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The detailed morphology of macrophages involved in the uptake and intracellular processing of low density lipoprotein (LDL) and, ultimately, formation of macrophage-derived foam cells of atherosclerotic lesions has long fascinated investigators. This study examined localization of LDL in subcellular compartments of macrophage-derived intimal foam cells in cardiac valves isolated from rabbits by diet-induced hypercholesterolemia and, as an in vitro model of formation of foam cells, in cultured human monocyte-macrophages incubated for 2;-120 h with aggregated LDL produced by vortexing or phospholipase C lipolysis. The quasi-three-dimensional morphology of macrophages involved in endocytosis was preserved by ultrarapid freezing and freeze-etch microscopy in conjunction with thin-section electron microscopy. This approach produced unique images of subcellular compartments in human monocyte-macrophages involved in the uptake and processing of aggregated LDL with a clarity not previously reported. Three-dimensional ultrastructural analyses revealed a complex network of coated and uncoated vesicles, surface-connected saclike compartments, and endosomal/lysosomal compartments including the labyrinth of vesicular/tubular lysosomes all enmeshed in the microtubular, microfilament cytoskeletal network. These dynamic views of subcellular structures at the high resolution of the electron microscope provide an additional framework to better understand how lipoprotein particles are transported into, and processed within, macrophages during foam cell formation in atherogenesis.

Background: Oxidized LDL has been found within the subendothelial space, and it exhibits numerous atherogenic properties, including induction of inflammatory genes. We examined the possibility that variations in endothelial response to minimally modified LDL (MM-LDL) constitute one of the genetic components in atherosclerosis., Methods and Results: By a novel explant technique, endothelial cells (ECs) were isolated from the aorta of inbred mouse strains with different susceptibilities to diet-induced atherosclerosis. Responses to MM-LDL were evaluated by examining the expression of inflammatory genes involved in atherosclerosis, including monocyte chemotactic protein-1 (MCP-1) and macrophage-colony-stimulating factor (M-CSF), an oxidative stress gene, heme oxygenase-1 (HO-1), and other, noninflammatory, genes. ECs from the susceptible mouse strain C57BL/6J exhibited dramatic induction of MCP-1, M-CSF, and HO-1, whereas ECs from the resistant strain C3H/HeJ showed little or no induction. In contrast, ECs from the 2 strains responded similarly to lipopolysaccharide., Conclusions: These data provide strong evidence that genetic factors in atherosclerosis act at the level of the vessel wall.

Malondialdehyde, a product of lipid peroxidation, produces threshold conversion of low density lipoprotein (LDL) to a form recognized by type I and type II scavenger receptors of monocyte-macrophages. To investigate whether localized domains of human apoB-100 protein provide recognition determinants, we tested the ability of several different apoB-bearing particles to interact with the scavenger receptor of human monocyte-macrophages. Genetically engineered, carboxyl-terminally truncated apoB proteins assembled into lipoprotein form were labeled by fluorescent dye. Fluorescence microscopy and quantitative fluorescent spectrophotometry showed that purified particles containing as little as 23% of the apoB amino-terminus were internalized by the scavenger receptor after, but not before, malondialdehyde modification. There was no recognition of the particles by the LDL receptor. Similar results were obtained with human plasma LDL homozygous for carboxyl-terminally truncated apoB-45.2. Liposome-incorporated fusion protein containing apoB residues 547-735 displayed specific uptake by the scavenger receptor without modification by malondialdehyde. In contrast, fusion proteins containing apoB residues 3,029-3,133 or a short amino terminal segment failed to interact. Thus, primary sequence presented by residues 1-1,084 sufficed to produce recognition of modified LDL by the scavenger receptor. These receptor-combining domains were sequestered when secreted in lipoprotein form and were expressed upon malondialdehyde modification. When packaged exogenously in liposome form, fusion protein containing apoB residues 547-735, containing approximately 4% of the primary sequence, mediated scavenger receptor-dependent uptake and hydrolysis. Our findings provide an additional function or the amino-terminal region of apoB and demonstrate that primary sequence presented by the first 2% of apoB-100 protein suffices to produce recognition on malondialdehyde-modified LDL by the scavenger receptor of human monocyte-macrophages.

By using fast protein liquid chromatography, we isolated from human plasma a minor electronegative LDL subfraction designated LDL(-). After immunoaffinity chromatography against apolipoprotein (apo)(a) and apo A-I, LDL(-) represented 6.7 +/- 0.9% (mean +/- SD; n = 18) of total LDL. Compared with the major LDL subfraction, designated LDL(+), LDL(-) contained similar amounts of thiobarbituric acid-reactive substances, conjugated dienes, and vitamin E and had a similar lipid/protein ratio and mean density. Moreover, the apo B of LDL(-) was not aggregated and its LDL receptor-binding activity was slightly increased. These results were consistent with the nonoxidized nature of LDL(-). LDL(-) showed increased contents of sialic acid (38.1 +/- 5.2 versus 28.9 +/- 3.3 nmol/mg protein; n = 7; P < .01), apo C-III (1.43 +/- 0.21% versus 0.14 +/- 0.04%; n = 7; P < .01), and apo E (1.64 +/- 0.26% versus 0.10 +/- 0.05%; n = 7; P < .0005). Compared with LDL(+), LDL(-) displayed enhanced cytotoxic effects on cultured human umbilical vein endothelial cells, as shown by lactate dehydrogenase assay (P < .003; n = 6), neutral red uptake (P < .02; n = 6), and morphological studies. We also studied the relationship of LDL(-) to age and plasma lipid levels in 133 subjects. The percentage of contribution of LDL(-) to total plasma LDL correlated with age (P < .05), total cholesterol (P < .05), and LDL cholesterol (P < .003). In conclusion, this study shows that LDL(-), a circulating human plasma LDL, is an electronegative native LDL subfraction with cytotoxic effects on endothelial cells. This subfraction, which correlates positively with common atherosclerotic risk factors, might induce atherogenesis by actively contributing to alteration of the vascular endothelium.

