No abstract

Incubation of mouse peritoneal macrophages with native human low density lipoprotein (LDL) did not cause any significant storage of Intracellular cholesteryl esters. However, when the LDL was subjected to brief (30-second) vortexlng, It formed self-aggregates that were rapidly Ingested and degraded by macrophages, converting them to cholesteryl ester-rich foam cells. Such aggregates were as potent as acetyt-LDL In stimulating cholesterol esterlflcatlon in the macrophages. The degradation of LDL aggregates was strongly inhibited by cytochalasln B (85%), whereas degradation of native LDL was only weakly Inhibited (23%), suggesting that uptake occurred by phagocytosis rather than plnocytosls. Several lines of evidence suggest that the phagocytlc uptake depends, in part, upon the LDL receptor and not the acetyl-LDL receptor: 1) soluble, native LDL and 0-VLDL (but not acetyl-LDL) competed for uptake and degradation of LDL aggregates; 2) reductive methylatlon of LDL before vortexlng reduced the effect of the aggregates on degradation and cholesterol esterlflcatlon; 3) heparln, which During studies of foam cell formation in cell culture, we noted marked differences among different LDL preparations in their ability to induce lipid storage in mouse peritoneal macrophages. While most preparations caused little accumulation of lipid-staining droplets, an occasional preparation caused striking lipid accumulation. The tubes containing these samples generally showed some precipitated material. We then tried to develop a reproducible way to effect the changes apparently caused by storage and found that simply vortexing LDL solutions at room temperature would cause aggregation. The ease with which LDL is denatured at surfaces has been recognized for some time. However, the extent of such denaturation during vortexing turns out to be surprising; vortexing for even 30 seconds caused about 50% of the LDL protein to become precipitable. The studies described below show that these LDL self-aggregates are avidly ingested by mouse peritoneal macrophages. The LDL aggregates were as effective as soluble, native LDL in competing for the degradation of soluble native 12S I-LDL. In addition, heparin reduced the degradation of both LDL aggregates and native LDL to nearly the same extent. We suggest that the common denominator in this, and the several other LDL complexes that are degraded more rapidly than LDL itself, 13 -18 is simply that they represent aggregates. The self-aggregation of LDL produced by vortexing does not involve the addition of new potential ligands and thus, clearly identifies the aggregation per se as the key variable.The remarkable ease and extent with which LDL undergoes denaturation during this simple physical handling at room temperature or at 4°C raises the possibility that some analogous process may occur in vivo at body temperature and contribute to foam cell formation during atherogenesis. Methods Isolation of Mouse Peritoneal MacrophagesPrimary cultures of mouse peritoneal macrophages were prepared as described...

Oxidative modification of low density lipoprotein (LDL) enhances its potential atherogenicity in several ways, notably by enhancing its uptake into macrophages. In vivo studies in the rabbit show that inhibition of LDL oxidation slows the progression of atherosclerotic lesions. In the present studies, rabbits were fed either a newly developed variant sunflower oil (Trisun 80), containing more than 80% oleic acid and only 8% linoleic acid, or conventional sunflower oil, containing only 20% oleic acid and 67% linoleic acid. LDL isolated from the plasma of animals fed the variant sunflower oil was highly enriched in oleic acid and very low in linoleic acid. These oleate-rich LDL particles were remarkably resistant to oxidative modification. Even after 16-hr exposure to copper-induced oxidation or 24-hr incubation with cultured endothelial cells, macrophage uptake of the LDL was only marginally enhanced. The results suggest that diets sufficiently enriched in oleic acid, in addition to their LDL-lowering effect, may slow the progression of atherosclerosis by generating LDL that is highly resistant to oxidative modification.

Oxidized low-density lipoprotein (LDL) is more atherogenic than native LDL. The initial step in the oxidation is the peroxidation of polyunsaturated fatty acids. Thus, decreasing the concentration of polyunsaturated fatty acids should reduce the susceptibility of LDL to oxidation. Therefore, we tested the possibility that diets enriched in oleate might result in LDL that is less susceptible to oxidative modification. LDL isolated from subjects consuming an oleate-enriched diet, compared with LDL from subjects on a linoleate-enriched diet, contained significantly more oleate (28.7% vs 11.5%) and less linoleate (31.9% vs 50.9%). Generation of conjugated dienes was significantly lower in the LDL from the oleate group. Most important, after incubation with endothelial cells, LDL from the oleate group underwent less degradation by macrophages. These studies demonstrate the feasibility of altering the diet in a way that will not raise LDL cholesterol concentrations and yet will decrease the susceptibility of LDL to oxidative modification.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-il. MFC plated in medium containing 0.5% FCS for 24 h contained -600 gg cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (fl-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonenal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol. (J. Clin. Invest. 1991. 87:90-99.)