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(Circulation. 2000;101:2342.)
© 2000 American Heart Association, Inc.

Brief Rapid Communications

Estrogen-Induced Vasoprotection Is Estrogen Receptor Dependent

Evidence From the Balloon-Injured Rat Carotid Artery Model

Stephen Bakir, MD; Tatsuhiko Mori, MD; Joan Durand, BS; Yiu-Fai Chen, PhD; J. Anthony Thompson, PhD; Suzanne Oparil, MD

From thr Vascular Biology and Hypertension Program, Division of Cardiovascular Disease, Department of Medicine (S.B., T.M., J.D., Y.-F.C., S.O.), and Division of Transplantation, Department of Surgery (J.A.T.), University of Alabama at Birmingham, Ala.

Correspondence to Stephen Bakir, MD, 1047 ZRB, 703 South 19th St, Birmingham, AL 35294. E-mail sbakir{at}uab.edu

	Abstract

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Background—Previous studies have shown that estrogen (E2) is vasoprotective in multiple animal models of vascular injury, including mice with homologous disruptions of either the or ß isoforms of the estrogen receptor (ER) gene, calling into question the ER dependency of the vasoprotective effect. This study used ICI 182,780, a nonselective ER antagonist, to test the hypothesis that the vasoprotective effect of E2 in the rat carotid injury model is ER mediated.

Methods and Results—Intact female Sprague-Dawley rats were divided into 4 groups and treated with the nonselective ER antagonist ICI 182,780 (ICI; 0.5, 1.5, or 5 mg · kg^-1 · d^-1, subcutaneously [S.C.]) or vehicle, beginning before balloon injury of the right common carotid artery and continuing for 14 days afterward. Four groups of ovariectomized rats (OVX) were treated with 17ß estradiol (E2) (20 µg · kg^-1 · d^-1, S.C.) alone or combined with ICI 5 mg · kg^-1 · d^-1, S.C.; with ICI 5 mg · kg^-1 · d^-1 alone; or with vehicle according to a similar protocol. Two weeks after injury, rats were killed, and the carotid arteries were evaluated for neointima formation using morphometric analysis. ICI 182,780 blunted the E2-related protective effect and increased neointima formation in injured carotid arteries of intact female rats in a dose-dependent fashion. ICI had no effect on neointima formation in OVX, but addition of ICI to E2 in OVX blocked the inhibitory effect of exogenous E2 on neointima formation.

Conclusions—These results indicate that the vasoprotective effect of E2 in the balloon-injured rat carotid artery model is mediated by ER.

Key Words: restenosis • hormones • carotid arteries • estrogen receptors

	Introduction

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Previous work from our laboratory has demonstrated a sexual dimorphism in the response to balloon injury of the rat carotid artery, with intact male rats developing a more robust neointimal response to injury than intact females.¹ Gonadectomy of female but not male rats resulted in increased neointima formation, and treatment with E2 but not testosterone inhibited neointima formation in gonadectomized rats of both sexes, indicating that the observed sexual dimorphism was E2-dependent. Studies using a mouse carotid injury model showed that E2 provided vascular protection in wild-type mice and in mice with homologous disruptions of either the or ß isoform of the ER.^{2 3} This suggests that expression of either of the 2 functionally distinct ERs is sufficient to protect against pathological responses to vascular injury, that a third, uncharacterised ER may be responsible for the vascular protective effects of E2, or that a nongenomic, nonreceptor mediated signaling mechanism is involved.

In this study, we used ICI 182,780, a nonselective ER antagonist, alone in intact female rats (INT) or alone or in combination with 17ß-estradiol in ovariectomized (OVX) rats to test the hypothesis that E2-dependent vasoprotection in this model is ER-mediated.

	Methods

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Ten-week-old female Sprague-Dawley rats obtained from Charles River Breeding Laboratories (Wilmington, MA) were randomized to undergo bilateral ovariectomy under ether anesthesia or to remain intact. Three days later, INT were divided into 4 groups and were treated with either the nonselective ER antagonist ICI 182,780 (0.5, 1.5, or 5 mg · kg^-1 · d^-1, subcutaneously [S.C.]) or vehicle (V) initiated 3 days before balloon injury of the right common carotid artery and continuing for 14 days afterward. Four groups of OVX rats were treated with either 17ß estradiol 20 µg · kg^-1 · d^-1, S.C.) (OVX+E2); ICI 5 mg · kg^-1 · d^-1 (OVX + ICI 5.0), combined E2 and ICI 5 mg · kg^-1 · d^-1 (OVX+E2+ICI 5.0); or vehicle (OVX+V) according to a similar protocol.

