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The Journal of Clinical Endocrinology & Metabolism 87(9):4341– 4349 Copyright © 2002 by The Endocrine Society doi: 10.1210/jc.2001-010588

17␤-Estradiol Up-Regulates Vascular Endothelial Growth Factor Receptor-2 Expression in Human Myometrial Microvascular Endothelial Cells: Role of Estrogen Receptor-␣ and -␤ CAROLINE E. GARGETT, MARINA ZAITSEVA, KRISTINA BUCAK, SIMON CHU, PETER J. FULLER, AND PETER A. W. ROGERS Centre for Women’s Health Research (C.E.G., M.Z., K.B., P.A.W.R.), Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, Clayton, Victoria 3168, Australia; and Prince Henry’s Institute of Medical Research (S.C., P.J.F.), Clayton, Victoria 3168, Australia Estrogen has a cardiovascular protective role in women due in part to its effect on the vasculature. The roles of the two estrogen receptors (ERs), ER␣ and ER␤, in the vascular actions of estrogen are unclear, as are effects of estrogen on microvascular endothelial cells (MEC) derived from sex steroid-responsive tissues. The present study demonstrates that 17␤-estradiol, but not progesterone, increases vascular endothelial growth factor (VEGF) receptor (VEGFR) expression on human myometrial MEC measured using biotin-recombinant human (rh) VEGF165 and flow cytometry. This response occurred in a time- and dose-dependent manner, with significantly increased rhVEGF165 binding at 3 h and maximal responses between 0.1 and 10 nmol/liter 17␤-estradiol, which was blocked by the antiestrogen ICI 182,780. Approximately 60% of samples demonstrated this response to 17␤-estradiol. All samples of myometrial MEC expressed both ER␤ mRNA

and protein demonstrated by semiquantitative RT-PCR and Western blotting. However, ER␣ mRNA and protein were expressed in only 13 of 21 MEC samples. There was a significant association between ER␣ expression in myometrial MEC and their ability to respond to 17␤-estradiol by increasing rhVEGF165 binding. 17␤-estradiol increased VEGFR-2 expression in ER␣-expressing MEC isolates, which also demonstrated increased rhVEGF165 binding, but failed to have these effects on ER␣ negative samples. Similarly, 17␤-estradiol augmented VEGF-induced MEC proliferation in ER␣-expressing MEC samples, which was blocked by ICI 182,780. These observations suggest that 17␤-estradiol increases VEGFR-2 expression on human myometrial MEC promoting endothelial cell proliferation, an effect that varies between subjects and appears to be mediated primarily by ER␣. (J Clin Endocrinol Metab 87: 4341– 4349, 2002)

T

The two ERs, ER␣ and ER␤, which are encoded by separate genes, are involved in the vascular actions of 17␤-estradiol, but their relative roles are unclear (1, 12). ER␣ and ER␤ bind 17␤-estradiol with similar high affinity causing receptor dimerization. They act as ligand-activated transcription factors for a range of estrogen target genes, binding to estrogen response elements or indirectly by interaction with other DNA binding proteins (13). However, ER␣ and ER␤ can mediate opposing transcriptional activities, dependent on the type of response element in target gene promoters and other cell specific factors such as presence or absence of coregulators (12). There are also differences in the tissue distribution of ER␣ and ER␤, although there is considerable overlap and heterodimerization may occur in cells where they are coexpressed. Vascular endothelial growth factor (VEGF) is a specific mitogen for EC, acting through two tyrosine-kinase receptors, VEGF receptor (VEGFR)-1 (flt-1) and VEGFR-2 (KDR), expressed almost exclusively on EC (14). Both receptors bind VEGF with high affinity, but signaling through VEGFR-2, rather than VEGFR-1, mediates the angiogenic effects of VEGF (14). Both VEGFRs are expressed at low levels in vivo and in quiescent cultured EC (15, 16) where they serve to promote EC survival (17) but are up-regulated during periods of angiogenesis (18). The factors regulating VEGFR ex-

HE UTERUS IS a classical sex steroid target organ, where both myometrial and endometrial cells express estrogen and progesterone receptors (ERs and PRs). The vascular system is also estrogen responsive (1, 2). Experimental studies show that 17␤-estradiol exerts a range of direct effects on endothelial cells (EC), which can be either rapid or long-term (3). These include up-regulation of endothelial nitric oxide synthase activity, modulation of adhesion molecule expression, and promotion of EC survival and angiogenic activity (3–7). In contrast, progesterone has proapoptotic effects and inhibits EC proliferation (8). Because ER and PR have been demonstrated in human large vessel EC (7–9) and in endometrial MEC (10), it is likely that sex steroid effects are mediated by these receptors, although estrogen also has nongenomic effects (11). Abbreviations: bFGF, Basic fibroblast growth factor; CDS, Cell Dissociation Solution; ch, charcoal stripped; EC, endothelial cell(s); ER, estrogen receptor; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; HS, human serum; MEC, microvascular endothelial cell(s); MFI, mean fluorescence intensity; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulphophenyl)-2H-tetrazolium; PE, phycoerythrin; PR, progesterone receptor; rh, recombinant human; RS, Spearman rank correlation coefficients; TBST, Tris-buffered saline-Tween 20; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; VSMC, vascular smooth muscle cells.

