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Y. Wen, Y.Y. Zhao, S. Li, M.L. Polan, B.H. Chen, Differences in mRNA and protein expression of small proteoglycans in vaginal wall tissue from women with and without stress urinary incontinence, Human Reproduction, Volume 22, Issue 6, June 2007, Pages 1718–1724, https://doi.org/10.1093/humrep/dem039
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Abstract
To investigate changes in mRNA and protein levels of biglycan (BGN), decorin (DCN) and fibromodulin (FMOD) in vaginal wall tissue from women with stress urinary incontinence (SUI) compared to menstrual-cycle matched continent women.
We determined mRNA expressions of BGN, DCN and FMOD by quantitative real-time PCR. They were localized in vaginal wall tissue by immunohistochemistry. We performed western blot analysis to examine protein expression.
BGN, DCN and FMOD co-localized with collagen and elastin in the extracellular matrix (ECM) of vaginal wall tissue from both groups. The mRNA expression of FMOD was significantly lower in cases versus controls in the proliferative phase (P = 0.03). DCN mRNA expression in cases was higher in the proliferative (P = 0.05) and secretory phases (P = 0.02) versus controls. BGN mRNA expression showed no significant differences in either phase. Protein expression of FMOD in cases was lower in the proliferative phase versus controls (six out of nine pairs), whereas DCN and BGN protein expression in the secretory phase in cases was higher (seven out of nine pairs).
BGN, DCN and FMOD expressions in vaginal wall tissue differ in women with SUI and are hormonally modulated. Differences in small proteoglycans may contribute to the altered pelvic floor connective tissues found in these women.
Introduction
Stress urinary incontinence (SUI) is defined as the involuntary leakage of urine on effort or exertion, such as when coughing or laughing. Together with other pelvic floor disorders—including pelvic organ prolapse and other types of urinary incontinence—SUI causes major health and quality-of-life problems in women (Bump and Norton, 1998; Harris et al., 1998; Cheater and Castleden, 2000; Keane and O'Sullivan, 2000; Romanzi, 2001). Effective closure of the urethra requires the concerted action of various pelvic floor structures, in addition to the proper function of urethral musculature (Liu et al., 2006). The pathophysiology of SUI involves defects in the supporting tissues that include the suburethral vaginal wall, pelvic floor muscles and pubo-urethral ligaments. The connective tissue linking these structures is also a key factor (Falconer et al., 1998). Altered collagen and elastin metabolism has been documented in tissues from women with SUI (Chen et al., 2005).
The pelvic floor-supporting tissues are composed mainly of connective tissue in which fibrous elements (collagen and elastic fibers) and visco-elastic matrix [proteoglycans (PGs)] are the predominant extracellular matrix (ECM) components. The ECM is a complex network of numerous macromolecules that fulfill a large number of mechanical, chemical and biological functions. Collagens and elastin fibers confer strength and elasticity to tissues, respectively, whereas structural PGs create tissue cohesiveness. PGs are subdivided into two groups: large molecules—such as aggrecan, versican and perlecan—and the small molecules, which consist of decorin (DCN), fibromodulin (FMOD), biglycan (BGN), lumican and chondroadherin (Levens et al., 2005). The significance of ECM PGs has become evident only recently. PGs interact with collagen and elastic fibers (Baccarani-Contri et al., 1990; Itabashi et al., 2005) to form a network of surrounding fibers, hold the fibers in place and resist compression by trapping water molecules (Hardingham and Fosang, 1992). They also bind growth factors, influence cell proliferation, migration and adhesion and create viscoelastic and turgor pressures (Kresse and Schönherr, 2001; Bosman and Stamenkovic, 2003). The large PGs are responsible for maintaining tissue hydration and contributing to overall structural scaffolding in the ECM, whereas small proteoglycans (SLRPs) play important roles in regulating collagen fibrillogenesis or acting as bridging molecules between various ECM components. DCN, FMOD and BGN are able to bind to type I collagen and they modulate transforming growth factor-β (TGF-β) by forming complexes with TGF-β (Hildebrand et al., 1994).
