Prog. Lipid Res. Vol. 31, No. 2, pp. 127-143, 1992 Printed in Great Britain. All fights reserved

0163-7827/92/$15.00 © 1992 Pergamon Press Ltd

ROLE OF OXIDIZED LOW DENSITY IN ATHEROGENESIS

LIPOPROTEIN

SAMPATHPARTHASARKrHYand SARA M. RANKIN 0613-D, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, U.S.A. CONTENTS I. INTRODUCTION II. LDL AND A ~ O S C L ~ O S l S A. Native LDL and the LDL receptor B. Modified LDL and the scavenger or acetyl-LDL receptor III. POST-SEc~TION MODIFICATIONSOF LDL IV. OXtOA1TCELYMODIFIED LDL A. Physicochemical changes in LDL during oxidation I. Physical changes 2. Chemical changes 3. Phospholipid hydrolysis 4. Oxidation of cholesterol 5. Fragmentation of apt) B 6. Changes in lysine and macrophage recognition B. Biological effects of oxidized LDL I. The uptake of oxidized LDL by macrophages 2. Oxidized LDL in recruitment and retention of monocytes/macrophages in the artery wall 3. Cytotoxicity of oxidized LDL 4. Other proatherogenic effects of oxidized-LDL C. A role for oxidized LDL in atherogenesis V. MECHANISMSOF LDL OXIOATIONBY CELLS A. Cellular oxidation of LDL I. The role of superoxide anions in cellular modification of LDL 2. The role of lipoxygenase in the cellular modification of LDL VI. EVIDENCEFOR THE PRESENCEAND ATHEROGENICITYOF OXIDIZED LDL IN wvo A. Methods used to determine LDL oxidation B. Lipid peroxidation and atherosclerosis 1. The presence of lipid peroxides in atherosclerotic lesions 2. The presence of lipid peroxides in plasma 3. Antioxidants and atherosclerosis 4. Isolation of oxidized LDL from biological tissue samples 5. Immunohistochemical demonstration of oxidation related lipid-protein adducts in atherosclerotic aorta C. Can oxidative modification of LDL occur in the plasma? VII. CONCLUSION REFERENCES

127 128 128 128 129 130 131 131 131 132 132 132 132 133 133 133 134 134 135 135 135 135 136 137 137 137 137 138 138 138 139 139 140 140

ABBREVIATIONS LDL Ox-LDL WHHL MDA BHT PtdCho Lyso PtdCho TBARS PMA

low density lipoprotein oxidized LDL watanabe heritable hyperlipidemic malondialdehyde butylatedhydroxytoluene phosphatidylcholine lysophosphatidylcholine thiobarbituric acid reactive substances phorbol myristate acetate

MM-LDL SOD PUFA PAF PLA2 TCA VLDL fl-VLDL 13-HODE

minimally modified LDL superoxide dismutase polyunsaturated fatty acid platelet activating factor phospholipase A2 trichloroacetic acid very low density lipoprotein p-migrating very low density lipoprotein 13 hydroxyoctadecadienoic acid

I. I N T R O D U C T I O N

Atherosclerosis is a disease that affects large arteries. The early atherosclerotic lesion, the fatty streak, is characterized by the build up of lipids in the intima. These lipids are derived from the major cholesterol carrier, namely the low density lipoprotein (LDL). 93 Both free and esterified cholesterol are present as cytoplasmic lipid droplets in the intimal 127

128

s. PARrI-ImARATh'Yand S. M. RANKIN

cells that, due to their appearance, are termed foam cells. Recent biochemical and immunohistochemical studies have established that mononuclear phagocytes are the predominant cellular origin of these cells, l'3Lr~'*°'67'127,la°'t52,155 In this review the role of lipid peroxidation in the formation of the fatty streak lesion will be discussed. In humans the progression of atherosclerosis is slow, occurring over decades. As a result, the disease was originally attributed to the natural process of ageing. In recent years, a number of specific risk factors have been identified that affect the progression of the disease. One of the major risk factors is the elevated levels of plasma cholesterol, particularly that associated with LDL. 64')5°The mechanism(s) by which LDL manifests its atherogenic properties has been the topic of intense investigation during the past decade. II. LDL AND ATHEROSCLEROSIS A. Native LDL and the LDL Receptor

LDL is a source of cholesterol for peripheral cells. The basic mechanism(s) involved in the cellular uptake of LDL by cells was elegantly elucidated by Goldstein, Brown and coworkers. 43 The LDL molecule is internalized by cells by way of a specific receptor that recognizes the apoprotein B~ooon the particle. 43 In vivo evidence suggests that the removal of more than two thirds of plasma LDL is mediated by this LDL receptor (apoprotein B/E receptor) and that this removal occurs predominantly in the liver. "°:" The LDL receptor is down-regulated when the cellular need for cholesterol is met. 12 This property suggests that the excessive accumulation of lipids by arterial foam cells may not be mediated by the LDL receptor, unless an aberrant metabolic regulation of the receptor exists. Other lines of evidence suggest that the LDL receptor may not be involved in foam cell formation. Goldstein and Brown found that when mouse peritoneal macrophages (commonly used as a model for arterial macrophages) were incubated with native human LDL, in vitro, it was poorly degraded and the cells did not accumulate cholesteryl esters) 3'45 This finding suggested that these cells have very few receptors for native LDL. Patients suffering from the homozygous form of familial hypercholesterolemia 44 and Watanabe heritable hyperlipidemic (WHHL) rabbits, ~4:27 despite a near complete absence of functional LDL receptors, exhibit markedly elevated plasma LDL levels and develop a severe form of atherosclerosis, with the characteristic macrophage-derived foam cells. More recent studies utilizing in situ hybridization techniques show little or no mRNA for the LDL receptor in the macrophage-rich lesions of human atherosclerotic aorta) 6° These observations suggested that receptor(s) other than the apoprotein B/E receptor, that recognizes active LDL, must be involved in the uptake and accumulation of LDL lipids by the macrophages. The importance of the LDL receptor, however, can not be ignored. A deficiency in functional LDL receptors may result in the imbalance of cholesterol homeostasis and the ensuing hypercholesterolemia. B. Modified LDL and the Scavenger or Acetyl-LDL Receptor

If LDL represents one of the major atherogenic lipoproteins and macrophages do not take up sufficient amounts of LDL to generate foam cells, then how do they accumulate cholesterol? Goldstein and Brown proposed that some form of post-transitional modification of the lipoprotein may be involved that would render the altered LDL susceptible to enhanced uptake and degradation by macrophages) 3'45 Subsequently they discovered that acetyl-LDL, in which the e-amino groups of the lysine residues are chemically acetylated (thus neutralizing the positive charge on the amino group) was avidly degraded by macrophages resulting in their accumulation of cholesteryl esters. 45 This degradation was mediated by a saturable, high affinity receptor and was competed for by a variety of polyvalent anionic molecules, both protein and non-protein in nature (e.g. polyvinyl sulfate and maleylated bovine serum albumin) and yet selective to a certain degree. H In accordance with the role of macrophages as scavengers the new pathway of modified LDL

Oxidized LDLs in atherogenesis

129

uptake was termed the scavenger pathway. The receptor was referred to as the scavenger receptor or more specifically the acetyl-LDL receptor. This receptor is distinct from the LDL receptor. In fact, native LDL is not recognized by this receptor. This receptor has subsequently been found to be expressed by macrophages of different origin, and by endothelial cells, particularly the sinusoidal endothelial cells of the liver, s~'~°9'~37It has also been described in cultured rabbit smooth muscle cells and fibroblasts. 1°8 The expression of this receptor in these cells is increased by products secreted from thrombin-stimulated platelets and by phorbol esters) °s A 220 kDa acetyl-LDL binding protein was originally identified by Via and coworkers in P388-D1 cells (a murine macrophage cell line)J 53 A protein composed of three monomers of about 77 kDa that binds acetyl LDL was then partially purified, from bovine liver membranes and subsequently from bovine lung membranes by monoclonal antibody affinity chromatography. 7° The purified protein in the trimeric form exhibited a molecular weight of about 220 kDa. A binding protein of the same molecular weight was also observed by ligand blotting on bovine alveolar macrophages and THP-1 cells (a human monocytic cell line) that had been stimulated with phorhol myristate acetate(PMA). 7° Two clones for the receptor have been isolated from bovine liver and P338-D1 cells and from a eDNA library for PMA treated THP-1 CCI1s. 37'69'70's2"124 One clone (type 1) encodes for a protein of 453 amino acids with a calculated M r of 49,975 suggesting glycosylation of the protein. The main difference between these two clones is that type I has a 110 amino acid, cysteine rich, C terminal region whereas the type II only has a 6 amino acid C terminal domain. Both have essentially the same affinity and broad substrate specificity. The presence of this receptor has been demonstrated by immunohistochemical and in situ hybridization techniques in human and rabbit atherosclerotic lesions. 82,j6° As expected, the acetyl-LDL receptor is localized predominantly in the macrophage-rich areas of the lesion. It is not known whether this is the only receptor involved in the uptake of modified lipoproteins or is one of a group of scavenger receptors. There is some evidence for other receptors recognizing distinct forms of modified LDL, as will be discussed later.2, a36 The normal physiological function of the scavenger receptor on macrophages is unknown and its physiological ligand has not been yet elucidated. Recently, however, the lipid A moiety of bacterial lipopolysaccharide has been reported to interact with the acetylLDL receptor. 49 III. POST-SECRETION MODIFICATIONS OF LDL In addition to acetylation, other modifications of LDL that increased its uptake by macrophages have been forthcoming. Fogelman et al. reported that malondialdehyde (MDA), known to be generated by aggregating platelets or from lipid pcroxidation, could covalenfly modify lysine residues of LDL and generate a negatively charged LDL capable of interacting with the scavenger receptor. 3: More importantly oxidation of LDL lipids, described in detail in this review, also generated a ligand(s) that showed increased uptake by macrophages. 54'55 Many of these modified forms of LDL, including acetoacetyl-LDL and MDA-LDL are also recognized by the acetyl-LDL receptor. 32's~ A list of modified forms of LDL that have been reported to result in enhanced uptake by macrophages, mediated by the scavenger receptor, is given in Table 1. It should be pointed out that these modifications, that presumably alter the charge on the lipoprotein by derivatizing the E-amino residues of lysine differ vastly in their ability to convert LDL to a form recognized by the scavenger receptor. For example, a considerable degree of acetylation (over 80%) is required for modification of LDL, while only 15-20% derivatization of the lysine amino groups is necessary for modification by MDA. 4s The latter, a bifunctional crosslinker, may also result in particle aggregation suggesting that changes in lipoprotein structure in addition to charge may contribute to scavenger receptor recognition. While the modification of E-amino groups of lysine residues of apoprotein B~eohas been the primary focus, the ability with which the receptor interacts with other polyanionic

