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(Circulation. 1996;94:1402-1407.)
© 1996 American Heart Association, Inc.

Articles

Expression of an Estrogen Receptor by Human Coronary Artery and Umbilical Vein Endothelial Cells

Seunghee Kim-Schulze, PhD; Kelly A. McGowan, MD; Susan C. Hubchak, BS; Maria C. Cid, MD; Mary Beth Martin, PhD; Hynda K. Kleinman, PhD; Geoffrey L. Greene, PhD; H. William Schnaper, MD

the Department of Pediatrics, Northwestern University Medical School, Chicago, Ill (S.K.-S., S.C.H., H.W.S.); the Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Md (K.A.M., H.K.K.); the Department of Internal Medicine, Fundacio Clinic, Barcelona, Spain (M.C.C.); the Lombardi Cancer Center, Georgetown University School of Medicine, Washington, DC (M.B.M.); and the Ben May Institute, University of Chicago (Ill) (G.L.G.).

Correspondence to Seunghee Kim-Schulze, PhD, Pediatrics W-140, 303 E Chicago Ave, Chicago, IL 60611-3008. E-mail ski057@nwu.edu.

	Abstract

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Background Premenopausal women have much lower susceptibility to coronary artery disease than do men or postmenopausal women. It has been proposed that estrogen plays a role in cardioprotection, but little information is available regarding the mechanism by which estrogen may help to protect the vasculature. Here, we describe an estrogen receptor (ER) in human coronary artery and umbilical vein endothelial cells.

Methods and Results Human umbilical vein endothelial cells and human coronary artery endothelial cells were cultured in hormone-free medium for 48 hours before experiments. Estradiol (3.7 nmol/L) added to cultures promoted proliferation by a mechanism that is inhibited by the specific ER antagonist ICI182,780. Estradiol-treated cells incorporated twice the [³H]thymidine of hormone-free cells; this increase was prevented by ICI182,780. Endothelial cells from both sources stained in a nuclear pattern with an ER-specific antibody. Ribonuclease protection assay detected mRNA for the ER. Ligand-binding studies estimated 2x10⁴ to 8x10⁴ receptors per cell and a K_d of 5 nmol/L. Interaction of ERs with a consensus estrogen response element was shown by an electrophoretic mobility shift assay. In addition, an antibody against the ER supershifted the protein-DNA complex.

Conclusions These studies define the presence of an ER in human coronary artery and umbilical vein endothelial cells. They support the hypothesis that cardioprotective effects of estrogen are mediated, at least in part, through a classic steroid hormone receptor mechanism.

Key Words: estrogen • endothelium • receptors • cells

	Introduction

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The incidence of cardiovascular disease in premenopausal women is lower than in men, but it increases after menopause.¹ It has thus been proposed that estrogen has a cardioprotective effect.² A number of studies have indicated that estrogen directly affects the vascular system^{3 4 5} and may promote reestablishment of normal structure after vascular injury.⁶ These protective effects are proposed to be mediated through an ER.

Estrogen generally has been characterized as a steroid hormone that acts on tissues relevant to gonadal function, such as breast, ovaries, and uterus. Indeed, the ER has been characterized most fully by evaluation of tumors from these target tissues. Classically, biological effects of estrogen are mediated through intracellular receptors that act as ligand-activated transcription factors. In many cases, the activated ER specifically binds to an ERE, consequently leading to altered target gene expression.^{7 8 9}

Several analyses of radioligand binding indicate that tissues not classically defined as estrogen targets also express ER, including VSMCs.^{10 11 12} Studies of the effects of estrogen on blood vessels have largely examined modulation of VSMCs. These effects may be direct, via altered smooth muscle cell cGMP activity,^{13 14} or indirect, via endothelial NO synthesis.¹⁵ Recently, ER was identified in VSMCs from coronary arteries of premenopausal women.¹⁶ Expression was much lower in atherosclerotic vessels than in normal arteries, suggesting a role for the ER or its deficiency in atherosclerosis. We have documented that estrogen enhances several human endothelial cell functions, including proliferation, attachment, migration, and formation of capillary-like tubes in vitro. Furthermore, estrogen increases angiogenesis in vivo in mice.¹⁷ However, an ER has not been fully characterized in human endothelial cells.

