concentrations caused adverse effects on endothelial barrier function by increasing EC permeability and disrupting cell-cell junction complexes [11,12,13]. In order to reconcile the difference in the reported effects of OxPAPC on endothelial cells from different vascular beds and examine the mechanism by which high doses of OxPAPC increased EC permeability, we used two different endothelial cell types isolated from pulmonary and systemic circulation, which were exposed to low and high OxPAPC concentrations. Previous studies by our group in human aortic endothelial cells (HAEC) demonstrated an activation of vascular endothelial growth factor receptor-2 (VEGFR2), as measured by increased tyrosine phosphorylation at Y1175,in response to high OxPAPC concen- trations. These high levels of OxPAPC led to the activation of SREBP and Erk-1,2 MAP Kinase signaling, and the expression of LDL receptor and inflammatory molecules- interleukin-8 and tissue factor [14]. There is evidence that VEGFR2 signaling may also lead to increased endothelial permeability in vivo and in vitro [15,16,17]. We present evidence for a role of VEGFR2 in cytoskeletal remodeling and increased EC monolayer permeability by high concentrations of OxPAPC via a Rho - Rho kinase - myosin light chain phosphorylation dependent mechanism. Results Dose-dependent effects of Ox-PAPC on permeability responses of pulmonary and aortic endothelial cell types Previous reports by our group characterized dose dependent effects of OxPAPC on barrier properties of human pulmonary artery endothelial cells (HPAEC). In these experiments, we examined whether differential barrier responses to low and high OxPAPC concentrations represent a general feature of endothelial cells regardless of their origins. We compared the time course and dose response effects of OxPAPC on barrier function in HAEC and HPAEC. The maximal barrier-enhancing effect was observed in HPAEC (Figure 1A) and HAEC (Figure 1B) treated with a similar range of OxPAPC concentrations (5–10 mg/ml). Higher OxPAPC concentrations did not further enhance the EC monolayer barrier properties, but instead decreased barrier function in both HAEC and HPAEC. The most prominent barrier disruptive response was observed in the 50–100 mg/ml range of OxPAPC. 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]. The TER increase observed at low OxPAPC concentrations developed in 5–15 min after stimulation, reached maximal levels by 30 min and lasted more than 3 hours. High OxPAPC doses caused rapid TER response, but in the opposite direction. In both HPAEC and HAEC, permeability increased and was sustained up to 4 hours when induced by high OxPAPC concentrations (50–100 mg/ml). Cytoskeletal remodeling induced by low and high Ox- PAPC doses in two endothelial cell types We next examined the effects of high and low OxPAPC doses on EC actin cytoskeletal arrangement using immunofluorescence staining of EC monolayers with Texas Red-conjugated phalloidin. Untreated HPAEC displayed generally random F-actin distribu- tion throughout the cells with some localization of actin filament bundles at the cell boundaries (Figure 2A, left panel). A Similar cytoskeletal arrangement, but with more even cellular F-actin, was observed in HAEC (Figure 2B, left panel) Treatment with barrier enhancing OxPAPC concentration (10 mg/ml) caused redistribu- tion of actin filaments to the cell periphery with formation of the VEGFR2 Mediates Endothelial Permeability by OxPAPC dense F-actin rings at 30 and 120 minutes of OxPAPC stimulation in both HPAEC and HAEC (Figure 2A,B). In turn, barrier disruptive response to 75 mg/ml OxPAPC manifested by forma- tion of paracellular gaps (shown by arrows) was observed in both HPAEC and HAEC (Figure 2A,B). Of note, the appearance of central stress fibers was slightly detectable after 30 minutes but strongly increased by 120 minutes of OxPAPC treatment (Figure 2A, right panels). Involvement of VEGFR2 in Ox-PAPC mediated decreases in barrier function We have previously shown that 50 mg/ml, but not 10 mg/ml OxPAPC significantly activated VEGFR2. We have also shown that VEGFR2 activation at 50 mg/ml OxPAPC leads to an increase in interleukin-8 (IL-8) and LDL receptor expression, while these effects were blocked by VEGFR2 depletion using gene- specific siRNA [14]. Our and other groups have demonstrated an important role for VEGF in regulating barrier permeability in microvascular and macrovascular EC [19,20,21,22,23]. In order to test potential involvement of VEGFR2 in decreased barrier function mediated by OxPAPC, HPAEC were transfected with siRNA to VEGFR2 or nonspecific RNA. We obtained an approximately 90% knockdown at the RNA and protein levels (Figure 3A). Transfection with non-specific RNA or siRNA to VEGFR2 did not affect the basal TER levels or the early phase of TER drop (before 30 min). However, the late phase of OxPAPC- induced TER decline (after 1 hour) was significantly attenuated in EC with depleted VEGFR2, as compared to cells transfected with nonspecific RNA (Figure 3B). These data suggest that the initial drop in TER induced by high OxPAPC dose is not mediated by VEGFR2, whereas the sustained drop requires VEGFR2 activity. The average of 6 separate TER measurements shows a strong suppression of EC monolayer barrier dysfunction observed in the EC monolayers with depleted VEGFR2 after 5 hours of high dose OxPAPC treatment (Figure 3C). Depletion of VEGFR2 attenuates stress fiber formation induced by high Ox-PAPC doses in pulmonary and aortic EC The effect of VEGFR2 depletion on F-actin remodeling was studied in HPAEC treated with high OxPAPC doses (75 mg/ml, 2 hrs). Vehicle treated cells transfected with either nsRNA or siVEGFR2 showed similar patterns of actin distribution (Figure 4A, left panels). High OxPAPC doses caused an increase in stress fibers and formation of paracellular gaps by 2 hours in cells transfected with nsRNA. These changes were reduced in the EC treated with VEGFR2 siRNA (Fig. 4A middle and right panels). Quantitative analysis of paracellular gap formation is presented in Figure 4B. A similar dramatic reduction of stress fiber formation induced by high OxPAPC doses was seen in HAEC treated with VEGFR2 siRNA (Figure 4C). Mechanism of VEGFR2 regulation of barrier function Stress fiber formation and gap formation in EC monolayers is often associated with the activation of myosin light chain (MLC) phosphorylation, leading to the stimulation of actomyosin contractility, EC retraction, and increased EC permeability [24]. RhoA GTPase signaling plays a pivotal role in the induction of MLC phosphorylation in pulmonary endothelium via phosphor- ylation and inactivation of myosin phosphatase (MYPT) by Rho associated kinase [25,26,27]. The high dose of OxPAPC stimulated the phosphorylation of MYPT and MLC in a time- dependent manner with a significant increase observed at 30 min PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30957