VEGFR2 Mediates Endothelial Permeability by OxPAPC Figure 8. Effect of high and low OxPAPC concentrations on Rac and Rho activation. A - Time course of Rac and Rho activation induced by low (10 mg/ml) and high (75 mg/ml) OxPAPC concentrations was monitored using Rac- and Rho- GTP pulldown assays. Western blot analysis of Rac and Rho content in total HPAE cell lysates was used as normalization control. B - Time course of PAK1-Thr423 and MYPT1-Thr850 phosphorylation in HPAEC induced by low (10 mg/ml) and high (75 mg/ml) OxPAPC concentrations was monitored by western blot with corresponding phospho-specific antibodies. b-Tubulin antibody was used as normalization control. C – Membrane translocation of Rac and Rho after 4 hrs of HAEC stimulation with 10 mg/ml or 50 mg/ml of OxPAPC was monitored by western blot with corresponding antibodies. Calnexin antibody was used as normalization control for membrane fractions. doi:10.1371/journal.pone.0030957.g008 vascular endothelial barrier and promote pathological processes such as atherosclerosis or lung injury [2,32]. Previous studies demonstrated barrier protective effects of low OxPAPC concen- trations on pulmonary vascular EC in culture [9,10,12] and in animal models of sepsis and lung injury induced by bacterial lipopolysaccharide or pathologic mechanical ventilation [1,6,7,8,33]. In contrast, high OxPAPC concentrations led to barrier dysfunction in pulmonary EC [11,12,13], which is associated in part with OxPAPC-induced reactive oxygen species (ROS) production and degradation of tight junction protein occludin [13]. Determination of the in vivo concentrations of OxPL under physiologic and pathologic conditions remains an open issue. Published measurements differ significantly and often represent different OxPL species measured in different body compartments. In two reports, combined concentration of all oxidized phospho- lipids in human plasma and tissue samples ranged from 5.4 to 51 mM [34,35]. OxPL levels in the normal human plasma have been estimated in the range of 0.1–1.0 mM [36], but local tissue concentrations in atherosclerotic regions of the vessels have been reported in the range of 10–100 mM/kg [18,37]. In the most recent studies, we used for the first time a ‘‘phospholipidomics’’ analysis by mass-spectrometry to characterize OxPL generation in the model of LPS-induced lung injury. Lungs were collected 24 and 60 hours after intratracheal LPS injection. The results demonstrated a significant elevation of OxPL products (both fragmented and oxygenated phosphatidylcholine derivates) and their presence in lung tissue samples at micromole levels (K. Birukov and V. Bochkov, unpublished data). Thus, cumulative OxPL levels in LPS-challenged lungs are consistent with 50– 100 mg/ml concentrations used in this study. Since OxPAPC accumulates in many disease settings, we thought it was important to obtain an understanding of the mechanism of its permeability regulation . This study examined whether the dose dependent effects of OxPAPC can be reproduced in EC from both systemic and pulmonary circulation. PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30957