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Cited by 138Endometriosis Research and TreatmentEndometrial and Cervical Cancer TreatmentsEctopic Pregnancy Diagnosis and Management

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Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

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Matsuzaki, Sachiko, Darcha, Claude·2013·PLoS ONE
DOI 10.1371/journal.pone.0076808PMC PMC3790725
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Abstract

BACKGROUND: During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified. OBJECTIVES: The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice. METHODS: Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice. RESULTS: Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis. CONCLUSION: Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.

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Figure 1 Effects of β-catenin siRNA on fibrotic markers in endometrial and endometriotic stromal cells from patients with endometriosis. Effects of β-catenin siRNA on the mRNA expression of αSMA, Col-I, CTGF, and FN in endometriotic (A) and endometrial (B) stromal cells with or without TGF-β1 stimulation. C: control siRNA-transfected cells; ß: ß-catenin siRNA-transfected cells. *: p<.05 versus control (C) cells without TGF-β1 stimulation. **: p<.05 versus control (C) cells with TGF-β1 stimulation. Numerical values are presented as the mean + SEM. Expression levels of αSMA, Col-I, CTGF, and FN mRNA are given relative to the expression level of the reference gene, GAPDH. Endometriotic stromal cells (n=10), endometrial stromal cells (n=10).
Figure 1 Effects of β-catenin siRNA on fibrotic markers in endometrial and endometriotic stromal cells from patients with endometriosis. Effects of β-catenin siRNA on the mRNA expression of αSMA, Col-I, CTGF, and FN in endometriotic (A) and endometrial (B) stromal cells with or without TGF-β1 stimulation. C: control siRNA-transfected cells; ß: ß-catenin siRNA-transfected cells. *: p<.05 versus control (C) cells without TGF-β1 stimulation. **: p<.05 versus control (C) cells with TGF-β1 stimulation. Numerical values are presented as the mean + SEM. Expression levels of αSMA, Col-I, CTGF, and FN mRNA are given relative to the expression level of the reference gene, GAPDH. Endometriotic stromal cells (n=10), endometrial stromal cells (n=10).CC BY · PubMed Central
Figure 2 Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers in endometrial and endometriotic stromal cells from patients with endometriosis. A, B: Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on the mRNA expression of αSMA, Col-I, CTGF, and FN in endometriotic (A) (n=10) and endometrial (B) (n=10) stromal cells with or without TGF-β1 stimulation. *: p<.05 versus vehicle-treated controls without TGF-β1 stimulation. **: p<.05 versus vehicle-treated controls with TGF-β1 stimulation. C: Representative photomicrographs of endometriotic stromal cells after treatment with vehicle or CGP049090 (6.25 µM) for 24 h and immunostained for αSMA (green) and nuclei (blue). Scale bar, 100 μm. D: Percentage of αSMA-positive endometriotic stromal cells (n=10) after treatment with vehicle, PKF 115-584 (6.25 µM), or CGP049090 (6.25 µM) for 24 h. *: p<.05 versus vehicle-treated controls. Numerical values are presented as the mean + SEM. Expression levels of αSMA, Col-I, CTGF, and FN mRNA are given relative to the expression level of the reference gene, GAPDH.
Figure 2 Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers in endometrial and endometriotic stromal cells from patients with endometriosis. A, B: Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on the mRNA expression of αSMA, Col-I, CTGF, and FN in endometriotic (A) (n=10) and endometrial (B) (n=10) stromal cells with or without TGF-β1 stimulation. *: p<.05 versus vehicle-treated controls without TGF-β1 stimulation. **: p<.05 versus vehicle-treated controls with TGF-β1 stimulation. C: Representative photomicrographs of endometriotic stromal cells after treatment with vehicle or CGP049090 (6.25 µM) for 24 h and immunostained for αSMA (green) and nuclei (blue). Scale bar, 100 μm. D: Percentage of αSMA-positive endometriotic stromal cells (n=10) after treatment with vehicle, PKF 115-584 (6.25 µM), or CGP049090 (6.25 µM) for 24 h. *: p<.05 versus vehicle-treated controls. Numerical values are presented as the mean + SEM. Expression levels of αSMA, Col-I, CTGF, and FN mRNA are given relative to the expression level of the reference gene, GAPDH.CC BY · PubMed Central
Figure 3 Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on stromal cell-mediated collagen gel contraction. A–F: Collagen gel contraction at 0, 4, 8, 12, and 24 h in matched endometrial and endometriotic stromal cells from the same patient (A), *: p<.05: EES:deep endometriosis versus EES: ovarian endometriosis, endometrial stromal cells of patients with and without endometriosis (B), *: p<.05: versus EuEs: patients with endometriosis, treated and vehicle-treated endometrial stromal cells of patients with (C), *: p<.05: versus treated EuEs, and without endometriosis (D), *: p<.05: versus treated EuEs, treated and vehicle-treated endometriotic stromal cells (E, F), *: p<.05: versus treated EES, and G, H: Representative photomicrographs of contracted gels taken at 24 h in endometriotic stromal cells with and without treatment. Numerical values are presented as the mean + SEM. EES: endometriotic stromal cells; EuES: endometrial stromal cells. Endo (-): endometrium of patients without endometriosis. Endo (+): Endometrium of patients with endometriosis. EuES: ovarian endometriosis (n=10); EES: ovarian endometriosis (n=10). EuES: deep endometriosis (n=10); EES: deep endometriosis (n=10). EuES: patients with endometriosis (n=20); EuES: patients without endometriosis (n=10).
Figure 3 Effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on stromal cell-mediated collagen gel contraction. A–F: Collagen gel contraction at 0, 4, 8, 12, and 24 h in matched endometrial and endometriotic stromal cells from the same patient (A), *: p<.05: EES:deep endometriosis versus EES: ovarian endometriosis, endometrial stromal cells of patients with and without endometriosis (B), *: p<.05: versus EuEs: patients with endometriosis, treated and vehicle-treated endometrial stromal cells of patients with (C), *: p<.05: versus treated EuEs, and without endometriosis (D), *: p<.05: versus treated EuEs, treated and vehicle-treated endometriotic stromal cells (E, F), *: p<.05: versus treated EES, and G, H: Representative photomicrographs of contracted gels taken at 24 h in endometriotic stromal cells with and without treatment. Numerical values are presented as the mean + SEM. EES: endometriotic stromal cells; EuES: endometrial stromal cells. Endo (-): endometrium of patients without endometriosis. Endo (+): Endometrium of patients with endometriosis. EuES: ovarian endometriosis (n=10); EES: ovarian endometriosis (n=10). EuES: deep endometriosis (n=10); EES: deep endometriosis (n=10). EuES: patients with endometriosis (n=20); EuES: patients without endometriosis (n=10).CC BY · PubMed Central
