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Oxidized low density lipoprotein causes impaired efflux of cholesterol from macrophages : implications… Dhaliwal, Baljinder Singh 1999

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OXIDIZED LOW DENSITY LIPOPROTEIN CAUSES IMPAIRED EFFLUX OF CHOLESTEROL FROM MACROPHAGES: IMPLICATIONS FOR ATHEROGENESIS by Baljinder Singh Dhaliwal B.Sc, Simon Fraser University, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medicine) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1999 © Baljinder Singh Dhaliwal, 1999 In presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of this thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Medicine The University of B r i t i s h Columbia Vancouver, Canada ABSTRACT Oxidation of low density lipoproteins (LDL) has been implicated as a causal factor in the pathogenesis of atherosclerosis. Oxidized LDL has been found to exhibit numerous potentially atherogenic properties, including receptor-mediated uptake by macrophages. The present study confirms that oxidized LDL is resistant to intracellular degradation and accumulates in a lysosomal compartment in macrophages. Continued accumulation of lipid by macrophages in the arterial intima results in transformation of these cells into lipid-laden "foam cells", which are one of the earliest signs of atherosclerosis. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. We investigated the relative rates of HDL-mediated cholesterol efflux in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. It was found that acetylated LDL-loaded cells released approximately one-third of their total cholesterol to HDL-containing medium, whereas oxidized LDL-loaded cells released only one-tenth of their total cholesterol. To determine whether the impairment of cholesterol efflux was due to modification of the apolipoprotein B or the lipid component of LDL, cells were loaded with either acetylated LDL that had been oxidized for 5 hours or native LDL that had been modified with oxidized arachidonic acid. It was found that both the oxidized lipid component and the modified apolipoprotein B contributed to impaired efflux of cholesterol. To ascertain the proportion of cholesterol efflux mediated by the apolipoprotein component compared to the lipid component of HDL, we measured the rate of efflux when the cells were treated with trypsinized HDL. Very little efflux occurred when the ii macrophages were treated with trypsinized HDL, underscoring the importance of intact apolipoprotein in HDL-mediated cholesterol efflux. We also investigated the subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells. Our findings suggest that in contrast to acetylated LDL-derived cholesterol, oxidized LDL-derived cholesterol accumulates within lysosomes. The observed impairment of cholesterol efflux in oxidized LDL-loaded macrophages may influence the generation of foam cells in vivo, and should, therefore, be considered one of the more important atherogenic effects of oxidized LDL. iii TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations ix Acknowledgements xii Dedication xii Chapter One Introduction 1.1 Plasma Lipoproteins and Cholesterol Homeostasis 1 1.2 HDL-mediated Cholesterol Efflux 8 1.3 The Pathogenesis of Atherosclerosis 13 1.4 Mechanisms of Oxidation and Structural Changes Associated with LDL 18 1.5 The Biological Effects of Oxidized LDL 20 1.6 Rationale 25 1.7 Objectives and Hypotheses 27 Chapter Two Materials and Methods 2.1 Chemicals and Reagents 29 2.2 LDL Preparations 30 2.2.1 LDL isolation 30 2.2.2 LDL labelling 31 2.2.3 LDL modifications 33 2.2.4 Characterization of LDL 34 2.3 HDL Preparations 35 2.3.1 HDL3 isolation 35 2.3.2 HDL labelling 36 2.3.3 Trypsinization of HDL3 37 iv 2.3.4 Apolipoprotein A-I isolation 37 2.3.5 Characterization of HDL and apo A-I 38 2.4 Cell Culture 39 2.5 Microscopy 40 2.6 Experimental Assays 41 2.6.1 Protein determination 41 2.6.2 Colorimetric cholesterol assay 41 2.6.3 Fluorometric cholesterol assay 42 2.6.4 Phosphorus assay 42 2.6.5 Degradation assay 43 2.6.6 ER and lysosome enzyme assays 43 2.7 Subcellular Fractionation 44 2.8 Thin Layer Chromatography 45 2.9 Statistical Analysis 46 Chapter Three Results and Discussion 3.1 Cholesterol content of modified LDL 48 3.2 Oxidized LDL causes impaired efflux of cholesterol from macrophages 51 3.3 Impairment of cholesterol efflux involves both lipid & protein modification of LDL67 3.4 Cholesterol delivered by oxidized LDL accumulates in lysosomes 79 3.5 HDL-mediated efflux of cholesterol 94 Chapter Four Conclusions 108 References 110 v List of Tables Table 1 Human plasma lipoproteins classified by density and composition 3 Table 2 Major human plasma apolipoproteins 4 Table 3 Classification of scavenger receptors 17 Table 4 Biologic actions associated with oxidized LDL 23 Table 5 Relative compositon and electrophoretic mobility of modified LDL 49 Table 6 Sterol efflux into the medium from oxidized LDL-loaded macrophages 74 vi List of Figures Figure 1 Structure of plasma low density lipoprotein 2 Figure 2 Pathways for lipoprotein metabolism in humans 5 Figure 3 Sequential steps in the LDL receptor pathway of mammalian cells 7 Figure 4 Overview of cellular cholesterol efflux 10 Figure 5 The LCAT reaction between free cholesterol and lecithin 11 Figure 6 Oxidized LDL and atherosclerosis 24 Figure 7 Agarose gel electrophoresis of labelled and unlabelled modified LDL's 51 Figure 8 Oxidized LDL concentration is proportional to lipid accumulation 52 Figure 9 Effect of incubation time on lipid accumulation and efflux 55 Figure 10 Concentration-dependence of HDL-mediated cholesterol efflux 58 Figure 11 Effect of HDL incubation time on cholesterol efflux 59 Figure 12 Comparison of cholesterol efflux in normal and Brefeldin A-treated cells 62 Figure 13 Total cholesterol content of cells after lipid-loading and exposure to HDL3...64 Figure 14 Decrease in total cholesterol after HDL3-mediated cholesterol efflux 66 Figure 15 Accumulation and efflux of aggregated LDL-derived cholesterol 68 Figure 16 125I-labelled modified LDL content of loaded and effluxed cells 70 Figure 17 Degradation of 125I-labelled modified LDL by macrophages 71 Figure 18 Accumulation & efflux of cholesterol from various modified LDL's 72 Figure 19 Decrease in cholesterol in cells loaded with NaBH4-treated & A-OP LDL. 78 Figure 20 Distribution of ER and lysosome enzyme markers in control cells 80 Figure 21 Distribution in oxidized LDL-loaded cells 81 Figure 22 Distribution in oxidized LDL-loaded cells after HDL-mediated efflux 82 Figure 23 Distribution in acetylated LDL-loaded cells 84 vii Figure 24 Distribution in acetylated LDL-loaded cells after HDL-mediated efflux 85 Figure 25 Macrophages loaded with NBD-G6-sphingomyelin-labelled oxidized LDL 89 Figure 26 Macrophages treated with fluorescent oxidized LDL followed by HDL3.... 91 Figure 27 Macrophages loaded with NBD-C6-sphingomyelin-labelled acetylated LDL 94 Figure 28 Decrease in total cholesterol after treatment with apo A-I or HDL3.... 96 Figure 29 SDS-PAGE of native HDL3 and trypsinized HDL3 97 Figure 30 Decrease in total cholesterol after treatment with trypsinized HDL3 99 Figure 31 Control macrophages treated with Texas Red HDL 100 Figure 32 OxLDL-loaded macrophages treated with Texas Red HDL 102 Figure 33 HDL retention and degradation by macrophages 105 viii List of Abbreviations ACAT acyl CoA-cholesterol acyltransferase acLDL acetylated low density lipoprotein aggreg. LDL vortex-aggregated LDL A-OP LDL arachidonic acid-oxidation product low density lipoprotein apo A-I apolipoprotein A-I apo A-II apolipoprotein A-II apo B apolipoprotein B BSA bovine serum albumin CE cholesterol ester CETP cholesteryl ester transfer protein Ci Curies d density Da daltons DMEM Dulbecco' s modified Eagle' s medium DMSO dimethyl sulfoxide dpm's disintegrations per minute £ epsilon EDTA ethylenediaminetetra-acetic acid ER endoplasmic reticulum FBS fetal bovine serum FC free (unesterified) cholesterol ix HDL high density lipoprotein IDL intermediate density lipoprotein kDa kilodaltons kPa kilopascals L litre LCAT lecithin:cholesterol acyltransferase LDL low density lipoprotein lecithin phosphatidylcholine LPDS lipoprotein-deficient serum (isolated from human plasma) LPS lipopolysaccharide lyso-PC lysophosphatidylcholine mCi milliCuries uCi microCuries pi microlitres mmol millimoles M molar MARCO macrophage receptor with collagenous structure NaBH4 sodium borohydride NBD- 6-((N-(7-nitrobenz-2-oxa-l, 3-diazol-4-yl)amino)hexanoyl)-nm nanometers oxLDL oxidized low density lipoprotein ox-acLDL acetylated LDL that had subsequently been oxidized PAF platelet activating factor x PBS phosphate-buffered saline PC phosphatidylcholine PDGF platelet-derived growth factor Rf relative electrophoretic mobility S.D. standard deviation SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electroph S.E. standard error of the mean SR-ALTI scavenger receptor class A type I/II SR-BI scavenger receptor class B type I SR-CI scavenger receptor class C type I TC total cholesterol VLDL very low density lipoprotein vol/vol volume per volume wt/vol weight per volume xi Acknowledgements I would like to thank my supervisor, Dr. Urs Steinbrecher, for giving me the opportunity to pursue lipoprotein research in his lab. His continued guidance and encouragement have been a constant source of support throughout this endeavour. I also wish to express my gratitude to Marilee Lougheed and Rajinder Hundal for their support and advice in the lab. Finally, I would like to acknowledge my fiancee, Shannon, for her assistance and understanding throughout my graduate studies. Dedication I dedicate this dissertation to my father, Jarnail Singh Dhaliwal, in appreciation of his support and wisdom throughout my life. xii Chapter 1 Introduction 1.1 Plasma Lipoproteins and Cholesterol Homeostasis Cholesterol is one of the essential lipid molecules for animal cells. In the membrane of animal cells, it modulates fluidity and maintains the barrier between cell and environment (1). Excessive levels of unesterified cholesterol in membranes have been shown to result in membrane dysfunction and toxicity (2). Cholesterol is also a precursor for the biosynthesis of various steroid hormones (3). Because unesterified cholesterol is insoluble in water, it is transported, in multicellular organisms, by esterifying the sterol with long-chain fatty acids and packaging these esters within the hydrophobic cores of plasma lipoproteins. With its polar hydroxyl group esterified, cholesterol remains sequestered within this core, which is essentially an oil droplet composed of cholesteryl esters and triglycerides, solubilized by a surface monolayer of phospholipid and unesterified cholesterol and stabilized by protein (Figure 1). The major classes of lipoproteins are: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Table 1 (4) classifies these proteins based on their flotation density (g/ml) and composition (% mass). 1 LDL Cholesterol • Phospholipid • Apolipoprotein B-100 • Free Cholesterol • Cholesteryl Ester • Triacylglycerol C H 3 - ( C H 2 ) n - d - 0 Cholesteryl esters o Figure 1 Structure of plasma low density lipoprotein Low density lipoprotein is a spherical particle with a mass of 3 X 106 Daltons and a diameter of 22 nm. Each LDL particle contains about 1500 molecules of cholesteryl ester in an oily core that is shielded from the aqueous plasma by an amphipathic coat composed of 800 molecules of phospholipid, 500 molecules unesterified cholesterol, and one molecule of a 400,000 Dalton protein called apolipoprotein B-100. In addition, approximately 3.5% of the total mass of LDL is composed of triacylglycerols (1). 