CONICET | Buscador de Institutos y Recursos Humanos

Introduction: Migration and invasion of extravillous trophoblast cells (EVT) through decidua and maternal uterine spiral arteries are crucial processes in placentation. We propose that symptomatic or asymptomatic Shiga toxin producing E. coli (STEC) infections during early pregnancy may cause feto-placental damages mediated by Shiga toxin type 2 (Stx2). We previously reported that Swan 71 trophoblast cells expresses globotriaosylceramide (Gb3), the Stx receptor. In addition, we demonstrated that different concentrations of Stx2 (1- 0.001 μg/ml) affect Swan 71 viability after 72 h of exposure but not after 24 h.Objective: The aim of this work was to evaluate the effects of Stx2 on migration and invasion of human EVT, and to analyze if aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase can prevent Stx2 effects.Methods: Swan 71 cell line was used as human EVT model. Cell migration was determined by the wound-healing assay in cells grown in 24-well plates and incubated with and without Stx2 (0.1 μg/ml). A vertical scratch was performed in the center of each well and was considered time 0. Images of the scratch were taken before and 24 h after treatment. The ratio of the final area relative to the initial wound area was calculated and the % of wound closure was determined. For invasion analysis Swan 71 cells were grown on 8 μm pore transwell inserts coated with 0.5 mg/ml Matrigel (Sigma, USA) and incubated for 24 h with Stx2 in presence or not of AG (100 μg/ml) added in the upper side of the insert. Control cells were incubated only with fresh media or AG. Cells that invaded the lower side of the insert were fixed with methanol, stained with DAPI and images of six random fields were taken for cell counting. Differences between groups were analyzed by Kruskal-Wallis statistical test. Results: Stx2 decreased significantly Swan 71 migration (68.0 ± 17.2 vs 26.3 ± 9.3% of bound closure, Stx2 vs Control, n=3, P< 0.05) and inhibited trophoblast invasion (38 ± 28 vs 123 ± 23 invading cells, Stx2 vs Control, n=3, P< 0.05). Furthermore, AG reversed the Stx2 effects on trophoblast invasion (118 ± 24 vs 38 ± 28 invading cells, Stx2 + AG vs Stx2, n=3, P< 0.05).Conclusions: These results suggest that Stx2 could alter the early placenta formation impairing migration and invasion of trophoblast cells. AG reverts the inhibition of Stx2 on trophoblast invasion, suggesting that NO overexpression may be involved in the detrimental effects of Stx2. These results support the hypothesis that Stx2 may be responsible for feto-placental damages causing pregnancy loss.