Scholarly article on topic 'Endometriotic mesenchymal stem cells significantly promote fibrogenesis in ovarian endometrioma through the Wnt/β-catenin pathway by paracrine production of TGF-β1 and Wnt1'

Endometriotic mesenchymal stem cells significantly promote fibrogenesis in ovarian endometrioma through the Wnt/β-catenin pathway by paracrine production of TGF-β1 and Wnt1 Academic research paper on "Biological sciences"

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Academic research paper on topic "Endometriotic mesenchymal stem cells significantly promote fibrogenesis in ovarian endometrioma through the Wnt/β-catenin pathway by paracrine production of TGF-β1 and Wnt1"

Hum. Reprod. Advance Access published March 22, 2016

Human Reproduction, Vol.0, No.0 pp. 1 -12,2016

doi:I0.I093/humrep/dew058

human reproduction

ORIGINAL ARTICLE Gynaecology

Endometriotic mesenchymal stem cells significantly promote fibrogenesis in ovarian endometrioma through the Wnt/p-catenin pathway by paracrine production of TGF-b 1 and Wntl

Jing Li^, Yongdong Dai^, Haiyan Zhu, Yinshen Jiang, and Songying Zhang*

Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, No. 3 Qingchun East Road, Jianggan District, Hangzhou 310016, China

^Correspondence address. Tel: +86-571-86002222; E-mail: zhangsongying@l26.com Submitted on November 25, 2015; resubmitted on February 23, 2016; accepted on March 1, 2016

study question: Are endometriotic mesenchymal stem cells (Ecto-MSCs) involved in the fibrosis of ovarian endometrioma? summary answer: Ecto-MSCs enhanced the fibrotic behavior of stromal cells in ovarian endometrioma through the Wnt/p-catenin c

pathway by paracrine production of transforming growth factor-p 1 (TGF-p 1) and Wntl. s

what is known ALreAdY: Endometriosis is characterized by ectopic outgrowth of endometrial stroma and glands surrounded by

dense fibrous tissues. The pathogenesis of endometriosis, especially ovarian endometrioma-associated fibrosis, is still unknown. 8

study design, size, duration: We analyzed endometrial samples from 15 patients of reproductive age with ovarian endometrioma 8

and normal menstrual cycles. A total of 54 nude mice received a single injection of proliferative endometrial fragments from 14 individuals without O

endometriosis.

participants/materials, setting, methods: Conditioned medium (CM) was collected from endometrial mesenchymal stem cells (Euto-MSCs) and Ecto-MSCs. The effects of CM on cell proliferation, migration, invasion and collagen gel contraction of endometrial and endometriotic stromal cells (Euto- and Ecto-ESCs) in ovarian endometrioma were evaluated by cell counting kit-8, transwell and collagen gel contraction assays. Effects of CM on fibrotic markers' expression [including a-smooth muscle actin, Type I collagen, connective tissue growth factor and fibronectin (FN)] in Euto- and Ecto-ESCs were determined by real-time reverse-transcription-polymerase chain reaction and western blotting. Additionally, fibrogenic effects of Ecto-MSC CM treatment on endometriotic implants were analyzed using a xenograft model of endometriosis in immunodeficient nude mice.

main results and the role of chance: Our results demonstrated that Ecto-MSC CM significantly promoted cell proliferation, migration, invasion and collagen gel contraction of Euto- and Ecto-ESCs from patients with ovarian endometrioma compared with control and Euto-MSC CM. Expression levels of fibrotic markers in Euto- and Ecto-ESCs were dramatically elevated after treatment with Ecto-MSC CM. Ecto-MSCs secreted higher levels ofTGF-p 1 and Wntl compared with Euto-MSCs. Furthermore, both TGF-p 1 and Wntl significantly increased expression of fibrotic markers in Euto- and Ecto-ESCs, which was reversed by an anti-TGF-p 1 antibody or Wntl negative regulator, Dickkopf-related protein 1 (Dkkl). Mechanistic studies demonstrated that Wnt/p-catenin signaling pathways in stromal cells were activated by Ecto-MSC CM. Animal experiments showed that TGF-p 1 and Wntl as well as Ecto-MSC CM markedly increased the expression of FN and collagen I, which enhanced the progression of fibrosis in endometriosis.

limitations, reasons for caution: To our knowledge, this is the first study to identify the role of Ecto-MSCs in the pathogenesis offibrosisin ovarian endometrioma. However, numerous other growth factors and cell types may also be involved in the pathogenesis. Therefore, further studies are required to elucidate the paracrine effects of cells in ovarian endometrioma.

wider implications of the findings: Ecto-MSCs may be involved in the pathogenesis offibrosis in ovarian endometrioma.

^These authors contributed equally to this work.

© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by the National Natural Science Foundation of China (81471505 and 81270657). No competing interests are declared.

Keywords: ovarian endometrioma / mesenchymal stem cells / fibrosis / TGF-ß 1 / Wntl

Introduction

Endometriosis, defined as the growth of endometrial tissue outside the uterine cavity, is a common gynecological disorder that affects 6- 10% of all women and 35-50% of women with pelvic pain and infertility (Sensky and Liu, 1980; Giudice and Kao, 2004). Ovarian endometrioma is a cyst composed of endometrial tissue, which is detected in 20-40% of women with endometriosis (Giudice and Kao, 2004). Endometriosis is characterized by ectopic outgrowth of endometrial stroma and glands surrounded by dense fibrous tissues (Linden and Centola, 1997). Excess fibrosis in endometriotic lesions may lead to pain, scarring and altered tissue functions. Because of the severe fibrosis, endometriotic cysts of ovarian endometrioma have a detrimental effect on the ovarian reserve over time and the surgical excision technique (Gordts et al., 2015).

