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Hum. Reprod. Advance Access originally published online on December 11, 2007
Human Reproduction 2008 23(2):387-393; doi:10.1093/humrep/dem370
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Is {alpha}2-macroglobulin important in female stress urinary incontinence?

Y. Wen1, W.C. Man, E.R. Sokol, M.L. Polan and B.H. Chen

Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, H333 Stanford, CA 94305, USA

1 Correspondence address. Tel: +1-650-723-9536; Fax: +1-650-723-7737; E-mail: yanwen{at}stanford.edu


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 Funding
 Acknowledgement
 References
 
BACKGROUND: Loss of mechanical stability of the urethra and bladder is thought to be important in the development of stress urinary incontinence (SUI). The vaginal wall is the main supporting tissue for pelvic organs and changes in components of supporting tissues are known to be involved in the pathophysiology of SUI.

METHODS: We evaluated changes in expression of {alpha}2-macroglobulin ({alpha}2-M), a protease inhibitor, in vaginal wall tissues from premenopausal women (aged 42–45 years) with SUI (n = 28) compared with menstrual cycle-matched continent women (controls, n = 29). The distribution of {alpha}2-M in vaginal wall tissues and fibroblasts was analysed by immunohistochemistry and immunofluorescence. Expression levels of {alpha}2-M mRNA and protein was determined by relative real-time quantitative PCR and enzyme-linked immunosorbent assay, respectively. Protease inhibition was measured to assess bioactivity.

RESULTS: Vaginal wall tissues do express {alpha}2-M. Expression of {alpha}2-M mRNA and protein was significantly higher in tissues from controls compared to women with SUI in both proliferative and secretory phases (P < 0.05). Protease inhibitory activity of {alpha}2-M was significantly higher in tissues from controls compared to women with SUI in the secretory phase (P < 0.05), but we found no difference in the proliferative phase between groups. {alpha}2-M protein level was lower in the proliferative phase than the secretory phase in both controls and SUI patients, while for {alpha}2-M mRNA this was found only in controls.

CONCLUSIONS: Decreased expression of {alpha}2-M mRNA and protein and protease inhibitory activity in the vaginal wall tissues of women with SUI may contribute to the development of SUI.

Key words: extracellular matrix/stress urinary incontinence/{alpha}2-macroglobulin/vaginal wall/proteases


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 Funding
 Acknowledgement
 References
 
Stress urinary incontinence (SUI) is a common, costly and distressing condition for women (Nygaard and Heit, 2004Go; Anger et al., 2006Go; Subak et al., 2006Go). Frequency of SUI increases with age, and peaks in prevalence first around menopause and then after age 65 (Hannestad et al., 2000Go). SUI is defined as the involuntary loss of urine which occurs when intra-abdominal pressure exceeds urethral pressure. Factors affecting urethral pressure include bladder neck mobility; urethral sphincter muscle and nerve integrity; urethral smooth muscle and vascular plexuses; and surrounding tissue support. The vaginal wall is the main supporting tissue and provides a stable base for the urethra and bladder neck, preventing urethral and bladder neck descent (Nygaard and Heit, 2004Go). The loss of mechanical stability of the urethra and bladder is thought to be an important factor in the development of SUI (Weber et al., 2004Go).

The vaginal wall comprises sequanous epithelium, sub-epithelium, smooth muscle and adventia. All elements are embedded in an extracellular matrix (ECM), composed of fibrous elements (collagen and elastic fibers) and a viscoelastic matrix (proteoglycans, glycoproteins). The collagens and elastin fibers confer tissue strength and elasticity, respectively, whereas structural glycoproteins create tissue cohesiveness. The ECM of the vaginal tissue largely determines its tissue tensile strength and its mechanical stability. Therefore, the relationship between production and degradation of the ECM components is crucial for maintaining mechanical integrity of pelvic supporting tissues. Abnormal quantity and quality of ECM components in pelvic supporting tissues have been reported to be involved in the pathophysiology of SUI and/or pelvic organ prolapse (Liapis et al., 2001Go; Ewies et al., 2003Go; Goepel et al., 2003Go; Liu et al., 2004Go; Karam et al., 2007Go; Wen et al., 2007Go).