2 Table 1 Human plasma lipoproteins classified by density and composition Density range Lipoprotein fraction Protein FC CE PL TG (g/ml) (%) (%) (%) (%) (%) 0.920<d<0.960 Chylomicron remnants 1.5 2.0 3.0 5.5 87.5 0.950<d< 1.006 Very low density lipoprotein 10.4 5.8 13.9 15.2 53.4 1.006<d<1.019 Intermediate density lipoprotein 17.8 6.5 22.5 21.7 31.4 1.019<d< 1.063 Low density lipoprotein 25.0 8.6 41.9 20.9 3.5 1.063<d<1.120 High density lipoprotein 2 42.6 5.2 20.3 30.1 2.2 1.120<d<1.210 High density lipoprotein 3 54.9 2.6 16.1 25.0 1.4 1.210<d<1.250 Very high density lipoprotein 62.4 0.3 3.2 28.0 4.6 Free cholesterol is abbreviated FC; cholesteryl ester is abbreviated CE; phospholipid is abbreviated PL; and triacylglycerol is abbreviated TG. The composition of each lipoprotein class is expressed as a percent of total mass. VLDL is also referred to as pre-[3-lipoprotein, LDL as p-lipoprotein and HDL as oc-lipoprotein, using an older system of nomenclature based on electrophoretic migration rate in agarose (4). Chylomicrons and VLDL are specialized for transport of triacylglycerols while the other lipoproteins carry cholesterol and cholesterol esters as their predominant neutral lipids. Chylomicrons carry absorbed dietary fat from the small intestine to other organs, whereas VLDL mainly carries triacylglycerol from the liver to other organs. VLDL is eventually converted to IDL and then to LDL, after the triacylglycerol is removed. IDL particles are at an important branchpoint in lipoprotein metabolism because they may be cleared from the circulation by the liver or they may be further processed to become LDL, which is the major transporter of cholesterol to human organs. HDL, on the other hand, is involved in 3 cholesterol efflux from tissue sites, as well as the transport of cholesterol from peripheral tissue to the liver for ultimate catabolism and excretion. HDL's role in "reverse cholesterol transport" is discussed in section 1.2. Since many of the biochemical properties of lipoproteins are a function of their apolipoprotein composition, a list of the apolipoproteins is provided in Table 2 (4). Table 2 Major human plasma apolipoprotein Apolipoprotein Protein molecular weight (kDa) Lipoprotein distribution Apo A-I 28.1 HDL, Chylomicrons Apo A-II 18.6 HDL Apo A-IV 43.3 HDL, Chylomicrons Apo B-48 240.8 Chylomicrons Apo B-100 512.7 VLDL, LDL Apo C-I 6.6 VLDL, HDL Apo C-II 8.2 Chylomicrons, VLDL, HDL Apo C-III 8.8 Chylomicrons, VLDL, HDL ApoD 19.3 HDL Apo E 34.2 Chylomicrons, VLDL, HDL In general, cells outside the liver and intestine obtain cholesterol from plasma LDL rather than by synthesizing it de novo. As depicted in Figure 2 (5), the lipoprotein metabolism pathways in humans can be divided into exogenous and endogenous pathways, dealing with the transport of dietary and hepatically derived lipids, respectively (4). 4 EXOGENOUS ENDOGENOUS Diet Bile _ J / Intestine Extrahepatic | Tissues [Chylomicrons! T i A ' s ; Remnants | Figure 2 Pathways for lipoprotein metabolism in humans The liver is the crossing point of the exogenous pathway dealing with dietary lipids and the endogenous pathway that starts with hepatically synthesized lipoproteins. The exogenous branch begins with production of chylomicrons, whereas the liver synthesizes VLDL. Apolipoproteins are denoted by E, C, B-48, B-100, A-I and A-II. Other abbreviations are as follows: Remnants, chylomicron remnants; LDL, low density lipoprotein; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LCAT, lecithinxholesterol acyltransferase; CETP, cholesteryl ester transfer protein; CMR, chylomicron remnant receptor; and LDLR, LDL receptor. (5) Regulatory mechanisms have evolved to maintain the concentration of free (unesterified) cholesterol in cell membranes within narrow limits. Receptors for LDL, which are located on the surface of cells, bind to LDL and carry it into the cell by receptor-mediated endocytosis. The internalized lipoproteins are delivered to lysosomes where their cholesteryl 5 esters are hydrolyzed by acid cholesterol ester hydrolase, thereby liberating free cholesterol (6-11). The free cholesterol crosses the lysosomal membrane and enters a cytosolic pool accessible to the endoplasmic reticulum enzyme acyl CoA-cholesterol acyltransferase (ACAT), as depicted in Figure 3 (1, 12). Some of the free cholesterol is transported to the plasma membrane, and the remainder is esterified by ACAT and deposited in lipid droplets (13). A dynamic equilibrium is maintained by a continually active neutral cholesterol ester hydrolase, that releases free cholesterol from the droplets. In addition to increasing ACAT activity, the free cholesterol content of cells having an active LDL pathway is regulated in two ways. First, the released free cholesterol (or an oxygenated derivative of it) suppresses the transcription of the gene for 3-hydroxy-3-methylglutaryl CoA reductase, thereby blocking de novo synthesis of cholesterol (14, 15). Second, the native LDL receptor itself is subject to feedback regulation. When cholesterol is abundant inside the cell, new LDL receptors are not synthesized, and so the uptake of additional cholesterol from native LDL is blocked (16, 17). This allows cells to adjust the number of LDL receptors to provide sufficient cholesterol for metabolic needs without causing cholesterol overaccumulation (18). Under normal conditions, these regulatory mechanisms allow cells to keep their level of unesterified cholesterol remarkably constant despite wide fluctuations in cholesterol requirements and exogenous supply. There are many important species differences in the activity of the cholesterol ester cycle, and it has been shown to be very rapid in murine cells, intermediate in the rat, and very slow in the rabbit (19). Comparisons of cholesterol ester turnover with human macrophages have not been reported, but it has been demonstrated that both THP-1 cells and human 6 LDL binding > Intarnal- —Lysosomal —J*- Regulatory IzaHon hydrolysis actions Figure 3 Sequential steps in the LDL receptor pathway of mammalian cells Acyl-CoAxholesterol acyltransferase is abbreviated as ACAT. 3-hydroxy-3-methylglutaryl CoA reductase is abbreviated as HMG CoA reductase. Vertical arrows indicate the directions of regulatory effects. (12) monocyte-derived macrophages express hormone-sensitive lipase (HSL) mRNA (20). HSL is capable of hydrolyzing cholesteryl esters at approximately the same rate as triacylglycerol. Isolated macrophages have few native LDL receptors and as a consequence, take up very little native LDL. As discussed in Section 1.3, some cells, including macrophages, express scavenger receptors on their cell surface, which recognize and take up modified LDL. Scavenger receptors, unlike the native LDL receptor, do not appear to be regulated by cellular sterol levels. Consequently, cholesterol delivery to these cells via scavenger receptors may depend solely on the presence and concentration of modified lipoproteins in the extracellular space. Studying the unregulated uptake of oxidized LDL by scavenger receptors on macrophages is of particular importance to atherosclerosis because such uptake can lead to cholesterol overaccumulation within these cells. 7 1.2 HDL-mediated cholesterol efflux Animal cells other than hepatocytes and steroidogenic cells are incapable of catabolizing cholesterol, with the exception of the partial contribution made by the enzyme sterol 27-hydroxylase (21, 22). Hence, most of the body's excess cholesterol must be removed from the cells in tissues and transported to the liver for biological degradation to bile acids. Moreover, cholesterol efflux from macrophages within the arterial intima is essential to inhibit progression and cause regression of atherosclerosis (23-25). HDL is thought to play a central role in "reverse cholesterol transport", the pathway by which cholesterol is transported from extrahepatic cells to the liver from which it may be recycled to extrahepatic cells or excreted into the intestine in bile (3, 26). Furthermore, numerous population studies have shown an inverse correlation between risk for cardiovascular disease and plasma HDL levels (27-29), leading to the widely held view that HDL protects against atherosclerosis. The removal of cholesterol from cells is the first and presumed rate-limiting step in reverse cholesterol transport. Reverse cholesterol transport is equally as important as forward cholesterol transport in maintaining intracellular cholesterol homeostasis in the animal cell. However, unlike the native LDL receptor pathway, the mechanism for the removal of cellular cholesterol has not been fully characterized (30). There are two proposed mechanisms of cholesterol efflux from cells (Figure 4) (3). The first is based on the fact that cholesterol is exchangeable between lipoproteins and cellular membranes by diffusion through the aqueous phase surrounding cells (31, 32). The net efflux of cholesterol can therefore be achieved by the gradient of cholesterol content between the cell's plasma 8 membrane and the extracellular HDL surface (33-35). This gradient is increased and maintained by the enzyme lecithinxholesterol acyltransferase (LCAT) (Figure 5) (36-39). LCAT, as part of plasma HDL, converts cholesterol to nondiffusable cholesteryl esters that cannot recycle back to the cell (40). This process of cholesterol efflux is achieved without direct contact between the HDL particle and the plasma membrane (41). Other structural factors such as membrane lipid and protein compositions and shape of the acceptor lipoprotein particles may also influence the rate of cellular cholesterol efflux by this pathway (42-49). The specific function of HDL would therefore be attributed to its reactivity to LCAT, and also to its unique structure that may allow the particle to accommodate more cholesterol than other lipoproteins. This view, however, has been challenged by a second proposed mechanism of cholesterol efflux from cells, which operates when HDL interacts with a specific binding site on the surface of the cell (50-53). Many attempts have been made to identify a binding site for HDL (54-56); it is thought that the major function of the murine scavenger receptor class B type I (SR-BI) is to mediate the uptake of HDL cholesterol ester by the liver and steroidogenic tissues (57, 58). According to this hypothesis, it is thought to function as an HDL receptor, to allow the selective uptake of HDL cholesterol esters, without having to internalize the entire HDL molecule (59, 60). It has also been demonstrated that the SR-BI promotes cellular cholesterol efflux (61, 62). Although no single membrane protein has been conclusively identified as a mediator for cellular cholesterol efflux, specific cellular responses to the interaction with HDL have been implicated, supporting the idea for the presence of a specific cellular site of interaction. These cellular responses include translocation of intracellular cholesterol to the cell surface 9 (63-66), activation of protein kinase C (67-69), and other signal transduction pathways (70-72). Diffusion/LCAT-mediated Efflux Apolipoprotein-mediated Figure 4 Overview of cellular cholesterol efflux The left half represents diffusion-mediated efflux. Free cholesterol (FC) diffuses through the aqueous phase between plasma membrane and HDL. Phospholipid also diffuses through the aqueous phase between plasma membrane and HDL, but to a much lesser extent than FC. Lecithin:cholesterol acyltransferase (LCAT) reduces free cholesterol (FC) on the HDL surface to cholesteryl ester (CE), in order to maintain the gradient of free cholesterol. The right half of the figure represents generation of pre-(3-HDL with extracellular apolipoproteins and cellular cholesterol and phospholipid. Apolipoproteins are thought to be released from HDL to interact with a specific site on the cell surface and intracellular cholesterol subsequently mobilized is utilized to generate pre-p-HDL by this reaction. Pre-p*-HDL generated can be converted to regular HDL by various reactions such as free cholesterol exchange with other lipoproteins, or the LCAT reaction followed by CETP-mediated lipid exchange. (3) 10 o O CHg-O-C-^ 4- R 0-C-O-CH O • c I n CH 2 -0-P-0-CH 2 ,CH 2 N + (CH 3 ) 3 O-LECITHIN CHOLESTEROL | o rry^ + H0-?H ? V J L J L J CH 2 -0-P-aCH 2 -CH 2 N + (CH 3 ) 3 CHOLESTERYL ESTER LYSOLECITHIN Figure 5 The LCAT reaction between free cholesterol and lecithin (phosphatidylcholine). Cholesteryl ester is retained in the lipoprotein core while lysolecithin (lysophosphatidylcholine) is released and subsequently binds to albumin. The relative contribution of the two mechanisms of HDL-mediated cholesterol efflux depends on the cholesterol content and growth state of cells. In rapidly proliferating cells that have been cholesterol-depleted, the number of apolipoprotein binding sites is at a minimum (51, 65, 73, 74) and apolipoprotein-mediated phospholipid and cholesterol efflux is relatively low (65, 75, 76). Conversely, arresting the growth of cells and loading them with cholesterol increases the number of apolipoprotein binding sites and enhances apolipoprotein-mediated cholesterol and phospholipid efflux (65, 73-75). Thus, with 11 proliferating cells grown in the presence of serum, the aqueous diffusion mechanism accounts for most of the cholesterol efflux that occurs in the presence of HDL, especially at high serum concentrations (77). However, with quiescent and differentiated cells, the apolipoprotein-mediated mechanism contributes significantly to the total HDL-mediated cholesterol efflux and may be the major process for clearing cholesteryl esters from cells (78). Since the ability to eliminate cholesterol from extrahepatic cells is fundamental to life, it would be surprising if the process depended on a single extracellular acceptor. It has been hypothesized that all subpopulations of HDL, and most HDL apolipoproteins, are able to function as acceptors of cell cholesterol. The HDL fraction in human plasma is heterogeneous, comprising several discrete subpopulations of particles of varying size, density, electrophoretic mobility and lipid and apolipoprotein composition. Nondenaturing gradient gel electrophoresis separates HDL on the basis of size into at least five distinct subpopulations. HDL may also be subdivided into three subpopulations of apolipoprotein-specific particles: one with particles containing apo A-I but no apo A-II (A-I HDL), another with particles containing both apo A-I and apo A-II (A-I/A-II HDL), and a minor subpopulation of particles containing only apo A-II (A-II HDL). In terms of electrophoretic mobility, most HDL are a-migrating but there are also minor subpopulations of particles with pre-(3 or y mobility. There is evidence that, in terms of effectiveness as acceptors of cell cholesterol, A-I HDL are superior to both A-I/A-II HDL and A-II HDL, and that pre-(3-HDL (and possibly y-HDL) are superior to a-HDL (38, 46, 77). Although apolipoproteins completely free of lipid have not been identified in vivo, those containing a relatively small amount of phospholipids (10-40% of the total weight) are present in plasma and lymph (77). 