Enhanced macrophage uptake of low density lipoprotein after self-aggregation.

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...

Three Types of Naturally Occurring Modified Lipoproteins Induce Intracellular Lipid Accumulation in Human Aortic Intimal Cells — The Role of Lipoprotein Aggregation

“…Here we provide evidence that LDL isolated from patients with coronary heart disease or diabetes mel- (40). Our earlier findings show (11) that in vitro modified LDL spontaneously aggregate under the conditions of cell culture and that his aggregation is a necessary step in the process of cholesteryl ester accumulation within cultured human aortic smooth muscle cells.…”

Phagocytosis of lipase-aggregated low density lipoprotein promotes macrophage foam cell formation. Sequential morphological and biochemical events.

“…8 -32 The usual ligand for the LDL receptor is apolipoprotein B-100, 2 which remains intact after modification of LDL with phospholipase C. 14 Methylation blocks the binding of LDL to its receptor 2 and also the uptake and degradation of LDL aggregated by vortexing. 9 Methylation of LDL before phospholipase C treatment inhibits the uptake of the aggregated lipoprotein by macrophages. The inhibition of 125 I-PLC-LDL degradation by prior methylation is reversed by apolipoprotein E, which interacts with the LDL receptor.…”

“…8 " 32 Phospholipase C treatment of LDL generates diacylglycerol, 28 a potent activator of protein kinase C. 33 However, protein kinase C is not known to play a role in phagocytosis. Khoo et al 9 have shown that LDL aggregated by vortexing is rapidly ingested by macrophages, and they proposed that the mechanism of uptake was LDL receptor-mediated phagocytosis. Vortexed LDL presumably lacks diacylglycerol.…”

“…Vortexed LDL is taken up by phagocytosis and rapidly degraded, releasing free cholesterol that promotes cholesteryl ester synthesis. 9 In contrast, complexes composed of LDL, heparin, fibronectin, and denatured collagen are poorly degraded by mouse peritoneal macrophages. 10 - 12 The cytoplasm of macrophages exposed to these insoluble particulates exhibits membrane-limited vacuoles that contain flocculent material resembling LDL-proteoglycan complexes, 1012 suggesting that the lipoprotein remains intact after ingestion.…”

Oxidized low‐density lipoprotein induces the production of superoxide by neutrophils

“…Acetyl-LDL was prepared by the method of Basu et al [20]. EDL self-aggregates were prepared from native LDL by vortexing for I min as described by Khoo et al [21].…”