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. 2024 May;29(5):75.
doi: 10.3892/mmr.2024.13199. Epub 2024 Mar 15.

Pigment epithelium‑derived factor inhibits proliferation, invasion and angiogenesis, and induces ferroptosis of extravillous trophoblasts by targeting Wnt‑β‑catenin/VEGF signaling in placenta accreta spectrum

Rui Li  1 , Xiaoying Weng  1 , Xuyang Hu  1 , Junjun Wang  2 , Lin Zheng  1
Affiliations

Pigment epithelium‑derived factor inhibits proliferation, invasion and angiogenesis, and induces ferroptosis of extravillous trophoblasts by targeting Wnt‑β‑catenin/VEGF signaling in placenta accreta spectrum

Rui Li et al. Mol Med Rep. 2024 May.

Abstract

Placenta accreta spectrum (PAS) is one of the most dangerous complications in obstetrics, which can lead to severe postpartum bleeding and shock, and even necessitate uterine removal. The abnormal migration and invasion of extravillous trophoblast cells (EVTs) and enhanced neovascularization occurring in an uncontrolled manner in time and space are closely related to the abnormal expression of pro‑angiogenic and anti‑angiogenic factors. The pigment epithelium‑derived factor (PEDF) is a multifunctional regulatory factor that participates in several important biological processes and is recognized as the most efficient inhibitor of angiogenesis. The present study aimed to explore the effects of PEDF on EVT phenotypes and the underlying mechanisms in PAS. HTR‑8/SVneo cells were transfected to overexpress or knock down PEDF. Cell proliferation and invasion were assessed using Cell Counting Kit‑8, 5‑ethynyl‑2'‑deoxyuridine and Transwell assays. In vitro angiogenesis was analyzed using tube formation assays. The degree of ferroptosis was assessed by evaluating the levels of lipid reactive oxygen species, total iron, Fe2+, malondialdehyde and reduced glutathione using commercial kits. The expression levels of biomarkers of ferroptosis, angiogenesis, cell proliferation and Wnt signaling were examined by western blotting. PEDF overexpression decreased the proliferation, invasion and angiogenesis, and induced ferroptosis of EVTs. Activation of Wnt signaling with BML‑284 and overexpression of vascular endothelial growth factor (VEGF) reversed the PEDF overexpression‑induced suppression of cell proliferation, invasion and tube formation. PEDF overexpression‑induced ferroptosis was also decreased by Wnt agonist treatment and VEGF overexpression. It was predicted that PEDF suppressed the proliferation, invasion and angiogenesis, and increased ferroptosis in EVTs by decreasing Wnt‑β‑catenin/VEGF signaling. The findings of the present study suggested a novel regulatory mechanism of the phenotypes of EVTs and PAS.