12 The best characterized of these is pre-(3l-HDL a small particle containing only apo A-I and some phospholipid (24). The intracellular space of large arteries is known to contain a relatively high concentration of pre-(3-HDL (79). Of the "classical" lipoprotein fractions, it has been proposed that HDL3, which is predominantly composed of A-I HDL and A-I/A-II HDL, is the major promoter of cholesterol efflux (80-82). It is for this reason that all of the experiments presented in this thesis used HDL3 as the cholesterol acceptor, unless otherwise stated. 1.3 The Pathogenesis of Atherosclerosis Atherosclerosis is a chronic progressive disease that affects large and medium-sized elastic and muscular arteries. The name is derived from the Greek athero meaning paste and sclerosis meaning hardening. In the early stages of the disease, lipid (principally cholesterol) is deposited in the arterial intima. Lipoprotein transport into this subendothelial space is a concentration-dependent process and does not require receptor-mediated endocytosis (83, 84). Once low density lipoprotein (LDL) particles become trapped by proteoglycans within the arterial intima (85), they can undergo progressive oxidation since the intima lacks many of the antioxidant defense mechanisms that are present in the bloodstream (86-90). Initially, macrophages play a protective role by removing the modified LDL molecules via scavenger receptor-mediated endocytosis (91-93). Continued uptake of this modified LDL results in macrophage foam cell formation (94-96). As these cells die, they can release potent cytokines and oxidizing agents which can lead to smooth muscle cell proliferation, continued oxidation of incoming LDL, and recruitment of monocytes and T-lymphocytes into the 13 expanding intimal space (97, 98). There is a complex interplay of cellular signals (cytokines, growth factors, etc.) generated between the cells of the atherosclerotic lesion which influence each step of lesion progression, including differentiation of monocytes to macrophages (99). In addition, smooth muscle cells migrate from the media into the intima, where they begin to take up lipoproteins (100) and secrete large amounts of extracellular matrix (101-103). According to this model, these and other inflammatory events culminate to exacerbate lesion progression, resulting ultimately in the formation of atheromatous plaques or atheromas. Such plaques obstruct the blood flow and weaken the vessel wall, leading to thrombosis, internal blood coagulation, and aneurysms, where the internal wall is locally dilated. Also, plaques can rupture from the arterial wall and be washed downstream, where they lodge in smaller arteries. When an artery that feeds the heart, brain, or peripheral vasculature is occluded, it can result in myocardial infarction, stroke, or gangrene, respectively. Atherosclerosis remains the principal cause of death in North America, Europe, and much of Asia (104, 105). Numerous risk factors that increase the likelihood of atherosclerosis have been identified. Four risk factors that can not be modified are age, estrogen deficiency (including male gender), family history of vascular disease, and personal history of symptomatic vascular disease. Some modifiable risk factors that are also causally related to atherosclerosis and its clinical sequelae include: smoking, hypertension, diabetes mellitus, obesity, sedentary lifestyle, psychosocial stress, and abnormal plasma lipoprotein levels (106). Investigating the mechanism of action of the risk factors and their interplay with a number of genes has increased our understanding of the complex interactions of circulating blood proteins, lipoproteins, growth factors, cytokines, vasoregulatory molecules, extracellular matrix proteins, and the cells of the artery wall (106). 14 Perhaps the most important and best studied of the major risk factors for atherosclerosis is hypercholesterolemia. In humans, LDL is the major transporter of cholesterol in blood, and it is the source of most of the cholesterol found in atherosclerotic lesions (1, 4, 11, 18, 107-113). In contrast, high density lipoprotein (HDL) acts as an extracellular cholesterol acceptor, thereby allowing extrahepatic cells to unload excess cholesterol, and this is consistent with the known protective effect of HDL cholesterol (3, 19, 30, 61, 65, 114, 115). In early lesions, much of the cholesterol is found as cytoplasmic lipid droplets within macrophages, and it is believed that cholesterol accumulation within these cells is an important initial step in the formation of more advanced lesions (116-122). The mechanism by which macrophages in the arterial intima accumulate massive amounts of cholesterol in patients with hypercholesterolemia is not readily apparent, as the activity of the LDL receptor is regulated by intracellular cholesterol content, and incubation of cultured macrophages with high concentrations of normal plasma LDL fails to bring about lipid accumulation (123, 124). However, subsequent studies showed that certain chemical modifications of LDL lysine residues, such as acetylation, converted it to a form that was recognized by a specific receptor on macrophages termed the acetyl LDL receptor or scavenger receptor class A type I/II (SR-AI/II) (123, 125). This scavenger receptor, unlike the native LDL receptor, is not down-regulated when the cholesterol content of the cell increases (123). Hence, continued uptake of modified LDL by this receptor allows massive cholesterol deposits to accumulate in macrophages in vitro (123, 126, 127). Because there is no evidence that acetylation of LDL occurs in vivo, the search began for a biologically plausible modification of LDL that could account for foam cell formation and the initiation, or at least acceleration, of the atherosclerotic process (128). "Physiologic" 15 modifications of LDL that have been reported to facilitate foam cell formation include oxidation (87, 88, 128-130), glycation (131-133), aggregation (134, 135), association with proteoglycans (136-138), and incorporation into immune complexes (139-145). While there is evidence that all of these modifications may occur in vivo, oxidative modification has been the most extensively studied and there is increasing evidence that it plays a significant role in the pathogenesis of atherosclerosis (97, 128). Arterial endothelial cells and smooth muscle cells are capable of inducing oxidative modification of LDL, and LDL modified in this fashion has been shown to bind with high affinity to the SR-AI/II on macrophages (86, 146-153). However, more recent studies have shown that uptake of oxidized LDL in macrophages is only partly attributable to the SR-AI/II. These studies have shown that as much as 70% of the uptake of oxidized LDL cannot be accounted for by the SR-AI/II alone (154). The search for additional receptors for oxidized LDL has lead to the identification of a number of cell membrane proteins that can bind oxidized LDL in both transfected cells and ligand blots (155). Table 3 lists some of these receptors along with the cell/tissue in which they are found, their putative functions, and their ligand specificity.. 16 Table 3 Classification of scavenger receptors CLASSIFICATION CELL/TISSUE PUTATIVE FUNCTION LIGAND FEATURES REFERENCES Class A SR SR-AI/II macrophages innate immunity, cell adhesion, oxidized LDL uptake poly anions, LPS (10, 125, 156-171) MARCO§ spleen macrophages innate immunity? acetyl LDL, bacterial components (172-174) Class B SR CD36 platelets, monocytes, endothelial cells, retinal pigment epithelium multiple native or modified lipoproteins (175-187) SR-BI/CLA-1 adrenal, gonads, liver cholesterol transport (57-60, 178, 181, 188-192) others SR-CI embryonic insect macrophages innate immunity? . phagocytosis of apoptotic cells? polyanions (193, 194) macrosialin/CD68 macrophages/ Kupffer cells abundant in lysosomes, function unknown polyanions, phosphatidyl serine liposomes, apoptotic cells (195-198) Fey receptor macrophages immune complex binding IgG (142, 143, 199-201) LOX-1* endothelium signaling? polyanions, oxLDL (202) SREC* endothelium signaling? acetyl LDL, oxLDL (203) 'macrophage receptor with collagenous structure 17 lectin-like oxidized LDL receptor *scavenger receptor expressed by endothelial cells Although all of these receptors can bind to and take up oxidized LDL, it is not known whether this is an important physiological function of any them in vivo. The efforts to isolate and characterize receptors for oxidized LDL have led us to a greater understanding of the potential role of such receptors in atherosclerosis. Identification of a receptor that functions primarily to take up oxidatively modified lipoproteins will afford the opportunity to further investigate the contribution of oxidized LDL to the development and progression of atherosclerosis. 1.4 Mechanisms of Oxidation and Structural Changes Associated with LDL The physico-chemical changes that occur in the LDL particle that impart recognition by one or more scavenger receptors for oxidized LDL are very complex and incompletely understood. Oxidation of LDL by cells involves a transition metal-catalyzed, free radical-mediated lipid peroxidation process in which the polyunsaturated fatty acyl residues in the LDL lipids are degraded to a variety of peroxidation products (204). These products include hydroperoxy and hydroxy fatty acids (205-208), aldehydes and hydroxyaldehydes (169, 209), and more complex aldehydes (169). The aldehydic lipid peroxidation products that are formed are highly reactive and can derivatize the free amino groups of apolipoprotein B (86, 210). Such derivatization of the lysine 6-amino groups on apolipoprotein B results in an increased negative charge on the LDL particle, which is believed to be responsible for the 18 interaction of oxidized LDL with the scavenger receptor class A type I/II. Furthermore, derivatization of these amino groups not only prevents interaction with the native LDL receptor but it also permits binding to and unregulated uptake by scavenger receptors on phagocytic cells (86, 169, 170, 211). During metal-catalyzed oxidation, apolipoprotein B is also modified through radical-mediated scission of peptide bonds, and probably also by direct reaction with fatty acyl hydroperoxides (212). Oxidation of LDL also is accompanied by oxidation of cholesterol esters and free cholesterol (87), and by hydrolysis of phosphatidylcholine (PC) to lysophosphatidylcholine through the action of a lipoprotein-associated phospholipase A 2 known as platelet activating factor (PAF) acylhydrolase (213). PAF acylhydrolase can utilize the PC from oxidized LDL as a substrate but cannot convert PC from native LDL (213). Some lipid oxidation products found in oxidized LDL such as oxysterols, oxidized fatty acids, and oxidized phospholipids (for example, lyso-PC) have been shown to be important mediators or regulators