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pression are not fully understood, although hypoxia and some cytokines and growth factors may be involved (14). 17␤-estradiol is known to modulate the expression of some growth factors and their receptors (19). Because 17␤-estradiol up-regulates basic fibroblast growth factor (bFGF)-induced angiogenesis (6, 7, 10) we postulated that 17␤-estradiol and progesterone would also modulate VEGF-induced angiogenesis through alteration of VEGFR expression. While the study of sex steroid effects on endothelium has largely focused on large vessels, there are few studies examining the effects of 17␤-estradiol and progesterone on MEC derived from classic sex steroid-responsive tissues such as the uterus. We have used MEC isolated from the human myometrium as an in vitro model to investigate the effects of estrogen on the microvascular system. Here we demonstrate that 17␤estradiol up-regulates VEGFR-2 expression in myometrial MEC. We also show that myometrial MEC express both ER␣ and ER␤ and that 17␤-estradiol up-regulation of VEGFR-2 is associated with ER␣ expression. Materials and Methods Materials Fetal calf serum (FCS) (CSL, Melbourne, Australia) and male human serum (HS) (Victorian Red Cross Blood Service) were charcoal-stripped (ch-FCS, ch-HS) for hormone experiments and contained less than 1 pg/ml 17␤-estradiol by RIA. 17␤-estradiol, progesterone, and Cell Dissociation Solution (CDS) were from Sigma-Aldrich (St. Louis, MO). Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN). Mouse antihuman CD31 was from DAKO Corp. (Carpinteria, CA), mouse antihuman ER␣ was from Novocastra Laboratories, Ltd. (Newcastle upon Tyne, UK) and rabbit antihuman ER␤ (catalog no. PAI-313) was from Affinity BioReagents, Inc. (Golden, CO) Phycoerythrin (PE)-labeled sheep anti-IgG (Fab’2 fragments) were from Silenus (Victoria, Australia), whereas horseradish peroxidase (HRP)-conjugated sheep anti-IgG was from Zymed Laboratories, Inc. (San Francisco, CA). Western blotting reagents, Blot-Quickblocker, femto/Trisbuffered saline-Tween 20 (TBST) and femtoLUCENT chemiluminescence detection system were from Chemicon (Temecula, CA). CellTitre 96 MTS [3-4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4sulphophenyl)-2H-tetrazolium] reagent was from Promega Corp. (Madison, WI). ICI 182,780 was kindly provided by Dr. Frances Sutcliffe (AstraZeneca, Cheshire, UK). VEGFR-1 (flt-1, clone MAFL6) and VEGFR-2 [kinase domain region (KDR), clone MAKD6] were kindly provided by Dr. Napoleon Ferarra from Genentech, Inc. (San Francisco, CA).

Human tissues Human myometrial tissue was obtained from 35 ovulating women (aged 27–53 yr) who had not taken exogenous hormones in the previous 3 months and underwent hysterectomy for uterine prolapse or fibroids (benign tumor of the myometrium). Informed consent was obtained from each patient, and ethical approval was obtained from Monash Medical Centre Human Research and Ethics Committee B. Many of the hysterectomy samples contained fibroids, which are easily recognized by their well circumscribed border, and were removed before processing. The tissue was collected in HEPES-buffered M199 medium, containing 10% FCS and antibiotic-antimycotic solution (final concentrations: penicillin 106 U/liter, streptomycin 106 U/liter and fungizone 2.5 mg/liter), stored overnight at 4 C, and then processed.

Microvascular EC culture Myometrial tissue was dissociated with collagenase and deoxyribonuclease, followed by a short trypsin treatment to produce single cell

Gargett et al. • ER␣/ER␤ and VEGFR-2 Expression in MEC

suspensions, and MEC were isolated by positive selection with Ulex europeaus agglutinin-1-coated Dynabeads (Dynal, Oslo, Norway) as described (20). MEC were then seeded into fibronectin-coated tissue culture flasks (10 mg/liter) and cultured in M199 medium containing 15% HS/5% FCS, 2 mmol/liter glutamine, 5 ␮g/liter bFGF, 100 mg/liter heparin, and antibiotic/antimycotic solution as described (20). On subsequent passages, MEC were repurified with Ulex europeaus agglutinin1-coated Dynabeads and were used for all analyses between passages 1–3; purity was more than 99% by flow cytometry of CD31-immunolabeled cells. Not all analyses were performed on each isolate, due to insufficient numbers of highly purified MEC obtained from some samples.

Flow cytometric analysis of rhVEGF165 binding to myometrial MEC Confluent MEC cultures in six-well plates were incubated for 48 h in hormone-free medium (phenol red-free M199 with 15% ch-HS/5% chFCS, 10 ng/ml VEGF165, and 100 mg/liter heparin) and then quiesced in HS-free phenol red-free M199 containing 5% ch-FCS, 10 ␮g/liter VEGF165 and 100 mg/liter heparin for 24 h. We have previously established that myometrial MEC do not proliferate in 5% FCS containing VEGF (20). MEC were then stimulated with 17␤-estradiol, progesterone, or 17␤-estradiol and progesterone in HS-free medium containing 0.5% BSA, 10 ␮g/liter VEGF165, and 100 mg/liter heparin. In some experiments, 5% ch-FCS was substituted for 0.5% BSA in the HS-free medium. Preliminary experiments showed that it was necessary to include VEGF in human serum-free medium to prevent MEC apoptosis and obtain basal rhVEGF165 binding. Control samples were incubated with vehicle. MEC were harvested in CDS, washed, and resuspended in RDF buffer. Aliquots (25 ␮l; 5 ⫻ 104 cells) were incubated with saturating amounts of biotinylated-rhVEGF165 (30 ng) and anti-CD31 (4 mg/liter) for 1 h at 4 C, followed by 10 ␮l avidin-FITC for 30 min at 4 C. Cells were washed and then incubated with PE-conjugated antimouse IgG (1/100) for 30 min at 4 C. Nonspecific binding was assessed by substituting biotinylated soybean trypsin inhibitor (same biotin:protein ratio) for biotinylated-rhVEGF165, and CD31 with isotype matched IgG at equivalent concentrations (negative control). Samples were analyzed using a MoFlo flow cytometer (Cytomation, Fort Hills, CO) equipped with an argon laser (Cytomation) and Cyclops Summit software (Cytomation). Two parameter histograms of FITC-VEGF and PE-CD31 were used to determine mean fluorescence intensity (MFI) of VEGF binding on more than 5000 CD31⫹ MEC per sample. The negative control MFI was subtracted from the respective sample MFI.

Analysis of mRNA for ER␣ and ER␤ by RT-PCR and Southern blotting Total RNA was prepared from highly purified cultured MEC (⬎99%) using the QIAGEN RNeasy Mini Kit. One microgram of total RNA was reverse transcribed and amplified using universal primers for both ER␣ and ER␤ (sense primer: 5⬘-CCG GAA TTC TTC/T GAC ATG CTC/G CTGG; antisense primer: 5⬘-GAT GC/TT CCA TGC CC/TT TGT TAC TC) and for ␤2-microglobulin (sense primer: 5⬘-TGA ATT GCT ATG TGT CTG GGT-3⬘; antisense primer: 5⬘-CCT CCA TGA TGC TGC TTA CAT3⬘) in a single stage PCR for 30 cycles as previously described (21). PCR products were probed with gene-specific 32P-labeled probes (ER␣ probe: 5⬘-GGT TGT GTG CCT CAA ATC TAT TAT TT; ER␤ probe: 5⬘-ATA TCT CTG TGT CAA GGC CAT GA) as described (21).

