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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0419
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 11 6113-6122
Copyright © 2005 by The Endocrine Society

Small Artery Endothelial Dysfunction in Postmenopausal Women: In Vitro Function, Morphology, and Modification by Estrogen and Selective Estrogen Receptor Modulators

Karolina Kublickiene, Eimantas Svedas, Britt-Marie Landgren, Milita Crisby, Nasrin Nahar, Henry Nisell and Lucilla Poston

Institution for Clinical Science, Intervention, and Technology (K.K., E.S., B.-M.L., H.N.), Department of Obstetrics and Gynecology, and Department of Neurotec (M.C., N.N.), Section for Experimental Geriatrics, Karolinska Institutet, Karolinska University Hospital-Huddinge, 14186 Stockholm, Sweden; and Maternal and Fetal Research Unit (L.P.), Division of Reproductive Health, Endocrinology, and Development, King’s College London, London SE1 7EH, United Kingdom

Address all correspondence and requests for reprints to: Karolina Kublickiene, M.D., Ph.D., Institution for Clinical Science, Intervention, and Technology, Department of Obstetrics and Gynecology, Karolinska Institute, Karolinska University Hospital-Huddinge Campus, 14186 Stockholm, Sweden. E-mail: karolina.kublickiene{at}klinvet.ki.se.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Objective: Our objective was to assess vascular endothelial function and morphology in resistance vasculature from healthy pre- and postmenopausal women in vitro and to determine potential mechanisms of vascular protection by estrogenic compounds.

Methods: Arteries (~220 µm) were dissected from sc fat biopsies obtained from healthy premenopausal and postmenopausal women. Flow-mediated dilatation, agonist-induced endothelium-dependent and -independent relaxation, and myogenic responses to changes in intraluminal pressure were evaluated before and after incubation (3 h) with 17ß-estradiol, propyl pyrazole triol [a selective estrogen receptor-{alpha} (ER{alpha}) agonist], raloxifene (a second-generation selective ER modulator), and the phytoestrogen genistein, using pressure myography technique. In addition, endothelial morphology was assessed in arteries from pre- and postmenopausal women, and distribution of ERs within the artery wall from postmenopausal women was evaluated.

Results: Functional and morphological disturbances of endothelial function were observed in small arteries from postmenopausal women. Incubation with 17ß-estradiol improved postmenopausal resistance artery function, an effect mimicked by propyl pyrazole triol but not raloxifene or genistein. Immunohistochemical staining revealed similar expression of ER{alpha} and ERß in the smooth muscle of arteries from postmenopausal women; however, ER{alpha} was dominant in endothelium.

Conclusions: The resistance arteries from postmenopausal women show functional and morphological abnormalities. ER{alpha} may contribute to vascular protection by estrogens in the peripheral resistance circulation in postmenopausal women. Selective ER{alpha} agonists warrant further investigation as therapeutic agents for prevention of cardiovascular disease in postmenopausal women.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE PIVOTAL ROLE of the vascular endothelium in cardiovascular health is well established. In larger arteries, endothelial cell activation and reduced dilator function contribute to the inflammatory process of atherogenesis, whereas in the resistance vasculature, blunted endothelium-dependent dilation may lead to elevation of peripheral resistance and hypertension (1). Endothelium-dependent dilation of the conduit arteries is reduced in women after menopause, as evidenced by reduced brachial artery dilatation to a hyperemic flow stimulus (2). Experimental determination of responses to flow such as this have provided a useful tool in the assessment of endothelial function because the physical stimulus of shear stress, the tangential force created by blood flow, is among the most influential of those contributing to regulation of endothelial function (3). To date, however, there has been no attempt to directly evaluate responses to shear stress in the resistance vasculature in women after menopause, although small artery endothelial dysfunction has been implied from blunted dilator responses to pharmacological agonists in in vivo studies using forearm venous occlusion plethysmography (4).

Estrogens are generally considered to provide protection against cardiovascular risk because cardiovascular disease is relatively uncommon before the menopause and increases dramatically thereafter (5). Studies in women who have received estrogen replacement either acutely (6) or in the long term (7) demonstrate improvement in parameters of endothelium-dependent dilatation, including enhanced responses to a flow stimulus in conduit arteries (8). Recent randomized trials, however, have produced equivocal results and questioned whether combined hormonal replacement therapy (HRT) would prevent later cardiovascular events (9, 10). Investigations of alternatives to HRT have suggested that selective estrogen receptor modulators (SERMs), including naturally occurring phytoestrogens, may confer cardiovascular protection, which in part may be attributable to selectivity for the estrogen receptor (ER) subtypes ER{alpha} and ERß (11).