DCN, FMOD and BGN have been identified in the para-urethral ECM, and their ratio to collagen is decreased in premenopausal women with SUI, although no change in the amount of small PG was observed (Iozzo and Murdoch, 1996; Falconer et al., 1998). However, neither women with SUI nor asymptomatic women in these studies were separated by menstrual cycle. Levens et al. (2005) showed that expression of FMOD was menstrual cycle-dependent and was suppressed by GnRH analogue in myometrium and leiomyoma. This suggests that FMOD expression may be regulated by ovarian steroids. Thus, the aim of this study was to determine the expression of DCN, FMOD and BGN in vaginal wall tissue from premenopausal women with SUI compared with menstrual cycle-matched asymptomatic controls.
Materials and Methods
Patient selection and tissue collection
This study was approved by the Institutional Review Board of Stanford University School of Medicine. We recruited a total of 62 participants: 15 (age 35–54) with SUI; 17 (age 36–54) were controls in the proliferative phase; 16 (age 30–53) with SUI, 14 (age 30–49) were controls in the secretory phase. Women with a history of endometriosis, gynaecologic malignancies, pelvic inflammatory conditions, connective tissue disorders, emphysema, prior pelvic surgery and advanced pelvic organ prolapse (greater than stage II by POP-Q) were excluded. We selected women with SUI and asymptomatic controls from both proliferative and secretory phases of the menstrual cycle for this study. The phase of cycle was confirmed by endometrial histology. After informed consent was obtained, ∼1 cm2 of full-thickness, peri-urethral vaginal mucosa was excised 1 cm lateral to the urethrovesical junction identified by a Foley balloon, from women undergoing surgery for SUI. Smaller, 0.5 cm2 biopsies of vaginal mucosa from a similar area were excised in continent, control women undergoing benign gynaecologic surgeries for fibroids, dysfunctional bleeding and ovarian cysts (Chen et al., 2004).
The epithelial layer was removed with a razor blade when the tissues were collected. A representative cross-section was fixed in 10% buffered formalin for 16 h, processed with paraffin embedding and used for immunohistochemistry. The remainder of the tissue was frozen immediately in liquid nitrogen and then stored at −80°C for further processing.
Comparative relative quantification real-time PCR
The expressions of DCN, BGN and FMOD mRNA were analysed by quantitative real-time PCR in cases and controls. The extraction of RNA from the tissue sample was carried out with the RNA-STAT-60 reagent (Tel-Test Inc., Friendswood, TX, USA) and cDNA was generated from total RNA, as described previously (Chen et al., 2004). The primers for FMOD, DCN and BGN, intending to flank at least intron, were designed by OLIGO Software (Table 1). Real-time quantification PCR (QPCR) was carried out on the Mx3005P Mutiplex Quantificative PXR System with MxPro QPCR software (Stratagene, La Jolla, CA, USA). Brilliant SYBR Green QPCR Master Mix (Stratagene) was used to perform PCR. The amplifications were done following a 10-min hot start at 95°C in a three-step protocol with 30 s of denaturation (94°C), 1 min of annealing (60°C) and extension at 72°C for 30 s. Forty cycles were performed. Following 1 min of incubation at 95°C for DNA melt, the dissociation curve was run. It started with 30 s of incubation at 55°C. This was followed by 40 successive 30-s plateaus in which the temperature was increased by 1.0°C for each plateau. All PCR reactions were performed in duplicate. Hypoxanthine phosphoribosyl-transferase 1 (HPRT1) was used as the endogenous reference (Wen et al., 2006) against which the different template values were normalized. Cycle of threshold methods was used for quantification. Relative quantification of the gene of interest (FMOD, DCN and BGN), corrected for the quantity of the normalizer gene (HPRT1), was divided by one normalized control sample value (calibrator sample) to generate the relative quantification to calibrator. The calculations were done by MxPro QPCR software. PCR products were sequenced to ensure that the correct gene sequence was amplified. The PCR products were also loaded on 2% agarose gel to confirm their size.
