Abstract
Oxidised low density lipoprotein (LDL) was considered to be important in the pathogenesis of atherosclerosis, but the large clinical trials of antioxidants, including the first one using probucol (the PQRST Trial), failed to show benefit and have cast doubt on the importance of oxidised LDL. We have shown previously that LDL oxidation can be catalysed by iron in the lysosomes of macrophages. The aim of this study was therefore to investigate the effectiveness of antioxidants in preventing LDL oxidation at lysosomal pH and also establish the possible mechanism of oxidation. Probucol did not effectively inhibit the oxidation of LDL at lysosomal pH, as measured by conjugated dienes or oxidised cholesteryl esters or tryptophan residues in isolated LDL or by ceroid formation in the lysosomes of macrophage-like cells, in marked contrast to its highly effective inhibition of LDL oxidation at pH 7.4. LDL oxidation at lysosomal pH was inhibited very effectively for long periods by N,N'-diphenyl-1,4-phenylenediamine, which is more hydrophobic than probucol and has been shown by others to inhibit atherosclerosis in rabbits, and by cysteamine, which is a hydrophilic antioxidant that accumulates in lysosomes. Iron-induced LDL oxidation might be due to the formation of the superoxide radical, which protonates at lysosomal pH to form the much more reactive, hydrophobic hydroperoxyl radical, which can enter LDL and reach its core. Probucol resides mainly in the surface monolayer of LDL and would not effectively scavenge hydroperoxyl radicals in the core of LDL. This might explain why probucol failed to protect against atherosclerosis in various clinical trials. The oxidised LDL hypothesis of atherosclerosis now needs to be re-evaluated using different and more effective antioxidants that protect against the lysosomal oxidation of LDL.
Highlights
Oxidation of low density lipoprotein (LDL) was widely believed to be a critical step in the pathogenesis of atherosclerosis, the underlying cause of myocardial infarctions and thrombotic strokes (Steinberg and Witztum, 2010)
Iron-induced LDL oxidation might be due to the formation of the superoxide radical, which protonates at lysosomal pH to form the much more reactive, hydrophobic hydroperoxyl radical, which can enter LDL and reach its core
We showed that 7 days after taking up mechanically-aggregated LDL, mouse J774 cells and human monocyte-derived macrophages (HMDM) generated ceroid in their lysosomes (Wen and Leake, 2007)
Summary
Oxidation of low density lipoprotein (LDL) was widely believed to be a critical step in the pathogenesis of atherosclerosis, the underlying cause of myocardial infarctions and thrombotic strokes (Steinberg and Witztum, 2010). To address the shortcomings in the conventional LDL oxidation theory, we proposed that nonoxidatively modified LDL is rapidly endocytosed by macrophages and oxidised within lysosomes (Wen and Leake, 2007). We showed that 7 days after taking up mechanically-aggregated (vortexed) LDL, mouse J774 cells (a macrophage-like cell line) and human monocyte-derived macrophages (HMDM) generated ceroid (an advanced lipid oxidation product present in human atherosclerotic lesions) in their lysosomes (Wen and Leake, 2007). There was an increased production of oxysterols (e.g. 7-ketocholesterol) in the J774 cells when incubated with nonoxidized acetylated LDL which is rapidly endocytosed by macrophages, and incubated in the absence of extracellular lipoproteins. It was further shown that the oxidation in lysosomes is mediated by iron which is highly effective in oxidising LDL at pH 4.5, the approximate pH of lysosomes, but very poor at doing so at pH 7.4 in a simple buffer (Satchell and Leake, 2012; Wen and Leake, 2007)
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