Eutopic and ectopic stromal cells from patients with endometriosis exhibit differential invasive, adhesive, and proliferative behavior

ORIGINAL ARTICLE: ENDOMETRIOSIS

Eutopic and ectopic stromal cells from patients with endometriosis exhibit differential invasive, adhesive, and proliferative behavior Ali-Akbar Delbandi, M.Sc.,a Mahmoud Mahmoudi, Ph.D.,a Adel Shervin, M.D.,b Elham Akbari, M.D.,b Mahmood Jeddi-Tehrani, Ph.D.,c Mojtaba Sankian, Ph.D.,a Somayeh Kazemnejad, Ph.D.,b and Amir-Hassan Zarnani, Ph.D.d,e a Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad; b Reproductive Biotechnology Research Center, c Monoclonal Antibody Research Center, d Reproductive Immunology Research Center, Avicenna Research Institute, ACECR; and e Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran

Objective: To study immunophenotype, differential proliferation capacity, invasiveness, adhesion, and cytokine production in ectopic and eutopic endometrial stromal cells (EESCs and EuESCs) from patients with endometriosis. Design: In vitro study. Setting: Academic research center. Patient(s): Patients with ovarian endometriosis (endometrioma) and nonendometriotic controls. Intervention(s): None. Main Outcome Measure(s): EESCs and EuESCs from 25 patients with endometrioma and ESCs from 20 nonendometriotic controls (CESCs) were isolated, and their immunophenotype, proliferation, invasion, adhesion, and cytokine production were assessed and compared. Result(s): Isolated ESCs from all three sources expressed markers specific for cells of mesenchymal origin but were negative for hematopoietic markers. EESCs exhibited a significantly lower proliferation rate in fibronectin-coated plates and less invasive capacity compared with CESCs or EuESCs. Among all stromal cell groups studied, EuESCs showed the highest invasive behavior. EESCs adhered more firmly to extracellular matrix than EuESCs or CESCs in all time intervals examined. The levels of interleukin (IL) -6 and IL-8 production by EESCs were significantly higher compared with those of EuESCs or CESCs. Conclusion(s): The results of the present study demonstrated that retrograde menstruation alone does not account for the pathogenesis of endometriosis as eutopic and ectopic counterparts of ESCs from patients with endometriosis exhibit differential invasive, adhesive, and proliferative behavior. (Fertil SterilÒ 2013;-: Use your smartphone -–-. Ó2013 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Endometrioma, eutopic, invasion, adhesion, proliferation, cytokine, immunoand connect to the phenotype Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/delbandiaa-endometriosis-invasion-adhesion/

E

ndometriosis, first identified in 1860 by von Rokitansky, is a chronic inflammatory gyneco-

logical condition characterized by the growth of endometrial glands and stroma outside the uterine cavity (1).

Received January 5, 2013; revised April 16, 2013; accepted April 25, 2013. A.-A.D. has nothing to disclose. M.M. has nothing to disclose. A.S. has nothing to disclose. E.A. has nothing to disclose. M.J.-T. has nothing to disclose. M.S. has nothing to disclose. S.K. has nothing to disclose. A.-H.Z. has nothing to disclose. This work was supported as a Ph.D. research project by grants from Avicenna Research Institute and Mashhad University of Medical Sciences. Reprint requests: Amir-Hassan Zarnani, Ph.D., Reproductive Immunology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 1936773493. (E-mail: [email protected]); and Mahmoud Mahmoudi, Ph.D., Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.04.041 VOL. - NO. - / - 2013

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As the most common gynecological disorder, it affects at least 10%–25% of reproductive-aged women in the United Kingdom and United States. The most common symptoms are severe dysmenorrhea, dyspareunia, pelvic pain, and infertility (2). Several theories have been proposed to explain the development of this disease such as coelomic metaplasia (Meyer's theory), vascular and lymphatic metastasis (Halban's theory), embryonic rest theory, and stem/progenitor cells theory (3, 4), but the most widely cited 1

