 pg/ml, p , 0.01; women, from 19.7 6 2.8 to 94.6 6 9.6 pg/ml, p , 0.01), and there was no difference between the men and the women (p 5 0.59) in the serum level of E2 after E2 supplementation (Fig. 4).
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Figure 2. Bar graph showing the percent increase in arterial diameter after sublingual nitroglycerin administration in men and women before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE.
Discussion The present study demonstrated that short-term E2 supplementation augmented dilation of the brachial artery during reactive hyperemia after transient occlusion and increased the serum level of nitrite/nitrate in the women but not in the men. The response of arterial diameter to nitroglycerin (an endothelium-independent vasodilator) did not change after E2 supplementation in either the men or the women. Furthermore, the response of arterial diameter to nitrovasodilators has been reported (20,22,25) to be unaffected by the physiologic level of serum estrogens. Coronary risk factors, such as serum lipid profiles and blood pressure, are known to affect flow-mediated arterial dilation (16,17). However, they are unlikely to have modified the present results, because they were not changed, regardless of E2 supplementation, in either
Figure 3. Bar graph showing the level of serum nitrite/nitrate in men and women before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE. *Significant effect of estradiol supplementation compared with values in male subjects (p , 0.05 by ANOVA).
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Figure 4. Bar graph showing the variation in serum E2 concentrations before (striped bars) and after (solid bars) E2 supplementation. See text for details. Data are expressed as mean value 6 SE.
the men or the women. Thus, the present results suggest that short-term E2 supplementation may possibly augment flowmediated endothelium-dependent dilation of the brachial arteries in women but not in men. Endothelium-dependent vasodilation has been shown to play a crucial role in the regulation of vascular tone (26 –29). Furthermore, blood flow and shear stress cause production and release of endotheliumderived NO, which plays an important role in flow-mediated endothelium-dependent arterial dilation (28 –31). Estrogens increase calcium-dependent NO synthase activity in vascular endothelium (9). In the present study, the serum level of nitrite/nitrate, stable metabolites of NO (15), was increased after E2 supplementation in the women. This finding is in agreement with a previous study (10) showing that basal release of NO from endothelium-preserved aortic rings of the female rabbits is regulated by ovarian hormones. Furthermore, we (20) previously reported that the flow-mediated endotheliumdependent vasodilation and the serum nitrite/nitrate level varied in parallel with serum estrogen levels during the menstrual cycle in premenopausal healthy women. Both endotheliumdependent vasodilation and serum nitrite/nitrate levels in postmenopausal women are lower than those in the follicular phase in premenopausal women. In the present study, the depressed arterial reactivity was normalized with increasing serum nitrite/nitrate levels when serum E2 levels were increased to follicular levels after E2 supplementation in the postmenopausal women but not in the men. Thus, our present findings strongly suggests that short-term estrogen supplementation causes induction of NO synthase and improves flowmediated endothelium-dependent dilation of the brachial arteries in women. Mechanisms of the gender differences in the improvement of endothelial functions after E2 supplementation. The precise mechanism for the gender differences in the improvement of flow-mediated endothelium-dependent vasodilation after E2 supplementation remains undetermined in the present study. Treatment with various antioxidant agents restores
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endothelium-dependent vasodilation, and estrogens have shown antioxidant activity in experimental studies (32,33). However, in the present study, flow-mediated endotheliumdependent vasodilation of the brachial arteries was increased after 36 h of E2 supplementation in the postmenopausal women but not in the men. Collins et al. (34) reported that short-term administration of E2 modulates coronary artery responses induced by acetylcholine (an endothelium-dependent vasodilator) in women but not in men. These findings of a gender-specific benefit of estrogens suggest that the rapid improvement in endothelial function cannot be accounted for by the proposed receptor-independent actions of estrogens, such as the antioxidant activity reported in experimental animal studies (32,33). Weiner et al. (9) reported that the calcium-dependent NO synthase activity was increased in female pigs but not in male pigs after 5 days of estrogen supplementation. They also reported that, although calciumdependent NO synthase activity was not increased in male pigs after 5 days of estrogen supplementation, it was increased after 10 days of