ECM undergoes continuous tissue remodeling in response to different environmental stimuli (Alperin and Moalli, 2006Go). Proteolysis regulates ECM assembly, degradation of excess ECM components, remodeling of ECM structures and the release of growth factors and bioactive fragments (Weber et al., 2004Go). Matrix metalloproteinases (MMPs) and serine protease (neutrophil elastase, NE) are capable of degrading ECM components (collagen, elastin, proteoglycans and glycoproteins). In previous studies, we documented that the relative activity of MMPs and NE are increased whereas their respective inhibitors [tissue inhibitor of metalloproteinase-1 (TIMP-1) and {alpha}1-antitrypsin (ATT)] is decreased in the vaginal wall tissues of women with SUI compared with controls (Chen et al., 2002Go; Chen et al., 2004Go; Chen et al., 2007Go). Other investigators have also observed accelerated remodeling of ECM in the pelvic supporting tissues in women with SUI and/or pelvic organ prolapse compared with controls (Moalli et al., 2005Go; Gabriel et al., 2006Go; Phillips et al., 2006Go). {alpha}-2macroglobulin ({alpha}2-M) has been characterized as an extracellular panproteinase inhibitor with the unique ability to inhibit almost all known proteases, regardless of their mechanistic classes (serine, metallo, cysteine and aspartate). Information from a knock-out mice study suggests that {alpha}2-M is involved in tissue remodeling during both pregnancy and wound repair (Umans et al., 1995Go). However, {alpha}2-M's role in the development of SUI has not been examined.

In this study, we sought to investigate possible changes in the expression of {alpha}2-M by measuring its expression level and inhibitory capacity in vaginal wall tissues from premenopausal women with SUI compared with controls in both the proliferative and secretory phases of the menstrual cycle.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 Funding
 Acknowledgement
 References
 
Patient selection and tissue collection
We selected women with SUI (n = 28) and controls (n = 29) from both the proliferative and secretory phases of the menstrual cycle for this study. Phase of cycle was confirmed by endometrial histology. Women with a history of endometriosis, gynecologic malignancies, pelvic inflammatory conditions, connective tissue disorders, emphysema, prior pelvic surgery and advanced pelvic organ prolapse (greater than stage II by pelvic organ prolapse-quantification) were excluded. The Institutional Review Board of Stanford University School of Medicine approved this study. Informed consents were obtained from all participants.

In women undergoing surgery for SUI, ~1 cm2 of full-thickness, peri-urethral vaginal mucosa was excised 1 cm lateral to the urethrovesical junction identified by a Foley balloon. Smaller, 0.5 cm2 biopsies of vaginal mucosa from a similar area were excised from controls undergoing benign gynecologic surgeries for fibroids, dysfunctional bleeding or ovarian cysts. At the time of tissue collection, the epithelial layers were removed with a razor blade. A representative cross-section was fixed in 10% buffered formalin for 16 h, processed with paraffin embedding and used for immunohistochemistry. Tissue from a patient was collected into a tube containing Dulbecco's modified Eagle's medium for the isolation of fibroblasts from vaginal wall tissue for immunofluorescence staining as described previously (Chen et al., 2004Go; Wen et al., 2006Go). The remainder of the tissue was frozen immediately in liquid nitrogen and then stored at –80°C for further processing.

Immunofluorescence staining for {alpha}2-M
Immunofluorescence staining of fibroblasts from the vaginal wall was performed as previously described (Chen et al., 2004Go). Briefly, fibroblast cells from the vaginal cuff were cultured in a 4-well chamber slide. The cells were fixed with 4% paraformaldehyde and treated with 5% Triton. After washing with Tris–HCl-Tween buffer (TBS-T) and blocking with 5% normal secondary serum, the slides were incubated overnight with rabbit anti-{alpha}2-M (1/200, Sigma, St Louis, MO, USA) primary antibody at 4ºC. Non-immune serum of the primary antibody was used as a negative control. Then, after washing, the slides were stained with goat anti-rabbit-immunoglobulin (Ig)G- fluorescein isothiocyanate (1/50, Sigma) at room temperature for 1 h. 4',6-Diamidino-2-phenylindole staining was used to observe nuclei. The slides were washed three times and mounted with Vectashield (Vector Laboratory, Burlingame, CA, USA).

