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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-2651
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 5 1874-1883
Copyright © 2008 by The Endocrine Society

Effects in Postmenopausal Women of Estradiol and Medroxyprogesterone Alone and Combined on Resistance Artery Function and Endothelial Morphology and Movement

Karolina Kublickiene1, Xiao-Dong Fu1, Eimantas Svedas, Britt-Marie Landgren, Andrea R. Genazzani and Tommaso Simoncini

Department of Clinical Science (K.K., E.S., B.-M.L.), Intervention and Technology, Section for Obstetrics and Gynecology, Karolinska Institute, Karolinska University Hospital-Huddinge Campus, 14186 Stockholm, Sweden; and Molecular and Cellular Gynecological Endocrinology Laboratory (X.-D.F., A.R.G., T.S.), Department of Reproductive Medicine and Child Development, University of Pisa, 56100, Pisa, Italy

Address all correspondence and requests for reprints to: Karolina Kublickiene, M.D., Ph.D., Department of Clinical Science, Intervention and Technology, Section for Obstetrics and Gynecology, Karolinska Institute, Karolinska University Hospital-Huddinge Campus, 14186 Stockholm, Sweden. E-mail: karolina.kublickiene{at}ki.se; or Tommaso Simoncini, M.D., Ph.D., Molecular and Cellular Gynecological Endocrinology Laboratory, Department of Reproductive Medicine and Child Development, University of Pisa, Via Roma, 67, 56100, Pisa, Italy. E-mail: t.simoncini{at}obgyn.med.unipi.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Endothelial dysfunction in resistance arteries after menopause is important for the development of high blood pressure and cardiovascular disease.

Objectives: Our objectives were to study the effects of different hormone replacement therapies on the function and morphology of isolated resistance arteries, and to look for their mechanistic basis.

Design and Setting: This was a randomized, placebo-controlled double-blind study in a University hospital, along with laboratory based studies.

Patients and Interventions: We isolated resistance arteries in sc biopsies from 55 postmenopausal women before and after 3-month therapy with estradiol (E2), medroxyprogesterone acetate (MPA), E2 plus MPA, or placebo. In addition, we studied isolated human endothelial cells.

Main Outcome Measures and Results: Artery flow-mediated dilatation was augmented after treatment with E2 or E2 plus MPA, whereas MPA or placebo had no effect. Pressure-induced myogenic tone was reduced by E2 plus MPA, whereas it was unchanged in the other groups. Scanning microscopy showed that E2 improved endothelial cell morphology and decreased signs of endothelial apoptosis, but the addition of MPA impaired these events. E2, MPA, or the combination all increased the expression and phosphorylation of the actin-binding protein, moesin and of the focal adhesion complex controller, focal adhesion kinase, and induced the rearrangement of cytoskeletal actin and vinculin fibers. All treatments promoted endothelial cell horizontal migration, with E2 inducing the strongest effect.

Conclusions: This study suggests that hormone replacement therapy with estrogens or in combination with MPA may benefit the function of resistance arteries and may preserve the morphological integrity of endothelial cells by regulatory actions on the cytoskeleton.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
The hormonal changes after the menopause increase the risk for cardiovascular diseases (1). Hormone replacement therapy (HRT) is believed to induce cardiovascular protection (2). However, clinical trials have challenged this assumption (3, 4). A recent Women's Health Initiative subgroup analysis shows that in women enrolled within 10 yr from the menopause receiving estrogens there is a significant reduction of a composite outcome of coronary events (5). This is consistent with the earlier results suggesting that HRT reduces cardiovascular risk only in young postmenopausal women (6).

Endothelial dysfunction gradually ensues after the menopause (7). Recently, a "healthy endothelium" concept has been proposed, hypothesizing that the cardiovascular effects of HRT may largely depend on normal endothelial function, whereas "diseased" endothelial cells may not respond to steroids (8). In agreement, the Cardiovascular Health Study shows favorable vascular effects of HRT only in patients without atherosclerosis (9).