ORIGINAL ARTICLE: ENDOMETRIOSIS theory is Sampson's theory of retrograde menstruation. According to this theory, endometrial tissues travel to their most common site of implantation, the peritoneal cavity, through the fallopian tubes during menstrual shedding, adhere to the peritoneal cavity wall, invade the extracellular matrix (ECM), proliferate, and form endometriotic lesions (5). Epidemiological data suggest that endometriosis does have malignant potential (6), and endometriosis is currently classified as a tumor-like lesion under the World Health Organization histological classification of ovarian tumors (7). Although the mechanisms of endometriosis progression remain to be elucidated, a rapidly growing body of evidence shows that the immune system has a pivotal role in endometriosis development (8). Recent studies have revealed that the number of activated macrophages increases in the peritoneal fluid (PF) of patients with endometriosis. Peritoneal macrophages synthesize and secrete different cytokines such as interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor (TNF)-a, and transforming growth factor (TGF)-b that may facilitate the adhesion, invasion, or proliferation of endometrial cells and the progression of endometriosis (9). Among others, IL-6 and IL-8 have been studied extensively and implicated in the pathogenesis of endometriosis (10–13). PF levels of IL-6 and IL-8 correlate well with the severity of endometriosis, and a high PF level of IL-6 in severe endometriosis is also accompanied by a decrease in IL-6 soluble receptor concentration (14, 15). Although propagation of ectopic endometrial cells leading to the development of endometriosis has been attributed by many researchers to the permissive inflammatory microenvironment of the peritoneal cavity of patients with endometriosis, little is known about the inherent differential capacity of such cells in patients with endometriosis to adhere to and invade the ECM of the peritoneal cavity. Indeed, despite the well-established role of peritoneal macrophages as key cellular constituents of PF that produce proinflammatory cytokines, the role of endometrial cells in producing such cytokines and thereby smoothing the way for their own implantation and propagation is still elusive. In the present study, we hypothesized that the endometrial stromal cells of patients with endometriosis have a differential capacity to produce proinflammatory cytokines and to adhere to and invade the ECM compared with those of nonendometriosis subjects. To this end, we investigated and compared the immunophenotype; production of IL-6, IL-8, TNF-a, IL-17A, TGF-b, and IL-10; proliferation; adhesion to ECM; and invasion to matrigel of ectopic (EESCs) and eutopic endometrial stromal cells (EuESCs) of patients with ovarian endometriosis and of endometrial stromal cells of controls (CESCs).

MATERIALS AND METHODS Patient Recruitment and Specimen Collection The studied groups comprised 25 women with ovarian endometriosis (endometrioma) undergoing laparoscopy and 20 control women with benign gynecological conditions and without any evidence of endometriosis, despite careful evaluation by a laparoscopic surgeon. All patients were 2

reproductive age (19–45 years old), at the proliferative phase of the menstrual cycle, and had regular menstrual cycles. Subjects had not received hormones or GnRH agonist therapy for at least 3 months before surgery and had no history of malignancy or autoimmune diseases. Patients with endometrioma were in stage III–IV according to the revised American Fertility Society system (16). The study was approved by the Institutional Review Board and the Ethics Committee for Medical Research of the Avicenna Research Institute, and all participants signed written informed consent before enrolling in the study. Ectopic endometrial samples were obtained through laparoscopic sampling, and eutopics were obtained through biopsy curette. Because a portion of patients with endometriosis were virgins, only endometrioma samples were obtained in these cases. Some cases were excluded from the study owing to culture contamination or inconsistent pathology report, but in each experiment the number of cases included was not below 10. The samples were immediately transferred on ice to the laboratory in tissue culture medium containing antibiotics, frozen in freezing medium containing Dulbecco's modified Eagle's medium (DMEM) -F12 (Sigma), 10% fetal bovine serum (FBS; Gibco), and 20% dimethyl sulfoxide (Sigma) at
80 C and stored in liquid nitrogen. A fraction of all samples was sent to the pathology laboratory to confirm endometrioma.

Endometrial Cell Culture and Purification of ESCs We isolated ESCs according to the preestablished protocol with high yield and purity (17–20). Under sterile conditions, ectopic and eutopic endometrial tissues of patients with endometriomas and normal endometrial tissues from control subjects were thawed and minced into small pieces of about 1 mm3. Digestion of all tissues was performed for 1.5–2 hours in an atmosphere of 5% CO2 at 37 C in the presence of 2 mg/mL collagenase A and 300 mg/mL DNAse (Roche) in DMEM-F12 containing 100 U/mL penicillin and 100 mg/mL Streptomycin (Sigma) with intermittent vortexing every 15 minutes. For removal of undigested tissues, cell suspension was filtered through 100 mm mesh (Becton Dickinson Biosciences). The endometrial glands and epithelial cells were separated at the next step by passing the cell suspension through a 40-mm sieve (Becton Dickinson Biosciences). The obtained single-cell suspension was then washed twice with culture medium. For complete purification, cell suspension was seeded in T25 culture flasks for 6 hours. Then nonadherent cells were removed by two washes with warm medium, and adherent stromal cells were allowed to propagate.

