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Expression of matrix metalloproteinases in ovarian endometriomas: immunohistochemical study and enzyme immunoassay
Life Sciences 71 (2002) 259 – 273 www.elsevier.com/locate/lifescie
Expression of matrix metalloproteinases in ovarian endometriomas: immunohistochemical study and enzyme immunoassay Hisanobu Mizumoto, Tsuyoshi Saito*, Koji Ashihara, Makoto Nishimura, Masaki Takehara, Ryoichi Tanaka, Eiki Ito, Ryuichi Kudo Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo 060-0061, Japan Received 10 July 2001; accepted 6 December 2001
Abstract Like carcinoma, endometriosis has the unique characteristics, of invasion and metastasis, though pathologically, it is a benign tumor. However, the mechanism of destruction of the surrounding tissue in endometriosis is still unclear. In this study, the expression and localization of matrix metalloproteinases (MMP)-1, -2, -3, -7, -9 and tissue inhibitors of metalloproteinases-1 (TIMP-1) were evaluated by immunohistochemistry for 20 cases and the amounts of MMP-1, TIMP-1 and MMP-1/TIMP-1 complex in the fluid of endometrioma, were analyzed by ELISA and western blotting for 20 cases, which were analyzed by immunohistochemical study. MMP-1, -2 and -9 were detected strongly in both stromal and epithelial cells and MMP-7 in the epithelial cells in the menstrual period. MMP-3 was mainly expressed in macrophage containing hemosiderin but the change of expression was not clear. TIMP-1 was intensively detected in both stromal and epithelial cells in the menstrual period but the expression decreased in other stages of the menstrual cycle. ELISA for MMP-1 also showed results similar to immunohistochemistry, suggesting that it was released to the cyst in the menstrual period when it was released to the extracellular space from the cytoplasm. The expression of TIMP-1 was not clearly changed during the menstrual cycle. From these results, it was suggested that the destruction of the surrounding matrix by endometriosis might be caused by various MMPs, which are mainly produced in stromal cells. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Endometriosis; Endometrioma; MMP; Menstrual cycle; Immunohistochemistry
*
Corresponding author. Tel.: +81-11-611-2111; fax: +81-11-614-0860. E-mail address: [email protected] (T. Saito). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 6 4 1 - 7
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Introduction Endometriosis is the designation applied to the condition in which tissue more or less perfectly resembling the uterine mucous membrane occurs aberrantly in various locations in the pelvic cavity [1]. In the ovary, endometriosis presents either in the form of small superficial islands or the more important form of endometrioma of various sizes. The endometrioma always adheres to surrounding tissues and sometime extends into surrounding organs, destroying them. Though it extends aberrantly, growing and invading, pathologically it is a benign tumor, and little information is currently available on the mechanism of invasion and destruction of the surrounding tissue. Matrix metalloproteinases (MMPs) are an important group of zinc enzymes that are responsible for degradation of extracellular matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling, and in many disease processes such as invasion of carcinoma [2–5]. Connective tissue cells can also produce tissue inhibitors of metalloproteinases (TIMPs), and the balance between the levels of MMPs and TIMPs is thought to be an important determinant of extracellular matrix breakdown in vivo [6]. These are generally secreted by the same cells that secrete the MMPs and are also transcriptionally controlled [7]. The human endometrium undergoes marked macroscopic and microscopic changes in its complex architecture in response to a cyclically changing hormonal environment [8]. In the endometrium, it has been reported that some MMPs play an important role in endometrial physiologic characteristics. Recent studies have shown that endometrial stromal, but not epithelial, cells secrete several MMPs, MMP-1 (interstitial collagenase), MMP-2 (gelatinase-A), MMP-3 (stromalysin-1) and MMP-9 (gelatinase-B) [8–10]. MMP-7 (matrilysin) is localized only to epithelial cells [11]. These MMPs are regulated by ovarian steroid hormones [12,13]. Furthermore, several reports recently showed that the regulation of MMPs is mediated by the cytokines interleukin-1 [8,14,15], transforming growth factor-h [16] and tumor necrosis factor-a [8]. At menstruation, the extracellular matrix is degraded, accompanied by hemorrhage and tissue sloughing, and it is known that the MMPs play an important role in this process [8,9,17]. The evidence suggests that MMPs play an important role in the destruction of the surrounding connective tissue by endometriosis as MMPs of the normal endometrium do. There is some evidence that MMP-1, -2, -3 and -9 play roles in the pathogenesis of endometriosis [18–20]. We therefore analyzed, in ovarian endometrioma, MMP-1, -2, -3, -7, -9 and TIMP-1 by immunohistochemistry and also MMP-1, TIMP-1 and MMP-1/TIMP-1 complex and other MMPs in the fluid of endometrioma by ELISA and western blotting.
Materials and methods Patients Twenty patients with endometrioma of the ovary were chosen for the study. The clinical diagnosis of endometrioma was confirmed by laparoscopy or laparotomy and the endometrial fluid and tissue of the cyst were collected in each case at Sapporo Medical University Hospital. Samples were obtained according to institutional guidelines (university hospital), and informed consent was obtained from patients. The diameters of the cysts were between 3 and 5 cm. The patients had not received exogenous hormones within the previous 6 months. Five cases were in days 1–5, 8 cases were in days 6–14, and 7
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cases were at day 15 and later (days 15 ) of the menstrual cycle. For the control group, we analyzed 10 normal-cycle endometrial tissues; three were in days 1–5, four in days 6–14 and three in days 15 , which were taken from the unaffected endometrium of normally menstruating females with myoma or adenomyosis of the uterus. All the affected tissue specimens were confirmed to be endometrioma by hematoxylin and eosin (H&E) staining of sections. Immunohistochemistry Tissues were fixed overnight in 10% buffered formalin, dehydrated, and embedded in paraffin. Five-micrometer serial sections of each sample were used in this study. Sections were cut, floated onto albumin-coated slides, dried at 56 jC, deparaffinized in xylene, rehydrated, and washed with phosphate-buffered saline (PBS) for 15 min at room temperature. Specimens were treated in a microwave oven in 0.01 mol/L citrate buffer (pH 6.0) for 30 min at 100 jC, slowly cooled to room temperature, and then washed with PBS for 5 min at room temperature. After quenching endogenous peroxidase with 3% hydrogen peroxide in PBS for 10 min at room temperature, the sections were incubated with a blocking solution (PBS containing 5% skimmed milk) for 60 min at room temperature. Then the slides were incubated overnight at 4 jC with a 1:100 dilution of antihuman MMP-1 (Fuji Chemical Ltd, Toyama, Japan), MMP-2 (Oncogene Science, Cambridge, MA), MMP-3 (Fuji Chemical Ltd), MMP-7 (Fuji Chemical Ltd), MMP-9 (Fuji Chemical Ltd) or antiTIMP-1 (Fuji Chemical Ltd). The optimal dilution of the monoclonal antibodies was decided to stain the normal endometrium of the proliferative phase. After several washes with PBS, they were incubated with a second antibody, a 1:200 dilution of anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark), for 2 hours. The color reaction was developed by the silver intensification procedure described previously [21]. For the negative control, the same dilution of non-immunized
