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Chemokine expression profiles of ovarian endometriotic stromal cells in three-dimensional culture
Journal of Reproductive Immunology 138 (2020) 103100
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Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jri
Chemokine expression profiles of ovarian endometriotic stromal cells in three-dimensional culture
T
Ruofei Zhua, Kaei Nasua,b,*, Yoko Aoyagia, Tomoko Hirakawaa, Kanetoshi Takebayashia, Hisashi Naraharaa a b
Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Oita, Japan Division of Obstetrics and Gynecology, Support System for Community Medicine, Faculty of Medicine, Oita University, Oita, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Endometriosis Chemokine Gene expression microarray 3-Dimenional culture
Endometriosis is a chronic inflammatory disease which is associated with aberrant chemokine expression. We have established a three-dimensional (3D) floating collagen gel culture of human endometriotic cyst stromal cells (ECSCs) as an in vitro model of early-stage fibrosis formation in endometriosis. We evaluated the gene expression profiles of 3D-cultured ECSCs using a gene expression microarray. We identified and confirmed with reverse transcription-polymerase chain reaction that mRNA levels of CXCL1, CXCL2, CXCL3, CXCL8, and CCL20 in 3D-cultured ECSCs were significantly higher than in 2D-cultured ECSCs. The protein levels of CXCL1, CXCL2, CXCL8, and CCL20 in the supernatant of 3D-cultured ECSCs were significantly higher than in 2D-cultured ECSCs. It has been suggested that the 3D-culture model of ECSCs is more suitable for in vitro endometriosis research than 2D-culture. This microarray data provides a new platform to identify the candidate genes involved in the pathogenesis of endometriosis which could be masked in conventional 2D-culture.
1. Introduction Endometriosis is a major contributor to pelvic pain and occurs in 6–10 % of women of reproductive age. (Giudice, 2010). Endometriosis is a chronic inflammatory condition accompanied by adhesions, fibrosis, neuronal infiltration, angiogenesis, scarring, and anatomical distortion (Bulun, 2009; Giudice, 2010). Activated lymphocytes, neutrophils, and macrophages are hallmarks of the inflammation in endometriotic tissues (Lebovic et al., 2001). Chemokines are small polypeptides characterized by their proinflammatory functions, and demonstrate chemotactic activity targeted to specific leukocyte populations (Zlotnik et al., 2006; Vandercappellen et al., 2008). More than 50 human chemokines have been identified and divided into four subfamilies based on the relative position of their cysteine residues (CC, CXC, C, and CXC3) (Nishida et al., 2011). Chemokines are produced in endometriotic tissues and have been implicated in the pathological process of endometriosis (Bulun, 2009; Nishida et al., 2011).
We have established a three-dimensional (3D) floating collagen gel culture of human endometriotic cyst stromal cells (ECSCs) as an in vitro model of early-stage fibrosis formation in endometriosis (Yuge et al., 2007; Nasu et al., 2009; Tsuno et al., 2009; Nasu et al., 2010a, b; Tsuno et al., 2011). In this experimental model, 3D-cultured ECSCs induce the contraction of floating collagen gels, which is comparable to the earlystage fibrosis formation in vivo. We have identified the involvement of mevalonate- Ras homology (Rho)/ Rho-associated coiled-coil-forming protein kinase (ROCK) pathway in the development of early-stage endometriosis-associated fibrosis. However, global gene expression profiles in 3D-cultured ECSCs have not yet been elucidated. In this study, we evaluated the gene expression profiles of 3D-cultured ECSCs using a gene expression microarray. Then, as the first-line analysis, we identified the aberrantly expressed chemokines and examined the roles of these molecules in the development of this disease.
Abbreviations: 2D, 2 dimensional; 3D, 3 dimensional; C, cysteine; CC, cysteine–cysteine; CCL, cysteine–cysteine ligand; CXC, cysteine–X–cysteine; CXCL, cysteine–X–cysteine ligand; CXC3, cysteine–X3–cysteine; DMEM, Dulbecco’s modified Eagle’s medium; ECSCs, endometriotic cyst stromal cells; ELISA, enzymelinked immunosorbent assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NESCs, normal endometrial stromal cells; Rho, Ras homology; ROCK, Rho-associated coiled-coil-forming protein kinase; RT-PCR, reverse transcription-polymerase chain reaction ⁎ Corresponding author at: Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Idaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan. E-mail address: [email protected] (K. Nasu). https://doi.org/10.1016/j.jri.2020.103100 Received 3 September 2019; Received in revised form 30 January 2020; Accepted 5 February 2020 0165-0378/ © 2020 Elsevier B.V. All rights reserved.
Journal of Reproductive Immunology 138 (2020) 103100
R. Zhu, et al.
2. Materials and methods
Conventional 2D-culture of ECSCs and NESCs were also performed in 35-mm culture plates in the presence of 10 % charcoal-stripped heatinactivated FBS. After 48 h incubation, the supernatants were collected for chemokine ELISA. Then, total RNA was extracted from the cells.
2.1. Human ECSC and normal endometrial stromal cell (NESC) isolation procedures and cell culture conditions
2.3. Gene expression microarray analysis
Ovarian endometriotic tissues were obtained from patients with regular menstrual cycles undergoing surgical treatment of ovarian endometriotic cysts (n = 8, age 31–48 yrs), as described previously (Nishida et al., 2004; Nasu et al., 2005; Hirakawa et al., 2016; Aoyagi et al., 2017). Eutopic endometrial tissues were obtained from premenopausal patients who had undergone hysterectomies for subserosal or intramural leiomyoma and had no evidence of endometriosis (n = 10, age 41–52 yrs), as described (Nishida et al., 2004). No patients had received hormonal treatments for at least two years prior to the surgery. All of the specimens were confirmed as being in the mid-to-late proliferative phases according to pathological observation and/or menstrual cycles. This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine, Oita University, and written informed consent was obtained from all patients. ECSCs and NESCs were isolated from ovarian endometrioma and eutopic endometrial tissues, respectively, by enzymatic digestion, as described previously (Nishida et al., 2004; Nasu et al., 2005; Hirakawa et al., 2016; Aoyagi et al., 2017). Briefly, the ovarian endometriotic and eutopic endometrial tissues were minced in Hanks’ balanced salt solution and digested with 0.5 % collagenase B (Thermo Fisher Scientific, Tokyo, Japan) in Dulbecco’s modified Eagle’s medium (DMEM, Nissui, Tokyo, Japan) at 37 C for 40 min. The dispersed cells were filtered through a 70-μm pore size nylon mesh to remove the undigested tissue pieces. The filtrated fraction was separated from epithelial cell clumps by differential sedimentation at unit gravity as follows. The cells were resuspended in 2 ml culture medium and layered slowly over 10 ml of the medium in a centrifuge tube. Sealed tubes were placed in an upright position at 37 °C in 5 % CO2 in air for 30 min. After sedimentation, the top 8 ml medium were collected. Finally, the medium containing stromal cells was filtered through a 40-μm pore size nylon mesh. Final purification was achieved by allowing stromal cells (which attach rapidly to plates) to adhere selectively to culture dishes for 30 min at 37 °C, followed by the removal of nonadhering epithelial cells. Isolated ECSCs and NESCs were cultured in DMEM supplemented with 100 IU/ ml penicillin (Gibco-BRL, Gaithersburg, MD, USA), 50 mg/ml streptomycin (Gibco-BRL), and 10 % charcoal-stripped heat-inactivated fetal bovine serum (FBS; Gibco-BRL) at 37 °C in 5 % CO2 in air. ECSC and NESC in monolayer culture after the third passage were more than 99 % pure, as analyzed by immunocytochemical staining with antibodies to vimentin (V9; Dako, Tokyo, Japan), CD10 (Abcam, Cambridge, UK), cytokeratin (Dako), factor VIII (Dako), and leukocyte common antigen (2B11 + PD7/26, Dako) (Nishida et al., 2004; Nasu et al., 2005) and were used for the following experiments. Each experiment was performed in triplicate and repeated at least three times with cells isolated from different patients.