130

S. P J u t ~ l " H Y

TABLE 1. Modifications o f LDL that Result in Enhanced Uptake by Macrophages 1. 2. 3. 4. 5. 6. 7. 8.

Acetylation 45 Malondialdehyde addition 32 Acetoecetylation81 Carbamylation 46 LDL-Dextran sulfate complex formation 5 Oxidation s4,55.t43 Glycation 77'8° Desialylation m

and S. M. RANKIN TASLE 2. Cell Types that Oxidat/v©ly Modify LDL 1. 2. 3. 4. 5. 6.

Endothelial cellss4,sS,sS'm Smooth muscle cellsst,Ss,S* Monocytes l~'ts Macrophages including arterial foam cells7(''°~m Fibroblasts m''41 Monocyte cell lines such as U937 t6'm

compounds alerts us to other potential sites of modifications.H In fact, the domain of LDL whose changes in structure or configuration lead to scavenger receptor recognition has yet to be ascertained. It should be noted that in these in vitro assessments not all lipoproteins were tested under identical conditions, using LDL or macrophages from the same source. A variety of cells that include resident mouse peritoneal macrophages, cultured macrophage cell lines, human monocytes cultured into macrophages and rabbit macrophagederived foam cells have been used in these studies. Some of these lipoproteins may be recognized by more than one receptor on these macrophages. In addition to these modified forms of LDL other types of modifications have also been demonstrated. For example, Khoo et al. reported that the mechanical disruption of LDL structure by vortexing resulted in the formation of an aggregated particle that showed enhanced degradation by macrophages3 5 A similar aggregated form of LDL was also generated by the treatment of LDL with phospholipase C t~ or with elastase, m These lipoproteins, however, appear to be phagocytosed subsequent to recognition by the LDL receptor on the macrophages. It is thought that the LDL receptor and the Fc receptor, as well as the scavenger receptor, may be involved in the uptake of these LDL aggregates. There is recent evidence that some form of aggregated lipoproteins may exist in the atberosclerotic artery. 36

IV. O X I D A T I V E L Y

MODIFIED

LDL

Despite the identification of a number of in vitro modifications, the processes that result in the conversion of LDL to a form recognized by the scavenger receptor/n vivo have not been clearly defined. Acetylation appeared to be improbable, since considerable numbers of the lysine residues need to be acetylated before macrophage recognition occurs. Modification by malondialdehyde, generated by aggregated platelets or during lipid peroxidation, appeared to be an attractive candidate. Platelets, however, do not produce the massive quantities of malondialdehyde required for the modification of LDL. In the past few years considerable evidence has accumulated to suggest that lipids of the LDL themselves might undergo peroxidation and generate reactive aldehydes including MDA and in that process generate a modified LDL. It was known for some time that when LDL was incubated with cultured endothelial cells it became cytotoxic. 53,ss'ss,89Henriksen et al. in 1981 demonstrated that this cell-incubated LDL was also modified to a form that was degraded by macrophages several times more rapidly than native LDL. u The uptake and degradation was specific, saturable and was not competed for by native LDL. u.55 The degradation was, however, competed for by unlabeled cell-modified LDL. Acetyl-LDL competed for the uptake and degradation of the endothelial cell-modified LDL and vice versa to some extent, showing that at least part of the uptake of the modified LDL was by the same receptor that recognizes acetyl-LDL. 55 More recent experiments have suggested that a considerable portion of the uptake of the cell-modified LDL occurs by an independent receptor that is not subject to competition by acetyl-LDL 136.In subsequent studies it was found that numerous other cell types were able to modify LDL in a similar manner. These are listed in Table 2. As discussed later, lipids of LDL readily undergo peroxidation even in the absence of cells. This property has been exploited considerably by several investigators. As a result

Oxidized LDLs in atherogenesis

131

a plethora of information is now available on the potential mechanism(s) that may be involved in the oxidative modification of LDL. A. Physicochemical Changes in LDL During Oxidation 1. Physical Changes LDL undergoes several physico-chemical changes during the long incubation (2 hr) with the cells. Even before the biochemical nature of the cell-induced modification was suspected, Henriksen et al. observed that LDL incubated with cells showed an increased electrophoretic mobility on agarose gel. 54'55This increased anodic mobility has afforded a convenient measure of the cells' ability to modify LDL. In addition to this increased negative charge, the buoyant density of the modified LDL particle increased from a mean density of 1.035 to 1.06. An interesting feature of these changes is that, distinct subpopulations of LDL, representing varying degrees of modification could not be demonstrated. This suggests that the changes affected the particles evenly or the perturbations are rapidly and evenly distributed among the LDL particles. 2. Chemical Changes The modification of LDL by cells occurred in certain media, such as Ham's F-10, but not in Dulbecco's Modified Eagle's medium. 143This property provided the first clue as to the mechanism that may be involved in the modification process. Steinbrecher et alJ 43 noting the differences in the content of trace metals such as iron and copper in the commercially available cell culture media suggested the involvement of metal catalyzed lipid peroxidation as an important step in the modification of LDL. They were able to correlate the formation of thiobarbituric acid reactive substances (TBARS) with the uptake of the incubated LDL by macrophages. ~43 Soon, Esterbauer and coworkers, studying the autoxidation of LDL, showed that during the early phase (first couple of hours) of oxidation of LDL there was a rapid depletion of LDL associated antioxidants such as vitamin E and carotenoid. 29 Following the depletion of these antioxidants the polyunsaturated fatty acids (PUFA) were oxidized. Similar changes have since been reported to occur during the modification of LDL by macrophages. 6~ Exogenously added antioxidants such as vitamin E, butylated hydroxy toluene (BHT) and a host of others inhibit the oxidation. 74's9,~43It is also inhibited by metal chelators such as ethylenediamine tetra acetic acid. 51'74'14a At present it is not known by what mechanism(s) the metals contribute to the cellular oxidation of LDL. They may initiate free radical production and lipid peroxidation by reacting with thiols in the medium) 2 In fact, it has been suggested that the role of cells may be restricted to the generation of thiols, particularly cysteine, containing free-SH groups. 9~ The known ability of metals to promote the decomposition of peroxidized fatty acids and the generation of aldehydes is likely to be important in the oxidation of LDL. Copper, at high enough concentrations ( > 1/~M), by itself, promotes lipid peroxidation and the modification of LDLJ 43 At these concentrations copper oxidizes LDL even in simple buffers such as phosphate buffered saline. 29This suggests that the metal may either react with intrinsic peroxides on the LDL molecule or may have the capacity to generate oxygen radicals directly by interacting with molecular oxygen, or by interaction with specific thiol groups of apo B. It has been shown that the loss of LDL linoleate and araehidonic acid is concomitant with the generation of their specific lipid hydroxy and hydroperoxy derivatives. 76Extensive fragmentation of the oxidized fatty acid then occurs generating a wide array of short and medium chain aldehydes that have potent biological properties. 29Water-soluble aldehydes such as free MDA readily dissociate from the LDL particle and can be removed by dialysis or filtration. 145 Some of these however, may be in covalent association with the amino groups of lipids or the apoprotein. Unbound lipophilic aldehydes such as hexanal, nonenal or their derivatives may reside in the core of the particle. JPLR 3 1 / 2 ~

132

S. P A R ~ X X - ~ and S. M. RAN~N

3. Phospholipid Hydrolysis The LDL particle contains phosphatidylcholine (PtdCho) at the surface and during the oxidative modification some of which undergoes hydrolysis to lysophosphatidylcholine (|yso Ptdcho). 1°4 Inhibition of this hydrolysis by antioxidants suggested that oxidation of the lipid precedes the fatty acid removal. '°4 This hydrolysis is due to the removal of the fatty acid at the 2-position of PtdCho (which is usually esterified to an unsaturated fatty acid) by a phospholipase As (PLA~) activity that is intrinsic to LDL. 97't°4 The removal of the oxidized fatty acids from the surface of the particle has been suggested to aid in the rapid propagation of lipid peroxidation into the core of the LDL particle. '°4 A unique PLA2 activity that hydrolyzes only oxidized phospholipids has since been demonstrated in LDL preparations? 7 This activity was found to undergo progressive inactivation during oxidation of LDL, and consequently fully Ox-LDL possesses very little activity. Inactivation of the enzyme correlated with the loss of histidine residues of apo B during LDL oxidation. 97p-Bromophenyacyl bromide a histidine modifier that inhibits the oxidation of LDL also substantially inhibited the phospholipase activity. 97 It has been suggested that plasma platelet activating factor (PAF) hydrolase is associated with LDL on isolation, and may be responsible for the hydrolysis of oxidized phospholipids? 44 However, large molecular weight fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of apo B,oo, were found to be distinct from PAF-hydrolase and yet shown to possess this PLA2 activity. 97 Thus at present there is still some controversy as to the identity of this phospholipase activity.

