Objective-Previous studies have shown that oxidized products of PAPC (Ox-PAPC) regulate cell transcription of interleukin-8, LDL receptor, and tissue factor. This upregulation takes place in part through the activation of sterol regulatory element-binding protein (SREBP) and Erk 1/2. The present studies identify vascular endothelial growth factor receptor 2 (VEGFR2) as a major regulator in the activation of SREBP and Erk 1/2 in endothelial cells activated by Ox-PAPC. Methods and Results-Ox-PAPC induced the phosphorylation of VEGFR2 at Tyr 1175 in human aortic endothelial cells. Inhibitors and siRNA for VEGFR2 decreased the transcription of interleukin-8, LDL receptor, and tissue factor in response to Ox-PAPC and the activation of SREBP and Erk 1/2, which mediate this transcription. We provide evidence that the activation of VEGFR2 is rapid, sustained, and c-Src-dependent. Conclusions-These data point to a major role of VEGFR2 in endothelial regulation by oxidized phospholipids which accumulate in atherosclerotic lesions and apoptotic cells.

Key Points CAP-PEs, a novel type of oxidatively modified phospholipids, are present in vivo. CAP-PEs can activate platelets via TLRs by inducing a cross-talk between innate immunity and integrin activation signaling pathways.

The generation of phospholipid oxidation products in atherosclerosis, sepsis, and lung pathologies affects endothelial barrier function, which exerts significant consequences on disease outcomes in general. Our group previously showed that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (OxPAPC) at low concentrations increases endothelial cell (EC) barrier function, but decreases it at higher concentrations. In this study, we determined the mechanisms responsible for the pulmonary endothelial cell barrier dysfunction induced by high OxPAPC concentrations. OxPAPC at a range of 5-20 mg/ml enhanced EC barriers, as indicated by increased transendothelial electrical resistance. In contrast, higher OxPAPC concentrations (50-100 mg/ml) rapidly increased EC permeability, which was accompanied by increased total cell protein tyrosine (Tyr) phosphorylation, phosphorylation at Tyr-418, the activation of Src kinase, and the phosphorylation of adherens junction (AJ) protein vascular endothelial cadherin (VE-cadherin) at Tyr-731 and Tyr-658, which was not observed in ECs stimulated with low OxPAPC doses. The early tyrosine phosphorylation of VE-cadherin was linked to the dissociation of VE-cadherin-p120-catenin/b-catenin complexes and VE-cadherin internalization, whereas low OxPAPC doses promoted the formation of VE-cadherin-p120-catenin/b-catenin complexes. High but not low doses of OxPAPC increased the production of reactive oxygen species (ROS) and protein oxidation. The inhibition of Src by PP2 and ROS production by N-acetyl cysteine inhibited the disassembly of VE-cadherin-p120-catenin complexes, and attenuated high OxPAPC-induced EC barrier disruption. These results show the differential effects of OxPAPC doses on VE-cadherin-p120-catenin complex assembly and EC barrier function. These data suggest that the rapid tyrosine phosphorylation of VE-cadherin and other potential targets mediated by Src and ROS-dependent mechanisms plays a key role in the dissociation of AJ complexes and EC barrier dysfunction induced by high OxPAPC doses.Keywords: endothelium; oxidized phospholipids; protein tyrosine phosphorylation; VE-cadherin; permeability Oxidative stress and the activation of phospholipases lead to the formation and accumulation of biologically active lipid oxidation products. Phospholipid oxidation products were shown to accumulate in a number of inflammatory diseases (1). An increase in oxidative stress and in phospholipid oxidation products was demonstrated in patients with diverse lung diseases, such as acute respiratory distress syndrome, ventilator-induced lung injury, and asthma, and in animal studies of lung injury (2-4). Oxidized phospholipids were also shown to accumulate in atherosclerosis and other chronic inflammatory diseases (5). The increased concentrations of oxidized phospholipids in the injured lung may influence the functions of pulmonary endothelial cells (ECs), including the modulation of pulmonary inflammatory response and EC barrier regulation (6-9).Oxidized phospholipids may induce ...

Rationale Platelet hyperreactivity, which is common in many pathological conditions, is associated with increased atherothrombotic risk. The mechanisms leading to platelet hyperreactivity are complex and not yet fully understood. Objective Platelet hyperreactivity and accelerated thrombosis, specifically in dyslipidemia, have been mechanistically linked to accumulation in the circulation of a specific group of oxidized phospholipids (oxPCCD36) that are ligands for the platelet pattern-recognition receptor CD36. In the current manuscript, we tested whether the platelet innate immune system contributes to responses to oxPCCD36 and accelerated thrombosis observed in hyperlipidemia. Methods and Results Using in vitro approaches, as well as platelets from mice with genetic deletion of MyD88 or TLRs, we demonstrate that TLR2 and TLR6 are required for the activation of human and murine platelets by oxPCCD36. oxPCCD36 induce formation of CD36/TLR2/TLR6 complex in platelets and activate downstream signaling via TIRAP-MyD88-IRAK1/4-TRAF6, leading to integrin activation via the SFK-Syk-PLCγ2 pathway. Intravital thrombosis studies using ApoE−/− mice with genetic deficiency of TLR2 or TLR6 have demonstrated that oxPCCD36 contribute to accelerated thrombosis specifically in the setting of hyperlipidemia. Conclusions Our studies reveal that TLR2 plays a key role in platelet hyperreactivity and the prothrombotic state in the setting of hyperlipidemia by sensing a wide range of endogenous lipid-peroxidation ligands and activating innate immune signaling cascade in platelets.

Previous studies from our group have demonstrated that oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) activates over 1000 genes in human aortic endothelial cell (HAEC). Prominent among these are genes regulating inflammation, cholesterol homeostasis, antioxidant enzymes, and the unfolded protein response. Previous studies from our lab and others suggested that transcriptional regulation by Ox-PAPC may be controlled, at least in part, by reactive oxygen species (ROS). We now present evidence that Ox-PAPC activation of NADPH oxidase 4 (NOX4) is responsible for the regulation of two of these important groups of genes: those controlling inflammation and sterol regulation. Our data demonstrate that Ox-PAPC increases reactive oxygen species formation in HAEC as seen by DCF fluorescence. NOX4 is the major molecule responsible for this increase since downregulation of NOX4 and its components (p22phox and rac1) blocked the Ox-PAPC effect. Our data show that Ox-PAPC did not change NOX4 transcription levels but did induce recruitment of rac1 to the membrane for NOX4 activation. We present evidence that vascular endothelial growth factor receptor 2 (VEGFR2) activation is responsible for rac1 recruitment to the membrane. Finally, we demonstrate that knockdown of NOX4 and its components rac1 and p22phox decrease Ox-PAPC induction of inflammatory and sterol regulatory genes, but do not affect Ox-PAPC transcriptional regulation of other gene of antioxidant and unfolded protein response. In summary, we have identified a VEGFR2/NOX4 regulatory pathway by which Ox-PAPC controls important endothelial functions.