Basal release of urokinase plasminogen activator, plasminogen activator inhibitor-1, and soluble plasminogen activator receptor from separated and cultured endometriotic and endometrial stromal and epithelial cells

Basal release of urokinase plasminogen activator, plasminogen activator inhibitor-1, and soluble plasminogen activator receptor from separated and cultured endometriotic and endometrial stromal and epithelial cells Christine Bruse, M.D., Ph.D.,a Yongmei Guan, M.D.,a,b Magdalena Carlberg, Ph.D.,a,c Kjell Carlström, Ph.D.,a and Agneta Bergqvist, M.D., Ph.D.a,d a

Department of Clinical Science, Unit of Obstetrics and Gynecology, Karolinska University Hospital, Stockholm, Sweden; Department of Obstetrics and Gynecology, The Second Hospital, Harbin Medical University, Beijing, China; c Stockholm Center of Public Health, Unit for Psychological Health, Stockholm, Sweden; and d Department of Obstetrics and Gynecology, Karolinska Institutet, Danderyds Hospital, Stockholm, Sweden b

Objective: To investigate whether separated and cultured endometriotic and endometrial stromal and epithelial cells release urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and soluble plasminogen activator receptor (suPAR) antigens in vitro. Design: In vitro study. Setting: University hospital clinic. Patient(s): Regularly menstruating women with and without endometriosis. Intervention(s): Tissue samples were collected at surgery performed for clinical reasons. Main Outcome Measure(s): The antigen concentrations of uPA, PAI-1, and suPAR in culture medium were assayed by enzyme-linked immunosorbent assay. Result(s): Both stromal and epithelial cells from endometriotic and endometrial tissue released the three types of antigens, but the release of PAI-1 was significantly higher from stromal cells in the three types of tissue than from epithelial cells. Furthermore, the release of PAI-1 was significantly higher from endometriotic cells than from endometrial stromal cells. Conclusion(s): This study has demonstrated the basic capacity of separated epithelial and stromal cells from all three types of tissue to release uPA, PAI-1, and suPAR without any paracrine influence, as in vivo. The higher release of PAI-1 from endometriotic stromal cells might have importance for the invasive growth. (Fertil Steril威 2005;83(Suppl 1):1155– 60. ©2005 by American Society for Reproductive Medicine.) Key Words: Endometriosis, endometrium, epithelial cells, stromal cells, PAI-1, suPAR, uPA

Endometriosis, defined as ectopic endometrium, has invasive properties. Regurgitated menstrual debris reaches the pelvic cavity and can locally implant any single structure in the pelvis (1). Endometriosis can also invade distantly. Endometriotic lesions are found outside the pelvic cavity, that is, in the intestines, diaphragm, kidney, somatic muscles, lungs, and pleural cavity, as well as in surgical wounds (2). Although not malignant, the tissue can invade and damage other tissues. Early endometriosis invades the extracellular matrix (3), and collagen gel invasion in vitro studies have shown that the invasive potential of endometriotic cells is comparable with that of cell lines from a metastatic carcinoma (4). Received April 14, 2004; revised and accepted September 30, 2004. Supported by grants from Anders Otto Swärd’s Stiftelse, Sigurd och Elsa Goljes Minne, Ragnhild och Einar Lundströms Minne, and the Swedish Medical Research Council (grant no. 17X-09511), Stockholm, Sweden. Reprint requests: Christine Bruse, M.D., Ph.D., Department of Obstetrics and Gynecology, Karolinska University Hospital, Huddinge, SE-141 86 Huddinge, Sweden (FAX: 46-8-585-875-75; E-mail: [email protected]).

