Hum. Reprod. Advance Access originally published online on September 9, 2005
Human Reproduction 2006 21(1):303-308; doi:10.1093/humrep/dei296
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-Estradiol suppresses proliferation of fibroblasts derived from cardinal ligaments in patients with or without pelvic organ prolapse
Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong
1 To whom correspondence should be addressed. E-mail: yipsk{at}cuhk.edu.hk
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Abstract |
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BACKGROUND: Estrogen replacement therapy (ERT) has been used in the treatment of pelvic organ prolapse (POP) but clinical results are inconclusive. The purpose of this study was to investigate the effect of 17
-estradiol (E2) on the proliferation of fibroblasts derived from cardinal ligaments in women with or without POP. METHODS: Fibroblasts were derived from seven patients with POP and seven age-matched controls. The growth rate of POP fibroblasts was compared with that of control by 3-(4,5,-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Four cell strains from each patient and control group were treated with different concentrations of E2 (10]4, 10]8, 10]9 and 10]10 mol/l). The effect of E2 on cell proliferation was then measured by MTT assay. RESULTS: The overall growth rate of POP fibroblasts was significantly slower than that of controls under normal culture conditions. Addition of E2 suppressed cell proliferation of all the fibroblasts, especially in POP fibroblasts. POP fibroblasts showed a significantly lower proliferative rate than that of controls at all E2 concentrations, with the most prominent inhibitory effect at physiological concentration (10.83 34.41% versus 81.56 48.10% at 10]8 mol/l). CONCLUSIONS: Our results suggest that decreased fibroblast turnover may contribute to the development of POP; and ERT may not be an effective POP treatment.
Key words:
17-estradiol/cardinal ligament/cell proliferation/fibroblasts/pelvic organ prolapse
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Pelvic organ prolapse (POP) is a common and distressing condition, particularly in the elderly. The lifetime risk of undergoing surgical intervention for POP by age 80 years is estimated to be
10% (Olsen et al., 1997
). About 50% of all parous women show some degree of pelvic floor laxity, with 10–20% being symptomatic (DeLancey, 1993). Recent research has identified the cardinal ligament as the major supportive component of the pelvic floor (Bartscht and DeLancey, 1998; DeLancey, 1992). The cardinal ligament is a condensation of subserous fascia arising from pelvic side walls (Lang et al., 2003). Collagen forms 70–80% of the dry weight of the ligament, and fibroblast is the major tissue component (Hart et al., 1995).
Ageing is thought to be the inevitable cause of POP (Bidmead and Cardozo, 1998; MacLennan, 2000). The incidence of POP increases after menopause (Harvey et al., 2001). This indicates that the hypoestrogenic state may contribute to its aetiology (Mokrzycki et al., 1997). However, the effect of the post-menopausal hormonal milieu on pelvic supportive structures has not been fully investigated. Previous studies have shown that estrogen can mediate two diverse cellular growth responses: inhibition or proliferation (Geraldes et al., 2002). For instance, estrogen has been shown to decrease cell proliferation in human and rabbit anterior cruciate ligament fibroblasts with decreased collagen synthesis (Liu et al., 1997; Yu et al., 1999). In contrast, other studies have shown that estrogen increased cell proliferation in rabbit mandibular and condylar cartilage cells, and porcine aortic endothelial cells (Geraldes et al., 2002; Cheung et al., 2003).
One of the non-surgical treatments deemed helpful for POP is estrogen replacement therapy (ERT), which is thought to help by limiting further weakening of the connective tissues that support the pelvic organs (Falconer et al., 1994). Although ERT has been used in the treatment of POP, results are inconclusive (Klute and Bergman, 1994) and the precise role of estrogen on the function of the pelvic floor, and the pathogenesis, prevention and treatment of POP are not fully understood.
The aim of this study was to elucidate the role of estrogen on cell proliferation of cardinal ligament fibroblasts in women with and without POP, which would further gain insights into the pathogenesis of this disease. Further characterization of the relationship between estrogen and cell proliferation would also allow them to be exploited in monitoring responses to hormonal therapy.