Cystectomy in young patients with endometrioma maybe particularly detrimental to the follicle reserve. The loss of ovarian parenchyma atthe time of surgery is related to the cyst diameter and degree offibrosis (Kita-jima et al., 2011). The current consensus is that, in infertile patients with ovarian endometrioma, surgery should not be performed unless the cyst size exceeds 3 cm (Gelbaya etal., 2010). Previous studies also show that the size of the cyst is a key criterion to judge both the severity of the disease and the indication for surgery (Garcia-Velasco and Somigliana, 2009). However, to the best of our knowledge, little is known about the influence and mechanism of fibrogenesis in ovarian endometrioma, especially a systematic survey. Therefore, we investigated the fibrotic development of endometrioma to assist in effective treatments.

Retrograde flow of menstrual blood cells during menstruation is considered as the main cause for the development of endometriosis. Current evidence suggests that endometrial-derived stem cells may be key players in the pathogenesis of endometriosis (Nikoo et al., 2014). Mesenchymal stem cells (MSCs) have been reported to contribute to wound healing by secretion of paracrine factors, including matrix metal-loproteinases (MMPs) and vascular endothelial growth factors (VEGFs) (Yang et al., 2011). The paracrine factors of MSCs promote epithelial edge ingrowth, collagen synthesis (Ma et al., 2011), differentiation into residentwound-healing cells and angiogenesis (Gluecketal., 2015). Conditioned medium (CM) obtained from MSCs significantly enhances the cell survival offibroblasts, promotes the production orsecretion of cytokines and extracellular matrix (ECM) proteins and suppresses collagenase (MMPI) expression (Salazar et al., 2009; Jeon et al., 2010). The influence between MSCs and fibroblasts is of great importance in the development of fibrosis.

Stem cell therapy has been considered as a novel and promising strategy with the potential to be effective in ameliorating intrauterine adhesions (Yang et al., 2011) and other fibrotic diseases (Baulier et al., 2014; Min et al., 2015). However, considering the unknown side effects, there is a great deal to be learned about the mechanisms through which MSCs play a role in the development of fibrotic diseases.

In this study, we collected CM from MSCs to determine whether the cells released factors that influence the proliferative and fibrogenic features of endometrial and endometriotic stromal cells (Euto- and Ecto-ESCs).

Previous studies show that MSCs release wingless-type MMTV integration site family members (Wnts), transforming growth factor-b (TGF-b), MMPs and VEGFs into the culture medium (Salazar et al., 2009; Dzafic et al., 2014; Hsu et al., 2014). There is considerable evidence that WntI and TGF-b I are key regulators offibroblast activation in fibrotic disease (Ver-recchia and Mauviel, 2007; Strieter and Mehrad, 2009; Hong et al., 2015). VEGF is the most potent angiogenesis promoter and plays a significant role in neovascularization (Liu et al., 2013). MMP2 and MMP9 are involved in the breakdown of collagen (Moroz et al., 2013). The balance between WntI, TGF-b I, VEGFs, MMPs and other cytokines is associated with the switch from angiogenesis to fibrosis (Van Geest et al., 2012; Zhang et al., 2015). For these reasons, we speculated that abnormal paracrine functions of MSCs might modulate the processes of fibrosis and the development of ovarian endometrioma lesions.

Understanding the mechanisms of MSC participation in the fibrogenesis of ovarian endometrioma will allow us to understand the pathogenesis and develop novel approaches for prevention and therapies of ovarian endometrioma.

Materials and Methods

Ethical approval

Informed written consent was obtained from each patient priorto tissue collection. This study was approved and monitored by the ethics committee of Sir Run Run Shaw Hospital, Zhejiang University. Animal experimental protocols were approved by the Committee of Animal Ethics, Zhejiang University.

Patients

Patients aged 2I -39 years undergoing laparoscopy for ovarian endometrioma were recruited at Sir Run Run Shaw Hospital, China. No patient had received hormonal treatments, such as gonadotrophin-releasing hormone agonists or sex steroids. The patients had not used intrauterine contraception for at least 6 months priorto surgery. The recruited patients had regular menstrual cycles (28-32 days) with confirmation of their menstrual history. Samples included ovarian endometriotic cyst tissues (ovarian endometrioma) (n = I5) and theireutopic proliferative phase endometrium (n = I5) obtained from women undergoing laparoscopic treatment for pain and/or infertility. Endometrial tissue biopsies were obtained just priorto surgery using an endometrial suction catheter (Lilycleaner, Ningbo, China). A specimen of endometriotic tissue was collected by laparoscopy. Endometrial tissue samples were carefully stripped from the lining inner cyst wall to avoid contamination by the ovarian cortex, as confirmed by histological evaluation. The surgical protocol allowed scheduling of all surgical interventions (laparoscopyand hysteroscopy) intheweekfollowingmenstrual bleeding. All patients were in the proliferative phase as confirmed by ultrasound (Severi et al., 2003) and histological criteria. The cyst diameter measured by ultrasound ranged from 20 to 80 mm. All patients showed Stage III or IV endometriosis according to the American Society for Reproductive Medicine classification. Samples of endometrial and endometriotic tissues were divided into two portions. The first tissue portion was immediately collected in Hank's balanced salt solution (Life Technologies, Beijing, China) for ESC

isolation. The second portion was fixed in l0% formalin-acetic acid and embedded in paraffin.

Isolation and culture of ESCs

ESCs were isolated from biopsy specimens of eutopic and ectopic endome-tria of women with ovarian endometrioma. The tissues were minced using sterile scissors and digested with Type I collagenase for 60 min in a 37°C incubator. After digestion, the cells were filtered through wire sieves with various pore sizes to remove cell aggregates and epithelial cells. The cells were collected and frozen for the following procedures.

