TLR2 Plays a Key Role in Platelet Hyperreactivity and Accelerated Thrombosis Associated With Hyperlipidemia.

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 the 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 article, 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 (myeloid differentiation factor 88) or TLRs (Toll-like receptors), 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 (Toll-interleukin 1 receptor domain containing adaptor protein)-MyD88-IRAK (interleukin-1 receptor-associated kinase)1/4-TRAF6 (TNF receptor-associated factor 6), leading to integrin activation via the SFK (Src family kinase)-Syk (spleen tyrosine kinase)-PLCγ2 (phospholipase Cγ2) 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.

Novel phosphatidylethanolamine derivatives accumulate in circulation in hyperlipidemic ApoE-/- mice and activate platelets via TLR2.

A prothrombotic state and increased platelet reactivity are common in dyslipidemia and oxidative stress. Lipid peroxidation, a major consequence of oxidative stress, generates highly reactive products, including hydroxy-ω-oxoalkenoic acids that modify autologous proteins generating biologically active derivatives. Phosphatidylethanolamine, the second most abundant eukaryotic phospholipid, can also be modified by hydroxy-ω-oxoalkenoic acids. However, the conditions leading to accumulation of such derivatives in circulation and their biological activities remain poorly understood. We now show that carboxyalkylpyrrole-phosphatidylethanolamine derivatives (CAP-PEs) are present in the plasma of hyperlipidemic ApoE(-/-) mice. CAP-PEs directly bind to TLR2 and induces platelet integrin αIIbß3 activation and P-selectin expression in a Toll-like receptor 2 (TLR2)-dependent manner. Platelet activation by CAP-PEs includes assembly of TLR2/TLR1 receptor complex, induction of downstream signaling via MyD88/TIRAP, phosphorylation of IRAK4, and subsequent activation of tumor necrosis factor receptor-associated factor 6. This in turn activates the Src family kinases, spleen tyrosine kinase and PLCγ2, and platelet integrins. Murine intravital thrombosis studies demonstrated that CAP-PEs accelerate thrombosis in TLR2-dependent manner and that TLR2 contributes to accelerate thrombosis in mice in the settings of hyperlipidemia. Our study identified the novel end-products of lipid peroxidation, accumulating in circulation in hyperlipidemia and inducing platelet activation by promoting cross-talk between innate immunity and integrin activation signaling pathways.

Differential regulation of endothelial cell permeability by high and low doses of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine.

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 µg/ml enhanced EC barriers, as indicated by increased transendothelial electrical resistance. In contrast, higher OxPAPC concentrations (50-100 µg/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/ß-catenin complexes and VE-cadherin internalization, whereas low OxPAPC doses promoted the formation of VE-cadherin-p120-catenin/ß-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.

Regulation of platelet function by class B scavenger receptors in hyperlipidemia.

Platelets constitutively express class B scavenger receptors CD36 and SR-BI, 2 closely related pattern recognition receptors best known for their roles in lipoprotein and lipid metabolism. The biological role of scavenger receptors in platelets is poorly understood. However, in vitro and in vivo data suggest that class B scavenger receptors modulate platelet function and contribute significantly to thrombosis by sensing pathological or physiological ligands, inducing prothrombotic signaling, and increasing platelet reactivity. Platelet CD36 recognizes a novel family of endogenous oxidized choline phospholipids that accumulate in plasma of hyperlipidemic mice and in plasma of subjects with low high-density lipoprotein levels. This interaction leads to the activation of specific signaling pathways and promotes platelet activation and thrombosis. Platelet SR-BI, on the other hand, plays a critical role in the induction of platelet hyperreactivity and accelerated thrombosis under conditions associated with increased platelet cholesterol content. Intriguingly, oxidized high-density lipoprotein, an SR-BI ligand, can suppress platelet function. These recent findings demonstrate that platelet class B scavenger receptors play roles in thrombosis in dyslipidemia and may contribute to acute cardiovascular events in vivo in hypercholesterolemia.

Activation of aortic endothelial cells by oxidized phospholipids: a phosphoproteomic analysis.

Previous studies have shown that oxidized products of the phospholipid PAPC (Ox-PAPC) are strong activators of aortic endothelial cells and play an important role in atherosclerosis and other inflammatory diseases. We and others have demonstrated that Ox-PAPC activates specific signaling pathways and regulates a large number of genes. Using a phosphoproteomic approach based on phosphopeptide enrichment and mass spectrometry analysis, we identified candidate changes in Ox-PAPC-induced protein phosphorylation of 228 proteins. Functional annotation of these proteins showed an enrichment of the regulation of cytoskeleton, junctional components, and tyrosine kinases, all of which may contribute to the phenotypic and molecular changes observed in endothelial cells treated with Ox-PAPC. Many changes in protein phosphorylation induced by Ox-PAPC are reported here for the first time and provide new insights into the mechanism of activation by oxidized lipids, including phosphorylation-based signal transduction.

Vascular endothelial growth factor receptor 2 plays a role in the activation of aortic endothelial cells by oxidized phospholipids.

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 Tyr1175 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.

Enzymatic synthesis of [3'-O-methyl-(3)H]malvidin-3-glucoside from petunidin-3-glucoside.

Malvidin-3-glucoside has been labeled by enzymatic synthesis in a single-step experiment. Catechol-O-methyl transferase catalyzed the B-ring methylation of petunidin-3-glucoside, and S-Adenosyl-L-[methyl-(3)H] methionine was the methyl donor. Solid phase extraction and preparative high-performance liquid chromatography were necessary to separate [3'-O-methyl-(3)H]malvidin-3-glucoside from an isomer and the starting material. The specific activity was 2.2 Ci mmol(-1), and the yield of incorporation was 1.1%. A possible application of the labeled material is the study of anthocyanin reactions in complex mixtures such as red wine where products are difficult to isolate and analyze.

Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin.

Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36.

Phosphoproteomic analysis of aortic endothelial cells activated by oxidized phospholipids.

Comprehensive identification of quantitative changes in protein phosphorylation using mass spectrometry is becoming a common tool in cell signaling studies. To date, most of these kinase network studies are conducted in stable cancer cell lines, yeasts, or other models that are not representative of cardiovascular disease. We describe methods based on phosphopeptide enrichment after tryptic digestion of cell lysates to study changes in protein phosphorylation of endothelial cells. We used this approach to study the activation of aortic endothelial cells by oxidized phospholipids, compounds important in atherosclerosis and other inflammatory diseases.

A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids.

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 cells (HAECs). 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. 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 those involved in sterol regulation. Our data demonstrate that Ox-PAPC increases reactive oxygen species formation in HAECs as seen by DCF fluorescence. NOX4 is the major molecule responsible for this increase because downregulation of NOX4 and its components (p22(phox) 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 p22(phox) decreases Ox-PAPC induction of inflammatory and sterol regulatory genes, but does not affect Ox-PAPC transcriptional regulation of other genes for antioxidants and the unfolded protein response. In summary, we have identified a VEGFR2/NOX4 regulatory pathway by which Ox-PAPC controls important endothelial functions.