Total RNA from 3D-cultured and 2D-cultured ECSCs were extracted with a miRNeasy Mini kit (Qiagen, Valencia, CA, USA) and subjected to gene expression microarray analyses with a human mRNA microarray (SurePrint G3 Human GE Microarray Kit 8 × 60 K Ver2.0, Agilent Technologies, Santa Clara, CA, USA), as previously described (Okamoto et al., 2015; Aoyagi et al., 2017). To identify up- and down-regulated genes, we calculated Z-scores and ratios from the normalized signal intensities of each probe for comparison between 3D-cultured and 2Dcultured ECSC samples (Okamoto et al., 2015; Aoyagi et al., 2017). We established the criteria for determining regulated genes: Z-score ≥ 2.0 and ratio ≥ 2.0-fold for up-regulated genes, and Z-score ≤ –2.0 and ratio ≤ 0.5 for down-regulated genes. All gene expression microarray data are available at Gene Expression Omnibus via the National Center for Biotechnology Information under Accession No. GSE136412 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136412). 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) for mRNA expression The expressions levels of chemokines in 3D-cultured and 2D-cultured ECSCs and NESCs were evaluated by quantitative RT-PCR, as described previously (Okamoto et al., 2015; Hirakawa et al., 2016; Aoyagi et al., 2017). CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, and CCL20 were identified by the gene expression microarray analysis. Briefly, 48 h after 3D-culture and 2D-culture, total RNA from ECSCs and NESCs was extracted as described above and subjected to quantitative RT-PCR with the following specific primers (all from Applied Biosystems): CXCL1 (Assay ID: Hs00236937_m1), CXCL2 (Assay ID: Hs00601975_m1), CXCL3 (Assay ID: Hs00171061_m1), CXCL6 (Assay ID: Hs00605742_g1), CXCL8 (Assay ID: Hs00174103_m1), CCL20 (Assay ID: Hs00355476_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID: Hs02758991_g1). The expression levels of candidate mRNAs relative to those of GAPDH mRNA were calculated using a standard curve. The data were calculated from triplicate samples and are presented as the percentage of the values of 2D-cultured NESCs. 2.5. Chemokine levels in the culture media To evaluate the production of candidate chemokines in 3D-cultured and 2D-cultured ECSCs and NESCs, we measured chemokine levels in the culture media by ELISAs. Briefly, 4 h before cessation of 2D- and 3D-culture, the supernatant was replaced with fresh culture medium containing 10 % charcoal-stripped heat-inactivated FBS. Then, the supernatant was collected and stored at −70 °C until the assay was conducted. The concentrations of CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, and CCL20 were determined in each supernatant with commercially available ELISA kits (CXCL1, CXCL2, CXCL6, CXCL8, and CCL20 from R&D Systems, Minneapolis, MN, USA and CXCL3 from Aviva Systems Biology, San Diego, CA, USA). The sensitivities of the assay were 1.5 pg/mL for CXCL1, 0.15 pg/mL for CXCL2, 0.6 pg/mL for CXCL3, 0.17 pg/mL for CXCL6, 0.5 pg/mL for CXCL8, and 0.12 pg/mL for CCL20.
2.2. 3D-culture in floating collagen gels and conventional 2D-culture 3D-culture of ECSCs and NESCs in floating collagen gel were performed as described previously (Yuge et al., 2007; Tsuno et al., 2011; Hirakawa et al., 2016). ECSCs and NESCs were embedded in collagen gel (Cellmatrix type I-A; Nitta Gelatin, Osaka, Japan) in 35-mm culture plates according to the manufacturer’s instructions and cultured in a three-dimensional matrix. After incubation for 48 h in the presence of 10 % charcoal-stripped heat-inactivated FBS, the supernatants were collected for chemokine enzyme-linked immunosorbent assays (ELISAs). Then, ECSCs and NESCs were isolated from contracted collagen gels by enzymatic digestion with collagenase type I (Sigma-Aldrich, St Louis, MO, USA), as previously described (Yuge et al., 2007; Tsuno et al., 2009). Then, total RNA was extracted from the isolated cells.
2.6. Statistical analysis All data were obtained from triplicate samples and are presented as percent values relative to the corresponding controls in the form of mean ± SD. Data were analyzed using the Bonferroni test with the 2
Journal of Reproductive Immunology 138 (2020) 103100
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Table 1 Effects of 3-D culture on the chemokine mRNA expression of ECSCs. Gene symbol
CXCL1 CXCL2 CXCL3 CXCL6 CXCL8 CCL20
Other name
GROα GROβ GROγ GCP-2 IL-8 MIP-3α
Regulation
Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated
Z-score
5.8 10.1 8.3 3.1 5.5 2.8
Ratio
83.7 755.6 670.2 16.2 152.7 88.2
Signal intensity 2D
3D
174.0 ± 104.1 101.0 ± 46.2 35.3 ± 33.7 84.5 ± 55.5 540.9 ± 882.3 14.7 ± 18.8
11603.3 ± 10005.3 71001.6 ± 30439.2 14472.3 ± 14687.8 1510.4 ± 1948.0 5833.9 ± 3368.4 350.8 ± 390.8
Genes with Z-score ≥2.0 and ratio ≥2.0-fold were defined as upregulated, and those with Z-score ≤-2.0 and ratio ≤0.5 were defined as down-regulated.
