Exposure to T&I and S1P receptor modulators modifies hBMVEC morphology and viability.

<p><b>a</b>. Immunostaining for the cytoskeleton component F-actin, shows a confluent monolayer characterized by fusiform morphology and strict cell-to-cell contacts. <b>b</b>. Exposure to inflammatory cytokines, TNFα (10 U/ml) and IFNγ (5 U/ml) (T&I), causes changes in hBMVEC morphology as consequence of reduced cell viability. In the presence of S1P (50 nM, <b>c</b>) or fingolimod phosphate (pFTY 50 nM, <b>d</b>), effects of T&I on hBMVEC morphology are lessened. Scale bar represent 10 μM. <b>e</b>. hBMVEC ability to metabolize MTT is reduced as consequence of exposure to T&I for 24 h, while in the presence of S1P (50 nM), this effect is reverted. Values represent absorbance at 570 nm, normalized to resting conditions, in which hBMVEC are considered maximally viable. Data are mean ±S.E.M. of four independent experiments, each run in triplicate. ** p<0.01 vs. T&I, ***p<0.001 vs. T&I. <b>f</b>. Trypan blue exclusion demonstrates increased hBMVEC death after 24 h exposure to T&I. S1P (50 nM) co-treatment is conversely able to reduce this effect. <b>g</b>. Exposure to T&I in the presence of either FTY (50 nM) or pFTY (50 nM), shows a reduction in hBMVEC death. Data are mean ±S.E.M. of four independent experiments, each run in triplicate, in which three to five fields per well were counted. ** p<0.01 vs T&I, ***p<0.001 vs T&I.