In addition, we investigated a mechanism of endothelial barrier dysfunction induced by high OxPAPC concentrations. One major finding of this study is the demonstration of similar barrier protective effects of low OxPAPC concentrations on pulmonary and aortic EC and barrier disruptive responses to high OxPAPC doses by both cell types. However, HAEC were slightly more sensitive to the barrier disrupting effects of OxPAPC. We previously identified the signaling mechanisms by which low OxPAPC increased the endothelial barrier and demonstrated a major role of Rac GTPase-dependent strengthening of the cytoskeleton and adherens junctions [9,38,39,40]. The current study focused on the effects of higher OxPAPC concentrations leading to increased EC permeability. Treatment with high concentrations of OxPAPC caused rapid barrier dysfunction (observed after 5–10 minutes). Increased permeability lasted for at least 5 hours without toxic effects on HPAEC monolayers (as tested by live/dead cell assay, data not shown). In a previous study in HAEC (using the same concentrations of OxPAPC as in the current study), survival experiments were performed using propidium iodide assay which showed a lack of toxicity [41]. High OxPAPC doses induced rapid and significant tyrosine phosphorylation of adherens junction protein VE-cadherin, which leads to VE-cadherin internalization and abrupt EC barrier dysfunction caused by the disassembly of adherens junctions [29]. In the present study, we turned our attention to the question of whether stress fiber formation and MLC phosphorylation might play a role in the late phase of barrier decrease mediated by high concentrations of OxPAPC. In contrast to enhancement of peripheral actin cytoskeletal rim and unchanged levels of phos- phorylated MLC observed in EC treated with barrier protective OxPAPC concentrations [11,42], treatment with high OxPAPC concentrations increased MLC phosphorylation by 30 minutes, which was maximal by one hour and remained elevated for at least 4 hours. The increase in stress fibers and gaps in the EC monolayers followed this time course suggesting that stress fiber formation contributed to the long-term decrease in barrier function caused by high concentrations of OxPAPC in pulmonary and aortic EC. In search of upstream mechanisms stimulating MLC phosphor- ylation and stress fiber formation by high OxPAPC concentrations, we examined the potential involvement of VEGFR2. Our previous studies have demonstrated that OxPAPC induced VEGFR2 activation and examined the mechanism by which Ox-PAPC activates VEGFR2. We found that high OxPAPC doses induce high levels of Src activation [29], and activation of VEGFR2 by OxPAPC can be mostly blocked by pre-treatment with Src kinase inhibitor PP2 [14]. These data strongly suggested Src-dependent mechanism of VEGFR2 transactivation by high OxPAPC concen- trations [14]. VEGFR2 activation by its canonical ligand, VEGF, leads to activation of RhoA GTPase [16,28,43]. The exact mechanism of VEGFR2-induced RhoA activation remains unclear, but may involve heterotrimeric protein Gq/11 and phospholipase C [43]. In turn, the activation of RhoA and Rho-associated kinase leads to the inactivation of myosin light chain phosphatase resulting in increased MLC phosphorylation, stress fiber formation and increased endothelial permeability [25]. Of note, the activation of VEGFR2 has been previously demonstrated after 5 minutes of stimulation with 40 mg/ml OxPAPC, and that activation was sustained for at least 4 hours [14]. Interestingly, we found that the rapid phase of TER decrease in the first 30 minutes of EC treatment with high OxPAPC concentrations did not involve activation of MLC phosphorylation and stress fiber formation and was not affected by VEGFR2 depletion. High OxPAPC concentrations increased VE-cadherin tyrosine phosphorylation at Y731 , known to be associated with EC VEGFR2 Mediates Endothelial Permeability by OxPAPC barrier dysfunction [44], but VEGFR2 depletion caused very little changes in VE-cadherin phosphorylation state. On the other hand, TER continued to gradually decrease over a period of 1– 5 hours of treatment with high OxPAPC concentration. This decrease was associated with increased stress fiber formation and MLC phosphorylation, while VEGFR2 depletion prevented further TER decline after 30 minutes of OxPAPC treatment. VEGFR2 depletion and pretreatment with Rho kinase inhibitor Y-27632 abolished stress fiber formation and MLC phosphoryla- tion observed at later times of EC stimulation with high OxPAPC doses. Taken together, these results support the notion that VE- cadherin phosphorylation likely contributes to the early phase of EC barrier dysfunction caused by high OxPAPC concentrations, and this phase is largely independent of VEGFR2. In turn, activation of VEGFR2 – Rho - Rho kinase pathway mediates the late phase of barrier dysfunction and cytoskeletal remodeling in HPAEC and HAEC in response to 50–75 mg/ml OxPAPC. The complexity of signaling pathways activated by different doses of oxidized phospholipids in endothelial cells still leaves some open questions to be addressed. Why is VEGFR2-dependent phase of Rho activation and MLC phosphorylation delayed and observed only after 30 min? One mechanism may involve negative Rac-Rho crosstalk, as Rac activation has been shown to be induced by OxPAPC [11,39]. Another plausible explanation is the previously described OxPAPC-induced stimulation of cAMP signaling [41,45], which reached peak activation at 5–15 min and declined by 30 min [42]. This increase in cAMP may suppress early Rho activation via direct and indirect effects on Rho and Rho kinase activities [46,47,48]. The other question is the role of Rac in the different effects of OxPAPC on endothelial barrier function. We have previously demonstrated Rac activation by both low and high concentrations of OxPAPC [11,30]. Since Rac has been shown by multiple groups to be a barrier protective molecule, why does high dose OxPAPC open the barrier in spite of high levels of Rac activation? We think this can be attributed to: a) the role of Rac in activation of NAPDH oxidase enzymatic complex by high OxPAPC concentrations, which triggers ROS production and leads to oxidative stress and EC barrier dysfunction; or b) the increase in Rho activation that occurs in response to VEGFR2 activation and counterbalances the Rac impact in cytoskeletal mechanisms of barrier enhancement. In summary, these studies suggest that VEGFR2 involvement in vascular endothelial barrier breakdown caused by high OxPAPC concentration is a general mechanism effective in both pulmonary and systemic circulation, which may help explain in part the reported activation of pathologic VEGFR2 signaling in both acute lung injury and atherogenesis. Beneficial effects of inhibition of VEGFR2 signaling in reduction of atherosclerosis in mice have been attributed to the reduction of neo-angiogenesis in the plaque [49]. However, VEGFR2 signaling may also contribute to a barrier dysfunction of endothelium from the large systemic vessels, as has been described in this study. Although the origin and pathogenic mechanisms resulting in high levels of oxidized phospholipids in the pulmonary circulation or in systemic arteries may be quite different, inhibition of VEGFR2 signaling may prove to be a reasonable strategy to suppress pathologic endothelial barrier dysfunction in a number of diseases where OxPAPC accumulates. Methods Reagents and cell culture Diphospho-MLC and b-tubulin antibodies, rabbit anti-human VEGFR2 antibody, HRP-linked anti-mouse and anti-rabbit IgG PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e30957