0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2004.09.033

The plasminogen-activating system, including the plasminogen activators (PAs) and their inhibitors (PAIs) and the urokinase plasminogen activator receptor (uPAR), is involved in tissue degradation and remodeling under both normal and pathological conditions (5). Besides being involved in tumor growth, invasion, and metastasis, the plasminogen-activating system also appears to be involved in other cancer cell– directed tissue-remodeling processes, such as angiogenesis, stimulation of fibroblast proliferation, and extracellular matrix (ECM) protein synthesis (6). The activation of plasminogen, leading to the formation of plasmin, is catalyzed by uPA when bound to uPAR or by tissue-type plasminogen activator (tPA). Urokinase PA binds to uPAR with high affinity, and receptor binding of uPA initiates pericellular proteolysis and cell migration, two processes that prepare for tissue invasion (5, 6). There are two types of uPA receptors, cell surface uPAR and soluble uPAR (suPAR), which arise by alternative splicing of the glycosyl phosphatidyl inositol anchor (7). Soluble uPAR has retained uPA-binding capacity and is a water-

Fertility and Sterility姞 Vol. 83, Suppl 1, April 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.

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soluble, secreted protein. The function of suPAR is still unknown, but some reports suggest that suPAR can increase the local availability and activity of uPA by retarding its inhibition by PAI-1 and its clearance (8). The highly potent protease plasmin is able to degrade a broad spectrum of matrix and basement membrane proteins, like fibronectin, laminin, and proteoglycan (9). Plasmin also activates zymogens of other matrix-degrading proteases, like collagenases, stromolysins, and elastases, that is, matrix metalloproteinases (MMPs) (10). Further, it catalyzes activation of latent transforming growth factor-␤ (TGF-␤), which is important for the up-regulation of PAI-1 (9, 11). We have previously shown significantly elevated levels of uPA and PAI-1 antigens in homogenates from endometriotic and endometrial tissue from women with endometriosis, compared with endometrial tissue from healthy women (12). The levels of uPA and PAI-1 antigen were even significantly higher in endometriotic tissue compared with endometrium from women with endometriosis. We have also recently found that uPA mRNA, PAI-1 mRNA, and uPAR mRNA are up-regulated in different ways in endometriotic tissue and endometrium from women with endometriosis compared with control endometrium (13). In the present study, we cultured separated and isolated epithelial and stromal cells, respectively, from endometriotic and endometrial tissue from women with and without endometriosis to find out what endogenous potential each of these cell types has to release uPA, PAI-1, and suPAR antigens without any influence of exogenous hormones or growth factors (GF) added, with the exceptions of manufactory, supplemented GFs in the culture medium or by other paracrine interactions. MATERIALS AND METHODS Tissue Sources Endometriotic samples were obtained at laparoscopy for clinical reasons from nine women (mean age, 39 years; range, 26 – 49 years) with stage III ovarian endometriomas according to American Fertility Society (AFS) revised classification (14). From six of these women (mean age, 40 years; range, 28 – 49 years), an endometrial sample was obtained simultaneously. Altogether, 13 endometrial samples from women with endometriosis, AFS stage III (mean age, 37 years; range, 26 – 49 years) were obtained by uterine curettage performed for clinical reasons. Eight of the women were in the proliferative phase, and five in the secretory phase. Thirteen endometrial samples were used for epithelial cell culture, and 11 samples were used for stromal cell culture. Control endometrial samples were obtained by uterine curettage from 14 healthy women (mean age, 39 years; range, 30 –52 years) undergoing laparoscopic sterilization. Eight of the women were in the proliferative phase, and six 1156

