Inhibiting MMP activity prevents the development of endometriosis in the chicken chorioallantoic membrane model

Annemiek W. Nap1, Gerard A.J. Dunselman1, Anton F.P.M. de Goeij2, Johannes L.H. Evers1 and Patrick G. Groothuis1,3

Research Institute Growth and Development (GROW), Departments of 1Obstetrics and Gynaecology and and 2 Pathology, University Maastricht and University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands

3 To whom correspondence should be addressed. Email: patrick.groothuis{at}path.unimaas.nl


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Matrix metalloproteinases (MMPs) are essential for extracellular matrix remodelling and may contribute to the development of endometriosis. Transplantation of endometrium onto the chicken chorioallantoic membrane (CAM) results in endometriosis-like lesion formation, a process that requires extensive tissue remodelling. We investigated the expression of a wide range of MMPs in menstrual endometrium, endometriosis-like lesions in CAMs, in peritoneal endometriosis and in endometriosis in the rectovaginal space, as well as the function of MMPs in early lesion formation in the CAM model. METHODS: Expression of MMPs was evaluated by immunohistochemistry and MMP function was studied in the CAM by inhibiting MMP activity during lesion formation. RESULTS: Nearly all MMPs were present in all tissues studied. No significant differences in the expression of a majority of MMPs were found in endometriosis-like lesions in CAMs when compared with human endometriosis. Inhibition of MMP-1, -2, -3, -7 and -13 activities significantly impaired endometriosis-like lesion formation in CAMs. CONCLUSIONS: The MMP expression profiles of experimentally induced endometriosis in CAMs and human endometriosis are similar. The prevention of endometriosis-like lesion formation in the CAM by inhibiting MMP activity strongly suggests that MMPs have a function in the early development of endometriotic lesions.

Key words: chorioallantoic membrane model/endometriosis/endometrium/invasion/matrix metalloproteinase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Endometriosis presents as lesions of endometrium with functioning glands and stroma that grow at locations outside the uterus, predominantly within the peritoneal cavity. It presumably develops after the ectopic implantation of endometrium, which has entered the peritoneal cavity via the Fallopian tubes during menstruation (Sampson, 1927Go). The initial phase of the disease is an invasive event, which requires extracellular matrix (ECM) breakdown (Spuijbroek et al., 1992Go; Bruner et al., 1997Go). Matrix metalloproteinases (MMPs) are essential in the remodelling of the ECM in normal development, growth and repair of tissues, and are implicated in cancer and in inflammatory and degenerative diseases.

MMPs, a large family of zinc-dependent, structurally related endoproteases, can be distinguished in five subclasses according to structure and function, i.e. collagenases, gelatinases, stromelysins, membrane-type MMPs (MT-MMPs) and other MMPs. A survey of the known MMPs, their substrates and their tissue inhibitors (TIMPs) is presented in Table I. The possible role of MMPs and TIMPs in endometriosis has been discussed in various reports (Osteen et al., 1996Go, 1999Go, 2002Go; Sillem et al., 1998Go; Sharpe-Timms and Cox, 2002Go). Table II presents an overview of recent studies with regard to the relationship between MMP expression and endometriosis. The results of these studies are difficult to compare since different techniques are used, and only a selection of MMPs is studied in different case and control groups. Moreover, the results of some of these studies are conflicting. Although the expression of a number of MMPs is described, information on the function of these enzymes in the initial steps of the development of endometriosis is lacking.


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Table I. MMPs and their substrates

 

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Table II. MMPs and endometriosis in literature

 
The early phases in endometriotic lesion formation, when MMP activity is supposed to be particularly essential, are difficult to study in humans in vivo. Recently, the functions of some MMPs in endometriosis have been evaluated in a murine model of experimentally induced endometriosis (Bruner et al., 1997Go, 2002Go). These studies demonstrated that endometriosis-like lesion formation in this model can be prevented by inhibition of MMPs. However, no specific information with regard to MMP types was obtained, since the inhibitory actions affected a wide range of MMPs, and the relevance of these studies for the human situation is limited.