Endothelial cells are primary targets of sex steroids. Although estrogens improve endothelial function (10), less is known about progesterone or synthetic progestins, whereas recent studies suggest that progesterone and medroxyprogesterone acetate (MPA) might result in different biological effects in endothelial cells (11).

Combined HRT enhances endothelium-dependent vasodilatation (12, 13). Our previous report indicates that small arteries from postmenopausal women display impaired responses to flow- and to the endothelium-dependent agonist bradykinin (BK), and that endothelial monolayers from these vessels show signs of endothelial dysfunction (14). In the present study, we investigated how different HRT regimens affect flow- and BK-induced dilatation in isolated sc resistance arteries from postmenopausal women. We also assessed the levels of pressure-induced myogenic tone, and used scanning electron microscopy to evaluate the morphological changes in the endothelial cell layers before and after HRT.

We recently showed that estradiol (E2) regulates endothelial migration by inducing prominent morphological changes via a remodeling of the actin cytoskeleton (15). Therefore, we explored if the morphological and functional changes observed in resistance arteries were associated with alterations of cytoskeletal-regulating molecules, of the cytoskeleton, and of cell migration in cultured human endothelial cells.


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

A total of 66 healthy postmenopausal female participants were enrolled and randomly assigned to four treatment groups for 3 months: the estrogen replacement group [n = 16, E2, Femanest (AstraZeneca Sverige AB, Södertälje, Sweden), 2 mg/d]; MPA replacement group [n = 18, MPA, Gestapuran (LEO Pharma AB, Malmö, Denmark), 5 mg/d]; combination group (n = 16, E2 plus MPA); and placebo group (n = 16).

Baseline characteristics of pre-HRT postmenopausal women randomized to each group are shown in Table 1Go.


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  		TABLE 1. Comparisons of demographical and clinical characteristics of postmenopausal women before treatment (pre-HRT) and after 3-month continuous per oral treatment with E2, MPA, E2 plus MPA, or control

 
Blood sampling and biochemical analyses

Venous blood samples were obtained, and biochemical analysis was performed using standard techniques.

Biopsy technique and fat tissue handling

Two sc fat biopsies (~2 x 1.5 x 1.5 cm) were obtained after application of 1% prilocaine (Citanest; AstraZeneca PLC, London, UK) for local anesthesia from the lower left (pre-HRT) and right (post-HRT) abdominal regions as previously reported (14).

Assessment of vascular function in isolated arteries

Subcutaneous small arteries (internal diameter ~220 µm, 2- to 3-mm length) were dissected from the biopsies and mounted on a pressure myograph (Living Systems Instrumentation Inc., Burlington, VT) as previously described (16, 17).

Flow-mediated and endothelium dependent/independent dilatation and pressure-induced myogenic tone

Flow-mediated and endothelium dependent/independent dilatation or pressure-induced myogenic tone was measured as previously described (14).

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)] for 24 h. They were then postfixed in 1% osmium tetroxide in sodium cacodylate buffer [0.15 M (pH 7.3)] containing 75 mM sucrose. The samples were then examined under a scanning electron microscope JEOL 820 (JEOL USA, Inc., Peabody, MA).

Cell cultures and treatments

Human umbilical vein endothelial cells (HUVECs) were cultured and treated as described (11, 15). E2, progesterone, and MPA were from Sigma-Aldrich (St. Louis, MO).

Cell immunofluorescence

HUVECs were grown on coverslips and exposed to treatments as previously described (15). Immunofluorescence was visualized and recorded using an Olympus BX41 microscopy and digital camera (Hamburg, Germany).

Immunoblottings

Cell lysates were separated by SDS-PAGE. Bands were visualized with enhanced chemiluminescence.

Endothelial cell migration assays

Endothelial cell migration was assayed with razor scrape assays (15).