of many biological effects associated with atherosclerosis, which will be discussed in Section 1.5. Endothelial cells, smooth muscle cells, T-lymphocytes, monocytes and macrophages have all been shown to be capable of enhancing the rate of oxidation of LDL, in vitro (86). The role of cells in the oxidative modification of LDL appears to be simply to accelerate the rate of peroxidation at low (nanomolar) free metal ion concentrations, either by providing thiols which can reduce transition metal ions to a catalytically active form (214, 215) or by "seeding" LDL in the medium with lipid hydroperoxides to initiate a peroxidation reaction (216). In fact, identical changes occur when LDL is oxidized in the absence of cells by incubation with micromolar concentrations of transition metal ions such as copper (86, 211). Typically, both cellular oxidation of LDL and transition metal ion oxidation of LDL result in 19 depletion of more than 70% of its linoleic acid and arachidonic acid content, complete depletion of its endogenous antioxidants, nearly half of its PC being converted to lyso-PC, and more than 30% of its lysine residues being derivatized by lipid peroxide decomposition products, resulting in an electrophoretic mobility greater than 2.5 times that of native LDL (210). In addition to these changes, oxidized LDL (as compared to native LDL) has an increased negative charge and an increased density (217). Lipoprotein oxidation by cells or transition metal in vitro is blocked by small concentrations of plasma or plasma proteins (218), such as albumin (219-224), and any oxidized lipoproteins that might appear in the plasma in vivo would be rapidly removed by the liver (225, 226), rather than be deposited into developing lesions within the arterial wall (227). As mentioned previously, oxidation of LDL is more likely to occur within the arterial wall, where increased levels of copper and iron ions are generally found and antioxidant levels are low (228). 1.5 The Biological Effects of Oxidized LDL Oxidation of LDL has been implicated as a causal factor in the pathogenesis of atherosclerosis (88). This has been supported by several lines of evidence, including the demonstration that oxidatively modified LDL exists in atherosclerotic lesions in vivo in both experimental animals and humans (89, 90, 229, 230). In addition, inhibition of oxidation by several antioxidants (for example, probucol, butylated hydroxytoluene, diphenylphenylenediamine, and vitamin E) can slow the progression of the disease in several animal models, including the LDL-receptor-deficient rabbit, the cholesterol-fed New Zealand White rabbit, the cholesterol-fed hamster, the cholesterol-fed cynomolgus monkey, the LDL-20 receptor-deficient mouse, and the apolipoprotein E-deficient mouse (231-239). Also, it was recently shown that the susceptibility of LDL to oxidation varied with the severity of coronary atherosclerosis, as evaluated by angiography (240). These observations prompted a number of investigators to explore the possible mechanisms by which oxidized LDL might contribute to atherogenesis. Oxidized LDL has been found to exhibit a number of interesting biologic properties that are absent in native (unmodified) LDL (Figure 6). However, it should be emphasized here that a given biologic action of oxidized LDL can vary not only in magnitude but even in direction depending on the nature and extent of oxidative modification of LDL. In some studies, the extent of LDL oxidation was not controlled and/or inadequately characterized, thereby making it difficult to present anything more than a simple catalogue of actions that have been attributed to oxidized LDL. Most of the reports describing the biological effects of oxidized LDL have used extensively oxidized LDL, but some used mildly oxidized LDL. Mildly oxidized or "minimally modified" LDL has an increased hydroperoxide content compared to native.LDL, and unlike extensively oxidized LDL it retains at least some of its.endogenous antioxidants. Furthermore, less than 5% of the unsaturated fatty acids are consumed in "minimally modified LDL", and it exhibits minimal derivatization of free amino groups on its apolipoprotein B component. The electrophoretic mobility of "minimally modified LDL" is not very different from that of native LDL. Biologic effects seen with mildly oxidized LDL include induction of monocyte chemotactic protein-1 expression by endothelial cells and smooth muscle cells (241), enhanced adhesiveness of cultured endothelial cells for monocytes (242), and expression of the inflammatory cytokines GM-CSF, M-CSF, G-CSF, GRO, and IL-ip (205, 243-245). 21 The remainder of this thesis will focus on the atherogenic properties of extensively oxidized LDL, and for simplicity, extensively oxidized LDL will be referred to as oxidized LDL. The physical characteristics and properties of oxidized LDL were discussed in Section 1.4. In vitro studies have revealed that it has many potentially atherogenic actions, which may account for the progression of atherosclerotic lesions (128). As already mentioned, one of the most important properties of oxidized LDL that makes it more atherogenic than native LDL is that it is recognized by scavenger receptors on the surface of macrophages and can therefore give rise to foam cells, which are one of the earliest hallmarks of atherosclerosis (147). Table 4 provides a catalog of some of the known biologic actions associated with oxidized LDL. The events listed in Table 4 would tend to increase foam cell formation and increase the number of macrophages in the arterial intima at sites of lesion formation. These findings suggest that oxidized LDL may play an important role in the development of early stage atherosclerotic lesions. 22 Table 4 Biologic actions associated with oxidized LDL Biologic Action References accelerated uptake by macrophages via scavenger receptors (210, 246, 247) foam cell formation (94, 217, 247) cytotoxicity (152, 248-253) apoptosis (254-261) increased susceptibility to form aggregates (262) resistance to lysosomal degradation (129) enhanced binding to collagen (221, 263) monocyte chemotaxis and inhibition of migration of differentiated (264-266) macrophages stimulation of leukocyte adherence to the microvascular endothelium in (267, 268) vivo induction of macrophage proliferation (269, 270) activation of T lymphocytes (271, 272) increased monocytic cell expression of interleukin-8 (273) increased monocytic cell release of interleukin-ip (274) induction of VCAM-1 and ICAM-1 expression in endothelial cells (275, 276) inhibition of PDGF and tumor necrosis factor-a expression (277-279) inhibition of lipopolysaccharide-induced interleukin-ip expression (280) increased growth factor gene expression in endothelial cells (275, 281) promotion of DNA synthesis, cell cycle entry and proliferation of (282-285) vascular smooth muscle cells 23 Figure 6 Oxidized LDL and atherosclerosis. Once in the intimal space, LDL can become trapped by proteoglycans and subsequently oxidized by a transition metal catalyzed, free radical mediated reaction that converts unsaturated fatty acids to reactive lipid hydroperoxides and aldehydes. These reactive lipid substances then further accelerate oxidation of the LDL particle, leading to the depletion of polyunsaturated fatty acids and antioxidant activity, and the fragmentation of apolipoprotein B. Oxidized LDL stimulates the expression of cell adhesion molecules (VCAM, ICAM-1, and GRO) and the secretion of MCP-1 and M-CSF from endothelial cells, causing monocytes to enter the arterial intima and differentiate into macrophages. Oxidized LDL also becomes endocytosed via scavenger receptors and other receptors for oxidized LDL on the surface of macrophages, resulting in lipid accumulation and ultimately foam cell formation. Oxidized LDL has also been shown to promote chemotaxis of vascular smooth muscle cells from the tunica media, as well as stimulate cellular proliferation of both smooth muscle cells and macrophages. 24 1.6 Rationale There is reason to believe that the simple model used to describe the native LDL receptor pathway may not adequately account for the distribution and metabolism of cholesterol in arterial foam cells. Ultrastructural studies of macrophage-derived foam cells within atherosclerotic lesions in animals suggest that a significant part of the lipid deposits in these cells remains in lysosomes rather than cytosolic lipid droplets (286-291). Similarly, in macrophage foam cells in human atherosclerotic lesions, a substantial proportion of lipid has been found within membrane-bound vacuoles, at least some of which appear to be of lysosomal origin (290-296). Several studies in J774 murine macrophages have revealed retention of free cholesterol in lysosomes after incubation of cells with cholesterol oleate droplets or oxidized LDL (92, 297-299). However, J774 cells also fail to efficiently esterify cholesterol delivered by acetylated LDL (300), and hence the results may not apply to primary macrophages. For instance, Kritharides et al. recently examined the subcellular distribution of sterols in murine macrophages incubated with oxidized LDL and they concluded that there was equilibration of cholesterol to all membrane pools (300). However, their data appear to show a significant increase in the proportion of cholesterol and oxysterol in the endosomal compartment, rather than the lysosomal compartment, and so this question remains unresolved. Moreover, the findings of Yancey et al. suggest that murine macrophages differ from both pigeon and THP-1 macrophages, in that murine macrophages stored most (71%) of their oxidized LDL-derived cholesterol within cytoplasmic inclusions, whereas pigeon and THP-1 macrophages stored most (60-90%) of it within lysosomes (301). In order to gain a more complete understanding of cholesterol metabolism in oxidized LDL-25 loaded murine macrophages, the subcellular localization of oxidized LDL-derived lipid, both before and after HDL-mediated cholesterol efflux, must be ascertained. Although much research has concerned possible mechanisms of reverse cholesterol transport, relatively little attention has been directed to the comparative release from lipid-loaded macrophages of free cholesterol derived from oxidized LDL or acetylated LDL. This may be of great significance since oxidized LDL has been shown to exhibit many potentially atherogenic actions, including accelerated uptake by macrophages (210, 246, 247) and macrophage foam cell formation (94, 217, 247). It has been demonstrated that extensively oxidized LDL is resistant to lysosomal enzymes (302), and also can inactivate lysosomal acid hydrolases (129, 303). Inactivation of lysosomal cathepsins by aldehydic adducts on oxidized LDL may be responsible for impaired efflux of cholesterol from oxidized LDL-loaded macrophages (302), but this hypothesis has never been tested directly. It has also been established that apolipoprotein B from oxidized LDL accumulates within the lysosomal compartment (129, 304, 305), and that oxidation-specific protein adducts (presumably representing undegraded oxidized apo B) are detectable within foam cells in atherosclerotic lesions (306). In contrast to acetylated LDL-derived in vitro foam cell models which accumulate free cholesterol and cholesteryl esters (307), oxidized LDL-derived foam cells contain much lower quantities of cholesteryl esters and contain a number of oxidized cholesterol compounds (308, 309). It has been demonstrated that there is a major difference in the ability of cells loaded with unoxidized cholesterol compounds (acetylated LDL) or oxidized cholesterol compounds (oxidized LDL) to release their respective lipid compound to an extracellular cholesterol acceptor (300). Further investigation into the nature of this phenomenon is valuable because impairment of cholesterol efflux from oxidized LDL-loaded 26 cells may influence the generation and persistence of the foam cell phenotype in vivo and may, therefore, contribute to the atherogenicity of oxidized LDL. The purpose of this study was to further characterize the mechanism(s) by which oxidized LDL causes impaired efflux of cholesterol from macrophages. To accomplish this goal, we assessed the degree of HDL-mediated cholesterol efflux impairment in cells loaded with either oxidized LDL or acetylated LDL, we determined the subcellular location of oxidized LDL-derived lipid in macrophages before and after HDL-mediated cholesterol efflux, and finally, we investigated whether the lipid moiety or the protein moiety of the lipoproteins was predominantly involved in the process of cholesterol accumulation and efflux. 