Table 1 Immunohistochemistry of MMPs in normal endometrium Epithelial cells MMP-1 days 1 – 5 (n = 3) days 6 – 14 (n = 4) days 15 – (n = 3)
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 2 2
0 2 1
3 0 0
3 4 3
0 0 0
0 0 0
0 4 3
3 0 0
0 0 0
0 0 0
0 3 3
3 1 0
0 0 2
0 4 1
3 0 0
0 3 3
0 1 0
3 0 0
n, negative; w, weak; i, intensive Stromal cells MMP-1 days 1 – 5 (n = 3) days 6 – 14 (n = 4) days 15 – (n = 3) n, negative; w, weak; i, intensive
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 2 1
0 2 2
3 0 0
0 0 0
0 3 3
3 1 0
0 4 3
3 0 0
0 0 0
3 4 3
0 0 0
0 0 0
0 1 1
0 3 2
3 0 0
0 0 0
0 4 3
3 0 0
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Table 2 Immunohistochemistry of MMPs in endometrioma Epithelial cells MMP-1 days 1 – 5 (n = 5) days 6 – 14 (n = 8) days 15 – (n = 7)
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 0 0
0 3 2
5 5 5
0 7 7
2 1 0
3 0 0
0 0 0
5 8 7
0 0 0
0 5 2
1 1 5
4 2 0
0 0 0
1 3 1
4 5 6
0 7 2
0 1 5
5 0 0
n, negative; w, weak; i, intensive Stromal cells MMP-1 days 1 – 5 (n = 5) days 6 – 14 (n = 8) days 15 – (n = 7)
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 1 1
1 5 6
4 2 0
0 4 0
1 3 7
4 1 0
1 6 2
4 2 5
0 0 0
0 8 7
5 0 0
0 0 0
0 2 4
1 4 3
4 2 0
0 6 2
1 2 5
4 0 0
n, negative; w, weak; i, intensive
mouse immunogloblin was used as for the first antibody. Staining evaluation was performed by two independent observers (H.M. and T.S.) without knowledge of clinical outcome. For each tissue sample, the intensity of immunostaining was graded negative, weak and intensive for epithelial cells and stromal cells. Enzyme-linked immunosorbent assay (ELISA) To confirm the expression of MMP-1, TIMP-1 and MMP-1/TIMP complexes in the fluid of the endometrioma, we used enzyme-linked immunosorbent assays, (ELISA) for cyst fluid using an MMP-1 ELISA system, TIMP-1 ELISA system and MMP-1/TIMP complex ELISA system (Amersham, Little Chalfont, UK) of a 96 well plate. The collected fluid was centrifuged at 10000 g for 90 min to exclude Fig. 1. Immunohistochemical findings for MMPs and TIMP-1 in normal endometrium in the menstrual period. MMP-1 (a) and MMP-9 (e) showed intensive staining in both epithelial cells and stromal cells. MMP-2 (b) was detected intensively in stromal cells but was not in epithelial cells. MMP-3 (c) was weakly detected both in stromal and epithelial cells. MMP-7 (d) was detected intensively in epithelial cells but was negatively detected in stromal cells. TIMP-1 (f) was intensively detected in both stromal cells and epithelial cells. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200. Fig. 2. Immunohistochemical findings for MMPs and TIMP-1 in normal endometrium in the proliferative phase. This is a sample of proliferative phase (days 12). MMP-1 (a) and MMP-9 (e) showed intensive staining in both epithelial cells and stromal cells. MMP-2 (b) was detected intensively in stromal cells but not in epithelial cells. MMP-3 (c) was weakly detected in epithelial cells. MMP-7 (d) was detected in epithelial cells but was negatively detected in stromal cells. TIMP-1 (f) was detected in both stromal cells and epithelial cells. However the positivity of immunostaining was clearly weaker than in the menstrual period. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200.
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Fig. 1.
hemosiderin; the supernatants were diluted with PBS to 1:10 and 100 Al applied was to each well. The reaction was performed according to the manufacturer’s protocol and stopped by adding 100 Al of 1 N H2SO4, and the absorbance at 450 nm was measured with an ELISA plate reader. The ELISA assay was
Fig. 2.
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performed in triplicate. For multiple group comparisons, homogeneity of variance was assessed by the Levene test. Parametric comparisons used ANOVA. Western blotting of MMPs for the fluid of endometrioma For Western blotting, fluid samples from 9 were analyzed; 3 cases were in days 1–5, 3 cases were in days 6–14, and 3 cases were from day 15 or later of the menstrual cycle. The centrifuged fluid was mixed with SDS electrophoresis sample buffer (10 mmol/L Tris-HCl, pH 7.8, 1 mmol/L EDTA, 3% sodium dodecyl sulfate, 5% glycerol, 10% mercaptoethanol), heated for 5 min at 95 jC, run in lanes on 9% polyacrylamide electropheresis gels (Mini-Protein II, Bio-Rad), and then blotted onto a polyvinylidene difluoride membrane (Bio-Rad, CA). The filters were blocked in 5% (w/v) dry milk in PBS and incubated for 1 h at room temperature in anti-MMP-1, -MMP-2, -MMP-3, -MMP-7 or -MMP-9 (Fuji Chemical Ltd) diluted 1:500 in PBS. After four washes with 0.1% Tween in PBS, the blots were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-mouse antibody diluted 1:2000 in PBS. They were then washed and treated with enhanced chemiluminescence western blotting detection reagents (Amersham, Little Chalfont, Bucks) and exposed to blue-light-sensitive autoradiographic film (Hyperfilm-ECL, Amersham). For negative controls, normal mouse serum was used as the primary antibody. The western blotting study was performed in triplicate. The densities of the positive bands were measured using NIH-image.
Results Immunohistochemistry The expression and localization of MMP-1, -2, -3, -7, -9 and TIMP-1 in 10 normal-cycle endometria and 20 endometriomas was immunohistochemically analyzed. The results are summarized in Table 1 for normal endometrium and Table 2 for endometrioma.
Fig. 3. Immunohistochemical findings for MMPs and TIMP-1 in endometrioma in the menstrual period. MMP-1 (a) and MMP-9 (e) showed intensive staining in both epithelial cells and stromal cells. MMP-2 (b) was detected intensively in both stromal cells and epithelial cells. MMP-3 (c) was mainly detected in macrophages containing hemosiderin (arrow). MMP-7 (d) was detected intensively in epithelial cells but was weakly detected in stromal cells. TIMP-1 (f) was intensively detected in both stromal cells and epithelial cells. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200. Fig. 4. Immunohistochemical findings for MMPs and TIMP-1 in endometrioma in days 6 – 14 of menstrual cycle. Though MMP-1 (a) and MMP-9 (e) were intensively detected in epithelial cells, the intensity of staining decreased in stromal cells. MMP-2 (b) was still rarely detected in epithelial cells and their expression in stromal cells decreased compared to the menstrual period. MMP-3 (c) was weakly detected both in stromal cells and epithelial cells but intensively in macrophages containing hemosiderin (arrow). The staining of MMP-7 (d) in epithelial cells also became weaker than in the menstrual period. The expression of TIMP-1 (f) clearly decreased both in epithelial cells and stromal cells compared to days 1 – 5. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200.
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Fig. 3.
Normal endometrium For days 1–5, during menstruation, MMP-1 (Fig. 1a) and MMP-9 (Fig. 1e) showed intensive staining in both epithelial cells (intensive staining of MMP-1, 3 of 3; MMP-9, 3 of 3) and stromal cells (intensive
Fig. 4.