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Materials and methods |
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Acquisition of human tissues
The authors’ institutional review board approved the study. Informed consent was obtained prior to tissue collection. Seven subjects with POP and seven normal subjects were recruited at the authors’ hospital. Those subjects who had concomitant malignant pelvic diseases or those receiving ERT were excluded. Cardinal ligament biopsy samples were obtained during vaginal hysterectomy in POP subjects, or abdominal hysterectomy performed for non-prolapse/non-malignant indications in controls. A standardized biopsy site was selected. Specifically, during vaginal hysterectomy the cardinal ligaments were identified after anterior and posterior colpotomy, and the urinary bladder was dissected away from the anterior surface of the uterus. A biopsy of each cardinal ligament was made 1 cm lateral to its cervical insertion site. The samples were freed from surrounding adipose tissues and blood vessels if present. Similarly, a biopsy of each cardinal ligament was made in normal subjects.
Culture reagents
Eagle’s minimum essential medium (MEM), Phenol Red-free MEM, streptomycin, penicillin-G, fetal bovine serum (FBS), trypsin–0.02% EDTA and Ca2+- and Mg2+-free phosphate-buffered saline were purchased from Gibco (Grand Island, NY, USA). Charcoal-treated FBS was purchased from HyClone (South Logan, UT, USA). Monoclonal anti-human fibroblast surface protein (Clone 1B10
[PDB]
) and 17-estradiol were purchased from Sigma (St Louis, MO, USA). Sheep XMS IgG (Fluor) was purchased from Chemicon (Temecula, CA, USA), 3-(4,5,-dimethyl thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Amersham International (Amersham, UK). SuperScriptTM First-Strand Synthesis kit was purchased from Invitrogen (Carlsbad, CA, USA). Ampli Taq GoldTM DNA polymerase was purchased from Applied Biosystems (Foster City, CA, USA). Trizol Reagent was purchased from Molecular Research Center Inc. (Cincinnati, OH, USA).
Cell culture techniques
Fourteen cell strains of cardinal ligament fibroblasts derived from patients with POP (P1 to P7) and control patients without POP (C1 to C7) were established as described by Yamamoto et al. (1997). In brief, small branches of the cardinal ligaments were extracted for cell culture. The cardinal ligament specimens were aseptically transferred into sterile MEM with streptomycin (100 µg/ml) and penicillin-G (100 IU/ml). The specimens were minced into 1 mm pieces with the blades of two scalpels. The tissue fragments were attached to collagen-coated dishes and cultured in 5 ml of MEM supplemented with 15% FBS at 37°C under humidified 5% CO2–95% air. The medium was refreshed every 3 or 4 days. Confluent cultures were passed with 0.25% trypsin–0.02% EDTA in Ca2+- and Mg2+-free phosphate-buffered saline and subcultured at a 1:2 split ratio in 100 mm Falcon dishes. Passages 2–4 were used for the study.
Characterization of cardinal ligament fibroblasts by immunofluorescence staining
Cells at passage 2 were plated at 30–40% confluence in 8-well chamber slides and incubated for 2 days. The cells were washed with TBS twice and then fixed with 200 µl of 4% paraformaldehyde for 20 min. The fixative was then removed and the cells were washed again with TBS twice. The cells in each well were then permeabilized with 200 µl of TBS with 0.05% Triton X for 15 min. The 10% sheep serum in TBS-T was added to the cells for 30 min at room temperature for blocking. The cells were incubated with monoclonal anti-human fibroblast surface protein (Clone 1B10
[PDB]
) at 1:75 dilutions at 4°C overnight. After the incubation, the cells were washed with TBS and then incubated with secondary antibody of Sheep XMS IgG (Fluor) for 2 h in the dark at room temperature. The cells were washed and stained with DAPI in TBS. The cells were washed with TBS and mounted for microscopic examination.
Cell proliferation assay
The growth rate of the cardinal ligament fibroblasts from patients with POP (P1 to P7) was compared with that of controls (C1 to C7) by MTT assay. The cells at passage 2 were cultured at a density of 5000 cells/well in a 24-well plate. The cell proliferation was monitored by MTT assay from day 1 (1 day after plating) to day 11. The cells from each well were added with 300 µl/well MTT reagent 2 h prior to harvest. The supernatant in all wells was removed, and the cells were treated with 400 µl/well dimethylsulphoxide for 10 min. Reabsorbance at 570 nm was recorded using an enzyme-linked immunosorbent assay plate reader. Cells in triplicate were assayed for each time-point.