Isolation and culture of MSCs

For isolation of Euto- and Ecto-MSCs, cells were isolated from biopsy specimens of the endometrium and seeded in triplicate at low density (^200 cells per l00 mm dish) in Dulbecco's modified Eagle's medium/Fl2 (DMEM/Fl2; Gino Biological, Hangzhou, China). After incubation for2l days, large colonies were obtained and trypsinized into single cells. The cells were diluted and seeded in 96-well plates at a density of approximately one cell per well. After culture for l4 days, clonally derived proliferating colonies were trypsinized individually and cultured in l00mm dishes. The cells were grown in DMEM/Fl2 with l0% fetal bovine serum (FBS) until near confluence. Early passage cells (Passages 2-4) were collected and frozen for the following experiments.

Flow cytometric analysis

Cells (l x l06) were incubated with l mgfluorescein isothiocyanate (FITC)-conjugated mouse anti-rat monoclonal antibodies specific for rat CD44, CD90, CD73, CD29, CDll7, CD 34, CD45, HLA-DRorFITC-conjugated isotype-matched immunoglobulin G (l:50; all from BD, Franklin Lakes, NJ, USA) for l h at 4°C. After washing with PBS, the samples were analyzed by an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, USA).

To verify that the stem cell phenotypes were maintained in subcultures of MSCs, the mesenchymal characteristics were evaluated using positive MSC markers CD44, CD90, CD73 and CD29, and negative MSC markers CDll7, CD34, CD45 and HLA-DR. The MSC positive markers were observed in more than 90% of the cells, and the MSC negative markers were observed in < l0% of the cells (Supplementary data, Fig. SlA).

Multipotent differentiation

Osteogenic differentiation

The multipotent differentiation ability of the MSCs for osteogenesis and adipogenesis was examined as described previously (Lee and Im, 20l2). In brief, to induce osteogenic differentiation, the cells were seeded at a density of 5 x l03 cells/cm2 and treated with l0 mM p-glycerol phosphate, 0.l mM dexamethasone and 50 mg/ml ascorbic acid (all purchased from Sigma-Aldrich, St Louis, MO, USA) for 2 weeks. Positive induction was detected by alizarin red and alkaline phosphatase staining.

Adipogenic differentiation

Cells were also cultured in DMEM supplemented with adipogenic stimulatory supplements (StemCell Technologies, Vancouver, Canada) for 2 weeks to induce adipogenic differentiation. Positive induction was detected by oil red staining of lipid vacuoles.

Immunofluorescent staining

ESCs were cultured on coverslips, and fixed with 4% paraformaldehyde. After permeabilization with PBS-T (0.l % Triton X-100 in PBS solution), cells were blocked for non-specific interaction with 5% bovine serum albumin (BSA) for 30 min and then incubated with primary antibodies against Vimentin (l:l 00; Abcam, Cambridge, MA, USA), Pan-Keratin (l:l 00; Cell Signaling

Technology, Boston, MA, USA), total p-catenin (l :200; Cell Signaling Technology) or a-smooth muscle actin (a-SMA; l:l00; Abcam) at 4°C overnight. Fluorescent-conjugated secondary antibody solution (l:l 00; MultiSciences, Hangzhou, China) was used to visualize the signal. 4', 6-Diamidino-2-phenylindole (DAPI) solution was used to stain cell nuclei.

An osteogenic differentiation assay confirmed that most of the cells had mineralized calcium deposits after osteogenic induction, as shown by alizarin red and alkaline phosphatase staining. After adipogenic induction, oil red O staining demonstrated that the cells had differentiated into adipogenic lineages (Supplementary data, Fig. SlB). To verify the ESC phenotypes, we confirmed positive cellular staining for vimentin and negative cellular staining for cytokeratin (Supplementary data, Fig. SlC). Taken together, our results confirmed that we successfully isolated MSCs and ESCs.

CM preparation

Subconfluent MSCs were trypsinized and plated in l00 mm dishes at a concentration of 4 x l05 cells perdish. Cells were cultured in l0 ml serum-free medium for48 h. Collected CM was filter sterilized and concentrated using Centricon Plus-20 centrifugal concentration tubes (l0000 NMWL; Milli-pore, Co., Cork, Ireland) and resuspended at a 5-fold concentration in DMEM/Fl2. Acid activation of cytokines in CM was achieved by acidifying the concentrated CM with l N HCl followed by incubation at room temperature for l0 min. The CM was then neutralized with l.2 N NaOH/0.5 M HEPES before resuspending in DMEM/Fl2. The 5-fold concentration corresponded to 2 x l05 MSCs/ml.

ESC treatments

ESCs were seeded into 96-well plates (2 x l03 cells per well) for cell proliferation analyses, 24-well plates (l x l04 cells per well) for RNA extraction and immunocytochemistry detection or 60 mm dishes (5 x l04 cells per dish) for protein extraction. Forcell proliferation analyses, cells were cultured at 37°C for l day and then incubated with CM containing 2% FBS or DMEM/ Fl2 containing 2% FBS. For RNA extraction, immunocytochemistry detection and protein extraction, cells were cultured at 37°C for 24 h and then starved for 24 h in serum-free culture medium. Subsequently, the cells were treated with CM or serum-free culture medium.

