Abstract
Background and aimsWe have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. We have now used sphingomyelinase-aggregated LDL, a more pathophysiological form of aggregated LDL, to study lysosomal oxidation of LDL and its inhibition by antioxidants, including cysteamine (2-aminoethanethiol), which concentrates in lysosomes by several orders of magnitude. We have also investigated the effect of cysteamine on atherosclerosis in mice. MethodsLDL was incubated with sphingomyelinase, which increased its average particle diameter from 26 to 170 nm, and was then incubated for up to 7 days with human monocyte-derived macrophages. LDL receptor-deficient mice were fed a Western diet (19–22 per group) and some given cysteamine in their drinking water at a dose equivalent to that used in cystinosis patients. The extent of atherosclerosis in the aortic root and the rest of the aorta was measured. ResultsConfocal microscopy revealed lipid accumulation in lysosomes in the cultured macrophages. Large amounts of ceroid were produced, which colocalised with the lysosomal marker LAMP2. The antioxidants cysteamine, butylated hydroxytoluene, amifostine and its active metabolite WR-1065, inhibited the production of ceroid. Cysteamine at concentrations well below those expected to be present in lysosomes inhibited the oxidation of LDL by iron ions at lysosomal pH (pH 4.5) for prolonged periods. Finally, we showed that the extent of atherosclerotic lesions in the aortic root and arch of mice was significantly reduced by cysteamine. ConclusionsThese results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and hence antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease.
Highlights
Oxidised low density lipoprotein (LDL) was at one time widely believed to be important in the pathogenesis of atherosclerosis [1,2], the underlying cause of the large majority of myocardial infarctions and thrombotic strokes
These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease
Two key problems with this conventional view are that oxidation is inhibited by low concentrations of interstitial fluid or serum [13,14] and that large clinical trials have failed to show that antioxidants protect against cardiovascular disease [15]. To account for these problems, we hypothesised that LDL nonoxidatively modified and aggregated by enzymes, such as sphingomyelinase [16], proteases [17] or secretory phospholipase A2 enzymes [18], in the extracellular space of atherosclerotic lesions [19] is rapidly endocytosed by macrophages and delivered to lysosomes, where it might be oxidised [20]. In support of this view, we showed that 7 days after taking up mechanically-aggregated LDL, mouse J774 macrophage-like cells and human monocyte-derived macrophages (HMDM) generated ceroid in their lysosomes
Summary
Oxidised LDL was at one time widely believed to be important in the pathogenesis of atherosclerosis [1,2], the underlying cause of the large majority of myocardial infarctions and thrombotic strokes. This was because oxidised LDL has numerous potential pro-atherosclerotic effects. We have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. Conclusions: These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease
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