Gender Difference in Improvement of Endothelium-Dependent Vasodilation After Estrogen Supplementation

1.1 Study subjects.

The study subjects were 15 healthy postmenopausal female volunteers (mean age 63.6 ± 2.6 years) and 15 healthy male volunteers matched for age and risk factors for atherosclerosis (mean age 63.1 ± 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.

1.2 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 endothelium-dependent 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 distance 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 ± 0.02 mm, and an intraobserver variability of 0.02 ± 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 ± 0.04 mm.

1.3 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-Ca²⁺/Ni²⁺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.

1.4 Statistical analysis.

The changes in variables were assessed by two-way analysis of variance with repeated measures followed by post hoc testing with the Scheffé test. Statistical significance was defined as p < 0.05.

3.1 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 endothelium-dependent vasodilation, and estrogens have shown antioxidant activity in experimental studies [32, 33]. However, in the present study, flow-mediated endothelium-dependent 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 calcium-dependent 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.

3.2 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.

3.3 Differences in endothelial functions between premenopausal and postmenopausal women.

The postmenopausal women in this study demonstrated an 8% increase in arterial 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.

3.4 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.