Here, we report that estrogen enhancement of human endothelial cell proliferation is reduced by an ER antagonist. The presence of ER is demonstrated by specific cell staining, radioligand binding, and EMSA. These studies define an ER in HCAECs and HUVECs.

	Methods

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Endothelial Cell Culture
HUVECs and HCAECs were grown in a standard cell culture medium consisting of RPMI 1640 medium (Life Technologies) supplemented with 20% bovine calf serum (Hyclone Laboratories), 100 U/mL penicillin/streptomycin, 50 µg/mL gentamicin, 2 mmol/L glutamine, 5 U/mL heparin, and 200 µg/mL endothelial cell growth supplement (Collaborative Research). For experiments, the cells were switched to phenol red–free RPMI 1640 medium prepared identically to the standard medium except that 20% charcoal-stripped serum was used.¹⁷ The absence of phenol red avoids potential estrogen-like effects of this compound.¹⁸

Cell Proliferation Assay
Cells were plated in 24-well plates in standard endothelial cell medium. After the cells had attached, the medium was replaced with hormone-free medium for 48 hours. The cells were then cultured with 1.0 ng/mL (3.7 nmol/L) 17ß-estradiol, alone or in the presence of the specific ER antagonist ICI182,780 (Zeneca Pharmaceuticals). As a control, similar studies were performed with cells that had been maintained in standard endothelial cell medium and therefore were not estrogen depleted. Triplicate wells of cells were lifted each day for consecutive days with 0.05% trypsin-EDTA and counted with a cell counter (Coulter Corp).

[³H]Thymidine Incorporation Assay
The [³H]thymidine incorporation assay was performed according to the method of Williams et al.¹⁹ Cells (5x10⁵/well) were allowed to attach overnight in standard medium, which was then replaced with hormone-free medium for 48 hours. Estradiol and antagonists were then added to the cultures. Cells were incubated with [³H]thymidine (3 µCi/well) at 37°C for 4 hours, then solubilized with 0.1% SDS at room temperature overnight. After digestion with cell lysis buffer (50 mmol/L Tris, pH 7.2, 100 mmol/L NaCl, mixture of protease inhibitors), DNA was precipitated with 10% trichloroacetic acid, extracted with 0.2 mol/L NaOH, and assayed for radioactivity in a liquid scintillation counter (Beckman Instruments).

Immunolocalization of the ER
Immunolocalization of the ER was performed as described²⁰ with minor modifications. Briefly, cells were grown in standard endothelial cell medium on gelatin-coated tissue culture slides (Lab-Tek). After 24-hour culture in hormone-free medium, the cells were treated with vehicle or estradiol (1 ng/mL) for 2.5 hours, then fixed with 3.7% formaldehyde in PBS, pH 7.4, followed by sequential permeabilization with methanol, then acetone at -20°C. The cells were blocked with 10% normal goat serum in PBS for 1 hour at room temperature before incubation overnight at 4°C with the primary antibody, ER-21, 2.5 mg/mL in PBS containing normal goat serum.²¹ This antibody recognizes both human and rodent ER and does not cross-react with progesterone or testosterone receptors. After being washed three times with 2% BSA in PBS, the cells were incubated with goat anti-rabbit IgG conjugated with biotin, diluted in PBS containing normal goat serum, for 30 minutes at room temperature. Endogenous peroxidase activity was blocked with 0.3% H₂O₂ in PBS for 10 minutes before incubation with streptavidin–horseradish peroxidase diluted 1:100 in PBS for 30 minutes at room temperature. The color was developed with Enhanced DAB (diaminobenzidine; Pierce). As a control, the specificity of ER-21 antibody was verified by incubation with a peptide corresponding to the amino-terminal sequence of the ER (2.5 mg/mL) before its application to cells. The method was validated in parallel experiments with the ER-positive breast cancer cell line MCF-7 cells as a positive control and ER-negative MDA-MB-231 cells as a negative control (not shown). MCF-7 and MDA-MB-231 cells were kindly provided by Dr S. Rosen (Lurie Cancer Center, Northwestern University Medical School).

