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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 10 4634-4641
Copyright © 2002 by The Endocrine Society

Original Article

Hormone Replacement Effects on Endothelial Function Measured in the Forearm Resistance Artery in Normocholesterolemic and Hypercholesterolemic Postmenopausal Women

Mitsuhiro Sanada, Yukihito Higashi, Keigo Nakagawa, Mikio Tsuda, Ichiro Kodama, Masashi Kimura, Kazuaki Chayama and Koso Ohama

Department of Obstetrics and Gynecology (M.S., M.T., I.K., K.O.) and First Department of Internal Medicine (Y.H., K.N., M.K., K.C.), Faculty of Medicine, Hiroshima University, Hiroshima 734-8551, Japan

Address all correspondence and requests for reprints to: Mitsuhiro Sanada, M.D., Ph.D., Department of Obstetrics and Gynecology, Faculty of Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail: msanada64{at}hotmail.com.

Abstract

We investigated whether forearm resistance artery endothelial function differed between hypercholesterolemic postmenopausal women (n = 41) and normocholesterolemic postmenopausal women (n = 37), both generally and in terms of effects of long-term hormone replacement therapy (HRT) on endothelial function. Both menopause and hypercholesterolemia are associated with endothelial dysfunction and increased coronary risk. Forearm blood flow (FBF) during reactive hyperemia and after sublingual nitroglycerin (NTG) administration was measured by strain-gauge plethysmography. Treated women received conjugated equine estrogen (0.625 mg) plus medroxyprogesterone acetate (2.5 mg) daily for 6 months. Nitrite/nitrate, angiotensin-converting enzyme, and lipids were measured in serum. FBF during reactive hyperemia as well as serum nitrite/nitrate concentrations were significantly lower in hypercholesterolemic than normocholesterolemic subjects. Increases in the FBF induced by NTG were similar in the two groups. HRT significantly increased estradiol, high-density lipoprotein cholesterol, and serum nitrite/nitrate, while decreasing circulating angiotensin-converting enzyme activity in both groups. Reduction in total and low-density lipoprotein cholesterol was seen only in hypercholesterolemic subjects. After 6 months of HRT, maximal FBF response during reactive hyperemia increased in both groups. Augmentation of this response was greater in hypercholesterolemic than in normocholesterolemic subjects (maximal FBF, 55.4 ± 11.2 vs. 25.9 ± 11.5%; P < 0.05). Changes in the FBF with NTG were not altered by HRT in either group. Long-term HRT augments endothelial function in forearm resistance artery. This beneficial effect is greater in patients with hypercholesterolemia.

ALTHOUGH LOW IN premenopausal women, the incidence of atherosclerotic disease rises after menopause but can be reduced by hormone replacement therapy (HRT) including various estrogen preparations with or without progestin (1, 2, 3, 4). Such a benefit was attributed to various effects of estrogens, including lipid-lowering and antioxidant properties (5, 6) as well as inhibition of fibrosis (7). On the other hand, a well controlled clinical trial, the Heart and Estrogen/Progestin Replacement Study (HERS), failed to show a reduction in coronary heart disease (CHD) in postmenopausal women with established coronary disease who received estrogen combined with progestin (8). Nonetheless, reports indicate that estrogen has a direct effect on the vessel wall and favorably influences the physiology of the endothelium and smooth muscle; in some studies, transdermally or orally administered estrogen augmented endothelium-dependent vasodilation in brachial and coronary arteries in postmenopausal women (9, 10, 11, 12).

Hypercholesterolemia is a risk factor for atherosclerosis and cardiovascular events, whereas cholesterol-lowering therapy is associated with a decrease in cardiac morbidity and mortality (13). Hypercholesterolemic patients appear to have a defect in bioavailability of NO that may explain impaired endothelium-dependent vasodilation, and increasing evidence suggests central involvement of the L-arginine/NO pathway in the pathogenesis of atherosclerosis in hypercholesterolemia (14).

After menopause, lipoprotein profiles in women become more atherogenic, including higher serum concentrations of total and low-density lipoprotein (LDL) cholesterol (15, 16). However, these postmenopausal changes do not attain hypercholesterolemic levels in all women. We previously reported that HRT might have a greater cholesterol-lowering effect in hypercholesterolemic than in normocholesterolemic postmenopausal women (17). However, little is known as to whether the effects of HRT on forearm resistance artery endothelial function differ between these groups.

