RESUMEN
Endothelial dysfunction is a risk factor for atherosclerosis and includes impaired endothelium-dependent vasodilatation. We have shown previously that low density lipoprotein (LDL) can be oxidized by iron in the lysosomes of macrophages. Macrophage lysis in atherosclerotic lesions might expose endothelial cells to this oxidized LDL and adversely affect their function. LDL was oxidized by ferrous sulfate (5 µM) for 24 h at pH 4.5 at 37 °C. Aortas from male Wistar rats were cut into rings and subjected to wire myography for isometric tension recording. The rings were incubated with or without oxidized LDL (50 µg protein/ml) for one hour, constricted with 100 nM phenylephrine and relaxation to acetylcholine (1 nM - 3 µM) was measured. There was about 50% less relaxation in the presence of this oxidized LDL. Endothelial-independent vasodilatation induced by sodium nitroprusside was less affected by oxidized LDL. Oxidized LDL increased the formation of reactive oxygen species by the aortic rings and by cultured human aortic and dermal microvascular endothelial cells, which might have inactivated nitric oxide. Acetylcholine increased the activatory phosphorylation of eNOS (ser-1177), but oxidized LDL had little effect on this activation in cultured human aortic endothelial cells. These findings raise the possibility that LDL oxidized in lysosomes and released from lysed macrophages might decrease vasodilatation in atherosclerotic arteries.
RESUMEN
Many cholesterol-laden foam cells in atherosclerotic lesions are macrophages and much of their cholesterol is present in their lysosomes and derived from low density lipoprotein (LDL). LDL oxidation has been proposed to be involved in the pathogenesis of atherosclerosis. We have shown previously that LDL can be oxidised in the lysosomes of macrophages. α-Tocopherol has been shown to inhibit LDL oxidation in vitro, but did not protect against cardiovascular disease in large clinical trials. We have therefore investigated the effect of α-tocopherol on LDL oxidation at lysosomal pH (about pH 4.5). LDL was enriched with α-tocopherol by incubating human plasma with α-tocopherol followed by LDL isolation by ultracentrifugation. The α-tocopherol content of LDL was increased from 14.4 ± 0.2 to 24.3 ± 0.3 nmol/mg protein. LDL oxidation was assessed by measuring the formation of conjugated dienes at 234 nm and oxidised lipids (cholesteryl linoleate hydroperoxide and 7-ketocholesterol) by HPLC. As expected, LDL enriched with α-tocopherol was oxidised more slowly than control LDL by Cu2+ at pH 7.4, but was not protected against oxidation by Cu2+ or Fe3+ or a low concentration of Fe2+ at pH 4.5 (it was sometimes oxidised faster by α-tocopherol with Cu2+ or Fe3+ at pH 4.5). α-Tocopherol-enriched LDL reduced Cu2+ and Fe3+ into the more pro-oxidant Cu+ and Fe2+ faster than did control LDL at pH 4.5. These findings might help to explain why the large clinical trials of α-tocopherol did not protect against cardiovascular disease.