Radioligand Binding Assay for the ER
HUVECs or HCAECs were seeded into 24-well plates at 3x10⁴ cells per well. After 30 hours, the medium was replaced with hormone-free medium. After 24 hours, this medium was aspirated and replaced with 0.5 mL warm (37°C), phenol red–free RPMI 1640 containing 0.1% BSA. Half the wells received 3 µg (final concentration, 20 µmol/L) unlabeled estradiol or diethylstilbestrol. After 5 minutes, various amounts of (2,4,6,7)-³H-17ß-estradiol (DuPont/NEN) were added, in quadruplicate, to the wells. The cells were cultured at 37°C for an additional 75 minutes, then quickly washed three times with PBS plus 0.5% BSA. The wells were aspirated dry, and 0.5 mL 70% ethanol was added to solubilize the ligand. After 60 minutes, the entire amount of ethanol was removed and assayed in a liquid scintillation counter. The number of specific counts bound was determined by subtracting the mean number of counts bound at each concentration in the presence of excess unlabeled estradiol from the mean number of counts bound in the absence of unlabeled estradiol.

RNase Protection Assay
Total cellular RNA was isolated by homogenization in 4 mol/L guanidinium isothiocyanate, 25 mmol/L sodium acetate, pH 5.2, with 10 mmol/L ß-mercaptoethanol, and then centrifugation through a 5.7 mol/L CsCl₂ cushion.²² Fluid-phase hybridization was performed with a Ribonuclease Protection Assay Kit II (Ambion) according to the manufacturer's instructions. Homogeneously ³²P-labeled antisense cRNA probes were synthesized in vitro from pOR300 (a 288-bp segment of the ER DNA sequence) and from p36B4 (a 220-bp segment of acidic ribosomal protein DNA sequence) with T7 polymerase. The acidic ribosomal protein was used as a control because its expression is not affected by estradiol.²³ The riboprobes (specific counts bound >1.0x10⁸ cpm/µL) were used to hybridize with 30 or 60 µg of various RNA preparations at 42°C for 16 hours. Samples were subjected to RNase digestion at 37°C for 30 minutes. They were then electrophoresed on an 8 mol/L urea/6% polyacrylamide gel at 250 V and visualized by autoradiography.

Electrophoretic Mobility Shift Assay
Complementary DNA strands corresponding to a human consensus ERE sequence of 31 nucleotides (5'-CTTCGAGGAGGTCACAGTGACCTGGAGCGG-3'; palindrome is underlined) were synthesized by the Biotechnology Facility at Northwestern University. The two complementary strands were annealed, and 5'-overhangs were used for [-³²P]dCTP labeling with DNA polymerase. Cytoplasmic and nuclear extracts were prepared according to Dimitrova et al²⁴ with minor modifications. Cells were washed twice with ice-cold Tris-buffered saline, collected, and then incubated in lysis buffer (in mmol/L: HEPES 10, pH 7.9; KCl 10; EDTA 0.1; EGTA 1; DTT 1; and PMSF 0.5; and 10 vol% NP-40) containing a mixture of protease inhibitors for 15 minutes on ice. Nuclei were collected by centrifugation at 14 000g for 30 minutes at 4°C. The pellet was resuspended in lysis buffer with 0.4 mol/L NaCl and used as nuclear extract. Five to 500 µg nuclear extract preparations from HUVECs or HCAECs were incubated for 20 minutes at 20°C with 2 µg poly(dI-dC) in binding buffer (in mmol/L: HEPES 25, pH 7.9; KCl 60; EDTA 0.1, pH 8.0; DTT 0.1; and PMSF 0.2; and 12 vol% glycerol). ³²P-labeled ERE probe was then added to the reaction mixture and incubated for 20 minutes at 20°C. Excess unlabeled ERE was added to the reaction mixture 10 minutes before ³²P-labeled ERE to compete for specific ERE-protein binding in selected reactions. To confirm the identity of the protein participating in formation of the complex, the ER-specific monoclonal antibody H222 (rat IgG1) was incubated with the reaction mixture for 30 minutes before addition of ³²P-labeled ERE. Reaction mixtures were electrophoresed through a nondenaturing polyacrylamide gel (5%) (acrylamide:bisacrylamide, 19:1) in TBE buffer (in mmol/L: Tris 90, pH 7.6; borate 90; EDTA 2, pH 8.0) at 120 V at room temperature. Gels were dried, and protein-DNA complexes were visualized by autoradiography.

