Effects of lysosomal low-density lipoprotein oxidation by ferritin on macrophage function.

We have previously demonstrated that low-density lipoprotein (LDL) can be oxidized by iron in the lysosomes of macrophages. Some of the iron content of lysosomes might be delivered through autophagy of ferritin (the main iron-storage protein in the body). We have now investigated the effects of ferritin-mediated LDL oxidation on macrophage function. The addition of ferritin to human THP-1 cells and human monocyte-derived macrophages increased lysosomal lipid peroxidation, as shown by LPO-Foam, a fluorescent probe targeted to lysosomes. Incubating THP-1 cells with ferritin and native LDL or LDL aggregated by sphingomyelinase, to allow their endocytosis and delivery to lysosomes, led to the formation of lysosomal ceroid (an advanced lipid oxidation product), indicative of lysosomal LDL oxidation. Incubating THP-1 cells with ferritin and LDL caused metabolic activation of the cells, as shown by increased extracellular acidification and oxygen consumption measured by a Seahorse analyzer. LDL oxidized by ferritin in lysosomes might be released from macrophages when the cells die and lyse and affect neighboring cells in atherosclerotic lesions. Adding LDL oxidized by ferritin at lysosomal pH (pH 4.5) to macrophages increased their intracellular reactive oxygen species formation, shown using dihydroethidium, and increased apoptosis. Ferritin might therefore contribute to LDL oxidation in the lysosomes of macrophages and have atherogenic effects.

Vitamins E and C do not effectively inhibit low density lipoprotein oxidation by ferritin at lysosomal pH.

Low density lipoprotein (LDL) might be oxidized by iron in the lysosomes of macrophages in atherosclerotic lesions. We have shown previously that the iron-storage proteinferritin can oxidize LDL at lysosomal pH. We have now investigated the roles of the most important antioxidant contained in LDL, α-tocopherol (the main form of vitamin E) and of ascorbate (vitamin C), a major water-soluble antioxidant, on LDL oxidation by ferritin at lysosomal pH (pH 4.5). We incubated LDL with ferritin at pH 4.5 and 37 °C and measured its oxidation by monitoring the formation of conjugated dienes at 234 n min a spectrophotometer. α-Tocopherol is well known to inhibit LDL oxidation at pH 7.4, but enrichment of LDL with α-tocopherol was unable to inhibit LDL oxidation by ferritin at pH 4.5. Ascorbate had a complex effect on LDL oxidation by ferritin at lysosomal pH and exhibited both antioxidant and pro-oxidant effects. It had no antioxidant effect on partially oxidized LDL, only a pro-oxidant effect. Ascorbate completely inhibited LDL oxidation by copper at pH 7.4 for a long period, but in marked contrast did not inhibit LDL oxidation by copper at lysosomal pH. Dehydroascorbate, the oxidation product of ascorbate, had a pronounced pro-oxidant effect on LDL incubated with ferritin at pH 4.5. The inability of α-tocopherol and ascorbate to effectively inhibit LDL oxidation by ferritin at lysosomal pH might help to explain why the large clinical trials with these vitamins failed to show protection against cardiovascular diseases.

Low density lipoprotein oxidation by ferritin at lysosomal pH.

Oxidation of low density lipoprotein (LDL) has been proposed to be involved in the pathogenesis of atherosclerosis. We have previously shown that LDL can be oxidised by iron in lysosomes. As the iron-storage protein ferritin might enter lysosomes by autophagy, we have investigated the ability of ferritin to catalyse LDL oxidation at lysosomal pH. LDL was incubated with ferritin at 37 °C and pH 4.5 and its oxidation monitored spectrophotometrically at 234 nm by the formation of conjugated dienes and by measuring oxidised lipids by HPLC or a tri-iodide assay. Iron released from ferritin was measured using the ferrous iron chelator bathophenanthroline and by ultrafiltration followed by atomic absorption spectroscopy. LDL was oxidised effectively by ferritin (0.05-0.2 µM). The oxidation at lysosomal pH (pH 4.5) was much faster than at pH 7.4. Ferritin increased cholesteryl linoleate hydroperoxide, total lipid hydroperoxides and 7-ketocholesterol. Iron was released from ferritin at acidic pH. The iron chelators, diethylenetriaminepentaacetate and EDTA, and antioxidant N,N׳-diphenyl-p-phenylenediamine inhibited the oxidation considerably, but not entirely. The antioxidant tempol did not inhibit the initial oxidation of LDL, but inhibited its later oxidation. Cysteamine, a lysosomotropic antioxidant, inhibited the initial oxidation of LDL in a concentration-dependent manner, however, the lower concentrations exhibited a pro-oxidant effect at later times, which was diminished and then abolished as the concentration increased. These results suggest that ferritin might play a role in lysosomal LDL oxidation and that antioxidants that accumulate in lysosomes might be a novel therapy for atherosclerosis.