Data analysis and statistics

Values in the text and figures are given as mean ± SEM or mean ± SD. Statistical differences between mean values were determined by ANOVA. All differences were considered significant at P < 0.05.

For a detailed procedure, or information on all the aforementioned experiments or data analysis and statistics, see the supplemental Subjects and Methods, which is published as supplemental data on The Endocrine Society's Journals Online web site at http://jcem.endojournals.org.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effects of different HRT regimens on blood pressure, plasma lipids, and endometrial thickness

Treatment with HRT for 3 months had no impact on blood pressure in postmenopausal women. A significant decrease of plasma low-density lipoprotein (LDL)-cholesterol was found only in the E2 plus MPA group (Table 1Go). The LDL to high-density lipoprotein (HDL) ratio was favorably altered by E2 plus MPA, however, no changes were found in the E2, MPA, and control groups. Concentrations of total cholesterol, fasting plasma triglyceride, P-HDL were similar in all groups before and after treatment (Table 1Go). Endometrial thickness increased only in the E2 group.

Effects of different HRT regimens on flow-induced dilatation in resistance arteries

Flow-mediated dilatation in sc arteries obtained from postmenopausal women at enrollment was similar among the four groups (Fig. 1Go) (P = 0.87). Flow-mediated dilatation was significantly increased in arteries from women treated with E2 or E2 plus MPA: maximal changes in diameter, pre-HRT 28 ± 7% vs. post-HRT 68 ± 15% in the E2 group (Fig. 1AGo); pre-HRT 24 ± 8% vs. post-HRT 70 ± 12% in the E2 plus MPA group (Fig. 1CGo). On the contrary, treatment with MPA or placebo had no effect.


Figure 1
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  		FIG. 1. Flow-mediated dilatation, expressed as the percent (%) change in diameter before and after 3 months with E2 (A), MPA (B), E2 plus MPA (C), or placebo (D) in small resistance arteries. *Statistical significance.

 
Effects of different HRT regimens on endothelium-dependent dilatation

Baseline endothelium-dependent dilatation induced with BK in resistance arteries did not differ in the four groups, and BK-mediated dilatation was not affected by any of the HRT regimens (for additional data, see supplemental Fig. 1, which is published as supplemental data on The Endocrine Society's Journals Online web site at http://jcem.endojournals.org).

We also tested the dilatory response to acetylcholine (Ach) and sodium nitroprusside (SNP). Neither Ach- nor SNP-mediated relaxation changed in association with the different HRT regimens (data not shown).

Effects of different HRT regimens on pressure-induced myogenic tone

Before treatment, arterial myogenic tone achieved at all pressure steps (60, 80, and 100 mm Hg) was not different among the different groups (Fig. 2Go). After 3-month treatment, the magnitude of increase of pressure-induced myogenic tone diminished significantly in the arteries from postmenopausal women treated with E2 plus MPA (e.g. increases of myogenic tone at 100 mm Hg: pre-HRT 20 ± 5% vs. post-HRT 9 ± 3%). In contrast, no change was found in the arteries of the other three groups (Fig. 2Go).


Figure 2
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  		FIG. 2. Effect of HRT on pressure-induced myogenic tone before and after treatment with E2, MPA, and E2 plus MPA. Myogenic tone was similar in the placebo group (data not shown in the figure) *, P < 0.01.

 
Endothelial morphology

Endothelial layers from postmenopausal women (in all baseline samples as well as in posttreatment samples from women administered placebo) displayed signs of endothelial cell death, as indicated by the presence of endothelial cell blebs, fractured cell membranes, and the loss of intercellular connections, partial denudation, detachment from the underlying basal lamina, and the attachment of platelets, lymphocytes, and protein aggregates (Fig. 3Go, A–C, upper boxes, and D).