	Results

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Effect of Estrogen on Endothelial Cell Proliferation
The effect of hormone-free medium and 17ß-estradiol supplementation on endothelial cell growth was measured. As shown in Fig 1A, control cultures of cells that had not been depleted of estrogen showed a linear increase in cell number beginning at culture initiation. In contrast, estrogen-depleted cells in hormone-free medium showed no growth for 48 hours. Thereafter, some growth was observed, but the rate of division was comparatively slow. Cells depleted of estrogen for 48 hours that received estradiol supplementation showed a lag time between culture initiation and the resumption of a growth rate identical to that of control cells. The most effective dose of estradiol was 3.7 nmol/L (titration not shown). The increase in growth rate was abolished by treatment with the specific ER antagonist ICI182,780 (10 nmol/L). Similar results were observed in three different experiments for each with either HUVECs or HCAECs.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of 17ß-estradiol on HUVEC proliferation. A, Effect on cell number. Cells were cultured in standard medium or in hormone-free (HF) medium in the presence or absence of 17ß-estradiol. The specific ER antagonist ICI182,780 was added to some cultures that had received exogenous estradiol. Each day, triplicate cultures were detached, and the number of cells was quantified by Coulter counter. ICI182,780 alone did not affect cell proliferation in the concentration used here (not shown). B, Effect on [3H]thymidine incorporation. HCAECs were cultured in HF medium in the presence or absence of estradiol, respectively, and with ICI182,780. [3H]Thymidine incorporation was determined at various times thereafter. Results with cells grown in standard medium are shown as a control. Significant differences compared with HF medium: standard medium for the cell number (Student's t test), P<.08, days 2 and 3; P<.05, day 4. Estradiol-supplemented medium (1 ng/mL), P<.03, day 2; P<.04, days 3, 4, and 5. Significant differences compared with HF medium: standard medium for the [3H]thymidine incorporation, P<.05, 96 hours; P<.02, 48 and 120 hours. Estradiol-supplemented medium (1 ng/mL), P<.02, 96 and 120 hours; P<.01, 24 and 72 hours.

Effect of Estrogen on [³H]Thymidine Incorporation
DNA synthesis by HUVECs and HCAECs was quantified by [³H]thymidine incorporation. The response to estradiol was similar in HCAECs (Fig 1B) and HUVECs (not shown). The greatest increase was observed after 24 to 30 hours. Estradiol-treated cells showed twice the incorporation of hormone-free cells. This increase in [³H]thymidine incorporation was prevented when the ER antagonist ICI182,780 was added to the culture medium at the same time as estradiol.

Detection of ER by Immunocytochemical Staining
A polyclonal antibody generated against the N-terminal peptide of human ER was evaluated for its ability to identify ER in human endothelial cells. A strong nuclear staining pattern was seen when cells were treated with ER-21 antibody (Fig 2). Cells treated with control rabbit IgG show a faint pattern of nuclear and cytoplasmic staining. Pretreatment of the ER-21 antibody with N-terminal ER peptide abolished most of this staining. Little specific staining was detected in the cytoplasm. ER staining of HUVECs/HCAECs was less intense than that of MCF-7 cells, used as a positive control (not shown). Estradiol treatment of cells did not appreciably change the intensity and pattern of staining of the cells (not shown). Similar ER staining was detected with antibody ER-21 in both HUVECs and HCAECs.