estrogen supplementation. Thus, one possible explanation for these observations is that the number or availability of estrogen receptors is initially too low in most tissues of the male pigs and requires a period of estrogen priming (35). The effect of long-term E2 supplementation on the modulation of arterial tone in men remains unclear from the present study. We need further study to clarify the effect of the long-term E2 supplementation on the modulation of arterial tone in humans. Reactive hyperemia after temporary interruption of the blood flow may result from an interplay between physical (myogenic) and local metabolic factors, including prostaglandins and adenosine (36,37). Increased wall shear stress due to the increase in blood flow results in the production of the endothelium-derived vasodilators, including NO (30,38). We and others (39,40) have shown that after transient arterial occlusion, NO plays a significant role in the duration of hyperemia or flow debt repayment but not in the peak reactive hyperemia. That may be why the increase in peak blood flow before and after E2 supplementation was comparable in the men and the women despite the gender differences in the flow-mediated endothelium-dependent vasodilation. Clinical implications. Endothelium-derived NO inhibits platelet aggregation and development of atherosclerosis (11– 15). Impairment of endothelium-dependent vasodilation has been shown (41– 43) to be related to the pathophysiology of vascular diseases. A previous report (18) showed a close relation in the magnitude of endothelium-dependent vasodilation between the coronary and brachial arteries. Thus, the present study suggests that, in women, E2 supplementation may prevent various cardiovascular diseases such as coronary artery spasm, which has been shown (44,45) to be partly mediated by the dysfunction of endothelium-regulated arterial tone. Differences in endothelial functions between premenopausal and postmenopausal women. The postmenopausal women in this study demonstrated an 8% increase in arterial
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diameter during reactive hyperemia before E2 supplementation, in contrast, despite similar serum E2 levels, the premenopausal women at the menstrual phase in our previous study (20) demonstrated only a 4.9% increase. Because progesterone and other hormones in addition to estrogens vary during the menstrual cycle in premenopausal women, we cannot deny the possibility of effects of the other hormones on the variation of endothelial functions in premenopausal women. In fact, Miller and Vanhoutte (46) reported that progesterone antagonized the stimulatory action of estrogens on the release of endothelium-derived NO in a canine model. Nevertheless, it is not clear from the present study why similar levels of estrogens in women did not result in similar increases in arterial diameter. Conclusions. The present study showed that E2 supplementation may increase NO production/release and improve flow-mediated endothelium-dependent vasodilation in women but not in men. Thus, there may be gender differences in the effects of estrogen therapy on endothelial functions and NO production/release in humans. We thank all the volunteers who participated in this study. We also thank the staff in the Division of Cardiology, Kumamoto University for their help.
References 1. British Heart Foundation Statistics Database. Coronary Heart Disease Statistics, 1992. London: The Prevention Group, 1992. 2. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. N Engl J Med 1987;316:1105–10. 3. Witteman JCM, Grobbee DE, Kok FJ, Hofman A, Valkenburg HA. Increased risk of atherosclerosis in women after the menopause. Br Med J 1989;298:642– 4. 4. Bush TL, Barrett-Connor E, Cowan LD, et al. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation 1987;75:1102–9. 5. Samaan SA, Crawford MH. Estrogen and cardiovascular function after menopause. J Am Coll Cardiol 1995;26:1403–10. 6. Stampfer MJ, Colditz GA, Willett WC, et al. Postmenopausal estrogen therapy and cardiovascular disease. N Engl J Med 1991;325:756 – 62. 7. Smith SC, Blair SN, Criqui MH, et al. Preventing heart attack and death in patients with coronary disease. Circulation 1995;92:2– 4. 8. Kauser K, Rubanyi GM. Gender difference in bioassayable endotheliumderived nitric oxide from isolated rat aortae. Am J Physiol 1994;267: H2311–7. 9. Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci U S A 1994;91:5212– 6. 10. Hayashi T, Fukuto JM, Ignarro LJ, Chaudhuri G. Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: implications for atherosclerosis. Proc Natl Acad Sci U S A 1992;89:11259 – 63. 11. Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987;2:1057– 8. 12. Durante W, Kroll MH, Vanhoutte PM, Schafer AI. Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C. Blood 1992;79:110 – 6. 13. Dubey RK, Overbeck HW. Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth. Cell Tissue Res 1994;275:133– 41. 14. Bath PMW, Hassall DG, Gladwin AM, Palmer RMJ, Martin JF. Nitric oxide and prostacyclin. Arterioscler Thromb 1991;11:254 – 60.