1.7 Objectives and Hypothesis The main objective of this study was to investigate the role of oxidized LDL in lipid accumulation in macrophages, since this is pertinent to the formation of macrophage foam cells. In order to achieve this objective, the following specific hypotheses were addressed: 1. Macrophages loaded with oxidized LDL exhibit impaired efflux of cholesterol to HDL, compared to macrophages loaded with acetylated LDL. 2. Impairment of HDL-mediated cholesterol efflux in macrophages loaded with oxidized LDL involves both the oxidized lipid component and the oxidized apolipoprotein B moiety. 27 3. Cholesterol delivered by oxidized LDL is sequestered in lysosomes and therefore is inaccessible to HDL-mediated cholesterol efflux. 28 Chapter 2 Materials and Methods 2.1 Chemicals and Reagents The Amplex Red reagent, NBD-C6-sphingomyelin, and Texas Red sulfonyl chloride were purchased from Molecular Probes (Eugene, Oregon, USA). Butylated hydroxytoluene (BHT) was from J.T. Baker (Toronto, Ontario, Canada). Percoll, Folin and Ciocalteu's phenol reagent, fatty acid free bovine serum albumin, horseradish peroxidase, cholesterol esterase, cholesterol oxidase, arachidonic acid, 4-methylumbelliferyl oc-D-glucoside, 4-methylumbelliferyl N-acetyl-P-D-glucosaminide, trypsin and trypsin inhibitor were purchased from Sigma. LPS-free sterile water was purchased from Baxter (Toronto, Ontario). Centriflo CF25 membrane cones were obtained from Amicon (Beverly, USA). Carrier-free Na125I was purchased from NEN/Mandel Scientific (Guelph, ON, Canada). [3H]-cholesteryl linoleate was purchased from Amersham Corporation (Piscataway, NJ, USA). Dimethyl sulfoxide (DMSO), aquasol scintillation fluid, Scintisafe scintillation fluid, methanol, isopropyl alcohol, and chloroform were purchased from Fisher Scientific (Vancouver, BC, Canada). Dulbecco's modified Eagle's medium (DMEM) and gentamicin were purchased from GIBCO (Mississauga, Ontario). Hyclone defined fetal bovine serum was supplied by Professional Diagnostics (Edmonton, Alberta, Canada). Tissue culture plates were purchased from Canlab (Vancouver, B.C.). Female CD-I Swiss mice were supplied by the Charles River animal breeding facility (Montreal, Quebec). Glass thin-layer chromatography plates were purchased from Mandel Scientific (Guelph, ON, Canada). Intralipid was purchased from Pharmacia (Dorval, PQ, Canada). The MPR 1 free cholesterol 29 assay kit, the MPR 1 total cholesterol assay kit, and the cholesterol standards were obtained •.ffkom.Boehringer Mannheim (Mannheim, Germany). All other chemicals were the highest grade available from Sigma Chemical (St. Louis, MO), BDH Chemical (Toronto, ON, Canada), Fisher Scientific (Vancouver, BC, Canada), or VWR Canlab (Vancouver, BC, Canada). 2.2 LDL Preparation 2.2.1 LDL isolation Plasma from fasting, normolipidemic human volunteers was collected into EDTA (final concentration 3 mM). The density of the plasma was adjusted with sodium bromide solutions containing 10 mM EDTA, and LDL (d = 1.019-1.063 g/ml) was isolated by sequential ultracentrifugation in a Beckman Ti-60 fixed angle rotor by the method of Havel et al. (310). In order to reduce the high sodium bromide concentration, LDL preparations were then dialyzed against two changes of Dulbecco's PBS containing 10 uM EDTA at pH 7.4 at 4°C, and then stored at 4°C in the dark. This concentration of EDTA was sufficient to Inhibit spontaneous oxidation on storage, but low enough to permit reproducible oxidation upon addition of 5 uM CuS04. Finally, the preparation was passed through a 0.22 um filter to remove aggregates and ensure sterility. 30 2.2.2 LDL labelling LDL was radio-iodinated using a modification of iodine monochloride method of McFarlane (311,312). Typically, 5 mg of LDL was mixed with an equal volume of ice-cold 0.6 M glycine/0.4 M NaOH, pH 10. Lipid labelling is minimized at this pH. In a fume hood, 1 mCi carrier-free Na125I followed by 125 pi freshly diluted 1 mM IC1 were then added with brief gentle vortexing. The mixture was transferred to a dialysis bag and dialyzed against four changes of Dulbecco's PBS with 10 uM EDTA. Finally, the preparation was passed through a 0.22 urn filter to remove aggregates and ensure sterility. Labelling efficiency was about 20% and specific radioactivities ranged from 80-140 cpm/ng. Over 99% of radioactivity in labelled LDL was precipitable with 10% trichloroacetic acid. Cholesteryl esters in LDL were labelled by exchange with a microemulsion containing [3H]-cholesteryl linoleate. The labelled microemulsion was obtained by cosonicating 5 pi of 10% Intralipid with 109 dpm [3H]-cholesteryl linoleate in 350 pi PBS for 30 seconds at 20°C using an Artek 150 sonicator with titanium microprobe set at 30% power. Native LDL (5 mg) was then incubated for 4 hours at 37°C with the labelled Intralipid together with 30 mg/ml d > 1.21 g/ml plasma fraction as a source of lipid transfer activity. LDL was re-isolated by sequential ultracentrifugation between d 1.020 and 1.063 g/ml, and then dialyzed against several changes of Dulbecco's PBS containing 10 pM EDTA. Finally, the preparation was passed through a 0.22 um filter to remove aggregates and ensure sterility. This method is nearly identical to that used by Zhang et al. (308), but this method promotes greater incorporation of [3H]-cholesteryl linoleate into the LDL molecule. In 31 addition to labelling of native LDL, this procedure can be used to label modified forms of LDL either before or after the chemical modification of the LDL, depending on the purpose. Acetylated LDL and oxidized LDL were fluorescently labelled with NBD-Q-sphingomyelin. NBD-C6-sphingomyelin is the common name for 6-((N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino)hexanoyl) sphingosyl phosphocholine. 2 mg of modified LDL was pipetted into a sterile conical tube, to which 2-4 ml of LPDS was added. NBD-Q-sphingomyelin was dissolved in methanol to create a 1 mg/ml stock solution. 400 pi of this was removed and dried down in a glass test tube under a stream of nitrogen. This was redissolved in 100 pi DMSO and then transferred to the conical tube containing the modified LDL/LPDS mixture. The tube was then inverted a number of times and then incubated for 8 hours at 37°C. Throughout the labelling procedure, the NBD-C6-sphingomyelin was protected from light as much as possible. Upon completion of the incubation, the density of the mixture was adjusted with sodium bromide solutions containing 10 mM EDTA, and the modified LDL was isolated by ultracentrifugation in a Beckman Ti-60 fixed angle rotor. Isolation of fluorescently labelled acetylated LDL required adjustment of the density of the mixture to 1.063 g/ml; isolation of fluorescently labelled oxidized LDL required adjustment of the density of the mixture to 1.100 g/ml. In order to reduce the high sodium bromide concentration, the fluorescently-labelled modified LDL preparations were then dialyzed against two changes of Dulbecco's PBS containing 10 pM EDTA at pH 7.4 at 4°C, and then stored at 4°C in the dark. Finally, the preparation was passed through a 0.22 pm filter to remove aggregates and ensure sterility. 32 2.2.3 LDL modification Acetylation of LDL was performed by sequential addition of acetic anhydride (123). Seven aliquots each of 1 pi of acetic anhydride were added at 15 min intervals to 4 mg of LDL in ice-cold 50% saturated sodium acetate with gentle stirring, and then dialyzed against Dulbecco's PBS containing 10 uM EDTA. Before addition of acetic anhydride, BHT (20 pM) was added to prevent oxidation. Finally, the preparation was passed through a 0.22 um filter to remove aggregates and ensure sterility. LDL was oxidized by incubation of 200 pg/ml LDL in EDTA-free Dulbecco's PBS containing 5 pM CuS04 for 24 hours, at 37°C (86, 210). Oxidation was arrested by refrigeration and addition of 50 pM BHT and 200 uM EDTA. The oxidized LDL was then washed with EDTA-free Dulbecco's PBS and reconcentrated on CF25 membrane cones. Finally, the preparation was passed through a 0.22 pm filter to remove aggregates and ensure sterility. To produce NaBH4-treated oxidized LDL, 500 pg of oxidized LDL (prepared as above) was incubated with 150 pi of a freshly prepared solution of 1 M NaBH4 in 0.1 M NaOH (302, 313). Following the incubation for 4 hours at room temperature, the oxidized LDL sample was dialyzed against Dulbecco's PBS containing 10 pM EDTA. Finally, the preparation was passed through a 0.22 pm filter to remove aggregates and ensure sterility. To produce acetylated LDL that is subsequently oxidized, we simply prepared acetylated LDL as described above, and then we oxidized it for either 5 hours or 24 hours, using the method for oxidation of native LDL, given above. 33 In order to produce arachidonic acid-oxidation product LDL, we first generated reactive fatty acid oxidation products (OP) by thermal autoxidation of arachidonic acid (129, 170). Arachidonic acid (10 mg) was oxidized by exposure to air at 37 °C for 72 hr in a glass vial. The yellow-brown products were dissolved in Dulbecco's PBS with 300 uM EDTA at pH 7.4, and aliquots containing the residue from 0.8-1.5 mg of oxidized fatty acid were added to 1 mg LDL, in 1 ml Dulbecco's PBS containing 300 pM EDTA and 50 pM butylated hydroxy toluene. Irreversible derivatization of proteins occurred within minutes at 20°C and was substantially complete within a few hours. After overnight incubation at 20 °C, reaction mixtures were washed repeatedly with PBS on Amicon Centricon 30 microconcentrators to remove soluble non-bound materials. Omission of this wash step resulted in cytotoxicity at higher concentrations of modified LDL during subsequent incubation with macrophages. Aggregated LDL was prepared by vortexing a 1 mg/ml native LDL solution for 15 seconds in a 15 ml conical tube with a benchtop vortex mixer at medium speed setting. The extent of aggregation (as reflected in turbidity) was monitored as absorbance increase at 680 nm and was typically 09-1.0 (314). BHT (20 pM) and EDTA (10 pM) were present throughout and there was no appreciable oxidation of these preparations. 2.2.4 Characterization of LDL Lipoprotein electrophoresis was performed using a Ciba-Corning apparatus and 1% Universal agarose film in 50 mM barbital buffer (pH 8.6) according to the manufacturer's instructions. Bovine serum albumin was added to lipoprotein samples to ensure reproducible migration distances. Lipoprotein bands were visualized by staining with Fat Red 7B. Native 34 (unmodified) LDL was used as a reference. The relative electrophoretic mobility of modified LDL was calculated by dividing the distance travelled during electrophoresis by the distance travelled by native LDL, and satisfactory acetylation or oxidation was confirmed if the relative electrophoretic mobility (Rf) was greater than or equal to 3. Protein was assayed by the method of Lowry (315) in presence of 0.05% sodium deoxycholate to minimize turbidity. Bovine serum albumin was used as the standard. 2.3 HDL Preparations 2.3.1 HDL3 isolation Plasma from fasting, normolipidemic human volunteers was collected into EDTA (final concentration 3 mM). The density of the plasma was adjusted with sodium bromide solutions containing 10 mM EDTA, and HDL3 (d = 1.125-1.210 g/ml) was isolated by sequential ultracentrifugation in a Beckman Ti-60 fixed angle rotor by the method of Havel et al. (310). In order to reduce the high sodium bromide concentration, HDL3 preparations were then dialyzed against three changes of Dulbecco's PBS containing 10 pM EDTA at pH 7.4 at 4°C, and then stored at 4°C in the dark. This concentration of EDTA was sufficient to inhibit spontaneous oxidation on storage. Finally, the preparation was passed through a 0.22 pm filter to remove aggregates and ensure sterility. 35 2.3.2 HDL labelling HDL3 was radio-iodinated using a modification of iodine monochloride method of McFarlane (311, 312). Typically, 5 mg of HDL3 was mixed with an equal volume of ice-cold 0.6 M glycine/0.4 M NaOH, pH 10. Lipid labelling is minimized at this pH. In a fume hood, 1 mCi carrier-free Na125I followed by 125 pi freshly diluted 1 mM IC1 were then added with brief gentle vortexing. The mixture was transferred to a dialysis bag and dialyzed against four changes of Dulbecco's PBS with 10 pM EDTA. Finally, the preparation was passed through a 0.22 pm filter to remove aggregates and ensure sterility. Labelling efficiency was about 20% and specific radioactivities ranged from 80-140 cpm/ng. Over 99% of radioactivity in labelled HDL3 was precipitable with 10% trichloroacetic acid. Native HDL3 was fluorescently labelled with Texas Red sulfonyl chloride. 