The chicken chorioallantoic membrane (CAM) model has been used to study the invasive, metastatic and angiogenic potential of neoplastic cells (Scher et al., 1976Go; Armstrong et al., 1982Go), and can be used to differentiate between non-invasive and invasive epithelial cells (Schroyens et al., 1989Go). Collagen types I and IV, laminin and fibronectin are present in the ECM of the CAM (Giannopoulou et al., 2001Go), which is similar to the ECM of the human peritoneum (Witz et al., 2001Go). We have reported earlier that transplantation of human endometrium onto the CAM results in the formation of endometriosis-like lesions with endometrial glands and stroma of human origin (Maas et al., 2001Go). The early phase of endometriosis-like lesion formation in this model is a rapid, proliferation-independent process, in which extensive tissue remodelling has been observed (Nap et al., 2003Go). Consequently, we hypothesize that the expression of MMPs and TIMPs in human endometriosis is reflected in experimental endometriosis-like lesions in the CAM and that the CAM model is a helpful tool to evaluate functional involvement of MMPs in the development of endometriosis.

To this end, the expression of MMP-1, -2, -3, -7, -8, -9, -10, -11, -13, -14, -15, -16 and -23, and of TIMP-1, -2 and -3 was evaluated in menstrual endometrium, in endometriosis-like lesions in the CAM and in human peritoneal and deep invasive endometriosis using semi-quantitative immunohistochemistry. Subsequently, the MMP expression profile of experimentally induced endometriosis in the CAM was compared with that of human endometriosis in order to see whether these profiles were similar. Finally, as expression of MMPs does not provide information about the activity of these enzymes, a functional study in the CAM was performed in which an MMP inhibitor was applied after transplantation of menstrual endometrium onto the CAMs. We selected a broad spectrum inhibitor which antagonizes the MMPs that were present prominently in the menstrual endometrium, the tissue that is thought to adhere and implant ectopically and to develop into endometriosis. The inhibiting effect on endometriosis-like lesion formation in the CAM was evaluated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Tissue
Human endometrium tissue
Endometrium was collected by biopsy from 20 women with normal ovulatory cycles, undergoing laparoscopy for benign conditions. No visible endometriosis was present. An Endobiops endometrium sampling device (Gynotec, Malden, The Netherlands) was used. Endometrium was collected during the menstrual phase of the cycle (1–3 days after the onset of bleeding). The use of human endometrium was approved by the Institutional Ethical Review Committee of the University Hospital Maastricht. All women participating in the study gave their written informed consent. Immediately after collection, blood clots were removed and the endometrium was carefully minced in fragments of 1–2 mm3 and kept in serum-free Dulbecco's modified Eagle' medium (DMEM)/HAM's F12 culture medium supplemented with 2 mmol/l L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B (all from Gibco Life Technologies, Breda, The Netherlands). Part of the biopsied menstrual endometrium was used for transplantation onto the CAM. Of each endometrium biopsy, tissue was embedded in paraffin, sectioned, haematoxylin and eosin (H&E) staining was performed for histological evaluation and to assess tissue integrity, and immunohistochemical analysis for MMP expression was performed. Tissue was dated according to the Noyes criteria (Noyes, 1952Go) by a pathologist, and adjusted finely according to clinical information.

Archival human endometriosis tissue
Paraffin blocks of red peritoneal lesions (n=5) and of endometriosis located in the rectovaginal space (n=5) collected from patients undergoing surgery in order to remove their endometriosis were obtained from the archives of the Department of Pathology of the University Hospital Maastricht. Sections were prepared and stained with H&E to confirm the presence of endometriosis lesions.

Chorioallantoic membrane model
Fertilized eggs of White Leghorn chickens were incubated and prepared as described previously (Nap et al., 2003Go). Fragments of biopsied menstrual endometrial tissue were transplanted onto the CAM. After 72 h of incubation, the area of the CAM containing the endometrium fragment was excised, fixed in 3.7% buffered formaldehyde and embedded in paraffin. The entire specimen was sectioned (4 µm, 150–200 sections). Every fifth section was stained with H&E for histological evaluation to identify the lesion.

Immunohistochemistry
Immunohistochemistry was performed on paraffin sections from biopsied menstrual endometrium, from endometriosis-like lesions in CAMs and from archival peritoneal and deep invasive endometriosis tissue.