compared to 2D-cultured ECSCs. However, mRNA expressions of CXCL1, CXCL2, CXCL3, and CXCL8 in the 3D-cultured ECSCs were significantly higher than those in the 3D-cultured NESCs. We additionally found that the protein levels of CXCL1, CXCL2, CXCL8, and CCL20 in the supernatant of 3D-cultured ECSCs were significantly higher than those in the 3D- and 2D-cultured NESCs. These results suggest that the expressions of some chemokines are attenuated in 2Dcultured ECSCs, which is conventionally used as the in vitro model. In 3D-culture, these chemokine expressions were restored. Endometriotic lesions are characterized by chronic inflammation (Bulun, 2009; Giudice, 2010). The inflammatory cells and soluble factors, such as proinflammatory cytokines and chemokines, in the diseased tissues, as well as the peritoneal fluid are thought to contribute to the development and progression of endometriosis (Lebovic et al., 2001). The increased production of chemokines may induce the recruitment of inflammatory cells from circulation into endometriotic tissues and the peritoneal fluid (Akoum et al., 2000). We have established a three-dimensional floating collagen gel culture of ECSCs as a model of early-stage fibrogenesis in endometriosis (Yuge et al., 2007; Nasu et al., 2009, 2010a; Nasu et al., 2010b; Tsuno et al., 2011). In this experimental model, ECSCs cultured in floating collagen gels induce the reorganization and compaction of collagen fibers, and the contraction of collagen lattices, which is comparable to the tissue contraction in early-stage fibrogenesis in endometriosis. Research on the cell biology of 3D-cultured ECSCs may provide new insights for the pathogenesis of endometriosis-associated fibrosis. Using this 3D-culture model, we demonstrated that 3D-cultured ECSCs possess greater ability to differentiate into α-smooth muscle actin-positive myofibroblasts and stronger contractility than 3D-cultured NESCs (Yuge et al., 2007; Nasu et al., 2009, 2010a; Nasu et al., 2010b; Tsuno et al., 2009, 2011). Activation of the mevalonate-Rho/ROCK-mediated pathway as well as induction of α-smooth muscle actin expression in ECSCs may be involved in this phenomenon (Yuge et al., 2007; Nasu et al., 2009; Tsuno et al., 2009; Nasu et al., 2010a, b; Tsuno et al., 2011). However, other putative mechanisms of fibrosis formation in the diseased tissues have yet to be established. In the present study, therefore, we planned to elucidate the potential causes of cellular functional changes during the endometriosis-associated fibrosis formation by using gene expression microarray analysis of 3D-cultured ECSCs. Previous studies have shown that several chemokines are produced by endometriotic stromal cells. These chemokines include CXCL1 (Nishida et al., 2004), CXCL5 (Nishida et al., 2004), CXCL8 (Akoum et al., 2001; Nishida et al., 2004), CXCL12 (Shi et al., 2014), CXCL16 (Manabe et al., 2011), CCL2 (Akoum et al., 1995; Boucher et al., 2000), CCL5 (Lebovic et al., 2001; Hornung et al., 2001b), CCL8 (Hornung et al., 1997, 2001a; Hornung et al., 2001b), CCL11 (Hornung et al., 2000), CCL17 (Bellelis et al., 2013), CCL20 (Hirata et al., 2010), and CX3CL1 (Hou et al., 2016). Several of these chemokine expressions have been shown to be higher in endometriotic tissues compared to the eutopic endometrial tissues of unaffected women, which may induce a series of inflammatory responses, such as the recruitment of
Statistical Package for Social Science software (IBM SPSS statistics 24; IBM, Armonk, NY, USA). p-values < 0.05 were considered significant. 3. Results 3.1. Detection of chemokine mRNAs differentially expressed in 3D- and 2Dcultured ECSCs by gene expression microarray The gene expression microarray revealed that six chemokine mRNAs were upregulated in 3D-cultured ECSCs compared to those in 2D-culture (Table 1). Interestingly, no chemokine mRNAs were downregulated in 3D-cultured ECSCs compared to 2D-culture. 3.2. Expression of chemokine mRNAs in 3D- and 2D-cultured ECSCs and NESCs To validate the results of our gene expression microarray data and to compare the chemokine expressions generated by ECSCs and NESCs, we evaluated mRNA expressions of six genes in 3D- and 2D-cultured ECSCs and NESCs using quantitative RT-PCR. As shown in Fig. 1, the relative mRNA levels of CXCL1, CXCL2, CXCL3, CXCL8, and CCL20 in the 3D-cultured ECSCs were significantly higher than those in the 2D-cultured ECSCs, as measured by RT-PCR. These results are consistent with the gene expression microarray data. The mRNA expressions of CXCL1, CXCL2, CXCL3, and CXCL8 in the 3D-cultured ECSCs were significantly higher than those in the 3D-cultured NESCs. In 2D-culture, however, only CXCL1 mRNA expression was higher in ECSCs than NESCs. The primer sets for CXCL6 mRNA did not work well (data not shown). 3.3. Chemokine levels in the supernatant of 3D- and 2D-cultured ECSCs and NESCs As shown in Fig. 2, the relative protein levels of CXCL1, CXCL2, CXCL8, and CCL20 in the supernatant of 3D-cultured ECSCs were significantly higher than those in the 2D-cultured ECSCs. In 2D-culture, only the CXCL1 protein level was higher in ECSCs compared to NESCs. Importantly, protein levels of CXCL3 and CXCL6 in the 3D- and 2Dcultured NESCs and ECSCs in this culture condition were below the detection levels (data not shown). 4. Discussion To investigate the global gene expression profiles during the earlystage of fibrosis formation in endometriosis, we performed the gene expression microarray analysis of 3D- and 2D-cultured ECSCs. As a firstline analysis, we identified six aberrantly expressed chemokines, namely CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, and CCL20. RT-PCR performed for these six chemokines revealed that mRNA levels of CXCL1, CXCL2, CXCL3, CXCL8, and CCL20 in 3D-cultured ECSCs were significantly higher than those in the 2D-cultured ECSCs. There was no chemokine mRNA that was downregulated in 3D-cultured ECSCs 3
Journal of Reproductive Immunology 138 (2020) 103100
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Fig. 1. Chemokine mRNA levels in the 2D-cultured and 3D-cultured NESCs and ECSCs. (A), CXCL1; (B), CXCL2; (C), CXCL3; (D), CXCL8; (E), CCL20. *p < 0.0005 vs. NESC-3D and ECSC-2D, **p < 0.05 vs. ECSC-2D, and ***p < 0.005 vs. NESC-2D (Bonferroni test). NESC-2D, 2D-culturred NESCs; NESC-3D, 3Dculturred NESCs; 2D-ECSC, 2D-culturred ECSCs; 3D-ECSC, 3D-culturred ECSCs. The data were obtained from triplicate samples and are presented as percent values relative to the NESC-2D in the form of mean ± SD. Representative results are shown.
inflammatory cells, angiogenesis, fibrogenesis, and nerve fiber infiltration into the diseased tissues for the progression of the disease. It is known that, compared to conventional 2D-culture, 3D-culture is more equivalent to in vivo tissue. Whereas, it is also known that endometriotis is a chronic infllammmatory disease. Therefore, it can be speculated that aberrant expressions of some molecules are masked in the conventional 2D-culture and that 3D-cultured ECSCs may restore their original characteristics in vivo including the enhanced inflammatory responses. Alternatively, the differentiation of ECSCs into myofibroblastic phenotype in 3D-culture may also involve in the enhanced chemokine expression in these cells. To our knowledge, there is only one report that compared the chemokine expression between 3Dand 2D-culture. Wells et al. (2013) compared the gene expression patterns of 3D- and 2D-cultured Madin-Darby canine kidney renal epithelial cells by using the gene expression microarray. They found the increased CXCL8 expression in 3D-cultured cells, which is consistent with our present findings. In this report, however, they did not evaluate other ckemokine expressions. Further study with the present microarray data may elucidate the turn-on mechanisms of chemokine
expression in 3D-cultured ECSCs. The present gene expression microarray revealed the upregulation of CXCL3 and CXCL6 mRNA expression. Whereas, CXCL3 and CXCL6 proteins were not detected in the supernatants of ECSCs and NESCs by ELISAs. Although the precise mechanisms of these phenomenon are unknown, some posttranscriptional inhibitory mechnisms may exist in the regulation of these chemokine expressions. The limitation of the present study is that the endometriotic cells were isolated from ovarian endometriomas. It has been suggested that ovarian endometrioma is a different entity than deep infiltrative and peritoneal endometriosis (Nisolle and Donnez, 1997). Therefore, it remains possible that endometriotic cells existing in other locations may show different responses to 3D-culture. The experiments in this study were performed only with the stromal cells, because the stromal cells are responsible for the tissue contraction. In follow-up studies, chemokine expression profiles should be evaluated in the epithelial components as well as in the infiltrated inflammatory cells in the endometriotic tissues. In summary, we utilized a gene expression microarray to identify 4
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Fig. 2. Chemokine levels in the supernatants of 2Dcultured and 3D-cultured NESCs and ECSCs. (A), CXCL1; (B), CXCL2; (C), CXCL8; (D), CCL20. *p < 0.0005 vs. NESC-3D and ECSC-2D, **p < 0.05 vs. NESC-3D and ECSC-2D, and ***p < 0.005 vs. NESC-2D (Bonferroni test). NESC-2D, 2D-culturred NESCs; NESC-3D, 3D-culturred NESCs; 2D-ECSC, 2Dculturred ECSCs; 3D-ECSC, 3D-culturred ECSCs. The data were obtained from triplicate samples and are presented as percent values relative to the NESC-2D in the form of mean ± SD. Representative results are shown.