Figure 4 Effects of Wnt3a treatment on profibrotic responses in endometrial stromal cells of patients without endometriosis. A: Axin2 mRNA expression in endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 0, 4, 8, 12, 24, 48, or 72 h (n=5). *: p<.05 versus vehicle-treated controls. B: Effects of β-catenin siRNA on the mRNA expression of αSMA, Col-I, CTGF, and FN in endometrial stromal cells treated with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) for 24 h (n=10). C: control siRNA-transfected cells; ß: ß-catenin siRNA-transfected cells. *: p<.05 versus control (C) cells without stimulation. **: p<.05 versus control (C) cells with Wnt3a stimulation. ***: p<.05 versus control (C) cells with TGF-β1 stimulation. C: Cell proliferation of endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 72 h (n=5). *: p<.05 versus vehicle-treated controls. D: Cell migration of endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 72 h (n=5). *: p<.05 versus vehicle-treated controls. E: Collagen gel contraction of stromal cells treated for 24 h with vehicle or Wnt3a (150 ng/mL) (n=5), and representative photomicrographs of contracted gels taken at 24 h in stromal cells treated with vehicle or Wnt3a (150 ng/mL). *:p<.05 versus vehicle-treated controls. F: Percentage of αSMA-positive cells treated for 24 h with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) (n=5). *:p<.05 versus vehicle-treated controls. G: Representative photomicrographs of endometrial stromal cells from the same patient after treatment with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) for 24 h and stained for f-actin (red) or αSMA (green) and nuclei (blue). Scale bar, 100 μm. Numerical values are presented as the mean + SEM. Expression levels of axin2, αSMA, Col-I, CTGF, and FN mRNAs are given relative to the expression of the reference gene, GAPDH.
Figure 4 Effects of Wnt3a treatment on profibrotic responses in endometrial stromal cells of patients without endometriosis. A: Axin2 mRNA expression in endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 0, 4, 8, 12, 24, 48, or 72 h (n=5). *: p<.05 versus vehicle-treated controls. B: Effects of β-catenin siRNA on the mRNA expression of αSMA, Col-I, CTGF, and FN in endometrial stromal cells treated with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) for 24 h (n=10). C: control siRNA-transfected cells; ß: ß-catenin siRNA-transfected cells. *: p<.05 versus control (C) cells without stimulation. **: p<.05 versus control (C) cells with Wnt3a stimulation. ***: p<.05 versus control (C) cells with TGF-β1 stimulation. C: Cell proliferation of endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 72 h (n=5). *: p<.05 versus vehicle-treated controls. D: Cell migration of endometrial stromal cells treated with vehicle or Wnt3a (150 ng/mL) for 72 h (n=5). *: p<.05 versus vehicle-treated controls. E: Collagen gel contraction of stromal cells treated for 24 h with vehicle or Wnt3a (150 ng/mL) (n=5), and representative photomicrographs of contracted gels taken at 24 h in stromal cells treated with vehicle or Wnt3a (150 ng/mL). *:p<.05 versus vehicle-treated controls. F: Percentage of αSMA-positive cells treated for 24 h with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) (n=5). *:p<.05 versus vehicle-treated controls. G: Representative photomicrographs of endometrial stromal cells from the same patient after treatment with vehicle, Wnt3a (150 ng/mL), or TGF-β1 (5 ng/mL) for 24 h and stained for f-actin (red) or αSMA (green) and nuclei (blue). Scale bar, 100 μm. Numerical values are presented as the mean + SEM. Expression levels of axin2, αSMA, Col-I, CTGF, and FN mRNAs are given relative to the expression of the reference gene, GAPDH.CC BY · PubMed Central
Figure 5 Effects of CGP049090 treatment on fibrosis in a mouse endometriosis model. A-D: Staining score for Sirius Red (A, B) or Masson Trichrome (C, D) staining in a time course study of fibrosis development (on days 0, 7, 14, 21, and 28) (A, C) and vehicle-treated and CGP049090 (2 mg/kg)-treated mice (Groups I and II: treated once a day between days 7 and 21 for 14 days; Groups III and IV: treated once a day between days 14 and 28 for 14 days) (B, D). *: p<.05 versus endometrium on day 0 and endometriotic implants on day 7. **: p<.05 versus vehicle-treated mice. Day 0 (endometrium) (n=10), Days 7 (n=10), 14 (n=10), 21 (n=10), or 28 (n=10). V: vehicle-treated mice; T: CGP049090 (2 mg/kg)-treated mice. G1: Group I (n=10), GII: Group II (n=10), GIII: Group III (n=10), GIV: Group IV (n=10). E-G: Representative photomicrographs of the endometrium stained with hematoxylin and eosin, Sirius Red, or Masson Trichrome on day 0 (E), or of endometriotic implants of mice treated for 14 days with vehicle alone (F) or with CGP049090 (G) at day 21. Scale bars (a-c, g-i, m-o: 200 μm; d-f, j-l, p-r: 50 μm).
Figure 5 Effects of CGP049090 treatment on fibrosis in a mouse endometriosis model. A-D: Staining score for Sirius Red (A, B) or Masson Trichrome (C, D) staining in a time course study of fibrosis development (on days 0, 7, 14, 21, and 28) (A, C) and vehicle-treated and CGP049090 (2 mg/kg)-treated mice (Groups I and II: treated once a day between days 7 and 21 for 14 days; Groups III and IV: treated once a day between days 14 and 28 for 14 days) (B, D). *: p<.05 versus endometrium on day 0 and endometriotic implants on day 7. **: p<.05 versus vehicle-treated mice. Day 0 (endometrium) (n=10), Days 7 (n=10), 14 (n=10), 21 (n=10), or 28 (n=10). V: vehicle-treated mice; T: CGP049090 (2 mg/kg)-treated mice. G1: Group I (n=10), GII: Group II (n=10), GIII: Group III (n=10), GIV: Group IV (n=10). E-G: Representative photomicrographs of the endometrium stained with hematoxylin and eosin, Sirius Red, or Masson Trichrome on day 0 (E), or of endometriotic implants of mice treated for 14 days with vehicle alone (F) or with CGP049090 (G) at day 21. Scale bars (a-c, g-i, m-o: 200 μm; d-f, j-l, p-r: 50 μm).CC BY · PubMed Central

