Polyoxygenated Cholesterol Ester Hydroperoxide Activates TLR4 and SYK Dependent Signaling in Macrophages

Soo-Ho Choi,Aaron Armando,Coleen A Mcnamara,Darren Dumlao,Yury I Miller,Angela M Taylor,Edward A Dennis,Amir Ravandi,Felicidad Almazan,Huiyong Yin,Joseph L Witztum,Jungsu Kim,Sotirios Tsimikas

doi:10.1371/journal.pone.0083145

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.

Highlights

Oxidized low density lipoprotein (LDL) is considered a major pathogenic factor in the development of atherosclerosis [1,2]
We further attempted to identify a specific, biologically active oxidized cholesterol esters (OxCEs) molecule, but the amount of OxCE material isolated from minimally oxidized LDL (mmLDL) proved to be insufficient for the detailed liquid chromatography (LC)/MS analysis and biological assays
The LC fractions were tested with J774 macrophages to assay their effects on cell spreading and phosphorylation of ERK1/2, the two robust effects of nonfractionated AA-CE/15LO and mmLDL [14]

Summary

Introduction

Oxidized low density lipoprotein (LDL) is considered a major pathogenic factor in the development of atherosclerosis [1,2]. Even though LDL in these studies was oxidized in vitro, a number of biologically active molecules identified in in vitro oxidized LDL have been found in plasma and/or atherosclerotic lesions of humans and experimental animals [3,5,6,7,8]. These findings suggest that the mechanisms of OxLDLinduced activation of macrophages, endothelial cells and vascular smooth muscle cells are relevant to the initiation and progression of atherosclerosis. We and others have documented accumulation of OxCE in the lesions of

Methods

Results

Conclusion