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in the secretory phase. Of these, 14 samples were used for stromal cell culture and 13 samples were used for epithelial cell culture. All women were regularly menstruating, and none had taken any sex steroid hormones or had been pregnant or breast-feeding the previous 3 months before surgery. The local ethics committee at Huddinge University Hospital approved the study, and the women gave their oral informed consent for the samples to be collected. Institutional Review Board approval is not required in Sweden. Sample Preparation and Cell Isolation Tissue sampling and separation in epithelial and stromal cells was performed as previously described by Guan et al. (15). Briefly, the endometriotic tissue was scraped with the back edge of a knife from the inside of the cysts, which were 2– 6 cm in diameter. The endometriotic and endometrial tissue samples were collected immediately after surgical extirpation and transported in cold phosphate-buffered saline (PBS) to the laboratory. Part of the fresh tissue was fixed in 4% formalin, paraffin embedded, sectioned, and stained with hematoxylin-eosin for light microscopic verification of the diagnosis and of the cycle phase, according to Noyes et al. (16). The fresh tissue was cut into small pieces of 1 mm3 and transferred into 0.25% type IA collagenase in Dulbecco’s Modified Eagle Medium nutrient mixture F-12 (DMEM/F12) without phenol red, containing 10% fetal bovine serum (FBS), 1% of 1,000 IU/mL penicillin, 1,000 ␮g/mL streptomycin, and 1% glutamine. Digestion was performed for 1 hour at 37°C under gentle shaking. After digestion, the suspension was filtered through a 100-␮m cell strainer to remove mucous material, debris, and undigested tissue. The cells were freed from collagenase by centrifugation (5 minutes, ⫻400 g). The pellet was resuspended in DMEM/F-12 and centrifuged at ⫻55 g for 2 minutes to pellet epithelial cells. The epithelial cell pellet was washed in DMEM/F-12 and centrifuged at ⫻55 g for 2 minutes, and the new pellet was resuspended in 5 mL DMEM/F-12. Filtration of the epithelial cell suspension was then carried out through a 40-␮m sieve (Becton Dickinson Lab Ware, Franklin Lakes, NJ), allowing passage of contaminating stromal cells as well as of single epithelial cells. The retained large glands were backwashed from the sieve with 5 mL DMEM/F-12 and again centrifuged at ⫻55 g for 2 minutes. Culture of Endometriotic and Endometrial Stromal Cells The supernatant, which contained isolated stromal cells, was transferred into a 25-cm2 plastic culture flask. After one passage to get rid of macrophages, the cells were seeded at 50,000 cells/well (24-well plates) and an adhesion period of 1 week followed when the cells were incubated with DMEM/F-12 containing 10% FBS. The cells were incubated

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in an atmosphere of 5% CO2/95% air, and the medium was changed every second day. After the completed adhesion period, the medium was collected after 24 hours. The media were centrifuged at ⫻2,860 g for 5 minutes to remove dead cells and stored at ⫺20°C until assayed for uPA, PAI-1, and suPAR. The stromal cell cultures were 99% pure when analyzed by immunocytochemistry using antibodies against vimentin and cytokeratin (ZYMED, San Francisco, CA). Culture of Endometriotic and Endometrial Epithelial Cells The final epithelial cell pellet was resuspended in defined keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) supplemented with insulin, epidermal growth factor (EGF 0.03 ␮M), and fibroblast growth factor (FGF). The epithelial cells were seeded with a density of 2–5 ⫻ 105 cells/cm2 on a transparent Transwell Clear membrane insert with a membrane pore size of 0.4 ␮M (Invitrogen). The membrane insert had previously been coated with 40 ␮L Matrigel (Invitrogen) diluted 1:3 with PBS and allowed to gel for 1 hour at 37°C. The inserts were placed in a 24-well tissue culture plate so that a dual-chambered system resulted, which provided the medium access to both sides of the membrane. The medium was replaced every second day, 0.25 mL in the apical compartment and 0.5 mL in the basal compartment. As this was a short-term study, we did not wait for cells growing into confluence. After an adhesion period of 4 days in an atmosphere of 5% CO2/95% air in 37°C, the culture medium from the basal compartment was collected, centrifuged at ⫻2,860 g for 5 minutes to dispose of dead cells, and stored at ⫺20°C until assayed for uPA, PAI-1, and suPAR. The epithelial cell cultures were 98% pure when analyzed by immunocytochemistry using antibodies against vimentin and cytokeratin (ZYMED). Macrophage Staining Both stromal and epithelial cell cultures were checked after the isolation for contamination of macrophages by the use of a primary mouse-antihuman monoclonal CD14 antibody as described elsewhere by Guan et al. (15, 17). Briefly, stromal and epithelial cells were incubated with the antibody in parallel with macrophages obtained from peripheral venous human blood. After incubation with the primary antibody, a detection kit (ZYMED) including a secondary antibody was used to identify macrophages. From cultured stromal cells, 0.1% of CD14 positive staining was obtained, and 1% was obtained from cultured epithelial cells compared with cultured macrophages from human peripheral venous blood. The degree of macrophage contamination in the cell cultures was thus very small and seems very unlikely to have affected the results. Urokinase PA, PAI-1 and suPAR Immunoassays Concentrations of uPA, suPAR, and PAI-1 in incubation media from cultured stromal and epithelial cells were meaFertility and Sterility姞

