9-oxononanoyl-cholesterol and Arteriosclerosis

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.

Topics: Aldehydes; Antibodies, Monoclonal; Aorta; Apolipoprotein B-100; Apolipoproteins B; Arteriosclerosis; Ascorbic Acid; Autoantigens; Cholesterol; Cholesterol Esters; Cholesterol, LDL; Humans; Iron; Ketocholesterols; Lipid Peroxidation; Lipoproteins, LDL; Lysine; Oxidation-Reduction; Schiff Bases

Oxidation products of cholesteryl esters have been shown to be present in oxidized low density lipoprotein and in atherosclerotic lesions. Monocyte adhesion to the endothelium is an initiating crucial event in atherogenesis. Here, we show that in vitro oxidized cholesteryl linoleate (oxCL) stimulated human umbilical vein endothelial cells (HUVECs) to bind human peripheral blood mononuclear cells as well as monocyte-like U937 cells but not peripheral blood neutrophils or neutrophil-like HL-60 cells. Among the oxidation products contained in oxCLs, 9-oxononanoyl cholesterol (9-ONC) and cholesteryl linoleate hydroperoxides stimulated U937 cell adhesion. OxCL-induced U937 cell adhesion was inhibited by an antibody against the connecting segment-1 region of fibronectin. Neither oxCL nor 9-ONC induced activation of the classical nuclear factor-kappaB pathway. In contrast, stimulation of HUVECs with oxCL resulted in phosphorylation of the extracellular signal-regulated kinase 1/2. Moreover, U937 cell adhesion induced by 9-ONC and oxCL was blocked by a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor and a protein kinase C inhibitor. Taken together, oxCLs stimulate HUVECs to specifically bind monocytes, involving endothelial connecting segment-1 and the activation of a protein kinase C- and mitogen-activated protein kinase-dependent pathway. Thus, oxidized cholesteryl esters may play an important role as novel mediators in the initiation and progression of atherosclerosis.

Topics: Arteriosclerosis; Cell Adhesion; Cholesterol; Cholesterol Esters; Endothelium, Vascular; Humans; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Monocytes; NF-kappa B; Oxidation-Reduction; Peptides; Phosphorylation; Protein Kinase C; U937 Cells

9-Oxononanoyl cholesterol, a cholesterol core-aldehyde formed during lipoprotein oxidation, was recently identified in advanced human atherosclerotic lesions. Here we present a rapid and sensitive HPLC method for 9-oxononanoyl cholesterol analysis. 9-Oxononanoyl cholesterol was converted to the corresponding fluorescent decahydroacridine derivative by reaction with 1,3-cyclohexanedione. The derivatives formed were purified by solid-phase extraction on C-18 columns, separated by reversed phase HPLC with isocratic elution, and detected by their fluorescence. Decahydroacridine derivatives of 9-oxononanoyl cholesterol were stable for at least 160 h. The limit of quantitation of the method presented is at the low (approximately 50) femtomole level, with an absolute limit of detection (signal: noise = 6) of 15 fmol. Intra-assay variation was < or = 5%, while inter-assay variations were between 5 and 15%, depending on the concentration of the analyte. Standard curves were linear over nearly three orders of magnitude (50 fmol-12.5 pmol). 9-Oxononanoyl cholesterol proved to be the major cholesterol core-aldehyde formed during t-BuOOH/FeSO4 oxidation of cholesteryl linoleate and Cu2+-induced LDL oxidation, findings confirmed by atmospheric pressure chemical ionization-mass spectrometry. Analysis of lipid extracts obtained from advanced human atherosclerotic lesions revealed the presence of 9-oxononanoyl cholesterol in all tissue samples analyzed (28+/-14 micromol/mol cholesterol, n = 9) despite the presence of alpha-tocopherol (4+/-1.2 mmol/mol cholesterol, n = 9).

Topics: Adult; Aldehydes; Aorta, Abdominal; Aorta, Thoracic; Arteriosclerosis; Cholesterol; Chromatography, High Pressure Liquid; Humans; Indicators and Reagents; Lipoproteins, LDL; Mass Spectrometry; Middle Aged; Molecular Structure; Sensitivity and Specificity