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staining of MMP-1, 3 of 3; MMP-9, 3 of 3). MMP-2 (Fig. 1b) was detected intensively in stromal cells (intensive, 3 of 3), but was not detected in epithelial cells (negative, 3 of 3). MMP-3 (Fig. 1c) was quite weakly detected both in stromal cells (weak, 3 of 3) and epithelial cells (weak, 3 of 3). MMP-7 (Fig. 1e) was detected intensively in epithelial cells (intensive, 3 of 3) but was not detected in stromal cells (negative, 3 of 3). TIMP-1 (Fig. 1f) was intensively detected in both stromal cells (intensive, 3 of 3) and epithelial cells (intensive, 3 of 3). The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 1h). For days 6–14, the intensity of staining of MMP-1 (Fig. 2a) and MMP-9 (Fig. 2e) decreased both in epithelial cells (weak staining of MMP-1, 2 of 4; MMP-9, 4 of 4) and in stromal cells (weak staining of MMP-1, 2 of 4; MMP-9, 3 of 4, negative staining of MMP-1, 2 of 4; MMP-9, 1 of 4). MMP-2 (Fig. 2b) was still not detected in epithelial cells (negative, 4 of 4) and its expression in stromal cells decreased compared to days 1–5 (intensive, 1 of 4; weak, 3 of 4). MMP-3 (Fig. 2c) was not detected in stromal cells (negative, 4 of 4) and was only weakly present in epithelial cells (weak, 4 of 4). The staining of MMP-7 (Fig. 2d) in epithelial cells also became weaker than in the menstrual period (intensive, 1 of 4; weak, 3 of 4). The expression of TIMP-1 (Fig. 2f) clearly decreased both in epithelial cells (weak, 1 of 4; negative, 3 of 4) and stromal cells (weak, 4 of 4) compared to days 1–5. The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 2h). On day 15 and later, MMP-1 and MMP-9 were rarely detected in stromal cells (negative staining of MMP-1, 1 of 3; weak, 2 of 3; negative staining of MMP-9, 1 of 3; weak, 2 of 3) or in epithelial cells (negative staining of MMP-1, 2 of 3; weak, 1 of 3; negative staining of MMP-9, 2 of 3; weak, 1 of 3). MMP-2 was not detected in epithelial cells (negative, 3 of 3) and was only present weakly in stromal cells (weak, 3 of 3). MMP-3 was not detected either in stromal cells (negative, 3 of 3) or in epithelial cells (negative, 3 of 3). MMP-7 was weakly detected in epithelial cells (weak, 3 of 3) and negative in stromal cells (negative, 3 of 3). TIMP-1 was negative in the epithelial cells (negative, 3 of 3) and weakly detected in stromal cells (weak, 3 of 3). Endometrioma For days 1–5, MMP-1 (Fig. 3a) and MMP-9 (Fig. 3e) showed intensive staining in both epithelial cells (intensive staining of MMP-1, 5 of 5; MMP-9, 4 of 5) and stromal cells (intensive staining of MMP-1, 4 of 5; MMP-9, 4 of 5). MMP-2 (Fig. 3b) was detected intensively in both stromal cells (intensive, 4 of 5) and epithelial cells (intensive, 3 of 5; weak, 2 of 5). MMP-3 (Fig. 3c) was mainly detected in macrophages containing hemosiderin. MMP-7 (Fig. 3d) was detected intensively in epithelial cells (intensive, 4 of 5; weak, 1 of 5) but was weakly detected in stromal cells (weak, 5 of 5). TIMP-1 staining (Fig. 3f) was intensive in both stromal cells (intensive, 5 of 5) and epithelial cells (intensive, 4 of 5; weak, 1 of 5). The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 3h). For days 6–14, though MMP-1 (Fig. 4a) and MMP-9 (Fig. 4e) were intensively detected in epithelial cells (intensive staining of MMP-1, 5 of 8; MMP-9, 5 of 8), the intensity of staining decreased in stromal cells (intensive staining of MMP-1, 2 of 8; MMP-9, 2 of 8). MMP-2 (Fig. 4b) was still rarely detected in epithelial cells (weak, 1 of 8; negative, 7 of 8) and its expression in stromal cells decreased compared to days 1–5 (intensive, 1 of 8; weak, 3 of 8; negative, 4 of 8). MMP-3 (Fig. 4c) was weakly detected both in stromal cells (weak, 2 of 8; negative, 6 of 8) and epithelial cells (weak, 8 of 8) but intensively in macrophages containing hemosiderin. The staining of MMP-7 (Fig. 4d) in epithelial cells also became weaker than in the menstrual period (intensive, 2 of 8; weak, 1 of 8;
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negative, 5 of 8). The expression of TIMP-1 (Fig. 4f) clearly decreased both in epithelial cells (weak, 1 of 8; negative, 7 of 8) and stromal cells (weak, 2 of 8; negative, 6 of 8) compared to days 1–5. The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 4h). On day 15 and later, though MMP-1 (Fig. 5a) and MMP-9 (Fig. 5e) were rarely detected in stromal cells (negative staining of MMP-1, 1 of 7; weak, 6 of 7; negative staining of MMP-9, 4 of 7; weak, 3 of 7), they were detected in epithelial cells (weak staining of MMP-1, 2 of 7; intensive, 5 of 7; weak staining of MMP-9, 1 of 7; intensive, 6 of 7). MMP-2 (Fig. 5b) was not detected in epithelial cells (negative, 7 of 7) and only weakly found in stromal cells (weak, 7 of 7). MMP-3 (Fig. 5c) was weakly detected both in stromal cells (weak, 5 of 7; negative, 2 of 7) and epithelial cells (weak, 7 of 7) but intensively in macrophages containing hemosiderin. MMP-7 (Fig. 5d) was negative or only weakly detected in epithelial cells (weak, 5 of 7; negative 2 of 7) and was not found in stromal cells (negative, 7
Fig. 5. Immunohistochemical findings for MMPs and TIMP-1 in endometrioma in days 15 of menstrual cycle. Though MMP-1 (a) and MMP-9 (e) were intensively detected in epithelial cells, the intensity of staining decreased in stromal cells. MMP-2 (b) was still rarely detected in epithelial cells and their expression in stromal cells decreased compared to the menstrual period. MMP-3 (c) was weakly detected both in stromal cells and epithelial cells but intensively in macrophages containing hemosiderin (arrow). The staining of MMP-7 (d) in epithelial cells also became weaker than in the menstrual period. The expression of TIMP-1 (e) clearly decreased both in epithelial cells and stromal cells compared to days 1 – 5. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200.
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of 7). TIMP-1 (Fig. 5f) was weakly detected in epithelial cells (weak, 5 of 7; negative, 2 of 7) and in stromal cells (weak, 5 of 7; negative, 2 of 7). The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 5h). ELISA To confirm that the MMP expressed in the endometriosis tissue also existed in the fluid of the endometrioma, we quantified MMP-1, TIMP-1 and MMP-1/TIMP-1 complexes by ELISA using the endometrioma fluid. The results are shown in Fig. 6. For days 1–5, during the menstruation, MMP-1 was highly expressed (706.0 F 417.9 (S.D.) ng/ml), whereas after menstruation the expression significantly decreased (days 6–14, 136.3 F 60.5 (p < 0.05); days 15 , 61.4 F 15.7 (p < 0.05)). TIMP-1 was also detected most in the menstrual period (235.0 F 108.4) and decreased after menstruation (days 6–14, 200.0 F 76.0; days 15 , 73.6 F 53.7). MMP-1/TIMP-1 complex showed expression similar to TIMP-1, in the menstrual period, being detected most at days 1–5 (308.0 F 78.9), and decreasing after menstruation (days 5–14, 123.1 F 52.7 (p < 0.05); days 15 , 97.8 F 36.0 (p < 0.05)). Western blotting In this study, we analyzed the expression of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9 in 9 samples from endometrioma; 3 from days 1–5 (lanes 1, 2, 3), 3 from days 6–14 (lanes 4, 5, 6), and 3 from days 15 or later of the menstrual cycle (lanes 7, 8, 9). The positive bands of MMPs (Fig. 7a) were measured using image-analysis software and summarized in a graph (Fig. 7b). In the endometrioma fluid samples, these MMPs showed similar expression patterns in the menstrual cycle. In the menstrual period (days 1–5), all the MMPs showed strong expression (Fig. 4a and 4b, lanes 1, 2 and 3), whereas for days
Fig. 6. ELISA of MMP-1, TIMP-1 and MMP-1/TIMP-1 complex in the fluids of endometrioma. The expression of MMP1, TIMP-1 and MMP-1/TIMP complexes in the fluids of the endometriomas was analyzed by ELISA for 20 cases; 5 cases were from in days 1 – 5, 8 cases were in days 6 – 14, 7 cases were from day 15 or later of the menstrual cycle. *, p < 0.05.
