Furthermore, in animals treated with L. obliqua venom it was also observed accumulation and extravasation of leukocytes, in parallel with E-selectin and VCAM-1 enrichment on the vessel wall, suggesting that venom exerts a direct selleckchem effect on leukocyte and endothelial cells, activating and increasing the expression of crucial molecules for the onset of inflammatory responses. Cell shape is governed by the cytoskeleton, which acts as a mechanical supporting framework, and alterations in actin dynamics

is a hallmark of cell activation (Prasain and Stevens, 2009). To evaluate L. obliqua venom effect on endothelial cell morphology we determined a time course JAK inhibitor review of actin stress fiber formation in endothelial cells stimulated with the venom in vitro. After different times of incubation with the venom (3 μg/ml), cells were fixed and stained with rhodamine-conjugated

phalloidin to visualize F-actin distribution. As seen in Fig. 3A, immediately after contact with cells (1–3 min) L. obliqua venom induces rapid and profound alterations in actin cytoskeleton reorganization, when the membrane skeleton and the cortical actin rim crowned the cell border and stress fibers course through the cytosol. After 5 min, actin fibers rearrangement lead to the spreading and polarity of endothelial cells, which show spiky, thin cell protrusions extended at the cell periphery. This phenotype, characteristic of contracting or migrating cells is maintained throughout the observation (15 min) ( Fig. 3A). Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that localizes at focal adhesion complexes, mediating integrin, growth factor, and mechanical stress signaling (Schlaepfer et al., 2004). The phosphorylation at Tyr397 residues correlates with increasing

catalytic activity and induces FAK interaction with actin cytoskeleton, controlling adhesion, proliferation and cell migration (Schaller, 2010). Endothelial Ergoloid FAK is essential for vascular network stability, regulating cell survival and morphology (Braren et al., 2006). Fig. 3 shows that in parallel with the changes in cytoskeleton, L. obliqua venom induces FAK activation ( Fig. 3B) and its association to actin ( Fig. 3C) in endothelial cells. Increasing FAK phosphorylation at Tyr397 occurs just after 1 min of treatment and was maintained for 5 min ( Fig. 3B), diminishing after 15 min. It was also observed a close correlation between the kinetics of FAK phosphorylation and its subsequent association to polymerized actin in the first 3 min after treatment ( Fig. 3C), suggesting a direct link between these two events. At the 5th min, although pFAK was increased, its association to actin declined, reaching control level after 15 min.