ER␣ expression by flow cytometry The presence of ER␣ receptors was determined in MEC suspensions by microwave permeabilization (22) and flow cytometry (23). MEC were harvested with trypsin (0.025%), washed and 106 cells fixed in 2% paraformaldehyde for 30 min at 4 C, washed and resuspended in 0.01 mol/ liter citrate buffer, pH 6.0, and microwaved for 30 sec on High (500W), washed and resuspended in PBS/1% FCS. Aliquots (50 ␮l; 5 ⫻ 104 cells) were incubated with 7 mg/liter anti-ER␣ in 10% goat serum for 60 min at 4 C, washed and incubated with PE-antimouse IgG in 10% goat serum for 30 min at 4 C and examined by flow cytometry. The MFI of single parameter histograms of more than 5000 cells was obtained, the MFI of IgG controls subtracted, and the percentage of positive cells with flu-


orescence intensity more than 98% of control cells determined. For the negative controls, mouse IgG1 (7 mg/liter) was substituted for primary antibody. ER␣ positive (MCF-7 or T47D) and negative (MDA-DB-453) control cells were analyzed in each batch.

Western blot analysis for ER␣ and ER␤ MEC lysates (20 ␮g protein) were denatured in SDS sample buffer, separated by 8% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Membranes were blocked in 5% Blot-QuickBlocker for 30 min at 22 C, incubated for 90 min at 22 C with ER␣ antibody (0.93 mg/liter) in femto/TBST buffer or ER␤ (1 mg/liter) in femto/TBST/2% Blot-QuickBlocker, followed by incubation for 60 min at 22 C with HRP-antimouse IgG (1/2000 in femto/PBS Tween 20) for ER␣ or HRP-antirabbit IgG (1/3000 in femto/TBST/2% Blot-QuickBlocker) for ER␤, and detected by femtoLUCENT chemiluminescence system.

Flow cytometric analysis of VEGFR-1 and VEGFR-2 expression on myometrial MEC MEC were cultured in T25 flasks, incubated with hormone-free medium, quiesced in human serum-free medium, and incubated with 0 (vehicle), 1, and 10 nmol/liter 17␤-estradiol as described for rhVEGF165 binding. MEC were harvested in CDS and analyzed for rhVEGF165 binding as described, as well as VEGFR-1 and VEGFR-2 expression by flow cytometry. Aliquots (50 ␮l; 5 ⫻ 104 MEC) were incubated with 20 mg/liter anti-VEGFR-1 or 10 mg/liter anti-VEGFR-2 in PBS containing 10% goat serum for 60 min at 4 C, washed, and incubated with PEantimouse IgG in 10% goat serum for 30 min at 4 C and examined by flow cytometry. The MFI of single parameter histograms of more than 5000 cells was obtained and the MFI of IgG controls subtracted. For negative controls, mouse IgG1 (10 or 20 ␮g/ml) was substituted for primary antibody. CEM-A7R cells served as negative control cells, and the MFI for both VEGFR-1 or VEGFR-2 antibodies was the same as the IgG control.

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was less than nonconfluent, proliferating MEC (Fig. 1A). MEC incubated with 17␤-estradiol (10 nmol/liter), but not progesterone (100 nmol/liter), bound more biotinylated-rhVEGF165 than vehicle-treated control cells (basal binding), demonstrated by an increase in fluorescence intensity (Fig. 1A). Figure 1B shows that 10 nmol/liter 17␤-estradiol significantly increased VEGF165 binding (P ⫽ 0.01). In contrast, progesterone (100 nmol/liter) alone had no effect on VEGF165 binding and when progesterone was incubated with 17␤estradiol, there was a nonsignificant reduction of approximately 60% in the 17␤-estradiol-induced increase. Basal rhVEGF165 binding and the 17␤-estradiol effect on rhVEGF165 binding to myometrial MEC were variable between isolates, with a significant proportion that were unresponsive (Table 1). For characterizing the effect of 17␤-estradiol on rhVEGF165 binding, our experiments were limited to 17␤-estradiol-responsive MEC isolates, and we did not further study the effect of progesterone. The 17␤-estradiol-induced increase in rhVEGF165 binding to myometrial MEC was time dependent, commencing after 1 h incubation, reaching significance at 3 h and maximal at 18 h (Fig. 2A). Incubation times greater than 18 h were not studied as MEC began to undergo apoptosis with prolonged incubation in HS-free conditions. 17␤-estradiol also increased rhVEGF165 binding in a dose-dependent manner,

MEC proliferation assay MEC proliferation was assessed using the CellTitre 96 MTS bioassay (Promega Corp.) as previously described (20). Briefly, MEC (4 ⫻ 103) were seeded in triplicate into wells of a 96-well plate, made quiescent, and then incubated at 37 C in 5% CO2 with or without 17␤-estradiol (10 nmol/liter) in phenol-red free M199 medium containing 2 ␮g/liter VEGF and 5% ch-HS/15% ch-FCS in the presence or absence of ICI 182,780 (1 ␮mol/liter) added 1 h before 17␤-estradiol, with medium changes every 2 d. After 6 d incubation, medium was changed to M199/5% ch-FCS, 20 ␮l MTS reagent added and incubated for 2 h at 37 C. Absorbance was then measured at 490 nm using a plate reader.

Statistical analysis Data were analyzed using two-tailed t test or one-way ANOVA, followed by Dunnett’s test for comparison between samples and control using PRISM (GraphPad Software, Inc., San Diego, CA). Correlations were performed using least squares regression analysis and Spearman rank correlation coefficients (RS) were determined using SPSS version 10.0 (SPSS, Inc. Australasia, North Sydney, Australia). The association between estrogen responsiveness and ER␣ expression was examined by Fisher’s exact test using SPSS.

Results Effect of sex steroids on rhVEGF165 binding to myometrial MEC

The effect of 17␤-estradiol and progesterone on VEGFR expression was examined on confluent, quiescent myometrial MEC in sex steroid hormone-free minimal medium using a ligand binding assay and flow cytometry. Under these conditions, the level of biotinylated-rhVEGF165 binding to myometrial MEC represented basal VEGFR expression and

FIG. 1. Effect of 17␤-estradiol and progesterone on rhVEGF165 binding to human myometrial MEC. A, Flow cytometry traces showing that (left panel) proliferating MEC bind more rhVEGF165 than quiescent MEC (basal binding) and (right panel) 17␤-estradiol (E2), but not progesterone (P4) increased basal rhVEGF165 binding. The negative control represents nonspecific binding. B, Quiescent MEC incubated with vehicle, 10 nmol/liter E2, 100 nmol/liter progesterone (P), and 17␤-estradiol ⫹ progesterone for 18 h at 37 C in the presence 10 ␮g/liter VEGF were harvested and rhVEGF165 binding measured. Results are mean ⫾ SEM from four experiments on separate MEC isolates. *, P ⫽ 0.01.