A part of this manuscript was presented at the 33rd Annual Meeting of Japan Society for Immunology and Reproduction.
the aberrantly expressed genes that were not detected in conventional 2D-culture model. We confirmed that the expression of several chemokines in ECSCs were enhanced by 3D-culture, which may be similar to in vivo levels. With respect to inflammatory diseases, understanding the chemokine network is vital to elucidate the pathogenesis of endometriosis, which may lead to the development of novel and more effective medical therapies. It has been suggested that the 3D-culture model of ECSCs is more suitable for the in vitro endometriosis research than the 2D-culture model. In addition to the aberrant chemokine expression, the present microarray data may provide a new platform to identify candidate genes involved in the pathogenesis of endometriosis.
References Akoum, A., et al., 1995. Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis. Le groupe d’investigation en gynecologie. Fertil. Steril. 63, 322–328. Akoum, A., et al., 2000. Estradiol amplifies interleukin-1-induced monocyte chemotactic protein-1 expression by ectopic endometrial cells of women with endometriosis. J. Clin. Endocrinol. Metab. 85, 896–904. Akoum, A., et al., 2001. Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol. Hum. Reprod. 7, 859–866. Aoyagi, Y., et al., 2017. Decidualization differentially regulates microRNA expression in eutopic and ectopic endometrial stromal cells. Reprod. Sci. 24, 445–455. Bellelis, P., et al., 2013. Transcriptional changes in the expression of chemokines related to natural killer and T-regulatory cells in patients with deep infiltrative endometriosis. Fertil. Steril. 99, 1987–1993. Boucher, A., et al., 2000. Effect of hormonal agents on monocyte chemotactic protein-1 expression by endometrial epithelial cells of women with endometriosis. Fertil. Steril. 74, 969–975. Bulun, S.E., 2009. Endometriosis. N. Engl. J. Med. 360, 268–279. Giudice, L.C., 2010. Clinical practice. Endometriosis. N. Engl. J. Med. 362, 2389–2398. Hirakawa, T., et al., 2016. miR-503, a microRNA epigenetically repressed in endometriosis, induces apoptosis and cell-cycle arrest and inhibits cell proliferation, angiogenesis, and contractility of human ovarian endometriotic stromal cells. Hum. Reprod. 31, 2587–2597. Hirata, T., et al., 2010. Recruitment of CCR6-expressing Th17 cells by CCL 20 secreted from IL-1β-, TNF-α-, and IL-17A-stimulated endometriotic stromal cells. Endocrinology 151, 5468–5476. Hornung, D., et al., 1997. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J. Clin. Endocrinol. Metab. 82, 1621–1628. Hornung, D., et al., 2000. Localization in tissues and secretion of eotaxin by cells from normal endometrium and endometriosis. J. Clin. Endocrinol. Metab. 85, 2604–2608. Hornung, D., et al., 2001a. Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol. Hum. Reprod. 7, 163–168. Hornung, D., et al., 2001b. Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: assessment of autocrine/paracrine regulation by in situ hybridization. Am. J. Pathol. 158, 1949–1954. Hou, X.X., et al., 2016. Fractalkine/CX3CR1 is involved in the pathogenesis of endometriosis by regulating endometrial stromal cell proliferation and invasion. Am. J. Reprod. Immunol. 76, 318–325.
Authorship statement R.Z. and K.N. participated in the study design, data analysis and interpretation, literature search, generation of figures, and writing and editing of the manuscript. Y.A., T.H., K.T., and H.N. executed the data/ case collection, experiments, data analysis, and interpretation. All authors approved the final version to be published. Funding This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 16K11093to K. Nasu, no. 18K16774to T. Hirakawa, no. 17K16857 to K. Takebayashi, and no. 15K10679 to H. Narahara). Declaration of Competing Interest The authors declare that they do not have any conflicts of interests. Acknowledgements We would like to thank Ms. Sawako Adachi and Ms. Nozomi Kai for their excellent technical assistance, Dr. Kaori Yasuda (Cell Innovator Co. Ltd., Fukuoka, Japan) for the microarray analysis, and Editage (www.editage.jp) for English language editing. 5
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Nisolle, M., Donnez, J., 1997. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil. Steril. 68, 585–596. Okamoto, M., et al., 2015. Enhanced miR-210 expression promotes the pathogenesis of endometriosis through activation of signal transducer and activator of transcription 3. Hum. Reprod. 30, 632–641. Shi, X., et al., 2014. The role of SRC1 and SRC2 in steroid-induced SDF1 expression in normal and ectopic endometrium. Reproduction 147, 847–853. Tsuno, A., et al., 2009. Decidualization attenuates the contractility of eutopic and ectopic endometrial stromal cells: implications for hormone therapy of endometriosis. J. Clin. Endocrinol. Metab. 94, 2516–2523. Tsuno, A., et al., 2011. Fasudil inhibits the proliferation and contractility and induces cell cycle arrest and apoptosis of human endometriotic stromal cells: a promising agent for the treatment of endometriosis. J. Clin. Endocrinol. Metab. 96, E1944–1952. Vandercappellen, J., et al., 2008. The role of CXC chemokines and their receptors in cancer. Cancer Lett. 267, 226–244. Wells, E.K., Yarborough 3rd, O., Lifton, R.P., Cantley, L.G., Caplan, M.J., 2013. Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8. Am. J. Physiol., Cell Physiol. 304, C966–975. Yuge, A., et al., 2007. Collagen gel contractility is enhanced in human endometriotic stromal cells: a possible mechanism underlying the pathogenesis of endometriosisassociated fibrosis. Hum. Reprod. 22, 938–944. Zlotnik, A., et al., 2006. The chemokine and chemokine receptor superfamilies and their molecular evolution. Genome Biol. 7, 243.
Lebovic, D.I., et al., 2001. IL-1beta induction of RANTES (regulated upon activation, normal T cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-kappaB site in the proximal promoter. J. Clin. Endocrinol. Metab. 86, 4759–4764. Manabe, S., et al., 2011. Expression and localization of CXCL16 and CXCR6 in ovarian endometriotic tissues. Arch. Gynecol. Obstet. 284, 1567–1572. Nasu, K., et al., 2005. Bufalin induces apoptosis and the G0/G1 cell cycle arrest of endometriotic stromal cells: a promising agent for the treatment of endometriosis. Mol. Hum. Reprod. 11, 817–823. Nasu, K., et al., 2009. Simvastatin inhibits the proliferation and the contractility of human endometriotic stromal cells: a promising agent for the treatment of endometriosis. Fertil. Steril. 92, 2097–2099. Nasu, K., et al., 2010a. Heparin is a promising agent for the treatment of endometriosisassociated fibrosis. Fertil. Steril. 94, 46–51. Nasu, K., et al., 2010b. Mevalonate-Ras homology (Rho)/Rho-associated coiled-coilforming protein kinase (ROCK)-mediated signaling pathway as a therapeutic target for the treatment of endometriosis-associated fibrosis. Curr. Signal Transduct. Ther. 5, 141–148. Nishida, M., et al., 2004. Down-regulation of interleukin-1 receptor type 1 expression causes the dysregulated expression of CXC chemokines in endometriotic stromal cells: a possible mechanism for the altered immunological functions in endometriosis. J. Clin. Endocrinol. Metab. 89, 5094–5100. Nishida, M., et al., 2011. Role of chemokines in the pathogenesis of endometriosis. Front. Biosci. S3, 1196–1204.