Immunophenotyping of ESCs Immunophenotyping of the cultured stromal cells was performed by immunofluorescent staining (17, 18) and flow cytometry. For immunofluorescent staining, 5  103 cells at 100 mL DMEM-F12 were seeded on a multiwell microscope slide and incubated for 18 hours at 37 C and 5% CO2 incubator; then adhesive cells were rinsed 2 times with warm Tris-buffered saline (TBS) and air dried. Cells were fixed in cold acetone for 2 minutes and air dried. After VOL. - NO. - / - 2013

Fertility and Sterility® additional TBS rinses, primary antibodies (mouse antivimentin, clone RV202, or rabbit anti-cytokeratin, both from Abcam) were added for 1.5 hours followed by fluorophore-labeled secondary antibodies (goat anti-mouse FITC for vimentin, Sigma or goat anti-rabbit FITC for cytokeratin; Rey-Biotec). Cells were then washed 3 times and 7-amino-actinomycin D (Becton Dickinson Biosciences) was applied for nucleus staining. Cell staining was visualized on a light microscope equipped with a fluorescent light source. For nestin staining, Alexa flour 568-labeled mouse anti-nestin (Avicenna Research Institute) was used followed by 4'-6-diamidino-2-phenylindole (Becton Dickinson Biosciences) for nucleus staining. Flow cytometry analysis was carried out according to the method we published recently (21, 22). To this end, cells were harvested, washed in cold phosphate-buffered saline (PBS) þ 1% FBS (stain buffer), and incubated for 30 minutes at 4 C in stain buffer containing the R-phycoerythrin (PE)conjugated mouse anti-human CD10, CD29, CD44, CD73, CD105, and CD133 or FITC-labeled mouse anti-human CD9, CD34, CD38, and CD45 antibodies (Becton Dickinson Biosciences). In all experiments, the corresponding isotypematched antibodies (Becton Dickinson Biosciences) were used as negative controls. Cells were washed twice in stain buffer. Signals were then read by Partech flow cytometer.

Cell Proliferation Assay The XTT proliferation assay was employed for evaluation of the proliferation property of ESCs. Briefly, cells were seeded in triplicate in 96-well tissue culture plates at a predetermined density of 7,500 cells/well in a final volume of 100 mL in phenol red-free DMEM-F12 medium (Sigma) with 5% FBS. The cultures were incubated in an atmosphere of 5% CO2 at 37 C for 48 hours, and then 50 mL of activated XTT (1 mg/mL XTT þ 5 mM phenazine methosulfate (PMS), 200:1 ratio; Sigma) were added to each well and incubation was continued for 2 hours. The optical density of the wells was read at 450 nm with 630 nm as the reference wavelength.

Cell Invasion Assay The invasive properties of EESCs, EuESCs, and CESCs were tested by cell invasion assay using matrigel invasion chambers (Becton Dickinson Biosciences). The invasive ovarian cancer cell line SKOV3 (National Cell Bank of Iran) served as a positive control. Briefly, 5  104 cells/ chamber were added to the matrigel invasion inserts in FBS-free DMEM-F12 medium. The lower chambers contained medium with 5% FBS as a chemoattractant. Chambers were incubated in an atmosphere of 5% CO2 for 36 hours at 37 C. Cells at the upper surface of the inserts were carefully removed by wiping off the matrigel 3 times using cotton swabs. The cells that had invaded to the lower surface of the membranes through 8 mm pores were fixed with absolute methanol and stained with 1% (w/v) toluidine blue in 1% (w/v) borax buffer for 5 minutes. The number of invading cells was enumerated in 50 random fields of light microscope (20) and averaged. VOL. - NO. - / - 2013