4. Oxidation of Cholesterol Although lipid peroxidation is often, in discussion, restricted to fatty acid peroxidation, there is considerable evidence that the cholesterol moiety of LDL is also similarly affected. In fact, several oxy-sterols have been identified that include 5,6-epoxy cholesterol, 7-hydroperoxy, 7-hydroxy and 7-keto cholesterol. In addition, oxidized cholesteryl esters have also been reported in Ox-LDLJ 63

5. Fragmentation of Apt B During the oxidative modification of LDL the apoB-100 is degraded into several smaller fragments. Based on the finding that antioxidants, but not proteolytic inhibitors, prevented this fragmentation, it was suggested to occur by direct oxidative scission of the polypeptide. 33 The oxidation process also decreases the content of histidine, lysine, proline and methionine of the apoprotein and results in an increase in aspartic acid. The loss of histidine is perhaps due to ring cleavage and its resultant conversion to aspartic acid may also contribute to the increased negative charge of the lipoprotein.

6. Changes in Lysine and Macrophage Recognition The question "what is the chemical nature of the receptor recognition site on the oxidatively modified LDL?" cannot be answered yet. However, considerable progress has been made in the elucidation of modifications that result in recognition by specific maerophage receptors. First, it is the apoprotein of the Ox-LDL that carries the recognition sites. Delipidated and resolubilized Ox-LDL protein was shown to be degraded by macrophages and this degradation was competed for by intact Ox-LDL. 98It is currently believed that aldehydes generated during lipid peroxidation form Schiff's bases, with the free amino groups of lysine residues of a p t B, that result in the generation of a negative charge. In agreement a decrease in lysine residues after oxidation has been demonstrated. ~4°'t45Steinbrecher et al. have also shown that '4C-labeled fatty acid fragments from LDL, after oxidation, are incorporated into the apoprotein. '42 A number of questions remain to be resolved. These include (a) relationship between apoprotein breakdown and

Oxidized LDLs in atherogenesis

4. 5. 6.

TABLE3. Properties of Ox-LDL Increased negativecharge and density54,55,143 Dec/easeduptake via LDL receptorss Increased uptake and degradation by scavenger receptor~4~5 Decreased vitamin E and antioxidants~'6t Decreased polyunsaturatedfatty acidsI°Lm Decreased PtdCho and increased lyso PtdCho

7. 8. 9. 10. 1 1. 12.

eontenttO4, t42 Increased cholesterol oxidation products 62,163 Formation o f fatty acid peroxidation products 29,~42 Fragmentation of apoB 33 Loss o f histidine, lysine and proline 7t Increased fluorescence of the apoprotein t62 Altered immunogenicity t62

1. 2. 3.

133

TABLE4. AtherogenicEffectsof Ox-LDL 1. Ox-LDL is degraded at a faster rate than native LDL by macrophagesand leads to their accumulation of cholesteryl

ester$54,55,102 Ox-LDL is chemotactic for monocytes n4m6 Ox-LDL inhibits macrophage ¢hemotaxis ns Ox-LDL is cytotoxic to cells ~6,s7 Ox-LDL inhibits endothelium dependent relaxation73,~6~ 6. M M - L D L enhances monocyte adhesion to endothelial cells7 7. M M - L D L induces endothelial cell expression of granulocyte and maerophage colony stimulating factor H7 8. M M - L D L stimulates maerophage chemotaetie peptide-I from endothelial cells and smooth muscle cells24

2. 3. 4. 5.

lysine modification (b) the role of particle aggregation during oxidative modification5s (c) role of phospholipase A2 reaction in the modification and (d) the involvement of other amino acid changes in the apoprotein. Identification of a specific domain(s) of L D L that is altered as a result of oxidative modification would facilitate understanding of the chemistry involved in the modification process. The properties of Ox-LDL are summarized in Table 3.

B. Biological Effects of Oxidized LDL The initial interest in oxidatively modified L D L focussed on the ability of macrophages to take up and degrade the lipoprotein. Subsequently an array of other potentially atherogenic effects have been identified (Table 4).

1. The Uptake of Oxidized LDL by Macrophages One of the effects first observed of cell-modified L D L was its increased uptake and degradation by macrophages. 54'55Cultured mouse macrophages degraded ~25I-Ox-LDL, on average 4-10-fold faster than unincubated native LDL. This was determined by the measurement of trichloroacetie acid (TCA) soluble radioactivity in the medium after incubation of labeled lipoprotein with macrophages for 5 hrs. Recent studies have shown that considerable amounts of Ox-LDL remain within the cell in the undegraded form. 7s,136 Thus the total uptake is considerably more than that represented by TCA-soluble radioactivity in the medium. This accumulation of undegraded Ox-LDL may be due in part to the reduced rate of proteolysis of Ox-LDL (as compared with native, or acetyl LDL) by macrophage proteases. 7s Incubation of macrophages with Ox-LDL in the presence of ~'C-oleate results in meager incorporation of the radioactivity into the cholesterol ester fraction. It has been suggested that the inefficient degradation and hence processing of Ox-LDL may account for its reduced ability to induce cholesteryl ester formation. 163 In contrast, when macrophages are incubated with ~25I-acetyl-LDL it is degraded efficiently and hence there is no significant accumulation of the undegraded lipoprotein within the cell. Indeed, when incubated with acetyl-LDL macrophages synthesize massive amounts of cholesteryl esters.

2. Oxidized LDL in the Recruitment and Retention of Monocytes/Macrophages The adherence of blood borne monocytes and their migration into the intima represents one of the earliest steps in the development of atherosclerosis. 39'4°Presumably one or more chemotactic factors are involved in the mononuclear cell movement. Since neutrophils are rarely seen in the atherosderotic aorta, monocyte specific chemoattractants are thought

134

S. PARTttASARA'mYand S. M. RANKIN

to be involved in this process. Oxidatively modified LDL has been shown to have components that are potent monocyte chemoattractants)~4 The activity is associated with the lipids of the Ox-LDL and is in part attributable to lyso PtdCho that is generated during the modification of LDL. "6 There is evidence to suggest that the chemotactic activity of Ox-LDL is dependent on the metabolism of lyso PtdCho by the target cells, n3 It is interesting that/~-very low density lipoprotein (VLDL) isolated from cholesterol-fed rabbit plasma, has been shown to contain high levels of lyso PtdCho and exhibit chemotactic activity for human monocytes) °3 The monocytes that migrate into the artery eventually become tissue macrophages. In contrast to observations made for acetyl-LDL, which is chemotactic to macrophages, oxidatively modified LDL is a potent inhibitor of both the basal and stimulated migration of macrophages)'3'~'4'115 Again the active component is localized in the lipid fraction of the modified lipoprotein. Native LDL does not affect the chemotactic movement of either monocytes or macrophages. So far Ox-LDL was referred to as a single homogeneous particle. It should be borne in mind that there may be a wide range of Ox-LDL particles distinct from each other with respect to their content of peroxidation products as well as the degree of apoprotein modification, dependent on the method used to oxidize the LDL. In fact, recent studies show that minimally modified LDL (MM-LDL) (oxidized to an extent that the particle still retains the ability of LDL to be recognized by the LDL receptor and does not lead to the recognition by the scavenger receptor) has potent biological properties. 7'24'u7 MM-LDL may contribute to the chemotactic recruitment of monocytes by means of inducing the cellular synthesis of specific chemoattractant proteins. It also stimulates the release of several growth factors "7 and increases the adhesiveness of monocytes to endothelial cell monolayers. 7 However, the specific components responsible for its potent biological actions have not been identified.

3. Cytotoxicity of Oxidized LDL Endothelial injury is an important component of atherosclerosis, ns However, denudation of the endothelium does not appear to occur before the onset of the fatty streak lesion. 26 Once the lesion is established, evidence of endothelial damage has been noted by several workersfl '39 Even before the recognition that Ox-LDL may play an important role in the formation of macrophage foam cells, the cytotoxic nature of Ox-LDL was well established. Chisolm and coworkers ~s,~ and Henriksen et al. 53 independently recognized that Ox-LDL was cytotoxic to cultured cells. Although Ox-LDL contains a variety of cytotoxins such as 2-alkenals and 4-hydroxy 2-alkenals and the oxidation products of fatty acids and cholesterol, there is suggestion that the cytotoxic process may manifest a certain degree of specificity. Cells appear to be most susceptible for cytolysis during their S-phase of the cell cycle.72

4. Other Proatherogenic Effects of Oxidized LDL There are other proatherogenic effects attributed to Ox-LDL. Noteworthy are the inhibition of the release of endothelium dependent relaxing factor 73'~6~and its stimulation of the induction of proteins responsible for the adhesion and chemotaxis of monocytes at the endothelial cell surfaceTM Not all the effects of Ox-LDL are proatherogenic. Some of the components associated with Ox-LDL have anti-atherogenic effects themselves or are capable of eliciting antiatherogenic responses from cells. For example, Ox-LDL has been reported to increase prostacyclin synthesis of cells which may impede plated aggregation) 49 Ox-LDL also inhibits the release of platelet derived growth factor which may decrease the proliferation of smooth muscle cells during atherogenesis. 35

Oxidized LDLs in atherogenesis

135

C. A Role for Oxidized LDL in Atherogenesis Based on the above discussion the following scenario of events can be postulated. In the presence of high levels o f L D L in the plasma the intimal L D L concentration will be increased. In the subendothelial space the L D L would undergo oxidative modification at a certain rate. The modified L D L would contribute to the chemotactic recruitment of circulating monocytes directly, as well as by indirect means, by inducing the synthesis of monocyte chemotactic proteins. Other growth factors and cytokines, such as colony stimulating factors, induced by various forms of Ox-LDL, would increase the number of mononuclear cells. Once the monocytes differentiate into tissue macrophages, their return to the plasma will be inhibited by the Ox-LDL. As monocytes/macrophages themselves can oxidatively modify LDL, there will be a further increase in the rate of L D L oxidation. The subendothelial macrophages take up the Ox-LDL by means of the scavenger receptor in an unregulated manner, thereby accumulating cholesterol esters and become foam cells. Thus high levels of plasma L D L and favorable oxidation conditions could accelerate the development of the fatty streak lesion.

