Effects of the Menopause, Gender, and Estrogen Replacement Therapy on Vascular Nitric Oxide Activity

The Journal of Clinical Endocrinology & Metabolism, Apr 2000

Changes in vascular nitric oxide (NO) activity may contribute to cardiovascular risk. We determined the effect of the menopause, gender, and estrogen replacement therapy on arterial vascular NO activity. Vascular NO activity and sensitivity were determined in 15 healthy premenopausal women (mean age, 48 yr), 12 postmenopausal women (51 yr), and 14 men (51 yr). The effects of 14 days of estrogen replacement therapy (625 μg conjugated estrogens) were studied in 20 healthy postmenopausal women (60 yr). Forearm blood flow responses to brachial arterial infusions of l-NG-monomethyl-arginine (L-NMMA), norepinephrine, glyceryl trinitrate (GTN), and serotonin were determined using venous occlusion plethysmography. Constrictor responses to L-NMMA were reduced in postmenopausal women (82 ± 14, summary response, mean ± sem) and men (89 ± 6) compared to premenopausal women (118 ± 10; P < 0.05). Constrictor responses to norepinephrine were increased in males (125 ± 13) compared to premenopausal (81 ± 8) and postmenopausal (88 ± 16) women (P < 0.05). No differences were observed in GTN or serotonin responsiveness. Constrictor responses to L-NMMA increased after estrogen replacement (132 ± 7 vs. 89 ± 14; P < 0.05), with no change in norepinephrine, GTN, or serotonin responses. The menopause and male gender were associated with reduced arterial NO activity. Two weeks of estrogen replacement therapy restored vascular NO activity to premenopausal levels. Changes in vascular NO activity may contribute to changes in cardiovascular risk associated with male gender, postmenopausal status, and estrogen replacement therapy. Increased α-adrenoceptor responsiveness may contribute to increased cardiovascular risk in males.

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Effects of the Menopause, Gender, and Estrogen Replacement Therapy on Vascular Nitric Oxide Activity

0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society Effects of the Menopause, Gender, and Estrogen Replacement Therapy on Vascular Nitric Oxide Activity N. G. MAJMUDAR 0 S. C. ROBSON 0 G. A. FORD 0 0 Departments of Pharmacological Sciences (N. G.M., G.A.F.), Obstetrics (S.C.R.), and Medicine (N.G.M., G.A.F.) , University of Newcastle Upon Tyne , NE2 4HH Newcastle Upon Tyne , United Kingdom Changes in vascular nitric oxide (NO) activity may contribute to cardiovascular risk. We determined the effect of the menopause, gender, and estrogen replacement therapy on arterial vascular NO activity. Vascular NO activity and sensitivity were determined in 15 healthy premenopausal women (mean age, 48 yr), 12 postmenopausal women (51 yr), and 14 men (51 yr). The effects of 14 days of estrogen replacement therapy (625 mg conjugated estrogens) were studied in 20 healthy postmenopausal women (60 yr). Forearm blood flow responses to brachial arterial infusions of L-NG-monomethyl-arginine (L-NMMA), norepinephrine, glyceryl trinitrate (GTN), and serotonin were determined using venous occlusion plethysmography. Constrictor responses to L-NMMA were reduced in postmenopausal women (82 6 14, summary response, mean 6 SEM) and men (89 6 6) compared to premenopausal women (118 6 10; P , 0.05). Constrictor responses - ARDIOVASCULAR disease is the leading cause of death in women in the developed world. Premenopausal women in particular have significantly reduced risk compared to men, although the incidence increases greatly after the menopause ( 1 ). Women who experience a surgical menopause without estrogen replacement have twice the risk of coronary heart disease compared with their premenopausal peers ( 2 ). In women undergoing natural menopause, each years of delay in age of onset of the menopause reduces cardiovascular mortality risk by 2% ( 3 ). The increased risk is thought to be due to withdrawal of estrogenic effects. Epidemiological studies suggest that estrogen replacement in postmenopausal women is associated with a 50% reduction in the incidence of cardiac events ( 4 ). However, the recent Heart and Estrogen/Progestin Study found no benefit from 4 yr of treatment with conjugated equine estrogens and medroxyprogesterone acetate in postmenopausal women with established coronary disease, and the results of other ongoing randomized controlled trials are awaited ( 5 ). Estrogen has several actions that may contribute to a cardioprotective effect: lowering of total and low density lipoprotein cholesterol, elevation of high density lipoprotein cholesterol ( 6 ), and reductions in fibrinogen and factor VII ( 7 ). However, lipid changes only appear to account for 25% of the protective effect ( 8 ). Nitric oxide (NO), synthesized by the vascular endothelium, plays a major physiological role in to norepinephrine were increased in males (125 6 13) compared to premenopausal (81 6 8) and postmenopausal (88 6 16) women (P , 0.05). No differences were observed in GTN or serotonin responsiveness. Constrictor responses to L-NMMA increased after estrogen replacement (132 6 7 vs. 89 6 14; P , 0.05), with no change in norepinephrine, GTN, or serotonin responses. The menopause and male gender were associated with reduced arterial NO activity. Two weeks of estrogen replacement therapy restored vascular NO activity to premenopausal levels. Changes in vascular NO activity may contribute to changes in cardiovascular risk associated with male gender, postmenopausal status, and estrogen replacement therapy. Increased a-adrenoceptor responsiveness may contribute to increased cardiovascular risk in males. (J Clin Endocrinol Metab 85: 1577–1583, 2000) maintaining basal vascular arterial tone, inhibiting platelet aggregation and adhesion ( 9 ), inhibiting the development of atherosclerotic plaques ( 10 ), and inhibiting vascular smooth muscle proliferation ( 11 ). Some studies suggest that estrogen replacement may increase stimulated NO release in the arterial vasculature of postmenopausal women. Estrogen replacement has been shown to augment endothelial dependent flow-mediated vasodilatation to reactive hyperemia in the brachial artery ( 12 ), and brachial artery infusion of 17bestradiol increases endothelium-dependent vasodilatation to acetylcholine ( 13 ). Changes in vascular NO activity may be an important mechanism mediating the detrimental effect of the menopause and male gender on cardiovascular mortality and the benefits of estrogen and premenopausal status in reducing cardiovascular risk. However, no data exist on vascular responses in age-matched pre- and postmenopausal women or on gender effects in middle age. The aims of the present study were to determine the effects of menopausal status, gender, and estrogen replacement on arterial NO activity, NO sensitivity, and stimulated NO release. Subjects and Methods Subjects For the menopause and gender studies, healthy female and male subjects, aged 45–55 yr, were studied: 15 regularly menstruating women (mean 6 sd age, 48 6 2 yr) with serum FSH levels below 30 IU/L, 12 postmenopausal women (51 6 3 yr) with at least a 12-months history of amenorrhea and serum FSH levels above 30 IU/L, and 14 male subjects (51 6 3 yr). The effects of estrogen replacement were studied in a separate group of 20 healthy postmenopausal subjects, aged 45– 65 yr or otherwise described as above 60 6 5 yr. All subjects were nonsmokers, were taking no regular medication, and had normal history, examination (blood pressure, ,150/90 mm Hg), 12-lead resting electrocardiogram, blood count, glucose, and electrolytes. For the menopause and gender studies, all subjects had a nonfasting cholesterol of less than 6.5 mmol/L. Written informed consent was obtained from the subjects, and studies were approved by the Newcastle joint ethics committee. Study drugs All study drugs needing reconstitution were dissolved in sterile 0.9% saline. The following drugs were administered: norepinephrine (Sanofi Pharmaceuticals, Inc., Winthrop, UK), used as a control vasoconstrictor not affecting NO release; l-NG-monomethyl-arginine (CalbiochemNovabiochem, Nottingham, UK), a nitric oxide synthase inhibitor, used to measure basal NO release; oral conjugated estrogens (Wyeth Laboratories, Maidenhead, UK); serotonin hydrochloride (CalbiochemNovabiochem), used as an endothelium-dependent NO-dependent vasodilator; and glyceryl trinitrate (David Bull Laboratories, Inc., Warwick, UK), used as a NO donor. Study protocol Studies were performed in a quiet, temperature-controlled laboratory (25–27 C) between 1000 –1300 h. Subjects refrained from caffeine and alcohol and consumed no food for at least 2 h before the studies and rested supine for 30 min before commencement. Premenopausal studies were performed between 7–12 days from the start of the last menstrual period. After rest, a 27-gauge cannula (Cooper’s Needle Works, Birmingham, UK) was inserted into the brachial artery of the nondominant arm after administration of 1% lignocaine. Drugs or physiological saline were infused continuously at 1.0 mL/min. Forearm blood flow (FBF) was measured at 10-min intervals during 30 min of saline infusion to establish baseline FBF values. For constrictor studies, norepinephrine was infused at three doses (40, 80, and 160 ng/min), each for 5 min. Saline was