Gene . | Oligo . | Sequence . | Accession number . |
---|---|---|---|
HPRT1 | Sense | TGACACTGGCAAAACAAYGCA | NM_000194 |
Anti-sense | GGTCCTTTTCACCAGCAAGCT | ||
FMOD | Sense | GGGGCAAGGACTGTTGGAGGAG | NM_002023 |
Anti-sense | CCAGGTCTGGAGCCAAGAACGTAGT | ||
DCN | Sense | TCCTGAGACCGCGACTT | NM_001920 |
Anti-sense | GAGTTGTGTCAGGGGGAAGA | ||
BGN | Sense | TGTTCCCTCCATCTCTCCGAACCTG | NM_001711 |
Anti-sense | GACCGCTGTCCCTGGGGTTTTG |
Gene . | Oligo . | Sequence . | Accession number . |
---|---|---|---|
HPRT1 | Sense | TGACACTGGCAAAACAAYGCA | NM_000194 |
Anti-sense | GGTCCTTTTCACCAGCAAGCT | ||
FMOD | Sense | GGGGCAAGGACTGTTGGAGGAG | NM_002023 |
Anti-sense | CCAGGTCTGGAGCCAAGAACGTAGT | ||
DCN | Sense | TCCTGAGACCGCGACTT | NM_001920 |
Anti-sense | GAGTTGTGTCAGGGGGAAGA | ||
BGN | Sense | TGTTCCCTCCATCTCTCCGAACCTG | NM_001711 |
Anti-sense | GACCGCTGTCCCTGGGGTTTTG |
Gene . | Oligo . | Sequence . | Accession number . |
---|---|---|---|
HPRT1 | Sense | TGACACTGGCAAAACAAYGCA | NM_000194 |
Anti-sense | GGTCCTTTTCACCAGCAAGCT | ||
FMOD | Sense | GGGGCAAGGACTGTTGGAGGAG | NM_002023 |
Anti-sense | CCAGGTCTGGAGCCAAGAACGTAGT | ||
DCN | Sense | TCCTGAGACCGCGACTT | NM_001920 |
Anti-sense | GAGTTGTGTCAGGGGGAAGA | ||
BGN | Sense | TGTTCCCTCCATCTCTCCGAACCTG | NM_001711 |
Anti-sense | GACCGCTGTCCCTGGGGTTTTG |
Gene . | Oligo . | Sequence . | Accession number . |
---|---|---|---|
HPRT1 | Sense | TGACACTGGCAAAACAAYGCA | NM_000194 |
Anti-sense | GGTCCTTTTCACCAGCAAGCT | ||
FMOD | Sense | GGGGCAAGGACTGTTGGAGGAG | NM_002023 |
Anti-sense | CCAGGTCTGGAGCCAAGAACGTAGT | ||
DCN | Sense | TCCTGAGACCGCGACTT | NM_001920 |
Anti-sense | GAGTTGTGTCAGGGGGAAGA | ||
BGN | Sense | TGTTCCCTCCATCTCTCCGAACCTG | NM_001711 |
Anti-sense | GACCGCTGTCCCTGGGGTTTTG |
Western blot analysis
Protein extraction was performed as previously described (Chen et al., 2004). Afterwards, the samples were centrifuged at 14 000 rpm (16 000g) for 30 min. The total protein concentrations were determined using the Bradford method (Bio-Rad, Hercules, CA, USA). To remove glycosaminoglycan side chains from the protein cores of DCN, BGN and FMOD, protein extracts (100 µg of total protein from each patient) were digested with 1 U/ml of chondroitin avidin-biotin-peroxidase (ABC) lyase and 2 mU of keratinase (Sigma, St Louis, MO, USA) in Tris–Hcl buffer, pH 8, containing 15 mM sodium acetate for 24 h at 37°C (Cs-Szabó et al., 1997). After glycosidase digestion, 4 × SDS sample buffer with 5% mercaptoethanol was added into the sample and boiled for 5 min. The samples were subjected to SDS–PAGE on 4–20% gradient polyacrylamide gels (Bio-Rad) and electrophoresed at 100 V. The gels were blotted onto nitrocellulose membranes (Pierce, Rockford, IL, USA) in an electrophoretic transfer cell (Bio-Rad). Blots were blocked with 5% non-fat milk at 4°C overnight and then probed with goat anti-fibromodulin (1:200), DCN (1:200) (Santa Cruz, Biotechnology Inc.) or BGN (0.2 µg/ml) (R & D