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Fig. 7. Western blotting of MMPs in the fluids of endometriomas. a, MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 were detected as positive bands of MW 45 000, 66 000, 45 000, 19 000 and 86 000 respectively. b. The positive bands were quantified by image-analysis software. In the menstrual period (days 1 – 5), all the MMPs showed strong expression (lanes 1, 2 and 3), whereas for days 6 – 14, their expression clearly decreased (lanes 4, 5 and 6). After day 15, these MMPs showed quite weak expression (lanes 7, 8 and 9).
6–14, their expression clearly decreased (lanes 4, 5 and 6). After day 15, these MMPs showed quite weak expression (lanes 7, 8 and 9).
Discussion In the endometrium, it has been reported that some MMPs play an important role in endometrial physiologic characteristics. Studies have shown that endometrial stromal, but not epithelial, cells secrete several MMPs, including MMP-1, -2, -3, and -9, which are regulated by ovarian steroid hormones [12,13]. At menstruation, the extracellular matrix is degraded, accompanied by hemorrhage and tissue
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sloughing, and it is known that in this process the MMPs play an important role [8,17]. From the evidence, it was expected that MMPs would play an important role in the destruction of the surrounding connective tissue in endometriosis as MMPs of the normal endometrium do. In this study, MMP-1 and -9 were detected both in epithelial and stromal cells, MMP-2 and -3 were detected only in stromal cells and MMP-7 was detected only in epithelial cells of normal endometrium. For normal endometrium, a number of studies have reported that these MMPs are secreted in endometrial stromal cells [8,12,14–17,22–24]. However, for epithelial cells, some studies reported that they are not secreted without MMP-7 [10,11,25]. Salamonsen et al demonstrated that cultured endometrial stromal cells release MMP-1, -2, -3, -9, TIMP-1 and -2, whereas production by epithelial cells is minimal [24]. The present study is essentially in accord with these previous studies except for MMP-1 and -9 in epithelial cells. This may be because we stained them using a high-sensitive technique, silver-enhancement of nickel-DAB staining. In this study, MMP-2 and -7 were detected in both epithelial and stromal cells in endometriomas, though they were detected only in stromal cells or epithelial cells in the normal endometrium. The positive expression in endometrial epithelial cells of MMP-2 and in stromal cells of MMP-7 may be a characteristic feature of endometriosis, as surrounding epithelial cells and stromal cells express MMP-2 and MMP-7 in response to endometriosis, respectively. Nissole et al. demonstrated that the high proliferative activity and the persistence of ERs and PRs in the stroma of red lesions and ovarian endometriomas emphasized the primordial role of the stroma in the development of endometriosis and suggested different mechanisms of proliferation control from those observed in eutopic endometrium [26]. Another study demonstrated that steroid hormone receptor expression in ovarian endometrioma remained constant throughout the menstrual cycle [27]. These differences of the ovarian steroid hormone status may be responsible for the differences of MMPs between the normal endometrium and ovarian endometrioma. By immunohistochemical observation, MMP-1, -2, -3, -9 and TIMP-1 were detected in both epithelial cells and stromal cells and MMP-7 was detected mainly in epithelial cells. However, 1) the majority of cells that expressed MMPs were stromal cells in endometrioma, 2) they were also detected in stromal cells where epithelial cells were detected and 3) usually the stromal cells were close to the surrounding connective tissue. Therefore it is reasonable to think that the MMPs were mainly produced in the stromal cells in endometrioma and MMPs derived from the stromal cells contributed to breaking down the surrounding connective tissue. In ELISA, free MMP-1 was detected most in the menstrual period. With the passage of time after the menstruation there was a tendency for the expression to decrease. On the other hand, though TIMP-1 was detected at relatively low levels during the menstrual period and was detected most in the proliferative phase, the level of MMP-1/TIMP-1 complex was significantly higher than in other phases. These results agreed with the immunohistochemical observations. Though it is uncertain whether the detected MMP-1 still has activity as an interstitial collagenase in the fluid of endometrioma, it is suggested that MMP-1 is released to the cyst at the same time, during the menstrual period, when it is released to the extracellular matrix from the cytoplasm. In western blotting of MMP-1, -2, -3, -7 and -9, the results were similar to those obtained by ELISA and also to those from immunohistochemistry. The western blotting results suggested that these MMPs were released in the cyst during the menstrual period. As mentioned above, several MMPs are produced in endometrial stromal cells and the expression is regulated by ovarian steroid hormones and cytokines [12,16,17,22,23]. Schatz et al. demonstrated that stromalysin-1 (MMP-3) mRNA was inhibited by estradiol plus medroxyprogesterone acetate (MPA);
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hence, it is accelerated by steroid withdrawal in vitro [12]. Irwin et al. reported that the expression of MMP-2 in the stromal cells is stimulated by progesterone withdrawal in vitro [17]. Salamonsen et al. demonstrated that production of MMP-1, MMP-2, MMP-3 and MMP-9, which are expressed in stromal cells, is accelerated by progesterone withdrawal, but there was no effect of progesterone withdrawal on the production of TIMP-1, TIMP-2 and TIMP-3 [13]. The stimulation of MMP expression is also regulated by chemical agents such as phorbol esters, and some cytokines such as interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a) and transforming growth factor-h (TGF-h) in stromal cells [8,16]. Considering this evidence, our finding that MMPs were detected most during the menstrual period in ovarian endometrioma is reasonable and suggests that some cytokines may also contribute to the expression of MMPs in endometriosis. Actually, recent studies have shown that patients with endometriosis have higher chemotactic activity in their peritoneal fluid [28,29] and endometriosis tissues [30]. Previously, Kokorine et al. analyzed the correlation of MMP-1 expression with activity of endometriotic tissue, suggesting its involvement in tissue remodeling and bleeding, and possibly in the secondary shedding and reimplantation of endometriotic lesions [31]. Recently, Gottschalk et al. demonstrated significantly higher protein expression of MMP-1 and TACE, in endometriotic tissue than in endometrium [19], Wenzl et al. reported on MMP-2 [20], Saito et al. on MMP-3 [32] and Chung et al. on MMP-9 [18]. These findings were in accord with the present results; furthermore, we showed the possibility that not only MMP-1, -2, -3, -9 but also MMP-7 contributed to destruction of the surrounding matrix. Clinical observations and in vitro experiments have indicated that endometriotic cells are invasive and able to metastasize [33], common phenomena characteristic of malignant cells. Previous studies reported that endometrial cancer cells themselves express MMP-1, -2, -3, -7 and -9, which contribute to the invasiveness [3–5,34]. On the other hand, though endometriosis tissues also expressed -1, -2, -3, -7 and 9 in this study, stromal cells mainly produced them. This is the critical difference between the destruction of the surrounding extracellular matrix in endometrial cancer and endometriosis.
Acknowledgements We thank Mr. M. Kim Barrymore for editing the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (No. 14571575 and 13214106).
