VEGFR2 Mediates Endothelial Permeability by OxPAPC or VEGFR2 siRNA; n = 4–6 per condition; *p,0.05. C - After 72 hrs of transfection with VEGFR2-specific or non-specific RNA HAECs were stimulated with OxPAPC (50 mg/ml, 120 min) or vehicle followed by immunofluorescence staining for F-actin. doi:10.1371/journal.pone.0030957.g004 strongly suggest that phosphorylation of MLC induced by high OxPAPC doses contributes to increased EC permeability and occurs via VEGFR2 dependent mechanism. It remains unclear what is the mechanism of the switch that converts the OxPAPC protective effect to a disruptive effect. Our previous studies show that the protective effect of low OxPAPC doses is mediated by Rac, while Rho signaling was not activated [11]. The present results show that delayed activation of Rho signaling contributes to the EC barrier disruptive response at high OxPAPC concentrations. We next addressed the question of whether the Rac protective effect is overcome by Rho, or whether Rac is not activated by high OxPAPC concentrations. To distinguish between these alternatives, we measured Rac and Rho activation in the same EC treated with high and low OxPAPC doses for different time periods. Low OxPAPC concentrations caused early activation of Rac and autophospho- rylation (at S423) of Rac effector PAK1 reflecting PAK1 activation, while Rho activity was not affected by low OxPAPC concentrations (Figure 8AB). High OxPAPC concentration also caused both the early Rac activation and PAK1 phosphorylation. However high OxPAPC caused a delayed activation of Rho and phosphorylation of Rho kinase target MYPT1 which was observed after 30 min (Figure 8AB). In agreement with these data, treatment of HAEC with low OxPAPC concentrations induced membrane translocation of Rac, but not Rho, while high OxPAPC concentrations caused membrane translocation of both Figure 5. Involvement of Rho pathway in cytoskeletal effects of high OxPAPC concentrations on endothelial cells. A - Time course of myosin light chain phosphatase (MYPT) and myosin light chain (MLC) phosphorylation induced by OxPAPC (75 mg/ml and 10 mg/ml) was examined by western blot with corresponding phospho-specific antibodies. b-Tubulin antibody was used as normalization control. B - HAEC were pretreated with vehicle or Y-27632 (2 mM, 30 min) prior to stimulation with OxPAPC (50 mg/ml, 2 hrs) followed by immunofluorescence staining for F-actin. doi:10.1371/journal.pone.0030957.g005 PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e30957