Uptake of Type III Hypertriglyceridemic VLDL by Macrophages Is Enhanced by Oxidation, Especially After Remnant Formation

We previously showed that hypertriglyceridemic VLDL (HTG-VLDL, Sf 60 to 400) from subjects with type III (E2/E2) hyperlipoproteinemia do not induce appreciable cholesteryl ester (CE) accumulation in cultured macrophages (J774A.1). In the present study, we examined whether oxidation of type III HTG-VLDL would enhance their uptake by J774A.1 cells. Type III HTG-VLDL were oxidized as measured by both conjugated-diene formation and increased electrophoretic mobility on agarose gels. Both LDL and type III HTG-VLDL undergo oxidation, albeit under different kinetic parameters. From the conjugated-diene curve, type III HTG-VLDL, compared with LDL, were found to have a 6-fold longer lag time, to take 6-fold longer to reach maximal diene production, and to produce a 2-fold greater amount of dienes but at half the rate (allP <.005). Incubation of macrophages with either native type III HTG-VLDL or LDL (50 μg lipoprotein cholesterol/mL media for 16 hours) caused small increases (4-fold and 2.7-fold, respectively) in cellular CE levels relative to control cells (bothP =.0001). After 24 hours of CuSO4 exposure, we found that oxidized type III HTG-VLDL and LDL caused a 9.4-fold and 10.5-fold increase, respectively, in cellular CE levels (P =.0001). We next examined whether extending the exposure period for type III HTG-VLDL to CuSO4 beyond 24 hours would further enhance its ability to induce macrophage CE accumulation. After 48 hours of CuSO4 exposure, type III HTG-VLDL and LDL caused 21.3-fold and 11.6-fold increases, respectively, in cellular CE levels (P =.0001). The cellular CE loading achieved with 48 hour–oxidized type III HTG-VLDL was significantly higher than either 24 hour–oxidized type III HTG-VLDL (2.3-fold,P =.003) or 48 hour–oxidized LDL (1.8-fold,P =.012). There was no significant difference between the CE loading achieved by incubation of cells with either 24 hour–oxidized type III HTG-VLDL, 24 hour–oxidized LDL, or 48 hour–oxidized LDL (P ≥.518). In this study, we also examined whether partial lipolysis (19% to 50% triglyceride hydrolysis) of type III HTG-VLDL to produce remnants would increase the susceptibility of the lipoprotein to oxidative modification and subsequent cellular CE loading. Forty-eight hour–oxidized type III VLDL-remnants stimulated CE accumulation 30.4-fold over baseline (P =.0001). In contrast, nonoxidized type III VLDL-remnants caused the same very low level of CE loading as did native type III HTG-VLDL (P =.680). The increase in cellular CE levels achieved with 48 hour–oxidized type III VLDL-remnants was significantly higher than that achieved with 48 hour–oxidized type III HTG-VLDL (P =.047). In conclusion, we have shown that oxidized type III HTG-VLDL will induce macrophage CE accumulation well above levels achieved with oxidized LDL. In addition, we also showed that by forming a VLDL-remnant before oxidative modification, we can further enhance macrophage CE accumulation. These results provide a potential mechanism for the atherogenicity of type III HTG-VLDL and their remnants.