Hum. Reprod. Advance Access originally published online on July 31, 2006
Human Reproduction 2006 21(12):3059-3067; doi:10.1093/humrep/del297
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Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis
1 Centre de Recherche, Hôpital Saint-François d’Assise, Centre Hospitalier Universitaire de Québec and 2 Département d’Obstétrique et Gynécologie, Faculté de Médecine, Université Laval, Québec, Canada
3 To whom correspondence should be addressed at: Unité d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, Centre Hospitalier Universitaire de Québec, 10 rue de l’Espinay, Local D0–711, Québec, Canada G1L 3L5. E-mail: ali.akoum{at}crsfa.ulaval.ca
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Abstract |
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BACKGROUND: Endometriosis is a disease where endometrial tissue implants in ectopic locations. Remodelling of the extracellular matrix (ECM) is a prerequisite for the implantation of this tissue to be possible. METHODS: In this study, we detected immunoreactive matrix metalloproteinase-9 (MMP-9) throughout endometrial tissue and identified von Willebrand factor (vWF)-positive endothelial cells, CD45-positive leukocytes, CD3-positive T lymphocytes and CD68-positive macrophages as cells expressing MMP-9 in the stroma. RESULTS: We found an increased expression of MMP-9 in the uterine endometrial tissue of women with endometriosis, as assessed by zymography and enzyme-linked immunosorbent assay (ELISA) (P < 0.05). However, RT–PCR did not show a statistically significant increase in MMP-9 mRNA expression in these tissues (P = 0.14). There was no significant difference between women with and without endometriosis in the expression of tissue inhibitor of MMPs (TIMP)-1, a known natural inhibitor of the pro- and active forms of MMP-9, whether tested by ELISA or by RT–PCR (P = 0.46 and 0.37, respectively). Interestingly, the ratio of MMP-9/TIMP-1 expression was significantly higher in women with endometriosis than in normal women both at the protein and the mRNA levels (P < 0.05). CONCLUSION: These findings make plausible the involvement of MMP-9/TIMP-1 imbalance in the invasiveness of the endometrial tissue of patients with endometriosis and the ectopic development of the disease.
Key words: endometriosis/endometrium/matrix metalloproteinase-9/tissue inhibitor of matrix metalloproteinase
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Introduction |
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Endometriosis is a gynaecological disorder characterized by the implantation and the proliferation of endometrial cells outside the uterus. The most widely accepted theory postulated to explain peritoneal endometriosis consists of Sampson’s theory of retrograde menstruation. According to this theory, the disease arises from ectopic implantation and growth of endometrial tissue that reaches the peritoneal cavity by tubal reflux (Sampson, 1927
). However, the reflux of the menstrual fluid has been observed in 90% of women with patent fallopian tubes (Halme et al., 1984), and endometriosis is present in about 10% of women during their reproductive lives (Strathy et al., 1982). Some intrinsic factors must therefore be differently expressed for the ectopic implantation and growth of endometrial tissue to take place. The endometrial tissue must attach itself to the host tissue, then invade it and derive the local vasculature to ensure its own blood supply (Giudice et al., 1998). Thus, the degradation of the extracellular matrix (ECM) consists of a primordial step for the formation of new vessels in angiogenesis and tissue remodelling (Moses et al., 1996; Pepper et al., 1996). Many factors are necessary for the degradation of the ECM needed for the implantation of endometrial tissue in ectopic sites, notably cathepsin D (Bergqvist et al., 1996; Suzumori et al., 2001), plasminogen (Sillem et al., 1997), urokinase plasminogen activator (Bruse et al., 1998; Guan et al., 2002) and matrix metalloproteinases (MMPs) (Kokorine et al., 1997; Bruner-Tran et al., 2002; Chung et al., 2002).