Dose-response of cell growth to E2
Four cell strains from each patient (P2, P3, P4 and P6) and control (C1, C2, C4 and C5) (age-matched) at passage 2 were treated with different concentrations of E2. Cell proliferation study of fibroblasts under the effect of estrogen was carried out by colorimetric MTT assay. The fibroblasts derived from the cardinal ligament of patients with POP and controls were isolated and seeded at a density of 15 000 cells/well in 24-well plates. After overnight culture, the cells became attached to the bottom of the plates, and the culture medium was replaced by FBS-free MEM and the cells were cultured for 24 h. The medium was then replaced by Phenol Red-free MEM plus 2% charcoal-treated FBS containing different concentrations of 17-estradiol (0, 10–4, 10–8, 10–9 and 10–10 mol/l). The control cultures contained absolute ethanol as a vehicle. Different concentrations of 17-estradiol were freshly prepared by dissolving the 17-estradiol powder in absolute ethanol and then serially diluted with medium. Each group of the experiment was done in triplicates. After culturing for 48 h, the cell viability was measured by MTT assay as described before.
RNA extraction and RT–PCR analysis
Total RNA was extracted from the 14-cell strains of cardinal ligament fibroblasts at passage 2 by Trizol Reagent. Cell pellet was homogenized in 1 ml of Trizol Reagent. After extraction with chloroform and isopropanol, the RNA pellet was washed with 1 ml of 75% ethanol and then dissolved in 15 µl of RNase-free water. The extracted RNA was reverse-transcribed to cDNA by SuperScriptTM First-Strand Synthesis kit. For each sample, 2 µg total RNA, 600 ng random primers, and 10 nmol/l dNTP in 12 µl reaction mixture were first heated at 65°C for 5 min. Then 4 µl 5xFirst-Strand Buffer, 0.2 µmol/l DTT and 40 IU RNaseOUT were added to the reaction mixture, which was then heated to 42°C for 2 min. A total of 200 IU SuperScript II reverse transcriptase was added to each reaction mixture. The reaction mixture was then heated to 25°C for 10 min, 42°C for 50 min and then 70°C for 15 min. To check the transcript expression of ER
and GAPDH, PCR was performed thereafter in a 20 µl volume of the final reaction solution containing 1 µl of the RT reaction product (diluted at 1:5x), 2.5 µl of 10xPCR buffer, 3 mmol/l MgCl2, 0.5 nmol dNTP, 5 pmol of each of the forward and reverse primers, and 0.8 IU Ampli Taq GoldTM DNA polymerase. The amplification condition was as follows: initial denaturation at 94°C for 10 min, followed by 35 cycles at 94°C for 45 s, 56°C for 45 s for ER and 55°C for GAPDH and 72°C for 45 s, with a final extension period at 72°C for 10 min. The PCR products were subjected to electrophoresis on 2% agarose gels and visualized by ethidium bromide staining.
The sequence of the forward primer for the RT–PCR analysis of ER was 5'-TGTGCAATGACTATGCTTCA-3' and that of the reverse primer was 5'-GCTCTTCCTCCTGTTTTTA-3', which were designed with the use of published sequences (Leygue et al., 1998). The sequence of the forward primer for GAPDH was 5'-GAAGGTGAAGGTCGGAGTC-3' and that of the reverse primer was 5'-GAAGATGGTGATGGGATTTC-3'. The PCR product derived from ER mRNA was expected to be 149 base pairs, whereas that of GAPDH mRNA was expected to be 226 base pairs.
