Macrophage Oxidation of Low Density Lipoprotein Generates a Modified Form Recognized by the Scavenger Receptor

Transcription

1 Macrophage Oxidation of Low Density Lipoprotein Generates a Modified Form Recognized by the Scavenger Receptor Sampath Parthasarathy, David J. Printz, Donna Boyd, Lorna Joy, and Daniel Steinberg Incubation of low density lipoprotein (LDL) with endothelial cells or smooth muscle cells overnight has resulted in an oxtdative modification of LDL that results in its recognition by macrophages by way of the acetyl LDL receptor. In the present study, we examined whether macrophages themselves can oxidize and modify LDL in a manner similar to that of endothelial cells. Incubation of 125 l-labeled LDL with resident or thioglycollate-elicited macrophages for 24 hours in Ham's F-10 medium resulted in the appearance of thiobarbituric acid (TBA) reactive materials and trichloroacetic acid (TCA) soluble radioactivity in the medium. The LDL harvested from these incubations showed increased electrophoretic mobility and was degraded rapidly when added to fresh macrophages as compared to LDL previously incubated in the absence of cells. These macrophage-induced modifications could be prevented if the first incubation was carried out in the presence of the antioxidant butylated hydroxytoluene (BHT) or in Dulbecco's modified Eagle's medium (DMEM). The degradation of 125 l-labeled macrophage-modified LDL by macrophages was competitively inhibited by unlabeled acetyl LDL or unlabeled endothelial cell-modified LDL but not by native LDL, indicating that the degradation was mediated by the acetyl LDL receptor. (Arteriosclerosis 6: , September/October 1986) T he accumulation of lipid-laden foam cells of monocvte origin in the aortic intima is an early event in the development of atherosclerosis. 1 " 3 Monocyte-macrophages take up and degrade native low density lipoprotein (LDL) by way of the classical LDL (B/E) receptor, but only at rather low rates. 4 On the other hand, chemically acetylated LDL and other chemically modified forms of LDL 5-6 are taken up much more rapidly by a distinct, alternative receptor, designated the acetyl LDL or scavenger receptor. 7 Incubation of macrophages with these chemically modified forms readily generates foam cells whereas it is difficult to generate foam cells by incubation with native LDL 4 unless very long incubation times are used. 8 Incubation of native LDL overnight with cultured endothelial cells has been shown to result in a modification that converts LDL to a form recognized by the same receptor that recognizes acetyl LDL. 9 " 11 This modification has been shown to depend upon the presence of trace metals in the medium, to involve extensive lipid peroxidation, and to require the ac- tion of a phospholipase A ' 13 The peroxidation of LDL lipids during such incubations has been shown to account for the cytotoxicity of LDL for cultured endothelial cells. 14 All of these changes can be blocked by the addition of alpha-tocopherol, butylated hydroxytoluene (BHT), or by probucol, a drug used in the management of hyperlipoproteinemia. 15 Since macrophages generate active oxygen species, which may play a role in their ability to scavenge and kill cells, it seemed likely that they might share the ability to oxidatively modify LDL. It was recently reported 16 ' 17 that LDL is oxidized by human monocytes and neutrophils and that the electrophoretic mobility of LDL is increased after incubation with cultured porcine monocytes. We report here that mouse peritoneal macrophages, like circulating human monocytes, can cause extensive oxidation of LDL lipids. We show further that the modified LDL is specifically recognized by the acetyl LDL receptor on the same cells that oxidized the LDL and, finally, that this macrophage-modified LDL competes with endothelial cellmodified LDL for uptake and degradation. From the Division of Endocrinology and Metabolism, Department of Medicine, M-013D, University of California, San Diego, La Jolla, California This work was supported in part by Grant HL from the National Heart, Lung, and Blood Institute and Training Grant HL Address for reprints: Daniel Steinberg, M.D., Department of Medicine, M-013D, University of California, San Diego, La Jolla, California Received December 23,1985; revision accepted May 21, Materials Methods Carrier free Na 125 l was purchased from Amersham (Arlington Heights, Illinois). Ham's F-10 was from Irvine Scientific (Santa Ana, California). Dulbecco's modified Eagles medium (DMEM) and a-minimum Essential medium (a-mem) were from GIBCO (Santa Clara, California). BHT was from J.T. Baker (Phillipsburg, New Jersey).