We therefore measured forearm blood flow (FBF) during reactive hyperemia (an index of endothelium-dependent vasodilation) and in response to nitroglycerin (NTG; an index of endothelium-independent vasodilation) in hypercholesterolemic and normocholesterolemic postmenopausal subjects before and after HRT.

Subjects and Methods

Study protocol 1: cholesterol status and forearm resistance artery endothelial function

We studied 37 Japanese postmenopausal women who were normocholesterolemic and 41 who were hypercholesterolemic as defined by the Japanese Atherosclerosis Society (total cholesterol, 5.69 mmol/liter or higher; LDL cholesterol, 3.62 mmol/liter or higher; Ref. 18). Age ranges in the respective groups were 48–57 yr and 47–57 yr. Each subject had experienced natural menopause for at least 1 yr but not longer than 10 yr. Each was of normal weight, with a body mass index (weight/height2, kg/m2) no greater than 25. Menopausal status was confirmed by a serum FSH concentration greater than 40 IU/liter and a serum estradiol concentration less than 73.4 pmol/liter. Excluded from the study were patients with hypertension or diabetes, cigarette smokers, those with clinical manifestations of atherosclerosis (CHD, peripheral artery disease, cerebrovascular disease), venous thromboembolism, liver disorders, unexplained vaginal bleeding, and a personal or family history of breast cancer. Before being enrolled in the study, each subject underwent a physical examination, including gynecological evaluation and mammography. None had received HRT, other steroid hormones, or any medication known to affect lipoprotein metabolism or blood pressure. The ethics committee of the Department of Obstetrics and Gynecology of Hiroshima University approved the study protocol. Informed consent for participation was obtained from each subject.

The vasodilator response to reactive hyperemia, an index of endothelium-dependent vasodilation, and to the sublingual administration of NTG, an index of endothelium-independent vasodilation, was evaluated in each subject at baseline and again after 3 and 6 months. This evaluation began at 0830 h. Each subject had fasted the previous night for at least 14 h. After overnight fasting, they rested supine in a quiet, air-conditioned room (constant temperature, 22–25 C). After the subject had rested for 30 min in that position, the basal FBF was measured as described below. Next, the effects of reactive hyperemia and of the sublingual administration of NTG on the FBF were evaluated by inflating a cuff over the left upper arm to 280 mm Hg for 5 min. After the cuff occlusion was released, the FBF was measured for 3 min. Next, a NTG tablet (0.3 mg) (Nihonkayaku Co., Tokyo, Japan) was administered sublingually, and the FBF was again measured for 3 min. These studies were performed in a randomized fashion. Each study proceeded after FBF had returned to baseline. In the preliminary study, we confirmed the reproducibility of the FBF response to reactive hyperemia and sublingual NTG on two separate occasions in 28 healthy male subjects (mean age, 27 ± 5 yr). The coefficients of variation were 4.3% and 2.8%, respectively.

The baseline fasting serum concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, creatinine, glucose, electrolytes, nitrite/nitrate, plasma rennin activity (PRA) and angiotensin-converting enzyme (ACE) activity were obtained after a 30-min rest period. The body weight, blood pressure, and heart rate were determined at baseline and again after 3 and 6 months of treatment.

Study protocol 2: effect of HRT on forearm resistance artery endothelial function

Twenty-four of the normocholesterolemic subjects and 26 of the hypercholesterolemic subjects who wished to undergo HRT received 0.625 mg conjugated equine estrogen (Premarin; Wyeth-Ayerst Laboratories, Philadelphia, PA) plus 2.5 mg medroxyprogesterone acetate (Provera, Pharmacia \|[amp ]\| Upjohn, Inc., Peapack, NJ). Six of the normocholesterolemic subjects and seven of the hypercholesterolemic subjects continued 6-month follow-up, not taking HRT. Medication was taken every morning for 6 months. All participants were followed for 6 months. Each subject was asked to avoid making any changes in lifestyle or dietary habits during the study. Vasodilatory responses to reactive hyperemia and sublingual NTG were evaluated using a protocol identical to that used in study protocol 1 just before beginning treatment and again after 3 and 6 months. A physical examination, including gynecological evaluation and mammography, was performed at the conclusion of the study.

