Increased Expression of Estrogen Receptor-ß mRNA in Male Blood Vessels After Vascular Injury

From the Molecular Cardiology and Tupper Research Institutes (S.K.K., R.H.K., M.E.M.) and the Surgical Research Division (S.K.K.), New England Medical Center, Tufts University School of Medicine, Boston, Mass; the Maine Medical Center (V.L.), Portland, Me; and the Department of Medical Nutrition and Center for Biotechnology (G.G.J.M.K., J.-Å.G.), Karolinska Institute, Uppsala, Sweden.

Correspondence to Michael E. Mendelsohn, MD, Molecular Cardiology Research Center, 750 Washington St, #80, Boston, MA 02111. E-mail michael.mendelsohn{at}es.nemc.org

	Abstract

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Abstract—Estrogen exerts direct effects on vascular endothelial and smooth muscle cells that are important for vascular protection. Estrogen receptor- (ER) is expressed in vascular cells from males and females and may mediate some of the effects of estrogen on vascular tissue. However, we recently found that estrogen is able to protect against vascular injury in ovariectomized female ER knockout mice. These mice express the newly described estrogen receptor-ß (ERß) in their aortas, suggesting that ERß may also mediate some of the direct effects of estrogen on the vasculature. In this study, the level of expression of ER and ERß mRNA in male rat aortas was examined before and after vascular injury using en face (Häutchen) preparations and in situ hybridization. Little or no change in ER expression was observed after vascular injury in either vascular endothelial or smooth muscle cells at any time point. In contrast, ERß mRNA was found to be expressed markedly after balloon injury. In endothelial cells, ERß was increased by 2 days after injury, and high levels of expression were maintained at 8 and 14 days. Furthermore, ERß expression was high in luminal smooth muscle cells at 8 and 14 days after injury and had decreased to low levels by 28 days after injury. These data demonstrate the presence of ERß in male vascular tissues and the induction of ERß mRNA expression after vascular injury, supporting a role for ERß in the direct vascular effects of estrogen.

Key Words: estrogen receptor • vasculature • knockout mouse • vascular injury • endothelium

	Introduction

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Ischemic cardiovascular events are the leading cause of morbidity and mortality in Western society. These diseases are uncommon in women before menopause, and postmenopausal estrogen replacement therapy decreases their incidence markedly, suggesting that estrogen protects against vascular disease.^{1 2 3} Although the beneficial effects of estrogen have been attributed previously to indirect effects of estrogen on classic risk factors, recent data suggest that these do not account for the majority of the protective cardiovascular effects of estrogen.^{4 5 6 7 8} Indeed, estrogen is now recognized to have direct effects on the blood vessel wall that are central to the beneficial effects of estrogen on vascular physiology and disease (reviewed in References 9 and 10^{9 10} ).

Many of the effects of estrogen on its nonvascular target cells are mediated through the first ER identified, a ligand-activated transcription factor now called ER (reviewed in References 11¹¹ to 13). Very recently, a second ER capable of regulating gene expression, ERß, was cloned from rat prostate tissue¹⁴ as well as from mice¹⁵ and humans.¹⁶ Although some domains of ER and ERß are homologous and share functional similarities, new data suggest differences in their tissue localization and in the mechanisms regulating their transcriptional activities.^{14 15 16 17} ER is known to be expressed^{18 19 20} and functional¹⁹ in vascular smooth muscle cells from male and female animals and humans, and functional ER has been demonstrated recently in vascular endothelial cells as well.^{21 22 23} However, ERß expression in vascular tissues after injury has not yet been studied.

At least 2 direct effects of estrogen on vascular function have been described: rapid effects on vasomotor tone and longer-term effects on vascular cell proliferation and atherosclerosis (reviewed in References 9 and 10^{9 10} ). Physiological estrogen replacement markedly suppresses the carotid arterial response to injury to the same degree in female wild-type and ERKO mice.^{24 25} These female ERKO mice express mRNA for ERß in their aortas,²⁵ suggesting that this receptor may mediate the protective effects of estrogen in the ERKO mice. In the present study, we sought to test the hypothesis that male animals also express ERß and to explore the expression of ER and ERß mRNA in male blood vessels after vascular injury.

