Molecular Human Reproduction, Vol. 8, No. 8, 770-775,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Estrogen receptor-
and -ß expression in microvascular endothelial cells and smooth muscle cells of myometrium and leiomyoma
1 Centre for Women's Health Research, Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre and 2 Prince Henry's Institute of Medical Research 246 Clayton Road, Clayton, Victoria, 3168, Australia
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Abstract |
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The two estrogen receptors, ER
and ERß, are likely to have roles in the pathophysiology of fibroid development. They have been detected in myometrial and leiomyoma (fibroid) tissue, but the cell types expressing ER and ERß have not been determined. ERs have also been detected in human endothelial cells. The aims of the present study were to determine whether pure populations of myometrial microvascular endothelial cells (MEC) express ER and ERß, to compare MEC ER/ERß expression with that of pure populations of myometrial smooth muscle cells (SMC) and to determine if ER/ERß are differentially expressed in MEC and SMC of myometrium and fibroids from nine paired samples. Using RT–PCR (for ER and ERß) and Western blotting (for ER only), we demonstrated that all cultures of early passage myometrial and fibroid SMC (>99% pure) expressed ER but not ERß, while myometrial and fibroid MEC (>99% CD31+) constitutively expressed ERß. However, both myometrial and fibroid MEC showed variable expression of ER, with ~60% of MEC samples expressing ER. While the majority (6/9) of MEC from myometrial and fibroid pairs demonstrated the same pattern of ER expression, 3/9 pairs showed discordant ER expression. These results show that ER and ERß are differentially expressed in SMC and MEC of human myometrium and fibroids. Since ER and ERß mediate opposing transcriptional activities, any effect of estrogen on the growth and development of fibroids is likely to be complex and may involve both SMC and MEC.
ER/ERß/fibroid/myometrium/microvascular endothelial cells/smooth muscle cells
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Introduction |
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Uterine fibroids or leiomyomata are the most common solid tumour in adult women, affecting at least 25% during their reproductive years (Vollenhoven, 1998
). However, the actual incidence is much higher, since 77% of hysterectomies examined in a systematic study contained fibroids, irrespective of indication for surgery (Cramer and Patel, 1990). Fibroids are the most common indication for hysterectomy, and in Australia 21.7% of all hysterectomies are performed for fibroids. As the prevalence of hysterectomy in Australia is 3.97 per 1000 women, it is one of the most common surgical procedures performed (Renwick and Sadhowski, 1991). Despite fibroids being such a major public health issue, little is known of their aetiology. Uterine leiomyomata are benign tumours of myometrial smooth muscle cells (SMC), that are well encapsulated, collagen-rich and contain a sparse vasculature (Scully, 1992; Casey et al., 2000). The sex steroid hormones estrogen and progesterone appear to play a central role in fibroid pathology. Hypoestrogenic states, such as menopause and GnRH analogue treatment induce fibroid shrinkage (Vollenhoven, 1998). Fibroids overexpress estrogen receptors (ER) (Andersen et al., 1995; Brandon et al., 1995; Englund et al., 1998) and show hypersensitivity to estrogen by up-regulating a number of estrogen target genes (Andersen and Barbieri, 1995).
There are two ERs, ER and ERß, which are encoded by separate genes (Foegh and Ramwell, 1998; Dechering et al., 2000). ER and ERß bind estrogen with similar high affinity, causing receptor dimerization. They act as ligand-activated transcription factors for a range of estrogen target genes by binding to estrogen response elements or indirectly by interaction with other DNA binding proteins (Paech et al., 1997). However, ER and ERß can mediate opposing transcriptional activities, depending on the type of response element in target gene promoters and on other cell-specific factors such as the presence or absence of co-regulators (Dechering et al., 2000). There are also differences in the cellular distribution of ER and ERß, although there is considerable overlap, and heterodimerization may occur in cells where they are co-expressed (Cowley et al., 1997).
It is well known that ERs are expressed in the smooth muscle tissue of both myometrium and fibroids. Recent studies have detected both ER and ERß in myometrium and uterine fibroid tissue (Brandon et al., 1995; Pedeutour et al., 1998; Benassayag et al., 1999; Wu et al., 2000), but the actual cells expressing ER and ERß in myometrium and fibroids have not been determined. ER have also been detected in human large vessel endothelial cells (Kim-Schulze et al., 1996; Venkov et al., 1996) and in microvascular endothelial cells (MEC) from human endometrium (Iruela-Arispe et al., 1999), although the type of ER was not determined in these studies. We hypothesized that myometrial MEC would express the two ER subtypes, ER and ERß, and that myometrial MEC and SMC would differ in their expression of ER and ERß. We also hypothesized that expression of ER and ERß in MEC and SMC would differ between myometrium and fibroids. The aims of the present study were to determine: (i) whether pure populations of cultured myometrial MEC express ER and ERß; (ii) to compare MEC ER and ERß expression with that of pure populations of cultured myometrial SMC; and (iii) to determine if ER and ERß are differentially expressed in MEC and SMC from paired samples of myometrium and fibroids. Here we show that myometrial and fibroid SMC constitutively express ER but not ERß, while myometrial and fibroid MEC constitutively express ERß, and that ER expression varies between subjects, and between myometrial and fibroid MEC in some individual subjects.