Systems, Minneapolis, MN, USA) at room temperature for 1 h. After three washes with PBS, pH 7.4 and 0.1% Triton (PBS-T), the membrane was then incubated in 1:5000 dilution of mouse anti-goat immunoglobulin G (IgG) (Amersham Pharmacia Biotech, Sunnyvale, CA, USA) and conjugated to horse-radish peroxidase for 1 h at room temperature, followed by three washes in PBS-T. Blots were developed by chemiluminescence. The band density was determined by Bio-Rad Quality One Software. We re-probed the membranes with rabbit anti-glyceraldehyde-3-phosphatedehydrogenase (GAPDH) and mouse anti-β-actin antibodies (Abcam, Cambridge, MA, USA) for loading control after stripping the membranes. However, we were unsuccessful in stripping-off the antibodies completely from the membranes with three kinds of stripping buffer, including the one from Pierce. GAPDH bands (37–40 kDa) and β-actin (43 kDa) were not clearly separated from three small PG bands. Because of this, we used three small PG forms (non-digested forms) on the same blots as the protein loading controls.
Immunohistochemistry
A representative cross-section from one patient in each group was fixed in 10% buffered formalin for 16 h, processed with paraffin embedding. Immunohistochemical staining for type I collagen, elastin, FMOD, BGN and DCN were performed on fixed embedded tissue using the ABC method to confirm the presence and distribution of these proteins in vaginal wall specimens. Paraffin-embedded specimens were cut into 5 µm sections, dewaxed in xylene and rehydrated through graded ethanol solutions. After washing with Tris–HCl Tween buffer (TBS-T), endogenous peroxidases were blocked with 3% H2O2 in TBS-T. Non-specific binding proteins were blocked with 1% BSA, 5% normal secondary antibody host serum in TBS-T at room temperature for 1 h. After rinsing with TBS-T, the slides were incubated with rabbit anti-tropoelastin (1/50, Elastin Products Company), goat anti-type I collagen (1/20), goat anti-FMOD (1/20) or goat anti-DCN (1/20, Santa Cruz) or goat anti-BCN (1/20, R & D System) primary antibody overnight at 4°C. Deletion of the primary antibody was used as a negative control. After rinsing with TBS-T, slides were incubated with a secondary antibody, goat anti-rabbit biotin conjugate or rabbit anti-goat biotin conjugate (1/50, Vector Laboratories Inc., Burlingame, CA, USA). The slides were incubated with Vectastatin ABC Kit (Vector Laboratories) reagent for 30 min at room temperature. Immunoreactive products were visualized by incubating slides with the substrate solution in 0.1 M Tris–HCl buffer with levamisole to block alkaline phosphatase activity. Once optical colour intensity was obtained, reaction was stopped by immersing the slides in distilled water. Slides were counterstained with 25% of haematoxylin and mounted with Permount (Fisher, Fair Lawn, NJ, USA). The slides were visualized and photographed with AxioCam (Zeiss, Oberkochen, Germany).