Figure 3
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  		FIG. 3. Scanning electron microscopy (1000-fold magnification) of endothelial cell layers in isolated resistance arteries from the same postmenopausal women at baseline and after treatment for 3 months with E2 (A), MPA (B), E2 plus MPA (C), or placebo (D). Inner layer of sc arteries collected present signs of endothelial cell death [e.g. blebs, "white & dry" cells indicating apoptosis (Ap)], denudation (De), attachments of platelets (Pl), white blood cells (WBC), and protein (Pro) aggregates, fractured basal membranes (Mb), loss of intercellular junctions (Jn), and crater-like defects (craters) in endothelial cells. E, Endothelial cell (EC) number per 50 µm2 area of endothelial cell layer was counted by Wilcoxon test after treatments for 3 months. F, The number of cells with signs of apoptosis was indicated with a cell death index after treatments for 3 months. E and F, Asterisks indicate significant differences vs. baseline. Please note that in E and F, all baseline results are pooled.

 
Treatment with E2 for 3 months was associated with a reversal of these morphological derangements, and the arteries displayed continuous layers of tightly connected endothelial cells and the absence of platelet or protein attachment (Fig. 3AGo, lower boxes), in association with an increased endothelial cell number (Wilcoxon test, endothelial cell count per 50 µm2 area of endothelial cell layer; n = 8; P < 0.05 vs. pre-HRT) (Fig. 3EGo). Moreover, treatment with E2 decreased the number of cells with signs of apoptosis (cytoplasmic blebs, cell shrinkage, and partial detachment from the basal lamina, Fig. 3AGo, lower boxes), with a reduced cell death index (n = 8; P < 0.05 vs. pre-HRT; Fig. 3FGo).

Combined treatment with E2 plus MPA or MPA alone did not result in improvements of endothelial morphology (Fig. 3Go, B and C). In addition, treatment with MPA was associated with an increased attachment of platelets and proteins to endothelial cells, and with the presence of crater-like defects (Fig. 3CGo).

Effects of different HRT regimens on endothelial actin cytoskeleton

At baseline, actin fibers were arranged longitudinally through the major axis of endothelial cells (Fig. 4AGo). After exposure to E2 (1 nM), MPA (10 nM), or E2 plus MPA for 48 h, cellular actin changed its spatial organization, concentrating at the cell membrane to form a typical cortical actin complex. Specialized membrane structures, such as focal adhesion complexes, pseudopodia could be observed. In parallel, increased expression of the actin-regulatory protein, moesin, as well as increased phosphorylation were found in cells treated with E2, MPA, or the combination (Fig. 4Go, B and C).


Figure 4
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  		FIG. 4. A, HUVECs were treated with E2 (1 nM), MPA (10 nM), or E2 plus MPA for 48 h. Immunofluorescence shows changes of actin fibers localization and the formation of specialized cell membrane structures (yellow arrows indicate pseudopodia, green arrows show ruffles, and light blue arrows indicate focal adhesion complexes). B, Protein extracts from endothelial cells treated with E2 (1 nM), MPA (10 nM), or E2 plus MPA for 48 h were assayed with Western analysis for moesin or Thr558-phosphorylated moesin (P-moesin). C, Band intensity of each protein was analyzed using a quantitative digital imaging system. *, P < 0.05 vs. corresponding control (CON).

 
Effects of different HRT regimens on endothelial focal adhesion complexes

Exposure of human endothelial cells to E2 (1 nM), MPA (10 nM), or E2 plus MPA for 48 h results in similar modifications of the intracellular localization of vinculin fibers, with a concentration of these fibers at the cell membrane, in actin-enriched areas where endothelial focal adhesions are formed (Fig. 5AGo).


Figure 5
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  		FIG. 5. A, HUVECs were treated with E2 (1 nM), MPA (10 nM), or E2 plus MPA for 48 h. Vinculin fibers, actins, and nuclei were stained (light blue arrows indicate focal adhesion complexes). B, Phospho-Tyr397 FAK (P-FAK), actin, and nuclei in HUVECs were stained after the indicated treatments (light blue arrows indicate active FAK at the cell membrane). C, After the indicated treatments, protein extracts from endothelial cells were assayed with Western analysis for their content of P-FAK, FAK, vinculin, eNOS, and actin proteins. D, Band intensity of each protein was analyzed. *, P < 0.05 vs. corresponding control (CON). #, P < 0.05 with corresponding E2 treatment.