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TABLE 1. 17␤-Estradiol-mediated increase in rhVEGF165 binding is associated with ER␣ expression E-mediated response Positive

Negative

6 2 P ⫽ 0.03

0 4

E-mediated response Positive

Negative

8 na

4 na

ER␣ mRNA

Positive Negative

ER␤ mRNA

Positive Negative


5 1 P ⫽ 0.025

Negative

1 6


Negative

5 na

5 na

ER␣ protein

Positive Negative

ER␤ protein

Positive Negative

Contingency table analysis of myometrial MEC responsiveness to 17␤-estradiol and ER␣ and ER␤ expression on cells from a total of 16 subjects. A significant relationship between ER␣ mRNA and ER␣ protein expression and 17␤-estradiol-mediated increase in rhVEGF165 binding was demonstrated (Fisher’s exact test, P ⫽ 0.03 and 0.025, respectively). Analyses could not be done for ER␤ because all samples were positive. Not all analyses were performed on all isolates due to inadequate numbers of MEC obtained from some tissue samples. E, 17␤-estradiol; na, not applicable.

reaching a maximum between 0.1 and 10 nmol/liter (Fig. 2B). The concentration-response curve was biphasic, and at higher 17␤-estradiol concentrations the effect was reduced. The 17␤-estradiol concentration giving maximal rhVEGF165 binding varied between isolates and would peak at 0.1, 1, or 10 nmol/liter 17␤-estradiol for any given MEC isolate. We therefore used two 17␤-estradiol concentrations (0.1 or 1, and 10 nmol/liter) to further examine 17␤-estradiol-stimulated increase in rhVEGF165 binding. Myometrial MEC express ER␣ and ER␤ mRNA and protein

To determine whether the 17␤-estradiol effect on rhVEGF165 binding was associated with ER expression, we examined highly purified responsive and nonresponsive MEC (⬎99% CD31⫹) by semiquantitative RT-PCR using primers specific for human ER␣ and ER␤. Figure 3 shows Southern blots of nine myometrial MEC isolates examined. All expressed ER␤ as the major transcript, whereas ER␣ was expressed at lower and more variable levels. Figure 3 shows that ER␣ is expressed in 5/9 of the MEC cultures, specifically samples 3–5, 7 and 9. This ER␣ expression was not due to contaminating myometrial smooth muscle cells, which only expressed ER␣ (24) because ER␣ expression was still observed after FACS sorting CD31-labeled MEC (Fig. 3, sample 9). MEC expression of ER␣ and ER␤ transcripts was observed from passages P1 to P7 in four different MEC isolates (24). The presence of ER␣ and ER␤ protein in 17␤-estradiolresponsive and -nonresponsive myometrial MEC was then examined by flow cytometry and Western blotting. Figure 4A shows flow cytometric traces from two representative MEC samples, one expressing ER␣ protein shown by a rightward shift in MFI. Low level and variable expression of ER␣ protein was observed in 4/8 samples (Fig. 4, A and B). Western blotting confirmed the variable expression of ER␣ in myometrial MEC isolates, showing a band at approximately 67 kDa, the expected molecular size for human ER␣ protein (Fig. 4C). ER␤ protein was examined by Western blotting, because highly specific ER␤ antibodies suitable for flow cytometry are not yet commercially available (25). In all MEC

FIG. 2. 17␤-estradiol increased rhVEGF165 binding to myometrial MEC in a time- and dose-dependent manner. A, Time course of rhVEGF165 binding to MEC incubated with 10 nmol/liter 17␤-estradiol. B, Concentration-response curve of MEC incubated with or without 17␤-estradiol for 18 h at 37 C. MFI of rhVEGF165 binding is reported as percentage of control (vehicle-treated cells). Results are mean ⫾ SEM from four experiments on separate MEC isolates for (A) and five isolates for (B), except for 10⫺11 and 10⫺6 mol/liter 17␤-estradiol, which were obtained from three of the five isolates. Significantly different from 0 h. *, P ⬍ 0.05, or vehicle **, P ⬍ 0.01.

FIG. 3. ER␣ and ER␤ mRNA expression in myometrial MEC. Representative Southern blot analysis of RT-PCR products of 9 MEC samples amplified using universal ER primers. mRNA from sample 9 was examined before and after FACS sorting CD31-labeled MEC. Before sorting MEC were 98.3% CD31⫹, after sorting 100%. ⫺, No RT; U, uterus; O, ovary.

isolates examined, the long form of ER␤ (26) was present, demonstrated by a band at around 59 kDa (Fig. 4C). There was correspondence in ER␣ expression in the 8/9 MEC samples examined at both mRNA and protein level (Figs. 3 and 4, and data not shown). 17␤-estradiol-mediated increase in rhVEGF165 binding is associated with ER␣ expression

There was a strong association between ER␣ expression in myometrial MEC and the ability of 17␤-estradiol to



17␤-estradiol increases VEGFR-2 expression in ER␣ positive myometrial MEC

rhVEGF165 binds to both VEGFR-1 and VEGFR-2, and we wished to determine whether the increased rhVEGF165 binding was due to 17␤-estradiol up-regulation of one or both receptors. We examined the effect of 17␤-estradiol on VEGFR-1 and VEGFR-2 surface protein expression by flow cytometry using a specific monoclonal antibody to each receptor and compared this expression with 17␤-estradiolinduced rhVEGF165 binding on four ER␣ positive and six ER␣ negative MEC samples. Figure 5A shows representative flow cytometry traces of VEGFR-2 expression in an ER␣expressing (ER␣⫹) MEC sample and an ER␣ negative

FIG. 4. ER␣ and ER␤ protein expression in myometrial MEC. A, Flow cytometric analysis of control cells, T47D (ER␣⫹), MDA-DB-453 (ER␣⫺) and two representative MEC samples, 6 (ER␣⫺) and 10 (ER␣⫹). CD31 for MEC is also shown and (B) summary of results from 8 MEC samples. C, Western blotting for ER␣ and ER␤ in control cells (T47D, MDA) and six representative MEC samples. Not all analyses were possible on all isolates due to inadequate numbers of low passage MEC available from some samples.

increase rhVEGF165 binding. 17␤-estradiol increased rhVEGF165 binding in 9 of the 16 MEC samples, and in 7 of these, ER␣ mRNA and/or protein was also expressed. Of the seven samples that failed to respond to 17␤-estradiol, six did not express ER␣ mRNA and/or protein, and only one was ER␣ positive. Contingency table analysis of this data (Table 1) demonstrates a significant relationship between 17␤-estradiol- mediated increase in rhVEGF165 binding in MEC for both ER␣ mRNA expression (P ⫽ 0.03) and ER␣ protein expression (P ⫽ 0.025). However, ER␤ mRNA and/or protein was expressed in all MEC samples examined, whether or not they responded to 17␤-estradiol (Table 1).

