Abstract
Oxidation of low-density lipoprotein (LDL) is one of the major causative mechanisms in the development of atherosclerosis. In previous studies, we showed that minimally oxidized LDL (mmLDL) induced inflammatory responses in macrophages, macropinocytosis and intracellular lipid accumulation and that oxidized cholesterol esters (OxCEs) were biologically active components of mmLDL. Here we identified a specific OxCE molecule responsible for the biological activity of mmLDL and characterized signaling pathways in macrophages in response to this OxCE. Using liquid chromatography - tandem mass spectrometry and biological assays, we identified an oxidized cholesteryl arachidonate with bicyclic endoperoxide and hydroperoxide groups (BEP-CE) as a specific OxCE that activates macrophages in a TLR4/MD-2-dependent manner. BEP-CE induced TLR4/MD-2 binding and TLR4 dimerization, phosphorylation of SYK, ERK1/2, JNK and c-Jun, cell spreading and uptake of dextran and native LDL by macrophages. The enhanced macropinocytosis resulted in intracellular lipid accumulation and macrophage foam cell formation. Bone marrow-derived macrophages isolated from TLR4 and SYK knockout mice did not respond to BEP-CE. The presence of BEP-CE was demonstrated in human plasma and in the human plaque material captured in distal protection devices during percutaneous intervention. Our results suggest that BEP-CE is an endogenous ligand that activates the TLR4/SYK signaling pathway. Because BEP-CE is present in human plasma and human atherosclerotic lesions, BEP-CE-induced and TLR4/SYK-mediated macrophage responses may contribute to chronic inflammation in human atherosclerosis.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biological Transport
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Cell Line
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Cholesterol / analogs & derivatives*
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Cholesterol / chemistry
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Cholesterol / isolation & purification
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Cholesterol / pharmacology
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Cholesterol, LDL / metabolism
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Gene Expression Regulation
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Humans
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Inflammation / genetics
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Inflammation / metabolism
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Inflammation / pathology
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Intracellular Signaling Peptides and Proteins / deficiency
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Intracellular Signaling Peptides and Proteins / genetics*
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JNK Mitogen-Activated Protein Kinases / genetics
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JNK Mitogen-Activated Protein Kinases / metabolism
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Lipoproteins, LDL / chemistry
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Lymphocyte Antigen 96 / genetics
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Lymphocyte Antigen 96 / metabolism
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MAP Kinase Kinase 4 / genetics
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MAP Kinase Kinase 4 / metabolism
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Macrophages / drug effects
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Macrophages / metabolism*
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Macrophages / pathology
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Mice
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Mice, Knockout
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Mitogen-Activated Protein Kinase 1 / genetics
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3 / genetics
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Mitogen-Activated Protein Kinase 3 / metabolism
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Oxidation-Reduction
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Plaque, Atherosclerotic / genetics*
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Plaque, Atherosclerotic / metabolism
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Plaque, Atherosclerotic / pathology
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Primary Cell Culture
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Protein Multimerization
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Protein-Tyrosine Kinases / deficiency
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Protein-Tyrosine Kinases / genetics*
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Signal Transduction
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Syk Kinase
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Toll-Like Receptor 4 / deficiency
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Toll-Like Receptor 4 / genetics*
Substances
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Cholesterol, LDL
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Intracellular Signaling Peptides and Proteins
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Lipoproteins, LDL
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Ly96 protein, mouse
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Lymphocyte Antigen 96
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Tlr4 protein, mouse
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Toll-Like Receptor 4
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oxidized low density lipoprotein
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cholesterol hydroperoxide
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Cholesterol
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Protein-Tyrosine Kinases
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SYK protein, human
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Syk Kinase
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Syk protein, mouse
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JNK Mitogen-Activated Protein Kinases
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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MAP Kinase Kinase 4