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Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jri
Chemokine expression profiles of ovarian endometriotic stromal cells in three-dimensional culture
T
Ruofei Zhua, Kaei Nasua,b,*, Yoko Aoyagia, Tomoko Hirakawaa, Kanetoshi Takebayashia, Hisashi Naraharaa a b
Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Oita, Japan Division of Obstetrics and Gynecology, Support System for Community Medicine, Faculty of Medicine, Oita University, Oita, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Endometriosis Chemokine Gene expression microarray 3-Dimenional culture
Endometriosis is a chronic inflammatory disease which is associated with aberrant chemokine expression. We have established a three-dimensional (3D) floating collagen gel culture of human endometriotic cyst stromal cells (ECSCs) as an in vitro model of early-stage fibrosis formation in endometriosis. We evaluated the gene expression profiles of 3D-cultured ECSCs using a gene expression microarray. We identified and confirmed with reverse transcription-polymerase chain reaction that mRNA levels of CXCL1, CXCL2, CXCL3, CXCL8, and CCL20 in 3D-cultured ECSCs were significantly higher than in 2D-cultured ECSCs. The protein levels of CXCL1, CXCL2, CXCL8, and CCL20 in the supernatant of 3D-cultured ECSCs were significantly higher than in 2D-cultured ECSCs. It has been suggested that the 3D-culture model of ECSCs is more suitable for in vitro endometriosis research than 2D-culture. This microarray data provides a new platform to identify the candidate genes involved in the pathogenesis of endometriosis which could be masked in conventional 2D-culture.
1. Introduction Endometriosis is a major contributor to pelvic pain and occurs in 6–10 % of women of reproductive age. (Giudice, 2010). Endometriosis is a chronic inflammatory condition accompanied by adhesions, fibrosis, neuronal infiltration, angiogenesis, scarring, and anatomical distortion (Bulun, 2009; Giudice, 2010). Activated lymphocytes, neutrophils, and macrophages are hallmarks of the inflammation in endometriotic tissues (Lebovic et al., 2001). Chemokines are small polypeptides characterized by their proinflammatory functions, and demonstrate chemotactic activity targeted to specific leukocyte populations (Zlotnik et al., 2006; Vandercappellen et al., 2008). More than 50 human chemokines have been identified and divided into four subfamilies based on the relative position of their cysteine residues (CC, CXC, C, and CXC3) (Nishida et al., 2011). Chemokines are produced in endometriotic tissues and have been implicated in the pathological process of endometriosis (Bulun, 2009; Nishida et al., 2011).
We have established a three-dimensional (3D) floating collagen gel culture of human endometriotic cyst stromal cells (ECSCs) as an in vitro model of early-stage fibrosis formation in endometriosis (Yuge et al., 2007; Nasu et al., 2009; Tsuno et al., 2009; Nasu et al., 2010a, b; Tsuno et al., 2011). In this experimental model, 3D-cultured ECSCs induce the contraction of floating collagen gels, which is comparable to the earlystage fibrosis formation in vivo. We have identified the involvement of mevalonate- Ras homology (Rho)/ Rho-associated coiled-coil-forming protein kinase (ROCK) pathway in the development of early-stage endometriosis-associated fibrosis. However, global gene expression profiles in 3D-cultured ECSCs have not yet been elucidated. In this study, we evaluated the gene expression profiles of 3D-cultured ECSCs using a gene expression microarray. Then, as the first-line analysis, we identified the aberrantly expressed chemokines and examined the roles of these molecules in the development of this disease.
Abbreviations: 2D, 2 dimensional; 3D, 3 dimensional; C, cysteine; CC, cysteine–cysteine; CCL, cysteine–cysteine ligand; CXC, cysteine–X–cysteine; CXCL, cysteine–X–cysteine ligand; CXC3, cysteine–X3–cysteine; DMEM, Dulbecco’s modified Eagle’s medium; ECSCs, endometriotic cyst stromal cells; ELISA, enzymelinked immunosorbent assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NESCs, normal endometrial stromal cells; Rho, Ras homology; ROCK, Rho-associated coiled-coil-forming protein kinase; RT-PCR, reverse transcription-polymerase chain reaction ⁎ Corresponding author at: Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Idaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan. E-mail address: [email protected] (K. Nasu). https://doi.org/10.1016/j.jri.2020.103100 Received 3 September 2019; Received in revised form 30 January 2020; Accepted 5 February 2020 0165-0378/ © 2020 Elsevier B.V. All rights reserved.
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2. Materials and methods
Conventional 2D-culture of ECSCs and NESCs were also performed in 35-mm culture plates in the presence of 10 % charcoal-stripped heatinactivated FBS. After 48 h incubation, the supernatants were collected for chemokine ELISA. Then, total RNA was extracted from the cells.
2.1. Human ECSC and normal endometrial stromal cell (NESC) isolation procedures and cell culture conditions
2.3. Gene expression microarray analysis
Ovarian endometriotic tissues were obtained from patients with regular menstrual cycles undergoing surgical treatment of ovarian endometriotic cysts (n = 8, age 31–48 yrs), as described previously (Nishida et al., 2004; Nasu et al., 2005; Hirakawa et al., 2016; Aoyagi et al., 2017). Eutopic endometrial tissues were obtained from premenopausal patients who had undergone hysterectomies for subserosal or intramural leiomyoma and had no evidence of endometriosis (n = 10, age 41–52 yrs), as described (Nishida et al., 2004). No patients had received hormonal treatments for at least two years prior to the surgery. All of the specimens were confirmed as being in the mid-to-late proliferative phases according to pathological observation and/or menstrual cycles. This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine, Oita University, and written informed consent was obtained from all patients. ECSCs and NESCs were isolated from ovarian endometrioma and eutopic endometrial tissues, respectively, by enzymatic digestion, as described previously (Nishida et al., 2004; Nasu et al., 2005; Hirakawa et al., 2016; Aoyagi et al., 2017). Briefly, the ovarian endometriotic and eutopic endometrial tissues were minced in Hanks’ balanced salt solution and digested with 0.5 % collagenase B (Thermo Fisher Scientific, Tokyo, Japan) in Dulbecco’s modified Eagle’s medium (DMEM, Nissui, Tokyo, Japan) at 37 C for 40 min. The dispersed cells were filtered through a 70-μm pore size nylon mesh to remove the undigested tissue pieces. The filtrated fraction was separated from epithelial cell clumps by differential sedimentation at unit gravity as follows. The cells were resuspended in 2 ml culture medium and layered slowly over 10 ml of the medium in a centrifuge tube. Sealed tubes were placed in an upright position at 37 °C in 5 % CO2 in air for 30 min. After sedimentation, the top 8 ml medium were collected. Finally, the medium containing stromal cells was filtered through a 40-μm pore size nylon mesh. Final purification was achieved by allowing stromal cells (which attach rapidly to plates) to adhere selectively to culture dishes for 30 min at 37 °C, followed by the removal of nonadhering epithelial cells. Isolated ECSCs and NESCs were cultured in DMEM supplemented with 100 IU/ ml penicillin (Gibco-BRL, Gaithersburg, MD, USA), 50 mg/ml streptomycin (Gibco-BRL), and 10 % charcoal-stripped heat-inactivated fetal bovine serum (FBS; Gibco-BRL) at 37 °C in 5 % CO2 in air. ECSC and NESC in monolayer culture after the third passage were more than 99 % pure, as analyzed by immunocytochemical staining with antibodies to vimentin (V9; Dako, Tokyo, Japan), CD10 (Abcam, Cambridge, UK), cytokeratin (Dako), factor VIII (Dako), and leukocyte common antigen (2B11 + PD7/26, Dako) (Nishida et al., 2004; Nasu et al., 2005) and were used for the following experiments. Each experiment was performed in triplicate and repeated at least three times with cells isolated from different patients.