Western blot analysis

Proteins were separated on sodium dodecyl sulfate-polyacrylamidegel electrophoresis mini-gels and transferred electrophoretically onto polyvinylidene fluoride membranes (Invitrogen, Carlsbad, CA, USA). Membranes were then probed with specific primary antibodies against a-SMA (l:l000; Cell Signaling Technology), connective tissue growth factor (CTGF, l:l000; Cell Signaling Technology), Wntl (l:l000; Abcam), TGF-pl (l:500; Abcam), total p-catenin (l:l000; Cell Signaling Technology) or Histone H4 (l:l000; Abcam). An anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (l:3000; Kangchen, Shanghai, China) was used to detect GAPDH as a loading control. A secondary anti-rabbit antibody (l:3000; Dawen, Hangzhou, China) and Immobilon Western Chemilumines-cent HRP Substrate (Millipore, Boston, MA, USA) were used to visualize immunoactive bands. ImageJ software (Wayne Rasband, National Institutes of Health, USA, http://imagej.nih.gov/ij) was used to evaluate the protein band densities. Protein expression levels were normalized to GAPDH.

RNA extraction and real-time reverse-transcription - polymerase chain reaction

Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentrations were quantified by a Nano-Drop 2000 (Nanodrop, Wilmington, DE, USA). Reverse transcription of l mg

total RNA was performed using a Quantscript RT kit (Tiangen Biotechnology, Beijing, China). mRNAexpression levels were determined by quantitative realtime polymerase chain reaction (PCR) usinga SYBR Green Master Mix Kit (DBI Bioscience, Ludwigshafen, Germany) and ABI 7500 RealTime PCR System. Human GAPDH was used as an internal control. Primers are listed in Supplementary data, Table SI. Melting curve analysis was performed to verify the specificity of the PCR after each run. The procedure was repeated independently six times to ensure the reproducibility of results. Samples with a cycle threshold coefficient of variation value of >5% were retested.

Cell proliferation assay

To test the effect of CM on cell growth, ESCs were seeded into96-well plates at a density of 2 x I03 cells per well. After 24 h of incubation, the growth medium was replaced with CM containing 2% FBS or phenol red-free DMEM/FI2 containing 2% FBS. The cell growth rate was measured using a cell counting kit-8 (CCK8) (Tojindo, Shanghai, China) according to the manufacturer's instructions.

Collagen gel contraction assay

The acid-soluble Type I collagen (Corning, Bedford, MA, USA) was prepared according to the manufacturer's instructions. Twenty-four-well culture plates were coated with I% BSA and incubated for I h at 37°C to create a non-stick surface that prevented gels from attaching to the dishes. To investigate the effects of CM treatment on ESCs, cells were pretreated for 48 h with CM or DMEM/FI2 containing 2% FBS. ESCs were seeded at a concentration of 2.5 x I0 cells/ml into a 2.0 mg/ml Type I collagen solution in PBS containing 0.023 N NaOH. The collagen/cell suspension was vortexed, and added to the BSA-coated plates at 500 ml/well. The solution was allowed to polymerize for 30 min at 37°C. Five hundred microliters of CM or DMEM/ FI2 containing 2% FBS were added to the three-dimensional solidified collagen gels. Plates were returned to the incubator and collagen gel contraction was monitored over 24 h. The surface area of the contracted gels was measured at 0, 4, 8, I2 and 24 h using Image J software. The experiment was performed six times.

ESCs migration and invasion assays

ESCs were treated with CM or control medium for 24 h priorto migration assays. The cells were trypsinized and resuspended at 5 x I04 cells per chamber for migration assays, and I x I05 cells per chamber for invasion assays in DMEM/FI2 (200 ml) containing I% FBS. The cell suspensions were added to the upper chambers with or without coatings of Matrigel in transwells (Corning, NY, USA) consisting of inserts containingan 8 mm pore-size polyethyleneterephthalate (PET) membrane. DMEM/FI2 (750 ml) containing I0% FBS was placed in the lowerchamber. CM was added to the lower chambers of the experimental group. After incubation for 24 h at 37°C, cells that remained in the upper chamberwere removed with a cotton swab, and the membrane was cut off. The side facing the lower chamber was stained with 0.I% crystal violet. The attached cells were counted undera light microscope. The experiment was performed six times.

Mouse model of endometriosis

Animals

Female athymic nude mice (6 weeks old) were purchased from Shanghai Laboratory Animal Co Ltd (Shanghai, China). Mice were maintained in a barrier unit in a well-controlled, pathogen-free environment with regulated cycles of light/dark (I2/I2 h, 23 -25°C). Two weeks of acclimation to the vivarium was allowed before performingany procedures. The nude mouse model of endometriosis was established as described previously (Matsuzaki and Darcha, 20I4). Briefly, human endometrial tissues were washed with sterile serum-free DMEM/F-I2. Nude mice received a subcutaneous injection of

proliferative endometrial fragments (1 -2 mm ) in 200 ml serum-free DMEM/F-I2. All procedures were performed under isoflurane anesthesia. Mice received a daily intraperitoneal injection of 200 mg/kg I7|-estradiol.

Treatment with TGF-b 1, Wnt1 and Ecto-MSC CM

To achieve higher local concentrations of TGF-| I, WntI or Ecto-MSC CM, subcutaneous injection was employed as the administration route based on previous animal experiments (Shinozaki et al., 1997). To investigate the time course offibrosis development in the animal model, endometrial tissues from six patients without endometriosis were implanted subcutaneously into 24 mice. The mice were then sacrificed on Days 7, 14,21 and 28. Next, we evaluated the effects of TGF-| I, WntI and Ecto-MSC CM treatment on fibrosis in endometriotic implants during the development of fibrosis on Day I4 based on our results and a previous study (Matsuzaki and Darcha, 20I3b). Endometrial tissues from eight patients without endometriosis were implanted subcutaneously into 30 mice (Supplementary data, Fig. S2). Mice were then randomly divided into five groups: control, TGF-|I, WntI, TGF-|I + WntI and Ecto-MSC CM. Intra-lesional injections were started on Day I after endometrial tissue implantation and were continued for I4 days. Mice were sacrificed on Day I4 for collection of endometriotic implants. The treatment doses were determined in preliminary experiments.