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15. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43:109 – 42. 16. Celermajer DS, Sorensen KE, Gooch VM, et al. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340:1111–5. 17. Celermajer DS, Sorensen KE, Bull C, Robinson J, Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol 1994;24:1468 –74. 18. Anderson TJ, Uehata A, Gerhard MD, et al. Close relation of endothelial function in human coronary and peripheral circulations. J Am Coll Cardiol 1995;26:1235– 41. 19. Corretti MC, Plotnick GD, Vogel RA. Technical aspects of evaluating brachial artery endothelium-dependent vasodilatation using high frequency ultrasound. Am J Physiol 1995;268:H1397– 404. 20. Kawano H, Motoyama T, Kugiyama K, et al. Menstrual cyclic variation of endothelium-dependent vasodilation of the brachial artery: possible role of estrogen and nitric oxide. Proc Assoc Am Physicians 1996;108:473– 80. 21. Pignoli P, Tremoli E, Poli A, Oreste P, Paoletti R. Intimal plus medial thickness of the arterial wall: a direct measurement with ultrasound imaging. Circulation 1986;74:1399 – 406. 22. Gilligan DM, Badar DM, Panza JA, Quyyumi AA, Cannon RO. Acute vascular effects of estrogen in postmenopausal women. Circulation 1994;90: 786 –91. 23. Noma A, Okabe H, Netsu-Nakayama K, Ueno Y, Shinohara H. Improved method for simultaneous determination of cholesterol in high- and lowdensity lipoproteins. Clin Chem 1979;25:1480 –1. 24. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and 15N nitrate in biological fluids. Anal Biochem 1982;126:131– 8. 25. Gilligan DM, Quyyumi AA, Cannon RO, Johnson GB, Schenke WH. Effects of physiological levels of estrogen on coronary vasomotor function in postmenopausal women. Circulation 1994;89:2545–51. 26. Vallance P, Collier J, Moncada S. Effect of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989;28:997–1000. 27. Sinoway LI, Hendrickson C, Davidson WR, Prophet S, Zelis R. Characteristics of flow-mediated brachial artery vasodilation in human subjects. Circ Res 1989;64:32– 42. 28. Nabel EG, Selwyn AP, Ganz P. Large coronary arteries in humans are responsive to changing blood flow: an endothelium-dependent mechanism that fails in patients with atherosclerosis. J Am Coll Cardiol 1990;16:349 –56. 29. Drexler H, Zeiher AM, Wollschla¨ger H, Meinertz T, Just H, Bonzel T. Flow-dependent coronary artery dilatation in humans. Circulation 1989;80: 466 –74. 30. Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endothelium-derived relaxing factor. Am J Physiol 1986;250:H1145–9.
KAWANO ET AL. GENDER DIFFERENCE IN ENDOTHELIAL FUNCTIONS
919
31. Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res 1994;74:349 –53. 32. Sugioka JM, Shimosegawa Y, Nakano MM. Estrogens as natural antioxidants of membrane lipid peroxidation. FEBS Lett 1987;210:37–9. 33. Keaney JF, Gaziano JM, Xu A, et al. 17-beta estradiol preserves endothelial vasodilator function and limits low-density lipoprotein oxidation in hypercholesterolemic swine. Circulation 1994;89:2251–9. 34. Collins P, Rosano GMC, Sarrel PM, et al. 17b-Estradiol attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease. Circulation 1995;92:24 –30. 35. Rosser M, Chorich L, Howard E, Zamorano P, Mahesh VB. Changes in rat uterine estrogen receptor messenger ribonucleic acid levels during estrogenand progesterone-induced estrogen receptor depletion and subsequent replenishment. Biol Reprod 1993;48:89 –98. 36. Sparkes HV, Belloni FL. The peripheral circulation: local regulation. Annu Rev Physiol 1978;40:67–92. 37. Carlsson I, Sollevi I, Wennmalm A. The role of myogenic relaxation, adenosine and prostaglandines in human forearm reactive hyperemia. J Physiol (Paris) 1987;389:147– 61. 38. Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 1986;8:37– 44. 39. Yamabe H, Okumura K, Ishizaka H, Tsuchiya T, Yasue H. Role of endothelium-derived nitric oxide in myocardial reactive hyperemia. Am J Physiol 1992;263:H8 –14. 40. Tagawa T, Imaizumi T, Endo T, Shiramoto M, Harasawa Y, Takeshita A. Role of nitric oxide in reactive hyperemia in human forearm vessels. Circulation 1994;90:2285–90. 41. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med 1990;323:27–36. 42. Dzau VJ, Gibbons GH, Cooke JP, Omoigui N. Vascular biology and medicine in the 1990s: scope, concepts, potentials, and perspectives. Circulation 1993;87:705–19. 43. Zeiher AM, Drexler H, Wollschla¨ger H, Just H. Modulation of coronary vasomotor tone in humans: progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 1991;83:391– 401. 44. Kugiyama K, Yasue H, Okumura K, et al. Nitric oxide production is deficient in spasm arteries of patients with coronary spastic angina. Circulation 1996;94:266 –72. 45. Yasue H, Horio Y, Nakamura N, et al. Induction of coronary spasm by acetylcholine in patients with variant angina: possible role of the parasympathetic nervous system in the pathogenesis of coronary artery spasm. Circulation 1986;74:955– 63. 46. Miller VM, Vanhoutte PM. Progesterone and modulation of endotheliumdependent responses in canine coronary arteries. Am J Physiol 1991;261: R1022–7.