Immunohistochemistry for {alpha}2-M
Immunohistochemical staining for {alpha}2-M was performed on fixed embedded tissue as described previously (Wen et al., 2006Go) to localize the expression of {alpha}2-M in vaginal wall specimens. Briefly, paraffin-embedded specimens were cut into 5 µm sections, de-waxed in Xylene and rehydrated through graded ethanol solutions. After washing with TBS-T, endogenous peroxidases were blocked with 3% H2O2 in TBS-T, and non-specific binding was blocked with 1% bovine serum albumin, 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-{alpha}2-M (1/20, Sigma) primary antibody overnight at 4°C. Omission 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 (1/50, Vector Laboratory). The slides were incubated with Vectastatin ABC Kit (Vector Laboratory) reagent for 30 min at room temperature. Immunoreactive products were visualized by incubating slides with the substrate solution in TBS-T buffer with levamisole to block alkaline phosphatase activity. Slides were counterstained with 25% of hematoxylin (Fisher, Fair Lawn, NJ, USA). They were visualized and photographed with AxioCam (Zeiss, Oberkochen, Germany).

Relative real-time quantitative PCR
Expression of {alpha}2-M mRNA was analysed by real-time quantitative PCR (QPCR) in SUI and control patients. The extraction of RNA from the tissue sample was carried out with the RNA-STAT-60 reagent (Tel-Test, Inc., Friendswood, TX, USA). The complementary DNA (cDNA) was generated from total RNA, as described previously (Chen et al., 2004Go). PCR primers used to amplify {alpha}2-M cDNA were forward: 5'-TTCGTAAACCCAAAATGTGTCCA-3' and reverse: 5'-GTGAGGCTCTTCAACATGCAC-3'. Real-time QPCR was carried on the Mx3005P Multiplex Quantificative PCR System with MxPro QPCR software (Stratagene, La Jolla, CA, USA). Brilliant SYBR Green QPCR Master Mix (Stratagene) was used to perform PCR. QPCR was performed as described previously (Wen et al., 2006Go). The amplifications were carried out following a 10-min hot start at 95°C in a three-step protocol with 30 s denaturation (94°C), 1 min annealing (55°C) and extension at 72°C for 30 s. Forty cycles were performed. Hypoxanthine phosphoribosyl-transferase 1 (HPRT1) was used as an endogenous reference (Wen et al., 2006Go) against which the different template values were normalized. All PCR reactions were performed in duplicate. Cycle of threshold (Ct) methods was used for quantification. Relative quantification of the gene of {alpha}2-M, 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 (Rel. Quant. to Cal.). The PCR products were sequenced to ensure that the correct gene sequence was amplified, and were also loaded on 2% agarose gel to confirm their size (134 bp).

We were only able to run 24 samples (two groups of 24, in duplicate) at a time for RT–PCR because the Mx3005P Multiplex Quantificative PCR System only has a 96-well blot. Samples from controls and women with SUI from the same phase of the menstrual cycle were run together. Then, samples from the controls or women with SUI from the different phases of the menstrual cycle were run together.

Western blot analysis
Protein extraction was performed as described previously (Chen et al., 2004Go). Total protein concentrations were determined using the Bradford method (Bio-Rad, Hercules, CA, USA). The samples were reduced with a sodium dodecyl sulfate (SDS) sample buffer containing 5% of 2-mercaptoethanol and boiled for 5 min. The samples were subjected to 8% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). 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, then probed with goat anti-{alpha}2-M (1:1000, Affinity Biologicals, Inc., Ontario, Canada) at room temperature for 1 h. After washing three times with phosphate buffered saline with 0.1% Triton, pH 7.4 (PBS-T), the membrane was then incubated in 1:10 000 dilution of mouse anti-goat IgG conjugated to horseradish peroxidase (HRP) (GE Healthcare, Pittsburgh, PA, USA) for 1 h at room temperature, followed by three washes in PBS-T. Blots were developed by chemiluminescence. The blots were re-probed with rabbit anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (1/2500, Abcam, Inc., Cambridge, MA, USA), then 1/10 000 dilution of donkey anti-rabbit IgG conjugated to HRP (GE Healthcare, Sunnyvale, CA, USA). The band density was determined by Bio-Rad Quality One Software (Bio-Rad).