V. MECHANISMS OF LDL OXIDATION BY CELLS Lipid peroxidation can be achieved by a variety Of means and there may not be a sole determinant factor that initiates lipid peroxidatiori o f the L D L molecule. Lipid peroxidation can be initiated by both enzymatic and non-enzymatic mechanisms, at least under in vitro conditions. Some of the conditions under which L D L can be oxidized in vitro are listed in Table 5.

A. Cellular Oxidation of LDL Enzymatic lipid peroxidation can be direct, as in the case of lipoxygenases, or indirect, for example by means of generation of oxygen free radical intermediates. When a suitable oxidation system is provided, such as the addition of a sufficiently high concentration of copper and/or iron to the medium containing L D L the cell-induced modification can be mimicked in the absence of cells. Addition of compounds containing a free thiol -SH also facilitate the conversion of L D L to a modified form. 96 These observations suggest that the contribution of the cells to the modification may be restricted to the enhancement o f the rate of oxidation. The cell may achieve this by (a) generating reactive oxygen species such as superoxide radicals or (b) by way of specific fatty acid oxygenases or (c) by secreting substances that provide oxidants into the extracellular medium (Table 6).

1. The Role of Superoxide Anions in Cellular Modification of LDL The exclusive involvement of superoxide anions in the oxidative modification of L D L appears unlikely. Superoxide radicals generated by pulse radiolysis 6 or by the xanthine/xanthine oxidase system t°° were ineffective in oxidizing L D L unless large amounts of copper were present. Moreover, a number of different superoxide dismutases (SOD) afforded only limited protection against L D L oxidation by both endothelial cells and TAeLE5. Conditions that Favor Oxidation of LDL TABLE6. Possible Mechanisms of Oxidation of LDL 1. Extensive dialysis in oxygen saturated buffers29 2. Incubation in the presence of cells~'55's7 1. Cell generated superoxide anions 3. Incubation with metals such as iron or copper~43 2. Extracellulargeneration of superoxide anions by thioi recycling 4. Exposure to ionizing radiations 5. Incubation with free radical generators such as 3. Lipoxygenasereactions AAPHt29 4. Lipoxygenase-dependentsuperoxide or other reactive oxygen generation 6. Incubation with iron/ADP33 5. Nitric oxide dependent oxidation 7. Treatment with enzymessuch as lipoxygenase~35 6. Hydrogen peroxide dependent oxidation 8. Treatment with H202and peroxidaseI~ 7. Free radicals generated from other sources 9. Incubation with thiol/metals~

136

S. PARTtt~aC~THY and S. M. RANKIN

macrophages. ~°5,H8 In contrast, SOD effectively inhibited PMA-stimulated monocytemediated oxidation of LDLJ 7'1s'57In addition the modification of LDL by fibroblasts was subject to significant inhibition by SOD. Hs 2. The Role of Lipoxygenase in the Cellular Modification of LDL

Two findings prompted the idea that cellular lipoxygenases may be involved in the modification of LDL. Firstly, several inhibitors of cellular lipoxygenase activity prevented LDL modification by cells, s4'~°s'Hs Secondly, incubation of LDL with purified soybean lipoxygenase in the presence of PLA2 generated a modified LDL with similar characteristics to the cell-modified LDL. 135Indirect evidence for the involvement of 15-1ipoxygenase came from the studies of Bailey and coworkers. Using minced sections of aortic intima, they showed that the activity of 15-1ipoxygenase and its products are increased in lesioned, as compared to normal areas, of aortas.134 In these studies the cellular origin of this increase was not investigated. Recently Ylti-Herrtuala et al. using in situ hybridization and immunohistochemical techniques provided evidence for the presence of 15-1ipoxygenase mRNA and protein in macrophage-rich haman and W H H L rabbit lesions, z59'16°There was no indication for the presence of 5- or 12-1ipoxygenases in the macrophage lesions. At present there are several lines of evidence to suggest that 15-1ipoxygenase activity may be involved in the oxidative process. These are summarized in Table 7. The mechanism(s) by which cellular lipoxygenases may contribute to the initiation of the oxidation of LDL has not been determined. As suggested by Steinberg et al. 94'139 several pathways can be visualized for the cellular modification of LDL. In one, the oxidized cell lipids can transfer to the LDL in the medium and initiate propagation reaction through metal catalyzed degradation. Secondly, the cell lipoxygenase may be capable of direct action on LDL lipids. This may require the release of the enzyme as a result of cell damage. Thirdly, the cellular generation of reactive oxygen may be tied to the action of lipoxygenase. 2° While further studies are necessary to pin point the specific role and mechanism of cellular oxidant, the direct interaction of LDL with the cells and continued presence of cells in the incubation medium appear to be necessary. Whatever the initiating mechanism is, once the LDL contains peroxidized lipids, rapid decomposition of the peroxides ensues (particularly in the presence of copper), thus propagating the chain reaction and in that process generating more and more aldehydes. The inevitable result is the covalent attachment of the aldehydes to the available amino groups of apoprotein B-100 fragments TXnLE 8. Methods Used for the Determination of LDL Oxidation

TABLE 7. Evidence for the Potential Involvement of 15-Lipoxygenase in Atherogenesis 1. Lipoxygenase inhibitors inhibit the oxidative modification of LDL l°5'm 2. Treatment of the LDL with lipoxygenases results in the oxidative modification of the lipoprotein t35 3. 15-Lipoxygenase activity and products are increased in atherosclerotic aorta I~ 4, 15-Lipoxygenase enzyme protein and mRNA could be demonstrated in the macrophage-rich areas of WHHL rabbit and human atherosclerotic lesions t~,le°

A. Methods based on lipid peroxidation 1. 0 2 consumption 1:9 2. Spin trapping 63 3. Generation of conjugated dienes 3° 4. Measurement of TBARS u,le 5. Generation of lipid peroxides29,tL,Ts 6. Loss of polyunsaturated fatty acids 29,j°~,t22 7. Loss of antioxidants2%6t 8. Measurement of specific aldehydes29 9. Measurement of cholesterol oxidation products62,t~3 B. Methods that rely on changes in apo B upon oxidation 1. Enhanced electrophoretic mobility 143 2. Changes in the immunoreactivity~2 3. Increased fluorescence7t C. Methods that measure the biological activity of Ox-LDL 1. Increa__~d degradation by macrophages ~,~5 2. Increased 14C.oleate incorporation 1°2 3. Cytotoxicity of Ox-LDL to cells t*

Oxidized LDLs in atherogenesis

137

VI. EVIDENCE FOR THE PRESENCE AND ATHEROGENICITY OF OXIDIZED LDL IN VIVO The long incubation conditions, requirement of special media and metals for the oxidative modification of LDL caused considerable doubts as to the existence, leave alone involvement, of Ox-LDL in the atherogenic process. Besides, LDL as isolated from human plasma contains considerable amounts of antioxidants such as vitamin E, carotenoid, ubiquinone and others. However, there are several lines of evidence that suggest that oxidatively modified LDL exists in vivo. Before attempts are made to isolate or identify Ox-LDL in vivo, it is essential to discuss specific methods of measuring the formation of Ox-LDL in vitro. A. Methods Used tO Determine L D L Oxidation

Although initial methods were restricted to the determination of increases in electrophoretic mobility, cytotoxicity and macrophage uptake, the identification of lipid peroxidation as a key step in the modification process, afforded other convenient methods. These vary from simple TBARS measurement to the elaborate time consuming determination of macrophage lipid accumulation. However, caution should be exercised in the use of any of these methods to quantify LDL oxidation. Individual preparations of LDL may vary in their antioxidant and PUFA content. Other factors such as concentration of LDL during storage, duration of storage, density range of isolated LDL, intrinsic PLA2 activity, method and cell used for oxidation may all affect oxidation in a irreproducible manner. The methods employed to determine the oxidative modification of LDL are summarized in Table 8. It should be emphasized that these methods may measure different aspects of the oxidative modification of LDL. B. Lipid Peroxidation and Atherosclerosis 1. The Presence o f Lipid Peroxides in Atherosclerotic Lesions