View larger version (102K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining for ER in HCAECs. Cells were treated with normal rabbit IgG (left panels) or with ER-21, a polyclonal antibody raised against a peptide representing the amino-terminal sequence of the ER (right panels). The antibody either received no pretreatment (Control, upper panels) or was pretreated with amino-terminal peptide of ER to absorb out the specific antibody (ER-Peptide, lower panels). Treatment of ER-21 with antigen eliminated the specific nuclear staining pattern. Identical staining was observed with HCAECs (not shown).

Identification of the ER via Radioligand Binding
Tritiated estradiol bound to both HUVECs and HCAECs in a reproducible and saturable manner. Binding was not inhibited by either testosterone or progesterone but was inhibited equally well by unlabeled estradiol or diethylstilbestrol (data not shown). By division of the inhibitable counts bound by the number of cells, the number of receptors per cell was estimated to be 2x10⁴ to 6x10⁴ in three different experiments. Scatchard analysis of radioligand binding (Fig 3, inset) showed a K_d of 5 nmol/L and 6x10⁴ to 8x10⁴ receptors per cell. Receptor saturation occurred at 20 nmol/L in the direct binding studies. In eight different experiments with HUVECs or HCAECs from male or female donors, all showed saturable binding with similar kinetics. Because the absolute number of inhibitable counts bound was relatively low, it was not possible to use this technique to determine whether there were small differences in receptor number or in binding characteristics between cells from these various sources.

View larger version (21K): [in this window] [in a new window]   Figure 3. ER analysis by radioligand binding assay. The curve generated by subtracting counts bound in the presence of excess cold estradiol from total cpm bound indicates that receptor binding was saturable and specific. A Scatchard plot generated for the data (inset) estimates the Kd to be 5 nmol/L and receptor number to be 7.5x104/cell. B/F indicates bound/free.

Detection of ER mRNA by RNase Protection Assay
We evaluated endothelial cell ER production via expression of ER mRNA. By RNase protection assay, the ER-positive MCF-7 cell (a positive control) shows a protected mRNA fragment that is consistent with the size of the ER riboprobe (Fig 4). The signal intensity of the protected RNA fragment increased proportionally with an increasing amount (30 to 60 µg) of total RNA. The ER-negative MDA-MB-231 breast cancer cell line did not show a similar protected fragment. RNA harvested from HUVECs exposed to estradiol (2 ng/mL) for 4 and 24 hours showed a protected band slightly smaller than that seen in MCF-7 cells. This band was not observed in endothelial cell preparations not exposed to estradiol. Expression of the acidic ribosomal protein mRNA (evaluated by use of a 220-bp riboprobe) was unaffected by estradiol treatment. These data provide evidence for the synthesis of the ER by endothelial cells.

View larger version (82K): [in this window] [in a new window]   Figure 4. RNase protection assay for ER mRNA in HUVECs. Cells were cultured in the presence or absence of estradiol. Riboprobes used in these assays corresponded to 288 bases of the 5' end of the coding region for ER and 220 bases of sequence from the acidic ribosomal protein (ARP) as control for loading. MCF-7 cells were used as an ER-positive control and MDA-MB-231 cells as an ER-negative control. A faint band is seen at 280 bp, corresponding to the protected fragment, in both estrogen-treated HUVEC RNA and MCF-7 RNA. RNase-treated ER-riboprobe and control riboprobe were completely digested, whereas undigested ER and the control riboprobes indicate the full-length protected segments of 288 and 220 bp, respectively.

Analysis of ER-ERE Interaction by EMSA
We then sought to determine whether this receptor was functionally similar to ER defined previously in other cell types. EMSA was performed with nuclear extracts of either HCAECs or HUVECs. A shifted DNA binding complex was seen in both HUVEC and HCAEC nuclear extracts, similar in size to but less intense than the shifted complex obtained from nuclear extracts of MCF-7 cells (Fig 5A). The endothelial band comigrated with the MCF-7 band, suggesting the presence of ER or an ER-like protein. Signal intensity for the ER-ERE complex from HUVECs was threefold to fivefold lower than that obtained with MCF-7 cells. There was no difference in signal intensity for shifted complex when extracts from estrogen-treated versus -untreated HUVECs were compared. The intensity of signal for the complex of the HUVECs decreased in a dose-dependent manner when increasing amounts of unlabeled ERE oligonucleotides were present in the reaction mixture (Fig 5B). In addition, binding was not competed for by an unrelated DNA sequence, indicating that the binding was specific (not shown). The binding complex was supershifted by incubation with antibody against ER, H222, confirming the presence of ER (Fig 5A), whereas nonimmune IgG had no effect on the mobility of the complex (not shown). Similar results were obtained with nuclear extracts of HCAECs.