	Materials and Methods

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Animal Studies
In situ studies were on tissues from 15 intact male Sprague-Dawley rats (400 g, 3 to 4 months old, Taconic, Germantown, NY). All studies were carried out with the approval of the Institutional Animal Care and Use Committee. All surgical procedures were carried out under general anesthesia by intraperitoneal injection of xylazine (2.2 mg/kg, AnaSed, Lloyd Laboratories) and ketamine (50 mg/kg body wt, Ketaset, Aveco Co, Inc). Early time points of wounded endothelium (10 hours and 2 days) were studied in the partially denuded rat aorta by passing an uninflated catheter along the vessel. Later time points (8 days, 2 weeks, and 4 weeks) were examined in vessels from which the endothelium was removed completely by passing an inflated embolectomy catheter along the vessel as described by Clowes et al.²⁶ We have previously published illustrations of this procedure,^{29 30} which in the aorta leads to outgrowth of endothelium from the intercostal arteries and the appearance of smooth muscle cells in the intima within 7 days after denudation. Since the normal rat aorta and carotid artery have no intimal smooth muscle cells, endothelial cells (and a rare inflammatory cell) are the only cell types present in the intima at time points earlier than 4 days. All rats were injected with Evans blue before they were killed to identify the areas of endothelial outgrowth. On en face preparations, endothelial cells were readily distinguished from smooth muscle cells by use of the following criteria: Endothelial cells are identifiable as a single coherent layer of cells originating from the intercostal arteries with oblong nuclei equidistant from each other and oriented with blood flow.^{29 30} Smooth muscle cells, on the other hand, appear anywhere within the denuded area, have randomly oriented nuclei that frequently overlap, and appear in multiple layers. The Häutchen technique reliably removes the entire luminal cell layer for examination; smooth muscle cells appear on the en face preparations as they migrate into the region of the lumen, which occurs only after day 4 in this model. The vasculature of all animals was perfusion-fixed with phosphate (0.1 mol/L, pH 7.4) buffered with 4% paraformaldehyde. For in situ hybridization and immunostaining, rats were killed at the indicated times after injury (between 4 hours and 4 weeks).

In Situ Hybridization
For en face preparations, vessel segments were cut open longitudinally, and the tissue was pinned out flat on polytetrafluoroethylene (Teflon) cards (luminal side facing up). Incubation with proteinase K (1 µg/mL, 37°C, Boehringer Mannheim Corp) took place for 15 minutes, followed by prehybridization for 2 hours with 0.3 mol/L NaCl/20 mmol/L Tris (pH 7.5)/5 mmol/L EDTA/1x Denhardt's solution/10% dextran sulfate/10 mmol/L dithiothreitol/50% formamide. T3 and T7 polymerase (Promega Corp) were used to generate both sense and antisense strand ³⁵S-UTP–labeled riboprobes. For ER, a 195-bp riboprobe corresponding to the 3' end (F domain) of the published rat sequence³¹ was generated from linearized pBluescript containing this B5TX1-EcoR1 fragment of the receptor. For ERß, a 400-bp riboprobe (EcoR1-AccI fragment) from the 5' untranslated region of the rat ERß¹⁴ was generated. This riboprobe has been validated by separate studies on nonvascular tissues and gives results similar to 2 separate and distinct ERß riboprobes in testis, prostate, and ovary tissue studies (see Reference 32³² ). For ER, 17 separate specimens were hybridized with the antisense probe, and 5 were hybridized with the sense probe. For ERß, 18 separate specimens were hybridized with the antisense probe, and 5 were hybridized with the sense probe. Photographs were taken from 14 different animals, with 8 preparations per aorta. After hybridization (at 55°C overnight), specimens were washed with 2x SSC/10 mmol/L ß-mercaptoethanol/1 mmol/L EDTA (twice for 10 minutes each) (1x SSC contains 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.0), treated with RNase A (20 µg/mL for 30 minutes at 37°C, Sigma Chemical Co), and washed in 2x SSC (as above), followed by a high-stringency wash at 55°C for 2 hours (0.1x SSC/10 mmol/L ß-mercaptoethanol/1 mmol/L EDTA). Subsequent steps followed the protocol as described.²⁹ The Häutchen procedure for en face preparations was carried out after the probe hybridization. Slides were coated with autoradiographic emulsion (Kodak, NTB2), exposed for 3 weeks, and then developed (Kodak, D-19). Vessel preparations were observed under the light microscope using dark-field, bright-field, and a combination of epiluminescence and bright-field illumination (reflective light). Estimates of ER and ERß mRNA were made by comparison of sense or background signal with those at each of the various time points and quantified according to an arbitrary scale (see Table legend) by an experienced observer (V.L.) blinded to probe identity and animal treatment.

