were obtained from Cell Signaling (Beverly, MA). 1-Palmitoyl-2- arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) was obtained from Avanti Polar Lipids (Alabaster, AL). PAPC was oxidized by exposure to air for 72 hours. The extent of oxidation was measured by positive ion electrospray mass spectrometry (ESI-MS) as previously described [37]. After completion of oxidation, the phospholipids were stored at 270uC dissolved in chloroform and were used within 2 weeks of MS analysis. .All oxidized and non- oxidized phospholipid preparations were analyzed by the limulus amebocyte assay (BioWhittaker, Frederick, MD) and shown negative for endotoxin. All reagents for immunofluorescence were purchased from Molecular Probes (Eugene, OR). Human lung pulmonary artery endothelial cells (HPAEC) were obtained from Lonza (Walkersville, MD), cultured according to the manufacturer’s protocol, and used at passages 5–7. Unless specified, biochemical reagents were obtained from Sigma (St. Louis, MO). Isolation and propagation of HAEC Human aortic endothelial cells (HAECs) were isolated as described previously [50] and cultured in M199 medium supplemented with FBS (20% v/v), penicillin (100 U/mL), streptomycin (100 mg/mL), sodium pyruvate (1 mmol/L), heparin (90 mg/mL), and endothelial cell growth supplement (20 mg/mL). VEGFR2 depletion using siRNA approach Two sets of VEGFR2-specific oligonucleotides (Stealth Select) were obtained from Invitrogen (Carlsbad, CA) and characterized in our previous study [14]. A GC% matched negative control siRNA (scrambled) was also obtained from Invitrogen. Transfection of EC with siRNA was performed as described previously [11,51]. After 72 hr of transfection cells were used for experiments or harvested for western blot verification of specific protein depletion. Measurement of transendothelial electrical resistance The cellular barrier properties were analyzed by measurements of transendothelial electrical resistance (TER) across confluent endothelial monolayers using an electrical cell-substrate imped- ance sensing system (Applied Biophysics, Troy, NY) as previously described [11,12]. Immunofluorescence staining Endothelial cells were grown to confluence, stimulated with agonist of interest, and immunofluorescence staining for F-actin was performed as described elsewhere [25]. Likewise, after 72 hours of transfection with nsRNA or VEGFR2 siRNA, EC were stimulated with Ox-PAPC followed by immunofluorescence staining for F-actin using Texas Red-conjugated phalloidin or visualization of diphosphorylated MLC using phosphospecific References 1. Berliner JA, Gharavi NM (2008) Endothelial cell regulation by phospholipid oxidation products. Free Radic Biol Med 45: 119–123. 2. Bochkov VN, Oskolkova OV, Birukov KG, Levonen AL, Binder CJ, et al. (2010) Generation and biological activities of oxidized phospholipids. Antioxid Redox Signal 12: 1009–1059. 3. Gargalovic PS, Gharavi NM, Clark MJ, Pagnon J, Yang WP, et al. (2006) The unfolded protein response is an important regulator of inflammatory genes in endothelial cells. Arterioscler Thromb Vasc Biol 26: 2490–2496. 4. Zimman A, Chen SS, Komisopoulou E, Titz B, Martinez-Pinna R, et al. (2010) Activation of aortic endothelial cells by oxidized phospholipids: a phosphopro- teomic analysis. J Proteome Res 9: 2812–2824. 5. Imai Y, Kuba K, Neely GG, Yaghubian-Malhami R, Perkmann T, et al. (2008) Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell 133: 235–249. 6. Ma Z, Li J, Yang L, Mu Y, Xie W, et al. (2004) Inhibition of LPS- and CpG DNA-induced TNF-alpha response by oxidized phospholipids. Am J Physiol Lung Cell Mol Physiol 286: L808–816. VEGFR2 Mediates Endothelial Permeability by OxPAPC antibody [25]. Images were processed with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) software. Quantitative analysis of paracellular gap formation in OxPAPC treated cells transfected with nsRNA or VEGFR2 specific siRNA was performed as previously described [25,52,53]. Western Blot Protein extracts were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed with antibodies of interest, as previously described [54,55,56]. GTPase activation assays GTPase activation assays were performed using commercially available assay kits purchased from Upstate Biotechnology (Bill- erica, MA). In brief, after stimulation, cell lysates were collected, and GTP-bound Rac or Rho were captured using pull-down assays with immobilized PAK1-PBD or rhotekin-RBD, respec- tively, according to the manufacturer protocol. The levels of activated small GTPases as well as total Rac or Rho content were evaluated by western blot analysis. Rac and Rho membrane translocation Cells were resuspended in buffer (10 mM HEPES, 40 mM KCl, 5 mM MgCl2, 1 mM, EDTA) containing protease and phospha- tase inhibitor cocktails, and PMSF (1 mM). Cells were disrupted by 10 passages through a 26 gauge needle, followed by freezing and thawing. Lysates were centrifuged for 10 min at 4000 rpm. The supernatant was centrifuged for 1 hr 15 min at 35,000 rpm. The final membrane pellets were resuspended in RAL buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2.5 mM MgCl2, 1% NP-40, 10% glycerol) containing protease and phosphatase inhibitor cocktails, and PMSF (1 mM). Statistical analysis Results are presented as mean 6 SD of three to six independent experiments. Stimulated samples were compared to controls by unpaired Student’s t-test. For multiple-group comparisons, a one- way analysis of variance (ANOVA), followed by the post hoc Tukey test, were used. P,0.05 was considered statistically significant. Author Contributions Conceived and designed the experiments: AB JB KB. Performed the experiments: SL VS TW TH JK. Analyzed the data: AB JB KB. Contributed reagents/materials/analysis tools: AB JB KB. Wrote the paper: AB JB KB. 7. Nonas S, Birukova AA, Fu P, Xing J, Chatchavalvanich S, et al. (2008) Oxidized phospholipids reduce ventilator-induced vascular leak and inflammation in vivo. Crit Care 12: R27. 8. Nonas SA, Miller I, Kawkitinarong K, Chatchavalvanich S, Gorshkova I, et al. (2006) Oxidized phospholipids reduce vascular leak and inflammation in rat model of acute lung injury. Am J Respir Crit Care Med 173: 1130–1138. 9. Birukova AA, Malyukova I, Poroyko V, Birukov KG (2007) Paxillin - {beta}- catenin interactions are involved in Rac/Cdc42-mediated endothelial barrier- protective response to oxidized phospholipids. Am J Physiol Lung Cell Mol Physiol 293: L199–211. 10. Birukova AA, Zebda N, Fu P, Poroyko V, Cokic I, et al. (2011) Association between adherens junctions and tight junctions via Rap1 promotes barrier protective effects of oxidized phospholipids. J Cell Physiol 226: 2052– 2062. 11. Birukov KG, Bochkov VN, Birukova AA, Kawkitinarong K, Rios A, et al. (2004) Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac. Circ Res 95: 892–901. PLoS ONE | www.plosone.org 12 January 2012 | Volume 7 | Issue 1 | e30957