MMPs form a multigene family of proteolytic enzymes that require zinc for their activation (Duncan et al., 1998; Rudolph-Owen et al., 1998; Nayak et al., 2000). These enzymes are first secreted in a latent form as proenzymes, but they can be later activated (Van Wart and Birkedal-Hansen, 1990). MMPs have different specificities, even if there is a considerable overlap, and together, they can degrade most components of the ECM, including the different types of collagens that compose the basement membrane (Freitas et al., 1999).
The degradation of the ECM by the MMPs is closely regulated by tissue inhibitors of MMPs (TIMPs) during normal physiological conditions such as tissue repair, embryogenesis and menstruation (Koks et al., 2000). Several recent studies have shown an increase in the expression of MMP-1 (Kokorine et al., 1997), MMP-2 (Wenzl and Heinzl, 1998), MMP-3 (Cox et al., 2001), MMP-7 (Rodgers et al., 1993) and MMP-9 (Chung et al., 2001; Liu et al., 2002) in endometriotic tissue. However, only a few have addressed a possible change in MMP expression in the eutopic endometrial tissue of endometriosis women compared with normal women. This hypothesis is supported by the finding that eutopic endometrium from patients with endometriosis may be more invasive and prone to peritoneal implantation because of higher expression of MMP-2 and membranous type 1 MMP and lower expression of TIMP-2 mRNA, compared with endometrium from women without endometriosis (Chung et al., 2002). Other studies from the same laboratory have shown that uterine endometrium from women with endometriosis expressed lower levels of TIMP-3 than endometrium from normal women (Chung et al., 2001). Chen et al. (2004) reported higher MMP-9 and lower TIMP-1 immunostaining in ectopic and eutopic endometrium. This is in keeping with our previous data demonstrating an increased release of proteolytic activity by the eutopic endometrium of women with endometriosis compared with normal women in which MMP-9 appeared to be involved, and a decreased release of its natural inhibitor, TIMP-1, in vitro in the culture medium of endometrial tissue (Collette et al., 2004).
The aim of the present study was to investigate MMP-9 and TIMP-1 expressions both at the protein and at the mRNA levels and to further evaluate MMP-9 forms in the eutopic endometrial tissue of women with endometriosis compared with normal women with no evidence of endometriosis at laparoscopy. Imbalance between MMP-9 and TIMP-1 occurring in the endometrial tissue may reflect in vivo the enhanced capacity of this tissue to break down the ECM in host tissues, thereby favouring its ectopic implantation and development.
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Materials and methods |
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Source and handling of tissue
The women recruited in this study provided informed consent for a research protocol approved by Saint-François d’Assise Hospital Ethics Committee on Human Research. Endometriosis was identified during laparoscopy or laparotomy in women consulting for infertility and/or pelvic pain. Patients with endometriosis (n = 18; mean age = 32.8 ± 4.9 years) otherwise had no other pelvic pathology. The stage of endometriosis was determined according to the revised classification of The American Fertility Society (1997). Normal women (n = 24; mean age = 36.4 ± 5.2 years) were fertile, requesting tubal ligation and having no visible evidence of endometriosis at laparoscopy. Endometriosis and normal women included in this study had not received any anti-inflammatory or hormonal medication during a period of at least 3 months before the intervention.
The cycle phase was determined based on the patients’ cycle history, serum progesterone and Noyes’s histological criteria (Noyes et al., 1975). For quantitative analysis and comparison of endometriosis and normal controls by zymography, enzyme-linked immunosorbent assay (ELISA) and RT–PCR, the distribution of subjects according to the menstrual cycle phase was as follows: for zymography, 14 patients with endometriosis (two at the early-mid proliferative phase, days 1–9, three at the late proliferative phase, days 10–14, five at the early-mid secretory phase, days 15–22 and four at the late secretory phase, days 23–28) and 15 normal controls (two at the early-mid proliferative phase, three at the late proliferative phase, six at the early-mid secretory phase and four at the late secretory phase) were included.