The aim of this study was to determine whether responses to shear stress are compromised in the resistance vasculature in postmenopausal women. We have employed an in vitro method, pressure myography, to directly evaluate flow-mediated responses in arteries obtained from biopsies of sc fat from postmenopausal women who had never received estrogen replacement or combined HRT and have made comparison with arteries from premenopausal women. We have also characterized the responses to endothelium-dependent and -independent pharmacological agonists and performed the morphological examination of the endothelium. Pressure-induced vasoconstriction (myogenic tone) was assessed because it may contribute to increased peripheral resistance in hypertension (12). Furthermore, in arteries from postmenopausal women, functional responses after prolonged exposure to 17ß-estradiol (17ß-E2), ICI 182,780 (ER antagonist), propyl pyrazole triol (PPT, a selective ER{alpha} agonist), raloxifene (a second-generation SERM with antiestrogenic effects on uterus and breast and agonistic effect on bone), and the phytoestrogen genistein (a predominant ERß agonist) were determined. The distribution of ER subtypes within the vascular wall of small arteries from postmenopausal women was evaluated.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The study was approved by the Ethical Committee at Huddinge University Hospital, Stockholm, Sweden. Subcutaneous fat biopsies (2 x 1.5 x 1.5 cm) were obtained from premenopausal women during laparoscopic surgery (n = 6) and elective abdominal surgery (n = 10) for sterilization and/or infertility treatment on fallopian tubes and uterus from volunteers under local anesthesia (n = 5) and from postmenopausal women volunteers under local anesthesia (n = 66) (1% prilokain; Citanest, AstraZeneca, Stockholm, Sweden). Biopsies were taken from the lower abdominal region. The tissue was immersed in cold physiological salt solution (PSS) and kept on ice. All postmenopausal women had been amenorrheic for at least 1.5 yr. Cigarette smokers and women with hypertension, diabetes mellitus, clinical manifestations of arteriosclerosis (coronary heart disease, peripheral artery disease, or cerebrovascular disease), venous thromboembolic disease, liver disorders, unexplained vaginal bleeding, and personal or family history of breast cancer were excluded. None had received HRT, other steroid hormones, or any medication known to affect lipoprotein metabolism or blood pressure. None of the premenopausal women had taken contraceptives during the 3 months preceding the study. Separate consent was obtained for blood samples for assessment of estradiol, progesterone, FSH, and lipid profile.

Assessment of vascular function in isolated arteries

Subcutaneous small arteries (~220 µm, 2–3 mm length) were dissected and mounted on a pressure myograph (LSI, Burlington, VT) as previously described (13). The organ bath was perfused (7 ml/min) with PSS (mM: NaCl 119, KCl 4.7, CaCl2 2.5, MgSO4 1.17, NaHCO3 25, KH2PO4 1.18, EDTA 0.026, and glucose 5.5; pH 7.4) at 37 C and gassed with 5% CO2 in O2. Arteries were discarded if they failed to maintain pressure, demonstrated incomplete occlusion of the lumen in response to extraluminal norepinephrine (NE, 10–6 M in potassium-substituted PSS (64 mM KCl in PSS), or failed to relax to bradykinin (BK, 10–6 M).

