Plasma LDL oxidation leads to its aggregation in the atherosclerotic apolipoprotein E-deficient mice.

Abstract

Two major modifications of low density lipoprotein (LDL) that can lead to macrophage cholesterol accumulation and foam cell formation include its oxidation and aggregation. To find out whether these modifications can already occur in vivo in plasma and whether they are related to each other, the oxidation and aggregation states of plasma LDL were analyzed in the apolipoprotein E-deficient (E degree) transgenic mice during their aging (and the development of atherosclerosis), in comparison to plasma LDL from control mice. Plasma LDL from the E degree mice was already minimally oxidized at 1 month of age in comparison to control mice LDL, and it further oxidized with age in the E degree mice but not in the control mice. At 6 months of age, the contents of the E degree mice LDL-associated cholesteryl ester hydroperoxides, thiobarbituric acid reactive substances, and conjugated dienes were higher by two, three, and twofold, respectively, in comparison to LDL from the young, 1-month-old E degree mice. We also investigated the LDL aggregation state in E degree mice. In the young E degree mice, LDL oxidation was shown in comparison to control mice, but in both groups of young mice their LDL was not aggregated. In the E degree mice, however, the LDL aggregation state substantially increased with age, by as much as 125% at 6 months of age compared to the 1-month-old mice, whereas no significant aggregation could be detected in plasma LDL from control mice at the same age. To question the possible effect of LDL oxidation on its subsequent aggregation, LDL oxidation was induced by either copper ions, or by the free radical generator 2,2-azobis-2-amidinopropane hydrochloride, or by hypochlorite. All these oxidative systems led to LDL oxidation (to different degrees) and resulted in a similar, substantial LDL aggregation. These oxidation systems also enhanced the susceptibility of LDL to aggregation (induced by vortexing) by 23%, 28%, or 40%, respectively. To further analyze the relationships between the lipoprotein oxidation and its aggregation, LDL (0.1 mg of protein/mL) was incubated with 5 mumol/L CuSO4 at 37 degrees C in the absence or presence of the antioxidant, vitamin E (25 mumol/L). In the absence of vitamin E, a time-dependent increment in LDL oxidation was noted, which reached a plateau after 2 hours of incubation. LDL aggregation, however, only started at this time point and reached a plateau after only 5 hours of incubation. In the presence of vitamin E, both LDL oxidation and its aggregation were reduced at all time points studied. We extended the vitamin E study to the in vivo situation, and the effect of vitamin E supplementation to the E degree mice (50 mg.kg-1.d-1 for a 3-month period) on their plasma LDL oxidation and aggregation states was studied. Vitamin E supplementation to these mice resulted in a 35% reduction in the LDL oxidation state and in parallel, the LDL aggregation state was also reduced by 23%. These reductions in LDL oxidation and aggregation states were accompanied by a 33% reduction in the aortic lesion area, in comparison to nontreated E degree mice. We conclude that in E degree mice, LDL oxidation, which already took place in the plasma, can lead to the lipoprotein aggregation. These modified forms of LDL were shown to be taken up by macrophages at an enhanced rate, leading to foam cell formation. Thus, the use of an appropriate antioxidant can inhibit the formation of both atherogenic forms of LDL.