Low Density Lipoprotein Undergoes Oxidation Within Lysosomes in Cells

Abstract

Abstract-The oxidized low density lipoprotein (LDL) hypothesis of atherosclerosis proposes that LDL undergoes oxidation in the interstitial fluid of the arterial wall. We have shown that aggregated (vortexed) nonoxidized LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidized intracellularly, as assessed by the microscopic detection of ceroid, an advanced lipid oxidation product. Confocal microscopy showed that the ceroid was located in the lysosomes. To confirm these findings, J774 macrophages were incubated with acetylated LDL, which is internalized rapidly to lysosomes, and then incubated (chase incubation) in the absence of any LDL. Key Words: atherosclerosis Ⅲ ceroid Ⅲ lysosome Ⅲ iron Ⅲ oxidized low density lipoprotein T he local oxidation of low density lipoprotein (LDL) within atherosclerotic lesions is widely believed to be of importance in the pathogenesis of atherosclerosis. 1 LDL is thought to be oxidized within the extracellular space of atherosclerotic lesions and then to be bound by scavenger receptors and taken up by macrophages, which become cholesterol-laden foam cells, a major feature of atherosclerotic lesions. 2 Among many other effects, oxidized LDL increases the expression of cellular adhesion molecules and chemokines, 3,4 increases the production of metalloproteinases, 5 which probably destabilize the fibrous caps over advanced lesions, and induces apoptosis in cells. 6 The mechanisms by which LDL is oxidized in atherosclerotic lesions remain uncertain, despite a great deal of work. 7 The oxidation hypothesis of atherosclerosis needs to address the high antioxidant capacity of extracellular fluids. Even a few percent of serum or interstitial fluid can inhibit greatly the oxidation of LDL by cells. 8,9 We postulated that LDL oxidation might occur not within the interstitial fluid of atherosclerotic lesions but within lysosomes in macrophages in atherosclerotic lesions. Materials and Methods LDL Isolation and ModificationBlood was taken from healthy volunteers with EDTA as the anticoagulant (final concentration 3 mmol/L). LDL (1.019 to 1.063 g/mL) was isolated from the plasma by sequential density ultracentrifugation at 4°C, as described previously. 10 LDL was stored in the dark under argon at 4°C and used within 1 month. Aggregation of LDL was achieved by vortexing 11 or acetylation. 12 Acetylation of LDL was confirmed by agarose gel electrophoresis (Paragon gels; Beckman), as seen by an increase of about 4.5 in electrophoretic mobility relative to native LDL. Cell CultureCell culture media (DMEM, RPMI 1640, and Ham's F-10) and phosphate buffered saline (PBS) (without calcium or magnesium) were obtained from Gibco Life Technologies. The media used in this study were supplemented with 20% (v/v) fetal calf serum, Glutamax (2 mmol/L), penicillin (50 IU/mL), streptomycin (50 g/mL), and amphotericin B (0.95 g/mL), unless otherwise stated. Humidified 95% air/5% carbon dioxide at 37°C was used for cell culture. J774 cells were regularly cultured in supplement...

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