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Metadata

Authors
Matsuzaki, S., & Darcha, C.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • Centre Hospitalier Universitaire de Clermont-Ferrand (FR)
Corresponding author
Matsuzaki, Sachiko
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-10-04
Volume
8
Issue
10
Pages
e76808
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0076808
PMC
PMC3790725
OpenAlex
W2105973884
Cited by
138
References
29
Citations by year
2026: 7 · 2025: 14 · 2024: 13 · 2023: 8 · 2022: 7 · 2021: 7 · 2020: 13 · 2019: 11 · 2018: 12 · 2017: 14 · 2016: 13 · 2015: 12 · 2014: 6
FWCI
12.52
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Topics
  • Health Sciences › Medicine › Reproductive Medicine › Endometriosis Research and Treatment
  • Health Sciences › Medicine › Obstetrics and Gynecology › Endometrial and Cervical Cancer Treatments
  • Health Sciences › Medicine › Obstetrics and Gynecology › Ectopic Pregnancy Diagnosis and Management
Keywords
endometriosis, fibrosis, Wnt/β-catenin signaling, endometrial stromal cells, Wnt3a, collagen gel contraction, type I collagen, alpha-smooth muscle actin, connective tissue growth factor, fibronectin, xenograft models, fibrosis prevention, chronic pelvic pain, scar formation, TGF-β1
MeSH terms
Actins, Adult, Animals, Collagen, Disease Models, Animal, Endometriosis, Female, Fibrosis, Humans, Perylene, Stromal Cells, RNA Interference, Wnt Proteins, beta Catenin, Connective Tissue Growth Factor, Young Adult, Wnt Signaling Pathway, Heterografts

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