The presence of lipid peroxides in tissue and cell lipids has long been assumed as a sign of abnormality. The first evidence of the presence of lipid peroxides in human atherosclerotic aortas was provided by Glavind et al. in 1952.42 They observed that in human lesions with increasing severity of the lesion the amount of peroxide in the lesion increased. Whether the presence of the peroxide had any significance to the development of the lesion could not be determined in these studies. Furthermore, the conditions under which the samples were obtained, their storage and handling techniques could not be ascertained. It is known that unsaturated lipids readily undergo oxidation under certain conditions. The correlation the authors obtained between peroxide values and the severity of the lesion could, therefore, very well have been due simply to the increased amounts of lipids found in the advanced lesions. Since then there have been sporadic reports of the identification and quantification of lipid peroxides in the plasma as well as in the arterial tissue. ~°'5°'91 The analytical methods employed in these studies varied from measurements of TBARS to sophisticated methods using HPLC separation of the lipid peroxides and quantification using chemiluminescence. Such studies have indicated, for example, a marginal increase in TBARS in the plasma and arteries of patients exhibiting atherosclerosis, as compared to the controls. 75 In a study of post mortem samples of diseased human aortas the lipid component was extracted and analyzed by TLC and GLC. Oxidized cholesterol and cholesterol linoleate hydroperoxide were found, levels correlating with the severity of the disease. 1°,5°The predominant oxidized lipid was cholesterol linoleate hydroperoxide. The presence of cholesterol ester hydroperoxides has since been observed in lipid droplets from atherosclerotic lesions of the thoracic aorta of W H H L rabbits. HPLC analysis of the saponified lipid demonstrated that it coeluted with 13 HODE. 9~ Ceroid is a yellowish-brown autofluorescent material, thought to contain lipid peroxides. 25.2v,2sExtensive studies have reported the presence of ceroid in arterial lesions, m

138

s. Pxga'm~ARArr~and S. M. RANmN

Mitchinson and coworkers have demonstrated that the ceroid is located intracellularly, predominantly in macrophages. 4,85It is thought that ceroid may be derived from Ox-LDL as antibodies that recognize ceroid partially cross-react with Ox-LDL. ~°7 These studies support the view that lipid peroxidation occurs during atherosclerosis; they do not, however suggest whether it is a cause or consequence of the disease process. More recently methods have been developed for the isolation of arterial foam cells from atherosclerotic lesions. These ceils have been shown to express scavenger receptors and are capable of oxidizing LDL. ~°'126Under controlled conditions these isolated foam cells may provide a unique opportunity to define the oxidative process. 2. The Presence o f Lipid Peroxides in Plasma

Despite the apparent strength of the plasma antioxidant defence there are reports of detectable levels of TBARS, 19'~'79'1s4 cholesterol ester hydroperoxides~57and phospholipid hydroperoxides3S, s6 in the plasma. It is not known, however, whether the oxidation of these lipids occurs within the plasma or extravascuiar domains. It should be emphasized that to date no correlation has been established between plasma lipid peroxide levels and the extent of atherosclerosis. 3. Antioxidants and Atherosclerosis

Several reports have appeared on the effect of antioxidants on the oxidation of LDL. In vitro studies have shown that LDL oxidation does not occur until all the associated

vitamin E has been depleted and that the addition of micromolar concentrations of vitamin E completely inhibits LDL oxidation.29'61'74'143'151 Due to its lipophilic nature vitamin E is carried in lipoproteins in the plasma s3 and would therefore appear to be a good candidate for the prevention of LDL oxidation in vivo. In vitro studies suggest that the resistance of LDL to oxidation is related to its vitamin E content. 29'61 In studies of vitamin E feeding in experimental animals that were fed a diet enriched in cholesterol both pro- and anti-atherogenic effects were noted as reviewed by Chisolm. 2~ Earlier it was shown that probucol, a hypolipidemic drug in clinical use had potent antioxidant properties and inhibited the in vitro oxidative modification of LDL. 1°6 It was also shown that LDL isolated from patients on conventional probucol therapy contained considerable amounts of probucol and was resistant to oxidation. ~°6Using WHHL rabbits Carew et aL ~5 and Kita et al. ~ demonstrated that probucol inhibited the extent of atherosclerotic lesion formation, far beyond the levels expected from its cholesterollowering effect. In addition, Carew et aL showed that probucol treatment prevented the degradation of the injected labeled LDL specifically in the macrophage-rich areas of the lesion, where there is a prerequisite for some type of modification.~5 Since then both probuco1147 and BHT s have been reported to be effective against atherosclerosis even in cholesterol-fed rabbits, suggesting that perhaps oxidation of some kind may be important in these animal models also where fl-VLDL is the predominant lipoprotein species. However, more studies are obviously needed as some investigators failed to note any improvement in atherosclerosis in cholesterol-fed animals. ~38 4. Isolation o f Oxidized L D L f r o m Biological Tissue Samples

As early as in 1971, human peripheral lymph was found to contain apoprotein B containing lipoproteins in a density range greater than 1.063, while in the plasma of the same subjects apo B was restricted to density of less than 1.063.123 Raymond and Reynolds isolated LDL from interstitial inflammatory fluid of the rabbit that exhibited properties similar to Ox-LDL, including changes in electrophoretic mobility, particle size, hydrated density and chemical composition. Hg,n°,m The isolated LDL contained very low levels of polyunsaturated fatty acids in its phospholipids and showed an enhanced clearance rate as compared to LDL isolated from plasma. Hoff and coworkers also reported the isolation

Oxidized LDLs in atherogenesis

139

of apo B containing lipoproteins from the atherosclerotic aorta that contained measurable amounts of peroxides. 23'59'9°Recently Yl~i-Herttuala et al. using mild extraction techniques isolated LDL fraction from atherosclerotic lesions of WHHL rabbits and showed that the isolated lipoprotein was chemotactic to monocytes and shared several other properties of Ox-LDL. 15s 5. Immunohistochemical Demonstration o f Oxidation Related Lipid-Protein Adducts in Atherosclerotic Aorta

MDA-LDL as well as Ox-LDL are atherogenic. In fact, autoantibodies to Ox-LDL are present in most humans and rabbits. 95 Antibodies generated against MDA-LDL and Ox-LDL recognize MDA and other aldehyde derivatized-lysine epitopes in Ox-LDL as well as in other lysine modified proteins. Using these antibodies Haberland et al. 47 and Palinski et al. 94 looked for the presence of modified LDL in the atherosclerotic lesion of WHHL rabbits. Using immunohistochemical techniques they demonstrated the presence of materials that cross-reacted with these antibodies in the macrophage-rich areas of the lesion. This work has subsequently been confirmed by others) Palinski et al. also used antibodies directed against Ox-LDL and 4-hydroxynonenal LDL and obtained similar results. 95 Thus there are at least four different lines of evidence to suggest the presence of OX-LDL in vivo. (1) Antioxidants prevent the development and progression of atherosclerotic lesion in WHHL rabbits. They also specifically inhibit the degradation of LDL in macrophage foam cells. (2) LDL isolated from atherosclerotic lesions has some of the characteristics of Ox-LDL. (3) Autoantibodies directed towards Ox-LDL are present in humans and rabbit plasma and (4) lysine-aldehyde adducts that may indicate the presence of Ox-LDL can be demonstrated in the macrophage-rich lesions of human and rabbit aorta. C. Can Oxidative Modification o f L D L Occur in the Plasma?

Ox-LDL is very rapidly removed from circulation by the liver. 92 Plasma clearance of Ox-LDL appears to be directly related to the extent of its oxidation. 145 However, a minimally modified(MM)-LDL, as has been described by Berliner et al. 7 that is not modified enough to be recognized by the scavenger receptor, may have a relatively longer half life in the plasma and when it enters the artery wall may suffer the fate of further oxidative degradation. In fact, serum antibodies to Ox-LDL and ceroid extracted from human aortas were detected in the human sera particularly of those with cardiovascular diseases and advancing years. 1°7 The presence of lipid peroxidation products in the LDL and VLDL fractions of plasma has been well documented. The toxicity of diabetic plasma has been attributed to lipid peroxidation products. Morel and Chisolm have demonstrated that VLDL/LDL fractions from streptozotocin induced diabetic rat plasma contained higher levels of peroxides and were toxic to cultured cells.22's7This reinforces the point that the detection of LDL oxidation products is not necessarily associated with its biological modification. The presence of such MM-LDL in the plasma is more credible and perhaps represents the subfractions of LDL that have been reported. Avagaro et al) have described the isolation of a more electronegative subfraction of LDL isolated by ion exchange chromatography. This fraction was degraded avidly by macrophages and showed evidence of apoprotein aggregation. In a more recent study Shimano et al.133 described the isolation of a subfraction of LDL (< 1% of the total LDL) that while exhibiting certain changes in its physico-chemical characteristics its cellular metabolism was not significantly different to that of native LDL. This LDL was, however, significantly more labile to oxidative modification. Whether this electronegative LDL represents oxidative modification occurring in the plasma or the acquisition of lipid peroxidation products from elsewhere in the body remains to be established. Due to the high antioxidant capacity of the plasma a more likely location for extensive oxidative modification of LDL may be in the subendothelial space, between cells in an antioxidant depleted, sequestered microenvironment. On the

140

S. PARTHASARATHYand S. M. RA~K~N

other hand, chronic depletion of plasma antioxidants may generate conditions in the plasma favorable to o x i d a t i o n J ~ VII. C O N C L U S I O N

The available evidence supports the contention that antioxidants may have anti-atherogenic effects. However, before these studies can be extrapolated to human atherosclerosis it should be pointed out that (a) no evidence for a protective effect of antioxidants against human atherosclerosis exists although epidemiological data suggests a negative correlation between plasma vitamin E levels and incidence of cardiovascular diseases~; (b) demonstration of a protective effect in humans is difficult because of the limitations of currently available techniques in identifying ongoing lipid peroxidation and establishing a correlation with the disease process; (c) difficulties in following the course of treatment and potential drawbacks in setting in vitro criteria of protection and (d) defining the right type and dose of antioxidants. Nevertheless, the prospect that antioxidants may provide yet another mechanism by which the progression of atherosclerosis may be slowed should not be ignored. REFERENCES

