A Role for VEGFR2 Activation in Endothelial Responses Caused by Barrier Disruptive OxPAPC Concentrations Anna A. Birukova1., Sangderk Lee2., Vitaliy Starosta1, Tinghuai Wu1, Tiffany Ho2, Jin Kim2, Judith A. Berliner2, Konstantin G. Birukov1* 1 Section of Pulmonary and Critical Medicine, Department of Medicine, Lung Injury Center, University of Chicago, Chicago, Illinois, United States of America, 2 Departments of Pathology and Medicine, University of California Los Angeles, Los Angeles, California, United States of America Abstract Introduction: Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion. Vascular endothelial growth factor receptor-2 (VEGFR2) is involved in the OxPAPC-induced EC inflammatory activation. This study examined a role of VEGFR2 in barrier dysfunction caused by high concentrations of OxPAPC and evaluated downstream signaling mechanisms resulting from the effect of OxPAPC in EC from pulmonary and systemic circulation. Methods: EC monolayer permeability in human pulmonary artery endothelial cells (HPAEC) and human aortic endothelial cells (HAEC) was monitored by changes in transendothelial electrical resistance (TER) across EC monolayers. Actin cytoskeleton was examined by immunostaining with Texas Red labeled phalloidin. Phosphorylation of myosin light chains (MLC) and VE-Cadherin was examined by Western blot and immunofluorescence techniques. The role of VEGFR2 in OxPAPC- induced permeability and cytoskeletal arrangement were determined using siRNA-induced VEGFR2 knockdown. Results: Low OxPAPC concentrations (5–20 mg/ml) induced a barrier protective response in both HPAEC and HAEC, while high OxPAPC concentrations (50–100 mg/ml) caused a rapid increase in permeability ; actin stress fiber formation and increased MLC phosphorylation were observed as early as 30 min after treatment. VEGFR2 knockdown dramatically decreased the amount of MLC phosphorylation and stress fiber formation caused by high OxPAPC concentrations with modest effects on the amount of VE-cadherin phosphorylation at Y731. We present evidence that activation of Rho is involved in the OxPAPC/VEGFR2 mechanism of EC permeability induced by high OxPAPC concentrations. Knockdown of VEGFR2 did not rescue the early drop in TER but prevented further development of OxPAPC-induced barrier dysfunction. Conclusions: This study shows that VEGFR2 is involved in the delayed phase of EC barrier dysfunction caused by high OxPAPC concentrations and contributes to stress fiber formation and increased MLC phosphorylation. Citation: Birukova AA, Lee S, Starosta V, Wu T, Ho T, et al. (2012) A Role for VEGFR2 Activation in Endothelial Responses Caused by Barrier Disruptive OxPAPC Concentrations. PLoS ONE 7(1): e30957. doi:10.1371/journal.pone.0030957 Editor: Cara Gottardi, Northwestern University Feinberg School of Medicine, United States of America Received August 11, 2011; Accepted December 28, 2011; Published January 31, 2012 Copyright: 2012 Birukova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NHLBI HL87823, HL76259, and PO1 HL58064 for KB; HL89257, HL107920 and the American Heart Association Midwest Affiliate Grant-in-Aid for AB; NHLBI PO1 HL030568 for JB and K99HL105577 for SL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kbirukov@medicine.bsd.uchicago.edu . These authors contributed equally to this work. Introduction Endothelial barrier dysfunction plays an important role in a number of chronic and acute inflammatory diseases such as atherosclerosis and lung pathologies including asthma, acute lung inflammation and its severe complication, acute respiratory distress syndrome (ARDS). Phospholipid oxidation products, specifically oxidized 1-palmi- toyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC), derived from lipoproteins and membranes of cells undergoing oxidative stress or apoptosis, have been shown to accumulate in a number of inflammatory diseases including atherosclerosis, lung inflammation and tissue injury [1,2]. OxPAPC has multiple effects on the vascular endothelium including a change in the expression of approximately 1500 genes [3] and the phosphorylation of 228 molecules regulating multiple pathways involved in inflammation, sterol regulation, coagulation, cell cycle and cell junctions [4]. Increased levels of oxidized phospholipids present in the injured lung may influence pulmonary endothelial cell (EC) functions including the modulation of pulmonary inflammatory response and EC barrier regulation [5,6,7,8]. Previous studies demonstrated that OxPAPC concentrations in the 5–20 mg/ml range enhanced endothelial monolayer barrier properties in vitro, and similar doses of intravenously injected OxPAPC protected lung barrier function and reduced inflamma- tion in models of acute lung injury caused by LPS injection or mechanical ventilation at high tidal volume [6,7,8]. Protective effects of OxPAPC involved enhancement of peripheral actin cytoskeleton, adherens junctions and tight junctions mediated by Rac and Rap1 GTPases [9,10]. In contrast, high OxPAPC PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30957