FIG. 5. 17␤-estradiol increases VEGFR-2 expression in ER␣⫹ expressing but not in ER␣⫺ myometrial MEC. Quiescent MEC in human serum-free medium were incubated for 18 h at 37 C in the presence of 0 (vehicle), 1 and 10 nmol/liter 17␤-estradiol and rhVEGF165 binding (f) and VEGFR-2 expression (䡺) then measured by flow cytometry. A, Representative flow cytometry histograms of VEGFR-2-FITC fluorescence for ER␣⫹ and ER␣⫺ samples. Negative control is mouse IgG1. B, ER␣⫹ expressing MEC. C, ER␣⫺ MEC. Results are expressed as percentage of control (vehicle only) rhVEGF165 binding or MFI of VEGFR-2 expression. Means ⫾ SEM from n ⫽ 4 (B) and n ⫽ 6 (C) separate MEC isolates. *, P ⬍ 0.05; **, P ⬍ 0.01.

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(ER␣⫺) sample treated for 18 h with vehicle or 1 nmol/liter 17␤-estradiol. A rightward shift of the fluorescence intensity histogram obtained from 5000 MEC was observed for the ER␣⫹ sample (left panel) compared with the vehicle control but not in ER␣⫺ MEC, indicating that 17␤-estradiol increased VEGFR-2 expression in the former but not the latter. Figure 5B shows that 17␤-estradiol (1 and 10 nmol/liter) significantly increased VEGFR-2 expression by approximately 25% compared with vehicle in ER␣⫹ MEC. Similarly, 17␤-estradiol significantly increased rhVEGF165 binding by 25%. In contrast, 17␤- estradiol had no stimulatory effect on rhVEGF165 binding or VEGFR-2 expression in ER␣ negative MEC samples (Fig. 5C). Rather, 17␤-estradiol decreased rhVEGF165 binding and VEGFR-2 expression in these ER␣ negative MEC samples. There was a significant correlation between the effect of 17␤-estradiol on rhVEGF165 binding and VEGFR-2 expression in both ER␣⫹ and ER␣⫺ MEC samples (RS ⫽ 0.66, P ⫽ 0.03, n ⫽ 10; and RS ⫽ 0.71, P ⫽ 0.047, n ⫽ 10) for 1 and 10 nmol/liter 17␤-estradiol, respectively. 17␤-estradiol had no effect on VEGFR-1 expression in ER␣ positive or negative MEC samples (results not shown). The ER antagonist ICI 182,780 inhibits 17␤-estradiolmediated increase in rhVEGF165 binding and 17␤-estradiol-augmentation of VEGF-induced MEC proliferation

The role of ER in 17␤-estradiol-mediated increase in rhVEGF165 binding was examined on 17␤-estradiol-responsive MEC isolates using an ER antagonist, ICI 182,780, which blocks the action of both ER␣ and ER␤ (27). MEC isolates were incubated with a maximal 17␤-estradiol concentration (0.1 or 10 nmol/liter) in the presence and absence of 100-fold molar excess ICI 182,780, and rhVEGF165 binding was measured. Figure 6A shows that ICI 182,780 significantly inhibited 17␤-estradiol-induced VEGF-binding (P ⬍ 0.05), suggesting that the increase in rhVEGF165 binding induced by 17␤-estradiol was mediated via ER. Because 17␤-estradiol increased both rhVEGF165 binding and VEGFR-2 expression, we examined whether 17␤-estradiol increased the proliferative response of ER␣⫹ myometrial MEC grown under suboptimal conditions (VEGF, 2 ␮g/liter and serum, 5% ch-HS/15% ch-FCS) (20). Figure 6B shows that 10 nmol/liter 17␤-estradiol augmented VEGFstimulated MEC proliferation, and this effect was also inhibited by 100-fold molar excess ICI 182,780. Discussion

A major finding of the present study was that 17␤estradiol, but not progesterone, increased VEGFR expression in human myometrial MEC as measured by changes in the level of ligand (rhVEGF165) binding. We found that this response to 17␤-estradiol was highly variable between subjects and was mediated by ER. A second major finding was that MEC isolated and cultured from human myometrium, a classic sex steroid-responsive tissue, consistently expressed ER␤, whereas the expression of ER␣ was low and variable between subjects. Thirdly, we found a strong association between ER␣, but not ER␤, expression in myometrial MEC and their ability to respond to 17␤-estradiol by increasing

FIG. 6. Antiestrogen ICI 182,780 inhibits 17␤-estradiol up-regulation of rhVEGF165 binding to myometrial MEC and 17␤-estradiol enhancement of VEGF-stimulated MEC proliferation. A, Quiescent MEC in serum-free medium containing 10 ␮g/liter VEGF were incubated for 18 h at 37 C with either 0.1 or 10 nmol/liter 17␤-estradiol in the presence or absence of 0.01 or 1 ␮mol/liter ICI 182,780 respectively and rhVEGF165 binding was then measured. Results are mean ⫾ SEM from three experiments on separate MEC isolates. *, P ⬍ 0.01; **, P ⬍ 0.05. B, Quiescent myometrial MEC (4 ⫻ 103) seeded in triplicate were incubated with 1 ␮mol/liter ICI 182,780 in the presence or absence of 10 nmol/liter 17␤-estradiol for 6 d at 37 C in phenol-red free M199 medium containing 2 ␮g/liter VEGF and 5% ch-HS and 15% ch-FCS, then MTS reagent added and absorbance measured. Results are presented as the change in absorbance over 6 d expressed as percentage of control MEC (vehicle only). Means (⫾ SEM) from five experiments on separate MEC isolates are shown. *, P ⬍ 0.01; **, P ⬍ 0.05.

rhVEGF165 binding. Finally, we demonstrated that the increased rhVEGF165 binding correlated with increased VEGFR-2 expression. 17␤-estradiol up-regulation of VEGFR-2 expression in human myometrial MEC