1. AQEL, N. M., BXLL, R. Y., WALDMAN,H. and MITCI~NSON,M. J. Atherosclerosis 53, 265-271 (1984). 2. A ~ , H., IOTA, T., YOKODE,M., NXPJ~nJIYA, S. and KAw~, C. Biochem. Biophys. Res. Commun. 1.59, 1375-1382 (1989). 3. AvooARo, P., CXZZOLA~, G and BITTOLOnON,G. Atherosclerosis 91, 163-171 (1991). 4. BALL,R. Y., Cx~EI~_~, K. L. H~ and MrremNsoN, M. J. Arch. Pathol. Lab. Med. 111, I134-1140 (1987). 5. BxSU, S. K., BROW~, M. S., HO, Y. K. and G o ~ s ~ , J. L. J. Biol. Chem. ?,54, 7141-7146 (1979). 6. BEDWELL,S., DEAN, R. T. and JEssuP, W. Biochem. J. 262, 707-712 (1989). 7. B ~ a ~ I ~ , J. A., Tr~uTo, M. C., S~'vxmxN, A., Po,mN, S., IOM, J. A., BX~SH~, B., ESTm~SON,M. and FOOELM~, A. M. J. Clin. Invest. 85, 1260-1266 (1990). 8. BJOmCH~M,I., HEmUr~oN-F~scHUSS, A., B R E ~ , O., DICZFALUS'~,U., B~ROLtr~D,L. and HEmur~sso~, P. Arteriosclerosis Thromb. 11, 15-22 (1991). 9. BOYD, H. C., GowN, A. M., WOLFnA~R, (3. and CH~T, A. Am. J. Pathol. 135, 815-825 (1989). I0. BROOKS,C. J. W., STEEL,G., G m ~ T , J. D. and HxPa.~D, W. A. Atherosclerosls 13, 223-237 (1971). I1. BgowN, M. S., Bxsu, S. K., FALCK,J. R., HO, Y. K. and GoLDSTmN,J. L. J. Supramol. Sir. 13, 67-81 (1980). 12. BRowN, M. S. and GoLDSTmN,J. L. Cell 6, 307-316 (1975). 13. BROwN, M. S. and GoLt)S~N, J. L. Annu. Rev. Biochem. 52, 223-261 (1983). 14. BUJA,L. M., IOTA,T., GoLDSTI~N,J. L., WATANABE,Y. and BROWN,M. S. Arteriosclerosis 3, 87-101 (1983). 15. CAREw, T. E., SCHWE~rOB,D. S. and S T E I ~ O , D. Proe. Natl. Acad. Sci. U.S.A. 84, 7725-7729 (1987). 16. CATHCART,M. K., CmsoI~, (3. M., MCNALLY, A. K. and MOREL, D. W. In vitro cell Dev. Biol. 24, 1001-1008 (1988). 17. CATHCART,M. K., MCNALLY,A. K., MOREL, D. W. and CHISOLM,G. M. J. lmmunol. 142, 1963-1969 (1988). 18. CA'mCAgT, M. K., MOREL, D. W. and CmSOLM, G. M. J. Leuk. Biol. 28, 341-350 (1985). 19. ~ U N , S., T/d.I.1NEAU,C., POh'TCHARRAUD,R., GUETTmR,A. and Pmtou, A. Biochim. Biophys. Acta 1042, 324-329 (1990). 20. ~ L r r R A T , W., HUOHF.S,M. F., ELINO,T. E. and MASON,R. P. Arch. Biochem. Biophys. 290, 153-159 (1991). 21. CmSOLMIII, (3. M. Clin. Cardiol. 4, 1-25-30 (1991). 22. CmSOLM,(3. M. and MOREL, D. W. Am. J. Cardiol. 62, 20B-26B (1988). 23. Ct£v~wcE, B. A., MORTON,R. E., WEST,G., DtrsE[, D. M. and Hovr, H. F. Arteriosclerosis 4, 196-207 (1984). 24. Cusx-I~O, S. D., BEgt.IN~, J. A., VAt~N~, A. J., TEREaXO,M. C., N^VAe, M., PARI, F., GF.RRn'Y,R., SCHWARTZ,C. J. and FOOEI.MAN,A. M. Proc. Natl. Acad. Sci. U.S.A. 87, 5134-5138 (1990). 25. DAM, H. and GgXNADOS,H. Science 103, 327-328 (1945). 26. DAVmS,P. F., Rlm3Y, M. A., GOODE, T. B. and BowY~, D. E. Atherosclerosis 25, 125-130 (1976) 27. DUTRXDE OLIV~VA, J. Ann. N.Y. Acad. Sci. $2, 125 (1949/50). 28. ENDICOTT,K. M. Arch. Pathol. 37, 49-53 (1944). 29. ~ u l ~ , H., JOROI~NS,G., Qi~braEgOEg, O. and KOLLER, E. J. Lipid Res. 28, 495-509 (1987). 30. ~ u l ~ , H., STRmOL, G., ~ E[,~luld ROTlt~IV~ER,M. Free Rad. Res. Commun. 6, 67-75 (1989). 31. FAoolo'rro, A., Ro~, R. and H~uUIIt, L/Arteriosclerosis 4, 323-340 (1984). 32. Fool~g~'q, A. M., Sl~ctrr~, I., S~,o~,t, J., HOKOM,M., Cmta3, J. S. and EDWARDS,P. A. Proc. Natl. Acad. $ci. U.S.A. 77, 2214-2218 (1980). 33. FONO, L. G., P A R ~ 4 Y , S., WrrzTu~, J. L. and S ~ e ~ o , D. J. Lipid Res. 28, 1466-1477 (1987). 34. FOWLER,S., Stllo, H. and HAtgY, N. J. Lab. Invest. 41, 372-378 (1979). 35. Fox, P. L., CI-nSOLM,G. M. and D~COIo.Lrm, P. E. J. Biol. Chem. 262, 6046-6054 (1987).

Oxidized LDI~ in athero[~n~is

141

36. FgAN]¢, J. S. and FoG~.*aq, A. M. J. Lipid Res. 30, 967-978 (1989). 37. ~ N , M., A,m~NAS, L, PJ~, D. J. G., K~GSLE¥, D. M., CO~LAh'D, N. G., JEh'rJNS, N. A. and KafFir.g, M. Proc. Natl. Acad. Sci. U.S.A. 87, 8810-8814 (1990). 38. Fnm, B., Y~J~oTO, Y., NICLAS, D. and l o ~ s , B. N. Ann. Biochem. 103, 744-746 (1988). 39. Glw,mTY, R. G. Am. J. Pathol. 103, 181-190 (1981). 40. GEggI~, R. G., N ~ r o , H. K., R]CH~C)SON, M. and SCh'WAgTZ, C. J. Am. J. PathoL 95, 775-792 (1979). 41. Gmr, K. F. Biblithaca nutr. Dieta. 37, 53-91 (1986). 42. GLAWND,J., HARTS,N, S., CLE)~qSEN, J., J ~ , K. E. and DAMT,H. Acta Pathol. Microbioi. Scand. 30, 1-6 (1952).

