VEGFR2 Mediates Endothelial Permeability by OxPAPC Figure 2. Dose dependent effects of OxPAPC on HAEC and HPAEC actin cytoskeletal remodeling and monolayer integrity. A – HPAEC, and B – HAEC monolayers grown on glass coverslips were stimulated with OxPAPC (10 mg/ml or 75 mg/ml) for 30 min or 120 min followed by immunofluorescence staining for F-actin. Arrows depict areas of intercellular gaps caused by treatment with 75 mg/ml OxPAPC in HPAEC or 50 mg/ml OxPAPC in HAEC. Shown are representative results of three independent experiments. doi:10.1371/journal.pone.0030957.g002 kinase abolished OxPAPC-induced stress fiber formation (Figure 5B). Previous reports demonstrated that the activation of VEGFR2 stimulates RhoA signaling, RhoA-mediated cytoskeletal remodel- ing, and actomyosin contractility, leading to an increase in permeability of endothelial monolayers [16,23,28]. We next examined whether VEGFR2 is involved in the increase of MLC phosphorylation observed in EC treated with high OxPAPC doses. HPAEC were transfected with either VEGFR2-specific siRNA or non-specific RNA. Consistent with results shown above, treatment of HPAEC with 75 mg/ml OxPAPC did not induce rapid MLC phosphorylation (no increase at 5 min). However, MLC phos- phorylation was seen at 30 and 120 minutes and was strongly inhibited by VEGFR2 depletion (Figure 6A). Quantitative analysis of MLC phosphorylation in control and VEGFR2 depleted HPAEC is presented in Figure 6B. Increased MLC phosphorylation in response to high OxPAPC dose was also observed in HAEC treated with nonspecific RNA, while depletion of VEGFR2 strongly inhibited MLC phosphorylation (Figure 6C). The kinetics of the VEGFR2 effect on TER suggested that there were different regulators of the early and late decreases in barrier function caused by high dose OxPAPC and that VEGFR2 activation mainly controls the later phase. The current results and our recent published studies [29] suggest that the phosphorylation of adherens junction proteins such as VE-cadherin may be involved in the early phase of junction breakdown and that an increase in MLC phosphorylation regulates the later phase. To test the involvement of VEGFR2 in OxPAPC-induced VE-Cadherin phosphorylation, we examined the effect of knockdown of VEGFR2 on VE-Cadherin phosphorylation (Figure 6A). Phos- phorylation of VE-Cadherin induced by high OxPAPC dose was rapid, reaching maximal levels by 5 minutes, and was only slightly attenuated by VEGFR2 knockdown in HPAEC (Figure 6B). In HAEC, VE-Cadherin phosphorylation was also increased by 5 minutes, and this increase was VEGFR2 independent (Figure 6C). We also examined the intracellular distribution of dipho- sphorylated MLC (ppMLC) by double immunofluorescence staining with diphospho-MLC specific antibody. Vehicle treated cells transfected with either nsRNA or VEGFR2 siRNA showed low levels of diphospho-MLC immunoreactivity (Figure 7A, left panels). High OxPAPC dose caused a strong increase in ppMLC aligned with stress fibers. In contrast to nsRNA, treatment siVEGFR2 dramatically reduced ppMLC immunoreactivity in OxPAPC-treated cells (Figure 7A, right panels). Quantitative analysis of MLC phosphorylation in control and VEGFR2 depleted HPAEC is presented in Figure 7B. These results PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30957