Measurement of FBF

FBF was measured with a mercury-filled Silastic strain-gauge plethysmograph (EC-5R, D. E. Hokanson, Inc., Issaquah, WA) as previously described (19, 20). The intraobserver coefficient of variation was 3.0 ± 1.6%. Four plethysmographic measurements were averaged to obtain the FBF at baseline, during reactive hyperemia, and after the administration of sublingual NTG.

Analytical methods

Samples of venous blood were placed in polystyrene tubes containing sodium EDTA (1 mg/ml). The EDTA-containing tubes were immediately chilled in an ice bath. Next, the plasma was separated by centrifugation at 3100 rpm at 4 C for 10 min. Serum was separated at 1000 rpm at room temperature for 10 min. Samples were stored at -80 C until assayed. Routine chemical methods were used to determine the serum concentrations of total cholesterol, HDL cholesterol, triglycerides, creatinine, glucose, and electrolytes. The serum concentration of LDL cholesterol was determined by Freidewald’s method (21). Serum concentrations of FSH and estradiol were analyzed by a RIA. Nitrite/nitrate concentrations were measured with an autoanalyzer (flow injection analyzer, TCI-NOX1000, Tokyo Kasei Kogyo, Tokyo, Japan), which uses a protocol based on the Griess reaction (22). Serum ACE activity (in international units per liter at 37 C) was measured with ACE color (Fujirebio Co., Ltd., Tokyo, Japan). PRA was assayed by RIA.

Statistical analysis

Results are presented as the mean ± SEM. The Mann-Whitney U test was used to compare baseline clinical parameters in the normocholesterolemic and hypercholesterolemic groups. Comparisons of parameters before and after HRT were performed for adjusted means by analyses of covariance with the baseline data used as covariates. Time-course curves of the FBF during reactive hyperemia were compared by two-way ANOVA for repeated measures. Univariate linear regression analysis was used to evaluate the relationship between serum nitrite/nitrate concentrations and maximal FBF during reactive hyperemia in hypercholesterolemic subjects. Comparisons of variables, including maximal FBF during reactive hyperemia or maximal FBF response to NTG, were performed by one-way ANOVA, followed by the Bonferroni correction. A P level less than 0.05 was accepted as statistically significant. Data were processed using the software package Statview IV (Brainpower), or Super ANOVA (Abacus Concepts, Berkeley, CA).

Results

Study protocol 1: cholesterol status and effects of reactive hyperemia and sublingual NTG on FBF

Baseline clinical variables in normocholesterolemic and hypercholesterolemic groups are summarized in Table 1Go. Serum total cholesterol, LDL cholesterol, and apolipoprotein B concentrations were significantly higher in the hypercholesterolemic group than in the normocholesterolemic group. Serum nitrite/nitrate concentrations were significantly lower in hypercholesterolemic than in normocholesterolemic subjects. Other parameters were similar in the two groups. The FBF during reactive hyperemia in hypercholesterolemic subjects was significantly lower than in normocholesterolemic subjects (Fig. 1Go). In contrast, the increase in the FBF after sublingual administration of NTG was similar in the two groups (Fig. 2Go). Maximal FBF response to reactive hyperemia correlated positively with serum nitrite/nitrate concentrations in the hypercholesterolemic group (r = 0.39; P < 0.05; Fig. 3Go).


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  		Table 1. Clinical variables in postmenopausal normocholesterolemic and hypercholesterolemic women

 


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  		Figure 1. Time curves show FBF at rest and during reactive hyperemia in normocholesterolemic (n = 37) and hypercholesterolemic (n = 41) postmenopausal women. Reactive hyperemia was relatively impaired in the latter group. Results are presented as mean ± SEM.

 


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  		Figure 2. The bar graph presents maximal FBF after sublingual administration of NTG in normocholesterolemic (n = 37) and hypercholesterolemic (n = 41) postmenopausal women. NTG-induced vasodilation was similar between the two groups. Results are presented as mean ± SEM.