 
All the active treatments (E2, MPA, or E2 plus MPA) result in the increased intracellular content of Tyr397-phosphorylated focal adhesion kinase (FAK) and in the localization of active FAK to the cell membrane at adhering sites (Fig. 5BGo). This is confirmed by Western analysis, showing increased FAK protein expression and Tyr397-phosphorylation (Fig. 5Go, C and D). Interestingly, the endothelial content of the endothelial nitric oxide synthase (eNOS) was increased exclusively by E2 and not MPA, and the addition of MPA to E2 prevented the increase in eNOS expression. As a control, cellular actin expression did not change under any of the treatments (Fig. 5Go, C and D).

Effects of different HRT regimens on endothelial cell movement

HUVECs treated with all hormone regimens (E2 1 nM, MPA 10 nM, or E2 plus MPA) displayed an enhanced migration toward the cell-denuded area in comparison to vehicle-treated cells, with the maximal effect exerted by E2 (Fig. 6Go).


Figure 6
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  		FIG. 6. A, HUVECs were treated with E2 (1 nM), MPA (10 nM), or E2 plus MPA for 48 h. Representative migration images are shown. B, Cell migration distances were measured, and values are presented as percentage (%) of migration distance from the starting line of vehicle-treated cells ± SD. *, P < 0.01 vs. control (CON).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study provides new insights on the vascular effects of different HRT preparations. In particular, different hormonal combinations result in distinct effects on flow-mediated dilatation, pressure-induced myogenic tone, and endothelial cell morphology in isolated resistance arteries from postmenopausal women. These actions are coupled with specific effects on actin remodeling and endothelial cell movement related to the recruitment of the actin-regulatory protein, moesin, as well as of the controller of the formation of focal adhesion complexes, FAK. These results support the role of HRT for cardiovascular protection (6, 18).

Our study is the first to show effects of different HRTs on the function of peripheral resistance arteries of postmenopausal women. Previous studies from our laboratory have shown effects of E2 on flow- and BK-mediated dilatation, and pressure-induced myogenic tone after prolonged in vitro incubations (14). This study shows that the addition of MPA to E2 does not impair the improvement in flow-mediated dilatation in resistance arteries but also that MPA alone has no effect. Several mechanisms have been proposed by which estrogens may up-regulate nitric oxide (NO) release in response to shear stress, such as activation of eNOS (19, 20) or other factors that could augment the bioavailability of NO (21).

Although flow-mediated dilatation in these vessels was enhanced by E2, dilatation induced by NO-related agonists, such as BK and Ach or by the NO donor SNP, was not affected by any of the HRT regimens. This suggests that the ability to respond to endothelium-dependent agonists and to NO donors is not impaired in the vessels of these young postmenopausal women and that the addition of estrogens does not alter the response to these stimuli, as previously suggested (22). The range of flow rates used in the present investigation is compatible to that used in other investigations (17, 23). It is difficult to relate the shear stress values achieved in such types of studies with those in vivo in the peripheral circulation because those have yet to be established.

Contrary to the results on flow-mediated vasodilatation, myogenic tone was significantly reduced in patients receiving combined E2 plus MPA. Although we have not compared myogenic tone in arteries after the removal of endothelium, MPA has had some specific actions on smooth muscle cells, at least on cell proliferation (24).

Interestingly, only E2 administration turns into a normalization of endothelial morphology. This finding is consistent with studies demonstrating effects of estrogen on re-endothelialization in animal vascular injury models (25), or protective effects of estrogens on hypoxia (26) or oxidative stress-induced endothelial cell apoptosis (27). However, the addition of MPA to E2 obstructed these actions, suggesting that this progestin might alter some structural features of endothelial cells, which may eventually be relevant for endothelial function.