Cell Attachment Assay The attachment capacity of EESCs, EuESCs, and CESCs was examined by cell attachment assay using fibronectin-coated 96-well attachment plates (Becton Dickinson Biosciences). Briefly, the predetermined optimal cell number of 5  104 ESCs were seeded in each well of attachment plates in a final volume of 100 mL DMEM-F12 containing 5% FBS. After incubation for 1, 1.5, or 2 hours in an atmosphere of 5% CO2 at 37 C, the wells were washed gently with 150 mL warm PBS 5 times, and adhered cells were fixed with 100 mL 96 C ethanol for 10 minutes. Cells were then stained with 100 mL of 0.1% (w/v) crystal violet for 30 minutes. Wells were rinsed with distilled water 4 times to remove excess dye. For extraction of crystal violet, the cells were lysed with 100 mL of 10% (v/v) acetic acid on orbital shaker for 5 minutes. The extent of adhesion was evaluated by measuring the optical density at 570 nm (23).

Cytokine Assay The concentration of IL-4, IL-6, IL-8, TNF-a, TGF-b1, IL-10, IFN-g (Becton Dickinson Biosciences), and IL-17A (eBioscience) in the supernatant of cultured ESCs was measured by ELISA. In brief, EESCs, EuESCs, and CESCs were seeded onto 6-well plates at 8  105 cells/well in 2 mL DMEM-F12 medium containing 5% FBS. The cultures were incubated in an atmosphere of 5% CO2 at 37 C for 24 hours. Then cells were treated with 0.1 ng/mL IL-1b (Sigma) for 24 hours. Cell culture supernatants were harvested and centrifuged at 10,000 g. Then cell-free supernatants were analyzed for cytokine levels by sandwich ELISA according to the protocol provided by the manufacturer. The minimum detection levels for IL-4, IL-6, IL-8, TNF-a, IL-17A, TGF-b1, IL-10, and IFN-g were 7.8, 4.7, 3.1, 7.8, 7.8, 125, 7.8, and 4.7 pg/mL, respectively.

Data Analysis Data were presented as median with range. To compare invasion, adhesion, proliferation, and levels of cytokine production, the Mann-Whitney U-test was used. P< .05 was considered statistically significant.

RESULTS Immunophenotyping of ESCs ESCs exhibited flat, spindle-shaped fibroblastic-like morphology. The purity and immunophenotype of cultured ESCs at third passage were analyzed by immunofluorescent and flow cytometry assessment of markers specific to cells of mesenchymal or hematopoietic origin. The results clearly showed that isolated ESCs from all three sources express vimentin (a stromal cell cytoskeletal marker) and nestin but fail to express the epithelial marker cytokeratin (Fig. 1A). Flow cytometric analysis of expanded cells showed that ESCs typically express the surface antigens associated with mesenchymal origin such as CD9, CD29, CD44, CD73, and CD105 but were negative for any of the other markers examined including CD34, CD38, CD133, and CD45 (Fig. 1B). These results clearly demonstrate that isolated 3

ORIGINAL ARTICLE: ENDOMETRIOSIS

FIGURE 1

Immunophenotyping of ESCs, which were isolated from ectopic and eutopic endometrial samples of patients with endometriosis and nonendometriotic controls and characterized by immunofluorescent staining and flow cytometry analysis. (A) Representative immunofluorescent staining of ESCs showing the expression of vimentin (a) and nestin (b) and negative immunoreactivity for cytokeratin (c). Delbandi. Eutopic and ectopic endometriotic stromal cells. Fertil Steril 2013.

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FIGURE 1 Continued ESCs from all three sources exhibited the same pattern. Panels d, e, and f show negative reagent controls for vimentin, nestin, and cytokeratin, respectively. (B) Representative flow cytometric analysis of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, and CD133 in ectopic ESCs (a), eutopic ESCs (b), and control ESCs (c). Data are presented as mean  SD. Delbandi. Eutopic and ectopic endometriotic stromal cells. Fertil Steril 2013.

ESCs were pure with no epithelial, endothelial, or bone marrow–derived cell contamination.

with EESCs (median ¼ 0.795; P< .01). EuESCs, in turn, showed higher invasiveness in comparison with CESCs in terms of matrigel invasion (P< .05; Fig. 2C).