In the present study, we showed that approximately 60% of human myometrial MEC samples responded to 17␤estradiol with an increase in rhVEGF165 binding. This effect of 17␤-estradiol was both time and concentration dependent. 17␤-estradiol elicited a maximal increase in rhVEGF165 binding that includes physiological plasma concentrations for reproductive age women (0.1–2.2 nmol/liter). The response to 17␤-estradiol concentration was biphasic, and the peak concentration varied between individual MEC isolates. Similarly, Suzuma et al. (28) demonstrated that 17␤-estradiol specifically increased VEGFR-2 mRNA expression in a biphasic manner over a similar time frame in bovine retinal EC. Data from the present study using VEGFR-specific antibod-


ies suggest that 17␤-estradiol up-regulates VEGFR-2 rather than VEGFR-1 expression. 17␤-estradiol enhancement of VEGF-induced MEC proliferation observed in the present study and that of Suzuma et al. (28) may be a direct consequence of an estrogen-mediated increase in VEGFR-2 expression because EC proliferation is dependent on VEGFR-2 rather than VEGFR-1 signaling (29). Alternatively, 17␤estradiol may have an indirect effect on VEGFR-2 expression by increasing VEGF secretion from EC (28) because VEGF gene transcription is regulated by 17␤-estradiol (30). VEGF up-regulates its own receptor, VEGFR-2 (31), although this response was only observed after 24 h (18, 28, 31). 17␤estradiol augmentation of bFGF-stimulated EC proliferation is an indirect response, resulting from 17␤-estradiol stimulation of bFGF secretion by EC (32). Together, these studies suggest that 17␤-estradiol enhances angiogenesis by augmenting EC proliferation stimulated by two of the most significant angiogenic growth factors, VEGF and bFGF, although different mechanisms maybe involved (6, 28, 32, 33). The 17␤-estradiol enhancement of VEGFR-2 expression and subsequent proliferative effect mediated by VEGF in myometrial MEC may be important for the expansion of the myometrial microvasculature that undoubtedly occurs during myometrial growth associated with pregnancy and in vascular remodeling associated with placentation. It is uncertain whether 17␤-estradiol promotion of angiogenesis is limited to vascular beds of sex steroid-responsive tissues or occurs throughout the vasculature. This effect of 17␤estradiol has been observed in a range of EC in vivo and in vitro. For example, 17␤-estradiol augments EC proliferation in human coronary artery EC, umbilical vein EC (6, 7), myometrial MEC, and bovine retinal EC (28). 17␤-estradiol also promotes angiogenesis in bFGF-impregnated Matrigel plugs implanted sc in mice, where dermal MEC sprout, proliferate and migrate into the plug (6, 33). The cardiovascular protection afforded by estrogen replacement therapy may be to promote an EC proliferative response by up-regulating VEGFR-2 when vessel endothelium is damaged. This effect may not just be confined to large vessels but may contribute to protection of endothelium in other tissues, sensitizing EC to low levels of angiogenic growth factors by up-regulating VEGFR-2. In fact, topical 17␤-estradiol promotes wound healing in the aged, in whom it is typically delayed and in whom circulating 17␤-estradiol levels are low (34). The lack of response by myometrial MEC to progesterone, together with the diminished response observed with 17␤-estradiol and progesterone, may explain why the inclusion of progesterone in hormone replacement therapy is not as beneficial as a cardiovascular protective agent as estrogen alone (35). ER␣ and ER␤ expression in human myometrial MEC

The second major finding of this study was that cultured myometrial MEC constitutively express ER␤, whereas ER␣ expression was variable between subjects. This is the first study that has examined ER␣ and ER␤ expression in purified human MEC in a cohort of subjects large enough to detect the level of variability observed for ER␣ expression. We found that ER␣ (when present) and ER␤ mRNA expression was


consistently demonstrated for up to seven passages. Others have demonstrated ER in human large vessel and endometrial MEC, but the specific type of ER was not determined (7, 9, 10). In an immunohistochemical study of human tissues, the great vessels were both ER␣ and ER␤ positive (36), whereas others found cultured umbilical vein EC expressed ER␤, but not ER␣ mRNA (37, 38). However, in immunohistochemical studies of myometrial tissue, ER␤ but not ER␣ was demonstrated in rat EC (39), but in mouse and rhesus monkey, both ER␣ and ER␤ were expressed (40, 41). Together, these studies indicate that ER␤ expression may be constitutive in large and microvessel EC and that ER␣ expression is variable and may depend on EC phenotype and the conditions under which they were evaluated. Role of ER␣ and ER␤ in 17␤-estradiol up-regulation of VEGFR-2 in human myometrial MEC

A major finding of the present study was the significant association between ER␣ expression and the ability of myometrial MEC to functionally respond to 17␤-estradiol by increasing rhVEGF165 binding and VEGFR-2 expression. While our data on ER expression is only semiquantitative, it is striking that in general those samples expressing ER␣ responded to 17␤-estradiol, whereas those lacking ER␣ failed to respond. This, together with the time taken (several hours) for 17␤-estradiol to elicit this response, and its inhibition by the antiestrogen, ICI 182,780, provides three lines of evidence suggesting that the 17␤-estradiol-mediated increase in VEGFR-2 expression is genomic and mediated by ER, most likely ER␣. It is uncertain whether the 17␤-estradiol-induced increase in VEGFR-2 expression in bovine retinal EC was mediated by ER, because the effect of an ER antagonist was not examined (28). It is possible that 17␤-estradiol via ER␣ directly regulates VEGFR-2 expression, because the VEGFR-2 promoter contains 5 Sp1 binding sites (42) and is regulated by the Sp1 transcription factor (43). Furthermore, 17␤-estradiol activates gene transcription through interaction with Sp1 proteins via ER␣, but not ER␤ (44). The variable expression of ER␣ in myometrial MEC, as well as the variability in functional response to 17␤-estradiol, suggests that other factors may influence these parameters. However, we have found no relationship between age of patient, indication for hysterectomy (mainly fibroids), stage of menstrual cycle when hysterectomy was performed, or the ability of MEC to grow well in culture, and ER␣ expression. Although MEC were obtained from the inner two thirds of the myometrium (20), because the inner layers show greater sex steroid responsiveness (45), it is possible that sampling different sites of the uterine wall may influence ER␣ expression. It will be important to determine whether similar variability in ER␣ expression occurs in other vascular beds and in particular in the large vessels commonly affected by atherosclerosis. Similar variability in ER expression was observed in a study of cultured vascular smooth muscle cells (VSMC) from human coronary arteries, where 53% of subjects expressed ER (46). While this study could not examine for ER␣ and ER␤ expression, the authors did find a significant association between VSMC expression of ER and the absence of atherosclerosis in premenopausal women. These