sured by enzyme-linked immunosorbent assay using commercial kits obtained from American Diagnostics (Greenwich, CT; suPAR and PAI-1) and from Biopool AB (Umeå, Sweden; uPA). The detection limits were for uPA and uPAR, 0.1 ␮g/L, and for PAI-1, 0.05 ␮g/L. Values below the detection limits were set to 50% of the detection limits in the calculations. Statistical Analyses The significance of differences in concentrations of uPA, PAI-1, and suPAR antigens between cell cultures from endometriotic tissue, endometrium from patients with endometriosis, and endometrium from control patients was tested by the Kruskal-Wallis test followed by post hoc analysis with the Mann-Whitney U test. Differences between pairs of stromal and epithelial cell cultures were analyzed by Wilcoxon’s signed rank test or t-test for paired observations according to distribution. Correlation calculations were performed by Spearman’s rank correlation test. The values were not normally distributed and are given as median and range. RESULTS The uPA, PAI-1, and suPAR concentrations in media from stromal and epithelial cell cultures obtained from the three tissue types are given in Table 1. The concentrations in media from epithelial cell cultures did not differ significantly with respect to type of tissue. In media from stromal cell cultures, only the concentration of PAI-1 differed between tissue types, being significantly higher in cultures from endometriotic tissue than in cultures from endometrium from patients with endometriosis as well being significantly higher in cultures from endometrium of control patients (P⬍.05 and P⬍.01, respectively). A paired comparison between endometriotic tissue and uterine endometrium from the same patient was only possible in six cases. Despite this small number of observations, the concentration of PAI-1 was significantly higher in endometriotic tissue than in uterine endometrium, 112 (38.0 –220) vs. 59.5 (23.7–167) ␮g/L (P⬍.01, t-test for paired observations). Comparisons between stromal and epithelial cells revealed significantly higher PAI-1 levels in culture medium from stromal cells in all three tissue types (P⬍.01; Table 1). Comparisons between the concentrations of suPAR and uPA in epithelial and stromal cell culture medium, respectively, showed a difference only in cultures from control endometrium, where the concentration of suPAR tended to be lower and the concentration of uPA tended to be higher in medium from stromal than from epithelial cells (P⬍.06). A significantly positive correlation between PAI-1 in epithelial and stromal cells from the same patient was found in culture medium from endometrium from controls (rs ⫽ 0.72, P⬍.05) and from patients with endometriosis (rs ⫽ 0.79, P⬍.05) but was completely absent in culture media from endometriotic tissue. No significant correlations between 1157

TABLE 1 Basal release in vitro of uPA, PAI-1, and suPAR from stromal and epithelial cells from endometriotic tissue (EOS), from endometrium of women with endometriosis (EMP), and from controls (EMC). EOS uPA, ␮g/L Stroma Epithelium PAI-1, ␮g/L Stroma Epithelium suPAR, ␮g/L Stroma Epithelium

n

EMP

n

EMC

n

1.75 (0.05–5.24) 1.35 (0.05–4.31)

8 9

0.78 (0.05–5.00) 0.23 (0.05–5.33)

11 13

1.91 (0.43–6.29)a 0.73 (0.05–5.93)

14 13

115 (38.0–220)b,c,d 2.76 (0.25–34.1)

8 9

33.7 (10.0–145)d 0.47 (0.05–55.5)

11 13

32.4 (9.91–140)d 4.93 (0.25–44.5)

14 13

0.39 (0.05–14.5) 0.70 (0.05–14.5)

9 9

0.36 (0.05–7.37) 0.65 (0.05–11.8)

11 13

0.27 (0.05–3.46)a 0.63 (0.05–5.59)