Immunoflourescence staining
Immunofluorescence staining of fibroblasts from vaginal wall was performed and vimetin was used as a fibroblast marker to confirm the purity of cultured fibroblasts as previously described (Chen et al., 2004). Briefly, from each group we selected a patient whose vaginal wall sample contained enough tissue for RNA or protein extraction. We then cultured the selected tissue in a 4-well chamber slide. The cells were fixed with 4% paraformaldehyde and treated with 5% Triton. After washing with TBS-T and blocking with 5% normal secondary serum, the slides were incubated with goat anti-FMOD (1/20) or goat anti-DCN (1/20) or goat anti-BGN (1/20) and mouse anti-type I collagen (1/20; Sigma) or rabbit anti-tropoelastin (1/50) primary antibody at 4°C overnight. Non-immune serum of the primary antibody was used as a negative control. After washing, the slides were double stained with goat anti-mouse IgG–fluorescein isothiocyanate (FITC) (1/50; Sigma) and rabbit anti-goat IgG–tetramethylrhodamine isothiocyanate (TRITC) (1/50; Sigma) or goat anti-rabbit IgG–FITC (1/50, Sigma) and rabbit anti-goat-IgG-TRITC (1/50) at room temperature for 1 h. DAPI staining was used to observe nuclei. The slides were washed three times and mounted with Vectashield (Vector Inc., Foster City, CA, USA).
Statistical analysis
Statistical analysis was performed using unpaired t-test. The level of significance was set at P < 0.05. A non-parametric analysis (Wilcoxon/Kruskal–Wallis test) was also used because of the small sample size. Both parametric and non-parametric analysis produced equivalent significant results.
Results
Immunofluorescence and immunohistochemistry
The expression of BGN, DCN and FMOD in fibroblasts from vaginal wall has not been previously reported. Therefore, we stained fibroblasts cultured from the vaginal wall tissue from a control woman with double colour immunoflourescence method to confirm that the cultured fibroblasts could express BGN, DCN and FMOD (red). They were expressed in association with both type I collagen and elastin fibers (green) (Figs 1 and 2, respectively). The cultured fibroblasts from the case group were also stained with same antibodies and they showed the same expression pattern as that of the controls (data not shown). Immunoreactive BGN, DCN and FMOD were localized in vaginal wall tissue sections with staining associated with connective tissue fibroblasts as well as connective tissue ECM (Fig. 3). When compared with the fibroblasts, more intense staining was localized in the tissue ECM where both type I collagen and elastin were also abundantly expressed (Fig. 3). Expression patterns and anatomic locations of these three PGs in vaginal wall tissues were similar between cases and controls (data not shown).
Real-time PCR
The expressions of BGN, DCN and FMOD in case and control groups from the proliferative and secretory phases of menstrual cycle were evaluated using relative quantitative real time PCR (Table 2). The expression level of DCN mRNA in the case group, although being not statistically significant (P = 0.05), was about three times higher than in the control group during the proliferative phase, and the level of DCN mRNA in the case group was ∼8 times higher (P = 0.02) than in the control group during the secretory phase. FMOD mRNA expression was a mean of 2.5 times lower in the case than in the control group during the proliferative phase (P = 0.03), but did not show a difference in the secretory phase. The expression levels of BGN mRNA were similar between case and control groups in either phase of the menstrual cycle.