For ELISA, 10 patients with endometriosis (one at the early-mid proliferative phase, three at the late proliferative phase, three at the early-mid secretory phase and three at the late secretory phase) and nine normal controls (two at the early-mid proliferative phase, two at the late proliferative phase, two at the early-mid secretory phase and three at the late secretory phase) were included.
For RT–PCR, 11 patients with endometriosis (two at the early-mid proliferative phase, two at the late proliferative phase, three at the early-mid secretory phase and four at the late secretory phase) and nine normal controls (two at the early-mid proliferative phase, two at the late proliferative phase, two at the early-mid secretory phase and three at the late secretory phase) were included.
Endometrial biopsies were obtained using a sterile pipelle (Unimar Inc., Prodimed, Neuilly-en-Thelle, France). Samples were placed at 4°C in sterile Hanks’ balanced salt solution (Gibco BRL, Burlington, Ontario, Canada) containing 100 IU/ml of penicillin, 100 µg/ml of streptomycin and 0.25 µg/ml of amphotericin, and they were immediately transported to the laboratory. Biopsies used in this study were devoid of any visible blood contamination.
Immunohistochemistry
Immunohistochemical staining of MMP-9 in endometrial tissue sections was performed using a sheep polyclonal anti-human MMP-9 antibody [1:50 dilution in phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA)/0.01% Tween-20 (PBS/BSA/Tween)] (EMD Biosciences Inc., San Diego, CA, USA), a biotin-conjugated donkey anti-sheep antibody (1:1000 in PBS/BSA/Tween) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), peroxidase-conjugated streptavidin (1:1000 dilution in PBS/BSA/Tween) and diaminobenzidine (DAB) as chromogen (3 mg DAB/0.03% H2O2 in PBS). Sections were counterstained with haematoxylin, mounted in Mowiol (Calbiochem-Novabiochem Corp., La Jolla, CA, USA) and observed using a Leica microscope (Leica mikroskopie und systeme GmbH, Model DMRB; Postfach, Wetzlar, Germany) connected to an image analysis system (ISIS, Metasystems, Altlussheim, Germany). Sections incubated with goat immunoglobulins (IgGs) at the same concentration as the primary antibody were used as negative controls in all experiments.
Dual immunofluorescence
Dual immunofluorescent staining was performed using the same sheep polyclonal anti-human MMP-9 antibody used for immunohistochemistry (1:50 dilution in PBS/BSA/Tween) and one of the following antibodies: mouse monoclonal anti-human von Willebrand factor (vWF) to detect endothelial cells (1:200 dilution in PBS/BSA/Tween) (Dako Diagnostics Inc., Mississauga, Ontario, Canada), mouse monoclonal anti-human CD45, also known as the leukocyte common antigen, to detect leukocytes (1:50 dilution in PBS/BSA/Tween) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse monoclonal anti-human CD3 to detect T lymphocytes (1:100 dilution in PBS/BSA/Tween) (gift from Dr W. Mourad, Laval University) and mouse monoclonal anti-human CD68 to detect macrophages (1:50 dilution in PBS/BSA/Tween) (DAKO Diagnostics). Tissue sections were then incubated with a biotin-conjugated donkey anti-sheep antibody (1:1000 dilution in PBS/BSA/Tween) then simultaneously with Alexa Fluor 488-conjugated streptavidin (1:100 dilution in PBS/BSA/Tween) and Alexa Fluor 568-conjugated goat anti-mouse antibody (1:1000 dilution in PBS/BSA/Tween) (Invitrogen Life Technologies, Burlington, Ontario, Canada). Slides were mounted in Mowiol containing 10% para-phenylenediamine (Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada), an anti-fading agent and observed under a Leica microscope equipped for fluorescence with a 100-W UV lamp (Leica mikroskopie und systeme GmbH) connected to an image analysis system.