Proliferation of ESCs To test the differential proliferative capacity of the stromal cells from different origins, the XTT proliferation test was performed in untreated and fibronectin-coated plates (Fig. 2). In the absence of fibronectin, EuESCs exhibited a significantly lower proliferation rate compared with CESCs (P< .001) or EESCs (P< .01). More importantly, the proliferative capacity of EESCs was comparably lower than that of CESCs (P< .001; Fig. 2A). In a fibronectin-coated plate, a similar trend was achieved except that EuESCs exhibited higher proliferation compared with EESCs (P< .05; Fig. 2B).

Invasion of ESCs to Matrigel Invasiveness was measured as the ability of cells to digest matrigel and migrate through the membrane (Figs. 2 and 3). EuESCs (median ¼ 16.43) and CESCs (median ¼ 7.895) exhibited significantly higher invasive properties compared

Attachment of ESCs to ECM To evaluate the adhesive properties of stromal cells derived from ectopic or eutopic endometrium, their attachment to the fibronectin-coated wells at different time points was assessed and compared (Fig. 2). EESCs showed higher adhesive properties to ECM compared with CESCs in the first hour (P< .01). This superiority was further highlighted in the 1.5-hour postseeding (P< .001). After 1.5 hours of postseeding, EuESCs showed lower capacity of cell attachment compared with EESCs (P< .05). Although EuESCs showed higher adhesion compared with CESCs at all time intervals examined, this did not reach statistical significance (Fig. 2D).

Cytokine Production We hypothesized that besides immune cells, ESCs may per se serve a role in shaping the local immune responses through

FIGURE 2

Comparative proliferation, invasion, and adhesion analysis of EESCs, EuESCs, and CESCs. Proliferation of isolated stromal cells was assessed by XTT assay in untreated (A) and fibronectin-coated (B) plates. Invasive capacity of stromal cells was evaluated by matrigel invasion assay at 200 magnification (C). Stromal cell attachment was measured at different time intervals (D). Each bar represents the median with a range of 10–24 different samples. EESCs ¼ stromal cells from ectopic site (endometrioma); EuESCs ¼ stromal cells from the eutopic endometrium of patients with endometriosis; CESCs ¼ stromal cells from nonendometriotic controls; OD ¼ optical density. *P<.05, **P<.01, ***P<.001. Delbandi. Eutopic and ectopic endometriotic stromal cells. Fertil Steril 2013.

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FIGURE 3

EuSCs and EESCs show different invasive capacity. EESCs (B), EuESCs (C), and CESCs (D) were isolated, and their matrigel invasive capacity was assessed. Ovarian cancer cell line (SKOV3) (A) was used in parallel as a positive control. EESCs ¼ stromal cells from ectopic site (endometrioma); EuESCs ¼ stromal cells from the eutopic endometrium of patients with endometriosis; CESCs ¼ stromal cells from nonendometriotic controls. Scale bar ¼ 50 mm. Delbandi. Eutopic and ectopic endometriotic stromal cells. Fertil Steril 2013.

the production of a different array of cytokines. To this end, EuESCs, EESCs, and CESCs were stimulated with IL-1b, and secreted cytokines were measured in culture supernatants. The results showed that, overall, the majority of stromal cells from aforesaid origins failed to produce detectable levels of IL-4, TNF-a, IL-17A, TGF-b1, IL-10, and IFN-g. Owing to scattered production of these cytokines among groups of study, no statistical analysis was performed. However, very high amounts (on a nanogram scale) of IL-6 and IL-8 were produced by stromal cells, whatever the origin was (Fig. 4). The levels of IL-6 production by EESCs were significantly

higher compared with production by EuESCs (P< .001) or CESCs (P< .01), while no significant differences were observed between EuESCs and CESCs (Fig. 4A). Likewise, EESCs produced comparably higher amounts of IL-8 than EuESCs and CESCs (P< .01; Fig. 4B).

DISCUSSION In this paper we analyzed some properties of EuESCs from patients with endometriosis in reference to their ectopic counterpart and ESCs from nonendometriotic patients. We

FIGURE 4

Cytokine production by EESCs, EuESCs, and CESCs. Stromal cells from 18 patients with endometriosis and 14 nonendometriotic controls were cultured in DMEM-F12 medium, and the levels of interleukin (IL)-8 and IL-6 were measured by capture ELISA. IL-6 (A) and IL-8 (B). EESCs ¼ stromal cells from ectopic site (endometrioma); EuESCs ¼ stromal cells from the eutopic endometrium of patients with endometriosis; CESCs ¼ stromal cells from nonendometriotic controls. **P<.01, ***P<.001. Delbandi. Eutopic and ectopic endometriotic stromal cells. Fertil Steril 2013.