1 ml of 0.5 M sodium phosphate buffer (pH 8) was added to 10 mg of native HDL3 in a vial wrapped in aluminum foil (to protect from light). 1 mg of Texas Red sulfonyl chloride was dissolved in 50-100 pi of 100% ethanol, and then added directly to the vial containing the buffer and the HDL3. A stir bar was added to the vial and it was stirred in the dark at the slowest speed (to prevent aggregation) for 7-8 hours at 4°C. Upon completion of the incubation, the density of the mixture was adjusted to 1.210 g/ml with sodium bromide solutions containing 10 mM EDTA, and the labelled HDL3 was isolated by ultracentrifugation in a Beckman Ti-60 fixed angle rotor. In order to remove sodium bromide and unreacted Texas Red sulfonyl chloride, the fluorescently-labelled HDL3 preparation was then dialyzed against three changes of Dulbecco's PBS containing 10 pM EDTA at pH 7.4 at 4°C, and then passed through a 0.22 pm filter to remove aggregates and ensure sterility. The labelled product was 36 stored at 4°C in the dark. Texas Red sulfonyl chloride reacts via a displacement reaction with primary amines such as the amino acid, lysine; it forms a stable sulfonamide linkage to the side chain of this amino acid. 2.3.3 Trypsinization of HDL3 Trypsinized HDL3 was prepared by isolating HDL3 as described above and then treating 5 mg of HDL3 with 125 pg of trypsin for 1 hour at 37 °C (316, 317). The reaction was stopped with excess trypsin inhibitor. Trypsin and trypsin inhibitor were removed from HDL3 by dialysis against Dulbecco's PBS containing 10 uM EDTA. Finally, the preparation was passed through a 0.22 pm filter to remove aggregates and ensure sterility. 2.3.4 Apolipoprotein A-I isolation HDL3 was obtained from human plasma as described above. After delipidating it with ethanol : diethyl ether (2 : 1) at -20°C (318), apolipoprotein A-I was isolated by chromatography at 4°C on a Superose 12 Column (Pharmacia) in 0.01 M Tris-HCl, 6 M Urea, pH 8.0 (319). Each fraction collected was analyzed for protein content and run on SDS-PAGE to identify the purest fraction. The purest fractions were pooled and dialyzed at 4°C against 0.1 M ammonium bicarbonate and then thoroughly dialyzed against distilled water. Lyophilized protein was stored at -20°C under nitrogen atmosphere. Before use, lyophilized apo A-I was dissolved in Dulbecco's PBS at 4°C by stirring overnight. The 37 dissolved apolipoprotein was then passed through a 0.22 pm filter to remove aggregates and ensure sterility. 2.3.5 Characterization of HDL and apo A-I Purity of HDL3 and apolipoprotein A-I preparations was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a Bio-Rad mini-gel apparatus (320). The separating or resolving gel that we used contained 10% acrylamide. 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 0.1% (wt/vol) bromophenol blue, and 2% (vol/vol) (3-mercaptoethanol were mixed with samples of either HDL3, trypsinized HDL3, apolipoprotein A-I, or column fractions containing apolipoprotein A-I. This mixture was then heated to 100°C for 2 minutes. The amount of trypsinized HDL3 applied to the well was equivalent to the amount of native HDL3, as assessed by phosphorus content. As for the fractions collected from the Superose 12 column, an equal volume was applied in order to accurately compare the content of purified apolipoprotein A-I in each fraction. Nonradioactive low range molecular weight markers (range, 14,400 Da to 97,400 Da; Bio-Rad) were run concurrently for calibration. The bands were visualized by staining for protein with Coomassie brilliant blue R-250. Apo A-I migrated as a single band (321). Gels were run in 10 mmol/L Tris-glycine buffer (pH 8.3) for 50 minutes at 150 Volts and were fixed in ethanol/acetic acid/water (38:10:52, vol/vol/vol). Protein was assayed by the method of Lowry (315) in presence of 0.05% sodium deoxycholate to minimize turbidity. Bovine serum albumin was used as the standard. 38 2.4 Cell Culture Procedures were followed in accordance with ethical guidelines of the University of British Columbia and the Medical Research Council of Canada. Resident peritoneal macrophages were obtained from female CD-I Swiss mice (after asphyxiation using carbon dioxide gas) by peritoneal lavage with ice-cold Dulbecco's PBS (86, 123). The isolated cells were pelleted, and then resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal bovine serum (FBS) and gentamicin (50 pg/ml). Next, the cells were plated either in 35 mm diameter (Falcon) or 100 mm diameter (Corning) tissue culture plates at a density of either 6.0 X 106 cells/plate or 2.0 X 107 cells/plate, respectively. The larger plates were used for the subcellular fractionation experiments as well as all experiments in which the colorimetric cholesterol assay was used. The smaller plates were used in all experiments in which the fluorimetric cholesterol assay was used. The freshly plated cells were incubated at 37°C for 1 to 2 hours, and then washed four times with prewarmed Dulbecco's PBS to remove nonadherent cells. After washing, cells were cultured overnight in DMEM containing 10% FBS and gentamicin (50 pg/ml) prior to use in experiments. Cells were washed with serum-free DMEM and then, unless otherwise specified, 50 pg/ml modified LDL was added in DMEM containing gentamicin (50 pg/ml) and 0.5 mg/ml fatty acid free bovine serum albumin (tissue culture grade) to minimize toxicity. After 24 hours of incubation with modified LDL, cell were washed with warm Dulbecco's PBS three times to remove LDL-containing medium. At this point, representative cultures were then taken for analysis. The remaining cultures were then incubated for a further 12 hours in DMEM (with gentamicin and BSA as specified above) 39 containing 75 ug/ml HDL3. Upon completion of this "efflux" incubation, these cultures were also taken for analysis. The cells that were removed for analysis both before and after the 12 hour "efflux" incubation were lysed by incubating in 1 ml of cold 0.1 M NaOH for 15 minutes at 4°C. The contents of the plate were then scraped off and collected in a glass test tube. Of this, 0.1 ml was removed and stored at -20°C for later protein assay, and the remainder was lipid extracted with chloroform and methanol according to the method of Bligh and Dyer (322). Lipid extracts were stored at -20°C until cholesterol analysis, which was usually performed within 24 hours. In the experiments in which we measured the retention of radiolabeled lipid or apoproteins, cells were dissolved in NaOH, scraped, and assayed for protein content as described above. The radioactivity of these cells was counted on either an LKB 1282 gamma spectrometer or a Beckman LS5000CE scintillation spectrometer, depending on whether radioactivity was emitted by 125I or 3H, respectively. In some cases, medium was collected and counted on the beta counter to measure the counts of 3H-cholesterol effluxed into the medium. Furthermore, degradation of 125I-labelled lipoproteins was measured using the degradation assay, described in section 2.6.6. 2.5 Microscopy Cells treated with fluorescently-labelled lipoproteins were cultured on sterile coverslips. Upon completion of the experiment, cells were washed and then fixed with PBS containing Ca2+ and 2% formaldehyde. A microscope slide was prepared by adding 7 pi of a Glycerol/PBS/DABCO solution to it before mounting the cover slip on it. The edges of the 40 coverslip were sealed with clear nail polish. These slides were then viewed and photographed using a Zeiss Axioskop fluorescence microscope for transmitted light and incident light fluorescence microscopy. For experiments related to retention of NBD-Q-sphingomyelin, cells were examined with a fluorescein filter set. A rhodamine filter set was used to image Texas Red sulphonyl chloride-labelled HDL3. Morphological observations of cultured macrophages were made by inverted phase-contrast microscopy. Cell viability was established by trypan blue staining at the end of either the 24 hour "loading" incubation or the 12 hour "efflux" incubation. Counting was preformed twice in representative plates, and the result was averaged in each case. Both acetylated LDL-loaded cells and oxidized LDL-loaded cells showed a viability of 90%. 2.6 Experimental Assays 2.6.1 Protein determination Protein was assayed by the method of Lowry (315) in presence of 0.05% sodium deoxycholate to minimize turbidity. Bovine serum albumin was used as the standard. 2.6.2 Colorimetric cholesterol assay Lipid extracts were dissolved in isopropanol and 50 pi aliquots were assayed for total cholesterol and free cholesterol using MPR 1 colorimetric enzymatic kits (Boehringer Mannheim; catalog numbers 1 442 341 and 310 328) with a final reaction volume of 1 ml. 41 Absorbances of the colored product were read at 500 nm using an LKB 4054 Ultrospec Plus spectrophotometer. Cholesterol standards were purchased from Boehringer Mannheim. Both of these assays are based on the assay method originally described by Trinder (323, 324). 2.6.3 Fluorimetric cholesterol assay Lipid extracts were dissolved in DMSO and 50 pi aliquots were assayed for total cholesterol and free cholesterol using the Amplex Red cholesterol assay (Molecular Probes; catalog number A-12216) with a final reaction volume of 2 ml. After excitation at 540 nm, the fluorescence emission of the product, resorufin, was measured at 585 nm using a Turner Model 450 fluorometer, as previously described (325). Cholesterol standards were purchased from Boehringer Mannheim. 2.6.4 Phosphorus assay The phosphorus content of both HDL3 and trypsinized HDL3 was assayed according to the method described by Rouser et al. (326). Briefly, lipoprotein samples were lipid extracted with chloroform and methanol (322), and the organic phase was dried under nitrogen. The extracted lipid was dissolved in 0.45 ml perchloric acid by heating at 180°C for 30 minutes, and subsequently treated with 2.5 ml distilled water, 0.5 ml of 2.5% ammonium molybdate, and 0.5 ml of 10% ascorbate. Samples were then incubated in a 37°C 42 water bath for 15 minutes, and the absorbances read at 820 nm using an LKB 4054 Ultrospec Plus spectrophotometer. Na2HP04 was used as the standard. 2.6.5 Degradation assay The degradation of lipoproteins by cultured macrophages was measured. Resident peritoneal macrophages harvested from CD-I Swiss mice were cultured as previously described (86, 123). To measure the degradation of various modified LDL's, the LDL-containing medium was harvested at the end of the 24 hour "loading" incubation. To measure the degradation of HDL3, the HDL3-containing medium was harvested at the end of the 12 hour "efflux" incubation. Lipoprotein degradation was estimated as the amount of trichloroacetic acid-soluble noniodide radioactivity in the medium. Undegraded lipoprotein was precipitated with 10% trichloroacetic acid in the presence of 3% BSA as a carrier. Free iodine was removed by precipitation with 2% AgN03. Radioactivity in the supernatant, representing iodotyrosine and peptide fragments, was counted in an LKB 1282 gamma spectrometer. The cells were washed and dissolved in 0.1 M NaOH. Aliquots were taken for determination of cell-associated radioactivity and protein content. Degradation rates were corrected by subtraction of values obtained in cell-free control dishes incubated in parallel. 2.6.6 ER and lysosome enzyme assays In the subcellular fractionation experiments, endoplasmic reticulum and lysosomes were identified in density gradient fractions by fluorescent assay of neutral a-glucosidase 43 and N-acetylglucosaminidase activities, respectively, using the corresponding 4-methylumbelliferyl substrates (327, 328). Latency of N-acetyl (3-glucosaminidase was determined as the ratio of activity in post-nuclear supernatant without Triton X-100 to that with 0.1% Triton X-100. 