CONICET | Buscador de Institutos y Recursos Humanos

Introduction: Migration and invasion of extravillous trophoblast cells(EVT) through decidua and maternal uterine spiral arteries are crucialprocesses in placentation. We propose that symptomatic or asymptomatic Shigatoxin producing E. coli (STEC)infections during early pregnancy may cause feto-placental damages mediated byShiga toxin type 2 (Stx2). We previously reported that Swan 71 trophoblast cellsexpresses globotriaosylceramide(Gb3), the Stx receptor. In addition, we demonstrated that differentconcentrations of Stx2 (1- 0.001 μg/ml) affect Swan 71 viability after 72 h ofexposure but not after 24 h.Objective: The aim of this workwas to evaluate the effects of Stx2 on migration and invasion of human EVT, and to analyze ifaminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase(iNOS) can prevent Stx2 effects.Methods: Swan 71 cell line wasused as human EVT model. Cell migration was determined by the wound-healingassay in cells grown in 24-well plates and incubated with and without Stx2 (0.1μg/ml). A verticalscratch 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 ofthe final area relative to the initial wound area was calculated and the % ofwound closure was determined. For invasion analysis Swan 71 cells were grown on8 μm pore transwell inserts coated with 0.5 mg/ml Matrigel (Sigma, USA) andincubated for 24 h with Stx2 in presence or not of AG (100 μg/ml) added in the upper chamber. Control cells wereincubated only with fresh media or AG. Cells that invaded the lower side of thechamber were fixed with methanol, stained with DAPI and images of six randomfields for cell counting. Differences between groups were analyzed byKruskal-Wallis statistical test. Results:Stx2 decreased significantly Swan 71 migration (15.4 ± 4.2 vs 29.1 ± 2.3% of bound closure, Stx2 vs Control, *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, n.s.).Conclusions: These results suggest thatStx2 could alter the early placenta formation impairing migration and invasionof trophoblast cells. AG reverted the inhibition of Stx2 on trophoblastinvasion, suggesting that NO overexpression may be involved in the detrimentaleffects of Stx2. These results support the hypothesis that Stx2 may beresponsible for feto-placental damages causing pregnancy loss.

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