View this table: [in this window] [in a new window]   Table 1. Summary of In Situ Hybridization Experiments for ER and ERß Expression in Balloon-Injured Aortas From Male Rats

	Results

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ER and ERß mRNA Expression in Male Animals After Vascular Injury
We have shown previously using RT-PCR methods that aortic vessels from female wild-type and ER-disrupted (ERKO) mice express mRNA for ERß at qualitatively similar levels.²⁵ To examine the expression of mRNA for ER and ERß after vascular injury, the surface cells of uninjured or balloon-injured rat aortas were studied using the en face (Häutchen) technique.²⁹ This procedure allows study of the surface endothelial and intimal smooth muscle cells migrating into the region as a single monolayer and provides a sensitive method for studying gene expression in these 2 cell populations. The en face technique was used to study expression of both ER and ERß in uninjured endothelial cells (the intact and normal vascular lining) as well as in endothelial cells localized to the advancing endothelial cell edge at various time points after injury. Since smooth muscle cells are observed in such preparations only at 4 to 8 days after injury,²⁹ ER and ERß expression in smooth muscle cells in the en face section were examined and compared at 8, 14, and 28 days after balloon injury. The Table summarizes the level of expression of ER and ERß in endothelial cells and smooth muscle cells in these experiments. ER expression by vascular endothelial cells was very modest in all cases and was barely above background at all time points examined (Table and Figure 1). Similarly, smooth muscle cells also expressed very little ER mRNA after injury (Table and Figure 1).

View larger version (140K): [in this window] [in a new window]   Figure 1. Photomicrographs of en face preparations of male rat aorta showing ER mRNA expression in endothelial and smooth muscle cells after balloon injury. Representative in situ hybridization using 35S-labeled riboprobes for ER is shown. A, ER sense control, 2 weeks, endothelial cells. B, ER antisense, 2 weeks, endothelial cells. C, ER sense control, 2 weeks, smooth muscle cells. D, ER, antisense, 2 weeks, smooth muscle cells. Bar=20 µm. Arrows depict leading edge of endothelial cells advancing to cover the wound. See also the Table.

In contrast, ERß was clearly expressed in both vascular endothelial cells and smooth muscle cells in the aortas of male rats after injury. For endothelial cells, ERß mRNA was moderately increased at the leading edge of endothelial cells by 2 days after balloon injury, and by 8 days leading edge endothelial cells showed high levels of expression of ERß mRNA (Table and Figure 2). This high level of expression of ERß in endothelial cells at the leading edge persisted at day 14 after injury. Expression levels of ERß behind the leading edge were similar to those seen in normal (uninjured) endothelium in sections examined (data not shown).

View larger version (188K): [in this window] [in a new window]   Figure 2. Photomicrographs of en face preparations showing ERß mRNA expression in vascular endothelial cells in male rat aorta after balloon injury. In situ hybridization using 35S-UTP-labeled riboprobes for rat ERß is shown. Arrows depict leading endothelial cell edge. A, Sense probe is seen in uninjured en face preparation of endothelial cell monolayer. B, Antisense probe in uninjured endothelial cell monolayer detects no ERß mRNA in the uninjured endothelium. C to F, ERß mRNA also was not increased in the endothelial cells at the wound edge at 10 hours (C) but was clearly increased at 2 days after injury (D) and was markedly increased at 8 days (E) and 2 weeks (F) after injury. All photomicrographs are representative examples obtained from 10 to 30 high-power fields (x400). Bar=20 µm.

Expression of ER and ERß in luminal smooth muscle cells was also examined. The en face technique does not allow determination of baseline gene expression in smooth muscle cells of normal vessels and allows study only of smooth muscle cells that migrate into the region of the luminal surface 4 days after vascular injury. This population of smooth muscle cells thus differs from other intimal or medial smooth muscle cell populations in the vessel wall. However, once smooth muscle cells have appeared on the luminal surface (ie, 8 days and 2 and 4 weeks), comparison of ER and ERß mRNA expression between time points is possible. After injury, ERß mRNA was moderately abundant in luminal smooth muscle cells at both 8 and 14 days, and in all cases, ERß message was significantly greater than that for ER in these cells (Table and Figure 3). By 28 days, the level of expression of ERß mRNA in the smooth muscle cell population had decreased to very low levels compared with those seen at the 8-day and 2-week time points.