Measurement of total {alpha}2-M
We used an enzyme-linked immunosorbent assay (ELISA) kit (Assaypro, St Charles, Mo, USA) to measure total {alpha}2-M in the vaginal wall tissue extracts prepared as described above for the Western blot. A 96-well plate was pre-coated with a polyclonal anti-{alpha}2-M antibody. Fifty microliters of standard or samples were added into the wells in duplicates for 2 h at room temperature. After washing, anti-{alpha}2-M biotinylated polyclonal antibody was added to the plates for 1 h at room temperature. Plates were then incubated with 50 µl of streptavidin-peroxidase conjugate for 30 min at room temperature. Substrate was added to the wells for ~15 min for color development, and then terminated by addition of 0.5 N HCl. The absorbance at 450 nm was measured on a plate reader (Molecular Devices, Sunnyvale, CA, USA). According to the manufacturer, intra-assay and inter-assay coefficients of variation are 5.1% and 7.8%, respectively. The level of total {alpha}2-M in the samples was normalized by protein concentration.

The {alpha}2-M protease (thermolysin) inhibitory activity assay
The ability of {alpha}2-M in vaginal wall tissue extracts to inhibit proteases was assessed using QuantiCleave Protease Assay Kit (Pierce) (Wu and Pizzo, 2001Go). Briefly, 50 µl of the tissue extracts or {alpha}2-M standard were added in duplicate to 96-well plates containing 50 µl of thermolysin (0.02 mg/ml, Sigma) per well, and pre-incubated at 37°C for 30 min. After pre-incubation, 100 µl of Tris–HCl buffer (0.01 M Tris–HCl, 0.002 M CaCl2, pH 8.2, control blank) or 100 µl of succinylated casein (2 mg/ml) was added into each well and incubated at 37°C for 1 h. Finally, 50 µl of trinitrobenzene sulfonate solution was added and incubated at room temperature for 20 min. The plates were read on a microplate reader at 450 nm. According to the manufacturer's protocol, {alpha}2-M attributable thermolysin inhibitory activity was calculated for the change in absorbance at 450 nm by subtracting the absorbance at 450 of the control blank from that of the corresponding succinylated casein well. The {alpha}2-M protease inhibitory activity was then normalized by total protein concentration.

Statistical analysis
Statistical analysis was performed using unpaired t-tests. The level of significance was set at P < 0.05. JMPIN software version 5.1 was used (SAS Institute, Inc., Cary, NC, USA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 Funding
 Acknowledgement
 References
 
We recruited 57 participants: 14 (mean age, 43.2 ± 1.6 years) with SUI, and 14 (mean age, 45.6 ± 1.4 years) controls in the proliferative phase, as well as 14 (mean age, 45.6 ± 1.2 years) with SUI and 15 (mean age, 42.7 ± 1.0 years) controls in the secretory phase of the menstrual cycle. There were no significant differences in mean age and in number of vaginal deliveries between SUI and control groups in both phases of the cycle. The small tissue sample size limited our ability to perform all the assays on each specimen. Where tissue samples were large enough, we performed all assays. Where tissue samples were too small for the entire panel of assays, we included more participants from each group. This accounts for the discrepancy in the number of tissue samples included in each experiment.

Identification and localization of {alpha}2-M expression in vaginal wall tissue
The presence of {alpha}2-M in cultured human embryonic lung fibroblasts has been reported previously (Ma et al., 2004Go). Our immunoflouresence study shows that {alpha}2-M is also expressed in the fibroblasts isolated from vaginal wall tissue (Fig. 1). The immunohistochemistry results further indicate that {alpha}2-M is expressed in both cells and ECM of the vaginal wall tissue, but the expression is primarily localized in the cells (Fig. 2).