 


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  		Figure 3. The scatter plot depicts the relationship between serum nitrite/nitrate concentrations and maximal FBF during reactive hyperemia in hypercholesterolemic subjects (n = 41). Maximal FBF response to reactive hyperemia correlated significantly with serum nitrite/nitrate concentrations in this group.

 
Study protocol 2: effect of HRT on baseline clinical variables

The effect of a 6-month course of HRT was compared in the normocholesterolemic and hypercholesterolemic (Table 2Go) subjects. Before HRT, serum total and LDL cholesterol and apolipoprotein B concentrations were significantly higher in the hypercholesterolemic than in the normocholesterolemic group. Other parameters were similar in the four groups. Significant decreases in serum total and LDL cholesterol and apolipoprotein B concentrations were seen after HRT only in the hypercholesterolemic group. Significant and similar increases in serum HDL cholesterol, apolipoprotein AI, and estradiol concentrations as well as significant decreases in ACE activity, were found in both groups. Although significant increases in serum nitrite/nitrate concentrations and PRA after 6 months of HRT were found in both groups, changes in these parameters were greater in hypercholesterolemic than in normocholesterolemic subjects (nitrite/nitrate, 44.4 ± 3.8 vs. 19.3 ± 3.1%; PRA, 57.8 ± 7.4 vs. 29.7 ± 6.2%; P < 0.05). In contrast, these parameters were unchanged in the control groups. Blood pressure, heart rate, and basal FBF were similar at baseline and after 6 months in all four groups.


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  		Table 2. Clinical variables in postmenopausal normocholesterolemic and hypercholesterolemic women before and after HRT

 
Study protocol 2: effect of HRT on forearm resistance artery endothelial function

Maximal FBF response to reactive hyperemia was lower in the hypercholesterolemic HRT group than in the normocholesterolemic HRT group (P < 0.05; Fig. 4Go). After 6 months of HRT, the maximal FBF response to reactive hyperemia had increased from 29.8 ± 2.1 to 43.9 ± 3.1 ml/min per 100 ml tissue (P < 0.01) in the hypercholesterolemic group and from 37.9 ± 2.8 to 44.5 ± 3.0 ml/min per 100 ml tissue (P < 0.05) in the normocholesterolemic group (Fig. 4Go). Augmentation of the FBF response to reactive hyperemia by HRT was greater in hypercholesterolemic than in normocholesterolemic subjects (maximal FBF, 55.4 ± 11.2 vs. 25.9 ± 11.5%; P < 0.05). In contrast, there were no changes in the control groups (Fig. 4Go). Changes in the FBF after sublingual administration of NTG were similar between measurements taken at baseline and after 6 months in all four groups (Fig. 5Go).



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  		Figure 4. The bar graphs show maximal FBF during reactive hyperemia in HRT groups [normocholesterolemic (n = 24) and hypercholesterolemic (n = 26) women] and control groups [normocholesterolemic (n = 6) and hypercholesterolemic (n = 7) women]. Before treatment, reactive hyperemia in the hypercholesterolemic HRT group was less significant than in the normocholesterolemic HRT group (*, P < 0.05). An HRT-induced increase in FBF response during reactive hyperemia was greater in the hypercholesterolemic than in the normocholesterolemic group. Results are presented as mean ± SEM.

 


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  		Figure 5. The bar graphs depict maximal FBF after sublingual administration of NTG in HRT groups [normocholesterolemic (n = 24) and hypercholesterolemic (n = 26) women] and control groups [normocholesterolemic (n = 6) and hypercholesterolemic (n = 7) women]. Similar changes in FBF were induced by sublingual administration of NTG in the four groups, both before and after 6 months of observation. Results are presented as mean ± SEM.

 
Both treatment groups consistently reported no side effects. Two hypercholesterolemic women showed mild, reversible elevations in serum aminotransferases, and one normocholesterolemic woman had a minor, reversible elevation of creatine kinase. No occurrences of abnormal endometrial histology, including hyperplasia or carcinoma, were detected in either group. No cases of breast cancer were noted during the study.