Determination of specific protein expression
Total protein extracts from each cardinal ligament fibroblast sample and MCF-7 breast cancer cell line were prepared with lysis buffer (50 mmol/l Tris–HCl, 0.3 mol/l NaCl, 1 mmol/l EDTA, 1 mmol/l dithiothreitol, and 1 mmol/l phenylmethylsulphonyl fluoride). Equal amounts (50 µg) of cell extracts were separated on sodium dodecyl sulphate (SDS)–polyacrylamide gels and subsequently transferred onto a nitrocellulose membrane (Amersham, Buckinghamshire, UK) in trans-buffer (25 mmol/l Tris; 129 mmol/l glycine; 10% methanol; 0.05% SDS). These filters were blocked overnight with ovalbumin (Sigma)-saturated TBS [50 mmol/l Tris (pH 7.6) and 150 mmol/l NaCl]. Primary antibodies at 5 µg/ml in TBST (50 mmol/l Tris, 150 mmol/l NaCl, and 0.1% Tween-20) were incubated for 1 h at 37°C. These are monoclonal antibodies to ER (F-10; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and actin (C4; Roche Molecular Biochemicals). The filters were then incubated with secondary antibodies (anti-mouse horseradish peroxidase; Amersham) in TBST at room temperature for 1 h and treated with an enhanced chemiluminescence (ECL) sensitization kit (Amersham), according to the manufacturer’s protocol. The band volume was quantified by computer, using the quantitation software (Quantity One; Bio-Rad).
Data analysis
The statistical analyses were performed using the computer software Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) for Windows version 10. P < 0.05 was considered statistically significant. The statistical comparisons were performed using analysis of variance.
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Results |
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Characterization of cardinal ligament fibroblasts
Fibroblasts derived from the cardinal ligaments showed long, needle-shaped and fibroblast-like morphology under light microscopy (Figure 1A). Microscopically, no difference in morphology was observed between patient and control cardinal ligament fibroblasts. Immunofluorescence study of fibroblasts derived from patient and control showed strong cytoplasmic expression of fibroblast surface protein, indicating the fibroblast origin of cell type (Figure 1B and C). To determine the expression of ER
in the culture fibroblast, we performed RT–PCR on all samples before treatment with E2. All patient and control fibroblasts expressed mRNA of ER (Figure 1D). Semiquantitative analysis was performed and normalization with the housekeeping gene of GAPDH showed no significant difference in ER expression between fibroblasts derived from controls and patients. We found no significant difference in ER expression by semiquantitative RT–PCR or western blot analysis (Figure 1E), although a weak ER protein expression was noticed when compared to their respective mRNA levels. This could be due to post-translational alteration of ER.
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Cell proliferation assay
To determine whether there were differences in cell proliferation between patients and controls, cell growth study of both patients (P1 to P7) and controls (C1 to C7) were conducted by MTT assay (Figure 2A and B). The growth rate of all the seven patient fibroblasts was lower than that of control fibroblasts (except C2) by comparing the slope of the growth curve in the log phase. The age of patients with POP was 65.3 ± 10.5 years old (mean ±SD), while that of controls was 49.4 ± 10.1 years old.
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Dose-response of cell growth to E2
To eliminate the age difference between patients and controls, we selected four cell strains each from patients (P2, P3, P4 and P6) and controls (C1, C2, C4 and C5) with similar age (patient age: 65.8 ± 12.0 versus control age: 60.1 ± 7.4, P > 0.05) to investigate the effect of estrogen in cell proliferation. Fibroblasts were treated with different concentrations of E2.
In this dose–response study, both patient and control fibroblasts showed increased cell proliferation at placebo control. However, the percentage of increase in cell number in patient fibroblasts (32.20% ± 23.84) (percentage mean ± SD) was significantly lower than that of controls (91.94% ± 43.14) (P < 0.001). Treatment with E2 suppressed cell growth in both patient and control fibroblasts as shown in Figure 3. At superphysiological E2 concentration (10–4 mol/l), both patient and control fibroblasts showed a significant decrease in cell number. Morphological changes were also observed in both groups when cardinal ligament fibroblasts were treated with 10–4 mol/l E2 for 48 h (Figure 4). The cells became flat, round and showed intracellular debris accumulation. In addition, there was a decrease in cell number (7.29 ± 29.51%) of patients’ fibroblasts. Conversely, the controls showed a proliferative response (16.54 ± 31.38%) (Figure 3).