Histology

Endometrium and endometriotic implants were collected, fixed in I0% formalin -acetic acid and then embedded in paraffin for histopathological examination. Paraffin-embedded tissue sections were stained with Masson trichrome and subjected to immunohistochemisty (IHC) according to common protocols (Zuo et al., 2002). IHC was performed with a rabbit monoclonal antibody against human collagen I (Col-I; I:I500; Abcam). The staining scores of Masson trichrome and IHC were calculated using a computerized image analysis system as described previously (Matsuzaki and Darcha, 20I3b). Parameters were computed per sample in endometriotic implants for Masson trichrome staining: the percentage of stained surface (compared with the counterstained surface), the mean staining intensity and the staining score (percentage of stained surface x mean staining intensity). The density of the mesenchymal Col-I-positive area was calculated to quantify the staining. The entirefield ofthe endometriotic implant in each section was analyzed for Masson trichrome and Col-I staining.

Statistical analysis

The SPSS program version I6 was used for statistical analysis. Comparisons between different groups were made using Student's t-test, one-way analysis of variance or general linear model repeated measures following Scheffe's method. Statistical significance was defined as P < 0.05.

Results

Ecto-MSC CM promotes cell proliferation, migration, invasion and cell-mediated collagen gel contraction

A hallmark of fibrosis is aggressive overgrowth of stromal cells. To determine the effect of Ecto-MSC CM and Euto-MSC CM on stromal cells, we performed in vitro cell-based assays. CCK8 assays showed that Ecto-MSC CM treatment significantly increased the proliferation of Euto- and Ecto-ESCs compared with control and Euto-MSC CM treatment (Fig. IA). Both Euto- and Ecto-ESC-mediated contraction of collagen gels were markedly accelerated by Ecto-MSC CM compared with the control and Euto-MSC CM (Fig. IB). Moreover, treatmentwith Ecto-MSC CM significantly increased cell migration and invasion of Euto- and Ecto-ESCs (Fig. IC).

Figure 1 CM from MSCs promotes cell proliferation, migration, invasion and cell-mediated collagen contraction in Euto- and Ecto-ESCs from patients with ovarian endometrioma. (A) Effects of low-serum medium (Dulbecco's modified Eagle's medium/FI2 with 2% FBS), Euto-MSC CM with 2% FBS and Ecto-MSC CM with 2% FBS on the growth of endometrial stromal cells (Euto-ESCs) (n = 6) and endometriotic stromal cells (Ecto-ESCs) (n = 6) were compared using a cell counting kit-8 (CCK8) assay. (B) Effects of CM from MSCs on Euto-ESC and Ecto-ESC-mediated collagen gel contraction. Collagen gel contraction at 0,4, 8, 12 and 24 h in Euto-ESCs (n = 6) and Ecto-ESCs (n = 6). Representative photomicrographs of contracted gels at 8 h in Euto- and Ecto-ESCs treated with Euto-MSC CM, Ecto-MSC CM or control medium are shown. (C) Number of migrated cells/mm2 in control-and MSC CM-treated Euto-ESCs (n = 6) and Ecto-ESCs (n = 6) of the same patients. Representative photomicrographs of cell migration in control- and or MSC CM-treated Euto- and Ecto-ESCs of the same patients are shown (upper panels). Number of invasive cells/mm2 for control- and MSC CM-treated Euto-ESCs (n = 6) and Ecto-ESCs (n = 6). Representative photomicrographs of cell invasion in control-, Euto- and Ecto-MSC CM-treated Euto- and Ecto-ESCs of the same patients are shown (lower panels). *P < 0.05 versus control-treated Euto-ESCs; $P < 0.05 versus control-treated Ecto-ESCs; §P < 0.05, Ecto-MSC CM-treated cells versus Euto-MSC CM treated cells; #P < 0.05 control-treated Ecto-ESCs versus control-treated Euto-ESCs. Data are presented as the mean + SEM.

Ecto-MSC CM enhances fibrotic gene expression in ESCs from patients with ovarian endometrioma

Another hallmark of fibrosis is overproduction of collagen. To determine the effect of Ecto-MSC CM on fibrogenesis in stromal cells, CM was concentrated, serially diluted and applied to stromal cells for48 h. Expression offibrotic markers was assessed by real-time reverse-transcription-PCR (RT-PCR), western blotting (WB) and immunofluorescence staining. Ecto-MSC CM significantly up-regulated a-SMA, Col-I, CTGF and fibro-nectin (FN) expression in stromal cells compared with the control and Euto-MSC CM, and their expression levels in Ecto-ESCs were even higher than those in Euto-ESCs (Fig. 2A and B). Serial dilutions of CM promoted fibrogenesis at various degrees (Supplementary data, Fig. S3). Immunofluorescence staining showed that Ecto-MSC CM significantly increased the percentage of a-SMA-positive ESCs compared with the control (Fig. 2C).

Ecto-MSCs secrete higher levels of profibrotic factors TGF-pl and Wntl than Euto-MSCs from the same patients

To identify the fibrotic factors secreted by MSCs, mRNA and protein as well as concentrated CM were collected from Euto- and Ecto-MSCs of 15 ovarian endometrioma patients. Real-time RT-PCR and WB showed that Ecto-MSCs expressed higher levels of TGF-p 1 and Wntl compared with Euto-MSCs from the same patients (Fig. 3A and B), while no difference in Wnt3, MMP2, MMP3, MMPI0, VEGFA, VEGFB or VEGFC mRNA levels was found between Euto- and Ecto-MSCs, MMP9 and MMP7were undetectable in most samples (Supplementary data, Fig. S4).

