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 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 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 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 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).