Figure 1
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  		Figure 1: Fibroblasts from the vaginal wall of a control patient were cultured

The cells were fixed and subjected to immunofluorescence staining analysis using anti-{alpha}2-M antibody (green). 4',6-Diamidino-2-phenylindole was used to stain nuclei (blue)

 

Figure 2
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  		Figure 2: Immunohistochemical localization of {alpha}2-M, which was detected in the cells and throughout the ECM of vaginal wall tissue

 
Expression levels of {alpha}2-M mRNA in vaginal wall tissue from controls and women with SUI
The expression levels of {alpha}2-M in tissues from women with SUI and control groups from the proliferative and secretory phases of menstrual cycle were evaluated using relative real-time QPCR. The RT–PCR fragments of {alpha}2-M run on 2% agarose gels confirmed their size (134 bp, Fig. 3A). The expression level of {alpha}2-M mRNA in the control group (n = 8) was ~11 times higher than that in the SUI group (n = 8) during the proliferative phase (P = 0.03) and the level of {alpha}2-M mRNA in the continent control group (n = 9) was ~8 times higher (P = 0.01) than that in the SUI group (n = 9) during the secretory phase (Fig. 3B).


Figure 3
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  		Figure 3: Relative real-time QPCR analyses were performed to determine and compare the mRNA expression levels of {alpha}2-M in vaginal wall tissue from both the controls and SUI patients in the proliferative and the secretory phases

Relative quantification of the gene of {alpha}2-M, normalized to the internal control (hypoxanthine phosphoribosyl-transferase 1), was divided by one normalized control sample value (calibrator sample) to generate the relative quantification to calibrator (Rel.Quant. to Cal.). (A) The RT–PCR fragments of {alpha}2-M shown on a 2% agarose gel confirm their size (134 bp). (B) The bars represent the mean ± SEM. {alpha}2-M mRNA expression level was higher in both control groups than in the SUI groups during the proliferative phase (11-fold increase, P = 0.03) and secretory phase (8-fold increase, P = 0.01). Relative Quantity to Calibrator Average (baseline-corrected, reference dye—normalized fluorescence) [Rel. Quant. to Cal. Avg (dRn)]

 
To examine the in vivo ability of reproductive hormones to alter the mRNA expression levels of {alpha}2-M in vaginal wall tissue from both SUI and control groups, we compared the expression levels of {alpha}2-M mRNA between the proliferative and the secretory phases of the menstrual cycle in both groups. The expression level of {alpha}2-M mRNA in the control group was ~3 times higher (P = 0.01) in the secretory phase (n = 12) compared to the level in the proliferative phase (n = 9) (Fig. 4). However, the expression level of {alpha}2-M mRNA in the SUI group was similar during the proliferative (n = 10) and the secretory phases (n = 10) (Fig. 4).


Figure 4
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  		Figure 4: Comparative relative real-time QPCR analyses were performed to determine the ability of reproductive hormones to modulate mRNA expression of {alpha}2-M in vaginal wall tissue from women with SUI and the continent controls in both the proliferative and the secretory phases

The bars represent the mean ± SEM. The expression level of {alpha}2-M mRNA in SUI groups shows no difference between the proliferative (n = 10) and secretory phases (n = 10).The expression level of {alpha}2-M mRNA in the continent control group was ~3 times higher (P = 0.01) in the secretory phase (n = 12) compared with the level in the proliferative phase (n = 9)

 
Characterization of proteolytic {alpha}2-M fragments in the vaginal wall tissues from continent controls and women with SUI
{alpha}2-M may be proteolytically degraded during severe inflammatory processes, resulting in decreased inhibitory function and increased proteolysis. This proteolytic process is characterized in reducing SDS–PAGE and Western blot as the appearance of additional {alpha}2-M fragment bands (smaller than 90 kDa) rather than 180 and 90 kDa bands (Wu and Pizzo, 2001Go). Figure 5 shows Western blot analysis of the proteolytic state of {alpha}2-M in the tissue extracts of both control and SUI groups in the secretory phase. In controls, {alpha}2-M shows a similar protein pattern as the SUI group, consisting of the 180 kDa subunits and the 90 kDa fragments resulting from {alpha}2-M bait region proteolysis, together with the 50 kDa fragments from {alpha}2-M secondary proteolysis. Densitometric analysis shows that the 180/90 kDa and 180/50 kDa ratios for {alpha}2-M were similar for control and SUI groups. Both groups also had equivalent {alpha}2-M (180 kDa) /GAPDH (40 kDa) ratios. Western blot analysis of the proteolytic state of {alpha}2-M in the tissue extracts of both controls and SUI groups in the proliferative phase shows similar results (data not shown).