TGF-bl and Wntl induce fibrosis via nuclear translocation of b-catenin and activation ofthe Wnt/b-catenin pathway

To determine whether MSC-secreted TGF-p I and Wntl contribute to fibrogenesis, human recombinantTGF-p I and Wntl, TGF-p l-neutraliz-ing antibody and the Wntl negative regulator Dickkopf-related protein I (Dkkl) were used to stimulate stromal cells. Our data showed that TGF-pl, Wntl and Ecto-MSC CM significantly increased the mRNA levels of fibrotic markers including a-SMA and CTGF, with a similar trend toward increased protein levels. Interestingly, both mRNA and protein expression levels of a-SMA were higher in the combination of TGF-pl and Wntl group compared with the Ecto-MSC CM group (Fig. 4A and B). However, the profibrotic property of Ecto-MSC CM was attenuated by the TGF-p l-neutralizing antibody or Dkkl. Furthermore, compared with control, blocking TGF-pl or Wntl partially blocked the stimulatory effect of Ecto-MSC CM on a-SMA and CTGF expression (Fig. 4A and B).

Activation of the Wnt/p-catenin pathway manifests as nuclear accumulation of p-catenin and subsequently elevated transcriptional levels of Axin-2. To explore the potential molecular mechanism ofthe profibrotic effect of Ecto-MSC CM on ESCs, we characterized the Wnt/p-catenin signaling pathway in ESCs. Immunofluorescence was used to visualize the localization of p-catenin at various time points (0, l, 2 and 6 h) after incubation with Ecto-MSC CM. As shown in Fig. 4C, p-catenin was mainly localized on the membrane of ESCs before incubation with

Ecto-MSC CM. After l h incubation, membranous p-catenin started to enter the cytoplasm of cells. After incubation with Ecto-MSC CM for 2 h, p-catenin was translocated into the nucleus. Interestingly, p-catenin appeared in the membrane of ESCs again after 6 h of incubation with Ecto-MSC CM (Fig.4C). Similarly, increased expression ofintra-nuclear p-catenin was detected at l and 2 h after Ecto-MSC CM treatment by WB analysis (Fig. 4D). No increase in p-catenin expression was observed in control ESCs. To investigate whether the nuclear translocation of p-catenin could up-regulate its downstream targets, Axin-2 mRNA level was compared in Ecto-MSC CM-treated and control cells by real-time RT-PCR. The results showed that Ecto-MSC CM significantly increased the expression of Axin-2 in ESCs (Fig. 4E).

Ecto-MSC CM promotes fibrosis in a mouse model of endometriosis

The in vitro results demonstrated that Ecto-MSCs promoted the formation of fibrosis. Thus, we further assessed this effect by in vivo mouse experiments. All mice developed endometriotic lesions showing typical characteristics of endometriosis with glandular structures and stroma. To monitor the overall wellbeing of mice treated with the vehicle alone, TGF-p, Wnt l, TGF-p plus Wnt l or Ecto-MSC CM, the mice were monitored daily, and body weights were recorded. After the experimental period, the mice were sacrificed, and cysts were collected for Masson tri-chrome staining and Col-I IHCto determine the fibrosis degree. No difference was found in thegrowth rates of cysts between each group. Scores for Masson staining and Col-l IHC were increased rapidly in endometriotic implants on Day l4 (Fig. 5A). Thus, we evaluated the effects of the treatments on the fibrosis of endometriotic implants during this key period of time when fibrosis was establishing rapidly. The treatments were initiated on Day l. The staining scores were significantly higher in cytokine- and Ecto-MSC CM-treated mice than in control mice (Fig. 5B).

Discussion

Endometriosis is characterized by dense fibrous tissue surrounding the endometrial glands and stroma (Giudice and Kao, 2004). The post-surgery follicle reserve of young patients with ovarian endometrioma depends on the cyst diameter and degree of fibrosis (Kitajima et al., 20ll). A previous study showed that high proportions of nerve encapsulation in fibrosis of deep-infiltrating endometriosis result in high preoperative pain scores (Anaf et al., 2000). Ovarian parenchymal loss at the time of surgery, which is crucial to ovarian reserve, is related to the cyst diameter and degree offibrosis (Kitajima etal., 20ll). Understanding the mechanisms of fibrogenesis is of great importance in endometriosis, especially ovarian endometrioma.

ESCs are thought to be the most common cell type in endometriotic tissue, which deposit the collagen matrix in endometriotic implants. Our results demonstrated that Ecto-MSC CM promoted cell proliferation, migration, invasion and collagen gel contraction of Euto- and Ecto-ESCs, which are critical aspects of fibrogenesis (Ding etal., 20l3; Matsuzaki and Darcha, 20l4). Previous reports have demonstrated inhibitory effects of MSCs on the growth of target cells (Jones et al., 2007; Li et al., 2009). However, our results are consistent with many studies showing that MSCs induce target cell proliferation and migration (Chen et al., 2008; Chuah et al., 20l5; Wajid et al., 20l5).