Total RNA from 3D-cultured and 2D-cultured ECSCs were extracted with a miRNeasy Mini kit (Qiagen, Valencia, CA, USA) and subjected to gene expression microarray analyses with a human mRNA microarray (SurePrint G3 Human GE Microarray Kit 8 × 60 K Ver2.0, Agilent Technologies, Santa Clara, CA, USA), as previously described (Okamoto et al., 2015; Aoyagi et al., 2017). To identify up- and down-regulated genes, we calculated Z-scores and ratios from the normalized signal intensities of each probe for comparison between 3D-cultured and 2Dcultured ECSC samples (Okamoto et al., 2015; Aoyagi et al., 2017). We established the criteria for determining regulated genes: Z-score ≥ 2.0 and ratio ≥ 2.0-fold for up-regulated genes, and Z-score ≤ –2.0 and ratio ≤ 0.5 for down-regulated genes. All gene expression microarray data are available at Gene Expression Omnibus via the National Center for Biotechnology Information under Accession No. GSE136412 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE136412). 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) for mRNA expression The expressions levels of chemokines in 3D-cultured and 2D-cultured ECSCs and NESCs were evaluated by quantitative RT-PCR, as described previously (Okamoto et al., 2015; Hirakawa et al., 2016; Aoyagi et al., 2017). CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, and CCL20 were identified by the gene expression microarray analysis. Briefly, 48 h after 3D-culture and 2D-culture, total RNA from ECSCs and NESCs was extracted as described above and subjected to quantitative RT-PCR with the following specific primers (all from Applied Biosystems): CXCL1 (Assay ID: Hs00236937_m1), CXCL2 (Assay ID: Hs00601975_m1), CXCL3 (Assay ID: Hs00171061_m1), CXCL6 (Assay ID: Hs00605742_g1), CXCL8 (Assay ID: Hs00174103_m1), CCL20 (Assay ID: Hs00355476_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID: Hs02758991_g1). The expression levels of candidate mRNAs relative to those of GAPDH mRNA were calculated using a standard curve. The data were calculated from triplicate samples and are presented as the percentage of the values of 2D-cultured NESCs. 2.5. Chemokine levels in the culture media To evaluate the production of candidate chemokines in 3D-cultured and 2D-cultured ECSCs and NESCs, we measured chemokine levels in the culture media by ELISAs. Briefly, 4 h before cessation of 2D- and 3D-culture, the supernatant was replaced with fresh culture medium containing 10 % charcoal-stripped heat-inactivated FBS. Then, the supernatant was collected and stored at −70 °C until the assay was conducted. The concentrations of CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, and CCL20 were determined in each supernatant with commercially available ELISA kits (CXCL1, CXCL2, CXCL6, CXCL8, and CCL20 from R&D Systems, Minneapolis, MN, USA and CXCL3 from Aviva Systems Biology, San Diego, CA, USA). The sensitivities of the assay were 1.5 pg/mL for CXCL1, 0.15 pg/mL for CXCL2, 0.6 pg/mL for CXCL3, 0.17 pg/mL for CXCL6, 0.5 pg/mL for CXCL8, and 0.12 pg/mL for CCL20.
2.2. 3D-culture in floating collagen gels and conventional 2D-culture 3D-culture of ECSCs and NESCs in floating collagen gel were performed as described previously (Yuge et al., 2007; Tsuno et al., 2011; Hirakawa et al., 2016). ECSCs and NESCs were embedded in collagen gel (Cellmatrix type I-A; Nitta Gelatin, Osaka, Japan) in 35-mm culture plates according to the manufacturer’s instructions and cultured in a three-dimensional matrix. After incubation for 48 h in the presence of 10 % charcoal-stripped heat-inactivated FBS, the supernatants were collected for chemokine enzyme-linked immunosorbent assays (ELISAs). Then, ECSCs and NESCs were isolated from contracted collagen gels by enzymatic digestion with collagenase type I (Sigma-Aldrich, St Louis, MO, USA), as previously described (Yuge et al., 2007; Tsuno et al., 2009). Then, total RNA was extracted from the isolated cells.
2.6. Statistical analysis All data were obtained from triplicate samples and are presented as percent values relative to the corresponding controls in the form of mean ± SD. Data were analyzed using the Bonferroni test with the 2
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Table 1 Effects of 3-D culture on the chemokine mRNA expression of ECSCs. Gene symbol
CXCL1 CXCL2 CXCL3 CXCL6 CXCL8 CCL20
Other name
GROα GROβ GROγ GCP-2 IL-8 MIP-3α
Regulation
Upregulated Upregulated Upregulated Upregulated Upregulated Upregulated
Z-score
5.8 10.1 8.3 3.1 5.5 2.8
Ratio
83.7 755.6 670.2 16.2 152.7 88.2
Signal intensity 2D
3D
174.0 ± 104.1 101.0 ± 46.2 35.3 ± 33.7 84.5 ± 55.5 540.9 ± 882.3 14.7 ± 18.8
11603.3 ± 10005.3 71001.6 ± 30439.2 14472.3 ± 14687.8 1510.4 ± 1948.0 5833.9 ± 3368.4 350.8 ± 390.8
Genes with Z-score ≥2.0 and ratio ≥2.0-fold were defined as upregulated, and those with Z-score ≤-2.0 and ratio ≤0.5 were defined as down-regulated.