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data, together with our own, raise interesting clinical questions of whether such a correlation exists between MEC expression of ER␣ in our cohort of patients, and whether the vascular response of these women to estrogen replacement therapy would also vary. With respect to the myometrial microvasculature, variable ER␣ expression may result in the variable degrees of uterine atrophy observed in women taking hormone replacement therapy, because the microvasculature may regulate tissue mass (47). We have demonstrated a significant association between ER␣ expression and the functional response mediated by 17␤-estradiol, but definitive proof awaits the use of ligands or antagonists with sufficient specificity for ER␣ and ER␤. Much research is being devoted to the identification of such specific ligands; however, those currently available are not of sufficient specificity (48) for testing in MEC coexpressing both ER␣ and ER␤. Because ER␣ and ER␤ can heterodimerize (49), and ER␤ can operate as a negative regulator of target gene transcription in a promoter-dependent manner (25, 49), the ratio of ER␤:ER␣ may be more important in determining the final outcome of 17␤-estradiol effects on myometrial MEC and EC in general. In human VSMC, where ER␤ predominates, the ratio of ER␤:ER␣ varied considerably between individuals (25). It remains to be determined whether the ER␤:ER␣ ratio is similar for EC and VSMC in a given vascular bed for an individual and whether the function of target genes in these two vascular cell types are altered similarly. Differences already exist, because 17␤-estradiol has opposing activities on EC and VSMC proliferation (2, 6, 7, 28, 41, 50). Studies from ER␣ knockout mice suggest that 17␤estradiol-dependent angiogenesis and re-endothelialization involve ER␣ (33, 51), but that ER␤ is important in vascular response to injury, which involved both VSMC and EC (50, 52). It is likely that the cardiovascular protective effect of estrogen results from multiple effects of 17␤-estradiol on different cellular components of the blood vessel wall, which in turn are dependent on complex interactions between ER␣ and ER␤, the response elements present in target genes and the complement of coregulators in both cell types. Although 17␤-estradiol may have different functional responses on EC and VSMC, these actions may together contribute to the cardiovascular protection afforded by 17␤-estradiol. The level of protection may be quite different between individuals, depending on the level of endothelial ER␣ expression, which is expected to affect uterine MEC and may involve the entire vasculature. Acknowledgments We thank the gynecologists at Monash Medical Centre for provision of hysterectomy tissue, Nancy Taylor for collection of tissue, Mal Forsyth and the Australian Red Cross Blood Service, Melbourne for the provision of male human serum, and Paul Hutchinson, Department of Immunology, for assistance with the flow cytometry. Received April 9, 2001. Accepted May 28, 2002. Address all correspondence and requests for reprints to: Dr. Caroline Gargett, Centre for Women’s Health Research, Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: caroline.gargett@med.monash.edu.au. This study was supported by the Australian National Health and Medical Research Foundation Grant 124331 (to P.A.W.R.).


References 1. Foegh ML, Ramwell PW 1998 Cardiovascular effects of estrogen: implications of the discovery of the estrogen receptor subtype ␤. Curr Opin Nephrol Hyperten 7:83– 89 2. Mendelsohn ME, Karas RH 1999 The protective effects of estrogen on the cardiovascular system. New Engl J Med 340:1801–1811 3. Nasr A, Breckwoldt M 1998 Estrogen replacement therapy and cardiovascular protection: lipid mechanisms are the tip of an iceberg. Gynecol Endocrinol 12:43–59 4. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn RE, Shaul PW 1999 Estrogen receptor-␣ mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103:401– 406 5. Razandi M, Pedram A, Levin ER 2000 Estrogen signals to the preservation of endothelial cell form and function. J Biol Chem 275:38540 –38546 6. Morales DE, McGowan KA, Grant DS, Maheshwari S, Bhartiya D, Cid MC, Kleinman HK, Schnaper HW 1995 Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model. Circulation 91:755–763 7. Kim-Schulze S, McGowan KA, Hubchak SC, Cid MC, Martin MB, Kleinman HK, Greene GL, Schnaper HW 1996 Expression of an estrogen receptor by human coronary artery and umbilical vein endothelial cells. Circulation 94: 1402–1407 8. Vazquez F, Rodriguez-Manzaneque JC, Lydon JP, Edwards DP, O’Malley BW, Iruela-Arispe ML 1999 Progesterone regulates proliferation of endothelial cells. J Biol Chem 274:2185–2192 9. Venkov CD, Rankin AB, Vaughan DE 1996 Identification of authentic estrogen receptor in cultured endothelial cells. A potential mechanism for steroid hormone regulation of endothelial function. Circulation 94:727–733 10. Iruela-Arispe ML, Rodriguez-Manzaneque JC, Abu-Jawdeh G 1999 Endometrial endothelial cells express estrogen and progesterone receptors and exhibit a tissue specific response to angiogenic growth factors. Microcirculation 6:127–140 11. Wehling M 1997 Specific, nongenomic actions of steroid hormones. Annu Rev Physiol 59:365–393 12. Dechering K, Boersma C, Mosselman S 2000 Estrogen receptors ␣ and ␤: two receptors of a kind? Curr Med Chem 7:561–576 13. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson JÅ, Kushner PJ, Scanlon TS 1997 Differential ligand activation of estrogen receptors ER␣ and ER␤ at AP1 sites. Science 277:1508 –1510 14. Ferrara N, Davis-Smyth T 1997 The biology of vascular endothelial growth factor. Endocr Rev 18:4 –25 15. Hewett PW, Murray JC 1996 Coexpression of flt-1, flt-4 and KDR in freshly isolated and cultured human endothelial cells. Biochem Biophys Res Commun 221:697–702 16. Jakeman L, Winer J, Bennett GL, Altar CA, Ferrara N 1992 Binding sites for vascular endothelial growth factor are localised on endothelial cells in adult rat tissues. J Clin Invest 89:244 –253 17. Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N 1998 Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3⬘-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 273:30336 –30343 18. Kremer C, Breier G, Risau W, Plate KH 1997 Up-regulation of flk-1/vascular endothelial growth factor receptor 2 by its ligand in a cerebral slice culture system. Cancer Res 57:3852–3859 19. Smith CL 1998 Cross-talk between peptide growth factor and estrogen receptor signaling pathways. Biol Reprod 58:627– 632 20. Gargett CE, Bucak K, Rogers PAW 2000 Isolation, characterization and longterm culture of human myometrial microvascular endothelial cells. Hum Reprod 15:293–301 21. Chu S, Mamers P, Burger HG, Fuller PJ 2000 Estrogen receptor isoform gene expression in ovarian stromal and epithelial tumors. J Clin Endocrinol Metab 85:1200 –1205 22. Lan HY, Hutchinson P, Tesch GH, Mu W, Atkins RC 1996 A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry. J Immunol Meth 190:1–10 23. Schutte B, Scheres HME, de Goeij AFPM, Rousch MJM, Blijham GH, Bosman FT, Ramaekers FCS 1992 Flow cytometric steroid receptor analysis. Prog Histochem Cytochem 26:68 –76 24. Gargett CE, Bucak K, Chu S, Fuller PJ, Rogers PAW, Human myometrial and fat microvascular endothelial cells express ER␣ and ER␤. Proceedings of the 10th International Congress of Endocrinology, Sydney, Australia, 2000, p 377 (Abstract P1128) 25. Hodges YK, Tung L, Yan XD, Graham JD, Horwitz KB, Horwitz LD 2000 Estrogen receptors ␣ and ␤. Prevalence of estrogen receptor ␤ mRNA in human vascular smooth muscle and transcriptional effects. Circulation 101:1792–1798 26. Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, Muramatsu M 1998 The complete primary structure of human estrogen receptor ␤ (hER␤) and its heterodimerization with ER ␣ in vivo and in vitro. Biochem Biophys Res Commun 243:122–126 27. Hermenegildo C, Cano A 2000 Pure anti-oestrogens. Hum Reprod Update 6:237–243