Western blotting
Protein extraction from endometrial tissue was performed according to our previously described procedure (Kats et al., 2002), and total protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories Ltd, Mississauga, Ontario, Canada). Briefly, samples were denatured and separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) in 10% acrylamide slab gels then transferred onto 0.45 µm nitrocellulose membranes (Protean, Schleicher & Schuell, Keene, NH, USA). A monoclonal mouse anti-human MMP-9 antibody (Oncogene Research Products, Boston, MA, USA) at 2 µg/ml of blocking solution [0.1 M Tris buffer, 0.9% NaCl/0.05% Tween-20 containing 5% non-fat dry milk (w/v)] was used for the detection of MMP-9, and a monoclonal mouse anti-human TIMP-1 antibody (R&D Systems, Minneapolis, MN, USA) at 2 µg/ml of blocking solution was used for the detection of TIMP-1. Equivalent concentrations of normal mouse IgGs instead of the primary antibodies were used as negative controls. Membranes were then successively incubated with a biotin-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories) diluted 1:10 000 in the blocking solution, peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories) diluted 1:5000 in the blocking solution and BM chemiluminescence blotting substrate (POD) (Roche Diagnostics, Laval, Québec, Canada) and exposed to Kodak BioMax film.
Zymography
Frozen endometrial tissue samples were directly homogenized by using a microscale tissue grinder (Kontes, Vineland, NJ, USA) in a buffer containing 0.5 M Tris–HCl pH 7.6, 0.2 M NaCl, 10 mM CaCl2 and 1% (w/v) Triton X-100. Tissue homogenate was then incubated at 4°C for 45 min under gentle shaking and centrifuged at 12 000 g for 30 min at 4°C to recover the soluble extract. Total protein concentration was determined by using the Bio-Rad DC Protein Assay. Proteins (5 µg) were analysed by zymography on 7.5% SDS–PAGE gels containing 0.5 mg/ml of gelatin (Sigma-Aldrich Canada Ltd) under non-reducing conditions. Culture supernatant from the human fibrosarcoma HT1080 cell line known for releasing elevated proteolytic activity was used as control (a gift from Dr Éric Petitclerc, Québec City, PQ). After electrophoresis, gels were washed with post-electrophoretic buffer (50 mM Tris, 5 mM CaCl2, 0.02% NaN3) containing 2.5% Triton X-100 (2 x 20 min) then incubated with a post-electrophoretic buffer containing 1% Triton X-100 (20 min at room temperature) and finally incubated overnight at 37°C. Gels were stained with 0.25% Coomassie brilliant blue G-250 dye for 30 min and destained in 30% methanol/10% glacial acetic acid. The quantification of detectable gelatinases was achieved by computer-assisted densitometry (BioImage, Visage 110s, Genomic Solutions Inc., Ann Arbor, MI, USA).
ELISA
MMP-9 and TIMP-1 concentrations in extracted proteins were measured using ELISA procedures developed in the laboratory (Collette et al., 2004; Bellehumeur et al., 2005). The sensitivity limit of these assays was 600 pg/ml for MMP-9 and 400 pg/ml for TIMP-1.