Small proteoglycans . | Menstrual phase . | |||||
---|---|---|---|---|---|---|
Proliferative . | Secretory . | |||||
SUI . | Control . | P-value . | SUI . | Control . | P-value . | |
BGN | 4.12 ± 2.7 (N = 8) | 4.56 ± 1.6 (N = 9) | 0.85 | 2.56 ± 1.12 (N = 8) | 5.77 ± 1.5 (N = 8) | 0.12 |
DCN | 1.19 ± 0.28 (N = 8) | 0.43 ± 0.076 (N = 11) | 0.05 | 2.16 ± 0.74 (N = 9) | 0.26 ± 0.5 (N = 9) | 0.02 |
FMOD | 15.6 ± 5.2 (N = 8) | 40.8 ± 8.9 (N = 9) | 0.03 | 16.03 ± 6.4 (N = 9) | 16.5 ± 4.2 (N = 9) | 0.95 |
Small proteoglycans . | Menstrual phase . | |||||
---|---|---|---|---|---|---|
Proliferative . | Secretory . | |||||
SUI . | Control . | P-value . | SUI . | Control . | P-value . | |
BGN | 4.12 ± 2.7 (N = 8) | 4.56 ± 1.6 (N = 9) | 0.85 | 2.56 ± 1.12 (N = 8) | 5.77 ± 1.5 (N = 8) | 0.12 |
DCN | 1.19 ± 0.28 (N = 8) | 0.43 ± 0.076 (N = 11) | 0.05 | 2.16 ± 0.74 (N = 9) | 0.26 ± 0.5 (N = 9) | 0.02 |
FMOD | 15.6 ± 5.2 (N = 8) | 40.8 ± 8.9 (N = 9) | 0.03 | 16.03 ± 6.4 (N = 9) | 16.5 ± 4.2 (N = 9) | 0.95 |
SUI, stress urinary incontinence.
Relative quantification of the gene of interest (BGN, DCN and FMOD), corrected for the quantity of normalizer gene [hypoxanthine phosphoribosyl-transforase 1 (HPRT1)], was divided by one normalized control sample value (calibrator sample) to generate the relative quantification to calibrator. The results shown are the mean ± SEM. The numbers in the bracket represent the numbers of patients in each group.
Small proteoglycans . | Menstrual phase . | |||||
---|---|---|---|---|---|---|
Proliferative . | Secretory . | |||||
SUI . | Control . | P-value . | SUI . | Control . | P-value . | |
BGN | 4.12 ± 2.7 (N = 8) | 4.56 ± 1.6 (N = 9) | 0.85 | 2.56 ± 1.12 (N = 8) | 5.77 ± 1.5 (N = 8) | 0.12 |
DCN | 1.19 ± 0.28 (N = 8) | 0.43 ± 0.076 (N = 11) | 0.05 | 2.16 ± 0.74 (N = 9) | 0.26 ± 0.5 (N = 9) | 0.02 |
FMOD | 15.6 ± 5.2 (N = 8) | 40.8 ± 8.9 (N = 9) | 0.03 | 16.03 ± 6.4 (N = 9) | 16.5 ± 4.2 (N = 9) | 0.95 |
Small proteoglycans . | Menstrual phase . | |||||
---|---|---|---|---|---|---|
Proliferative . | Secretory . | |||||
SUI . | Control . | P-value . | SUI . | Control . | P-value . | |
BGN | 4.12 ± 2.7 (N = 8) | 4.56 ± 1.6 (N = 9) | 0.85 | 2.56 ± 1.12 (N = 8) | 5.77 ± 1.5 (N = 8) | 0.12 |
DCN | 1.19 ± 0.28 (N = 8) | 0.43 ± 0.076 (N = 11) | 0.05 | 2.16 ± 0.74 (N = 9) | 0.26 ± 0.5 (N = 9) | 0.02 |
FMOD | 15.6 ± 5.2 (N = 8) | 40.8 ± 8.9 (N = 9) | 0.03 | 16.03 ± 6.4 (N = 9) | 16.5 ± 4.2 (N = 9) | 0.95 |
SUI, stress urinary incontinence.
Relative quantification of the gene of interest (BGN, DCN and FMOD), corrected for the quantity of normalizer gene [hypoxanthine phosphoribosyl-transforase 1 (HPRT1)], was divided by one normalized control sample value (calibrator sample) to generate the relative quantification to calibrator. The results shown are the mean ± SEM. The numbers in the bracket represent the numbers of patients in each group.