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Compared to vehicle control (0 mol/l E2), both patient and control fibroblasts showed a decreased cell number even under physiological E2 concentrations (10–8, 10–9and 10–10 mol/l) (Figure 3). Interestingly, cells derived from patients showed a significantly lower proliferative rate than that of controls under physiological E2 concentrations (10–8, 10–9and 10–10 mol/l) tested. The inhibitory effect ranged from 60 to 81% in the patient group versus 10 to 38% in the control group (P < 0.001).
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Discussion |
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In this study, we have demonstrated that the overall growth rate of patient cardinal ligament fibroblasts was slower than that of controls, and this difference was not solely due to age difference. Fibroblast is the major cell component in ligaments, playing an important role in producing collagen, elastic fibres and other extracellular matrix (ECM) proteins (Hart et al., 1995
). Alteration in cell growth may affect the metabolism of collagens and ECM, such as the rate of synthesis or turnover, assembly, cross-linking and remodelling. In addition, changes in ECM proteins have been shown in the ligaments of prolapsed uteri, characterized by a higher expression of collagen III and tenascin (Ewies et al., 2003) and suppression of the expression of elastin (Yamamoto et al., 1997; Ewies et al., 2003). Taking these observations together with our findings, it suggests that slower fibroblast proliferation would imply changes in collagen and ECM synthesis and turnover, which may further contribute to the development of POP.
Our results demonstrated that all cells studied expressed ER receptors and that superphysiological E2 suppressed cell proliferation of cardinal ligament fibroblasts derived from patients with POP and controls. This inhibitory effect is consistent with a previous study in rabbit anterior cruciate ligament fibroblasts (Liu et al., 1997). Another study also showed that estrogen suppressed porcine smooth muscle cells by inhibiting platelet-derived growth factor (PDGF)-BB and subsequent suppression of phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK (Geraldes et al., 2002). However, the mechanism of this inhibitory effect has not been fully addressed.
The cell growth of patient cardinal ligament fibroblasts is slower than that of controls at 0 mol/l E2 condition, which is consistent with our cell proliferation study (Figure 2). We also demonstrated that E2 suppresses cell proliferation in both patient and control cardinal ligament fibroblasts at physiological E2 concentrations. It is also interesting to note that this inhibitory effect is more prominent in POP patient-derived fibroblasts. Previous study has demonstrated that the expression of ER was 1.5–2.5 times more in cardinal ligaments of prolapsed uteri when compared to the controls with no difference in the ER expression (Ewies et al., 2004). Hence, Ewies et al. suggested that the increase in estrogen receptor protein expression is associated with tissue injury or tissue repair process, but possibly helps to enhance the sensitivity of tissue to the respective estrogen ligands. However, we did not find a significant difference in ER expression by semiquantitative analysis. As a result, the sensitivity of fibroblasts of the cardinal ligaments in our patients may respond differently to E2 and subsequently cell growth was inhibited more than that of the controls. We could not exclude the possibility that one of the important roles of E2 in the cardinal ligament is to induce differentiation mechanisms endowed with characterization of the protein and proteoglycan constituents of the ECM as exemplified by the changes observed by Ewies et al. (2003, 2004). But estrogen could be equally important in regulating the proliferation of cells.
ERT has been thought to be useful in managing women with urinary incontinence (Robinson and Cardozo, 2003). However, in a recent study by Hendrix et al. (2005), conjugated equine estrogen with or without progestin was shown to increase the risk of urinary incontinence among continent women. Therefore Hendrix et al. suggested that estrogen should not be prescribed for the prevention or treatment of urinary incontinence in menopausal women. The results of Hendrix et al.’s study provided clinical evidence to corroborate our laboratory findings. Nevertheless, the clinical effects of estrogen are multiple, and it is possible that estrogen treatment may still be useful in other circumstances.
In conclusion, although ERT has been used in the treatment of urinary stress incontinence and POP, results are inconclusive. Our findings showed that cell proliferation of cardinal ligament fibroblasts from patients with POP is lower than that of controls. This finding could be explained by two possible mechanisms: firstly, slower fibroblast proliferation may contribute to the development of POP; secondly, the lower turnover is an effect rather than a cause of POP. E2 suppresses cell proliferation of cardinal ligament fibroblasts derived from patients with POP and controls.
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Submitted on June 6, 2005; resubmitted on August 10, 2005; accepted on August 12, 2005.
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