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Expression of matrix metalloproteinases in ovarian endometriomas: immunohistochemical study and enzyme immunoassay Hisanobu Mizumoto, Tsuyoshi Saito*, Koji Ashihara, Makoto Nishimura, Masaki Takehara, Ryoichi Tanaka, Eiki Ito, Ryuichi Kudo Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo 060-0061, Japan Received 10 July 2001; accepted 6 December 2001
Abstract Like carcinoma, endometriosis has the unique characteristics, of invasion and metastasis, though pathologically, it is a benign tumor. However, the mechanism of destruction of the surrounding tissue in endometriosis is still unclear. In this study, the expression and localization of matrix metalloproteinases (MMP)-1, -2, -3, -7, -9 and tissue inhibitors of metalloproteinases-1 (TIMP-1) were evaluated by immunohistochemistry for 20 cases and the amounts of MMP-1, TIMP-1 and MMP-1/TIMP-1 complex in the fluid of endometrioma, were analyzed by ELISA and western blotting for 20 cases, which were analyzed by immunohistochemical study. MMP-1, -2 and -9 were detected strongly in both stromal and epithelial cells and MMP-7 in the epithelial cells in the menstrual period. MMP-3 was mainly expressed in macrophage containing hemosiderin but the change of expression was not clear. TIMP-1 was intensively detected in both stromal and epithelial cells in the menstrual period but the expression decreased in other stages of the menstrual cycle. ELISA for MMP-1 also showed results similar to immunohistochemistry, suggesting that it was released to the cyst in the menstrual period when it was released to the extracellular space from the cytoplasm. The expression of TIMP-1 was not clearly changed during the menstrual cycle. From these results, it was suggested that the destruction of the surrounding matrix by endometriosis might be caused by various MMPs, which are mainly produced in stromal cells. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Endometriosis; Endometrioma; MMP; Menstrual cycle; Immunohistochemistry
*
Corresponding author. Tel.: +81-11-611-2111; fax: +81-11-614-0860. E-mail address: [email protected] (T. Saito). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 6 4 1 - 7
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Introduction Endometriosis is the designation applied to the condition in which tissue more or less perfectly resembling the uterine mucous membrane occurs aberrantly in various locations in the pelvic cavity [1]. In the ovary, endometriosis presents either in the form of small superficial islands or the more important form of endometrioma of various sizes. The endometrioma always adheres to surrounding tissues and sometime extends into surrounding organs, destroying them. Though it extends aberrantly, growing and invading, pathologically it is a benign tumor, and little information is currently available on the mechanism of invasion and destruction of the surrounding tissue. Matrix metalloproteinases (MMPs) are an important group of zinc enzymes that are responsible for degradation of extracellular matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling, and in many disease processes such as invasion of carcinoma [2–5]. Connective tissue cells can also produce tissue inhibitors of metalloproteinases (TIMPs), and the balance between the levels of MMPs and TIMPs is thought to be an important determinant of extracellular matrix breakdown in vivo [6]. These are generally secreted by the same cells that secrete the MMPs and are also transcriptionally controlled [7]. The human endometrium undergoes marked macroscopic and microscopic changes in its complex architecture in response to a cyclically changing hormonal environment [8]. In the endometrium, it has been reported that some MMPs play an important role in endometrial physiologic characteristics. Recent studies have shown that endometrial stromal, but not epithelial, cells secrete several MMPs, MMP-1 (interstitial collagenase), MMP-2 (gelatinase-A), MMP-3 (stromalysin-1) and MMP-9 (gelatinase-B) [8–10]. MMP-7 (matrilysin) is localized only to epithelial cells [11]. These MMPs are regulated by ovarian steroid hormones [12,13]. Furthermore, several reports recently showed that the regulation of MMPs is mediated by the cytokines interleukin-1 [8,14,15], transforming growth factor-h [16] and tumor necrosis factor-a [8]. At menstruation, the extracellular matrix is degraded, accompanied by hemorrhage and tissue sloughing, and it is known that the MMPs play an important role in this process [8,9,17]. The evidence suggests that MMPs play an important role in the destruction of the surrounding connective tissue by endometriosis as MMPs of the normal endometrium do. There is some evidence that MMP-1, -2, -3 and -9 play roles in the pathogenesis of endometriosis [18–20]. We therefore analyzed, in ovarian endometrioma, MMP-1, -2, -3, -7, -9 and TIMP-1 by immunohistochemistry and also MMP-1, TIMP-1 and MMP-1/TIMP-1 complex and other MMPs in the fluid of endometrioma by ELISA and western blotting.
Materials and methods Patients Twenty patients with endometrioma of the ovary were chosen for the study. The clinical diagnosis of endometrioma was confirmed by laparoscopy or laparotomy and the endometrial fluid and tissue of the cyst were collected in each case at Sapporo Medical University Hospital. Samples were obtained according to institutional guidelines (university hospital), and informed consent was obtained from patients. The diameters of the cysts were between 3 and 5 cm. The patients had not received exogenous hormones within the previous 6 months. Five cases were in days 1–5, 8 cases were in days 6–14, and 7
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cases were at day 15 and later (days 15 ) of the menstrual cycle. For the control group, we analyzed 10 normal-cycle endometrial tissues; three were in days 1–5, four in days 6–14 and three in days 15 , which were taken from the unaffected endometrium of normally menstruating females with myoma or adenomyosis of the uterus. All the affected tissue specimens were confirmed to be endometrioma by hematoxylin and eosin (H&E) staining of sections. Immunohistochemistry Tissues were fixed overnight in 10% buffered formalin, dehydrated, and embedded in paraffin. Five-micrometer serial sections of each sample were used in this study. Sections were cut, floated onto albumin-coated slides, dried at 56 jC, deparaffinized in xylene, rehydrated, and washed with phosphate-buffered saline (PBS) for 15 min at room temperature. Specimens were treated in a microwave oven in 0.01 mol/L citrate buffer (pH 6.0) for 30 min at 100 jC, slowly cooled to room temperature, and then washed with PBS for 5 min at room temperature. After quenching endogenous peroxidase with 3% hydrogen peroxide in PBS for 10 min at room temperature, the sections were incubated with a blocking solution (PBS containing 5% skimmed milk) for 60 min at room temperature. Then the slides were incubated overnight at 4 jC with a 1:100 dilution of antihuman MMP-1 (Fuji Chemical Ltd, Toyama, Japan), MMP-2 (Oncogene Science, Cambridge, MA), MMP-3 (Fuji Chemical Ltd), MMP-7 (Fuji Chemical Ltd), MMP-9 (Fuji Chemical Ltd) or antiTIMP-1 (Fuji Chemical Ltd). The optimal dilution of the monoclonal antibodies was decided to stain the normal endometrium of the proliferative phase. After several washes with PBS, they were incubated with a second antibody, a 1:200 dilution of anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark), for 2 hours. The color reaction was developed by the silver intensification procedure described previously [21]. For the negative control, the same dilution of non-immunized