Rats were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg). The right carotid artery was isolated by a middle cervical incision, suspended on ties, exposed, and injured with an inflated 2F Fogarty balloon catheter (Baxter V. Mueller), as previously described.¹

Two weeks after injury, rats were killed with an overdose of sodium pentobarbital (75 mg/kg) and perfused with 10% formalin at a pressure of 120 mm Hg. The uterus was removed, blotted dry, and weighed to assess E2 effects. The carotid arteries were fixed and processed for morphometric examination as previously described.¹ Uteri were processed similarly for histologic examination. Morphometric analysis was performed with a Bioquant II Morphometric system by a single examiner, who was blinded with respect to the experimental group to which each sample belonged. Neointima formation in the injured artery was expressed as the absolute area of intima and the ratio of the intimal area to the medial area. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985).

At the time of sacrifice, a 1 mL blood sample was removed from the abdominal aorta. Serum 17ß-estradiol levels were determined by commercially available radioimmunoassay kits (Diagnostic Products Corp) with an 8 pg/mL sensitivity.

Results were expressed as mean±SEM, and data were analyzed with StatView 4.0 software. Statistical comparisons of body weight, uterine weight, serum estradiol level, neointimal area, medial area, and ratio of the intimal area to the medial area among experimental groups were performed with a 1-way ANOVA. When significant differences were identified, the Sceffé’s multiple range test was applied to determine the level of significance. P<0.05 was considered to be significant.

	Results

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Neointima formation in INT rats was greatly attenuated compared with OVX rats (Figures 1D and 2). Treatment of INT rats with ICI increased neointima formation in a dose-dependent manner, and the neointimal area in the INT+ICI 5.0 group was not significantly different from that of the OVX+V rats (Figures 1E and 2). Neointima formation was robust in OVX rats (Figures 1A and 2) and was not altered by ICI administration. Treatment with E2 inhibited neointima production by 61% (Figures 1B and 2). Addition of ICI 5.0 to OVX rats treated with E2 completely blocked the E2-mediated reduction in neointima formation (Figures 1C and 2).

View larger version (109K): [in this window] [in a new window]   Figure 1. Representative light micrographs of injured carotid arteries from OVX rats at 14 days after balloon injury. A, OVX rats treated with V. B, OVX rats treated with E2. C, OVX rats treated with E2 and ICI 182,780. D, INT treated with V. E, INT rats treated with ICI 182,780. F, OVX rats treated with ICI 182,780.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of E2 (20 ìg · kg-1 · d-1, S.C.) and ICI 182,780 (ICI; 0.5, 1.5, and 5.0 mg · kg-1 · d-1, S.C.) to INT, or of combined E2 and ICI treatment to OVX rats on neointima formation at 14 days after balloon injury of the carotid artery. Ratios of neointimal to medial area are presented as means±SEM. #P<0.05, ##P<0.01, compared with the INT+V group. **P<0.01, compared with the OVX+V group.

Morphometric analysis showed that the neointimal area of carotid arteries from INT+V rats was 0.072±0.010 mm², a 48.9% reduction from the OVX+V group (0.141±0.010 mm²). Administration of ICI to INT rats dose dependently increased neointima formation (Table), and in the highest ICI dose group, neointimal area (0.122±0.015 mm²) was similar to that seen in OVX+V rats. Neointimal area in OVX+E2 rats was 0.051±0.004 mm², a 61% reduction compared with OVX+V rats (Table). Treatment with ICI 5.0 did not alter neointimal area in OVX rats but did abolish the protective effect of E2 (neointima 0.124±0.017 mm²) in these animals. In this model of vascular injury, the medial area was not altered, so intima/media ratios (Figure 2) closely reflected absolute neointimal area.

View this table: [in this window] [in a new window]   Table 1. Physiologic Indices 14 Days After Balloon Injury in INT and OVX Rats Treated with V, E2, or Varying Concentrations of ICI

Serum 17-ß estradiol levels were similar in OVX+E2 and INT+V rats (Table); ICI treatment in the INT rats increased estradiol levels dose dependently. In the highest ICI dose group, 17-ß estradiol levels were 2-fold higher than in INT+V. In OVX+E2 rats, ICI produced a nonsignificant increase in serum 17-ß estradiol levels compared with OVX+E2 (P=0.95, Table). Serum 17-ß estradiol concentration in OVX+V rats was at the level of sensitivity of the assay.