Figure 5
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  		Figure 5: Western blotting of {alpha}2-M in vaginal wall tissues from 9 SUI and 10 continent control patients in the secretory phase

{alpha}2-M shows three main bands on the blot; the 180 kDa subunit and the 90 kDa fragments resulting from {alpha}2-M bait region proteolysis, together with the 50 kDa fragments from {alpha}2-M secondary proteolysis. ‘M’ represents protein marker. ‘C’ represents controls. ‘S’ represents women with SUI. Densitometric analysis showed that the 180/90 kDa or 180/50 kDa and {alpha}2-M (180 kDa) / glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (40kDa) ratios were similar between the two groups

 
Total concentrations of {alpha}2-M protein in vaginal wall tissues determined by ELISA
We measured {alpha}2-M concentrations in the tissue extracts using ELISA (Table I). For controls (n = 7) in the proliferative phase, the mean concentration of total {alpha}2-M was 2-fold higher than for women with SUI (n = 8, P = 0.048, Table I). For controls (n = 8) in the secretory phase, the mean concentration of total {alpha}2-M was roughly 3-fold higher than for women with SUI (n = 7, P = 0.044, Table I). Within the SUI group, the mean concentration of total {alpha}2-M was also 2-fold higher in the secretory phase compared with the mean concentration in the proliferative phase (P = 0.049, Table I). Within controls, the mean concentration of total {alpha}2-M was 2-fold higher in the secretory phase compared with the mean concentration in the proliferative phase (P = 0.015, Table I).


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  		Table I. Total {alpha}2-M concentrations in vaginal wall tissues (µg/mg/ml).

 
Protease inhibitory activity of {alpha}2-M in vaginal wall tissues
Having shown that {alpha}2-M protein levels are increased in controls compared with women with SUI in both proliferative and secretory phases, we sought to correlate these differences with the ability of {alpha}2-M to inhibit proteases (Table II). We specifically measured the {alpha}2-M-attributable thermolysin inhibitory activity and found a significantly higher mean protease inhibitory level in the controls (n = 8) compared to women with SUI (n = 8) during the secretory phase (P = 0.02, Table II). We saw no significant difference between the mean protease inhibitory level in controls (n = 8) compared to women with SUI (n = 9) during the proliferative phase (P = 0.64, Table II). Within each group, there was no significant difference between the proliferative and secretory phases.


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  		Table II. {alpha}2-M attributable-thermolysin inhibitory activity was measured by QuantiCleave Protease Assay Kit.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
Funding
 Acknowledgement
 References
 
In the present study, we investigated the expression and protease inhibitory capacity of {alpha}2-M in the vaginal wall tissues of women with and without SUI during the proliferative and secretory phases of the menstrual cycle. We found that the expression levels of {alpha}2-M mRNA and protein in vaginal wall tissues are significantly higher in controls than in women with SUI during both proliferative and secretory phases. The capacity of {alpha}2-M to inhibit protease activity in vaginal wall tissues is also higher in controls than in women with SUI during the secretory phase. In the proliferative phase, we did not observe a significant difference between the SUI and control groups in {alpha}2-M protease inhibitory capacity, which differs from our PCR and ELISA data. This may be due to the small number of patients in each group or because the assay was not sensitive enough to pick up small differences. This could also be due to cyclic variation in other factors which affect enzyme activity.

Collagen and elastin play a major role in maintaining tissue tensile strength and the mechanical stability of pelvic supporting tissues. Their functions are complementary, with collagen being involved with tensile strength and elastin with deformation and recoil. The quantity and quality of collagen, elastin and other components in the pelvic supporting tissues are maintained through a precise balance between proteolytic and {alpha}2-M inhibitory activity in the ECM. {alpha}2-M has a broad-specificity proteinase inhibitory ability in the ECM (Jensen and Sottrup-Jensen, 1986Go). Decreased {alpha}2-M expression and protease inhibitory activity could result in higher proteolytic activity in the ECM. Excessive degradation of collagen, elastin and other components in the ECM then weakens the pelvic supporting tissues.