Immunoblot
Due to small tissue sample sizes, biopsies from half of the patients in each group analysed by competitive PCR were no longer available for western blot analysis. Samples from other additional patients were added for immunoblot studies. Protein expression of DCN, BGN and FMOD in vaginal wall tissues from both case and control groups in the proliferative phase (N = 9 pairs) and the secretory phase (N = 9 pairs) were evaluated by western blot (Fig. 4). BGN core protein appears as two bands, 40 and 48 kDa, on immunoblots. Expression of BGN in seven pairs showed higher density in case samples than in control samples in the secretory phase (Figure 4A and B), whereas no visual difference was observed in the proliferative phase (Fig. 4A). DCN core protein appears as two bands, 36 and 45 kDa, on immunoblots. Expression of DCN in seven pairs showed a higher density in case compared with control vaginal wall samples in the secretory phase (Fig. 4A and B). No difference was observed in the proliferative phase (Fig. 4A). FMOD core protein presents as two bands with masses ∼60 and ∼50 kDa. When compared with vaginal tissues from controls, the FMOD core protein density appeared lower in women with SUI in six matched tissue pairs (Fig. 4A and B). No difference was seen in the secretory phase (Fig. 4A). These immunoblot data for DCN and FMOD were consistent with the differences observed in mRNA expressions.
Discussion
Any hypothesis to explain the etiology of SUI must address ECM remodelling of pelvic floor supporting tissues. ECM architecture is stabilized by PGs, which play an important role in the spatial arrangement of structural polymers (Bosman and Stamenkovic, 2003). Besides regulating collagen assembly, PGs have a key function in cellular interactions and growth factor storage (Ruoslahti, 1989). DCN, BGN and FMOD all bind TGF-β1, β2, and β3 and function as antifibrotic molecules in the ECM (Hildebrand et al., 1994). Although the role of collagen and elastic fiber homeostasis in the etiology of SUI has been studied by many (Falconer et al., 1998; Ulmsten and Falconer, 1999; Romanzi, 2001; Chen et al., 2004; Liu et al., 2006; Wen et al., 2006), a few reports exist examining small PGs in the ECM of pelvic floor supporting tissues.
In this study, we showed that fibroblasts cultured from vaginal wall tissues express BGN, DCN and FMOD. Vaginal wall tissues from women with clinical SUI in the proliferative phase have significantly lower FMOD mRNA compared with vaginal wall tissues from controls, although no differences in FMOD expression levels were observed in the secretory phase. We also demonstrated that SUI vaginal wall tissues have higher DCN mRNA expression during the proliferative phase and significantly higher DCN mRNA during the secretory phase.
Falconer et al. (1998) reported no change in PG amount or composition in paraurethral connective tissue in premenopausal women with SUI. However, these patients were not separated into proliferative and secretory phases. In contrast to their data, our study demonstrates that mRNA and protein expression levels of DCN and FMOD in tissues from cases are different from those from the control group. This discrepancy could be due to the differences in quantifying methodologies and the grouping of women by phase of the menstrual cycle done in this study. Levens et al. (2005) reported that FMOD was expressed at significantly higher levels in leiomyoma compared with myometrium from the proliferative phase, but not from the secretory phase (Levens et al., 2005). Our data support this observation and strengthen the possibility that the expression of small PGs may be regulated by ovarian steroids in the female reproductive tract.
In the rat model, the transition from scarless fetal-type repair to scar-forming adult-type repair occurs between days 16 and 18 of gestation (Soo et al., 2000). The FMOD mRNA level declined dramatically in fetuses at day 18 (adult-type repair with scar) compared with fetuses at day 16 (scarless fetal repair). FMOD expression was down-regulated at 12 h and minimally up-regulated between days 3 and 5 during adult wound healing. DCN mRNA was up-regulated in day 18 fetuses compared with day 16 fetuses and also in adult repair. Soo et al. (2000) also showed that FMOD protein expression significantly increased 36 h after injury in gestation day 16 fetuses, but not in gestation day 19 fetuses. These data suggest that high FMOD levels are associated with scarless fetal repair, whereas adult-type fetal repair with scar formation is associated with relatively low levels of FMOD. This, as well as our observations that SUI vaginal wall tissues have lower FMOD and higher DCN expressions, suggest that tissues from women affected with SUI may respond to injury similar to adult-type fetal repair and adult wound repair.