The parallel findings of altered actin and vinculin fiber localization during the administration of E2, MPA, or the combination further confirm this hypothesis, highlighting the presence of specific actions of these steroidal compounds on the structure/function of endothelial cells. We find that E2 and MPA similarly activate moesin as well as of FAK. These effects are coupled with visible actions on the remodeling of actin and vinculin fibers, which are concentrated at the cell membrane during exposure to these steroids, supporting the formation of pseudopodia and focal adhesion complexes.

Because these structures are implicated in cell movement, we also show that in the presence of E2, MPA, or the combination, endothelial cells are driven to migrate in horizontal wound-healing assays. However, E2 alone is more effective than MPA, and the addition of MPA results in a partial reduction of the effect of E2, indicating that MPA might in part hinder the positive effects of E2 on endothelial morphology and movement, in line with the electron microscopy data. However, the basis for this interference is currently unknown and will be the target of future studies.

Our findings of favorable actions of HRT on some markers of peripheral vascular function in postmenopausal women may seem inconsistent with the outcomes from the Women's Health Initiative trial (4). However, this trial did not take aging into proper account, and most of the participants missed the alleged "window of opportunity" to start and benefit from HRT (28). In addition, the progressive worsening of the status of the vessels might add to the aging process because a recent study showed that an average of 3.2-yr treatment with unopposed estrogen or estrogen plus progestins in elder postmenopausal women with established coronary heart disease did not result in significant improvements in endothelial vasodilatation (29). Overall, these findings suggest that there are age-related alterations in endothelial function that are probably not reversible by HRT.

It must also be acknowledged that our study has some limitations. We used measurements of flow-mediated and agonist-mediated dilatation to study the effects of HRT on vasodilatation. Such techniques are widely accepted for evaluation of endothelial function in vivo and in vitro (30, 31), as well as for studies in isolated arteries (32), but other methods could possibly provide slightly different results. In addition, we used HUVECs for the cell culture experiments, and not endothelial cells from the microcirculation. This difference might explain some internal discrepancy, such as the evidence that MPA abolishes the E2-dependent increase in eNOS expression in HUVEC, whereas it does not reduce the E2-induced flow-mediated dilatation in isolated arteries.

In summary, this study suggests that HRT with estrogens or in combination with MPA may benefit the function of resistance arteries and may preserve the morphological integrity of endothelial cells. These actions may be important for the prevention of overt endothelial malfunctions, which in these vessels may be implicated in the development of high blood pressure and other cardiovascular diseases in postmenopausal women. Because the effects on the studied targets vary significantly based on the combination of hormones, further studies on other compounds currently in use for HRT could be important to understand better the vascular actions of these therapies.


   Acknowledgments
 
We thank Margareta Ström, Lena Ydenius, and Maria Karlsson for assistance recruiting participants at the Department of Women's Health, and Professor Kjell Carlström for handling hormone analysis.


   Footnotes
 
Supported by grants from the Swedish Heart and Lung Foundation, Swedish Society of Medicine and Center for Gender Medicine at Karolinska Institutet (to K.K.), and by the Research Project of National Interest Grant 2004057090_007 by the Italian University and Scientific Research Ministry (Ministero dell'Università e della Ricerca) (to T.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 4, 2008

1 K.K. and X.-D.F. contributed equally to this work. Back

Abbreviations: Ach, Acetylcholine; BK, bradykinin; eNOS, endothelial NO synthase; E2, estradiol; FAK, focal adhesion kinase; HDL, high-density lipoprotein; HRT, hormone replacement therapy; HUVEC, human umbilical vein endothelial cell; LDL, low-density lipoprotein; MPA, medroxyprogesterone acetate; NO, nitric oxide; SNP, sodium nitroprusside.

Received November 30, 2007.

Accepted February 25, 2008.


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 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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