Flow-mediated dilatation

Unless stated, all protocols were carried out in arteries from premenopausal (n = 21) and postmenopausal (n = 66) women. After equilibration (60 mm Hg for 40 min), intraluminal pressure was set to 80 mm Hg, and the internal diameter (ID) recorded. The artery was constricted with NE (10–6 M) until a stable diameter was achieved (30 min). Intraluminal flow was then initiated via the flow pump. Flow was incrementally increased (25, 60, 100, 130, 175, and 204 µl/min) at 5-min intervals and ID recorded at each flow rate. Arteries were then incubated in 17ß-E2 (10–7 or 10–8 M, 3 h), followed by washing in PSS (30 min) and the flow protocol repeated. This approach avoided the confounding influence of acute effects of 17ß-E2. During incubation, intraluminal flow was stopped and NE absent in the superfusate for 3.5 h. In separate arteries, the nitric oxide synthase inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME 10–4 M, 30 min) was added after incubation with 17ß-E2 (10–7 M, 3 h) and before estimation of the second flow response. Flow responses were also evaluated in separate arteries from postmenopausal women before and after incubation with 17{alpha}-E2 (10–7 M). Because 17ß-E2 is a mixed ER{alpha}/ERß agonist, and to aid in characterization of the receptor subtype involved in estrogen-mediated responses, we have also investigated flow responses to a selective ER{alpha} agonist, PPT (10–8 M), as well to raloxifene (5 x 10–8 M) and genistein (10–8 M). As a control, in different arteries, the second flow response was carried out after preincubation with vehicle alone and after 3.5-h incubation (washout period intervening) with PSS alone for time control. In other experiments, the ER antagonist ICI 182,780 (10–6 M) was added through the superfusate during the flow response after preincubation with 17ß-E2; these experiments were carried out at the lower concentration of 17ß-E2 (10–8 M), because previous protocols had shown equivalent effects of 17ß-E2 at 10–7 and 10–8 M. Only one incubation condition was tested in any given artery from any participant.

Endothelium-dependent and -independent dilatation

Responses to BK (10–9 to 10–6 M) and to sodium nitroprusside (SNP, 10–9 to 10–5 M) after preconstriction with NE (10–6 M) were compared in arteries from post- and premenopausal women. Concentration-response curves in postmenopausal arteries were determined after preincubation with 17ß-E2 (10–8 M) and after preincubation with L-NAME, ICI 182,780, PPT, raloxifene, and genistein, as appropriate.

Pressure-induced myogenic tone

After equilibration (60 mm Hg for 40 min), intraluminal pressure was set to 20 mm Hg (15 min) and thereafter increased stepwise in 20-mm Hg increments from 20–120 mm Hg every fifth minute and ID recorded after achievement of steady-state diameter. The role of NO and ERs in modulation of myogenic tone after incubation with 17ß-E2 (10–8 M) was determined by measuring pressure responses with addition of L-NAME or ICI 182,780. Pressure responses were also compared before and after incubation with PPT, raloxifene, and genistein. To enable calculation of myogenic tone, the responses to incremental pressure steps were repeated in Ca2+-free PSS substituted with EGTA (10–3 M) and papaverine (10–4 M).

Expression of ER{alpha} and ERß by immunohistochemistry

Arteries from postmenopausal women were embedded in OCT (Histolab, Göteborg, Sweden), frozen on dry ice, and stored (–70 C). Sections (8 µm) were taken on SuperFrost glass slides and fixed in ice-cold acetone (10 min). After washing in 1% PBS, sections were blocked with endogenous peroxide (0.3% hydrogen peroxide, 30 min), washed twice in 1% PBS, and then blocked with BSA (60 min). After overnight incubation (40 C) with primary antibody ER{alpha} or ERß (Santa Cruz Biotechnology, Santa Cruz, CA) (1:10 and 1:20, respectively) and after washing (1% PBS), sections were incubated with rabbit antimouse secondary antibody and swine antirabbit secondary antibody (Dako, Copenhagen, Denmark) (1:200 and 1:300, respectively; 60 min). After washing, the sections were incubated with avidin-biotin (30 min) and exposed to 0.001% of 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., Stockholm, Sweden) with 0.03% hydrogen peroxide followed by counterstaining with hematoxylin.

Hormone assays

Venous blood samples were taken after a 12-h overnight fast at baseline. Total plasma cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides were measured in the hospital’s clinical chemistry laboratory. Serum concentrations of 17ß-E2, FSH, and progesterone were determined by RIA using commercials kits (Diagnostic Products Corp., Los Angeles, CA).

Scanning electron microscopy

Dissected arteries were rinsed in saline and immediately immersed in 2.5% glutaraldehyde in sodium cacodylate buffer (0.15 M, pH 7.3, 24 h). They were then postfixed in 1% osmium tetroxide in sodium cacodylate buffer (0.15 M, pH 7.3) containing 75 mM sucrose. After dehydration in acetone and drying in a critical point drier with CO2, the samples were mounted, coated with gold palladium, and examined.

Chemicals

17ß-E2, PPT, raloxifene, and ICI 182,780 were dissolved in 100% ethanol to a concentration of 10 mM (17ß-E2), 1 mM (PPT), 10 mM (raloxifene), 10 mM (ICI 182,780), and 10 mM (genistein) in dimethylsulfoxide. Subsequent dilutions of these solutions were made with PSS. Preliminary experiments revealed that ethanol or dimethylsulfoxide (vehicle) had no effect on the responses. NE, BK, and SNP were dissolved in distilled water. PPT, ICI 182,780, and genistein were obtained from Tocris Ltd. (Avonmouth, UK). Raloxifene was a gift from Stefan Nilsson at Karo Bio AB (Huddinge, Sweden). Other chemicals were from Sigma.