Gargett et al. • ER␣/ER␤ and VEGFR-2 Expression in MEC 28. Suzuma I, Mandai M, Takagi H, Suzuma K, Otani A, Oh H, Kobayashi K, Honda Y 1999 17␤-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells. Invest Ophthalmol Vis Sci 40:2122–2129 29. Petrova TV, Makinen T, Alitalo K 1999 Signaling via vascular endothelial growth factor receptors. Exp Cell Res 253:117–130 30. Mueller MD, Vigne JL, Minchenko A, Lebovic DI, Leitman DC, Taylor RN 2000 Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors ␣ and ␤. Proc Natl Acad Sci USA 97:10972–10977 31. Shen BQ, Lee DY, Gerber HP, Keyt BA, Ferrara N, Zioncheck TF 1998 Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro. J Biol Chem 273:29979 –29985 32. Kim-Schulze S, Lowe WLJ, Schnaper HW 1998 Estrogen stimulates delayed mitogen-activated protein kinase activity in human endothelial cells via an autocrine loop that involves basic fibroblast growth factor. Circulation 98: 413– 421 33. Johns A, Freay AD, Fraser W, Korach KS, Rubanyi GM 1996 Disruption of estrogen receptor gene prevents 17␤ estradiol-induced angiogenesis in transgenic mice. Endocrinology 137:4511– 4513 34. Ashcroft GS, Greenwell-Wild T, Horan MA, Wahl SM, Ferguson MJ 1999 Topical estrogen accelerates cutaneous wound healing in aged humans associated with an altered inflammatory response. Am J Pathol 155:1137–1146 35. Barrett-Connor E, Grady D 1998 Hormone replacement therapy, heart disease, and other considerations. Annu Rev Public Health 19:55–72 36. Taylor AH, Al Aawizz F 2000 Immunolocalisation of oestrogen receptor ␤ in human tissues. J Mol Endocrinol 24:145–155 37. Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried G, Nordenskjo¨ ld M, Gustafsson JÅ 1997 Human estrogen receptor ␤-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab 82:4258 – 4265 38. Stefano GB, Prevot V, Beauvillain JC, Cadet P, Fimiani C, Welters I, Fricchione GL, Breton C, Lassalle P, Salzet M, Bilfinger TV 2000 Cell-surface estrogen receptors mediate calcium-dependent nitric oxide release in human endothelia. Circulation 101:1594 –1597 39. Wang H, Masironi B, Eriksson H, Sahlin L 1999 A comparative study of estrogen receptors ␣ and ␤ in the rat uterus. Biol Reprod 61:955–964 40. Wu WX, Ma XH, Smith GCS, Nathanielsz PW 2000 Differential distribution


41. 42. 43. 44. 45.

46. 47. 48. 49. 50.

51. 52.

of ER ␣ and ER ␤ mRNA in intrauterine tissues of the pregnant rhesus monkey. Am J Physiol 278:C190 –C198 Zhang L, Fishman MC, Huang PL 1999 Estrogen mediates the protective effects of pregnancy and chorionic gonadotropin in a mouse model of vascular injury. Arterioscler Thromb Vasc Biol 19:2059 –2065 Patterson C, Perrella MA, Hsieh CM, Yoshizumi M, Lee ME, Haber E 1995 Cloning and function analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. J Biol Chem 270:23111–23118 Hata Y, Duh E, Zhang K, Robinson GS, Aiello LP 1998 Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem 273:19294 –19303 Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustaffson JÅ, Safe S 2000 Ligand-, cell-, and estrogen receptor subtype (␣/␤)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:5379 –5387 Noe M, Kunz G, Herbertz M, Mall G, Leyendecker G 1999 The cyclic pattern of the immunocytochemical expression of oestrogen and progesterone receptors in human myometrial and endometrial layers: characterization of the endometrial-subendometrial unit. Hum Reprod 14:190 –197 Losordo DW, Kearney M, Kim EA, Jekanowski J, Isner JM 1994 Variable expression of the estrogen receptor in normal and atherosclerotic coronary arteries of premenopausal women. Circulation 89:1501–1510 Folkman J 1998 Is tissue mass regulated by vascular endothelial cells? Prostate as the first evidence. Endocrinology 139:441– 442 Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson JA, Nilsson S 1998 Differential response of estrogen receptor ␣ and estrogen receptor ␤ to partial estrogen agonists/antagonists. Mol Pharmacol 54:105–112 Cowley SM, Hoare S, Mosselman S, Parker MG 1997 Estrogen receptors ␣ and ␤ form heterodimers on DNA. J Biol Chem 272:19858 –19862 Iafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan Jr TR, Lubahn DB, O’Donnell Jr TF, Korach KS, Mendelsohn ME 1997 Estrogen inhibits the vascular injury response in estrogen receptor ␣-deficient mice. Nat Med 3: 545–548 Brouchet L, Krust A, Dupont S, Chambon P, Bayard F, Arnal JF 2001 Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-␣ but not estrogen receptor-␤. Circulation 103:423– 428 Lindner V, Kim SK, Karas RH, Kuiper GM, Gustafsson JA, Mendelsohn ME 1998 Increased expression of estrogen receptor-␤ mRNA in male blood vessels after vascular injury. Circ Res 83:224 –229