Expression of MMPs was evaluated by immunohistochemistry using the commercially available antibodies against human MMPs which were suitable to stain paraffin-embedded tissue (Oncogene, La Jolla, CA). These included MMP-1, -2, -3, -7, -8, -9, -10, -11, -13, -14, -15, -16 and -23, and TIMP-1, -2 and -3. All antibodies were monoclonal mouse antibodies, except anti-MMP-23, which was a polyclonal rabbit antibody. The antibody dilutions were optimized for application on routinely fixed, paraffin-embedded tissue sections using breast cancer, colon cancer and placental tissue as positive controls. For each MMP, all tissues were included in one staining procedure in order to keep variability to a minimum. In short, paraffin sections were deparaffinized in xylene twice for 2 min and rehydrated in alcohol series. Endogenous peroxidase activity was blocked by incubation with 0.25% hydrogen peroxide in methanol for 20 min. Sections were rinsed three times in phosphate-buffered saline (PBS). For incubation with primary antibodies against MMP-1, -3, -7 and -23, antigen retrieval in Tris EDTA buffer (20 min at 95°C) was required. After rinsing again in PBS, sections were incubated overnight at 4°C (except for the antibodies against MMP-10 and -16 which were incubated for 2 h at room temperature) with the appropriately diluted primary antibodies, i.e. MMP-15 and -16,: 1:10; MMP-3, -8, -10 and -23, 1:20; MMP-2, -9 and -13, TIMP-2 and TIMP-3, 1:50; TIMP-1, 1:60; MMP-7 and -11, 1:100; MMP-14, 1:125; and MMP-1, 1:400. After three rinses in PBS, sections were exposed to Envision Chemmate (DAKO, Glostrup, Denmark) for 30 min. After rinsing again in PBS, antibody binding was visualized with 3',3-diaminobenzidine (DAB). Sections were washed and counterstained with haematoxylin, washed again, dehydrated and mounted for light microscopic evaluation. Sections in which the primary antibody was left out of the immunostaining procedure served as negative controls.

Evaluation of immunostaining
Immunostaining was assessed semi-quantitatively using a staining index. The staining was evaluated by three independent observers at a magnification of 400x. Menstrual endometrium specimens and the CAM lesions were evaluated entirely. In the tissue containing endometriosis, a representative lesion was selected and evaluated by all observers.

The semi-quantitative staining index was calculated according to the following equation: proportion of stained cells x staining intensity. The proportion of stained cells was expressed as 0, 1, 2 or 3 (0%, <10%, 10–50% or >50% of cells, respectively), and intensity of staining as 1, 2 or 3 (weak, moderate or strong, respectively). The minimum score was 0 and the maximum score was 9. The threshold for positive staining was set at a staining index of 1. The average of the scores of the three observers was calculated and taken as the staining index for that particular section. Intra- and interobserver variability was 9%. Since all MMPs except MMP-7 are present predominantly in the stroma (Hulboy et al., 1997Go), staining indices were calculated in the stromal compartment for all MMPs except for MMP-7. MMP-7 is known to be expressed in the epithelium (Hulboy et al., 1997Go), in which tissue the staining index for MMP-7 was calculated.

Inhibition of MMP activity
The effect of the inhibition of MMP activity on endometriosis-like lesion formation was studied by the administration of an MMP inhibitor that inhibits MMP-1, -2, -3, -7 and -13 (MMP inhibitor III, catalogue number 444264; Calbiochem, La Jolla, CA) to the menstrual endometrium fragments after transplantation onto the CAM. MMP inhibitor III was dissolved in dimethylsulphoxide (DMSO) and diluted 1:1000 in normal saline (NaCl) to reach a final concentration of 1 µmol/l (stock). Prior to the first application of the MMP inhibitor, endometrium fragments were allowed to attach to the CAMs for 1–2 h in order to avoid mechanical removal of the fragments by flushing during the administration of the MMP inhibitor. Vehicle (diluted DMSO) was applied to 10 CAMs and did not change or damage the CAMs. The MMP inhibitor was administered twice on days 0, 1 and 2 (65 µl of the 1 µmol/l stock). After incubation for 72 h, CAMs were fixed, embedded in paraffin, sectioned and stained as described above. Endometriosis-like lesion formation was assessed in 28 CAMs treated with the MMP inhibitor and 86 control CAMs treated with the vehicle.