then reinfused until baseline blood flows were reestablished, and then l-NG-monomethyl-arginine (L-NMMA) was infused at 3 doses (250, 500, and 1000 mg/min), each for 5 min. For both infusions, blood flows were measured during the last 2 min of each infusion period. The dilator studies were performed in 12 pre- and 10 postmenopausal women who had participated in the constrictor studies, at least 7 days after the initial studies. The above protocol was used with FBF responses measured to the infusion of 3 doses of glyceryl trinitrate (GTN) (250, 500, and 1000 ng/min), each for 5 min, followed by saline until baseline blood flows were reestablished, and then 3 doses of serotonin (18, 60, and 180 ng/min), each for 5 min. During serotonin infusion, FBF responses were measured during the first 2 min of infusion. Similarly, for the gender studies, the above protocols for constrictor and dilator studies were used on 14 men. Cross-over study designs were not used for either constrictor or dilator studies because of persisting vasoconstriction and vasodilatation with, respectively, L-NMMA and serotonin, whereas the effects of norepinephrine and GTN reversed within 20 min after continuing saline infusion. Initial studies of constrictor responses to norepinephrine used doses of 10, 20, and 40 ng/min in 3 pre- and 4 postmenopausal subjects. However, responses were consistently lower than that seen at the lowest dose of L-NMMA. To obtain constrictor responses to norepinephrine comparable to those seen with L-NMMA, the subsequent 12 pre- and 8 postmenopausal and male subjects were studied at doses of 40, 80, and 160 ng/min. Data for these groups are presented. Estrogen replacement studies were performed on 2 separate occasions. FBF constrictor responses to norepinephrine and L-NMMA were determined in 10 women, and dilator responses to serotonin and GTN were determined in a separate group of 10 women, before and after 14 days of treatment with oral conjugated estrogen (625 mg daily) using the above protocol. FBF measurements FBF (milliliters per 100 mL/min) was measured simultaneously in both arms by venous occlusion plethysmography, according to the method of Whitney ( 14 ), using galidinium in SILASTIC brand (Dow Corning Corp., Midland, MI) strain gauges. During recording, the hands were excluded from the circulation by inflation of the wrist cuffs to 200 mm Hg. The upper arm cuffs were then inflated to 50 mm Hg for 10 s in each 15-s cycle. Data were recorded directly onto computer using a MacLab3 system (AD Instruments Pty. Ltd., Castlehill, Australia) with on-line slope analysis to determine FBF. The average of five consecutive measurements for each measurement period was derived to determine FBF. Data analysis Forearm vascular resistance was derived from mean arterial pressure and baseline FBF. Differences in baseline heart rate, blood pressure, FBF, and forearm vascular resistance between and within groups were compared by Student’s t test. Within-subject differences in FBF in the control and infused arms were assessed using two separate repeated measures ANOVA. Further analysis was undertaken if ANOVA suggested a statistically significant change in FBF over time. FBF responses were expressed as the percentage of FBF during baseline infusion of saline. The overall drug response in each subject was assessed by the maximal response (percentage) and a summary response, calculated as the summation of the percentage of constrictor or dilator responses for the three doses of the infused drug (arbitrary units). Data are expressed as the mean 6 sem. Between-group comparisons were undertaken using initial repeated measures of ANOVA. Within-group comparisons were undertaken using paired Student’s t test. P , 0.05 was considered statistically significant. Results Menopause and gender studies Subject details are shown in Table 1. Mean age was higher in postmenopausal subjects, but blood pressure, weight, body mass index, cholesterol, and baseline FBF were similar. Men were taller, but had similar body mass indexes as women. There was no significant change in FBF in the control arm during the course of the studies. Constriction in response to L-NMMA (Fig. 1A) was increased in premenopausal women (maximum, 47 6 3%; summary, 118 6 10; mean 6 sem) compared to that in postmenopausal women [maximum, 33 6 6% (P , 0.05); summary, 82 6 14 (P , 0.05)] and males (maximum, 37 6 2%; summary, 89 6 6; P , 0.05), with no difference between the latter two groups. In contrast, constrictor responses to norepinephrine (Fig. 1B) were increased in males (maximum, 47 6 6%; summary, 125 6 13) compared to those in both premenopausal [maximum, 30 6 3% (P , 0.05); summary, 81 6 8 (P , 0.05)] and postmenopausal [maximum, 31 6 6% (P , 0.05); summary, 88 6 16 (P , 0.05)] groups, with no difference between the latter two groups. Responses to GTN and serotonin (Fig. 2, A and B) were not significantly different in the three groups (GTN: premenopausal: maximum, 142 6 18%; summary, 296 6 37; postmenopausal: maximum, 162 6 29%; summary, 363 6 63; male: maximum, 129 6 21%; summary, 281 6 31; serotonin: premenopausal: maximum, 99 6 11%; summary, 210 6 15; postmenopausal: maximum, 82 6 11%; summary, 190 6 23; male: maximum, 67 6 11%; summary, 160 6 23). Estrogen replacement study Subject details are shown in Table 2. Cholesterol decreased after estrogen treatment. FBF did not change in the control arm during the course of the studies. Constriction to LNMMA increased after estrogen [maximum, 54 6 3% vs. 40 6 5% (P , 0.05); summary, 132 6 17 vs. 89 6 14 (P , 0.05); Fig. 3A]. The constriction response to norepinephrine was unchanged [maximum, 45 6 6% vs. 38 6 6% (P 5 0.28); summary, 117 6 14 vs. 97 6 14 (P 5 0.27); Fig. 3B]. Dilatation responses to GTN and serotonin were unchanged after estrogen therapy [GTN: maximum, 96 6 18% vs. 101 6 14% (P 5 0.80); summary, 214 6 38 vs. 219 6 29 (P 5 0.88); serotonin: maximum, 92 6 26% vs. 74 6 12% (P 5 0.17); summary, 199 6 26 vs. 162 6 25 (P 5 0.19)]. Before After Before After Constrictor studies (n 5 10) This study is the first to show reduced constrictor responses to an NO synthase inhibitor in postmenopausal women and males compared to age-matched premenopausal peers, indicating a reduction in basal NO activity associated with the menopause. This change appears to be specific, with no change in the responsiveness of smooth muscle to norepinephrine in postmenopausal women. Two weeks of estrogen replacement therapy in postmenopausal women restores basal vascular NO activity to levels seen in premenopausal women. However, in our studies we found no alteration in stimulated NO release or smooth muscle sensitivity in association with the menopause or with estrogen replacement. We have also demonstrated men have basal vascular NO activity comparable to that in age-matched postmenopausal women, but have enhanced responsiveness to norepinephrine. Effects of the menopause and estrogen replacement on vascular NO activity Animal studies suggest that basal vascular NO activity may be reduced after the menopause ( 15 ), and that estrogen increases agonist-dependent NO activity ( 16 ). A study of NO metabolites in postmenopausal women receiving hormone replacement therapy observed a 50% increase in nitrite levels after 1 yr of estrogen therapy, but no increase with the estrogen/progesterone combination, suggesting that unopposed estrogen increases NOS activity ( 17 ). Interestingly, Sorenson et al. recently reported no increase in flow-mediated brachial artery dilatation in postmenopausal women taking combined estradiol and northisterone replacement therapy ( 18 ). These findings suggest that progesterone may attenuate the beneficial effects of unopposed estrogen on endothelial vascular function. Some studies suggest that the menopause and estrogen replacement may alter stimulated NO release in response to endothelium-dependent vasodilators, with reduced vasodilatation to acetylcholine at around the age of the menopause described ( 19 ). Acute infusion of estradiol in postmenopausal women increases the vasodilator response to acetylcholine ( 13 ). The effects of chronic oral and transdermal estrogen administration on stimulated NO release are unclear. Lieberman et al. reported an increased flow-mediated response to reactive hyperemia after 9 weeks of estrogen therapy in postmenopausal women ( 12 ). In contrast, two other studies found no change in response to acetylcholine ( 20, 21 ). Vasodilator responses to acetylcholine are not solely mediated via NO, and alterations in the release of bradykinin or other mediators may have confounded interpretation of these studies. One small study has examined the effects of estrogen therapy on basal NO activity, showing an increased response to L-NMMA, a NO synthase inhibitor, in six perimenopausal women after 8 weeks of therapy (21). Responses to a control vasoconstrictor were not performed in this study. Our observations suggest that the L-NMMA effect is specific, and it is not due to a generalized increase in smooth muscle responsiveness to constrictor agents. The estrogen replacement group we studied were older and had greater cholesterol concentrations than the postmenopausal group studied in the menopause/ gender studies. It is possible the effects of estrogen replacement therapy would differ in a