Treatment of rabbit aortic endothelial cells, human umbilical vein endothelial cells, and human aortic endothelial cells for 4 hours with minimally oxidized low-density lipoprotein (MM-LDL) induced the adhesion of monocytes but not neutrophils or lymphocytes to these cells. This induction was blocked by inhibitors of glycoprotein synthesis (cycloheximide and tunicamycin), and binding was abolished by treatment of cells with low levels of trypsin, suggesting that the binding molecule(s) is a protein. There was no increase in binding of antibodies to E-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) after treatment of cells with MM-LDL. Treatment of endothelial cells with Fab fragments of antibody to monocyte chemotactic protein-1 or to fibronectin did not block monocyte binding. Several sugars (lactose-1-phosphate, maltose-1-phosphate, and N-acetylglucosamine) inhibited monocyte binding to cells treated with MM-LDL, but binding was not blocked by mannose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, or glucose-6-phosphate. EDTA or EGTA treatment inhibited binding, which was restored by adding either calcium or magnesium. We conclude that the binding of monocytes to endothelial cells induced by a 4-hour treatment with MM-LDL is caused by a binding molecule(s) other than E-selectin, VCAM-1, or ICAM-1 and that carbohydrate chains on the monocytes or the endothelium play a role in binding.

Lipoprotein (a) (Lp(a)) is known to be an independent risk factor for cardiovascular disease, but the mechanisms by which it contributes to this disease remain unclear. Current evidence indicates that the closely related plasma particle, low-density lipoprotein (LDL), may initiate atherosclerosis through deposition in the arterial wall. This study has compared the ability of both lipoproteins to enter and accumulate within the arterial wall. Experiments were conducted in vivo with animals from two strains of mice: C57BL/6 mice, which develop fatty streak lesions upon challenge by a high-fat diet, and C3H/HeJ mice, which are resistant to lesion formation. Animals from both strains were maintained up to 16 weeks either on chow or high-fat diet. The mice were intravenously injected with 125I-labeled human Lp(a) or 125I-labeled human LDL in equimolar amounts and the lipoprotein allowed to circulate in vivo for 2 or 24 h. Transverse sections of the aortic root including sites of predilection for lesion formation at the commissures of the valve were prepared and examined after autoradiography. The autoradiographic grains over lesions and histologically uninvolved areas were enumerated and compared after normalization. Both Lp(a) and LDL demonstrated nearly ten times greater accumulation in lesions compared with histologically uninvolved areas from C57BL/6 mice. Analyses of histologically uninvolved areas from both strains of mice showed a significantly higher accumulation of Lp(a) than LDL. Finally, significantly higher accumulations of both Lp(a) and LDL occurred in the histologically uninvolved intima and subintima of lesion-prone C57BL/6 mice as compared with lesion-resistant C3H/HeJ mice after 5 weeks on the diets. We propose that enhanced accumulation of Lp(a) in the arterial wall accounts, in part, for the increased risk of cardiovascular disease.

We have recently shown that cultured human monocyte-macrophages degraded 125I-labeled low density lipoprotein (125I-nativeee-LDL) by a saturable high-affinity process with maximal velocity at 25-30 microgram protein/ml (Fogelman et al., 1980, Proc. Nat. Acad. Sci. USA. 77:2214-2218). We now describe studies of the binding of 125I-native-LDL at 4 degrees C and the effects of chloroquine, Ca2+ concentration, and reductive methylation on high-affinity 125I-native-LDL degradation that indicate that native-LDL is processed by the monocyte-macrophages via the classic LDL receptor pathway. The high-affinity degradation of 125I-native-LDL increased substantially when monocyte-macrophages were exposed to the lipoprotein deficient-fraction of serum (LPDS) for periods as brief as 4 hours, and was 25-fold greater than that of lymphocytes. Freshly isolated monocytes that had never been exposed to LPDS also demonstrated high-affinity degradation of 125I-nativ-LDL. When these monocytes were cultured for 7 days in a medium containing native-LDL at a concentration (186 microgram protein/ml) greatly in excess of that apparently needed to saturate the high-affinity process, there was more than a 10-fold increase in 125I-native-LDL high-affinity degradation. LDL modified by treatment with malondialdehyde was processed by a second high-affinity cell surface receptor that appears identical to the "scavenger" receptor that processes acetylated LDL (Goldstein, et al., 1979, Proc. Nat. Acad. Sci. USA. 76: 333-337).