2 506 ARTERIOSCLEROSIS VOL 6, No 5, SEPTEMBER/OCTOBER 1986 Procedures LDL (d = to 1.063) was isolated from pooled normal human plasma and was radioiodinated as described elsewhere. 12 Acetyl LDL was prepared by using acetic anhydride/sodium acetate l-tc-labeled LDL was prepared as described by Pittman et al. 19 All lipoprotein preparations were dialyzed against phosphate-buffered saline containing 0.01% EDTA. The LDL concentration is expressed as micrograms of protein in the incubation systems. Cells Peritoneal macrophages were harvested from female Swiss-Webster mice (2 to 3 months old weighing 25 to 35 g) with or without prior thioglycollate stimulation (2 ml of 3% thioglycollate solution aged for 1 to 2 months and injected intraperitoneally 3 days before harvest by peritoneal lavage). The cells were plated on either 60 mm plastic dishes (LUX, Laboratory Tek, Miles Laboratories, Incorporated, Naperville, Illinois) at 1 x 10 7 cells/dish or in a 12- well dish at 2.5 x 10 s cells/well (Co Star, Cambridge, Massachusetts) in a-mem containing 10% fetal calf serum. After overnight culturing, nonadherent cells were removed by washing and the cells were used for LDL modification or degradation studies. Unlabeled endothelial cell-modified LDL was prepared by using confluent rabbit aortic endothelial cells as described earlier. 9 ' 12 The endothelial cell-modified LDL showed a thiobarbituric acid (TBA) value of 4.96 nmol malondialdehyde (MDA)/ml (49.6 nmol/mg LDL protein). x: CM 10 r D I 6 i- 4 O) <D Q Not No-Cell Incu- Control bated F-10 F-10 DME t +BHT, Thioglycollate- Elicited Cells Macrophage Modification of Low Density Lipoprotein Macrophage modification of LDL was accomplished by incubating 12S l-labeled LDL (100 /xg/ml) for 24 hours with macrophages in 60 mm culture dishes in 2 ml of serumfree Ham's F-10 medium at 37 C. Control dishes were incubated under identical conditions, in the absence of cells. The medium was removed after 24 hours, and an aliquot was removed for measuring trichloroacetic acid (TCA)-soluble radioactivity, 9 for measuring TBA-reactive materials, and for agarose gel electrophoresis. The remaining sample was subjected to overnight dialysis and then tested for rate of degradation by a fresh culture of macrophages, as described below. Macrophage Degradation of Native and of Macrophage-Modified Low Density Lipoprotein Dialyzed samples from the 24-hour macrophage incubation, containing 10 /xg of macrophage-modified LDL, were added to fresh macrophages in 12-well dishes in 1 ml of DMEM and incubated at 37 C for 5 hours. The medium was then analyzed for TCA-soluble radioactivity. 9 Degradation of native 12S I-LDL or 125 I-LDL that had been incubated 24 hours in the absence of cells was measured in the same way. Chemical Assays Lipid peroxidation was measured by determining the amount of TBA-reactive products as described elsewhere. 12 Protein was determined by the method of Lowry F-10 F-10 DME +BHT Resident Cells CO «< 5 Q ^ o O g c QC en Figure 1. Comparison of unincubated 125 I-LDL, 125 I-LDL incubated for 24 hours in the absence of cells (no-cell control), or with either thioglycollate-eliclted or resident mouse peritoneal macrophages. Cells were incubated either in Ham's F-10 medium without additions (F-10), Ham's F-10 plus 20 nm butylated hydroxytolene (BHT) (added in 10jtl ethanol), or in Dulbecco's modified Eagle's medium (DMEM) without additions in a total volume of 2 ml, containing 100 jig/ml LDL protein. After 24 hours the medium was analyzed for thiobarbtturic acid (TBA)-reactive materials (shaded bars) and accumulated trichloracetic acid (TCA)-soluble 125 l-radioactivity which was expressed as the equivalent amounts of 125 I-LDL degraded (open bars). Values shown are means of duplicate determinations from a representative experiment. CO