Figure 2 CM from MSCs promotes fibrotic marker expression in ESCs from patients with ovarian endometrioma. (A) Effects of CM on mRNA expression of a-smooth muscle actin (a-SMA), collagen I (Col-I), CTGF and FN in Euto-ESCs (n = 6) and Ecto-ESCs (n = 6). *P < 0.05 versus control-treated Euto-ESCs; $P < 0.05 versus control-treated Ecto-ESCs; §P < 0.05, Ecto-MSC CM-treated cells versus Euto-MSC CM-treated cells; #P < 0.05 control-treated Ecto-ESCs versus control-treated Euto-ESCs. Data are presented as the mean + SEM. Expression levels of a-SMA, Col-I, CTGF and FN mRNA are relative to the expression level of the reference gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). (B) Effects of CM on protein levels of a-SMAand CTGF in Euto-ESCs and Ecto-ESCs were assessed by WB.The upper panels show WB data, andthelowertwo panels show histograms of protein expression showing the densitometry value. (C) Immunostainingof a-SMAand nuclear staining in Euto-and Ecto-ESCs untreated ortreated with Ecto-MSC CM for 48 h. Scale bars, 100 mm.

In ourstudy, treatment of Euto-and Ecto-ESCs with Ecto-MSC CM significantly enhanced expression of genes involved in fibrogenesis compared with Euto-MSC CM and control. The high fibrotic gene expression suggests a potential for enhanced collagen synthesis by

ESCs. CM-treated MSCs had clearly visible a-SMA-positive stress fibers, demonstrating that CM induced myofibroblast differentiation (Tomasek et al., 2002). Our results are consistent with a previous study demonstrating that treatment with MSC CM

Figure 3 Detection of TGF-ß 1 and Wntl expression in Euto-and Ecto-MSCs from patients with ovarian endometrioma. (A) Paired comparison showed increased TGF-ß 1 andWntl expression in Ecto-MSCs (n = 15) compared with that in Euto-MSCs (n = 15) as detected by real-time RT-PCR. *P < 0.05, paired t-test. (B) TGF-ß 1 and Wntl expression was determined in four cases of matched Euto- and Ecto-MSCs by WB. The upper panels show WB data, and the lower panel shows histograms of protein expression showing the densitometry values. GAPDH was used as an endogenous control.

significantly increases cell proliferation and Col-I expression (Salazar et al., 2009).

At least 34 genes are associated with collagen production (Matsuzaki and Darcha, 2013b). Previous studies have reported that MSCs release Wnts, TGF-ß, MMPs and VEGFs that might be associated with the development of fibrosis (Salazar et al., 2009; Dzafic et al., 2014; Hsu et al., 2014). The most biochemically active endometriosis lesions are the proposed early lesions and, with disease progression, the endometriotic lesions undergo remodeling and become less biochemically active (Ueda et al., 2002). MMPs are endopeptidases whose function is to degrade ECM. The MMP system plays a critical role during the progression of endometriosis by favoring cell invasion and ECM remodeling (Sharpe-Timms and Cox, 2002). However, little is known about the expression profile of MMPs in MSCs of endometriosis. A recent study showed that Ecto-MSCs and Euto-MSCs express MMP2, MMP3 and MMP9 (Koippallil etal., 2015).

In our present study, both profibrotic factors and antifibrotic MMPs were detected in MSCs. Moreover, the levels of profibrotic factors TGF-ßl andWntl werehigherin Ecto-MSCs than in Euto-MSCs, suggesting a possible contribution tofibrogenesis in patients with ovarian endometrioma. Some reports have indicated that TGF-ß and Wntl may be involved in the molecular and cellular mechanisms of fibrogenesis in endometriosis (Matsuzaki and Darcha, 20 l3a,b) and other fibrotic diseases (Salazar et al., 2009; Akhmetshina et al., 2012). The CM collected from Ecto-MSCs, which contained secreted TGF-ßl and Wntl, promoted the expression of fibrotic markers, whereas the TGF-ß l-neutralizing

antibody and Wntl negative regulator Dkkl decreased their expression. The combined effect of TGF-ßl and Wntl tended to be the most potent stimulant for fibrogenesis compared with control and CM. This may be due to the presence of antifibrotic MMPs in CM, although no expression difference was found between Euto-MSCs and Ecto-MSCs. Our findingssuggestthat TGF-ßl andWntl secreted by Ecto-MSCs modulate fibrotic development. Compared with Euto-MSC, higherlevels ofsecreted TGF-ß l and Wnt l in Ecto-MSCs may disrupt the balance between fibrogenesis and fibrolysis, resulting in endometriosis fibrosis. To our knowledge, few studies have focused on MSCs in endometriosis (Kao et al., 20 ll ; Koippallil etal.,20l 5). This is thefirst study to demonstrate the paracrine function of MSCs in ovarian endometrioma. Our results indicate unique paracrine characteristics of Ecto-MSCs, which may underlie the pathogenesis of ovarian endometrioma.

To identify how the unique paracrine characteristics of Ecto-MSCs induce fibrosis, we focused on TGF-ßl and Wntl. Previous studies have demonstrated that both TGF-ßl and Wntl activate Wnt/ ß-catenin signaling (Akhmetshina et al., 20l2). The Wnt/ß-catenin pathway is involved in cell development and tissue self-renewal (Grigor-yan et al., 2008; Klaus and Birchmeier, 2008; Wend et al., 20l0). Pathologically activated canonical Wnt signaling has been implicated in various fibrotic diseases (Konigshoff et al., 2008; Henderson et al., 20l0; Akhmetshina et al., 20l2).