43. GOLDSTEIN,J. L. and BROWN, M. S. Annu. Rev. Biochem. 47, 897-930 (1977). 44. GOLDS~r.IN,J. L. and BROWN, M. S. J. Biol. Chem. 249, 5153-5162 (1974). 45. GOLDSTEIN,J. L., HO, Y. K., BASU, S. K. and BROWN, M. S. Proc. Natl. Acad. Sci. U.S.A. 76, 333-337 (1979). 46. GONEN, B., COLE, T. and H~n~, K. S. Biochim. Biophys. Acta 754, 201-207 (1983). 47. HABERLAND,M. E., FONO, D. and OmNO, L. Science 241, 215-218 (1988). 48. HAm~RI~NI),M. E., OLC8, C. L. and FOGEL~N, A. M. J. Biol. Chem. 259, 11305-11311 (1984). 49. HAMPTON,R. Y., GOt~qBOCK, D. T., I ~ N , M., KRIEOER,M. and RAL~Z, C. R. Nature 303, 342-344 (1991). 50. HARLAND,W. A., GILBEgT, J. D., ST~L, G. and BROOKS,C. J. W. Atherosclerosis 13, 239-246 (1971). 51. HFJNECKE,J. W., ROSES, H. and ~ T , A. J. Clin. Invest. 74, 1890-1894 (1984). 52. H ~ C K E , J. W., ROSEN, H., SuzuKI, L. A. and Ct~rr, A. J. Biol. Chem. 262, 10098-10103 (1987). 53. HENgIKSEN,T. EWN~N, S. A. and CARLANDER,B. Scand. J. Clin. Lab. Invest. 39, 361-368 (1979). 54. HE~JKSEN, T., MAI-IO~'¢, E. M. and S~INBERO, D. Proc. Natl. Acad. Sci. U.S.A. 78, 6499-6503 (1981). 55. I-I~NIUKSEN,T., M~O~EY, E. M. and S ~ , m ~ o , D. Arteriosclerosis 3, 149-159 (1983). 56. HmSI.~, J. R., ROe~RTSON,A. L. Js. and CmSOLM, G. M. Atherosclerosis 32, 213-229 (1979). 57. HIRAMATSU,K., ROSEN, H., HEINECKE,J. W., WOLFBAUER,G. and CHAIT,A. Arteriosclerosis 7, 55-60 (1987). 58. HOFF, H. F. and O'NEIL, J. Arteriosclerosis Thromb. Vll, 1209-1222 (1991). 59. How, H. F., O'NEIL, J. and COLE, T. B. Exp. Molec. Pathol. 54, 72-86 (1991). 60. JAAKKOLA,O., YL~,-HERTTUALA,S., SgRKIOJA, T. and NIKKARI,T. Atheroselerosis 79, 173-182 (1989). 61. JessuP, W., R~IoZq, S. M., De WHALLEY,C. V., HOULT, J. R., SCOTT,J. and L~AK~, D. S. Biochem. J. 265, 399-405 (1990). 62. JIALAL,I., FICSEMAN,D. A. and GRUNDY, S. M. Arteriosclerosis and Thromb. 11, 482-488 (1991). 63. KALYANARAM~,B., AWrHOUNE,W. E. and PAgTHAS~THY, S. Biochim. Biophys. Acta 1035, 286-292 (1990). 64. ~ L L , W. B., CAS~LLI, W. P., GORDON,T. and McNAM~'L~,,P. M. Ann. Intern. Med. 74, 1-12 (1971). 65. I ~ o o , J. C., M~LLER,E., McLouom.~s, P. and S~INnF.RO, D. Arteriosclerosis 8, 348-358 (1988). 66. Krr~, T., NAO~O, Y., YOKODS,M., Ism~, K., Ku-s~, N., OosI-I~, A., YOSmOA,H. and I~wA~, C. Proc. Natl. Acad. Sci. U.S.A. 84, 5928-5931 (1987). 67. KLURFEtO,D. M. Arch. Pathol. Lab. Med. 109, 445-449 (1985). 68. K~aowr, J. A., S m ~ , S. E., IO~,~ER, V. E. and A~STALL,H. B. Clin. Chem. 33, 2289-2291 (1987). 69. KODAMA,T., FREEMAN,M., ROHRER,L., ZABRECKY,J., MATSUDAIRA,P. and ICduZG~,M. Nature (Lond.) 343, 531-535 (1990). 70. KODXMA,T., R~DY, P., K~nMOTO, C. and K~moER, M. Proc. Natl. Acad. Sci. U.S.A. 85, 9238-9242 (1988). 71. KOLI~R, E., ~ E R G E ~ , O., JURGENS, G., WOLFBEIS,O. S. and F ~ a a ^ u ~ , H. FEBS Lett. 198, 229-234 (1986). 72. K~UG~, K., MO~L, D. W., D~COR~TO, P. E. and Cm~LM, G. M. J. Cell. Physiol. 130, 311-320 (1987). 73. KUO~YAMA,K., KS~NS,S. A., MoRms~r, J. D., ROe~RTS,R. and HENRY,P. D. Nature 344, 160-162 (1990). 74. Lr~r~, D. S. and R ~ N , S. M. Biochem. J. 270, 741-748 (1990). 75. L ~ w o z ~ v , A., MlCHA~, J., S~pm~, A. and K~Z~OL~, A. Clinica Chemica Acta 155, 275-283 (1986). 76. LENZ, M. L., H u o ~ s , H., MXTC~LL,J. R., V~, D. P., GuYroN, J. R., TAYLOR,A. A., ~ JR, A. M. and SMITH, C. V. J. Lipid Res. 31, 1043-1050 (1990). 77. LOP~-Vm~LL^, M. F., KLmN, R. L., LYONS,T. J., S~WNSON, H. C. and WffZTUM, J. L. Diabetes 37, 550-557 (1988). 78. LOUGI4~n, M., ZHANO, H. and ST~qeP.zctmg, U. P. ,I. Biol. Chem. 266, 14519-14525 (1991). 79. L u o ~ , P. V., S~n~o~a~, J., KORPEI~, H., Rxtrno, A., SOTA~rm~a,E. A., Suv~rro, E. and MAaN~a, J. J. Intern. Med. 227, 287-289 (1990). 80. LYOSS, T. J., KLIL~, R. L., BAY~es, J. W., S~wsso~, H. C. and LOpes-Vt~J~Lt~,,M. F. Diabetologia 30, 916-923 (1987). 81. MAm.~Y, R. W., I ~ T ¢ , T. L., W r a s s e , K. H. and OH, S. Y. J. Clin. Invest. 64, 743-750 (1979). 82. MXTSUMOTO,A., N~ro, M., I T ~ K ~ , H., Ird~oTo, S., AS~OK~, H., I-D,Y~o,w^, I., IC~N~d~O~, H., ABURATANI,H., TAKAKU,F., SUZUKI,H., Koa~a, Y., Mn~d, T., T ~ g _ , a t ~ , K., CottoN, E. H., WYDgO, R., HOUSMAN,D. E. and KODAMA,T. Proc. Natl. Acad. Sci. U.S.A. 87, 9133-9137 (1990). 83. McCo~ncK, E. C., C o m ~ v ~ , D. G. and BROWN, J. B. J. Lipid Res. 1, 221-228 (1960). 84. McNALLY,A. K., C3mOLM,G. M., MOREL,D. W. and CA~C~T, M. K. J. lmmanol. 145, 254-259 (1990). 85. MITCmNSON,M. J., HOTHEKS~L,D. C., BROOKS,P. N. and De B ~ , C. Y. J. Pathol. 145, 177-183 (1985). 86. MffAZ~WA, T., Y~SU~A, K., FuJmc¢o, K. and KA~D~, T. Y. Biochem. 103, 744-746 (1988). 87. MO~L, D. W. and Cmsot~, G. M. J. Lipid Res. 30, 1827-1834 (1989). 88. MO~L, D. W., DlCO~a~ro, P. E. and CI~OLM, G. M. Arteriosclerosis 4, 357-364 (1984). 89. MOI~L, D. W., HeUlaSR, J. R. and C I m O ~ , G. M. J. Lipid Res. 24, 1070-1076 (1983). 90. MORTON, R. E., WEST, G. A. and Ho~, H. F. J. L/pid Res, 27, 1124-1134 (1986). 91. Mow~, H., C m ~ , K., OHKUMA,S. and T ~ o , T. Biochem. Int. 12, 347-352 (1986).