2.7 Subcellular Fractionation Resident peritoneal macrophages harvested from CD-I Swiss mice were cultured as previously described (86, 123). Upon completion of the 24 hour "loading" incubation in the presence of either radiolabeled acetylated LDL or radiolabelled oxidized LDL, the cells were washed three times with Dulbecco's PBS and representative cultures were removed and prepared for subcellular fractionation. The remaining plates were incubated for a further 12 hours in the presence of HDL3, as previously described. Finally, these plates were also washed three times with Dulbecco's PBS and prepared for subcellular fractionation. In order to prepare cells for subcellular fractionation, they were washed twice with ice-cold Dulbecco's PBS and incubated with Dulbecco's PBS containing 0.02% EDTA for 10 minutes at 4°C. Macrophages were disrupted and fractionated essentially as described by Dean (329). Cells were gently removed from the plates using cell lifters and sedimented at 1100 X g for 10 minutes at 4°C. Between 18 and 20 million cells were resuspended in 2 ml cold 10 mM Tris, 0.25 M sucrose, 1 mM EDTA (pH 7.5) and disrupted by nitrogen cavitation at 445 kPa for 15 minutes in a precooled Kontes mini cell disruption chamber (Mandel Scientific, Edmonton, AB). Nuclei, mitochondria and intact cells were removed by centrifugation at 100 X g for 5 minutes at 4°C. An aliquot of the resultant post-nuclear supernatant was reserved for latency determination and the remainder was mixed with 10 mg of BSA and layered over 10 ml of 40% Percoll in 10 mM Tris, 0.25 M sucrose, 1 mM EDTA 44 (density 1.070 g/ml) on a cushion of 0.5 ml 2 M sucrose. Self-forming gradients were generated by centrifugation at 20,000 rpm for 3 hours at 4°C in a Beckman SW-40 Ti rotor. Fractions of 0.5 ml were collected from the bottom of the gradient and assayed for radioactivity using a Beckman LS5000CE scintillation spectrometer. Individual fractions were frozen at -20°C and assayed the following day for neutral a-glucosidase and N-acetylglucosaminidase. Density profiles of blank gradients spun in parallel were measured using a digital densitometer (Paar, Austria). 2.8 Thin Layer Chromatography In order to determine whether free or esterified cholesterol was effluxed into the medium. Macrophages were loaded with either [3H]-acetylated LDL or [3H]-oxidized LDL, washed, and then treated with HDL3-containing medium. The efflux medium was lipid extracted (322) and dried under nitrogen. Lipids were separated by thin-layer chromatography (TLC) on silica TLC plates using a solvent system of hexane : diethyl ether : acetic acid (80 : 20 : 1, vol/vol). Bands were visualized with iodine vapor and zones corresponding to cholesterol, cholesterol oleate, and 7-ketocholesterol were scraped, mixed with Aquasol scintillation fluid, and counted in a Beckman LS5000CE scintillation spectrometer. 45 2.9 Statistical Analysis Statistical significance was determined using a two-sample student's t-test for paired data. A value of p < 0.05 was considered to indicate a significant difference. Unless otherwise indicated, all data were expressed as the mean ± standard deviation of the mean. 46 Chapter 3 Results and Discussion There is substantial evidence suggesting a role for LDL oxidation in the development of atherosclerosis. The biologic effects of oxidized LDL on macrophages include accelerated uptake of oxidized LDL (210, 246, 247), lipid accumulation (128, 238) and ultimately, foam cell formation (94, 217, 247). As described in the introduction, a hallmark feature of atherosclerotic lesions is the presence of oxidized LDL-containing foam cells in the arterial intima. Most of the lipid within foam cells is derived from plasma LDL cholesterol (11). The first step in reverse cholesterol transport is the movement of cholesterol out of cells onto lipoprotein acceptors in the interstitial fluid. When compared to macrophages loaded with acetylated LDL, cells loaded with oxidized LDL exhibit impaired efflux of cholesterol to an extracellular acceptor such as HDL (300). The objective of this thesis was to gain a better understanding of the mechanism whereby oxidized LDL causes impaired efflux of cholesterol from macrophages. After determining the degree of cholesterol efflux in cells loaded with either oxidized LDL or acetylated LDL, we investigated the relative importance of the lipid component and the protein component of the lipoproteins involved in the process of lipid accumulation and efflux. In addition, the subcellular location of both oxidized LDL-derived and acetylated LDL-derived cholesterol was identified in macrophages before and after HDL-mediated cholesterol efflux. 47 3.1 Cholesterol content of modified LDL Acetylation and 24-hour copper oxidation are well-described LDL modifications that are known to result in recognition by scavenger receptors and foam cell formation (217). Copper-mediated oxidation of LDL results in profound alterations of both the lipid and protein compositions of LDL, including fragmentation of apolipoprotein B, hydrolysis of phosphatidylcholine, and loss of esterified cholesterol (210, 330). Consequently, cells loaded with acetylated LDL accumulated large quantities of cholesterol esters, whereas cells loaded with oxidized LDL accumulated a much smaller proportion of esterified cholesterol. As mentioned in the introduction, studies with oxidized LDL have used either mildly oxidized LDL or extensively oxidized LDL. There is a lack of evidence for the presence of extensively oxidized LDL in human atherosclerotic plaques. Mildly oxidized LDL has been extracted from human atheroma (331), but it was found to contain 7-ketocholesterol, which is a product of extensive, not mild, LDL oxidation (330, 332, 333). Such findings have made it difficult to deduce the degree of in vitro LDL oxidation that is most physiologically relevant. The oxidized LDL discussed in this thesis is extensively oxidized. The composition of modified LDL's determines their atherogenicity. For this reason, it is useful to analyze the protein and cholesterol content of the principal modified LDL's discussed in this thesis (Table 5). 48 Table 5 Relative composition and electrophoretic mobility of modified LDL FC/protein TC/protein Rf value (ug cholesterol (ug cholesterol (relative to native LDL) per mg protein) per mg protein) native LDL 0.39±0.04 1.64±0.10 1 unlabelled acetylated LDL 0.40±0.06 1.38+0.25 4.77 radiolabeled acetylated LDL 0.37±0.09 1.55+0.19 4.77 unlabelled oxidized LDL 0.39±0.03 0.87±0.11* 4.09 radiolabeled oxidized LDL 0.28±0.07* 0.56±0.14* 3.09 FC indicates Free Cholesterol; TC indicates Total Cholesterol; and R f is the relative electrophoretic mobility in agarose. In this instance, the modified LDL's were radiolabeled after being either acetylated or oxidized; the radiolabeled LDL's contained [3H]-cholesteryl linoleate. Protein and cholesterol contents were determined in the manner described in Chapter 2. Data represent the mean ± S.D. of three analyses of a single lipoprotein preparation. * p < 0.01 when compared to native LDL (by paired two-sample student's t-test). From the values in Table 5, it can be seen that the composition of acetylated LDL is not significantly different from the composition of unmodified LDL. In agreement with previous reports, oxidized LDL was found to contain significantly less cholesterol ester than both native and acetylated LDL (211, 309, 334). The loss of this hydrophobic core of lipids results in a shift of the average hydrated density from 1.033 g/ml to 1.070 g/ml (211). The proportion of free cholesterol remains essentially unchanged in the unlabelled oxidized LDL. It is possible that a portion of the difference in the cholesterol ester content of oxidized LDL compared to native LDL is due not only to an actual difference in cholesterol ester content but also due to a difference in detection. The enzymatic assays that were used to measure cholesterol do not measure oxidized cholesterols as well as they detect unmodified 49 cholesterol. In fact, we have found that the assay underestimates the 7-ketocholesterol content by a factor of about eight. Acetylated and oxidized LDL's have a greater Rf value than native LDL because these chemical modifications involve derivatization of lysine groups by adducts that neutralize the positively charged e-amino groups on apolipoprotein B (11, 210). The Rf value of acetylated LDL did not change after being labelled with [3H]-cholesteryl linoleate. However, labelling oxidized LDL particles by this technique does alter them to some extent. Radiolabeled oxidized LDL ran as a broad band with a lower Rf value than unlabelled oxidized LDL (Figure 7). This suggests that the radiolabelling procedure, which involves exchange with a microemulsion (308), may strip some of the oxidatively modified apo B fragments off the LDL particles. We observed a similar effect when oxidized LDL was incubated with native LDL, so the microemulsions themselves do not appear to be the cause of this. Moreover, the uptake of radiolabeled oxidized LDL by macrophages was only slightly lower than that of unlabelled oxidized LDL, and the cholesterol from radiolabeled oxidized LDL was retained within the cells to the same extent that unlabelled oxidized LDL was. Hence, oxidized LDL that is radiolabeled in this fashion can serve as a useful tool both to measure the degree of cholesterol efflux from oxidized LDL-loaded cells and to determine the subcellular localization of cholesterol delivered by oxidized LDL. 50 Figure 7 Agarose gel electrophoresis of labelled and unlabelled modified LDL's Abbreviations are as follows: LDL, low density lipoprotein; acLDL, acetylated LDL; 3H-acLDL, acetylated LDL that has been labelled with [3H]-cholesteryl linoleate; oxLDL, oxidized LDL; and 3H-oxLDL, oxidized LDL that has been labelled with [3H]-cholesteryl linoleate. The R, values for these lipoproteins are given in Table 5. 3.2 Oxidized LDL causes impaired efflux of cholesterol from Macrophages In order to observe meaningful levels of cholesterol efflux, we found that at least a two-fold increase in total cholesterol compared to the baseline levels was required. To establish optimal conditions for cholesterol-loading of macrophages and subsequent measurement of cholesterol efflux, cells were loaded with oxidized LDL for 24 hours and then subsequently treated for an additional 12 hours with 75 pg/ml HDL3. Figure 8 shows the results of a representative experiment out of three. 51 ug of Total Cholesterol per mg of cell protein Figure 8 E2 Cholesterol Ester • Free Cholesterol 300 2501 200 1 150 100 B Concentration of oxidized LDL used (in ug/ml) Oxidized LDL concentration is proportional to lipid accumulation. Each bar represents the micrograms of total cholesterol per milligram of cell protein; within each bar the solid area represents the free cholesterol content of the cells, and the hatched area represents the cholesterol ester content of the cells. Panel A depicts macrophages that were loaded for 24 hours with 0, 5, 10, 25 or 50 ug/ml of oxidized LDL. Panel B represents macrophages that were treated in the same manner as the cells in Panel A, only they underwent a further 12 hour incubation in the presence of 75 ug/ml HDL 3 . At these levels, the greater the oxidized LDL concentration, the greater the cholesterol accumulation within the cells. With respect to HDL-mediated cholesterol efflux, there was no significant decrease in total cholesterol in any of the plates, except for the plates that were loaded with 25 ug/ml oxidized LDL (* p < 0.05) compared to its counterpart which was not exposed to HDL 3. The values are mean ± S.D. for triplicate plates from one of three experiments with similar results. 