Stimulation with CM induced nuclear accumulation of ß-catenin and increased expression of its downstream target Axin-2 in cultured ESCs. Activation of the canonical Wnt pathway appears to be a general feature

Figure 4 TGF-p l and Wntl in Ecto-MSC CM induce fibrosis via nucleartranslocation of p-catenin and activation of the Wnt/p-catenin pathway. (A and B) Real-time RT-PCRand WB analyses of a-SMA and CTGF expression in Euto-ESCs and Ecto-ESCs treated by vehicle alone, TGF-p l (5 ng/ml), Wntl (l00 ng/ml), Ecto-MSC CM, a TGF-p l-neutralizing antibody or Wntl negative regulator Dickkopf-related protein l (Dkkl, 250 ng/ml). The lower panel (B) shows WB data, and the upper panel shows histograms of protein expression showing the densitometry values. *P < 0.05 versus control-treated cells. #P < 0.05 versus Ecto-MSC CM-treated cells. (C) Representative immunofluorescence images showing translocation of p-catenin from the membrane to the nucleus in ESCs treated with Ecto-MSC CM compared with cells at 0 h. Green indicates p-catenin; blue indicates DAPI-stained nuclei. Scale bars, l0 mm.

(D) Representative WB showing translocation of p-catenin from the membrane to the nucleus in ESCs treated with Ecto-MSC CM but not in the control at the indicated time points. The upper panel shows WB data, and the lower panel shows histograms of protein expression showing the densitometry values.

(E) Axin-2 mRNA expression in ESCs untreated or treated with Ecto-MSC CM for0,4, 8, l2, 24,48 or 72 h (n = 6). *P < 0.05 versus untreated controls.

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V TGF-P Wnt1 TGF-ß+Wnt1 CM

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Figure 5 TGF-b 1, Wnt1 and Ecto-MSCCM promote fibrogenesis in a mouse model of endometriosis. (A) Staining scores of Massontrichrome and Col-I IHC in a time course offibrosis development (Days 0,7, 14,21 and 28) in control-treated mice (treated every otherday between Days 1 and 28). (B) Staining scores of Massontrichrome and Col-I IHC in mice treated by vehicle, TGF-b l,Wntl, TGF-b 1 plus Wnt1 orEcto-MSC CM on Day 14 (treated every other day between Days 1 and 14). *P < 0.05 versus control-treated mice on Day 14. V, vehicle-treated mice (n = 6). Scale bars, 100 mm.

of fibrotic diseases. It occurs in both systemic fibrotic diseases and isolated organ fibrosis (He et al., 20(0 ; Wei et al., 2011; Akhmetshina et al., 2012). Wnt proteins act on target cells by binding to cell surface receptors that transduce a signal to ß-catenin via disheveled proteins (Tamai et al., 2000). In normal cells, most ß-catenin is localized at the plasma membrane. ß-Catenin is inactivated by a multimolecular complex containing the adenomatous polyposis coli protein, axin and glycogen synthase kinase-3b (GSK-3b). Canonical Wnt signaling inhibits ß-catenin degradation by inacti-vation of GSK-3b. Hypophosphorylated ß-catenin then translocates to the nucleus, where it binds to transcription factors and initiates transcription of target genes such as cyclin DI (CCNDI), v-myc avian myelocytomatosis viral oncogene homolog (MYC) and Axin-2 (Fu et al., 201 I). TGF-ß also

mediates b-catenin translocation from the plasma membrane into the nucleus and indirectly activates Wnt/p-catenin signaling pathways (Labbe etal.,200 ; Liu etal., 2006), although the mechanism is not fully understood. The overexpression of fibrotic genes in Euto- and Ecto-ESCs treated with CM containing TGF-b 1 andWntl may result from activation of the canonical Wnt pathway with nuclear accumulation of b-catenin and increased Axin-2 level. Activation of the canonical Wnt pathway and its potent profi-brotic effects suggest that the Wnt pathway might be a potential target for novel antifibrotic approaches.

The animal experiments showed that treatment with TGF-b 1, Wntl or Ecto-MSC CM promoted the progression offibrosis during the development of endometrioma. Thus, our results demonstrated that TGF-b 1

and Wntl were involved in the progression of fibrosis. These findings are in accordance with a previous study conducted in a mouse model of skin fibrosis (Akhmetshina et al., 20l2). Co-administration of ICG-00l, a selective inhibitor of Wnt/p-catenin-CBP-dependent transcription and bleomycin, prevents fibrosis, and late administration is even able to reverse established fibrosis (Henderson et al., 20l0). These findings and our present results support an important role of Ecto-MSCs and the Wnt/p-catenin signaling pathway in the pathogenesis offibrosis.

In conclusion, our study showed that treatment with Ecto-MSC CM promoted proliferation, migration, invasion and collagen gel contraction of Ecto-ESCs in ovarian endometrioma, which are all critical aspects of fibrogenesis. Ecto-MSC CM significantly increased the expression of genes involved in fibrogenesis in ESCs through the Wnt/p-catenin pathway by paracrine production ofTGF-p l and Wntl. Animal experiments showed that TGF-p l, Wntl and Ecto-MSC CM promoted the progression of fibrosis. The present results provide further evidence that Ecto-MSCs are key participants in the development of ovarian endometrioma. Before clinical treatment using MSCs for traumatic diseases such as intrauterine adhesion, the possibility of more serious adhesion should be taken into consideration. Confirmation of the involvement of stem cells in ovarian endometrioma fibrogenesis would indicate that surgery may be needed in the early stages of ovarian endometrioma to prevent fibrosis progression.

Supplementary data

Supplementary data are available at http://humrep.oxfordjournals.org/.

Acknowledgements

We are most grateful to all ofthe patients who participated in the present study.

Authors' roles

J.L. and Y.D. were involved in sample collection, experiments, acquisition, analysis and interpretation of data and drafting the article. Y.J., H.Z. and S.Z. were involved in concept and design and critical revision ofthe article. All authors read and approved the final version ofthe paper.

Funding

This study was supported in part by the National Natural Science Foundation of China (8l47l505 and 8l270657).

Conflict of interest

None declared.

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