Simvastatin and benznidazole prevent cell adhesion molecule expression on endothelial cells during Trypanosoma cruzi infection.

Endothelial-like (EA.hy926) and HUVEC cells were incubated with 5 μM simvastatin or 20 μM benznidazole dissolved in DMSO 0.025% v/v final concentration. After 24 hours, the medium was replaced, and the cells were infected for 16 hours with a 1:10 ratio of T. cruzi trypomastigotes. E-Selectin, ICAM-1, and VCAM-1 expression in EA.hy926 cells (A, C) and HUVECs (B, D) was determined at the end of the infection time. Flow cytometry was performed using human antibodies conjugated with PE, FITC, and APC for E-Selectin, ICAM-1, and VCAM-1, respectively. Panels A and B represent mean fluorescence intensity (MFI) calculations and panels C and D demonstrate representative histograms. Each group was compared with the respective IgG1 and IgG2A isotype match and normalized. In E and F, EA.hy926 cells were washed with PBS and incubated with IgG anti human E-selectin, ICAM-1, and VCAM-1 antibodies. Subsequently, the cells were incubated with anti-IgG mouse FITC-conjugated secondary antibodies. E. Representative immunofluorescence images (40X) and F. Quantitative analysis of relative fluorescence for each adhesion molecule. All controls were incubated with DMSO vehicle alone. The data are expressed as the mean ± SD from three independent experiments. The data represent the mean ± SD of MFI values (A and B) from four independent experiments. Comparisons were made by two-way ANOVA and Tukey’s post-test analysis. *** p<0.001; ** p<0.01; ‡ p< 0.001 (F).