Table 1 Immunohistochemistry of MMPs in normal endometrium Epithelial cells MMP-1 days 1 – 5 (n = 3) days 6 – 14 (n = 4) days 15 – (n = 3)
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 2 2
0 2 1
3 0 0
3 4 3
0 0 0
0 0 0
0 4 3
3 0 0
0 0 0
0 0 0
0 3 3
3 1 0
0 0 2
0 4 1
3 0 0
0 3 3
0 1 0
3 0 0
n, negative; w, weak; i, intensive Stromal cells MMP-1 days 1 – 5 (n = 3) days 6 – 14 (n = 4) days 15 – (n = 3) n, negative; w, weak; i, intensive
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 2 1
0 2 2
3 0 0
0 0 0
0 3 3
3 1 0
0 4 3
3 0 0
0 0 0
3 4 3
0 0 0
0 0 0
0 1 1
0 3 2
3 0 0
0 0 0
0 4 3
3 0 0
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Table 2 Immunohistochemistry of MMPs in endometrioma Epithelial cells MMP-1 days 1 – 5 (n = 5) days 6 – 14 (n = 8) days 15 – (n = 7)
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 0 0
0 3 2
5 5 5
0 7 7
2 1 0
3 0 0
0 0 0
5 8 7
0 0 0
0 5 2
1 1 5
4 2 0
0 0 0
1 3 1
4 5 6
0 7 2
0 1 5
5 0 0
n, negative; w, weak; i, intensive Stromal cells MMP-1 days 1 – 5 (n = 5) days 6 – 14 (n = 8) days 15 – (n = 7)
MMP-2
MMP-3
MMP-7
MMP-9
TIMP-1
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
n
w
i
0 1 1
1 5 6
4 2 0
0 4 0
1 3 7
4 1 0
1 6 2
4 2 5
0 0 0
0 8 7
5 0 0
0 0 0
0 2 4
1 4 3
4 2 0
0 6 2
1 2 5
4 0 0
n, negative; w, weak; i, intensive
mouse immunogloblin was used as for the first antibody. Staining evaluation was performed by two independent observers (H.M. and T.S.) without knowledge of clinical outcome. For each tissue sample, the intensity of immunostaining was graded negative, weak and intensive for epithelial cells and stromal cells. Enzyme-linked immunosorbent assay (ELISA) To confirm the expression of MMP-1, TIMP-1 and MMP-1/TIMP complexes in the fluid of the endometrioma, we used enzyme-linked immunosorbent assays, (ELISA) for cyst fluid using an MMP-1 ELISA system, TIMP-1 ELISA system and MMP-1/TIMP complex ELISA system (Amersham, Little Chalfont, UK) of a 96 well plate. The collected fluid was centrifuged at 10000 g for 90 min to exclude Fig. 1. Immunohistochemical findings for MMPs and TIMP-1 in normal endometrium in the menstrual period. MMP-1 (a) and MMP-9 (e) showed intensive staining in both epithelial cells and stromal cells. MMP-2 (b) was detected intensively in stromal cells but was not in epithelial cells. MMP-3 (c) was weakly detected both in stromal and epithelial cells. MMP-7 (d) was detected intensively in epithelial cells but was negatively detected in stromal cells. TIMP-1 (f) was intensively detected in both stromal cells and epithelial cells. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200. Fig. 2. Immunohistochemical findings for MMPs and TIMP-1 in normal endometrium in the proliferative phase. This is a sample of proliferative phase (days 12). MMP-1 (a) and MMP-9 (e) showed intensive staining in both epithelial cells and stromal cells. MMP-2 (b) was detected intensively in stromal cells but not in epithelial cells. MMP-3 (c) was weakly detected in epithelial cells. MMP-7 (d) was detected in epithelial cells but was negatively detected in stromal cells. TIMP-1 (f) was detected in both stromal cells and epithelial cells. However the positivity of immunostaining was clearly weaker than in the menstrual period. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200.
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Fig. 1.
hemosiderin; the supernatants were diluted with PBS to 1:10 and 100 Al applied was to each well. The reaction was performed according to the manufacturer’s protocol and stopped by adding 100 Al of 1 N H2SO4, and the absorbance at 450 nm was measured with an ELISA plate reader. The ELISA assay was
Fig. 2.
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performed in triplicate. For multiple group comparisons, homogeneity of variance was assessed by the Levene test. Parametric comparisons used ANOVA. Western blotting of MMPs for the fluid of endometrioma For Western blotting, fluid samples from 9 were analyzed; 3 cases were in days 1–5, 3 cases were in days 6–14, and 3 cases were from day 15 or later of the menstrual cycle. The centrifuged fluid was mixed with SDS electrophoresis sample buffer (10 mmol/L Tris-HCl, pH 7.8, 1 mmol/L EDTA, 3% sodium dodecyl sulfate, 5% glycerol, 10% mercaptoethanol), heated for 5 min at 95 jC, run in lanes on 9% polyacrylamide electropheresis gels (Mini-Protein II, Bio-Rad), and then blotted onto a polyvinylidene difluoride membrane (Bio-Rad, CA). The filters were blocked in 5% (w/v) dry milk in PBS and incubated for 1 h at room temperature in anti-MMP-1, -MMP-2, -MMP-3, -MMP-7 or -MMP-9 (Fuji Chemical Ltd) diluted 1:500 in PBS. After four washes with 0.1% Tween in PBS, the blots were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-mouse antibody diluted 1:2000 in PBS. They were then washed and treated with enhanced chemiluminescence western blotting detection reagents (Amersham, Little Chalfont, Bucks) and exposed to blue-light-sensitive autoradiographic film (Hyperfilm-ECL, Amersham). For negative controls, normal mouse serum was used as the primary antibody. The western blotting study was performed in triplicate. The densities of the positive bands were measured using NIH-image.
Results Immunohistochemistry The expression and localization of MMP-1, -2, -3, -7, -9 and TIMP-1 in 10 normal-cycle endometria and 20 endometriomas was immunohistochemically analyzed. The results are summarized in Table 1 for normal endometrium and Table 2 for endometrioma.
Fig. 3. Immunohistochemical findings for MMPs and TIMP-1 in endometrioma in the menstrual period. MMP-1 (a) and MMP-9 (e) showed intensive staining in both epithelial cells and stromal cells. MMP-2 (b) was detected intensively in both stromal cells and epithelial cells. MMP-3 (c) was mainly detected in macrophages containing hemosiderin (arrow). MMP-7 (d) was detected intensively in epithelial cells but was weakly detected in stromal cells. TIMP-1 (f) was intensively detected in both stromal cells and epithelial cells. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200. Fig. 4. Immunohistochemical findings for MMPs and TIMP-1 in endometrioma in days 6 – 14 of menstrual cycle. Though MMP-1 (a) and MMP-9 (e) were intensively detected in epithelial cells, the intensity of staining decreased in stromal cells. MMP-2 (b) was still rarely detected in epithelial cells and their expression in stromal cells decreased compared to the menstrual period. MMP-3 (c) was weakly detected both in stromal cells and epithelial cells but intensively in macrophages containing hemosiderin (arrow). The staining of MMP-7 (d) in epithelial cells also became weaker than in the menstrual period. The expression of TIMP-1 (f) clearly decreased both in epithelial cells and stromal cells compared to days 1 – 5. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200.
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Fig. 3.
Normal endometrium For days 1–5, during menstruation, MMP-1 (Fig. 1a) and MMP-9 (Fig. 1e) showed intensive staining in both epithelial cells (intensive staining of MMP-1, 3 of 3; MMP-9, 3 of 3) and stromal cells (intensive
Fig. 4.