Discussion

In the present investigation, we found that the FBF response to reactive hyperemia in forearm resistance arteries was more impaired in hypercholesterolemic than in normocholesterolemic postmenopausal women, whereas the FBF response to NTG was similar in the two groups. Although 6 months of HRT augmented the FBF response to reactive hyperemia in both groups, augmentation was significantly greater in the hypercholesterolemic than in the normocholesterolemic group. In contrast, 6 months of HRT did not alter the FBF response to NTG in either group.

Serum cholesterol and forearm resistance artery endothelial function

It is well known that hypercholesterolemia is closely linked with impaired endothelial function. Hypercholesterolemia decreases NO production by promoting interaction between caveolin and endothelial NO synthase as well as by increasing expression of adhesion molecules in endothelium and aggregability and adhesion in platelets (23, 24, 25). Hypercholesterolemia has been suggested to stimulate endothelial generation of superoxide radicals (26). Superoxide directly inactivates NO and may also increase subsequent oxidation of LDL particles by formation of peroxynitrite. In the present study, changes in circulating nitrite/nitrate were used as an index of changes in NO synthesis. Although endogenous NO is thought to be the primary source of circulating nitrite/nitrate, dietary nitrates also must be considered as a possible source. Dietary nitrates are excreted in the urine within 18 h of ingestion (27), and in the present study serum nitrite/nitrate was measured after overnight fasting (approximately 14 h after the most recent meal). Thus, most dietary nitrates would have been eliminated by the time of blood sampling.

We found that serum concentrations of nitrite/nitrate were significantly lower in hypercholesterolemic than in normocholesterolemic subjects, and that these concentrations correlated significantly with the maximal FBF response to reactive hyperemia in hypercholesterolemic women. Our present results showed that hypercholesterolemia in postmenopausal women exacerbated endothelial dysfunction, resulting in decreased production and release of NO.

Effect of HRT on forearm resistance artery endothelial function

Several possible mechanisms could account for augmentation of the FBF response to reactive hyperemia by 6 months of HRT. Reactive hyperemia has been shown to be mediated largely by release of NO (28). Some studies have shown that estrogen directly up-regulates expression of endothelial NO synthase mRNA and protein, resulting in increased endothelial NO synthase activity (29, 30). This estrogen-dependent increase in endogenous NO production could enhance vascular reactivity after HRT in postmenopausal women. Another possible mechanism of enhancement is based on the observation that ACE inhibition resulting from HRT may improve overall endothelial function. We recently reported that inhibition of ACE activity by estrogen therapy might be one basis for an endothelial benefit from estrogen (31, 32). In the present study, HRT decreased serum ACE activity and increased PRA in postmenopausal women. These results are consistent with previous reports (33, 34). The increase in PRA presumably occurs in response to a decrease in angiotensin II, the product of ACE activity. Angiotensin II increases vascular superoxide production through the activation of membrane-associated nicotinamide adenine dinucleotide reduced/nicotinamide adenine dinucleotide phosphate reduced oxidase, resulting in NO inactivation and toxic peroxynitrite production (35). Accordingly, ACE inhibition by HRT may increase NO by inhibiting angiotensin II production. Although we did not directly measure serum angiotensin II concentrations, increased PRA may have been due to an increase in the serum angiotensinogen concentration induced by HRT rather than by a decrease in the angiotensin II concentration. Inhibition of ACE also delays degradation of bradykinin, a substance that increases NO release and increases production of prostacyclin and endothelium-derived hyperpolarizing factor (36). For some or all of these reasons, long-term HRT improved forearm resistance artery endothelial function in our postmenopausal subjects.

We presently found that restoration of forearm resistance artery endothelial function evoked by long-term HRT was greater in hypercholesterolemic women than in normocholesterolemic women. This difference can be explained based on the general observation that the cholesterol-lowering effect of HRT may improve overall endothelial function. Some studies have reported that cholesterol-lowering therapy improved endothelial function in hypercholesterolemic patients through increased bioavailability of NO (37, 38). A reduction in serum cholesterol can reduce excessive oxygen-derived free radical production (39). Oxidized LDL, on the other hand, interferes with the formation of NO and even directly inactivates NO (40). Although increases in HDL cholesterol-induced HRT were similar in both groups, significant reductions in serum total and LDL cholesterol concentrations were seen only in hypercholesterolemic subjects. Indeed, increases in serum nitrite/nitrate concentrations were greater in the hypercholesterolemic group. The greater effect of HRT on vascular reactivity in hypercholesterolemic subjects may reflect this differential cholesterol-lowering effect of HRT. In addition, although serum ACE activity was decreased in both groups, the increase in PRA was greater in hypercholesterolemic than in normocholesterolemic subjects. We suspect that the former group might have particularly reduced angiotensin II concentrations after HRT, resulting in greater FBF response to reactive hyperemia.