Data analysis

Values in the text and figures are given as mean ± SEM. Flow-, BK-, and SNP-mediated dilatations were calculated as a percent change in ID from initial preconstriction with NE. Overall differences in responses to flow, agonists, and intraluminal pressure obtained in arteries from premenopausal and postmenopausal women and under different experimental conditions were compared by use of two-way repeated-measures ANOVA (StatSoft), using agonist concentration, flow rate, or intraluminal pressure as a within-subject factor and group membership or incubation condition as a between-subject factor.

The differences in ID of the arteries at different pressure steps before and after equilibration in Ca2+-free PSS (Ca2+-free PSS with EGTA and papaverine) provided an estimate of pressure-induced myogenic tone. This was calculated as the percentage decrease of the ID of arteries in Ca2+-free PSS from the following equation:

(1)
The evaluation of ER expression was performed using semiquantitative analysis. The staining was scored blindly: +, less than 5% staining; ++, 5–25%; +++, 25–50%; ++++, 50–70%; +++++, more than 70%. {chi}2 statistics were applied for analysis. Quantitative analysis of the calculated number of endothelial cells per 50 x 50-µm area that corresponds to the area in an original 1000-fold magnification picture of an arterial sample was performed and compared between the groups using Student’s t test. Significant difference in all comparisons was assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Patients

Premenopausal and postmenopausal women had similar anthropometric parameters and arterial blood pressure (Table 1Go).


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  		TABLE 1. Anthropometric parameters and arterial blood pressure in post- and premenopausal women

 
Flow-induced responses

Flow led to progressive dilation in arteries from both pre- and postmenopausal women, but the dilatory response was significantly reduced in postmenopausal (n = 59) compared with premenopausal (n = 21) vessels (P < 0.001) (Fig. 1Go). The phase of the menstrual cycle had no influence on flow-mediated dilatation in premenopausal arteries [maximal change in diameter, 75 ± 25% proliferative (n = 7) vs. 83 ± 24% secretory phase (n = 10); P = not significant (NS)].



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  		FIG. 1. Flow-mediated dilatation, expressed as the percentage change in ID after initial preconstriction, in isolated small arteries from postmenopausal (n = 59) compared with premenopausal (n = 21) women. *, P < 0.001.

 
17ß-E2 (10–7 M) preincubation did not affect flow-mediated dilatation in premenopausal arteries (maximal change in diameter, 80 ± 26% in PSS, vs. 78 ± 17% after 17ß-E2; n = 13; P = NS). However, 17ß-E2 improved flow-induced dilation in postmenopausal vessels (n = 17; P =0.001) (Fig. 2Go). In contrast, neither 17{alpha}-E2 (10–7 M, n = 3) nor vehicle (n = 3) had any effect on relaxation to flow (data not shown).



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  		FIG. 2. Flow-mediated dilatation in arteries from postmenopausal women in PSS ({blacksquare}) and after preincubation (3 h) with 17ß-E2 ({square}) (n = 17; *, P = 0.001) and in a separate study after preincubation with 17ß-E2 ({circ}) and with addition of L-NAME (•) (n = 6; §, P = 0.014) before the second flow response.

 
L-NAME inhibited 17ß-E2-enhanced (10–7 M) relaxation (n = 6; P = 0.014) (Fig. 2Go). Stimulation of flow-mediated dilatation also occurred after preincubation of arteries from postmenopausal women with a lower concentration of 17ß-E2 (10–8 M) (P = 0.001; n = 6) (Fig. 3AGo). Addition of ICI 182,780 prevented 17ß-E2-induced (10–8 M) dilatation (n = 7) (Fig. 3AGo). PPT also stimulated flow-mediated relaxation in postmenopausal arteries (n = 6; P = 0.002) (Fig. 3BGo). Raloxifene (n = 9) and genistein (n = 8) were without effect (Fig. 3BGo). In postmenopausal women, there was no difference in the initial flow-mediated relaxation in PSS between the subgroups of arteries randomly exposed to different incubations.