3 MACROPHAGE OXIDATION OF LDL Parthasarathy et al. 507 et al. 20 Cholesterol was determined by an enzymatic fluorometric assay. 21 Agarose gel electrophoresis was performed by using lipoprotein samples of identical radioactivity at 30 ma and 300 V for 2 hours with barbitol buffer. 9 " 12 The gel was fixed in 5% acetic acid in 70% ethyl alcohol for 1 hour, was dried, and was subjected to autoradiography. Results These studies involved two sequential incubations: 1) a 24-hour macrophage-modificatfon incubation in which native LDL was incubated with macrophages to allow them to oxidatively modify it; and 2) a test incubation to evaluate the extent to which LDL harvested from the macrophagemodificatjon incubation was modified with respect to its uptake and degradation by fresh macrophages. We first assessed the extent of LDL oxidation and the extent of LDL degradation during the 24-hour macrophage-modification incubation. Resident peritoneal macrophages and thioglycollate-elicited macrophages were compared for their relative efficiencies for oxidation and degradation. As shown In Figure 1, the results with these two preparations were similar. When the incubation was carried out in Ham's F-10 medium (containing 0.01 / x M copper and 1.53 nm iron), there was extensive peroxidatton (shaded bars). However, the addition of BHT reduced peroxidation to a level comparable to that seen in native LDL (possibly even slightly lower than that in LDL incubated in the absence of macrophages). As in the case of our previous studies with endothelial cells, 12 incubation in DMEM, which contains much lower concentrations of metal ions (no ^im copper and 0.28 /im iron), caused little or no peroxidation. As shown in Figure 2, the electrophoretic mobility of LDL incubated with the macrophages in F-10 medium was markedly increased, as in the case of endothelial cell-modified LDL. 11 While the nonlncubated native LDL migrated to a distance of 1.7 cm, the LDL that was incubated with macrophages moved to a distance of 3.6 cm from the origin. B Figure 2. Electrophoretic mobility of LDL samples treated as in the experiment of Figure 1. A. Unincubated 1& I-LDL B. 12S I- LDL incubated without cells. C to E. Incubated with thioglycollate-elicited macrophages (C In Ham's F-10 medium; D in Ham's F-10 medium plus 20 /I.M butylated hydroxytolene (BHT); E in DMEM). F to H. Incubated with resident peritoneal macrophages (Fin Ham's F-10 medium; G in Ham's F-10 medium plus 20 FIM BHT; H in DMEM). H Shown also in Figure 1 are the data for the amount of TCA-soluble 125 I generated during the 24-hour incubation of 12S I-LDL with the macrophages. The results for degradation were again similar whether the cells were resident macrophages or thioglycollate-elicited macrophages. The degradation in F-10 medium without additions was about twice that in F-10 medium containing BHT or in DMEM. This suggested that oxidative changes occurring during the course of the 24 hours accounted for the increased degradation, as in the case of endothelial cell-modified LDL The 12 5I-TCA soluble radioactivity in DMEM or in F-10 medium containing BHT might indicate degradation by way of the native LDL receptor, as macrophage-like cells do seem to interact with native LDL. 8 Endothelial cell modification has been shown to occur slowly but progressively. No clear increase in macrophage degradation of endothelial cell-modified LDL was detected before 6 to 8 hours, and a maximum was reached only at about 18 to 24 hours. 11 Thus, it was possible that the difference between oxidized, macrophage-modified LDL and the LDL incubated in the presence of BHT might be still greater If the measurement of degradation were made at the end of the 24-hour Incubatiop rather than being integrated over the entire 24 hours. Thus, to evaluate the difference between oxidized and unoxidized LDL more critically, an aliquot of the medium containing macrophage-modified LDL after 24 hours was dialyzed against phosphate-buffered saline containing 0.01% EDTA to remove the accumulated degradation products, and this was added to a fresh preparation of macrophages for an additional 5 hours of incubation (test incubation). At the end of this time, the degradation was again assessed. As shown in Figure 3, the rate of degradation of the oxidized LDL that was modified by a previous 24-hour incubation in F-10 medium was more than five times the degradation of unoxidized or minimally oxidized LDL that had been incubated ^ 5 g = 3 Not No-Cell Incu- Control tmiod F-10 + BHT Thloglycol lateel lclted Cells n n F-10 F-10 Resident Cells Figure 3. Degradation of 12 SI-LDL harvested from the initial 24- hour modification-incubation with macrophages and then incubated for 5 hours with fresh macrophages (testincubation). Dialyzed samples (10 /ig LDL protein) were added to the fresh macrophages and incubated at 37 C for 5 hours in a total volume of 1 ml of DMEM. Values are means of duplicates from a representative experiment.