Soo et al. (2000) also demonstrated that mRNA expression of latent TGF-β binding protein-1 (LTBP-1)—which is involved both in the sequestration of latent TGF-β in the ECM and in the regulation of its activation in the extracellular environment—declined significantly in fetuses at gestation day 18 when compared with fetuses at gestation day 16. They hypothesized that this decrease in LTBP-1 expression in late-term fetuses may result in increased TGF-β activity and potentially account for scar formation. We previously reported a similar decrease in LTBP-1 expression in vaginal wall tissues from SUI compared with control women in the proliferative phase (Wen et al., 2006). This, in conjunction with the currently observed decrease in FMOD expression in SUI vaginal wall tissues, suggests that women with SUI may respond to pelvic injury by forming connective tissue with altered mechanical properties such as scar tissue. Epidemiologic data indicate that race and genetic factors, in addition to birth trauma, can be involved in the development of SUI. It is possible that women who are genetically predisposed to developing pelvic floor dysfunction, lack an injury-induced FMOD response.
DCN regulates ECM remodelling in rabbit synovial fibroblasts via induction of the matrix metalloproteinase (MMP)-1 (Huttenlocher et al., 1996). Over-expression of DCN regulated the expression of several MMPs and cytokines in gingival fibroblasts (Al Haj Zen et al., 2003) and suppressed cell growth in certain cell types by interacting with epidermal growth factor receptor (EGFR) (Iozzo et al., 1999). The EGFR activation has been shown to associate with the expression of matrix proteolytic enzymes, such as urokinase-type plasminogen activator (UPA) and its receptor as well as the MMPs (Rosenthal et al., 1998). Our group has not observed differences in UPA mRNA expression and its activity in vaginal wall tissues in case and control groups from either proliferative or secretory phase (unpublished data). We hypothesize that DCN may be involved in the etiology of the increase in MMP and elastase activities in vaginal wall tissues of women with SUI, as reported in our previous studies (Chen et al., 2004, 2005). Higher expression of DCN in tissues from women with SUI during the secretory phase may contribute to degradation of collagen and elastin fibrils, leading to impaired function.
We did not observe a difference in BGN mRNA expression between the two groups. However, our protein data showed higher BGN expression in case compared with controls (seven out of nine tissue pairs). This discrepancy may be due to the small sample size, or it is possible that BGN expression is regulated at the post-translational level. Future studies are necessary to address this.
In summary, we demonstrated that DCN, BGN and FMOD are secreted by vaginal wall tissue fibroblasts. They co-localize primarily with type I collagen and elastin in the ECM of vaginal wall tissues determined by immunohistochemistry, thus suggesting possible involvement in collagen and elastin metabolism of pelvic floor supportive tissues. Women with SUI expressed significantly lower levels of FMOD mRNA in their vaginal wall tissues compared with the control group during the proliferative phase with no change in the secretory phase. But women with SUI express significantly higher levels of DCN mRNA in their vaginal wall tissues compared with the control group during the secretory phase. Protein expression of these small proteoglycans, evaluated by visual differences on Western blot are consistent with these mRNA data. These findings suggest that differences in small PG expressions may contribute to the altered pelvic floor function of the connective tissues in women with SUI, and these differences appear to be mediated by ovarian hormones.
Acknowledgements
We thank Lorna Groundwater for her excellent editorial help. Supported by NIH grant AG 17907 to M.L.P, MD, PhD, MPH.