Oxidative modification converts low-density lipoprotein (LDL) into its atherogenic form and appears to be a necessary precondition for LDL uptake by macrophages during foam cell formation. Cellular lipoxygenases have been implicated in this process. We studied the interaction of purified mammalian lipoxygenases with human LDL in v i m and found that the arachidonate 15-lipoxygenases of rabbit and man are capable of oxygenating lipoproteins as indicated by oxygen uptake and by the formation of thiobarbituric-acid-reactive substances. Furthermore, oxygenated polyenoic fatty acids, such as 13-hydro(pero)xy-9Z, 11 E-octadecadienoic acid and 15-hydro(pero)xy-5,8,11,13 (Z,Z, Z,E)-eicosatetraenoic acid were detected in the lipid compartment of various lipoproteins classes after lipoxygenase treatment. More than 90% of the oxygenated polyenoic fatty acids were found in the ester-lipid fraction, particularly in the cholesterol esters, whereas only small amounts of free hydro(pero)xy polyenoic fatty acids were detected. Lipoxygenase-catalyzed oxygenation of LDL is not restricted to the lipid compartment but also leads to a cooxidative modification of the apoproteins as indicated by changes in the electrophoretic mobility and by the formation of carbonyl derivatives of amino acid side chains. The possible biological significance of lipoxygenase-induced oxidative modification of lipoproteins in the pathogenesis of atherosclerosis is discussed.Atherosclerosis is a multifactoral disease, the pathogenesis of which is not fully understood [l-31. It has been shown by morphological studies that the accumulation of lipid-loaded foam cells in the subendothelial space leads to the formation of fatty streaks, which are generally accepted as the early atherosclerotic lesions [l -41. Foam cells develop from monocyte-derived macrophages [5, 61 or from smooth muscle cells [7] by taking up modified low-density lipoproteins (LDL) via scavenger-receptor(s)-mediated pathways. The occurrence of oxidatively modified proteins in atherosclerotic lesions [8, 91 and the fact that oxidatively modified LDL is rapidly taken up by macrophages [lo] suggested an involvement of oxidative processes in the pathogenesis of atherosclerosis. However, the mechanism of oxidative modification in vivo remains unclear. In-vitro studies with cell-free systems indicated that copper-mediated oxidation converts LDL into its atherogenic form [ll] The oxygenation of lipoproteins by mammalian lipoxygenases has not been studied so far. The soybean lipoxygenase which largely differs from mammalian lipoxygenases with respect to its protein chemical and enzymic properties is capable of oxidizing LDL only in the presence of phospholipase A, which provides free polyenoic fatty acids by cleaving lipoprotein phospholipids [20]. This result is not surprising since the soybean lipoxygenase has been shown to effectively oxygenate ester lipids only in the presence of detergents [21]. However, the rabbit reticulocyte lipoxygenase is capable of oxygenating complex ester lipids such ...

The arachidonate 15-lipoxygenase which is expressed in atherosclerotic lesions is implicated in the oxidative modification of low density lipoproteins during atherogenesis. To obtain experimental in vivo evidence for this hypothesis, we analyzed the structure of oxygenated lipids isolated from the aorta of rabbits fed with a cholesterol-rich diet for different time periods and compared the pattern of oxygenation products with that isolated from low density lipoproteins treated in vitro with the pure rabbit 15-lipoxygenase and with oxygenated lipids isolated from advanced human atherosclerotic lesions. In early atherosclerotic lesions (12-wk cholesterol feeding), specific lipoxygenase products were detected whose structure was similar to those isolated from lipoxygenase-treated low density lipoproteins. The appearance of these products did coincide with the lipid deposition in the vessel wall. In later stages of atherogenesis (26-wk cholesterol feeding) the degree of oxidative modification of the tissue lipids did increase but the share of specific lipoxygenase products was significantly lower, suggesting an increasing overlay of the specific lipoxygenase products by nonenzymatic lipid peroxidation. In advanced human atherosclerotic lesions, large amounts of oxygenation products were detected whose structure suggests a nonenzymatic origin. These data suggest that the arachidonate 15-lipoxygenase is of pathophysiological importance during the early stages of atherogenesis. In later stages of plaque development nonenzymatic lipid peroxidation becomes more relevant.

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...