View larger version (94K): [in this window] [in a new window]   Figure 5. EMSA for ER. Nuclear extracts of HUVECs were incubated with 32P-labeled ERE and analyzed by nondenaturing PAGE. Cells were cultured in standard medium and then switched to hormone-free conditions for 48 hours. A, Control lanes show loading of the reaction mixture without labeled ERE and loading without nuclear extract (Ext). A strong signal is generated by shifted complex (arrow) with extract from MCF-7 cells, whereas little shifted complex is seen with MDA-MB-231 cells. Asterisk indicates extract obtained from estrogen-treated HUVECs. The complex is supershifted by pretreatment of the extracts with the monoclonal anti-ER H222. No supershift was seen with irrelevant antibody (not shown). B, Binding specificity of the ERE with cell extracts was further analyzed by incubation with increasing amounts of unlabeled ERE at molar ratios from 5x to 200x as indicated above the figure. No inhibition was seen with irrelevant-sequence unlabeled competitor (not shown). Identical results were obtained with five other preparations of nuclear extracts from either HUVECs or HCAECs.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Estrogen has been shown to regulate ER expression and total DNA synthesis in classic target tissues, including the breast, uterus, and pituitary gland.^{23 25} With increasing interest in a role for estrogen in maintaining normal vascular function, several groups have pursued a human vascular ER.^{26 27} Human VSMCs express an ER at both mRNA and protein levels.^{16 28} Estrogen treatment increases activity of an estrogen-responsive reporter construct transfected into human VSMCs.²⁸ In addition, atherosclerotic coronary artery tissue VSMCs show less staining for ER than do VSMCs in tissue from healthy, normal premenopausal woman.¹⁶ Thus, human VSMCs express a functional ER, and the level of expression correlates inversely with the incidence of atherosclerosis. No similar studies have been performed on endothelial cells. Radioligand-binding studies suggested the presence of an estrogen-binding protein in endothelial cells.²⁹ Furthermore, an ER-like binding protein was described in bone endothelial cells.³⁰ Previously, we showed that estrogen increases endothelial adhesion, migration, and proliferation and angiogenesis,¹⁷ indicating that an ER might be present in human macrovascular endothelial cells. Despite this indirect evidence, an endothelial cell ER has not been directly demonstrated.

In the present study, human endothelial cell [³H]thymidine incorporation and cell proliferation increased after treatment with a concentration of estradiol that is near the circulating level achieved during ovulation and the luteal phase of the menstrual cycle.³¹ The increase was blocked by the specific ER antagonist ICI182,780. Distinct nuclear immunostaining of HUVECs and HCAECs was similar to that in MCF-7 cells, although less intense. Preabsorption of the antibody with an ER peptide confirmed that the ER-21 antibody specifically recognizes ER in the endothelial cells. Direct measurement of saturable, inhibitable counts in radioligand-binding studies demonstrated 2x10⁴ to 6x10⁴ ERs per cell. Scatchard analysis indicated 6x10⁴ to 8x10⁴ ERs per cell and a K_d of 5 nmol/L. These findings are consistent with ER studies in human VSMCs.¹⁶ It is likely that the number of receptors demonstrated by Scatchard analysis represents an overestimate, because the numbers observed with direct measurement are more consistent with the intensity of immunostaining seen in MCF-7 cells. However, the absolute number of specifically bound counts was low compared with the total number of bound counts, deterring more accurate analysis of receptor-ligand interactions. Further investigation of this issue is in progress. At present, our observation of specific, saturable binding is consistent with the rest of the data indicating estrogen-ER interaction in endothelial cells. Thus, our findings suggest that HUVECs and HCAECs are specific estrogen targets.