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  		FIG. 3. A, Flow-mediated dilatation in isolated arteries from postmenopausal women in PSS (•) and after preincubation (3 h) with 17ß-E2 ({circ}) (n = 6; *, P = 0.001), and in a separate study in PSS ({blacksquare}) and after preincubation with a combination of ICI 182,780 plus 17ß-E2 ({square}) (n = 7; P = NS). B, Flow-mediated dilatation in isolated arteries from postmenopausal women in PSS (•) and after preincubation (3 h) with PPT ({circ}) (n = 6; *, P = 0.002), raloxifene ({square}) (n = 9; P = NS), and genistein (*) (n = 8; P = NS). All flow-mediated dilatation curves obtained in PSS before the incubation are pooled for presentation purposes, although analyses were carried out on paired data.

 
BK- and SNP-mediated dilatation

BK-mediated relaxation was reduced in postmenopausal (n = 38) compared with premenopausal (n = 10) women’s arteries (P = 0.017) (Fig. 4AGo) but was improved by preincubation with 17ß-E2 (n = 10; P = 0.012) (Fig. 4BGo). L-NAME prevented this 17ß-E2-induced up-regulation of BK dilatation in postmenopausal arteries (n = 10; P = 0.01) (Fig. 4BGo) as ICI 182,780 did (n = 4; P = 0.035) (Fig. 4BGo). 17ß-E2 had no effect on BK responses in premenopausal arteries (n = 5) (data not shown). In postmenopausal arteries, PPT induced a nonsignificant increase in BK dilatation (n = 6; NS) (Fig. 4CGo). Raloxifene had no effect (n = 10; NS) (Fig. 4CGo), and genistein (10–8 M) induced a nonsignificant reduction in dilatation (n = 8; NS) (Fig. 4CGo).



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  		FIG. 4. A, Endothelium-dependent dilatation to BK, expressed as the percentage change in diameter after initial preconstriction, in arteries from postmenopausal (n = 38) compared with premenopausal (n = 10) women. *, P < 0.017. B, Endothelium-dependent dilatation to BK in arteries from postmenopausal women in PSS ({blacksquare}) and after preincubation (3 h) with 17ß-E2 ({square}) (n = 10; *, P = 0.01); in 17ß-E2 ({square}) and after 17ß-E2 plus L-NAME (•) (n = 10; #, P = 0.01); and after 17ß-E2 plus ICI 182,780 (*) (n = 4; P = NS). SEM is excluded in the relaxation curve to BK in the presence of 17ß-E2 plus ICI 182,780 to simplify the presentation of the figure. C, Dilatation to BK in arteries from postmenopausal women in PSS ({blacksquare}) and after incubation with PPT ({square}) (n = 6; P = NS), raloxifene ({circ}) (n = 10; P = NS), and genistein (*) (n = 8; P = NS). All relaxation response curves to BK obtained in PSS before the incubation are pooled for presentation purposes, although analyses were carried out on paired data.

 
Concentration-response curves to SNP were similar in pre- and postmenopausal arteries [10–6 M: maximal change in diameter, 54 ± 13% (n = 5) vs. 62 ± 25% (n = 5)]. 17ß-E2 (10–8 M) had no effect on relaxation to SNP in postmenopausal arteries (42 ± 12% vs. 41 ± 25% after 17ß-E2; n = 5).

Pressure-induced tone

An increment in pressure from 20–120 mm Hg led to a similar and significant (P = 0.01 within each group) rise in myogenic tone in arteries from postmenopausal (n = 46) and premenopausal (n = 9) women (e.g. 120 mm Hg: 22 ± 2% vs. 21 ± 7%; NS). 17ß-E2 (10–8 M) preincubation blunted the myogenic response in postmenopausal arteries (n = 15; P =0.001) (Fig. 5Go), but this was reversed by addition of L-NAME (n = 11; P = 0.001) (Fig. 5Go). Addition of ICI 182,780 also inhibited the response (n = 8) (Fig. 5Go). In separate arteries from postmenopausal women, PPT and raloxifene, but not genistein, reduced myogenic tone [e.g. 120 mm Hg: 15 ± 3% PSS vs. 6 ± 1% after PPT (n = 6; P < 0.05), 33 ± 6% PSS vs. 14 ± 5% after raloxifene (n = 10; P < 0.05), and 15 ± 3% PSS vs. 12 ± 2% after genistein (n = 6; NS)]. Figure 5Go represents myogenic tone at a physiological pressure of 80 mm Hg.