142

S. P A R ~ T H Y

and S. M. RANgas

92. NAGELKERKE,J. F., H^WKES, L., VANHINSBERGH,V. W. arid VANBERKEL,T. J. Arteriosclerosis 4, 256--264 (1984). 93. NEVV~, H. A. I. and ZaLW.SSmT,D. B. J. Biol. Chem. 237, 2078-2084 (1962). 94. PALINSKI,W., RO~I/NFELD,M. E., YLX-HERTrUALA,S., G u g ~ g , G. C., Sooting, S. S., Btrrt~g, S. W., PAR~3~d~, S., CAREW,T. E., STEINSERG,D. and WITZTU~,J. L. Proc. Natl. Acad. Sci. U.S.A. 86, 1372-1376 (1989). 95. PALINSKI,W., YLX-HERTTUALA,S., ROSENFELD,M. E., BUTLER,S. W., S O C k , S. A., P A g ~ T m ' , S., CUgTISS, L. K. and WITZ'rUM,J. L. Arteriosclerosis 10, 325-335 (1990). 96. PARTHASARATHY,S. Biochim. Biophys. Acta 917, 337-340 (1987). 97. PARTHASARATHY,S. and BAR~mTT,J. Proc. Natl. Acad. Sci. U.S.A. 87, 9741-9745 (1990). 98. P A R ~ T H Y , S., FONG, L. G., OTERO,D. and ST~NnERG,D. Proc. Natl. Acad. Sci. U.S.A. 84, 537-540 (1989). 99. PARTHASARATHY,S., FONG, L. G., QUINN, M. T. and STEINBERG,D. Eur. Heart J. 11, (Suppl. E) 83-87 (1990). 100. PAR~tASARATHY,S., FOUG, L. G. and S~JNnERG, D. In: Lipid Peroxidation in Biological Systems, pp. 225-235 (SEVA~N, A., ed.) (1988). 101. PARTHASARATh'Y,S., KHOO,J. C., MILLER,E., BARh~Yr,J., WITZTUM,J. L. and S ~ o , D. Proc. Natl. Acad. Sci. U.S.A. 87, 3894-3898 (1990). 102. PAR~SARATh~, S., ~ Z , D. J., BOYD,D., JOY, L. and STEINBERG,D. Arteriosclerosis 6, 505-510 (1986). 103. PARTHASARATHY,S., Qun~q, M. T., SCHWENKE,D. C., CAREW, T. E. and STEINBERG,D. Arteriosclerosis 9, 398-404 (1989). 104. PAR~dASARATh~,S., ST~NeP,ECHER,U. P., BARNETT,J., WITZTUM,J. L. and STF~qSERQ,D. Proc. Natl. Acad. Sci. U.S.A. 82, 3000-3004 (1985). 105. PAgTHXSARA~h'Y,S., WmL^~, E. and STEINnERG,D. Proc. Natl. Acad. Sci. U.S.A. 86, 1046-1050 (1989). 106. PAR~.~SAgA~Pi, S., YOLr~G, S. G., WITZTU~,J. L., PITDaAN, R. C. and S~JNeERo, D. J. Clin. Invest. 77, 641-644 (1986). 107. PARUm, D. V., BROWN, D. L. and MITCmNSON,M. J. Arch. Pathol. Lab. Med. 114, 383-387 (1990). 108. PIT^S, R. E. £ Biol. Chem. 265, 12722-12727 (1990). 109. PIT^S, R. E., BOYLES,J., MAHLEY, R. W. and B1SSELL,D. M. J. Cell Biol. 100, 103-117 (1985). 110. PITTMAN, R. C., ATTm, A. D., CAREW, T. E. and STEII~ERG, D. Proc. Natl. Acad. Sci. U.S.A. 76, 5345-5349 (1979). 111. PITTMAN,R. C., CAR~W,T. E., AT'tin, A. D., W1TZTUM,J. L., WATANABE,Y. and ST~NBERG,D. J. Biol. Chem. 257, 7994--8000 (1982). 112. POLACEg,D., BYPd~, R. E. and SCANU,A. M. J. Lipid Res 29, 797-808 (1988). 113. QU1NN, M. T., KONDRATENKO,N. and PARTHASARATHY,S. Biochim. Biophys. Acta 1082, 293-302 (1991). 114, QUINN, M. T., PARTHASARATHY,S., FONG, L. G. and STEINBERG,D. Proc. Natl. Acad. Sci. U.S.A. 84, 2995-2998 (1987). 115. QU1NN,M. T., PARTHASARATBY,S. and STEINnERG,D. Proc. Natl. Acad. Sci. U.S.A. 82, 5949-5953 (1985). 116. QUINN,M. T., PARTIO,SARATW/,S. and STEINnERG,D. Proc. Natl. Acad. Sci. U.S.A. 85, 2805-2809 (1988). 117. RAJ^VASmSHT,T. B., ANDALml,A., TERRITO,M. C., BERLINER,J. A., NAVAa, M. and LUSIS,A. J. Nature 344, 254-257 (1990). 118. RAlcgn% S. M., PARTRASAgATHY,S. and STEINBERG,D. J. Lipid Res. 32, 449-456 (1991). 119. RAYMOND,T. L. and REYNOLDS,S. A. J. Lipid Res. 24, 113-119 (1983). 120. RAYMOND,T. L. and REYNOLDS,S. A. Inflammation 8, 337-342 (1984). 121. RAYMOND,T. L., RE~OLDS, S. A. and SW^NSON, J. A. Inflammation 10, 93-98 (1986). 122. R£AWN, P., PARTHASAgATHY,S., GRASSE,B. J., MILLER, E., ALMAZ^N,F., M^TTSON,F. H., KJaoo, J. C., STWNBERG,S. and WITZTUM,J. L. Am. J. Clin. Nutr. 54, 701-706 (1991). 123. RF.ICHL,D., MYANT, N. B. and PFLUG, J. J. Biochim. Biophys. Acta 489, 98-105 (1977). 124. ROI-IR~, L., F ~ N , M., KODAMA,T., PENMAN,M. and KRmGER,M. Nature (Lond.) 343, 570-572 (1990). 125. ROS~FELD, M. E., I~oo, J. C., MILLER, E., P^RTHASARATHY,S., P^LINSKL W. and W1TZTUM,J. L. J. Clin. Invest. 87, 90-99 (1991). 126. ROSENF~LD,M. E., P^L~NSKI,W., YL/GHERTTU^L^,S., BUTLER,S. W. and W~TZTUM,J. L. Arteriosclerosis 10, 336-349 (1990). 127. RO~NFELD, M. E., TSUKADA,T., GOWN, A. M. and ROSS, R. Ateriosclerosis 7, 9-23 (1987). 128. Ross, R. and GLOMS~T, J. A. N. Engl. J. Med. 295, 369-377, 420-425 (1976). 129. S^TO, K., NmL E. and SmMmKL H. Arch. Biochem. Biophys. 279, 402-405 (1990). 130. ~ , T., TAYLOR,K., BARTU¢CI,E. J., FISCHER-DZOGER,K., BEESON,J. H., GLAGOV,S. and WISSLER, R. W. Am. J. Pathol. 100, 57-80 (1980). 131. Schta,~R, E., HUBER, L., FRtmms, J., SCHULZ, I., Zm~I.ER, R. and Dg~SEL, H. A. Atherosclerosis 82, 261-265 (1990). 132. SCHORN^GEL,H. E. J. Pathol. Bacteriol. 72, 267-272 (1956). 133. SmMAso, H., YAMADA,N., IsmBm~n, S., MoKuso, H., Morn, N., GOTOD^, T., H ~ ^ , K., AgA~Lr~A, Y., MURAS~,T., Y^ZAKI, Y. and TAKAKU,F. J. Lipid Res. 32, 763-773 (1991). 134. S~MON,T. C., MAKIIV.J^,A. N. and BAILEY,J. M. Atherosclerosis 75, 31-38 (1989). 135. SPAggOW, C. P., P , ~ g ~ ' ~ ' , S. and S'r~NB~ItG, D. J. Lipid Res. 29, 745-753 (1988). 136. S p ~ o w , C. P., P~t~a~,gA~h'~, S. and STEINnER~, D. J. Biol. Chem. 7,64, 2599-2604 (1989). 137. S3"~q, O. and STr~, Y. Biochim. Biophys. Acta 620, 631-635 (1980). 138. STEIN,O., STEn%Y., DELPLANQUE,B., FESMIRE,J. D., LEE, D. M. and ALAUPOVIC,P. Atherosclerosis 75, 145-155 (1989). 139. STVa~mERG,D., PAR~L*,SARATh'Y,S., CAR~W,T. E., 104oo, J. C. and WITZTUM,J. L. N. Engl. J. Med. 320, 915-924 (1989). 140. STEn~p.£cl-mit,U. P. J. Biol. Chem. 262, 3603-3608 (1987). 141. Srr~l,mR~CI-lER,U. P. Biochim. Biophys. Acta. 959, 20-30 (1988).

Oxidized LDLs in atherogenesis

143

142. STSI~V.CH~, U. P., LOUGI-IE~,M., KW^N, W.-C. and DIn.KS,M. J. BioL Chem. 264, 15216-15223 (1989). 143. STeINaRECH~, U. P., PARTHASARATHY,S., LEAF.E,D. S., WITZTUM,J. L. and ST~NnERG,D. Proc. Natl. Acad. Sci. U.S.A. 81, 3883-3887 (1984). 144. STmNn£CHER, U. P. and PmTCHARD,P. H. J. Lipid Res. 30, 305-315 (1989). 145. STIm~P.ECH~, U. P., WITZTUM,J. L., PAgTHASARATHY,S. and ST~NnERG,D. Arteriosclerosis 7, 135-143 (1987). 146. SuiTs, A., CHArt, A., AVmAM,M. and HFJNECKE,J. W. Proc. Natl. Acad. S¢i. U.S.A. 86, 2713-2717 (1989). 147. T^WARA, K., ISmHARA,M., OGAWA,H. and TOMIKAWA,M. Japan. J. Pharmacol. 41, 211-222 (1986). 148. T~TOV, V. V., SOSF.NIN,I. A., TONEVrrsKY,A. G., OREKHOV,A. N. and S~nRNov,V. N. Biochem. Biophys. Res. Commun. 167, 1122-1127 (1990). 149. TRIAU,J. E., MEYDANI,S. N. and SCHAEFER,E. J. Arteriosclerosis 8, 810-818 (1988). 150. TYROLER,H. A. Hypercholesterolemia and Atherosclerosis, pp. 99-116, Churchill Press, New York (1987). 151. VAN HINSBI~RGH,V. W. M., SCHEFFE,M., HAVEKES,L. and KEMPER,H. J. M. Biochim. Biophys. Aeta 878, 49-64 (1986). 152. VEDELER,C. A., NYLAND, H. and MATRE, R. Acta. Pathol. Mierobiol. Immunol. Scand. C. 92, 133-137 (1984). 153. VIA, D. P., DRESEL,H. A., CI~NG, S-L. and G o . o , A. M. J. Biol. Chem. 260, 7379-7386 (1985). 154. W/d3~, C. R. and RxJ, A. M. Life Sei. 43, 1085-1093 (1988). 155. WATANABE, T., HIRATA, M., YOSHIKAWA,Y., NAGAFUCHI,Y., TOYOSmMA, H. and WATANABE, T. Lab. Invest. 53, 80-90 (1985). 156. WW.LAND,E., PARTHASARATHY,S. and STFJNnERG,D. Proc. VIIPh Int. Atheroselerosis 1025, (1988). 157. YAMAMOTO,Y. and NIKI, E. Biochem. Biophys. Res. Comman. 165, 988-993 (1989). 158. YL)[-HERTTUALA,S., PALINSKI,W., ROSENFELD,M. E., PARTHASAgATHY,S., CAREW, T., BUTLER,S. W., WITZTUM,J. L. and STEINBERG,D. J. Clin. Invest. 84, 1086-1095 (1989). 159. YLg-HERTTUALA,S., ROSENTELD,M. E., PARTHASARATHY,S., GLASS,C. K., SIGAL,E., WITZTUM,J. L. and ST~NeERG, D. Proc. Natl. Acad. Sci. U.S.A. 87, 6959-6963 (1990). 160. YLg-HERTTUALA,S., ROSENFELD,M. E., PARTHASARATHY,S., SARKIOJA,T., WITZTUM,J. L. and ST~ItceERO, D. J. Clin. Invest. 87, 11146-11152 (1991). 161. YOKOYAMA,M., HmATA,K.-I., MIYAKE,R., AKITA,H., ISHIKAWA,Y. and FUKUZAKI,H. Biochem. Biophys. Res. Comman. 168, 301-308 (1990). 162. ZAWADXrd,Z., MILNE, R. W. and MARCEL,Y. U J. Lipid Res. 30, 885-891 (1989). 163. ZHAN~, H., BASRA,H. J. K. and STEINBR~CHER,U. P. J. Lipid Res. 31, 1361-1369 (1990).