Blood-borne human monocytes and macrophages derived from human monocytes in vitro express an active low density lipoprotein (LDL) receptor and an active receptor for negatively charged proteins, the scavenger receptor. When less than 15% of the lysine residues of human LDL were modified by malondialdehyde while the lipoprotein was in solution, recognition and uptake of the modified lipoprotein occurred via the LDL receptor. Further modification resulted in threshold recognition and uptake by the scavenger receptor with concomitant loss of recognition by the LDL receptor. The rate of degradation via the LDL receptor pathway was inversely related to the degree of modification whereas that mediated by the scavenger receptor was independent of the extent of incorporation of malondialdehyde once threshold recognition was achieved. In contrast to the interaction of LDL with malondialdehyde in solution, modification of less than 15% of the lysine residues of LDL adsorbed to heparin-Sepharose resulted in recognition and uptake by the scavenger receptor. The scavenger receptor-mediated uptake of malondialdehyde-modified LDL may be dependent on formation of recognition sites involving specific modified lysine residues or changes in the conformation of LDL induced by neutralization of specific lysine residues of the apoB polypeptides or both.

A comparison of the receptor-mediated interaction of malondialdehyde-low density lipoprotein and maleyl-albumin has been examined in human monocytes during differentiation in vitro. The recognition of both ligands by the scavenger receptor of these cells has been confirmed. We now report that human monocytes express a second cellular surface receptor for maleyl-albumin that is distinct from the scavenger receptor. The activity of the maleyl-albumin receptor, determined by both binding and lysosomal hydrolytic assays, substantially exceeds that of the scavenger receptor in freshly isolated monocytes. A dramatic and rapid decline in the activity of the maleyl-albumin receptor occurs within 72 to 96 h during differentiation in vitro. At day 7, while only 5-10% of the original activity of the maleyl-albumin receptor remains, it is similar to that of the maximally expressed scavenger receptor. Both the binding and hydrolysis of ligand mediated by the maleyl-albumin receptor are specifically inhibited by alpha-casein and alkaline-treated albumin; neither of these proteins is recognized by the scavenger receptor. The occurrence of the exceptionally active maleyl-albumin receptor on freshly isolated human monocytes suggests that it participates in processes necessary to the function of the cells that diminish in importance after differentiation of the monocytes into macrophages in vitro. Furthermore, while maleyl-albumin is a useful adjunct to studies of cellular events mediated by the scavenger receptor, the presence of a second receptor for maleyl-albumin must be taken into account as a potential contributing and complicating event.

Improved techniques of cell isolation resulted in 90 to 100 million monocytes from a single donor. Addition of low density lipoprotein (LDL) to to cultures of these cells resulted in the down regulation of LDL receptor activity. Addition of malondialdehyde-altered LDL. which enters the cell through a receptor for negatively charged proteins (the scavenger receptor), produced an even greater down regulation of the LDL receptor, indicating that both receptors are present on the same cell. Within hours of adherence of the cells, there was a dramatic decrease in the activity of both receptors. LDL receptor activity was highest during the first week in culture and then declined, despite the maintenance of a constant LDL concentration in the medium. Scavenger receptor activity surpassed LDL receptor activity by the 6th day and was maximally expressed during the second week. Increasing cell density resulted in a slight increase in the activity of the LDL receptor and a dramatic increase in scavenger receptor activity. Insulin had no significant effect on either receptor. Removing serum from the culture medium for 48 hr resulted in a 3.5-fold increase in LDL receptor activity and a 2-fold decrease in scavenger receptor activity. Twenty-four hr after the cells were re-exposed to serum, the activities of both receptors essentially returned to base line. Heat-inactivation of serum was associated with an increased cholesteryl ester content of the cells and depressed receptor activities. Scavenger receptor activity appears related to the maturation of monocytes into macrophages and is promoted by increasing cell density and serum factors that are heat labile.

The ability of the scavenger receptor of human monocyte macrophages to recognize human low density lipoproteins (LDL) progressively modified by three lysine-specific reagents, malondialdehyde, acetic anhydride, or succinic anhydride, has been investigated. Regardless of the reagent utilized, receptor-mediated uptake was dependent upon modification of greater than 16% of the peptidyl lysines rather than upon the net negative charge of derivatized LDL. Rates of lysosomal hydrolysis of acetyl-LDL and succinyl-LDL increased as a function of progressive modification and reflected the amount of derivatized LDL binding to the receptor. Succinylation or acetylation of greater than 60% of the lysines was necessary to attain maximal ligand binding, internalization, and degradation. In contrast, modification of only 16% of the peptidyl lysines by malondialdehyde resulted in maximal levels of binding, uptake, and hydrolysis. The expression of receptor recognition site(s) appears to depend upon the charge modification of critical lysine residues of the LDL protein rather than the net negative charge of the lipoprotein complex. Malondialdehyde, a bifunctional reactant, may modify surface and sequestered lysines concomitantly and thus promote efficient formation of the recognition site(s).