compared to 2D-cultured ECSCs. However, mRNA expressions of CXCL1, CXCL2, CXCL3, and CXCL8 in the 3D-cultured ECSCs were significantly higher than those in the 3D-cultured NESCs. We additionally found that the protein levels of CXCL1, CXCL2, CXCL8, and CCL20 in the supernatant of 3D-cultured ECSCs were significantly higher than those in the 3D- and 2D-cultured NESCs. These results suggest that the expressions of some chemokines are attenuated in 2Dcultured ECSCs, which is conventionally used as the in vitro model. In 3D-culture, these chemokine expressions were restored. Endometriotic lesions are characterized by chronic inflammation (Bulun, 2009; Giudice, 2010). The inflammatory cells and soluble factors, such as proinflammatory cytokines and chemokines, in the diseased tissues, as well as the peritoneal fluid are thought to contribute to the development and progression of endometriosis (Lebovic et al., 2001). The increased production of chemokines may induce the recruitment of inflammatory cells from circulation into endometriotic tissues and the peritoneal fluid (Akoum et al., 2000). We have established a three-dimensional floating collagen gel culture of ECSCs as a model of early-stage fibrogenesis in endometriosis (Yuge et al., 2007; Nasu et al., 2009, 2010a; Nasu et al., 2010b; Tsuno et al., 2011). In this experimental model, ECSCs cultured in floating collagen gels induce the reorganization and compaction of collagen fibers, and the contraction of collagen lattices, which is comparable to the tissue contraction in early-stage fibrogenesis in endometriosis. Research on the cell biology of 3D-cultured ECSCs may provide new insights for the pathogenesis of endometriosis-associated fibrosis. Using this 3D-culture model, we demonstrated that 3D-cultured ECSCs possess greater ability to differentiate into α-smooth muscle actin-positive myofibroblasts and stronger contractility than 3D-cultured NESCs (Yuge et al., 2007; Nasu et al., 2009, 2010a; Nasu et al., 2010b; Tsuno et al., 2009, 2011). Activation of the mevalonate-Rho/ROCK-mediated pathway as well as induction of α-smooth muscle actin expression in ECSCs may be involved in this phenomenon (Yuge et al., 2007; Nasu et al., 2009; Tsuno et al., 2009; Nasu et al., 2010a, b; Tsuno et al., 2011). However, other putative mechanisms of fibrosis formation in the diseased tissues have yet to be established. In the present study, therefore, we planned to elucidate the potential causes of cellular functional changes during the endometriosis-associated fibrosis formation by using gene expression microarray analysis of 3D-cultured ECSCs. Previous studies have shown that several chemokines are produced by endometriotic stromal cells. These chemokines include CXCL1 (Nishida et al., 2004), CXCL5 (Nishida et al., 2004), CXCL8 (Akoum et al., 2001; Nishida et al., 2004), CXCL12 (Shi et al., 2014), CXCL16 (Manabe et al., 2011), CCL2 (Akoum et al., 1995; Boucher et al., 2000), CCL5 (Lebovic et al., 2001; Hornung et al., 2001b), CCL8 (Hornung et al., 1997, 2001a; Hornung et al., 2001b), CCL11 (Hornung et al., 2000), CCL17 (Bellelis et al., 2013), CCL20 (Hirata et al., 2010), and CX3CL1 (Hou et al., 2016). Several of these chemokine expressions have been shown to be higher in endometriotic tissues compared to the eutopic endometrial tissues of unaffected women, which may induce a series of inflammatory responses, such as the recruitment of
Statistical Package for Social Science software (IBM SPSS statistics 24; IBM, Armonk, NY, USA). p-values < 0.05 were considered significant. 3. Results 3.1. Detection of chemokine mRNAs differentially expressed in 3D- and 2Dcultured ECSCs by gene expression microarray The gene expression microarray revealed that six chemokine mRNAs were upregulated in 3D-cultured ECSCs compared to those in 2D-culture (Table 1). Interestingly, no chemokine mRNAs were downregulated in 3D-cultured ECSCs compared to 2D-culture. 3.2. Expression of chemokine mRNAs in 3D- and 2D-cultured ECSCs and NESCs To validate the results of our gene expression microarray data and to compare the chemokine expressions generated by ECSCs and NESCs, we evaluated mRNA expressions of six genes in 3D- and 2D-cultured ECSCs and NESCs using quantitative RT-PCR. As shown in Fig. 1, the relative mRNA levels of CXCL1, CXCL2, CXCL3, CXCL8, and CCL20 in the 3D-cultured ECSCs were significantly higher than those in the 2D-cultured ECSCs, as measured by RT-PCR. These results are consistent with the gene expression microarray data. The mRNA expressions of CXCL1, CXCL2, CXCL3, and CXCL8 in the 3D-cultured ECSCs were significantly higher than those in the 3D-cultured NESCs. In 2D-culture, however, only CXCL1 mRNA expression was higher in ECSCs than NESCs. The primer sets for CXCL6 mRNA did not work well (data not shown). 3.3. Chemokine levels in the supernatant of 3D- and 2D-cultured ECSCs and NESCs As shown in Fig. 2, the relative protein levels of CXCL1, CXCL2, CXCL8, and CCL20 in the supernatant of 3D-cultured ECSCs were significantly higher than those in the 2D-cultured ECSCs. In 2D-culture, only the CXCL1 protein level was higher in ECSCs compared to NESCs. Importantly, protein levels of CXCL3 and CXCL6 in the 3D- and 2Dcultured NESCs and ECSCs in this culture condition were below the detection levels (data not shown). 4. Discussion To investigate the global gene expression profiles during the earlystage of fibrosis formation in endometriosis, we performed the gene expression microarray analysis of 3D- and 2D-cultured ECSCs. As a firstline analysis, we identified six aberrantly expressed chemokines, namely CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, and CCL20. RT-PCR performed for these six chemokines revealed that mRNA levels of CXCL1, CXCL2, CXCL3, CXCL8, and CCL20 in 3D-cultured ECSCs were significantly higher than those in the 2D-cultured ECSCs. There was no chemokine mRNA that was downregulated in 3D-cultured ECSCs 3
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Fig. 1. Chemokine mRNA levels in the 2D-cultured and 3D-cultured NESCs and ECSCs. (A), CXCL1; (B), CXCL2; (C), CXCL3; (D), CXCL8; (E), CCL20. *p < 0.0005 vs. NESC-3D and ECSC-2D, **p < 0.05 vs. ECSC-2D, and ***p < 0.005 vs. NESC-2D (Bonferroni test). NESC-2D, 2D-culturred NESCs; NESC-3D, 3Dculturred NESCs; 2D-ECSC, 2D-culturred ECSCs; 3D-ECSC, 3D-culturred ECSCs. The data were obtained from triplicate samples and are presented as percent values relative to the NESC-2D in the form of mean ± SD. Representative results are shown.
inflammatory cells, angiogenesis, fibrogenesis, and nerve fiber infiltration into the diseased tissues for the progression of the disease. It is known that, compared to conventional 2D-culture, 3D-culture is more equivalent to in vivo tissue. Whereas, it is also known that endometriotis is a chronic infllammmatory disease. Therefore, it can be speculated that aberrant expressions of some molecules are masked in the conventional 2D-culture and that 3D-cultured ECSCs may restore their original characteristics in vivo including the enhanced inflammatory responses. Alternatively, the differentiation of ECSCs into myofibroblastic phenotype in 3D-culture may also involve in the enhanced chemokine expression in these cells. To our knowledge, there is only one report that compared the chemokine expression between 3Dand 2D-culture. Wells et al. (2013) compared the gene expression patterns of 3D- and 2D-cultured Madin-Darby canine kidney renal epithelial cells by using the gene expression microarray. They found the increased CXCL8 expression in 3D-cultured cells, which is consistent with our present findings. In this report, however, they did not evaluate other ckemokine expressions. Further study with the present microarray data may elucidate the turn-on mechanisms of chemokine
expression in 3D-cultured ECSCs. The present gene expression microarray revealed the upregulation of CXCL3 and CXCL6 mRNA expression. Whereas, CXCL3 and CXCL6 proteins were not detected in the supernatants of ECSCs and NESCs by ELISAs. Although the precise mechanisms of these phenomenon are unknown, some posttranscriptional inhibitory mechnisms may exist in the regulation of these chemokine expressions. The limitation of the present study is that the endometriotic cells were isolated from ovarian endometriomas. It has been suggested that ovarian endometrioma is a different entity than deep infiltrative and peritoneal endometriosis (Nisolle and Donnez, 1997). Therefore, it remains possible that endometriotic cells existing in other locations may show different responses to 3D-culture. The experiments in this study were performed only with the stromal cells, because the stromal cells are responsible for the tissue contraction. In follow-up studies, chemokine expression profiles should be evaluated in the epithelial components as well as in the infiltrated inflammatory cells in the endometriotic tissues. In summary, we utilized a gene expression microarray to identify 4
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Fig. 2. Chemokine levels in the supernatants of 2Dcultured and 3D-cultured NESCs and ECSCs. (A), CXCL1; (B), CXCL2; (C), CXCL8; (D), CCL20. *p < 0.0005 vs. NESC-3D and ECSC-2D, **p < 0.05 vs. NESC-3D and ECSC-2D, and ***p < 0.005 vs. NESC-2D (Bonferroni test). NESC-2D, 2D-culturred NESCs; NESC-3D, 3D-culturred NESCs; 2D-ECSC, 2Dculturred ECSCs; 3D-ECSC, 3D-culturred ECSCs. The data were obtained from triplicate samples and are presented as percent values relative to the NESC-2D in the form of mean ± SD. Representative results are shown.