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Fertility and Sterility® first analyzed the immunophenotype of EESCs, EuESCs, and CESCs and found that they are largely similar. All cell types were found to be positively stained for markers specific to the cells of mesenchymal origin, whereas they failed to express hematopoietic markers. This similarity may be viewed as a common origin of EuESCs and EESCs. Similar phenotype has also been reported recently in stromal cells isolated from human endometrium (24) and also in the colony-forming units of EUSCs and EESCs (25). Interestingly, we also recently showed that stem cells derived from menstrual blood exhibit the same phenotypic markers with multilineage differentiation potency (21, 26, 27). Whether these endometrial stem cells are responsible for the development of endometriosis lesions has been the focus of recent research (4). Our findings on the similar phenotype of EESCs and EuESCs are also consistent with embryonic rest theory, which proposes that endometriosis stems from ectopic endometrium that is displaced in the peritoneal cavity during embryogenesis (28). Next we hypothesized that in addition to the role that the local immune system may play in the course of endometriosis, the inherent differential behavior of ESCs in patients with endometriosis is the key element in the pathogenesis of this disease. In line with this assumption, our results clearly showed that EuESCs of patients with endometriosis differ from their normal counterparts in terms of invasiveness and proliferative capacity. In fact, EuESCs exhibited considerably higher capacity to invade the ECM and had a lower proliferation rate compared with controls. But there were no significant differences in the adhesive capacity of EuESCs and controls. Studies on the differential proliferative capacity of EuESCs have produced conflicting results. Depending on the culture condition, assay used for assessment of proliferative capacity, menstrual phase in which the endometrial biopsy was obtained, severity of endometriosis, culture period, and number of subjects enrolled in the study, EuESCs exhibit great variability in their proliferation behavior (29–34). The results of the present study show that in the presence of ECM (fibronectin), EuESCs and CESCs have comparable proliferative behavior, but in its absence, EuESCs proliferated much less. Similar results were also obtained by Burlev et al. (33) and Jones et al. (31), who employed the in situ proliferation assessment technique (Ki67 immunohistochemistry) where the cells are in close proximity with ECM. These results imply that ESCs from women with endometriosis are more dependent on ECM for proliferation than their normal counterpart. Nevertheless, there are some reports showing that EuESCs from women with endometriosis display an increased proliferative capacity compared with ESCs from normal controls in the presence or absence of ECM (35, 36). Although the reason behind this discrepancy is not clear to us, such factors as the stage of endometriosis may be determining. Interestingly, the proliferation rate of ESCs was also influenced by the type of ECM and more importantly by whether cells were seeded on immobilized or soluble ECM (36). EESCs showed significantly lower proliferation compared with CESCs regardless of their culture condition. These findings are in line with previous reports on the considerably reduced VOL. - NO. - / - 2013