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  		FIG. 5. Myogenic tone at 80 mm Hg of intraluminal pressure before (solid bar) and after incubation (3 h) (cross-hatched bar) with the following compounds: in PSS and after 17ß-E2 (n = 15; *, P = 0.001); 17ß-E2 and after L-NAME (n = 11; *, P = 0.001); in PSS and after ICI 182,780 (ICI) plus 17ß-E2 (n = 8; P = NS); in PSS and after PPT (n = 6; *, P < 0.05); in PSS and after raloxifene (Ralox) (n = 10; *, P = 0.05); in PSS and after genistein (Gen) (n = 6; P = NS).

 
Endothelial morphology

Scanning electron micrographs (x1000 and 1500-fold magnification) showing endothelial morphology of small (~250 µm) arteries from post- and premenopausal women are demonstrated in Fig. 6Go. The endothelial cell layer in postmenopausal women (A–C) showed signs of endothelial cell death (indicated by endothelial cell blebs), partial denudation, and attachments of red blood cells, platelets, lymphocytes, and protein aggregates. Fractured cell membranes and loss of intercellular connections were apparent. This morphological profile contrasted with arteries from premenopausal women (D–F), which were characterized by a continuous layer of tightly connected endothelial cells and thick plasma membranes without any visible signs of endothelial death. The number of endothelial cells in an area of 50 x 50 µm, as calculated from the micrographs, was reduced in arteries from post- vs. premenopausal women (24 ± 3, n = 10, vs. 67 ± 3, n = 7; P < 0.05).



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  		FIG. 6. a, Scanning electron micrographs (x1000) of the endothelial cell layer in isolated arteries from healthy postmenopausal women (A–C) showing evidence of endothelial cell death (arrows point to apoptotic cells, Ap), denudation (De), and attachments of red blood cells (RBC), platelets (Pl), and protein (Pro) aggregates. Micrographs of arteries from premenopausal women (D–F) show a continuous layer with tightly connected endothelial cells. b, Micrographs (x1500) of the endothelial cell layer in isolated arteries from healthy postmenopausal women (A–C) showing fractured basal membranes (Mb), loss of intercellular junctions (Jn), signs of endothelial cell death (blebs, indicated as Ap), denudation, and attachments of platelets, lymphocytes (Lf), and protein aggregates. Micrographs of arteries from premenopausal women (D–F) show a continuous layer of endothelial cells and thick plasma membranes.

 
Immunohistochemistry

ER{alpha} and ERß expression was observed in all arteries from postmenopausal women. ER{alpha} and ERß staining was localized to the endothelial layer and in the media (Fig. 7Go). In the media, expression of ER{alpha} and ERß was similar [3 ± 1 vs. 2.7 ± 0.5 (mean ± SD); n = 7; P = 0.23]. ER{alpha} expression was higher than ERß in the endothelium (2.7 ± 0.5 vs.1.6 ± 0.5; n = 7; P = 0.013).



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  		FIG. 7. Immunohistochemical staining for ER{alpha} (A) and ERß (B) in media and endothelium (EC) in sc small arteries from healthy postmenopausal women. C, Negative control.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study is, to our knowledge, the only investigation to show that isolated small arteries from postmenopausal women demonstrate an impaired dilatation to a flow stimulus. Healthy postmenopausal women who have never received HRT, therefore, demonstrate a disorder of the peripheral circulation, which, in larger vessels, is strongly associated with cardiovascular risk (14). Generality of endothelial dysfunction was also evident from the blunted responses to BK, a physiologically relevant endothelium-dependent agonist used for in vitro and in in vivo assessment of endothelial function (13, 15). Investigation of the endothelial cell layer showed that the functional abnormalities were accompanied by cellular disruption and damage. The study has also demonstrated that 17ß-E2 and PPT but not raloxifene and genistein may improve endothelial function ex vivo and implicate the importance of ER{alpha} in vascular responses. These data have potentially important implications for the etiology and prevention of cardiovascular disease in postmenopausal women.

The small arteries of the skin provide a useful model for investigation of vascular dysfunction in the peripheral circulation. Whereas studies in vivo have demonstrated impaired flow responses in the brachial artery of postmenopausal women (7, 8), none has investigated flow-mediated dilatation in the resistance vasculature. The observation of poor responses to the infusion of endothelium-dependent dilators in the forearm circulation indirectly suggests compromised function of the resistance vasculature (4, 16) and provides supportive evidence. We have reported that NO is the primary mediator of vasodilatation to flow in human sc arteries (17). Because NO not only serves as a physiological vasodilator but also contributes to a wealth of other antiatherogenic pathways (18), the absence of flow-mediated NO release has the potential to promote a vascular phenotype to atherogenesis.