RT–PCR
Total RNA was extracted from endometrial tissue with Trizol reagent (Life Technologies), and complementary DNA (cDNA) was synthesized using the Gene Amp PCR core kit (Perkin-Elmer Corp., Foster City, CA, USA) according to the manufacturers’ instructions and as described previously (Kats et al., 2002). For PCR analysis, we used 10 and 2.5% of the RT reaction volume as template for MMP-9 and TIMP-1, respectively, in a final volume of 50 µl. About 50 pmol of each MMP-9 (forward primer, 5'-GAGGAATACCAGTACCGCTATG-3'; reverse primer, 5'-CAAACCGAGTTGGAACCACG-3'; amplimer size, 518 bp) or TIMP-1 (forward primer, 5'-CTGTTGTTGCTGTGGCTGATA-3'; reverse primer, 5'-CCGTCCACAAGCAATGAGT-3'; amplimer size, 481 bp), 0.2 mmol/l dNTP and 2.5 IU of Taq DNA polymerase (Qiagen, Santa Clarita, CA, USA) were used. Amplification was performed for 30 cycles composed of 45 s denaturation at 95°C, 1 min annealing at 69°C and 2 min primer extension at 69°C for MMP-9 and 72°C for TIMP-1. As internal control, glyceraldehyde phosphate dehydrogenase (GAPDH) amplification was used. For PCR analysis, we used 5% of the RT reaction volume as template in a final volume of 50 µl with 25 pmol of each primer (forward primer, 5'-TGATGACATCAAGAAGGTGGTGAAG-3'; reverse primer, 5'-TCCTTGGAGGCCATGTGGGCCAT-3'; amplimer size, 240 bp), 0.2 mmol/l dNTP and 1 IU Vent DNA polymerase. Amplification was performed for 30 cycles of 30 s denaturation (at 95°C), 30 s annealing (at 60°C) and 1 min primer extension (at 72°C). These optimal conditions were determined by performing linearity tests with different percentage of the RT reaction volume and amplification cycles. The amplification of genomic DNA with these primers did not produce a signal, suggesting that the amplification sites crossed at least one intron/exon boundary. The specificity of the amplification process was verified by Southern blot hybridization as described previously (Kats et al., 2002). A negative control (PCR in the absence of cDNA) as well as a positive control (cDNA preparation from human fibrosarcoma HT1080 cell line known for producing MMP-9 and TIMP-1, provided by Dr Éric Petitclerc) was included in each series of MMP-9, TIMP-1 or GAPDH amplification. The intensity of the hybridization signals was determined by computer-assisted densitometry, using BioImage, Visage 110s. The quantity of the PCR products was determined by densitometric analysis of the intensity of the hybridization signal. The relative level of MMP-9 or TIMP-1 mRNA normalized to GAPDH mRNA was calculated, and the results were expressed as percentage of control (positive control).
Statistical analysis
Data followed a parametric distribution, and they were therefore expressed as means (SEM). The comparison of two groups was performed using the unpaired t-test, whereas one-way analysis of variance and the Bonferroni’s test post hoc were used for multiple comparisons. Differences were considered as statistically significant whenever a P-value <0.05 occurred.
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Identification and quantification of MMP-9
The immunohistochemical analysis of MMP-9 expression showed a specific brownish immunostaining throughout endometrial tissue, both in the glandular and in the stromal compartments (Figure 1A). The incubation of tissue sections with normal sheep IgGs used at concentration equivalent to that of the primary sheep polyclonal anti-MMP-9 antibody (negative control) did not result in any non-specific immunostaining (Figure 1B). The specificity of the primary antibody was also verified by pre-absorption with an excess of recombinant human MMP-9 (10 µg/ml) (data not shown).
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To identify cells expressing MMP-9 in the stroma, we performed dual immunofluorescent analysis using antibodies specific to MMP-9 and to vWF, CD45, CD3 and CD68. Representative photomicrographs exhibited in Figure 2 show a marked expression of MMP-9 in vWF-positive endothelial cells, CD45-positive leukocytes, CD3-positive T lymphocytes and CD68-positive macrophages.
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Western blot analysis was then used to further assess the presence of MMP-9 in proteins extracted from endometrial biopsies. Protein extracts were separated by SDS–PAGE, and immunoblotting for MMP-9 was performed. The 170 kDa band might correspond to the putative dimeric MMP-9 pro-form described previously (Watari et al., 1999
), whereas the 92 and 86 kDa bands are consistent with the MMP-9 latent and active forms, respectively (Figure 3).