4 508 ARTERIOSCLEROSIS VOL 6, No 5, SEPTEMBER/OCTOBER 1986 Table 1. Macrophage Modification of Low Density Llpoproteln Labeled with the Trapped Ligand 125 l-tyramlne Celloblose Previous LDL treatment Unincu bated Incubated 24 hrs without cells Incubated 24 hrs with cells in F-10 medium Incubated 24 hrs with cells in F-10 medium + BUT (20 Incubated 24 hrs with cells in DMEM TBA-reactive materials (nmol malondialdehyde/ml) F Degradation by fresh macrophages (jj.g/5 hrs/mg protein) ftg/ml of 12S l-tc-labeled LDL was Incubated with resident peritoneal macrophages In Ham's F-10 or in Dulbecco's modified Eagles medium (DMEM) for 24 hours in 60 mm plastic culture dishes. Butylated hydroxytoluene (BHT) was added in 10 /xl ethanol; ethanol only was added to control dishes. One aliquot of medium was used for measurement of total thiobarbituric acid (TBA)-reactive materials. A second aliquot was dialyzed against phosphatebuffered saline containing 0.01% ethylenedinitrilo-tetraacetic acid (EDTA) to remove degradation products and then added to fresh macrophages for measurement of degradation over 5 hours, as discussed in the text. Results are averages of duplicates from a representative experiment. in the presence of the antioxidant or in DME. Again, there was little difference between resident cells and thioglycollate-elicited cells. Because macrophages are known to secrete a number of proteolytic enzymes, we had to consider the possibility that a part of the TCA-soluble radioactivity in the medium might reflect degradation occurring extracellulariy. This was addressed by utilizing LDL labeled with 12S l-tyramine cellobiose (TC) as described by Pittman et al. 19 The tyramine cellobiose is covalently linked to the apoprotein B and enters the cell with it. However, after lysosomal degradation of the protein, the tyramine cellobiose remains trapped in the lysosome because it cannot readily cross the lysosomal membrane. Consequently, the accumulation within the cells of 12S l-tyramine cellobiose is a measure of the number of labeled molecules that have been taken up and degraded over the course of the experiment. The 12S I-TC- LDL was first incubated for 24 hours with resident peritoneal macrophages to modify it, as discussed above. As shown in Table 1, this modifying incubation in F-10 medium led to significant peroxidation, and this was prevented when BHT was added or when the incubation was carried out in DMEM instead of F-10. Aliquots of the medium at the end of the 24-hour modification incubation were dialyzed, which removed TCA-soluble radioactivity as well as more than 75% of the TBA-reactive materials. Samples containing 10 ^g of the dialyzed lipoprotein were then added to fresh preparations of resident peritoneal macrophages, and degradation over a 5-hour period was determined (test incubation). Using the TC label, we assessed the degradation by determining the total radioactivity trapped within the cells at the end of the 5 hours. As shown in Table 1, the LDL oxidized and modified by previous incubation with macrophages in F-10 medium was degraded 10 times more rapidly than LDL that had been incubated in the presence of BHT or in DMEM rather than in F-10 medium. These results show that the enhanced degradation observed is not attributable to proteolytic enzymes released into and acting in the medium. To test whether the increased macrophage degradation of macrophage-modified LDL was occurring by way of the acetyl LDL receptor, the competition studies shown in Figure 4 were carried out. Both unlabeled acetyl LDL and unlabeled endothelial cell-modified LDL were effective competitors for the degradation of macrophage-modified LDL, decreasing specific degradation by as much as 80%. Unincubated native LDL, on the other hand, competed very poorly, indicating that most of the degradation observed was occurring by way of the acetyl LDL or scavenger receptor. co O Q.O O <D to B 5 c I CD 5! CD H- Q. C CO O l «Q T3-1 CO "O o> C D ^ Concentration of Unlabeled Competing Lipoprotein (M9/ml) Figure 4. Effects of increasing concentrations of unlabeled acetyl LDL (o), unlabeled endothelial cell-modified LDL ( ), and native LDL ( ) on macrophage degradation of 125 I-LDL previously modified by a 24-hour incubation with macrophages. Each point represents the mean of duplicate determinations.

