Abstract
IntroductionOxidation 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.MethodsEC 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.ResultsLow OxPAPC concentrations (5–20 µg/ml) induced a barrier protective response in both HPAEC and HAEC, while high OxPAPC concentrations (50–100 µg/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.ConclusionsThis 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.
Highlights
Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion
We examined whether differential barrier responses to low and high oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) concentrations represent a general feature of endothelial cells regardless of their origins
The dose range that we employed for high OxPAPC in the present study was approximately one-fifth of the levels we previously demonstrated to be present in the vessel wall of hypercholesterolemic rabbits [18]
Summary
Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion. Phospholipid oxidation products, oxidized 1-palmitoyl-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]. 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 inflammation in models of acute lung injury caused by LPS injection or mechanical ventilation at high tidal volume [6,7,8]. High OxPAPC concentrations caused adverse effects on endothelial barrier function by increasing EC permeability and disrupting cell-cell junction complexes [11,12,13]
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