The Rabbit 15-Lipoxygenase Preferentially Oxygenates LDL Cholesterol Esters, and This Reaction Does Not Require Vitamin E

Abstract

The oxidation of low density lipoprotein (LDL) by mammalian 15-lipoxygenases (15-LOX) was implicated in early atherogenesis. We investigated the molecular mechanism of 15-LOX/LDL interaction and found that during short term incubations, LDL cholesterol esters are oxygenated preferentially by the enzyme. Even when the LDL particle was loaded with free linoleic acid, cholesteryl linoleate constituted the major LOX substrate. In contrast, only small amounts of free oxygenated fatty acid isomers were detected, and re-esterification of oxidized fatty acids into the LDL ester lipid fraction was ruled out. When LDL was depleted from ␣-tocopherol, specific oxygenation of the cholesterol esters was not prevented, and the product pattern was not altered. Similar results were obtained at low (LDL/LOX ratio of 1:1) and high LOX loading (LDL/LOX ratio of 1:10) of the LDL particle. During long term incubations (up to 24 h), a less specific product pattern was observed. However, when the hydroperoxy lipids formed by the 15-LOX were immediately reduced by the phospholipid hydroperoxide glutathione peroxidase, when the reaction was carried out with vitamin E-depleted LDL, or when the assay sample was diluted, the specific pattern of oxygenation products was retained over a long period of time.These data suggest that mammalian 15-LOX preferentially oxidize LDL cholesterol esters, forming a specific pattern of oxygenation products. During long term incubations, free radical-mediated secondary reactions, which lead to a more unspecific product pattern, may become increasingly important. These secondary reactions appear to be suppressed when the hydroperoxy lipids formed are immediately reduced, when ␣-tocopherol-depleted LDL was used, or when the incubation sample was diluted. It may be concluded that 15-LOXinitiated LDL oxidation constitutes a dual-type oxygenase reaction with an initial enzymatic and a subsequent nonenzymatic phase. The biological relevance of this dual-type reaction for atherogenesis will be discussed. (15). (vi) A 15-LOX inhibitor that apparently lacks major antioxidative properties prevented lipid deposition in the aorta of cholesterolfed rabbits (16). These data are consistent with a proatherogenic activity of the enzyme. On the other hand, transgenic rabbits that overexpress the 15-LOX specifically in monocyte/ macrophages (17) develop significantly less atherosclerotic lesions when fed a cholesterol-rich diet or when cross-bred with LDL receptor-deficient Watanabe rabbits (18), suggesting an anti-atherogenic action of the enzyme.For a better understanding of the processes involved in LDL oxidation in vivo, the in vitro interaction of 15-LOXs with human LDL has been studied. Sparrow et al. (6) have shown that the 15-LOX from soybeans is capable of oxidizing LDL in the presence of phospholipase A 2 . Later on, we reported that the native rabbit (19) and the recombinant human 15-LOX (20) specifically oxidize LDL ester lipids in the absence of lipidhydrolyzing enzymes. Comparison of various LOX isoenzymes ind...

Low Density Lipoprotein Receptor-related Protein-mediated Membrane Translocation of 12/15-Lipoxygenase Is Required for Oxidation of Low Density Lipoprotein by Macrophages

“…It is shown that cholesterol ester is selectively transferred from the LDL particle to the plasma membrane via LRP in Y1 murine adrenocortical cells (14) and in our 12/15-lipoxygenase-expressing cells 2 and that linoleic acid esterified to cholesterol in the LDL particle is regio-and stereospecifically oxygenated by the 12/15-lipoxygenase-expressing cells (12). Thus, the above observations strongly support our contention that the 12/15-lipoxygenase associated with the membranes oxygenates cholesterol ester transferred to the membrane, because self-catalyzed inactivation of the 12/15-lipoxygenase, which should be observed in the enzyme reaction with cholesterol ester in the membrane, is known to occur (23). This would explain the much lower enzyme activity in membranes.…”

“…However, the reduction of the enzyme activity may be due to other mechanisms unrelated to suicide inactivation such as poor substrate availability or conformational changes of the enzyme. It is reported that 12/15-lipoxygenase preferentially oxygenates cholesterol ester in the LDL particle, whereas phospholipids or even free fatty acids are not oxygenated, although they are present on the surface of the LDL particle (23). These results suggest that the specific oxygenation of cholesterol ester transferred to the plasma membrane by 12/15-lipoxygenase could take place.…”

“…Regio-and stereospecific oxygenation of linoleic acid esterified to cholesterol in LDL by 12/15-lipoxygenase-expressing cells indicates that cholesterol ester is enzymatically oxygenated by the cells (12). The enzymatic oxygenation is presumed to be the first key step for the generation of the completely oxidized LDL, which is made in the subsequent steps, including non-enzymatic radical chain reaction (12,23). The fact that 12/15-lipoxygenase translocation takes place in a very short period such as 5-15 min supports this notion (Fig.…”

Critical Role of Macrophage 12/15-Lipoxygenase for Atherosclerosis in Apolipoprotein E–Deficient Mice

“…1 12/15-Lipoxygenase (12/15-LO) is a nonheme ironcontaining dioxygenase that forms 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE and oxidizes esterified fatty acids in lipoproteins (cholesteryl esters) and phospholipids. 2,3 On the basis of its product from arachidonic acid, it is classified as 15-lipoxygenase (15-LO) in humans and rabbits 4,5 and as "leukocyte-type" 12-lipoxygenase (12-LO) in pig, rat, and mouse. 6 12/ 15-LO can also produce 13-hydroperoxy-octadecadienoic acid (13-HPODE) from linoleic acid.…”

Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation

“…15-and 12͞15-LOXs play a central role in vascular disease, because (i) 15-LOX mRNA, protein, and lipid products are found in atheroma, (ii) inhibition of 15-LOX prevents atherosclerosis in rabbits, and (iii) functional inactivation of the 12͞15-LOX gene slows down aortic lipid deposition in apo E-deficient mice, and inhibits streptozotocin-induced diabetes (19)(20)(21)(22)(23)(24)(25). Furthermore, in vivo, 12͞15-LOX expression is required for neointimal thickening in ballooninjured rat aortae (26,27).…”