Keywords: Wnt; angiogenesis; extravillous trophoblasts; ferroptosis; invasion; vascular epidermal growth factor; β‑catenin.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
PEDF inhibits the proliferation, migration and angiogenesis of EVTs in vitro. EVTs were transfected with OE-PEDF or siPEDF. Cell viability and proliferation were assessed using. (A) Cell Counting Kit-8, (B) 5-ethynyl-2′-deoxyuridine (red, proliferative cells; blue, nuclei) and (C) colony formation assays. (D) Cell invasion was assessed using a Transwell assay. (E) Cell migration was detected using a wound healing assay. (F) Protein expression levels of E-cadherin, vimentin, FGF, VEGF, PDGF and PCNA were examined by western blotting. (G) In vitro angiogenesis was examined using a tube formation assay. Scale bar, 50 µm. Data are presented as the mean ± SD, n=3. *P<0.05, **P<0.01 vs. NC or siPEDF. EVT, extravillous trophoblast cell; FGF, fibroblast growth factor; NC, negative control; OD, optical density; OE-NC, control empty vector for overexpression; OE-PEDF, PEDF overexpression vector; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PEDF, pigment epithelium-derived factor; siNC, small interfering RNA negative control; siPEDF, small interfering RNA targeting PEDF; VEGF, vascular endothelial growth factor.
Figure 2.
Figure 2.
PEDF overexpression induces ferroptosis of extravillous trophoblast cells transfected with OE-PEDF. Levels of (A) lipid ROS, (B) total iron, (C) Fe2+, (D) MDA and (E) GSH were examined using the respective kits. (F) Protein expression levels of GPX4 and SLC7A11 determined by western blotting. Data are presented as the mean ± SD, n=3. **P<0.01 vs. OE-NC. GPX4, glutathione peroxidase 4; GSH, reduced glutathione; MDA, malondialdehyde; MFI, mean fluorescence intensity; OE-NC, control empty vector for overexpression; OE-PEDF, PEDF overexpression vector; PEDF, pigment epithelium-derived factor; ROS, reactive oxygen species; SLC7A11, solute carrier family 7 member 11.
Figure 3.
Figure 3.
PEDF suppresses the Wnt-β-catenin/VEGF signaling pathway in extravillous trophoblast cells. (A) Protein expression levels of Wnt and β-catenin were determined by western blotting. Relative (B) mRNA and (C) protein expression levels of VEGF were examined by reverse transcription-quantitative PCR and western blotting. Data are presented as the mean ± SD, n=3. **P<0.01 vs. OE-NC or OE-PEDF. OE-NC, control empty vector for overexpression; OE-PEDF, PEDF overexpression vector; PEDF, pigment epithelium-derived factor; VEGF, vascular endothelial growth factor.
Figure 4.
Figure 4.
PEDF regulates phenotypes of EVTs via the Wnt-β-catenin/VEGF signaling pathway. EVTs were transfected with OE-PEDF and treated with Fer-1 or BML-284 or transfected with OE-VEGF. Cell viability and proliferation were examined using (A) Cell Counting Kit-8, (B) 5-ethynyl-2′-deoxyuridine (red, proliferative cells; blue, nuclei) and (C) colony formation assays. Cell (D) invasion and (E) migration were assessed by Transwell and wound healing assays. (F) Angiogenesis was analyzed using a tube formation assay. (G) Expression levels of E-cadherin, vimentin, FGF, VEGF, PDGF and PCNA were examined by western blotting. Scale bar, 50 µm. Data are presented as the mean ± SD, n=3. *P<0.05 and **P<0.01 vs. OE-NC or OE-PEDF. EVT, extravillous trophoblast cell; Fer-1, ferrostatin-1; FGF, fibroblast growth factor; OD, optical density; OE-NC, control empty vector for overexpression; OE-PEDF, PEDF overexpression vector; OE-VEGF, VEGF overexpression vector; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PEDF, pigment epithelium-derived factor; VEGF, vascular endothelial growth factor.
Figure 5.
Figure 5.
PEDF induces ferroptosis of EVTs by suppressing the Wnt-β-catenin/VEGF pathway. EVTs were transfected with OE-PEDF and OE-VEGF or transfected with OE-PEDF and treated with Fer-1 or BML-284. The levels of (A) lipid ROS, (B) total iron, (C) Fe2+, (D) MDA and (E) GSH were examined using the respective kits. (F) Protein expression levels of GPX4 and SLC7A11 were determined by western blotting. Data are presented as the mean ± SD, n=3. **P<0.01 vs. OE-NC or OE-PEDF. EVT, extravillous trophoblast cell; Fer-1, ferrostatin-1; GPX4, glutathione peroxidase 4; GSH, reduced glutathione; MDA, malondialdehyde; MFI, mean fluorescence intensity; OE-NC, control empty vector for overexpression; OE-PEDF, PEDF overexpression vector; OE-VEGF, VEGF overexpression vector; PEDF, pigment epithelium-derived factor; ROS, reactive oxygen species; SLC7A11, solute carrier family 7 member 11; VEGF, vascular endothelial growth factor.
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Grants and funding

This study was supported by Fujian Province Health and Health Science and Technology Program Projects (grant no. 2020GGB006).