{alpha}2-M is not only a broad-specificity proteinase inhibitor, but also a carrier of tissue repair growth factors, such as TGF-β (Ma et al., 2004Go). TGF-β1, a profibrotic factor, enhances collagen and elastin production (McGowan and McNamer, 1990Go; Gleizes et al., 1997Go). It also suppresses ECM degradation by down-regulating the expression of proteinases, such as collagenase and elastase, and up-regulating proteinase inhibitors, such as the TIMPs (Overall et al., 1991Go). Low level of {alpha}2-M in SUI vaginal tissue may also cause decreased synthesis of ECM components through a decrease in TGF-β1. As a result, the weakened connective tissues lose their tensile strength and mechanical stability, accelerating the development of SUI.

The bands of 180 and 85–90 kDa of {alpha}2-M can normally be observed by reducing SDS–PAGE. The enhanced degradation of {alpha}2-M by secondary proteolysis could produce the smaller fragments of {alpha}2-M (lower than 85–90 kDa), as has been observed in rheumatoid arthritis synovial fluids and the plasma of patients with severe and lethal acute pancreatitis (Wu and Pizzo, 2001Go; Bísaro de Lorenc et al., 2005Go). Our Western blot data show that the protein pattern of {alpha}2-M in the SUI group is similar to that in the control group with three forms of {alpha}2-M fragments. The ratios of 180/90 kDa and 180/50 kDa bands are not significantly different between the SUI and control groups, indicating that higher secondary protease activity involved in {alpha}2-M cleavage is not observed in vaginal wall tissues from women with SUI or controls. This suggests that SUI is a chronic, as opposed to an acute, inflammatory process that gradually develops years after an injury resulting from vaginal childbirth and/or pregnancy.

{alpha}2-M is synthesized by granulosa cells of follicles in the rat ovary after the LH surge (Gaddy-Kurten and Richards, 1991Go). In our present study, we observed a significant increase in {alpha}2-M mRNA expression in vaginal wall tissues during the secretory phase compared with the proliferative phase in the control group. However, {alpha}2-M mRNA expression did not change in the SUI group. This difference between groups suggests that {alpha}2-M expression may be regulated by reproductive hormones, and that pregnancy may favor the development of SUI in women with a genetic predisposition to SUI, through a relative decrease in {alpha}2-M expression compared with controls. Given the scope of this study, we are unable to differentiate whether the observed difference in {alpha}2-M expression is the cause or the result of SUI. This requires further study.

Our study suggests that the imbalance of ECM synthesis and degradation in vaginal wall tissues of women with SUI could be caused in part by decreased levels of {alpha}2-M expression and a corresponding decrease in protease inhibitory activity. These data, combined with our previous observation of low expression of the protease inhibitors, ATT and TIMP-1, strongly suggest that decreased expression of protease inhibitors in ECM may contribute to the development of SUI. This information may lead to the discoveries of new methods to prevent and treat SUI and/or pelvic organ prolapse.


   Funding
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Funding
Acknowledgement
 References
 
National Institute of Health (AG 17907); Mary Lake Polan Transition Fund from Stanford University.


   Acknowledgement
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Funding
 Acknowledgement
References
 
The authors thank Lorna Groundwater for her excellent editorial help.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Funding
 Acknowledgement
 References
 
Alperin M, Moalli PA. Remodeling of vaginal connective tissue in patients with prolapse. Curr Opin Obstet Gynecol (2006) 18:544–550.[Web of Science][Medline]

Anger JT, Saigal CS, Madison R, Joyce G, Litwin MS. Increasing costs of urinary incontinence among female Medicare beneficiaries. J Urol (2006) 176:247–251. discussion 251.[CrossRef][Web of Science][Medline]

Bísaro de Lorenc L, Ramos AM, Sánchez MC, Montenegro R, Chiabrando GA. Structural evaluation of plasma alpha2-macroglobulin in acute pancreatitis. Clin Chem Lab Med (2005) 43:1183–1189.[CrossRef][Web of Science][Medline]

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Submitted on August 17, 2007; resubmitted on October 15, 2007; accepted on October 19, 2007.


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