Previous in vivo studies of premenopausal women during the menstrual cycle have demonstrated variability in cardiovascular responses, including flow- and agonist-mediated dilatation between different phases of the menstrual cycle (19, 20), but we found no association and concur therefore with one report showing similar forearm blood flow between groups of women at different phases of the menstrual cycle (21). Additionally, and in agreement with our study, responses to nitrovasodilators have been found to be similar in pre- and postmenopausal women, using the forearm techniques (16, 22). Both approaches argue against a role for reduced smooth muscle sensitivity to NO, but for a primary defect in NO synthesis at the level of the endothelial cell.

The impairment in shear-mediated dilatation in sc arteries from the postmenopausal women may be attributable to age and/or the concomitant decline in estrogens. Age is associated with reduction in endothelium-dependent dilatation in men and women (23, 24). In women, however, a steep decline commences around the menopause, indicating that estrogen withdrawal is a major factor (24). Endothelial dysfunction has also been reported in young ovariectomized women (5) and in isolated arteries from ovariectomized animals (25). In the present study, there was no relation between the age of either premenopausal or postmenopausal women and flow-mediated dilatation, which argues in favor of a role for estrogen decline.

Myogenic tone reflects the constrictor response to physiological transmural pressure, mediated via circumferential stretch. It shows vascular bed heterogeneity and is modulated by basal synthesis of NO (26). We found no evidence for increased myogenic responses in postmenopausal women. However, in vivo, myogenic tone can be amplified by local vasoconstrictors, e.g. endothelin-1, angiotensin II, and angiotensin-converting enzyme, the concentrations of which are raised in postmenopausal women (27), or by sympathetic tone, which may also be increased (28), and a role of myogenic response in increased peripheral vascular tone in postmenopausal women cannot be entirely discounted.

The disruption of the morphology of the endothelium in arteries from postmenopausal women with no evidence of overt cardiovascular disease could contribute to the functional defects observed. The vessels demonstrated fractured endothelial cell membranes, denuded areas of vascular intima covered with adherent proteins and cells, distorted cellular attachment to the basal membrane, and disrupted intercellular and myoendothelial connections resulting in the formation of gaps that in vivo could facilitate passage of cells and proteins from the intravascular space to the underlying tissue. All could be pertinent to endothelial cell activation. Intercellular communications play an important role in signal transduction, including responses to flow, endothelium-dependent agonists, and pressure (29), and these could be compromised by the cellular disorganization observed (30). Morphological signs (i.e. blebs) of endothelial cell death would be compatible with apoptosis and with estrogen decline, because estrogens have been implicated in the prevention of endothelial apoptosis (31) and oxidative stress (32). Because endothelial NO synthase (eNOS) is harbored within the plasma membrane caveolae (29), cell membrane disruption might also affect the critical interplay between eNOS and relevant intracellular signaling pathways.

This study has suggested that 17ß-E2 provides cardiovascular protection through up-regulation of flow-induced and endothelium-dependent NO-mediated dilatation and supports previous studies in vivo, reporting that 17ß-E2 increases flow-mediated dilatation in the brachial artery of postmenopausal women, generally attributed, although not proven to be the result of, NO stimulation (8, 33). Although in vivo studies have shown that estrogens increase the basal release/availability of NO (22), our data provide the first direct demonstration of a role for NO in 17ß-E2-mediated up-regulation of flow responses in postmenopausal women. Animal studies from our laboratory and others support this observation because 17ß-E2 enhanced flow-mediated dilatation via the NO pathway in arteries from prepubertal female rats and in isolated small arteries from women with preeclampsia (13, 34). 17ß-E2 also enhanced BK-mediated dilatation in the postmenopausal arteries via the NO pathway, because L-NAME abolished 17ß-E2-induced up-regulation of the BK-mediated response.

Several studies indicate that a 3-h incubation period is adequate to evoke a genomic response to estrogens (35). Target genes other than eNOS include prostacyclin synthase and inducible NO synthase (36). The NO pathway could also indirectly be influenced by estrogen-induced inhibition of reduced nicotinamide adenine dinucleotide phosphate oxidase with a resultant reduction in superoxide synthesis or though regulation of caveolin and heat shock protein 90 expression (37).

