Telocyte-Derived Exosomes Provide an Important Source of Wnts That Inhibits Fibrosis and Supports Regeneration and Repair of Endometrium

Abstract

Intrauterine adhesions (IUAs) often occurred after common obstetrical and gynecological procedures or infections in women of reproductive age. It was characterized by the formation of endometrial fibrosis and prevention of endometrial regeneration, usually with devastating fertility consequences and poor treatment outcomes so far. Telocytes (TCs), as a novel interstitial cell type, present in female uterus with in vitro therapeutic potential in decidualization-defective gynecologic diseases. This study aims to further investigate the role of TC-derived Wnt ligands carried by exosomes (Exo) in reversal of fibrosis and enhancement of regeneration repair in endometrium. IUA cellular and animal models were established from endometrial stromal cells (ESCs) and mice, followed with treatment of TC-conditioned medium (TCM) or TC-derived Exo. In cellular model, fibrosis markers (collagen type 1 alpha 1 [COL1A1], fibronectin [FN], and α-smooth muscle actin [α-SMA]), angiogenesis (vascular endothelial growth factor [VEGF]), and pathway protein (β-catenin) were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting (WB), and immunofluorescence. Results showed that, TCs (either TCM or TC-derived Exo) provide a source of Wnts that inhibit cellular fibrosis, as evidenced by significantly elevated VEGF and β-catenin with decreased fibrotic markers, whereas TCs lost salvage on fibrosis after being blocked with Wnt/β-catenin inhibitors (XAV939 or ETC-159). Further in mouse model, regeneration repair (endometrial thickness, number of glands, and fibrosis area ratio), fibrosis markers (fibronectin [FN]), mesenchymal-epithelial transition (MET) (E-cadherin, N-cadherin), and angiogenesis (VEGF, microvessel density [MVD]) were studied by hematoxylin-eosin (HE), Masson staining, and immunohistochemistry. Results demonstrated that TC-Exo treatment effectively promotes regeneration repair of endometrium by relieving fibrosis, enhancing MET, and angiogenesis. These results confirmed new evidence for therapeutic perspective of TC-derived Exo in IUAs.

Keywords: endometrial stromal cells (ESCs); exosome (Exo); fibrosis; intrauterine adhesions (IUAs); telocytes (TCs).

Figure 1.

Morphology and immunophenotype of mouse uterine ESCs and TCs observed by immunofluorescence staining and inverted fluorescent microscope. Nuclei were counterstained with DAPI (blue). (A) Immunofluorescence staining of ESCs was positive for Vimentin (green) and negative for cytokeratin. Scale bar = 100 μm. (B) TCs were typical mesenchymal cells with a characteristic oval cellular body and long extensions named telopodes (Tps), composed of alternating thin (podomer) and thick (podom) segments under high-magnification fields. Double positive for Vimentin (green) and CD34 (red). In the merged image, both immunofluorescence signals overlapped each other along entire cellular body and Tps, with the podomer, and podom indicated. Scale bar = 20 μm. (C) Immunofluorescence staining of TCs was positive for Vimentin (green) and CD34 (red) under low-magnification fields. Scale bar = 100 μm. ESC: endometrial stromal cell; TC: telocyte; DAPI: 4’6-diamidino-2-phenylindole.

Figure 2.

Time and dose-dependent effects of TGF-β1 on fibrosis markers expression (COL1A1, FN, and α-SMA) in regular ESCs analyzed by WB. β-Tubulin served as loading control for WB. Error bars indicated standard deviation (SD) from three independent experiments (biological replicates). ^#P < 0.05 compared with the control group (0 ng/ml or 0 h). *P < 0.05, by one-way ANOVA with Tukey’s post hoc test, whereas ns indicates non-significant. (A) WB analysis showed that three markers in regular ESCs reach peak value at 15 ng/ml TGF-β1, with no statistical difference at 20 ng/ml. (B) 15 ng/ml TGF-β1 can yield peak value for three markers at 48 h, with no statistical difference at 72 h. COL1A1: collagen type 1 alpha 1; FN: fibronectin; α-SMA: α-smooth muscle actin; ESC: endometrial stromal cell; WB: Western blotting; ANOVA: analysis of variance; TGF-β1: transforming growth factor-beta 1.

Figure 3.

TCM exposure reversed and downregulated fibrosis markers (COL1A1, FN, and α-SMA) in TGF-β1-treated ESCs. Error bars indicated SD from three independent experiments (biological replicates). *P < 0.05, by one-way ANOVA with Tukey’s post hoc test. mRNA (A) and protein (B) expression decreased significantly in TCM group (TGF-β1-ESCs + TCM), as compared with control (TGF-β1-ESCs + DMEM/F12) and blank cells (regular ESCs + DMEM/F12). Relative mRNA expression was determined by normalizing to GAPDH levels for qRT-PCR. β-tubulin served as loading control for WB. (C) Immunofluorescence staining demonstrated the down-expression of three proteins. Scale bar = 100 μm. TCM: TC-conditioned medium; COL1A1: collagen type 1 alpha 1; FN: fibronectin; α-SMA: α-smooth muscle actin; ESC: endometrial stromal cell; SD: standard deviation; WB: Western blotting; ANOVA: analysis of variance; mRNA: messenger RNA; qRT-PCR: quantitative real-time polymerase chain reaction; TGF-β1: transforming growth factor-beta 1; DMEM: Dulbecco’s modified eagle medium.