14 13

Note: Values are median (range). a Difference between stromal and epithelial cells, P⬍.06. b Significant difference from EMP, P⬍.05. c Significant difference from EMC, P⬍.01. d Significant difference between stromal and epithelial cells, P⬍.01. Bruse. uPA, PAI-1, and suPAR in culture medium. Fertil Steril 2005.

stromal and epithelial cell cultures were found for suPAR and uPA. Regarding the effect of the menstrual cycle phase, we found that in culture medium from control endometrium the concentration of uPA from stromal cells was significantly higher in the proliferative phase than in the secretory phase, 3.03 (1.46 – 6.29) vs. 1.19 (0.43– 4.91) ␮g/L (P⬍.05). The concentration of uPAR was significantly higher in culture medium from epithelial cells in the proliferative than in the secretory phase, 1.41 (0.30 –5.59) vs. 0.43 (0.05– 0.69) ␮g/L (P⬍.05). There was no significant difference between cycle phases for PAI-1. No cycle phase–related differences were found in endometriotic tissue or in endometrial tissue from women with endometriosis. DISCUSSION This in vitro study has demonstrated the basal capacity of isolated stromal and epithelial cells from endometriotic tissue and endometrium to release uPA, PAI-1, and suPAR without any paracrine influence from other cell types, as in vivo. Although both stromal and epithelial cells from the three types of tissue released the three antigens, significant differences were only found for PAI-1. The higher release of PAI-1 from endometriotic stromal cells compared with stromal cells from the two types of endometrium was notable. The medium used for culture of endometriotic and endometrial stromal cells contained 10% FBS. It has been reported that the PAI-1 mRNA level increases rapidly after serum stimulation (18). However, the PAI-1 release was not 1158

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increased in endometrial stromal cells, although the same medium was used. Moreover, we have shown in a previous study that PAI-1 is released from endometriotic stromal cells also when serum-free culture medium is used (16). Thus, the addition of FBS to the culture medium does not explain the enhanced release of PAI-1. The significantly higher release of PAI-1 from endometriotic stromal cells compared with the two types of endometrial stromal cells might be a result of endogenous upregulation of the PAI-1 gene. Known inducers of the gene are several growth factors such as TGF-␤, EGF, bFGF, and IGF-1, inflammatory cytokines such as IL-l and TNF-␣, and hormones such as corticosteroids and insulin (19). Posttranscriptionally, TGF-␤ insulin, and IGF-1 increase the levels of PAI-1 by stabilizing its mRNA (20, 21). Many of these factors have been reported to be elevated in peritoneal fluid from women with endometriosis (22–26), and we have previously shown significantly higher concentrations of IL-1␤ in endometriotic tissue homogenates compared with endometrial tissue homogenates both from women with endometriosis and without (27). Of special interest is TGF-␤, a peptide family of five homodimers with multifunctional effects on cellular growth and differentiation, which is also a potent chemoattractant for monocytes and an inducer of angiogenesis, capable of inhibiting the activity of T and B lymphocytes as well as Natural Killer (NK) cells (28). It also promotes local tumor progression (28), is produced by activated macrophages, and is elevated in peritoneal fluid from women with endometriosis (22, 23). Thus, TGF-␤ may be important in the pathophysiology of endometriosis. Chegini et al. (29) showed that