17ß-E2 may evoke NO-mediated vascular responses within minutes via calcium-dependent and -independent pathways, including activation of tyrosine kinase, MAPK pathways, heat shock protein 90, and Akt/protein kinase B signaling (38). Recent studies implicate endothelial membrane-specific ERs (ER{alpha} and ERß) in these acute responses (36, 38). By 3-h preincubation, and removal of the estrogens by washing, we attempted to avoid these rapid responses.

The responses to estrogens are abolished by the ER inhibitor ICI 182,780 (39), and this compound also blocked flow-, BK-, and pressure-mediated responses to 17ß-E2 in the present study. Specific activation of ER{alpha} was strongly implicated because PPT, an ER subtype-selective ligand, which binds to ER{alpha} with a 400-fold greater affinity than for ERß (40), evoked similar responses to 17ß-E2. Recent findings also suggest that ER{alpha} is involved in estrogen protection of the female vascular wall by acceleration of reendothelialization (41), NO stimulation (42), or protection from injury (43). The predominance of endothelial ER{alpha} expression observed in the postmenopausal arteries strengthens the suggestion that 17ß-E2 effects on vascular function may be mediated through ER{alpha}.

Because of the lack of apparent cardiovascular benefit of HRT in the Women’s Health Initiative and Heart and Estrogen-Progestin Replacement Study trials (9, 10), the second-generation SERM raloxifene and the phytoestrogen genistein have been proposed as alternatives to conventional HRT, e.g. the Raloxifene Use for the Heart trial (44) and the Soy Health Effects study (45). Both compounds enhance NO release in arteries and cultured endothelial cells via increase of eNOS mRNA expression (46, 47) and preserve endothelial function in ovariectomized animals (48, 49). Phytoestrogens also reduce vascular cholesterol content (50) and restore NO-mediated dilatation after chronic hypoxia (51), although raloxifene may reverse vascular injury (52) and reduce leukocyte adhesion to endothelial cells (53). In healthy postmenopausal women, prolonged administration of genistein (54) and resveratrol (55) improves flow-mediated dilatation in brachial artery, but these findings are not universal, because several studies (56, 57, 58) produced an opposing result. There is, however, no report on the prolonged effects of these ligands on the resistance vasculature of healthy postmenopausal women. In our study, neither genistein nor raloxifene affected flow- or BK-mediated dilatation in postmenopausal arteries, although the reduction of myogenic tone induced by raloxifene could have resulted from stimulated NO release (49). Such treatment alternatives might therefore be of minor importance, at least for resistance artery function in these women. The absence of responses to genistein and raloxifene compared with 17ß-E2 and PPT may reflect the relative predominance of ER{alpha} vs. ERß, because ERß has most frequently been associated with responses to genistein (59), although responses to raloxifene might be related to mixed agonist/antagonist action on both receptors in a tissue-selective manner (60). ERß has, however, also been implicated in the role of 17ß-E2 in the arterial response to injury (61) and in endothelium-dependent arterial responses in aging animals (62).

In conclusion, we have demonstrated functional and morphological evidence for impaired endothelial function in isolated resistance arteries from healthy postmenopausal women. The beneficial effects of 17ß-E2 on resistance-artery function seem to be mediated through ER{alpha}, because only PPT but not raloxifene or genistein reproduced the effect. The identification of a novel endothelial cell-specific ER{alpha} agonist may prove to be a useful therapeutic agent for cardiovascular protection in postmenopausal women.


   Acknowledgments
 
We are grateful to personnel at the Department of Women’s Health for assistance with recruiting participants.


   Footnotes
 
This work was supported by the Swedish Heart and Lung Foundation, the Swedish Society of Medicine, and the Gender-Medicine Center from Karolinska Institute.

First Published Online August 30, 2005

Abbreviations: BK, Bradykinin; 17ß-E2, 17ß-estradiol; eNOS, endothelial NO synthase; ER, estrogen receptor; HRT, hormone replacement therapy; ID, internal diameter; L-NAME, N{omega}-nitro-L-arginine methyl ester; NE, norepinephrine; NS, not significant; PPT, propyl pyrazole triol; PSS, physiological salt solution; SERM, selective estrogen receptor modulator; SNP, sodium nitroprusside.

Received February 28, 2005.

Accepted August 24, 2005.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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