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Endometrial tissue protein extracts were then analysed by gelatinolytic zymography. As shown in Figure 4A, five distinct bands of gelatinase activity at 170, 92, 86, 72 and 66 kDa were found. The 170 kDa band might correspond to the putative dimeric MMP-9 pro-form described previously (Watari et al., 1999
). The 92 and 86 kDa bands are consistent with the MMP-9 latent and active forms, respectively, whereas the 72 and 66 kDa bands are consistent with the latent and active forms of another gelatinase, MMP-2. It is noteworthy that the 86 kDa active MMP-9 form was rarely detected in endometrial tissue protein extracts by zymography. The densitometric analysis of MMP-9 lysis bands showed a significant increase in pro-MMP-9 levels in women with endometriosis compared with normal women (P < 0.05) and a tendency for an increase in putative dimeric MMP-9 levels (P = 0.09, Figure 4B).
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To further quantify the expression of MMP-9 in endometrial tissues from normal controls and women with endometriosis, we measured MMP-9 concentrations in the total protein extracts by ELISA and determined the levels of mRNA in the same tissues using RT–PCR analysis. As shown in Figure 5A, MMP-9 protein concentrations were significantly higher in women with endometriosis than in normal controls (P < 0.05). The analysis of MMP-9 mRNA expression showed no statistically significant difference between normal and endometriosis women (P = 0.14, Figure 5B). A Southern blotting showing specific amplification of MMP-9 and the internal control GAPDH is shown in Figure 6.
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Identification and quantification of TIMP-1
The finding of an increased MMP-9 protein expression in endometriosis women-derived endometrial tissues prompted us to assess the presence of TIMP-1, a known natural inhibitor of MMP-9, and to quantify its secretion in women with and without endometriosis. Western blot analysis showed a 31-kDa band, which corresponds to the known molecular weight of the inhibitor, and a faint band of 20 kDa, which could be seen in the recombinant human TIMP-1, used as reference and possibly represent some form of TIMP-1 degradation (Figure 7).
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31 and 20 kDa. The 31 kDa band corresponds to the known MW of TIMP-1, and the 20 kDa could possibly represent some TIMP-1 degradation.The measurement of TIMP-1 protein concentrations in endometrial tissue protein extracts by ELISA and statistical analysis of the data showed no statistically significant difference between women with endometriosis and normal controls (P = 0.46, Figure 8A). Also, no significant difference in TIMP-1 mRNA levels between endometriosis and normal women was noted (P = 0.37, Figure 8B). A Southern blotting showing specific amplification of TIMP-1 is shown in Figure 6. However, the analysis of the ratio of MMP-9/TIMP-1 expression in endometrial tissue showed that endometriosis women had a significantly higher ratio than normal women, either at the protein (P < 0.05, Figure 9A) or at the mRNA (P < 0.05, Figure 9B) level.
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The analysis of MMP-9 and TIMP-1 protein and mRNA levels according to the menstrual cycle phase (proliferative and secretory) showed no statistically significant difference between normal and endometriosis women. However, the ratio of MMP-9/TIMP-1 protein and mRNA levels was found to be statistically significant in the secretory phase (P < 0.05).
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Discussion |
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Many proteases are implicated in the dynamic processes of regeneration/degeneration occurring in the human endometrium during the normal menstrual cycle. Several recent studies have also underlined the critical role of proteolysis in the capability of endometrial tissue to invade the host tissues and develop endometriosis (Sharpe-Timms et al., 1998
; Cox et al., 2001). An increased expression of different proteases including MMPs has been found in endometriotic lesions (Rodgers et al., 1993; Bergqvist et al., 1996; Kokorine et al., 1997; Bruse et al., 1998; Wenzl and Heinzl, 1998; Chung et al., 2001; Cox et al., 2001; Liu et al., 2002). Brunner et al. (2002) have shown that the suppression of MMPs inhibits the establishment of ectopic lesions by human endometrium in nude mice. However, only a few studies addressed possible changes in protease expression in the eutopic endometrium of endometriosis patients (Bruse et al., 1998; Chung et al., 2001; Chung et al., 2002; Chen et al., 2004).