5 MACROPHAGE OXIDATION OF LDL Parthasarathy et al. 509 During the 24-hour exposure of macrophages to native LDL, there was a marked increase in the cell cholesterol ester content. In the presence of 100 /ig/ml of native LDL, the cholesterol content increased from 1.9 up to 10.5 jtg/mg cell protein. However, this increase was much less when BHT had been included during the modification incubation; in that case, the cell cholesterol ester increased only to 3.6 /ig/mg protein. The results imply that most of the increase in cell cholesterol content resulted from the conversion of the native LDL to the oxidized modified form followed by uptake via the acetyl LDL receptor. right and proper job, namely, scavenging, when it takes up peroxidized LDL. Our studies in cell culture tell us things that may happen; only after these processes have been evaluated in vivo will we know their true significance. It is interesting that while both circulating monocytes and resident macrophages are able to oxidize LDL, only the latter appear to have the ability to degrade the oxidize LDL, because freshly isolated monocytes possess fewer scavenger receptors. 25 ' x Discussion Previous studies showed that endothelial cells and arterial smooth muscle cells can modify LDL to a form recognized by the acetyl LDL receptor. 9 " 11 With the addition of the monocyte-macrophage to the list of cells able to catalyze this modification, it appears that all three of the major cell types in the artery wall share this modification capacity to some extent. The interesting possibility is raised that there may be a kind of "autocatalytic" LDL uptake and degradation by the macrophage because of its ability to modify LDL and thus speed its own uptake of the molecule (and also perhaps the uptake by the endothelial cells). To what extent these processes contribute to lipid accumulation in vivo remains to be established by appropriate studies of the intact artery. Cathead et al. 16 recently showed that both human monocytes and neutrophils can oxidize LDL and thus produce a potentially cytotoxic molecule. They made the interesting suggestion that the oxidized LDL may represent a longerlived, transportable cytotoxin that could have implications for cell death in developing atherosclerotic lesions and more generally in inflammation and cytotoxicity. Still another effect of oxidatively modified LDL that may be relevant to atherogenesis is its ability to inhibit the motility of macrophages, as recently reported by Quinn et al. 22 It was shown that LDL modified by incubation with endothelial cells (or oxidized in the presence of 5 /xm copper in the absence of cells) strongly inhibited both the basal motility and chemotactically stimulated motility of the resident mouse peritoneal macrophage. The suggestion was made that monocytes entering the subendothelial space (in response to some as yet unidentified chemoattractants) might then be prevented from exiting because of this inhibitory effect of oxidatively modified LDL The effect would be analogous to that of the macrophage inhibitory factor (MIF) at sites of inflammation. 23 The present findings suggest a scenario by which the macrophage, by oxidatively modifying LDL in its immediate environment, may immobilize itselfl Although it seems quite reasonable to propose that the accumulation of lipids in foam cells contributes to atherogenesis, that still has not been experimentally established, as discussed elsewhere. 24 It is conceivable, for example, that oxidized LDL, if not taken up rapidly by way of the macrophage acetyl LDL receptor, might be even more deleterious through its cytotoxicity in the subendothelial space. In other words, the macrophage may be doing its Acknowledgments We thank Dr. Ray C. Pittman and Simone Green for providing 12S l-tc-labeled LDL. We also thank Joellen Bamett for technical assistance and Anita Fargo tor the preparation of the manuscript. References 1. Fowler S, Shlo H, Haley WJ. Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. IV. Investigation of macrophage-like properties of aortic cell populations. Lab Invest 1979;41: Schaffner T, Taylor K, Bantuccl EJ, et al. Arterial foam cells with distinctive immunomorphologic and hlstochemical features of macrophages. Am J Pathol 1980;100: Qerrlty RG. The role of the monocyte In atherogenesis. Am J Pathol 1981;103: Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoproteins, producing massive cholesterol deposition. Proc Nat! Acad Sci USA 1979;76: Mahley RW, Innerarlty TL, Welsgrabor KH, Oh SY. Altered metabolism (in vtvo and In vitro) of plasma lipoproteins after selective modification of lysine residues of the apoprotelns. J Clin Invest 1979:64: Fogelman AM, Schechter I, Seager J, Hokom M, Child JS, Edwards PA. MalondlaJdehyde alteration of low density lipoproteins leads to cholesteryl ester accumulation In human monocyte macrophages. Proc Natl Acad Sci USA 1980; 77: Brown MS, Basu SK, Falck JR, Ho YK, Goldstein JL The scavenger cell pathway for lipoprotein degradation: specificity of the binding site that mediates the uptake of negativelycharged LDL by macrophages. J Supramol Str 1980;13: Tabas I, Welland DA, Tall AR. Unmodified low density lipoprotein causes cholesteryl ester accumulation in J774 macrophages. Proc Natl Acad Sci USA 1985;82: Henrlksen T, Mahoney EM, Steinberg D. Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: Recognition by receptors for acetylated low density lipoproteins. Proc Natl Acad Sci USA 1981;78: Henriksen T, Mahoney EM, Steinberg D. Interactions of plasma lipoproteins with endothelial cells. Ann NY Acad Sci USA1982;401: Henrlksen T, Mahoney EM, Steinberg D. Enhanced macrophage degradation of biologically modified low density lipoprotein. Arteriosclerosis 1983;3: Stelnbrecher UP, Parthasarathy S, Leak DS, WHztum JL Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidatlon and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci USA 1984;81: Parthasarathy S, Stelnbrecher UP, Bamett J, Wltztum JL, Steinberg D. Essential role of phospholipase A 2 activity in endotheliai cell induced modification of low density lipopro

6 510 ARTERIOSCLEROSIS VOL 6, No 5, SEPTEMBER/OCTOBER 1986 tein. Proc Natl Acad Sci USA 1985;82: Hessler JR, Morel DW, Lewis LJ, Chisolm GM. Lipoprotein oxidation and lipoprotein-induced cytotoxicity. Arteriosclerosis 1983:3: Parthasarathy S, Young SG, Witztum JL, Pittman RC, Steinberg D. ProbUcol inhibits oxidative modification of low density lipoprotein. J Clin Invest 1986;77: Cathcart MK, Morel DW, Chisolm GM. Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic. J Leuk Biol 1985:38: Vermeer BJ, Van Der Schroeff JG, Emeis JJ, Ponec M, Havekes L. Mechanisms of cholesterol ester accumulation in cultured monocytes. Br J Dermatol 1984;lll (Suppl 27): Basu SK, Goldstein JL, Anderson RGW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci USA 1976; 73: Pittman RC, Crew TE, Glass CK, Green SR, Taylor CA Jr, Attie AD. A radioiodinated intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J 1983;212: Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951:193: Gamble W, Vaughan M, Kruth HS, Avigan J. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. J Lipid Res 1978:19: Quinn MT, Parthasarathy S, Steinberg D. Endothelial cellderived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein. Proc Natl Acad Sci USA 1985:82: Nathan CF, Kamowsky ML, David JR. Alterations of macrophage functions by mediators from lymphocytes. J Exp Med 1971;133: Steinberg D. Lipoproteins and atherosclerosis: A look back and a look forward. Arteriosclerosis 1983:3: Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci USA 1979:76: Knight BL, Soutar AK. Changes in the metabolism of modified and unmodified low density lipoproteins during the maturation of cultured blood monocyte/macrophages from normal and homozygous familial hypercholesterolemic subjects. Eur J Biochem 1982;125: Index Terms: atherosclerosis foam cells acetyl-ldl receptor free radicals lipid peroxidation