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staining of MMP-1, 3 of 3; MMP-9, 3 of 3). MMP-2 (Fig. 1b) was detected intensively in stromal cells (intensive, 3 of 3), but was not detected in epithelial cells (negative, 3 of 3). MMP-3 (Fig. 1c) was quite weakly detected both in stromal cells (weak, 3 of 3) and epithelial cells (weak, 3 of 3). MMP-7 (Fig. 1e) was detected intensively in epithelial cells (intensive, 3 of 3) but was not detected in stromal cells (negative, 3 of 3). TIMP-1 (Fig. 1f) was intensively detected in both stromal cells (intensive, 3 of 3) and epithelial cells (intensive, 3 of 3). The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 1h). For days 6–14, the intensity of staining of MMP-1 (Fig. 2a) and MMP-9 (Fig. 2e) decreased both in epithelial cells (weak staining of MMP-1, 2 of 4; MMP-9, 4 of 4) and in stromal cells (weak staining of MMP-1, 2 of 4; MMP-9, 3 of 4, negative staining of MMP-1, 2 of 4; MMP-9, 1 of 4). MMP-2 (Fig. 2b) was still not detected in epithelial cells (negative, 4 of 4) and its expression in stromal cells decreased compared to days 1–5 (intensive, 1 of 4; weak, 3 of 4). MMP-3 (Fig. 2c) was not detected in stromal cells (negative, 4 of 4) and was only weakly present in epithelial cells (weak, 4 of 4). The staining of MMP-7 (Fig. 2d) in epithelial cells also became weaker than in the menstrual period (intensive, 1 of 4; weak, 3 of 4). The expression of TIMP-1 (Fig. 2f) clearly decreased both in epithelial cells (weak, 1 of 4; negative, 3 of 4) and stromal cells (weak, 4 of 4) compared to days 1–5. The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 2h). On day 15 and later, MMP-1 and MMP-9 were rarely detected in stromal cells (negative staining of MMP-1, 1 of 3; weak, 2 of 3; negative staining of MMP-9, 1 of 3; weak, 2 of 3) or in epithelial cells (negative staining of MMP-1, 2 of 3; weak, 1 of 3; negative staining of MMP-9, 2 of 3; weak, 1 of 3). MMP-2 was not detected in epithelial cells (negative, 3 of 3) and was only present weakly in stromal cells (weak, 3 of 3). MMP-3 was not detected either in stromal cells (negative, 3 of 3) or in epithelial cells (negative, 3 of 3). MMP-7 was weakly detected in epithelial cells (weak, 3 of 3) and negative in stromal cells (negative, 3 of 3). TIMP-1 was negative in the epithelial cells (negative, 3 of 3) and weakly detected in stromal cells (weak, 3 of 3). Endometrioma For days 1–5, MMP-1 (Fig. 3a) and MMP-9 (Fig. 3e) showed intensive staining in both epithelial cells (intensive staining of MMP-1, 5 of 5; MMP-9, 4 of 5) and stromal cells (intensive staining of MMP-1, 4 of 5; MMP-9, 4 of 5). MMP-2 (Fig. 3b) was detected intensively in both stromal cells (intensive, 4 of 5) and epithelial cells (intensive, 3 of 5; weak, 2 of 5). MMP-3 (Fig. 3c) was mainly detected in macrophages containing hemosiderin. MMP-7 (Fig. 3d) was detected intensively in epithelial cells (intensive, 4 of 5; weak, 1 of 5) but was weakly detected in stromal cells (weak, 5 of 5). TIMP-1 staining (Fig. 3f) was intensive in both stromal cells (intensive, 5 of 5) and epithelial cells (intensive, 4 of 5; weak, 1 of 5). The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 3h). For days 6–14, though MMP-1 (Fig. 4a) and MMP-9 (Fig. 4e) were intensively detected in epithelial cells (intensive staining of MMP-1, 5 of 8; MMP-9, 5 of 8), the intensity of staining decreased in stromal cells (intensive staining of MMP-1, 2 of 8; MMP-9, 2 of 8). MMP-2 (Fig. 4b) was still rarely detected in epithelial cells (weak, 1 of 8; negative, 7 of 8) and its expression in stromal cells decreased compared to days 1–5 (intensive, 1 of 8; weak, 3 of 8; negative, 4 of 8). MMP-3 (Fig. 4c) was weakly detected both in stromal cells (weak, 2 of 8; negative, 6 of 8) and epithelial cells (weak, 8 of 8) but intensively in macrophages containing hemosiderin. The staining of MMP-7 (Fig. 4d) in epithelial cells also became weaker than in the menstrual period (intensive, 2 of 8; weak, 1 of 8;
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negative, 5 of 8). The expression of TIMP-1 (Fig. 4f) clearly decreased both in epithelial cells (weak, 1 of 8; negative, 7 of 8) and stromal cells (weak, 2 of 8; negative, 6 of 8) compared to days 1–5. The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 4h). On day 15 and later, though MMP-1 (Fig. 5a) and MMP-9 (Fig. 5e) were rarely detected in stromal cells (negative staining of MMP-1, 1 of 7; weak, 6 of 7; negative staining of MMP-9, 4 of 7; weak, 3 of 7), they were detected in epithelial cells (weak staining of MMP-1, 2 of 7; intensive, 5 of 7; weak staining of MMP-9, 1 of 7; intensive, 6 of 7). MMP-2 (Fig. 5b) was not detected in epithelial cells (negative, 7 of 7) and only weakly found in stromal cells (weak, 7 of 7). MMP-3 (Fig. 5c) was weakly detected both in stromal cells (weak, 5 of 7; negative, 2 of 7) and epithelial cells (weak, 7 of 7) but intensively in macrophages containing hemosiderin. MMP-7 (Fig. 5d) was negative or only weakly detected in epithelial cells (weak, 5 of 7; negative 2 of 7) and was not found in stromal cells (negative, 7
Fig. 5. Immunohistochemical findings for MMPs and TIMP-1 in endometrioma in days 15 of menstrual cycle. Though MMP-1 (a) and MMP-9 (e) were intensively detected in epithelial cells, the intensity of staining decreased in stromal cells. MMP-2 (b) was still rarely detected in epithelial cells and their expression in stromal cells decreased compared to the menstrual period. MMP-3 (c) was weakly detected both in stromal cells and epithelial cells but intensively in macrophages containing hemosiderin (arrow). The staining of MMP-7 (d) in epithelial cells also became weaker than in the menstrual period. The expression of TIMP-1 (e) clearly decreased both in epithelial cells and stromal cells compared to days 1 – 5. Negative control (h) did not show any positive staining. a, MMP-1; b, MMP-2; c, MMP-3; d, MMP-7; e, MMP-9; f, TIMP-1; g, H&E; h, negative control Original magnification, 200.
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of 7). TIMP-1 (Fig. 5f) was weakly detected in epithelial cells (weak, 5 of 7; negative, 2 of 7) and in stromal cells (weak, 5 of 7; negative, 2 of 7). The negative control, which used normal mouse serum as the primary antibody, did not show any positive staining (Fig. 5h). ELISA To confirm that the MMP expressed in the endometriosis tissue also existed in the fluid of the endometrioma, we quantified MMP-1, TIMP-1 and MMP-1/TIMP-1 complexes by ELISA using the endometrioma fluid. The results are shown in Fig. 6. For days 1–5, during the menstruation, MMP-1 was highly expressed (706.0 F 417.9 (S.D.) ng/ml), whereas after menstruation the expression significantly decreased (days 6–14, 136.3 F 60.5 (p < 0.05); days 15 , 61.4 F 15.7 (p < 0.05)). TIMP-1 was also detected most in the menstrual period (235.0 F 108.4) and decreased after menstruation (days 6–14, 200.0 F 76.0; days 15 , 73.6 F 53.7). MMP-1/TIMP-1 complex showed expression similar to TIMP-1, in the menstrual period, being detected most at days 1–5 (308.0 F 78.9), and decreasing after menstruation (days 5–14, 123.1 F 52.7 (p < 0.05); days 15 , 97.8 F 36.0 (p < 0.05)). Western blotting In this study, we analyzed the expression of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9 in 9 samples from endometrioma; 3 from days 1–5 (lanes 1, 2, 3), 3 from days 6–14 (lanes 4, 5, 6), and 3 from days 15 or later of the menstrual cycle (lanes 7, 8, 9). The positive bands of MMPs (Fig. 7a) were measured using image-analysis software and summarized in a graph (Fig. 7b). In the endometrioma fluid samples, these MMPs showed similar expression patterns in the menstrual cycle. In the menstrual period (days 1–5), all the MMPs showed strong expression (Fig. 4a and 4b, lanes 1, 2 and 3), whereas for days
Fig. 6. ELISA of MMP-1, TIMP-1 and MMP-1/TIMP-1 complex in the fluids of endometrioma. The expression of MMP1, TIMP-1 and MMP-1/TIMP complexes in the fluids of the endometriomas was analyzed by ELISA for 20 cases; 5 cases were from in days 1 – 5, 8 cases were in days 6 – 14, 7 cases were from day 15 or later of the menstrual cycle. *, p < 0.05.