A part of this manuscript was presented at the 33rd Annual Meeting of Japan Society for Immunology and Reproduction.
the aberrantly expressed genes that were not detected in conventional 2D-culture model. We confirmed that the expression of several chemokines in ECSCs were enhanced by 3D-culture, which may be similar to in vivo levels. With respect to inflammatory diseases, understanding the chemokine network is vital to elucidate the pathogenesis of endometriosis, which may lead to the development of novel and more effective medical therapies. It has been suggested that the 3D-culture model of ECSCs is more suitable for the in vitro endometriosis research than the 2D-culture model. In addition to the aberrant chemokine expression, the present microarray data may provide a new platform to identify candidate genes involved in the pathogenesis of endometriosis.
References Akoum, A., et al., 1995. Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis. Le groupe d’investigation en gynecologie. Fertil. Steril. 63, 322–328. Akoum, A., et al., 2000. Estradiol amplifies interleukin-1-induced monocyte chemotactic protein-1 expression by ectopic endometrial cells of women with endometriosis. J. Clin. Endocrinol. Metab. 85, 896–904. Akoum, A., et al., 2001. Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol. Hum. Reprod. 7, 859–866. Aoyagi, Y., et al., 2017. Decidualization differentially regulates microRNA expression in eutopic and ectopic endometrial stromal cells. Reprod. Sci. 24, 445–455. Bellelis, P., et al., 2013. Transcriptional changes in the expression of chemokines related to natural killer and T-regulatory cells in patients with deep infiltrative endometriosis. Fertil. Steril. 99, 1987–1993. Boucher, A., et al., 2000. Effect of hormonal agents on monocyte chemotactic protein-1 expression by endometrial epithelial cells of women with endometriosis. Fertil. Steril. 74, 969–975. Bulun, S.E., 2009. Endometriosis. N. Engl. J. Med. 360, 268–279. Giudice, L.C., 2010. Clinical practice. Endometriosis. N. Engl. J. Med. 362, 2389–2398. Hirakawa, T., et al., 2016. miR-503, a microRNA epigenetically repressed in endometriosis, induces apoptosis and cell-cycle arrest and inhibits cell proliferation, angiogenesis, and contractility of human ovarian endometriotic stromal cells. Hum. Reprod. 31, 2587–2597. Hirata, T., et al., 2010. Recruitment of CCR6-expressing Th17 cells by CCL 20 secreted from IL-1β-, TNF-α-, and IL-17A-stimulated endometriotic stromal cells. Endocrinology 151, 5468–5476. Hornung, D., et al., 1997. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J. Clin. Endocrinol. Metab. 82, 1621–1628. Hornung, D., et al., 2000. Localization in tissues and secretion of eotaxin by cells from normal endometrium and endometriosis. J. Clin. Endocrinol. Metab. 85, 2604–2608. Hornung, D., et al., 2001a. Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid. Mol. Hum. Reprod. 7, 163–168. Hornung, D., et al., 2001b. Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: assessment of autocrine/paracrine regulation by in situ hybridization. Am. J. Pathol. 158, 1949–1954. Hou, X.X., et al., 2016. Fractalkine/CX3CR1 is involved in the pathogenesis of endometriosis by regulating endometrial stromal cell proliferation and invasion. Am. J. Reprod. Immunol. 76, 318–325.
Authorship statement R.Z. and K.N. participated in the study design, data analysis and interpretation, literature search, generation of figures, and writing and editing of the manuscript. Y.A., T.H., K.T., and H.N. executed the data/ case collection, experiments, data analysis, and interpretation. All authors approved the final version to be published. Funding This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 16K11093to K. Nasu, no. 18K16774to T. Hirakawa, no. 17K16857 to K. Takebayashi, and no. 15K10679 to H. Narahara). Declaration of Competing Interest The authors declare that they do not have any conflicts of interests. Acknowledgements We would like to thank Ms. Sawako Adachi and Ms. Nozomi Kai for their excellent technical assistance, Dr. Kaori Yasuda (Cell Innovator Co. Ltd., Fukuoka, Japan) for the microarray analysis, and Editage (www.editage.jp) for English language editing. 5
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Nisolle, M., Donnez, J., 1997. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil. Steril. 68, 585–596. Okamoto, M., et al., 2015. Enhanced miR-210 expression promotes the pathogenesis of endometriosis through activation of signal transducer and activator of transcription 3. Hum. Reprod. 30, 632–641. Shi, X., et al., 2014. The role of SRC1 and SRC2 in steroid-induced SDF1 expression in normal and ectopic endometrium. Reproduction 147, 847–853. Tsuno, A., et al., 2009. Decidualization attenuates the contractility of eutopic and ectopic endometrial stromal cells: implications for hormone therapy of endometriosis. J. Clin. Endocrinol. Metab. 94, 2516–2523. Tsuno, A., et al., 2011. Fasudil inhibits the proliferation and contractility and induces cell cycle arrest and apoptosis of human endometriotic stromal cells: a promising agent for the treatment of endometriosis. J. Clin. Endocrinol. Metab. 96, E1944–1952. Vandercappellen, J., et al., 2008. The role of CXC chemokines and their receptors in cancer. Cancer Lett. 267, 226–244. Wells, E.K., Yarborough 3rd, O., Lifton, R.P., Cantley, L.G., Caplan, M.J., 2013. Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8. Am. J. Physiol., Cell Physiol. 304, C966–975. Yuge, A., et al., 2007. Collagen gel contractility is enhanced in human endometriotic stromal cells: a possible mechanism underlying the pathogenesis of endometriosisassociated fibrosis. Hum. Reprod. 22, 938–944. Zlotnik, A., et al., 2006. The chemokine and chemokine receptor superfamilies and their molecular evolution. Genome Biol. 7, 243.
Lebovic, D.I., et al., 2001. IL-1beta induction of RANTES (regulated upon activation, normal T cell expressed and secreted) chemokine gene expression in endometriotic stromal cells depends on a nuclear factor-kappaB site in the proximal promoter. J. Clin. Endocrinol. Metab. 86, 4759–4764. Manabe, S., et al., 2011. Expression and localization of CXCL16 and CXCR6 in ovarian endometriotic tissues. Arch. Gynecol. Obstet. 284, 1567–1572. Nasu, K., et al., 2005. Bufalin induces apoptosis and the G0/G1 cell cycle arrest of endometriotic stromal cells: a promising agent for the treatment of endometriosis. Mol. Hum. Reprod. 11, 817–823. Nasu, K., et al., 2009. Simvastatin inhibits the proliferation and the contractility of human endometriotic stromal cells: a promising agent for the treatment of endometriosis. Fertil. Steril. 92, 2097–2099. Nasu, K., et al., 2010a. Heparin is a promising agent for the treatment of endometriosisassociated fibrosis. Fertil. Steril. 94, 46–51. Nasu, K., et al., 2010b. Mevalonate-Ras homology (Rho)/Rho-associated coiled-coilforming protein kinase (ROCK)-mediated signaling pathway as a therapeutic target for the treatment of endometriosis-associated fibrosis. Curr. Signal Transduct. Ther. 5, 141–148. Nishida, M., et al., 2004. Down-regulation of interleukin-1 receptor type 1 expression causes the dysregulated expression of CXC chemokines in endometriotic stromal cells: a possible mechanism for the altered immunological functions in endometriosis. J. Clin. Endocrinol. Metab. 89, 5094–5100. Nishida, M., et al., 2011. Role of chemokines in the pathogenesis of endometriosis. Front. Biosci. S3, 1196–1204.
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