Several additional findings regarding ER protein and mRNA expression are noteworthy. A protected band in the RNase protection assay was observed only with estrogen-treated endothelial cells. In MCF-7 cells, estrogen downregulates ER expression.²³ However, estrogen rapidly increases expression of ER protein in uterine epithelial cells.²⁵ Also notable in our studies is that the size of the protected endothelial RNA fragment consistently appears to be slightly smaller than that obtained with MCF-7 RNA. Since our finding of estrogenic enhancement of ER mRNA expression also differs from results with MCF-7 cells, our data raise the possibility that human endothelial cells use an alternative promoter, transcription initiation site, and/or splicing variant, resulting in truncated ER isoforms, as has recently been reported in osteoblasts.³² We are investigating this possibility. Finally, the extremely low abundance of ER mRNA, even in target tissue cells, is emphasized by the marked difference in signal intensity comparing the protected ER mRNA fragment with the control mRNA fragment. The faint signal for ER mRNA in Fig 4 reflects the need to limit the duration of exposure to avoid overexposing the higher-intensity control band.

In several experiments, estrogen treatment of human endothelial cells enhanced ER detection by EMSA by 20% to 30% (data not shown). However, this finding was not observed consistently. One possible mechanism for signal intensification is that estrogen enhances detection by concentrating the receptor in the nucleus. Another is that estradiol activates the receptor, increasing its ability to bind to the ERE. The literature is divided regarding whether estrogen binding is required for interaction between the ER and the ERE.^{33 34}

To our surprise, the intensity of immunostaining for ER was similar in male and female donor cells. Furthermore, neither EMSA nor ligand-binding studies showed reproducible sex differences in ER expression. Although it is not certain that such differences exist, technical limitations of both immunostaining and the radioligand experiments are such that further studies will be required to determine whether small but consistent differences might be identified between cells from donors of different sexes or among cells isolated from vessels in different tissues.

The mechanism by which ER binding influences endothelial cell behavior and, subsequently, vascular function remains to be elucidated. Classically, ligand-bound ER translocated to the nucleus regulates gene transcription. Our previous studies suggest that estrogen enhances endothelial cell activity stimulated by other factors rather than directly initiating new activities.¹⁷ Estrogen-ER binding may lead to complex interactions with other transcription factors known to regulate cell attachment, migration, and proliferation, such as those between the glucocorticoid receptor and the c-fos protein in cell growth.^{35 36} The ER has been shown to interact directly with the c-fos/c-jun complex (AP-1)³⁷ or to act through accessory molecules.³⁸ Thus, multiple mechanisms are possible and, indeed, may act in concert to moderate the effects we have observed on endothelial function. Considerable work remains to be done in this area. Regardless of the mechanism of estrogen modulation of endothelial cell activity, our studies demonstrating an ER in human endothelial cells suggest that genomic effects of ER binding could play a role in the cardioprotective effects of estrogen on endothelial function in premenopausal women.

	Selected Abbreviations and Acronyms

 

EMSA = electrophoretic mobility shift assay ER = estrogen receptor ERE = estrogen response element HCAEC = human coronary artery endothelial cell HUVEC = human umbilical vein endothelial cell VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by grants HL-53918 from the National Heart, Lung, and Blood Institute; DK-49362 from the National Institute of Diabetes and Digestive and Kidney Diseases; and AR-30692 from the National Institute of Arthritis and Musculoskeletal Disease (Dr Schnaper); CA-02897 from the National Cancer Institute and DAMD17-94-5-4228 from the Department of Defense (Dr Greene); and FIS 96/2145 (Dr Cid). Dr McGowan was a Howard Hughes Medical Institute–National Institutes of Health Research Scholar. We appreciate provision of the ER antagonist ICI182,780 by Zeneca Pharmaceuticals and of MCF-7 and MDA-MB-231 cells by Dr Steven Rosen. We are grateful to Drs Chris Waters and Mira An for many helpful discussions.

Received December 20, 1995; revision received March 27, 1996; accepted April 15, 1996.

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