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Fig. 7. Western blotting of MMPs in the fluids of endometriomas. a, MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 were detected as positive bands of MW 45 000, 66 000, 45 000, 19 000 and 86 000 respectively. b. The positive bands were quantified by image-analysis software. In the menstrual period (days 1 – 5), all the MMPs showed strong expression (lanes 1, 2 and 3), whereas for days 6 – 14, their expression clearly decreased (lanes 4, 5 and 6). After day 15, these MMPs showed quite weak expression (lanes 7, 8 and 9).
6–14, their expression clearly decreased (lanes 4, 5 and 6). After day 15, these MMPs showed quite weak expression (lanes 7, 8 and 9).
Discussion In the endometrium, it has been reported that some MMPs play an important role in endometrial physiologic characteristics. Studies have shown that endometrial stromal, but not epithelial, cells secrete several MMPs, including MMP-1, -2, -3, and -9, which are regulated by ovarian steroid hormones [12,13]. At menstruation, the extracellular matrix is degraded, accompanied by hemorrhage and tissue
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sloughing, and it is known that in this process the MMPs play an important role [8,17]. From the evidence, it was expected that MMPs would play an important role in the destruction of the surrounding connective tissue in endometriosis as MMPs of the normal endometrium do. In this study, MMP-1 and -9 were detected both in epithelial and stromal cells, MMP-2 and -3 were detected only in stromal cells and MMP-7 was detected only in epithelial cells of normal endometrium. For normal endometrium, a number of studies have reported that these MMPs are secreted in endometrial stromal cells [8,12,14–17,22–24]. However, for epithelial cells, some studies reported that they are not secreted without MMP-7 [10,11,25]. Salamonsen et al demonstrated that cultured endometrial stromal cells release MMP-1, -2, -3, -9, TIMP-1 and -2, whereas production by epithelial cells is minimal [24]. The present study is essentially in accord with these previous studies except for MMP-1 and -9 in epithelial cells. This may be because we stained them using a high-sensitive technique, silver-enhancement of nickel-DAB staining. In this study, MMP-2 and -7 were detected in both epithelial and stromal cells in endometriomas, though they were detected only in stromal cells or epithelial cells in the normal endometrium. The positive expression in endometrial epithelial cells of MMP-2 and in stromal cells of MMP-7 may be a characteristic feature of endometriosis, as surrounding epithelial cells and stromal cells express MMP-2 and MMP-7 in response to endometriosis, respectively. Nissole et al. demonstrated that the high proliferative activity and the persistence of ERs and PRs in the stroma of red lesions and ovarian endometriomas emphasized the primordial role of the stroma in the development of endometriosis and suggested different mechanisms of proliferation control from those observed in eutopic endometrium [26]. Another study demonstrated that steroid hormone receptor expression in ovarian endometrioma remained constant throughout the menstrual cycle [27]. These differences of the ovarian steroid hormone status may be responsible for the differences of MMPs between the normal endometrium and ovarian endometrioma. By immunohistochemical observation, MMP-1, -2, -3, -9 and TIMP-1 were detected in both epithelial cells and stromal cells and MMP-7 was detected mainly in epithelial cells. However, 1) the majority of cells that expressed MMPs were stromal cells in endometrioma, 2) they were also detected in stromal cells where epithelial cells were detected and 3) usually the stromal cells were close to the surrounding connective tissue. Therefore it is reasonable to think that the MMPs were mainly produced in the stromal cells in endometrioma and MMPs derived from the stromal cells contributed to breaking down the surrounding connective tissue. In ELISA, free MMP-1 was detected most in the menstrual period. With the passage of time after the menstruation there was a tendency for the expression to decrease. On the other hand, though TIMP-1 was detected at relatively low levels during the menstrual period and was detected most in the proliferative phase, the level of MMP-1/TIMP-1 complex was significantly higher than in other phases. These results agreed with the immunohistochemical observations. Though it is uncertain whether the detected MMP-1 still has activity as an interstitial collagenase in the fluid of endometrioma, it is suggested that MMP-1 is released to the cyst at the same time, during the menstrual period, when it is released to the extracellular matrix from the cytoplasm. In western blotting of MMP-1, -2, -3, -7 and -9, the results were similar to those obtained by ELISA and also to those from immunohistochemistry. The western blotting results suggested that these MMPs were released in the cyst during the menstrual period. As mentioned above, several MMPs are produced in endometrial stromal cells and the expression is regulated by ovarian steroid hormones and cytokines [12,16,17,22,23]. Schatz et al. demonstrated that stromalysin-1 (MMP-3) mRNA was inhibited by estradiol plus medroxyprogesterone acetate (MPA);
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hence, it is accelerated by steroid withdrawal in vitro [12]. Irwin et al. reported that the expression of MMP-2 in the stromal cells is stimulated by progesterone withdrawal in vitro [17]. Salamonsen et al. demonstrated that production of MMP-1, MMP-2, MMP-3 and MMP-9, which are expressed in stromal cells, is accelerated by progesterone withdrawal, but there was no effect of progesterone withdrawal on the production of TIMP-1, TIMP-2 and TIMP-3 [13]. The stimulation of MMP expression is also regulated by chemical agents such as phorbol esters, and some cytokines such as interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a) and transforming growth factor-h (TGF-h) in stromal cells [8,16]. Considering this evidence, our finding that MMPs were detected most during the menstrual period in ovarian endometrioma is reasonable and suggests that some cytokines may also contribute to the expression of MMPs in endometriosis. Actually, recent studies have shown that patients with endometriosis have higher chemotactic activity in their peritoneal fluid [28,29] and endometriosis tissues [30]. Previously, Kokorine et al. analyzed the correlation of MMP-1 expression with activity of endometriotic tissue, suggesting its involvement in tissue remodeling and bleeding, and possibly in the secondary shedding and reimplantation of endometriotic lesions [31]. Recently, Gottschalk et al. demonstrated significantly higher protein expression of MMP-1 and TACE, in endometriotic tissue than in endometrium [19], Wenzl et al. reported on MMP-2 [20], Saito et al. on MMP-3 [32] and Chung et al. on MMP-9 [18]. These findings were in accord with the present results; furthermore, we showed the possibility that not only MMP-1, -2, -3, -9 but also MMP-7 contributed to destruction of the surrounding matrix. Clinical observations and in vitro experiments have indicated that endometriotic cells are invasive and able to metastasize [33], common phenomena characteristic of malignant cells. Previous studies reported that endometrial cancer cells themselves express MMP-1, -2, -3, -7 and -9, which contribute to the invasiveness [3–5,34]. On the other hand, though endometriosis tissues also expressed -1, -2, -3, -7 and 9 in this study, stromal cells mainly produced them. This is the critical difference between the destruction of the surrounding extracellular matrix in endometrial cancer and endometriosis.
Acknowledgements We thank Mr. M. Kim Barrymore for editing the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (No. 14571575 and 13214106).
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