In INT rats, ICI treatment decreased uterine weight in a dose-dependent fashion (Table). Uterine weights in OVX rats were significantly increased by E2 treatment to values similar to those in INT; addition of ICI completely blocked the trophic effect of E2 on the uterus, as reflected in both uterine weights and the histologic appearance of uterine epithelial (endometrial) cells (data not shown).

	Discussion

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Our results demonstrated the following: (1) ICI blocked the vasoprotective effects of endogenous E2 on neointima formation in INT rats in a dose-dependent fashion. (2) When ICI was combined with exogenous E2 in a dose that resulted in physiological levels of circulating E2, vasoprotective effects of E2 were abolished in OVX rats. (3) ICI administration did not alter neointima formation in OVX rats. (4) ICI administration to both INT and E2-treated OVX rats decreased uterine weight and reduced endometrial hyperplasia. Thus, the nonspecific ER antagonist ICI 182,780 (antagonist of both ER and ERß) inhibited the vasoprotective effects of endogenous E2 in INT, and of exogenous E2 in OVX rats, indicating for the first time that the vasoprotective effects of E2 are ER-dependent.

Studies in animal models of atherosclerosis and vascular injury, as well as in human subjects, have revealed a variety of mechanisms by which E2 promotes normal vascular function and attenuates the cellular response to noxious stimuli.^{4 5} These include indirect systemic effects (alterations in plasma lipoprotein profiles, improvement in hemodynamic parameters, antioxidant actions, and beneficial effects on coagulation and fibrinolytic systems) and direct effects on the vessel wall.^{4 5 6 7} Many of the direct vascular effects of E2 involve modulation of gene programs associated with vascular growth and migration/proliferation of a variety of vascular cell types. These E2-induced alterations in gene expression are ER-dependent and are mediated by the classic ER signaling pathway, which involves entry of E2 into the cell, translocation to the nucleus, binding to the nuclear ER ( or ß subtype), binding of the resultant complex to E2 responsive target genes, and transcriptional regulation of these genes.⁸ This signaling mechanism requires hours to achieve its final effects. In addition, E2 acts via more rapid nongenomic mechanisms to mediate more immediate vascular responses, eg, inhibition of inward Ca²⁺ current in vascular smooth muscle cells and stimulation of endothelium-dependent vasodilation. Whether these nongenomic responses are ER-dependent is a matter of controversy.

The rapid nongenomic responses to E2 do not require gene transcription and have classically been thought not to involve interactions with ERs. Only recently has it been shown that both ER and ERß are expressed in cell membranes in addition to their classical nuclear positions and that short-term effects of E2 relevant to cardiovascular function can be mediated by classical ERs functioning in a novel, nongenomic manner.^{9 10}

This study provided the first direct evidence that the vasoprotective effects of E2 in the rat carotid injury model are mediated through ERs. Together with the previous observations of Iafrati et al² and Karas et al³ , these findings suggest that expression of either the ER or ERß subtype in injured blood vessels is sufficient to activate the signaling mechanism(s) responsible for E2-mediated vasoprotection or that a novel, not yet discovered, ER may be responsible. Whether these vasoprotective effects are genomically and/or nongenomically mediated and dependent on nuclear and/or membrane-bound ERs remains to be determined, as does the signaling pathway involved. A clearer understanding of these E2-related effects may facilitate translation of the benefits seen in animal models into effective preventative or therapeutic strategies for cardiovascular disease in humans.

	Acknowledgments

 
This work was supported in part by Grants HL-07457, HL-45990, HL-57270, HL-64614, and DK-51629 from the National Institutes of Health and a Grant-in-Aid (97-50665N) from the American Heart Association. The authors thank Sandra Camp for assistance in the preparation of the article and Dr Qiang Li for technical assistance.

Received December 22, 1999; revision received March 27, 2000; accepted March 30, 2000.

	References

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3. Karas RH, Hodgins JB, Kwoo, et al. Estrogen inhibits the vascular injury response in estrogen receptor ß deficient mice. Proc Natl Acad Sci USA. 1999;96:15133–15136.[Abstract/Free Full Text]

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bakir, S. Articles by Oparil, S. Search for Related Content PubMed PubMed Citation Articles by Bakir, S. Articles by Oparil, S. Related Collections Restenosis Smooth muscle proliferation and differentiation Receptor pharmacology