VEGFR2 Mediates Endothelial Permeability by OxPAPC Figure 7. Effect of VEGFR2 depletion on intracellular localization of phophorylated MLC in HPAEC treated with high OxPAPC concentration. HPAEC transfected with VEGFR2-specific siRNA or non-specific RNA were stimulated with OxPAPC (75 mg/ml) or vehicle. A – Immunofluorescence staining was performed using diphospho-MLC antibodies as described in Methods. B - Quantitative image analysis of MLC phosphorylation was performed as described in Methods. All experiments were repeated three times. Values are mean 6 SD, * p,0.05 vs. control. doi:10.1371/journal.pone.0030957.g007 Rac and Rho GTPases (Figure 8C). Taken together with experiments using inhibition of Rho pathway (Figure 5B), our results suggest that at high OxPAPC concentrations, the barrier protective effect of Rac activation is overcome by Rho signaling, which contributes to the late phase of EC barrier failure via activation of stress fiber formation and actomyosin contractility. In addition, pronounced and sustained Rac activation caused by high OxPAPC concentrations may stimulate NADPH oxidase activity and cause oxidative stress, which may also contribute to EC barrier compromise [30,31]. These mechanisms will be discussed below. Discussion Oxidized phospholipids play a critical role in vascular endothelial barrier regulation in health and disease. Pathologic elevation of oxidized phospholipids associated with hyperlipide- mia, organ failure, or acute lung injury may compromise the PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30957