In this study, we showed that MMP-9 was detectable throughout endometrial tissue, in epithelial glands as well as in the stroma in a variety of cell types. Available data regarding the distribution of MMP-9 throughout the endometrial tissue are inconsistent. Whereas immunoreactive MMP-9 was found in endometrial epithelial and stromal cells (Li et al., 2000) or in different cell types throughout the cycle, especially in foci of stromal cells (Cornet et al., 2005) with predominant immunostaining in epithelial cells (Skinner et al., 1999; Cornet et al., 2005), other studies reported no evidence for MMP-9 production by stromal cells during the cycle (Jeziorska et al., 1996). However, the reasons for such a discrepancy in the cellular localization of immunoreactive MMP-9 are difficult to ascertain.
Dual immunofluorescent analysis further identified endothelial cells and leukocytes as cells expressing MMP-9 in the stroma. Of leukocytes, T lymphocytes and macrophages were further identified, but this does not preclude the expression of MMP-9 in other immune cells that may infiltrate the endometrial tissue and express MMP-9 (Shi et al., 1995). It is well documented that endometriosis is associated with immuno-inflammatory changes in the eutopic endometrium (Lebovic et al., 2001; Sharpe-Timms, 2001) with, for instance, an increased autoimmune response (Lebovic et al., 2001), leukocyte infiltration (Witz et al., 1994; Ota et al., 1996) and secretion of proinflammatory mediators, such as complement component 3, monocyte chemotactic protein-1, interleukin (IL)-1 and IL-6 (Sharpe-Timms, 2001). Consequently, proteases can be secreted by endometrial cells (Cornet et al., 2005) as well as by immune cells, at least in response to local inflammatory stimuli.
In the present study, further analysis of endometrial tissue protein extracts by zymography showed an increased production of MMP-9 in endometriosis as assessed by densitometric analysis of the lysis band corresponding to the pro-MMP-9. The measurement of MMP-9 concentrations by ELISA corroborated the zymography data and showed a significant increase in MMP-9 protein levels in the endometrial tissue of women with endometriosis compared with normal women. These data are in keeping with our previous findings showing an increased release of proteolytic activity by the eutopic endometrial tissue of women with endometriosis in which MMP-9 appeared to be significantly involved (Collette et al., 2004) and further demonstrate an abnormal expression of MMP-9 occurring within the eutopic endometrial tissue of endometriosis women. Although MMP-9 protein levels, as assessed by zymography and ELISA, were significantly higher in women with endometriosis compared with normal women, our data did not show any statistically significant difference in MMP-9 levels between women with and without endometriosis in the proliferative or the secretory phase of the menstrual cycle. This may likely be due to the lack of statistical power (41–50%) when subjects were subdivided according to the cycle phase. Otherwise, there was an equal spread of samples obtained at the different times in the menstrual cycle in both groups of women.
MMP-9 has been found to be implicated in the invasiveness of tumours, and tumour metastasis was significantly reduced in MMP-9-knockout mice (Itoh et al., 1999). In endometriosis, an increased expression of MMP-9 was found in the ectopic endometrial tissue (Chung et al., 2001; Liu et al., 2002). Interestingly, MMP-9 has been shown to be involved in the down-regulation of IL-1 receptor type II (IL-1RII) levels in the eutopic endometrial tissue of women with endometriosis (Bellehumeur et al., 2005). This may enhance IL-1-mediated activation of endometrial cells and contribute to the local inflammatory and tissue remodelling processes observed in endometriosis patients, as IL-1RII is known for being a specific inhibitor of IL-1 (Mantovani et al., 2001; Mantovani et al., 2003). Therefore, taken together, our present and previous findings support an important role for MMP-9 in the capability of endometrial tissue to invade and develop into the host tissues.