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in endometrial implants in surgically induced rat endometriosis, inflammatory cells that infiltrated among endometriotic stromal cells contained the highest immunostaining for TGF-␤ isoforms 1–3, and Tamura et al. (30) concluded that inflammatory cells, identified as macrophages, that infiltrate stromal tissue in endometriotic cysts show the highest immunostaining for TGF-␤ isoforms 1–3 and their receptors. Stromal cells in normal endometrium and in endometriotic cysts were only faintly immunostained for TGF-␤ 1–3 and their receptors (30). In a previous study (17), we found that TGF-␤ 1 significantly enhances the release of PAI-1 from cultured endometriotic stromal cells but not from endometrial stromal cells from women with endometriosis or from controls. Besides its proteolytic properties, the PA system also plays a role in cell adhesion and migration. The binding of uPA to its cell surface receptor uPAR promotes cell adhesion by increasing the affinity of the receptor for the adhesive protein vitronectin and for integrins (31). Urokinase PAR, acting as an adhesion receptor, binds to the somatomedin B–like domain of vitronectin and shares this binding site with PAI-1 (32). Since PAI-1 has a higher affinity to vitronectin, PAI-1 competitively inhibits cell uPAR-vitronectin and cell uPARintegrin binding (31, 32). PAI-1 can also detach cells by disrupting uPAR-vitronectin and integrin-vitronectin interactions by binding to the uPA present in uPA-uPAR-integrin complexes on the cell surface (33, 34). Furthermore, Czekay and coworkers (34) showed in vitro that PAI-1 can act as a de-adhesion molecule and promote detachment of cells from extracellular matrixes not containing vitronectin, for example, fibronectin and type 1 collagen matrices, by inactivating integrins. This action by PAI-1 may promote cell migration and promote rather than inhibit cell invasion (33, 34). On the other hand, Palmieri and coworkers (35) found that endogenous expression of wild-type PAI-1, although low, in a breast carcinoma cell line, increased the adhesive properties of the cells to various substrates such as vitronectin, fibronectin, laminin, and type 1 collagen in vitro and increased the cell motility to vitronectin and fibronectin. Adhesion of human endometrial fragments to peritoneum in vitro has been demonstrated by Groothuis and coworkers (36). They observed, using transmission electron microscopy, the adhesion of menstrual endometrium at locations where the mesothelium was absent and the basement membrane was exposed. They hypothesized that damage to the peritoneal lining was a prerequisite for endometrial cell adhesion and that menstrual endometrium produces soluble factors that lead to mesothelial disruption and exposure of the underlying basement membrane. They suggested MMPs, cytokines, and growth factors that are increased in endometrium during menstruation as possible initiators of the ECM remodeling, which leads to invasion of the ectopic endometrium into the submesothelial space (37, 38). Fertility and Sterility姞

Later, Witz and coworkers (39, 40) showed, using confocal laser-scanning microscopy and transmission electron microscopy, that proliferative, secretory, and menstrual phase endometrium adheres to intact peritoneal mesothelium and that individually cultured endometrial stromal and epithelial cells, as well as fragments of menstrual endometrium, invade the intact mesothelium of the peritoneal explants (40). In these and later experiments, first showing adhesion to (39, 40) and later satisfactorily showing invasion (41, 42) through peritoneal mesothelium, the investigators used endometrium from normal menstruating women without endometriosis. It seems that in their invasion study (42) both stromal and epithelial cells had the same capacity to invade. The invasive capacity of endometrium from women with endometriosis remains to be studied in such a model. The classic role of PAI-1 is to act as an inhibitor of uPA and as a regulator of plasminogen-mediated degradation of ECM to inhibit invasion. On the contrary, high levels of PAI-1 in several types of cancer have been reported to relate to poor prognosis for patients (6). The reason is not clear, but one explanation might be that PAI-1–induced cell detachment facilitates migration and invasion, as described by Czekay and coworkers (32). Another explanation of a high expression of PAI-1 in tumors is that PAI-1 is presumed to protect the cancer tissue itself against the destructive effect of uPA (43). We can only speculate about the significance of the overexpression of PAI-1 in endometriotic tissue (12, 13) and the significance of the higher levels of PAI-1 released from endometriotic stromal cells shown in this study. However, the simultaneously higher expression of uPA in endometriotic tissue (12, 13) makes it tempting to compare endometriotic tissue with malignant tissue in its capacity to invade surrounding structures. The higher release of uPA and PAI-1 from stromal cells compared with epithelial cells and the higher release of suPAR from epithelial cells compared with stromal cells from the two types of endometrium are in agreement with the results from our previous studies on uPA, PAI-1, and uPAR mRNA in endometriotic and endometrial tissue (13). In conclusion, data from the present study suggest that the higher release of PAI-1 from endometriotic stromal cells might be of importance for the invasive growth. Acknowledgment: The authors thank Ms. Aili Aav for excellent technical assistance.

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uPA, PAI-1, and suPAR in culture medium

Vol. 83, Suppl 1, April 2005