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Materials and methods |
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Human tissues
Human myometrial and fibroid tissues were obtained from nine ovulating women (mean age 44.3 years, range 33–53) who had not taken exogenous hormones in the previous 3 months and who had undergone hysterectomy due to fibroids. Informed consent was obtained from each patient and ethical approval was obtained from Monash Medical Centre Human Research and Ethics Committee B. The stage of the menstrual cycle was determined by histological examination of formalin-fixed, H&E-stained endometrial sections by experienced histopathologists using established criteria for the normal menstrual cycle (Noyes et al., 1950
). The tissue was collected in HEPES-buffered M199 culture medium (Gibco BRL, Gaithersburg, MD, USA), containing 10% fetal calf serum (FCS; CSL, Melbourne, Australia) and antibiotic/antimycotic solution (Gibco), stored overnight at 4°C and then processed.
Isolation and culture of myometrial microvascular endothelial cells and smooth muscle cells
Normal myometrial tissue was dissected from the surrounding fibroid(s) and dissociated with collagenase and DNase type I, followed by a short trypsin treatment to produce single cell suspensions containing both MEC and SMC, as previously described (Gargett et al., 2000b). MEC were separated from SMC by positive selection with UEA-1-coated Dynabeads (Dynal, Oslo, Norway), seeded onto fibronectin-coated tissue culture flasks (10 µg/ml) and cultured in M199 medium containing 15% human serum (HS) and 5% FCS, 2 mmol/l glutamine, 5 ng/ml basic fibroblast growth factor (bFGF), 0.1 mg/ml heparin and antibiotic/antimycotic solution. MEC were repurified with UEA-1-coated Dynabeads on subsequent passage and just prior to extracting RNA for RT–PCR analysis and purity was assessed (see below). MEC were used for all analyses between the first and third passage.
The separated SMC were cultured on uncoated plastic flasks in M199 medium containing 10% FCS and used for all analyses between the first and fourth passage. SMC harvested for RT–PCR were pretreated with UEA-1-coated Dynabeads to remove any contaminating MEC and purity was determined (see below).
Isolation and culture of fibroid MEC and SMC
Fibroid MEC and SMC were isolated and cultured from fibroid tissue obtained from the same hysterectomy samples and processed using similar protocols described for myometrial MEC and SMC. However, a longer enzymatic dissociation (3 h) and higher seeding density for MEC (12–16x104/cm2) was required. The endothelial character of fibroid MEC was determined by immunohistochemical analysis of cells grown on coverslips using biotinylated UEA-1 and antibodies to CD31, Factor VIIIra and -smooth muscle actin (-SMA) as described for myometrial MEC (Gargett et al., 2000b).
Flow cytometric analysis of myometrial and fibroid MEC and SMC purity
Prior to each analysis, fibroid and myometrial MEC and SMC cultures were examined for purity by harvesting the cells with trypsin (0.025%), EDTA (0.25 mmol/l) and immunophenotyping using flow cytometry.
MEC (5x104) were incubated with mouse anti-human CD31 antibody (Dako, Carpintaria, CA, USA; 8 µg/ml) for 1 h at 4°C, followed by washing and incubation with R-phycoerythrin (PE)-conjugated secondary antibody (PE-anti-mouse IgG Fab'2 fragments, 1/100; Silenus, Boronia, Victoria, Australia) for 30 min at 4°C. MEC were washed and examined in a flow cytometer (Mo-Flo; Cytomation, Colorado, USA). For the negative control, mouse IgG1 (8 µg/ml) was substituted for the primary antibody. The mean fluorescence intensity (MFI) of single parameter histograms from >5000 cells were obtained, the MFI of the IgG1 control subtracted and the percentage positive cells with fluorescence intensity >98% of control cells determined. MEC were used for ER/ERß RT–PCR when >99% CD31+ and for all other analyses when >95% CD31+.