Figure 4.

Identification and characterization of TC-derived Exo. (A) Transmission electron micrographs of exosomes. Scale bar (from left to right): 500, 200, 100 nm, respectively. (B) NTA of TC-derived exosomes. Relationship between mean diameter (159.4 ± 54.9 nm) and original concentration of Exo (3.5 × 109 particles/ ml), with peak distribution when particle size being 138.6 nm. (C) WB was used to identify the expression of CD63 and TSG101, both were specific surface proteins pertaining to Exo. Lanes 1, 2, and 3 represent three repeats. TC: telocyte; NTA: nanoparticle tracking analysis; WB: Western blotting.

Figure 5.

Reversal of fibrosis and enhanced secretion of VEGF in TGF-β1-treated ESCs when exposed to TC-derived Exo. Error bars indicate SD from three independent experiments (biological replicates). *P < 0.05, by one-way ANOVA with Tukey’s post hoc test. (A) WB analysis demonstrated that TC-derived Exo treatment significantly decreased fibrosis markers (COLIA1, FN, and α-SMA) in TGF-β1-induced ESCs among three groups (blank group: regular ESCs; control group: TGF-β1-ESCs; TC-Exo group: TGF-β1-ESCs + TC-Exo). β-Tubulin served as loading control. VEGF mRNA (B) and protein (D) expression in control ESCs (TGF-β1-ESCs) decreased significantly than in blank control (regular ESCs), with significant elevation after TC-derived Exo exposure (TGF-β1-ESCs + TC-Exo). Relative mRNA expression was determined by normalizing to GAPDH levels for qRT-PCR and β-tubulin served as loading control for WB. (C) Total amount of VEGF in culture media showed statistical difference among three groups, with the highest in TC-Exo group. VEGF: vascular endothelial growth factor; ESC: endometrial stromal cell; TC: telocyte; SD: standard deviation; ANOVA: analysis of variance; WB: Western blotting; COL1A1: collagen type 1 alpha 1; FN: fibronectin; α-SMA: α-smooth muscle actin; mRNA: messenger RNA; TGF-β1: transforming growth factor-beta 1.

Figure 6.

Reversal of fibrosis in TGF-β1-treated ESCs was antagonized by ETC-159 or XAV939 through blocking of Wnt ligands in TCs or Wnt/β-catenin pathway in ESCs. β-tubulin served as loading control. Error bars indicated SD from three independent experiments (biological replicates). *P < 0.05 by ANOVA with Tukey’s post hoc test, whereas ns indicates non-significant. WB analysis detected significant increase of β-catenin, with decreased fibrosis markers (COL1A1, FN, and α-SMA protein) in TGF-β1-treated ESCs exposed to TC-derived Exo. Whereas, ETC-159 (A) and XAV939 (B) treatment blocked the salvage of TC-derived Exo on fibrosis, with opposite trends of these markers. ESC: endometrial stromal cell; TC: telocyte; SD: standard deviation; ANOVA: analysis of variance; WB: Western blotting; COL1A1: collagen type 1 alpha 1; FN: fibronectin; α-SMA: α-smooth muscle actin; TGF-β1: transforming growth factor-beta 1.

Figure 7.

Morphometric analyses of endometrial thicknesses, number of endometrial glands, and fibrotic areas in IUA mouse model after TC-derived Exo treatment. Scale bar (from upper to lower enlarged images): 200, 20 μm, respectively. (A) HE staining to observe endometrial thicknesses and glands (arrow). (B) Masson trichrome staining of endometrium, with blue-stained collagen fibers and red-stained mucosa, submucosa, muscles, and blood vessels. (C) Statistical analysis of endometrial thicknesses, number of endometrial glands, and fibrotic areas in each group. *P < 0.05, by ANOVA with Tukey’s post hoc test, whereas ns indicates non-significant. IUA: intrauterine adhesion; TC: telocyte; HE: hematoxylin–eosin; ANOVA: analysis of variance.

Figure 8.

IHC of FN, CD31, VEGF, E-cadherin, and N-cadherin in uterine tissues from IUA mouse model. (A) IHC of FN, CD31, VEGF, E-cadherin, and N-cadherin in endometrial tissues. Arrow: CD31-positive endothelial cells. Scale bar = 20 μm. (B) Semi-quantitative analysis of FN, MVD, VEGF, E-cadherin, and N-cadherin in each group. *P < 0.05, by ANOVA with Tukey’s post hoc test, whereas ns indicates non-significant. IHC: immunohistochemistry; FN: fibronectin; VEGF: vascular endothelial growth factor.