Statistical analysis
Minimum, maximum, median and range of all staining intensities were calculated. Differences between medians of staining indices were calculated using the Kruskal–Wallis test for more than two independent variables. The number of endometriosis-like lesions developed in CAMs after treatment with an MMP inhibitor and the control CAMs treated with vehicle were compared using {chi}2 tests. P-values <0.05 were considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
MMP and TIMP expression in menstrual endometrium, experimental endometriosis in the CAM and in human peritoneal and deep invasive endometriosis
The majority of the studied MMPs and TIMPs were expressed in the examined tissues, except MMP-8 which was absent in peritoneal endometriosis, and MMP-14 which was absent in all tissues. Representative examples of MMP staining are presented in Figure 1.



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Figure 1. Representative examples of immunohistochemical stainings of MMPs in menstrual endometrium, CAM lesions, peritoneal endometriosis and deep invasive endometriosis (200x).

 
The staining index (SI) for MMP and TIMP expression was used to compare the MMP expression between experimental endometriosis in the CAM and human endometriosis. Differences in SI were found between endometriosis-like lesions in the CAM and human endometriosis in three out of 16 MMPs and TIMPs. Expression of MMP-1 (P<0.05), MMP-16 (P<0.05) and TIMP-3 (P<0.01) was significantly lower in human endometriosis compared with experimental endometriosis in the CAM, as shown in Figure 2A.



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Figure 2. Staining indices of human endometriosis (dotted bars) and endometriosis-like lesions in the CAM (striated bars) (A), and of menstrual endometrium (B). *P<0.05; #P<0.01.

 
No differences were found between the SIs of menstrual endometrium and the endometriosis-like lesions in the CAM.

Effect of inhibition of MMP activity on endometriosis-like lesion formation in the CAM
MMP inhibitor III was selected to inhibit the activity of MMP-1, -2, -3, -7 and -13, in order to evaluate its effect on the formation of endometriosis-like lesions in the CAM. This choice was based on the observation that MMP-1, -2, -3, -7, -11, -13, -15 and -23 were the most abundantly expressed MMPs in menstrual endometrium (Figure 2B). For MMP-11, -15 and -23, no inhibitors were available. After 72 h, endometriosis-like lesions were present in 32% of CAMs to which the MMP inhibitor was administered, whereas endometriosis-like lesions were observed in 75% of control CAMs (P<0.05).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, we demonstrate for the first time the expression of the collagenases MMP-8 and -13, the stromelysin MMP-10, the membrane-type MMPs MMP-15 and -16, and MMP-23 in human endometrium and in endometriosis. Inhibition of MMP activity impairs the development of early endometriotic lesions in the CAM model.

In order to investigate whether the CAM was suitable to study expression and function of MMPs in endometriosis, we initially compared the MMP expression profile of peritoneal and deep invasive endometriosis with that of endometriosis-like lesions in CAMs. The expression profiles turned out to be very similar. In menstrual endometrial tissue, MMP-1, -2, -3, -7, -11, -13, -15 and -23 showed the relatively highest expression levels, indicated by the SI. Inhibition of the majority of these MMPs led to significant inhibition of endometriosis-like lesion formation in CAMs. This indicates that MMPs are involved in endometriosis-like lesion development and that the CAM model is a useful tool to evaluate the involvement of MMPs in the initial steps of lesion formation of endometrial tissue.

Lesion formation in the CAM was not completely prevented by the inhibitors used. This suggests that other MMPs or other mechanisms contribute to successful implantation of endometrium. Alternatively, it is possible that the inhibitor did not completely abolish all MMP activity.

Until recently, MMPs were thought to function primarily as regulators of ECM composition and structure and to facilitate cell migration and invasion by removing barriers such as collagen. It is now becoming evident that MMPs cleave a variety of substrates that are not ECM components, such as growth factors [i.e. pro-transforming growth factor-{beta} (TGF-{beta}), proHB-epidermal growth factor (EGF)] and their receptors [i.e. fibroblast growth factor (FGF) receptor 1], adhesion receptors (i.e. E-cadherin and {alpha}v{beta}3-integrin) and cell surface proteoglycans [i.e. CD44 and intercellular adhesion molecule-1 (ICAM-1)] (Stamenkovic, 2003Go). Therefore, MMPs may be implicated in the regulation of (latent) growth factors, cytokines and other MMP family members, angiogenesis, invasion, tissue organization and cell survival (Stamenkovic, 2003Go). The high expression of various MMPs in the endometriotic lesions may indicate the presence of an alternative self-sustaining mechanism of the endometriotic tissue, next to the local production of estrogens (Gurates and Bulun, 2003Go).