proliferation capacity of ovarian endometrioma stromal cells (29, 34). The finding of our study on the higher invasion capacity of EuESCs compared with CESCs is similar to previous reports (37–39). Collette et al. showed increased expression of matrix metalloproteinase-9 (MMP-9) in the EuESCs of women with endometriosis, while there was no significant difference between women with and without endometriosis in the expression of tissue inhibitor of MMPs (TIMP)-1, a known natural inhibitor of the pro- and active forms of MMP-9 (40). In an elegant work by Chung et al. (37), it was found that endometrium from women with endometriosis expressed higher levels of MMP-2 and membranous type 1 (MT1)-MMP and lower levels of TIMP-2 than did endometrium from normal women. Interestingly, it was found that the mRNA and protein levels of CD82 in normal ESCs from endometrium without endometriosis are significantly higher than those of EuESCs or EESCs. CD82 inhibits the invasiveness of ESCs by downregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase and integrin b1 signal pathway and in turn by upregulating the expression of TIMP1 and TIMP2 in an autocrine manner (39). These results imply that EuESCs may be more invasive and prone to peritoneal implantation compared with endometrium from women without endometriosis. Increased proteolytic activity may help to explain the invasive factors that result in endometriosis. But it should be kept in mind that expression of invasion markers in and thereby the invasive properties of the endometrial tissues of women with endometriosis may vary considerably in different forms of endometriosis (41). In contrast to EuESCs, EESCs exhibited significantly lower invasiveness compared with both CESCs and EuESCs. This finding is supported by a report in which considerably lower levels of MT1-MMP and MMP-2 were observed in the endometrioma of patients with advanced endometriosis (37). Based on the fact that most patients enrolled to this study had stage III–IV endometriosis, this finding might be expected. It seems that after the establishment of ovarian endometriosis, ESCs become sluggish in terms of proliferation and invasion. This assumption may explain why, in spite of the huge proliferation and regeneration capacity of ESCs during menstrual cycles, ovarian endometrioma develop over a very long period. In an attempt to investigate the role of ESC adhesiveness in the pathogenesis of endometriosis, the adhesion properties of ESCs from normal patients and patients with endometriosis were compared. Our results showed that EuESCs and in particular EESCs had a higher capacity to adhere to ECM compared with CESCs. The low invasion capacity of EESCs may be relevant to this observation as higher interaction with ECM may lead to lower invasiveness. In line with our findings, recent studies showed that EuESCs and EECSs from women with endometriosis exhibit an aberrant integrin profile in vitro and display increased adhesion to specific ECM components compared with stromal cells derived from healthy controls (8, 36). In an excellent work by Griffith et al., EESCs from women with endometriosis showed higher adherence to peritoneal mesothelial cells and were more likely to express certain splice variants of CD44 (42). Increased expression of 7

ORIGINAL ARTICLE: ENDOMETRIOSIS laminin g-1 (LAMC1), which promotes adhesion and migration of monocytes, has also been shown recently in the endometrium of patients with endometriosis compared with in healthy endometrium (43). This may explain the role of LAMC1 in endometriosis as it may promote the adhesion of the endometrium to the peritoneum. Having considered the prominent role of cytokines in the course of endometriosis, at the next step we examined the cytokine profile of ESCs in patients with endometriosis and normal controls. Very high amounts of IL-6 and IL-8 were produced by the stromal cells of all groups. EESCs produced significantly higher amounts of IL-6 compared with EuESCs or CESCs. Likewise, EESCs produced comparably higher amounts of IL-8 than EuESCs and CESCs. It has been shown that cell adhesion to ECM leads to gene expression and production of IL-8, which is mediated by integrins (44, 45). IL-8 stimulates the adhesion of ESCs to fibronectin in a concentration-dependent manner (46, 47). In our study EESCs showed higher adhesive properties to ECM proteins than EuESCs or CESCs, while no significant differences were observed between EuESC and CESC attachment. The trend of IL-8 production by ESCs in our study is in concordance with their adhesive capacity and supports the assumption that this cytokine may be relevant for the attachment of endometrial implants in the pathogenesis of endometriosis. Indeed, IL-8 has potent activity on neutrophil recruitment and induction of angiogenesis. Interestingly, high levels of IL-8 in the PF of patients with endometriosis are accompanied by accumulation of neutrophils, inflammation, and neovascularization (11–13, 48). IL-6 is a multifunctional proinflammatory cytokine (49) with profound effects on a wide variety of cells (50). One of the main sources of IL-6 secretion is endometrial cells, and this cytokine has been implicated in uterine function (51). Reportedly, the level of this cytokine is increased in the PF of patients with endometriosis compared with in the control group (10, 12). Although such cytokines as TNF-a, IL-17, IFN-g, and IL-10 have been proposed to play a major role in the pathogenesis of endometriosis (10, 52–55), in a minority of cases these cytokines were produced by ESCs, implying the involvement of other cell types in their production. In conclusion, the data presented here imply that regardless of similar immunophenotype, EESCs from patients with endometriosis differ from their eutopic counterparts in terms of proliferation, invasion, adhesion, and proinflammatory cytokine production, suggesting the involvement of epigenome in their behavior. Acknowledgments: The authors thank all the patients who participated in this study and colleagues at the Avicenna Research Institute, particularly Mrs. Jamileh Ghasemi and Mr. Mirzadegan, and the staff of Mashhad Immunology Research Center and Tehran Clinic Hospital.

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