SMC were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS; 106 cells/ml) for 30 min at 4°C, washed, resuspended in 0.01 mol/l citrate buffer, pH 6.0 and permeabilized by microwaving for 30–40 s on high power (500 W), cooled on ice for 10 min, washed and 5x104 SMC were then incubated with 3 µg/ml mouse anti-human -SMA (Dako) using a modification of a previously published method for detecting intracellular antigens (Lan et al., 1996). SMC were then washed, incubated with PE-labelled secondary antibody and analysed by flow cytometry as described for MEC. Mouse IgG2A (3 µg/ml) was used for the negative control. SMC were used for ER/ERß RT–PCR when >98% were -SMA+ and for Western blotting when >90% were -SMA+.
Analysis of mRNA for ER and ERß by RT–PCR and Southern blotting
Total RNA was prepared from highly purified cultured MEC and SMC using the Qiagen RNeasy Mini Kit. Total RNA of 1 µg was reverse transcribed for 90 min at 42°C and amplified using universal primers for both ER and ERß (sense primer: 5'-CCGGAATTCTTC/TGACATGCTC/GCTGG; antisense primer: 5'-GATGC/TTCCATGCCC/TTTGT TAC TC) and for ß2-microglobulin in a single stage PCR for 30 cycles as previously described (Chu et al., 2000). PCR products were visualized on 1.8% agarose gel, transferred to Hybond N+ membranes (Amersham) and probed with gene-specific 32P-labelled probes (ER probe: 5'-GGTTGTGTGCCTCAAATCTATTATTT; ERß probe: 5'-ATATCTCTGTGTCAAGGCCATGA) (Chu et al., 2000).
Western blot analysis for ER
Lysates of cultured MEC and SMC were prepared in PBS by freeze–thaw method and denatured in sodium dodecyl sulphate (SDS) sample buffer for 5 min at 95°C. Samples of 20 µg protein (determined by BCA method; Pierce, Rockford, IL, USA) were then separated by 10% SDS–PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% Blot-QuickBlockerTM (Chemicon International, Temecula, CA, USA) for 30 min at 22°C, incubated for 90 min at 22°C with mouse anti-human ER antibody (0.93 µg/ml; Novacastra, Newcastle upon Tyne, UK) in femto/tris buffered saline Tween-20 (TBST) buffer (Chemicon) then incubated for 60min at 22°C with horseradish peroxidase-anti-mouse IgG (1/2000; Zymed, San Francisco, CA, USA) and detected by femtoLUCENTTM chemiluminescence system (Chemicon). ER positive and negative control cells (T47D and MDA-DB-453 respectively) were included with each run.
ERß protein was not examined in MEC or SMC due to the lack of good quality commercial ERß antibodies for detecting wild-type ERß in Western blots (Pavao and Traish, 2001).
ER expression by flow cytometry
In some samples, ER expression was determined by flow cytometric analysis. Cells were harvested with trypsin:EDTA and 106 cells were fixed for 30 min at 4°C in 2% paraformaldehyde in PBS and resuspended in 0.01 mol/l citrate buffer, pH 6.0, and microwaved for 30 s. Cells were resuspended in PBS/1% FCS and aliquots (5x104cells) incubated with mouse anti-human ER antibody (7 µg/ml) for 60 min at 4°C, followed by PE-anti-mouse IgG antibody for 30 min at 4°C and examined by flow cytometry. The MFI of single parameter histograms of >5000 cells were obtained after subtracting the MFI of IgG negative controls (mouse IgG1 7 µg/ml). ER positive (T47D) and negative (MDA-DB-453) control cells were analysed in each batch.
Statistical analysis
Contingency table analysis was by Fisher's exact test for low sample numbers for comparison between myometrial and fibroid MEC for ER expression using SPPS version 10.0. P < 0.05 was considered significant.
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Results |
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Fibroid MEC cultures were established for the first time and were characterized for their endothelial character as described (Gargett et al., 2000b
). Figure 1 shows that fibroid MEC exhibited typical endothelial cobblestone morphology in culture, expressed CD31, FVIIIra, bound UEA-1 lectin and were -SMA negative.
