Cytotoxic effects of Shiga toxin-2 on human extravillous trophoblast cell lines

in Reproduction
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Shiga toxin (Stx2) producing Escherichia coli infections during early gestation may impair placentation through a Stx2 damage of extravillous trophoblast (EVT) cells. We have previously demonstrated that Stx2 injected in rats in the early stage of pregnancy causes spontaneous abortion by a direct cytotoxic effect in the highly perfused feto-uteroplacental unit. The main aim was to evaluate the effects of Stx2 on EVT in order to understand the possible adverse effects that the toxin may have on trophoblast cells during early pregnancy. Swan 71 and HTR-8 cell lines were used as human EVT models. The presence of Stx2 receptor, globotriaosylceramide (Gb3), on Swan 71 and HTR-8 cells was evaluated by thin layer chromatography. The effects of Stx2 on cell viability were evaluated by neutral red uptake, migration by wound-healing assay and invasion was determined by the ‘transwell chamber’ assay. Metalloproteinase activity (MMP-2) was evaluated by zymography and tubulogenesis was analyzed by counting the total tube length and the number of branch formation. We have demonstrated that Swan 71 expresses high levels of Gb3 compared to HTR-8 cells. Stx2 decreased significantly Swan 71 viability in a dose-dependent manner after 72 h of toxin exposure. Furthermore, Stx2 impaired migration, invasion and tube-like formation of Swan 71 cells and decreased the MMP-2 activity. These cytotoxic effects were partially prevented by aminoguanidine, an inducible nitric oxide synthase inhibitor. These studies demonstrate that the function and viability of EVT cells may be altered by Stx2 and suggest that NO overexpression may be involved in the detrimental effects.


    Society for Reproduction and Fertility

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    Gb3 expression on trophoblast cells and Stx2 cytotoxic effects. (A) Neutral glycolipids extracted from HTR-8, Swan 71 and Vero cells were subjected to TLC and then visualized with orcinol staining. (B) Gb3 glycolipid bands from 1 × 106 cells were quantified by densitometric analysis and compared to standard Gb3 (1 µg/lane, Matreya). (C) Cells were exposed to different concentrations of Stx2 (1 × 10−7–1 µg/mL) in growth-arrested conditions for 24 and 72 h. Then, cells were incubated with neutral red for an additional 1 h. Results are expressed as percentage of cell viability where 100% represents control cells without toxin treatment. Data are shown as means ± s.d. from at least five independent experiments performed in triplicate. *P < 0.05 for Swan 71 (72 h) vs Swan 71 (24), **P < 0.01 for Swan 71 (72 h) vs HRT-8 (24 h) and HTR-8 (72 h).

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    Stx2 inhibits trophoblast cell migration. (A) Cell migration was evaluated in Swan 71 cells by the wound-healing assay in cells grown in 24-well plates and incubated for 24 h with Stx2 (0.001–1 µg/mL) before wound formation. Images of the wound healing were captured at 0 h and 24 h using a light microscope (×200) (B) Cell migration was calculated and expressed as percentages of cell coverage to the initial cell-free zone. Values are presented as means ± s.d. Experiments were repeated three times. Significant differences (*P < 0.05) were found compared to the control.

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    Stx2 inhibits trophoblast cell invasion and MMP-2 activity. Swan 71 cells were grown on transwell insert (8 µm pore size) coated with 0.5 mg/mL Matrigel (Sigma) and incubated for 24 h with Stx2 (0.01 and 0.1 µg/mL). Cells that invaded the lower side of the insert were fixed with methanol and stained with DAPI. (A) Representative photographs of invading cells in each experimental condition (×200). (B) Stx2 at 0.1 µg/mL significantly decreased Swan 71 cell invasion compared to control. MMP-2 activity was evaluated by zymography assay in Stx2-treated and non-treated Swan 71 cells incubated with Stx2 for 24 h. (C) a representative zymography is shown. (D) The densitometric analysis of MMP-2 band intensity showed a significant decrease of MMP-2 activity on Stx2 treated compared to control. Values are presented as means ± s.d. Each experiment was repeated three times. *P < 0.05.

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    Stx2 inhibits tube-like formation of trophoblast cells. Swan 71 cell monolayers treated with Stx2 were seed in 96-well plates coated with Matrigel. Pictures were taken of ten random fields 6 h after tube-like formation (×100). (A) Representative images of tube-like formation. (B) Total tube length and (C) number of branches were quantified in control and Stx2-treated cells. Stx2 significantly decreases each one of the parameters involved in tube-like formation compared to control. Values are presented as means ± s.d. Each experiment was repeated three times. *P < 0.05, **P < 0.01.

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    Aminoguanidine (AG) partially prevents Stx2 effects on trophoblast cells. AG (100 µM) partially prevented the cytotoxic effects of Stx2 (0.1 µg/mL) on (A) migration, (B) invasion and (C) total tube length and (D) number of branches of Swan 71 cells. Data are shown as means ± s.d. from at least three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001.


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