Transplantation of menstrual endometrium onto the CAM did not lead to upregulation of MMP expression in the endometriosis-like lesions that developed in CAMs. Apparently, the inherent MMP expression in the endometrial tissue itself is an important determinant in the process of lesion formation, and not the local environment. This is in agreement with the study of Osteen and co-workers who stated that misregulation of the paracrine mediators of MMPs in the eutopic endometrium may be a central discriminating factor for the propensity of endometrium to implant ectopically, as a consequence of (endogenous or exogenous) steroid exposure, environmental toxin exposure, immunological disturbances or genetic predisposition (Osteen et al., 2002Go). This is also supported by the observation that members of all MMP subclasses as well as all TIMPs were present and that there were no dramatic differences in the expression of MMPs in eutopic and ectopic endometrium, and in experimentally induced endometriosis-like lesions in CAMs.

To date, the immunohistochemical study of a large series of MMPs was hampered by the limited availability of antibodies applicable for staining paraffin-embedded tissue. In this study, we have used all antibodies that were commercially available. Special care was taken to quantify the expression and to minimize bias and variability. Since the active forms of MMPs and TIMPs are usually located in the stromal compartments of tissues, we focused on the presence of MMPs and TIMPs in the stroma. However, substantial MMP staining was also observed in the epithelial cells. The expression of MMPs in epithelial cells is not uncommon and has been described previouslt in endometrial epithelial cells (Henriet et al., 2002Go), human keratinocytes (Pilcher et al., 1997Go) and mammary epithelium (Witty et al., 1995Go). The significance of these epithelial MMPs is not yet known.

The presence of the collagenases MMP-8 and -13, the stromelysin MMP-10, the membrane-type MMP-15 and -16, and of MMP-23 was not reported before in endometrium and in endometriosis, but has been described in a variety of other tissues and processes (Balbin et al., 1999Go; Pilcher et al., 1999Go; Rechtman et al., 1999Go; Sorsa et al., 1999Go; Velasco et al., 1999Go; Hernandez-Barrantes et al., 2002Go; Leeman et al., 2002Go). The present study shows that MMP-13 and -23 are highly expressed in menstrual endometrium, in endometriosis-like lesions in the CAM as well as in human endometriosis. As MMP-13 was one of the MMPs effectively inhibited by the MMP inhibitor, it is tempting to suggest that MMP-13 plays a role in the early pathogenesis of endometriosis. The SI for MMP-9 and MMP-14 was low, in menstrual endometrium as well as in human and experimental endometriosis. Ueda and co-workers did not find high MMP-9 and -14 expression in endometrium from women without endometriosis. However, in contrast to our findings, they detected high expression levels in ectopic endometrium from endometriosis patients (Ueda et al., 2002). The expression of MMP-1, -2, -3, -7 and -11 and TIMP-1, -2 and -3 is in accordance with previous reports (Osteen et al., 1996Go, 1999Go, 2002Go; Sillem et al., 1998Go; Sharpe-Timms and Cox, 2002Go; see Table II). Based on the present study and literature reports, we conclude that a combined action of MMPs of all subclasses may be responsible for the development of endometriosis.

In summary, we have assessed the expression of a broad range of MMPs and TIMPs in human endometrium and endometriosis, and in experimentally induced endometriosis-like lesions. The expression patterns of MMPs in these tissues appear to be similar. Also, it has been shown that inhibition of most of the abundantly expressed MMPs in menstrual endometrial tissue effectively reduces endometriosis-like lesion formation in the CAMs. Therefore, the CAM model was found suitable to study MMP expression and function in lesion development in endometriosis. Our results indicate functional involvement of MMP-1, -2, -3, -7 and -13 in this process; however, other MMPs or mechanisms cannot be excluded.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors acknowledge Mrs J.Schouten and Ms Q.Theunissen for excellent laboratory assistance, and Mrs L.Colautti-Duysens for her help with providing the chorioallantoic membrane model used for this study. A.W.N. was supported by an unrestricted grant from Ferring B.V., The Netherlands.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 20, 2003; resubmitted on April 19, 2004; accepted on June 16, 2004.