In the present study, HRT did not affect blood pressure in either normocholesterolemic or hypercholesterolemic women. Our results are consistent with most studies, which also found no change in blood pressure with HRT (41, 42). However, a small number of studies have reported that HRT does affect blood pressure (43, 44). These discrepancies may be accounted for by differences in the dose or type of estrogen administered.

The recent findings from HERS have challenged previous data regarding the protective effect of HRT in preventing cardiovascular events in women with preexisting cardiovascular disease. The surprising results of the HERS, which found no protective effect against CHD, raised questions about the safety and efficacy of this regimen as prophylactic therapy. In the present study, HRT had beneficial effects on endothelial function in both normocholesterolemic and hypercholesterolemic women, and the grade of improvement in the FBF response to reactive hyperemia was greater in hypercholesterolemic than in normocholesterolemic women. Although this finding provides insight into the mechanism by which HRT may reduce the risk of atherosclerosis, proof of cardiovascular benefit awaits completion of randomized clinical trials such as the Women’s Health Initiative.

Study limitation

The use of agonists to stimulate NO release, such as acetylcholine or bradykinin, as well as NO antagonists would allow us to draw more specific conclusions concerning the role of basal and stimulated NO production mediated by HRT in the forearm circulation. Recently, we have demonstrated that noninvasive methods, such as the measurement of the FBF response to reactive hyperemia, can be used for assessing resistance vessel endothelial function instead of the intra-arterial vasoactive agent infusion method (45). This technique is simple, reproducible, and caused no adverse effects. Although it is thought that NO contributes to the FBF response to reactive hyperemia, we cannot deny the possibility that other factors, including prostaglandin, endothelium-derived hyperpolarizing factor, and adenosine, are involved in estrogen-enhanced hyperemia. There was a large difference between the magnitudes of vasodilatory response to reactive hyperemia, an index of endothelium-dependent vasodilation, and to NTG, an index of endothelium-independent vasodilation. In the present study, the FBF response to reactive hyperemia was about five times greater than to sublingually administered NTG. We cannot obtain a greater response than this NTG-induced vasodilation using our noninvasive method. However, several investigators, including us, have demonstrated that HRT predominately improves endothelium-dependent vasodilation in postmenopausal women. We believe that endothelial cells, but not smooth muscle cells, are selectively restored by HRT.

The present study was not designed as a double-blind randomized placebo study. In addition, the number of subjects in each group was relatively small. Therefore, we cannot exclude the possibility that there is selection bias in the results. Recent data suggest that LDL cholesterol itself is not as injurious as oxidized LDL in producing impaired endothelial function (46). In the present study, we did not assess the particle size or the susceptibility of LDL oxidation, both of which are prone to induce endothelial dysfunction and may have influenced our findings.

In conclusion, the present study showed that augmentation of forearm resistance artery endothelial function induced by long-term HRT was greater in hypercholesterolemic than in normocholesterolemic postmenopausal women. These findings suggest that HRT may have a particularly favorable effect on endothelial function in the former group. The augmentation, which probably involves production of NO, should result in reduced risk of CHD.

Acknowledgments

We thank Hitoshi Nakagawa, M.D., for technical support and Hiromi Muraoka for her secretarial assistance.

Footnotes

This study was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (no. 09771279). Tokyo Kasei Kogyo also supported this study.

Abbreviations: ACE, Angiotensin-converting enzyme; CHD, coronary heart disease; FBF, forearm blood flow; HDL, high-density lipoprotein; HERS, Heart and Estrogen/Progestin Replacement Study; HRT, hormone replacement therapy; LDL, low-density lipoprotein; NTG, nitroglycerin; PRA, plasma rennin activity.

Received July 30, 2001.

Accepted July 3, 2002.

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