younger group if increased age or hypercholesterolemia was associated with altered responsiveness of the vessel wall to estrogen. The absence of any significant difference in baseline L-NMMA responses between the two postmenopausal groups suggests that basal NO activity was not appreciably affected by increasing age or hypercholesterolemia. Despite the changes in basal NO activity observed with the menopause and after estrogen replacement, no difference was observed in basal blood flow, perhaps suggesting that vasoconstrictor influences are decreased after estrogen withdrawal at the menopause. However, many studies describe reduced endothelin plasma levels or activity after estrogen replacement, suggesting that a reduction in endothelin activity after the menopause is unlikely ( 22–24 ). Effects of gender on vascular NO activity Our finding of reduced vascular NO activity in middleaged men compared to premenopausal female peers contrasts with observations in younger subjects, in whom no significant gender difference was observed in FBF responses to L-NMMA in healthy subjects in their twenties ( 25 ). The gender difference in L-NMMA responses observed in our studies could reflect a preferential age-associated decline in vascular NO activity in males, perhaps due to estrogenmediated preservation of vascular NO activity in women before the menopause. Alternatively, there may be gender differences in age-associated changes in structural differences in forearm vasculature that could influence the response to vasoconstrictors. Interestingly, studies of NO production as determined by conversion of l-arginine to nitrite, demonstrate reduced whole body NO production in young to middle-aged healthy males compared to premenopausal females ( 26 ). A number of animal studies have found gender differences in vascular NO activity. Basal release of NO from aortic rings is greater in female rabbits, and ovariectomy reduces basal NO release to male levels ( 15 ). Pressureinduced myogenic constriction of rat gracilis muscle arterioles is less pronounced in female rats because of enhanced NO release, mediated through estrogen increasing NO release in response to vascular wall shear stress ( 27, 28 ). Similar observations have been made in rat coronary arteries (29). Effect of gender on a-adrenergically mediated responses Our observation of enhanced vasoconstriction in response to norepinephrine, a nonselective a-adrenoceptor agonist, in middle-aged males, has been reported in healthy young subjects ( 22 ). This most likely reflects enhanced a-adrenergic vascular sensitivity in males. Differences in a-adrenergic responsiveness with gender and after ovariectomy have been described in some animal vascular beds ( 30 –32 ). These observations suggest that circulating sex hormones may modulate vascular adrenergic responsiveness. Norepinephrine acts through both a1- and a2-adrenoceptors in the forearm to produce vasoconstriction. Norepinephrine may also act on endothelial cell a2-adrenoceptors to release NO, which could attenuate the vasoconstriction produced by norepinephrine ( 33 ). Gender differences in NO release stimulated by norepinephrine could account for the increased responsiveness of males to norepinephrine. Further studies examining vascular responses to selective a1- and a2-adrenoceptors in the presence and absence of NO inhibition would be of interest in establishing the underlying mechanisms accounting for these gender differences. Although increased NO activity could blunt norepinephrine responses in premenopausal women, this would not account for the difference observed in our studies between postmenopausal females and males and the lack of any difference between pre- and postmenopausal women. Interpretation of study findings The increased response to L-NMMA in premenopausal women could be due to either increased NO synthesis or increased sensitivity of arterial smooth muscle to released NO. Our finding that vasodilator responses to GTN, a NO donor, are not altered after the menopause or after systemic estrogen replacement confirms the results of previous studies ( 12, 19 –21 ). In one of these studies a small increase in the vasodilator response to nitroprusside was observed after local intraarterial infusion of estradiol, but not after 3 weeks of transdermal estrogen replacement (20). Thus, the changes we found in NO activity at the menopause probably reflect a decrease in NO activity after the menopause, rather than a change in smooth muscle sensitivity to NO, which is reversed with estrogen therapy. The additional finding that there is an enhanced constrictor response to noradrenaline in males most likely relates to enhanced a-adrenergic responsiveness, but could be due to a generalized increase in vasoconstrictor responsiveness. However, the latter is unlikely, as there was no difference in the response to L-NMMA between males and postmenopausal women. Mechanisms underlying change in NO activity The mechanism responsible for increased NO activity in premenopausal women compared to that in postmenopausal women and males and after estrogen replacement is most likely increased endothelial NO synthase activity secondary to higher levels of circulating 17b-estradiol. 