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ER
/ERß expression in paired myometrial and fibroid MECHighly purified MEC isolated and cultured from paired samples of fibroid and host myometrial tissue were examined for ER
and ERß mRNA expression. Figure 2 shows a representative Southern blot analysis of RT–PCR products amplified from cultures derived from two samples, using universal ER and ERß primers. All fibroid and myometrial MEC samples examined in the present study expressed ERß mRNA (Figure 2, Table I). However, there was variable expression of ER mRNA between subjects for myometrial MEC (5/9 ER+) and for fibroid MEC (5/8 ER+). Figure 2 shows typical examples of ER+ (lanes 1 and 2) and ER– (lanes 5 and 6) myometrial and fibroid MEC pairs. In some samples (3/8) there were differences in ER mRNA expression between MEC isolated from myometrial and fibroid pairs (Table I). Of these three discordant pairs, one myometrial sample was ER+, while the paired fibroid MEC were ER–, and there were two ER– myometrial MEC samples, while the paired fibroid MEC were ER+. However, there was no significant difference in ER expression between myometrial and fibroid MEC (P = 0.52). ER (when present) and ERß transcripts were consistently demonstrated for up to seven passages in culture (n = 2 myometrial and n = 2 fibroid MEC samples).
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Figure 3
shows a representative Western blot of the four cell types isolated and cultured from two subjects. ER protein in myometrial and fibroid MEC showed the same variable expression between samples, and within the same three myometrial fibroid pairs, as demonstrated for mRNA (Figure 3, Table I
). An example of ER– myometrial MEC and ER+ fibroid MEC from a paired sample is shown in Figure 3, sample 8.
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ER
/ERß expression in paired myometrial and fibroid SMCAs expected, SMC from both myometrial and fibroid pairs expressed ER
mRNA, but failed to express ERß mRNA in all eight samples examined (Table I). All myometrial and fibroid SMC examined expressed ER protein (Figure 3, Table I). As has been reported by others (Brandon et al., 1995), an apparent variation in expression levels of SMC ER was observed between patients (Figure 3); however, these were not quantified. |
Discussion |
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The present study characterized for the first time ER
and ERß expression in MEC and SMC cultured from paired myometrial and fibroid tissues. A major finding was that MEC and SMC in both myometrium and fibroids differentially express ER and ERß transcripts. Our results demonstrate that constitutive expression of ER mRNA and protein occurs in highly purified cultures of myometrial and fibroid SMC, while ERß is not expressed. In contrast, pure cultures of myometrial and fibroid MEC constitutively express ERß mRNA, while the expression of ER varies between subjects. In contrast, Wu and colleagues demonstrated ERß mRNA and protein in primary cultures of myometrial SMC (Wu et al., 2000). However, there is no indication that contaminating MEC were removed from their myometrial cultures. In our experience, it was essential to purify both MEC and SMC cultures prior to RT–PCR analysis, since primary non-passaged SMC cultures contain a significant proportion of MEC (up to 13%, Gargett and Bucak, unpublished data), which survive several passages in SMC cultures. Purification of SMC cultures with Ulex-coated Dynabeads reduce contaminating CD31+MEC to <1% and traces of ERß mRNA were removed in most samples. In agreement with our data, ER, but not ERß, was demonstrated in rat myometrium by in-situ hybridization (Mowa and Iwanaga, 2000), but vascular expression was not studied. However, ERß expression was demonstrated in endothelial cells of cervical blood vessels in pregnant and non-pregnant women by immunohistochemistry (Wang et al., 2001a), and although the authors claimed that endothelial cells showed no immunoreactivity with ER antibodies, close examination of their images suggest that some endothelial cells were also ER positive. Similarly, in another study by the same authors comparing myometrial and fibroid ER/ERß expression, ERß positive and variable ER immunostaining could be seen in microvessel endothelium of both myometrium and fibroids, although these observations were not commented upon (Wang et al., 2001b). ER and ERß immunoreactivity has also been demonstrated in endothelial cells of the rat uterine artery, with ERß the predominant receptor (Andersson et al., 2001). All three studies detected ERß in SMC using the same antibody (Andersson et al., 2001; Wang et al., 2001a,b), in contrast to in-situ hybridization findings (Mowa and Iwanaga, 2000; Wang et al., 2001b), suggesting that the half-life of ERß mRNA is shorter than that of the ERß protein, or that while ERß is detectable in vivo, unlike ER, ERß expression in myometrial SMC is rapidly lost in culture.