Although a trend for an increase in MMP-9 mRNA steady-state levels was observed in the endometrial tissue of women with endometriosis compared with normal women, our present data did not detect any statistically significant endometriosis-related change in MMP-9 expression at the mRNA level. This is in keeping with what has previously been reported by Chung et al. (2001) who showed an increase in MMP-9 mRNA levels observed in the ectopic rather than the eutopic endometrial tissue of endometriosis women compared with normal women (Chung et al., 2001). This is suggestive of differences at the post-transcriptional level, which is currently under investigation in the laboratory.
Natural TIMPs are especially important for the regulation of ECM remodelling because they regulate MMP activity. The reduced expression of TIMPs appeared to be involved in cancer invasiveness (Khokha et al., 1989; Ponton et al., 1991). A small change in the levels of TIMP leads to a biologically significant change in the net protease activity (Ponton et al., 1991). Previous studies have shown that TIMPs are secreted in insufficient amounts before menstruation, resulting in an imbalance between the TIMPs and the MMPs that are responsible for the degradation of the endometrium (Marbaix et al., 1996). Interestingly, the concentration of TIMP-1 is decreased in the peritoneal fluid of women with endometriosis (Sharpe-Timms et al., 1998). TIMP-1 is implicated in the regulation of the active forms of MMP-1, MMP-3 and MMP-9, because the formation of a complex between the inhibitors with any of these MMPs leads to protease inactivation (Hanemaaijer et al., 1993). In addition, TIMP-1 has the capacity to form a complex with pro-MMP-9, thereby blocking the activation of the enzyme (Goldberg et al., 1992; Hanemaaijer et al., 1993). Our present data did not show any significant change in TIMP-1 protein or mRNA expression in the endometrial tissue of women with endometriosis, as compared with normal women. However, a significant decrease in the ratio of MMP-9/TIMP-1 expression was observed, either at the protein or at the mRNA level. Furthermore, such a decrease was statistically significant in the secretory phase of the menstrual cycle. This denotes an imbalance between MMP-9 and TIMP-1 levels in the secretory phase which may play an important role in the invasiveness of endometriosis women-derived endometrial tissue and its capability to implant in ectopic locations. Chung et al. (2001) also found an imbalance between MMP-9 mRNA levels in the eutopic endometrium of endometriosis patients and those of another of its natural inhibitors, TIMP-3, with an increased MMP-9/TIMP-3 mRNA ratio. This, together with our findings, suggests a deregulation of MMP-9 expression by the endometrial tissue in endometriosis patients, an inadequate control of its activity and a possible role for this proteolytic enzyme in the enhanced aptitude of endometrial tissue to invade and implant into host tissues.
In conclusion, the present study revealed a significant increase in MMP-9 protein expression in the eutopic endometrium in women with endometriosis as compared with normal women and further showed an imbalance between MMP-9 expression and that of its natural inhibitor TIMP-1 at both the protein and the mRNA levels. This may reflect in vivo the enhanced capability of this tissue to invade and break down the ECM in host tissues, thereby favouring its ectopic implantation and development.
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This work was supported by grant MOP-14638 to Ali Akoum from the Canadian Institutes for Health Research. A.A. is Chercheur National from the Fonds de la Recherche en Santé du Québec (FRSQ). The authors thank Drs François Belhumeur, Jean Blanchet, Marc Bureau, Simon Carrier, Elphège Cyr, Marlène Daris, Jean-Louis Dubé, Jean-Yves Fontaine, Céline Huot, Pierre Huot, Johanne Hurtubise, Rodolphe Maheux, Jacques Mailloux and Marc Villeneuve for patient evaluation and providing peritoneal fluid samples, Mahéra Al-Akoum, Nathalie Bourcier, Madeleine Desaulniers, Rouslan Kats, Monique Longpré and Johanne Pelletier for technical assistance and Dr Lucile Turcot-Lemay for her assistance with the statistical analyses.
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Submitted on February 2, 2006; resubmitted on April 30, 2006; resubmitted on June 13, 2006; accepted on June 23, 2006.
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