17b-Estradiol increases constitutive NO synthase activity in endothelial cells from human umbilical veins and bovine aortas ( 34 ). In pregnancy, a hyperestrogenic state, an increase in the expression of uterine artery NOS has also been found ( 35 ). In addition to the estrogenic effects on NOS, other mechanisms may account for the present observations. The phenomenon of increased NO release due to increased flow is well established ( 36 ). Interestingly, Sudhir et al. reported an increase in basal FBF after estrogen replacement ( 21 ). In contrast, we found no change in FBF with estrogen or the menopause, suggesting that alterations in flow do not contribute to the observed changes in NO activity. Blood pressure changes at the menopause or after estrogen replacement could affect basal NO activity ( 37 ). However, most studies have found no independent effect of the menopause or estrogen therapy on blood pressure, and our results confirm this ( 38 ). Oral estrogen typically increases high density lipoprotein cholesterol by 10% and reduces low density lipoprotein cholesterol by 15% ( 39 ). Changes in cholesterol and lipoprotein levels may also modify basal vascular NO activity ( 40 ). Although there was only a small (,5%) reduction in total serum cholesterol levels after estrogen therapy, it is possible that changes in high density lipoprotein and low density lipoprotein cholesterol may have indirectly contributed to the increased vascular NO activity, independent of any direct effect of estrogen on NO synthesis or release. Two weeks of estrogen treatment would not be expected to produce final steady state changes in serum lipid concentrations, and longer term estrogen treatment could result in greater increases in vascular NO activity through such changes. Insulin resistance may influence endothelial function. We did not determine insulin resistance in subjects. However, body mass index and blood pressure were similar in all groups. Clinical relevance of alterations in vascular NO activity to cardiovascular risk Alterations in basal vascular NO activity may account for some of the changes in cardiovascular risk seen with the menopause and estrogen replacement. Reduced vascular NO activity has been found in conditions associated with an increased risk of cardiovascular disease, such as hypertension ( 37 ), hypercholesterolemia ( 40 ), smoking ( 41 ), and diabetes ( 42 ). Treatment of these conditions reverses the decline in NO activity. Estrogen has direct beneficial vascular effects with reduction in platelet aggregation, inhibition of smooth muscle proliferation, and reduction in cholesterol influx ( 43 ), and these changes may be mediated through the enhanced production of NO. This may therefore be an important mechanism in any cardioprotective role of estrogen, in addition to its effect on lipids. Our findings in the forearm circulation may not directly mirror changes in the coronary and cerebrovascular arterial vascular beds, which are of primary relevance to cardiovascular risk, although increased vascular NO release in response to acetylcholine after intracoronary estradiol infusion has been described in postmenopausal women undergoing cardiac catheterization ( 44 ). Conclusions The present studies demonstrate that premenopausal status is associated with increased basal vascular NO activity in the human forearm arterial vasculature compared to that in postmenopausal female and male peers. Vascular NO activity is increased to premenopausal levels after 2 weeks of estrogen replacement. Males demonstrate increased sensitivity to norepinephrine compared to both pre- and postmenopausal female peers. These changes in NO activity may contribute to the increase in cardiovascular risk seen after the menopause and any cardioprotective effect of estrogen replacement therapy. 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Majmudar, N. G., Robson, S. C., Ford, G. A.. Effects of the Menopause, Gender, and Estrogen Replacement Therapy on Vascular Nitric Oxide Activity, The Journal of Clinical Endocrinology & Metabolism, 2000, 1577-1583, DOI: 10.1210/jcem.85.4.6530