View larger version (152K): [in this window] [in a new window]   Figure 3. Photomicrographs of en face preparations showing ERß mRNA expression in male rat aortic vascular smooth muscle cells after balloon injury. In situ hybridization using 35S-UTP–labeled riboprobes for rat ERß is shown. A, Sense probe in smooth muscle cells seen in the en face preparation 2 weeks after injury. B to D, Antisense probe demonstrating moderate to marked increases in ERß mRNA in vascular smooth muscle cells at the wound edge at 8 days (B) and 2 weeks (C) after injury. By 4 weeks (D), little or no increase in ERß mRNA was detectable in the smooth muscle cells. All photomicrographs are representative examples obtained from 10 to 30 high-power fields (x400). Bar=20 µm.

	Discussion

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The present study demonstrates the expression of ERß mRNA in male rat aortas before and after balloon denudation injury. The level of expression of mRNA for the ER and ERß after balloon injury of male rat aortas was strikingly different in these studies. ER mRNA was expressed only at very low levels in vascular endothelial and smooth muscle cells after injury. ERß mRNA expression, however, increased to high or very high levels in vascular endothelial cells after injury. In the smooth muscle cells that appeared at the luminal surface after injury, ERß mRNA was abundant at both 8 days and 2 weeks but declined to near baseline levels by 28 days after injury.

Estrogen inhibits the response to vascular injury in a variety of animal models.^{24 25 33 34 35 36 37 38 39 40 41} In particular, rat vascular tissues express functional estrogen,⁴² and estradiol inhibits myointimal proliferation after carotid balloon injury in gonadectomized male rats,^{39 40} the animal studied in these experiments. The high levels of ERß expression in vascular cells of injured male vessels in these studies, without significant changes in ER expression, are striking and raise the possibility that ERß may mediate some of the protective effects of estrogen in the setting of vascular injury. This hypothesis is consistent with the presence of ERß in the aortas of ERKO mice and the ability of estradiol to inhibit the vascular response to injury in these animals to the same degree as in their wild-type littermates.²⁵

Upstream regulatory regions of the ERß gene important to the observed induction of ERß message after vascular injury remain to be defined, and the potential growth factors or other stimuli induced by balloon injury that lead to increased ERß expression also are presently unclear. However, the observed increase in mRNA for ERß after balloon injury suggests that study of ERß may be important in the understanding of the direct vascular protective effects of estrogen. It will be important in subsequent studies to define carefully the relative abundance of ERß mRNA and protein in vascular cells and tissues from intact and ovariectomized female animals after injury. Furthermore, careful analysis of ER and ERß mRNA protein in vascular cells and tissues under various conditions will be important, including human cells and tissues from men, premenopausal women, and postmenopausal women, both untreated and on hormone replacement therapy. It will be especially important to examine cell culture expression of the ERs and to correlate in vitro with in vivo expression. Preliminary RT-PCR studies indicate that ERß is expressed in male and female vascular cells (data not shown), but it is already apparent that expression levels of ERß mRNA, like those of ER, vary with culture conditions, passage, and vascular bed of origin (see Reference 23²³ ). It will also be important to understand regional differences in the vascular expression of ERß in vivo and to examine specifically the role of ERß in mediating the vascular protective effects of estrogen. The recent development of ERß-specific antagonists and the ERß knockout mouse will allow this latter topic to be studied directly.

	Selected Abbreviations and Acronyms

 

ER = estrogen receptor ERKO = ER knockout PCR = polymerase chain reaction RT = reverse transcription

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants HL-30386 (Dr Karas) and HL-56069 (Dr Mendelsohn) and by a research fellowship from the Swedish Medical Research Council (Dr Kuiper). We are indebted to Patricia Nayak for expert preparation of the manuscript. Drs Lindner and Mendelsohn are Established Investigators of the American Heart Association.

Received December 29, 1997; accepted April 16, 1998.

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