The present study agrees with our earlier work demonstrating that ER expression varies in MEC isolated from different subjects (Gargett et al., 2000a), while ERß is constitutively expressed in both myometrial and fibroid MEC. The present study also demonstrates consistency in ER expression between myometrial and fibroid MEC in most paired samples in our cohort; however, there were discrepancies in three of nine myometrial and fibroid pairs. There was agreement between ER protein and mRNA expression in these three myometrial fibroid MEC pairs, suggesting that the differences were real. However, it is possible that these differences result from the culture process. While culture conditions were the same for myometrial and fibroid MEC, some fibroid MEC cultures were difficult to establish, grew at a slower rate and remained for longer periods in culture prior to analysis compared with paired myometrial cultures (Zaitseva and Gargett, 1999; unpublished data), and this may have contributed to the observed differences in ER expression. However, we detected consistent expression of ER and ERß mRNA over seven passages for both myometrial and fibroid MEC and SMC. Others have demonstrated functional ER in myometrial and fibroid SMC which had been cultured for 3 weeks and sometimes passaged (Andersen et al., 1995). In contrast, ER expression was shown to be rapidly lost from myometrial and fibroid explant cultures (Severino et al., 1996). These differences probably reflect variable culture conditions and suggest that ER expression is maintained better in monolayer than explant culture.
Recent studies of paired myometrial and fibroid tissue homogenates demonstrated ER and ERß expression in both tissues (Pedeutour et al., 1998; Benassayag et al., 1999), but the relative contribution of the vascular and muscular components of these tissues could not be distinguished. Our studies highlight the importance of identifying the cells responsible for ER and ERß expression, particularly if functional studies using estrogen are to be conducted on whole tissues comprising a variety of cell types, as is the case for fibroids.
The factors regulating fibroid growth are not well understood, although both estrogen and progesterone are likely to be important (Vollenhoven, 1998). Fibroid SMC cultures demonstrate exaggerated transcriptional responses to estrogen (Andersen et al., 1995), and ER (ER) mRNA expression is greater in fibroid than surrounding myometrial tissue (Andersen et al., 1995; Brandon et al., 1995; Englund et al., 1998), although others have detected no differences (Vollenhoven et al., 1994; Lessl et al., 1997). While we did not undertake a quantitative analysis of relative ER expression in myometrial and fibroid SMC, our data suggest that SMC ER, rather than ERß, may be more important in the pathophysiology of fibroid development and growth. The relative expression of steroid receptor co-activators or co-repressors may also be important in the exaggerated transcriptional responses of fibroids to estrogen.
Angiogenesis is the growth of new vasculature from existing vessels, a process involving MEC (Risau, 1997). While the role of angiogenesis in malignant tumour growth is well established (Folkman, 1995), little is known of its role in the development and growth of benign tumours, particularly fibroids. Microvessel density (MVD) of fibroids is significantly lower than in surrounding myometrial tissue and the distribution of vessels in fibroids is highly irregular, with large avascular regions (Casey et al., 2000). The variable expression of ERß reported in fibroid tissues (Pedeutour et al., 1998; Benassayag et al., 1999) may be due to this variable degree of vascularization. This irregular vascular growth may be due to relative distribution of the angiogenic promoters such as vascular endothelial growth factor (VEGF) or bFGF, both reported in fibroid SMC (Harrison-Woolrych et al., 1995; Wu et al., 2001), and angiogenic inhibitors. Estrogen, via ER, promotes both bFGF and VEGF-induced endothelial cells proliferation in vitro and in vivo (Morales et al., 1995; Johns et al., 1996; Suzuma et al., 1999). Estrogen also has a role in endothelial cell survival (Razandi et al., 2000). It is possible that estrogen may promote survival and/or angiogenic responses of myometrial MEC, promoting the growth of neovessels into adjacent growing fibroids, as well as during pregnancy. Although the relative roles of ER and ERß in mediating the effects of estrogen on endothelial cells are still unclear (Mendelsohn and Karas, 1999), there is some evidence suggesting that ER is more important than ERß (Brouchet et al., 2001). Thus it is possible that MEC survival and proliferation in a growing fibroid may be greater if the MEC express ER. It would be interesting to examine whether the variable MVD observed in fibroids correlates with ER expression of myometrial and fibroid MEC and with tumour size.
In conclusion, we have demonstrated that cultured myometrial and fibroid SMC constitutively express ER, but not ERß, while myometrial and fibroid MEC constitutively express ERß, and that ER expression varies between subjects. Since ER and ERß mediate opposing transcriptional activities, any effect of estrogen on the growth and development of fibroids is likely to be complex and may involve both SMC and MEC.
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Acknowledgements |
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We wish to thank the gynaecologists and histopathologists at Monash Medical Centre for provision of hysterectomy tissue and dating samples respectively, as well as Ms Debbie Plunkett for technical assistance with the immunohistochemistry. This study was supported by the Australian National Health and Medical Research Foundation Grant No. 124331.
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Notes |
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3 To whom correspondence should be addressed. E-mail: caroline.gargett{at}med.monash.edu.au
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Submitted on October 26, 2001; resubmitted on February 14, 2002; accepted on May 1, 2002.
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