52 At the end of the first 24 hour incubation in the presence of varying concentrations of oxidized LDL, half of the cells were extracted and assayed for protein and cholesterol content, and the remaining plates were washed and then treated with HDL3 for an additional 12 hours, after which they too were processed in the same manner as the first set. It can be seen that there was no significant increase in total cholesterol after 24 hours of incubation with 5 pg/ml oxidized LDL. The cells loaded with 10 pg/ml oxidized LDL exhibited a less than two-fold increase in total cholesterol; those loaded with 25 pg/ml oxidized LDL exhibited a nearly three-fold increase in total cholesterol; and finally, the macrophages loaded with 50 pg/ml oxidized LDL exhibited a greater than three-fold increase in total cholesterol. Although, the cholesterol efflux from the plates loaded with 25 pg/ml is statistically significant (p < 0.05), it still amounts to only 11.2% of the total cholesterol in the lipid-loaded cells. The degree of cholesterol efflux in the cells loaded with 10 pg/ml or 50 pg/ml oxidized LDL was also approximately 10% of the total cholesterol in the lipid-loaded cells. There was a 2.8% decrease in total cholesterol in the cells loaded with 5 pg/ml oxidized LDL. In order to keep the cells viable during these long incubations, the medium was supplemented with 0.5 mg/ml bovine serum albumin (BSA) in all experiments described in this thesis. It was found that 5% fetal bovine serum (FBS) was also effective in maintaining viability; however, FBS has the disadvantage of containing HDL, and it was found to be capable of bringing about efflux during the initial 24 hour "loading" of modified lipoproteins. Although lipoprotein-deficient serum has been used by others in such experiments (300), we did not find it to be effective in maintaining cell viability. Even with the BSA-supplementation of the medium, we found that oxidized LDL concentrations greater than 65 53 pg/ml appeared to be cytotoxic to macrophages and were associated with blebbing, cell detachment, and a decrease in the proportion of cells that excluded trypan blue (data not shown). Since 50 pg/ml of oxidized LDL was not cytotoxic to the cells and it resulted in massive accumulation of cholesterol, it was deemed to be the most appropriate concentration to be used in determining the efficiency of cholesterol efflux from these lipid-loaded cells. In all experiments discussed in this thesis, cells were plated either in 35 mm diameter (Falcon) or 100 mm diameter (Corning) tissue culture plates at a density of either 6.0 X 106 cells/plate or 2.0 X 107 cells/plate, respectively. The total protein per plate was similar for both the acetylated LDL and the oxidized LDL-treated cells. In many cases, the control plates had lower cell numbers and consequently lower protein values at the end of the 24 hour incubation compared to the plates loaded with modified lipoproteins. The protein values did not change appreciably during the final 12 hour incubation with HDL3. Although most of the control cells remained small and rounded throughout the experiment, the cells loaded with modified lipoproteins underwent morphological changes. Macrophages loaded with either acetylated LDL or oxidized LDL exhibited a substantial increase in size and they adhered to tissue culture plates better than control cells did. In addition, macrophages loaded with oxidized LDL developed long cytoplasmic projections. In order to determine the effect of incubation time on cholesterol accumulation and efflux, macrophages were loaded with 50 pg/ml of oxidized LDL for 0, 6, 12, 18, and 24 hours, and then subsequently treated with 75 pg/ml HDL3 for an additional 12 hours. Figure 9 shows the results of a representative experiment out of three. 54 Cholesterol Ester Free Cholesterol ug of Total Cholesterol per mg of cell protein Figure 9 Incubation time with 50 ug/ml oxidized LDL (in hours) Effect of incubation time on lipid accumulation and efflux. Each bar represents the micrograms of total cholesterol per milligram of cell protein; within each bar the solid b area represents the free cholesterol content of the cells, and the hatched area represents the cholesterol ester content of the cells. Panel A depicts macrophages that were loaded with 50 ug/ml oxidized LDL for 0, 6, 12, 18 or 24 hours. Panel B represents macrophages that were treated in the same manner as the cells in Panel A, only they underwent a further 12 hour incubation in the presence of 75 pg/ml HDL 3 . Maximal lipid accumulation occurred at 18 hours of lipid-loading, but the cholesterol concentration in the cells loaded for 24 hours was not significantly lower. With respect to HDL-mediated cholesterol efflux, there was a significant decrease in total cholesterol in two of the conditions. The plates loaded for 6 hours and then exposed to HDL 3 exhibited a significant decrease (* p < 0.01) in total cholesterol compared to the corresponding plates that were not treated with HDL 3 . Similarly, the plates loaded for 24 hours and then exposed to HDL 3 exhibited a significant decrease (** p < 0.001) in total cholesterol compared to the corresponding plates that were not treated with HDL 3 . The values are mean ± S.D. for triplicate plates from one of three experiment with similar results. 55 At the end of the first incubation in the presence of 50 pg/ml oxidized LDL, half of the cells were extracted and assayed for protein and cholesterol content, and the remaining plates were washed and then treated with HDL3 for an additional 12 hours, after which they were processed in the same manner as the first set. It can be seen from Figure 9 that cholesterol accumulation increases as a function of duration of exposure to oxidized LDL up to a maximum of 18 hours, where it plateaus. All of the cells loaded with oxidized LDL exhibited a two- to three-fold increase in total cholesterol. Compared to the cells that did not undergo the 12 hour "efflux incubation", the cells that were exposed to HDL3 after being loaded with oxidized LDL for 6, 12, 18, and 24 hours exhibited a decrease in total cholesterol of 3%, 11%, 12%, and 12%, respectively. However, statistically significant decreases in total cholesterol were observed only in the cells loaded for 6 hours (* p < 0.01) and 24 hours (** p < 0.001). Since an incubation time of 24 hours resulted in a massive accumulation of cholesterol, it was deemed to be the most appropriate concentration to be used in determining the efficiency of cholesterol efflux from these lipid-loaded cells. Longer incubations times, such as 48 hours, have been used for such experiments, under different conditions (335); however, we have found that increasing the incubation time beyond 24 hours did not result in a greater accumulation of cholesterol within these macrophages, but it did lead to a decrease in viability (data not shown). The ability of high density lipoproteins to remove excess cholesterol from cells is well established (31, 114), but how this occurs has been disputed (77, 336). However, it is becoming increasingly accepted that removal of cellular cholesterol occurs by at least two pathways: one involving aqueous diffusion, and another promoted by cellular interaction with apolipoproteins (114). The role of plasma HDL as the preferential physiological 56 acceptor for cholesterol from extrahepatic cells in vivo has been strongly supported by epidemiologic and in vitro studies (23, 25, 27, 28, 80, 249, 337-349). Compared to other HDL fractions separated by density, HDL3 has been proposed to be the major promoter of cholesterol efflux, and it is for this reason that HDL3 was used in all of the experiments presented in this thesis (80-82). In order to observe meaningful levels of cholesterol efflux, the HDL3 concentration must not be a rate-limiting step. To determine the most appropriate concentration of HDL3 to incubate the cells with, several different concentrations were used and the efflux of radiolabeled cholesterol into the medium from lipid-loaded. cells was measured (Figure 10). At the end of the 24 hour incubation in the presence of 50 pg/ml radiolabeled acetylated LDL or 50 pg/ml radiolabeled oxidized LDL, the cells were washed and then incubated for an additional 12 hours in the presence of varying concentrations of HDL3. From Figure 10, we can see that cholesterol efflux increases as a function of HDL3 concentration up to a maximum of 60 pg/ml HDL3, where it plateaus. The release of [3H]-cholesterol into the medium was paralleled by its loss from the cells (data not shown). We see impaired efflux of cholesterol from the cells loaded with oxidized LDL compared to those loaded with acetylated LDL. It is interesting to note that there was a small degree of [3H]-cholesterol efflux in the absence of HDL; it occurred undoubtedly by simple diffusion from the cell membrane to the BSA in the medium. Kritharides et al. reported that maximal efflux of cholesterol from lipid-loaded cells occurred at apolipoprotein A-I concentrations as low as 20 pg/ml (300). We, on the other hand, found that HDL3 concentrations of 40 to 60 pg/ml were required to achieve maximal cholesterol efflux from lipid-loaded cells. This difference may, in part, be due to the higher total cholesterol content of our cells prior to 57 treatment with HDL3. We deemed 75 |ig/ml HDL3 to be an appropriate concentration to be used in determining the efficiency of cholesterol efflux from these lipid-loaded cells. While this is more than an order of magnitude lower than the concentration of HDL in human plasma, it is well above the estimated for HDL-mediated cholesterol efflux in cultured murine macrophages (300). HDL concentration (in ug/ml) Figure 10 Concentration-dependence of HDL-mediated cholesterol efflux. Each point represents the mean dpm's of [3H]-cholesterol measured in the medium after 12 hours of exposure to HDL 3. In this experiment, cells were loaded with 50 ug/ml of either radiolabeled acetylated LDL or radiolabeled oxidized LDL. Then, the cells were washed and exposed to varying concentrations of HDL 3 for 12 hours, at the end of which, the radioactivity in the medium was measured. For this experiment, LDL was radiolabeled with [3H]-cholesteryl linoleate before being either acetylated or oxidized. The values are mean ± S.D. for triplicate plates from one representative experiment out of three with similar results. Error bars not visible are within the plot symbols. 58 In order to determine the effect of HDL incubation time on cholesterol efflux, macrophages were loaded for 24 hours with 50 pg/ml of either radiolabeled acetylated LDL or radiolabeled oxidized LDL, and then subsequently treated with 75 pg/ml HDL3 for 6, 12, and 24 hours; efflux of [3H]-cholesterol into the medium was measured. Figure 11 shows the results of one representative experiment out of two. Time (in hours) Figure 11 Effect of HDL incubation time on cholesterol efflux. Each point represents the mean dpm's of [3H]-cholesterol measured in the medium after exposure to HDL 3. In this experiment, cells were loaded with 50 ug/ml of either radiolabeled acetylated LDL or radiolabeled oxidized LDL. Then, the cells were washed and exposed to 75 pg/ml of HDL 3 for 6, 12 or 24 hours, at the end of which, the radioactivity in the medium was measured. For this experiment, LDL was radiolabeled with [3H]-cholesteryl linoleate before being either acetylated or oxidized. The values are mean ± S.D. for triplicate plates from one representative experiment out of two with similar results. Error bars not visible are within the plot symbols. At the end of the 24 hour incubation in the presence of 50 pg/ml radiolabeled acetylated LDL or 50 pg/ml radiolabeled oxidized LDL, the cells were washed and then incubated with 75 pg/ml HDL3 for an additional 6, 12 or 24 hours. From Figure 11, we can 59 see that there was a progressive, time-dependent appearance of [3H]-cholesterol in medium containing 75 pg/ml HDL3, and it did not appear to be complete by 24 hours. This finding is supported by the literature (300). Since an HDL incubation time of 12 hours resulted in a substantial efflux of cholesterol from acetylated LDL-loaded cells, whereas an incubation time or 24 hours was associated with a decrease in viability (data not shown), 12 hours was deemed to be the optimal duration to be used in determining the efficiency of cholesterol efflux from these lipid-loaded cells. The transport pathways through which cholesterol is recycled between the endoplasmic reticulum and the plasma membrane are poorly characterized (350-354). It has been suggested that cholesterol removed from cells by long-term incubations (> 6 hours) with HDL may pass through the Golgi apparatus (355, 356). In order to further characterize HDL-mediated cholesterol efflux, we explored the possible involvement of the Golgi by treating acetylated LDL-loaded macrophages with an inhibitor of Golgi transport before exposing them to HDL3. Essential to the many cellular functions that take place within the central vacuolar system (which consists of the ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic, which mediates the exchange of components between different organelles (357-359). Brefeldin A (BFA) inhibits anterograde transport from the endoplasmic reticulum to the Golgi apparatus (360) and in contrast, it enhances retrograde transport from the Golgi apparatus to the endoplasmic reticulum (361). Although these have been the best characterized actions of BFA, its effects are not limited to the Golgi apparatus. For example, one of the hallmarks of the morphological effects of BFA is the rapid and dramatic induction of tubules not only in the Golgi apparatus, but also in peripheral organelles including endosomes, lysosomes, and the trans-Golgi network (362-60 364). The effects of BFA are concentration dependent, as high concentrations of BFA are cytotoxic to cells. In our experiments, we found that concentrations greater than 3.5 pg/ml were cytotoxic. In this experiment, macrophages were loaded for 24 hours with no lipoprotein (control) or 50 pg/ml acetylated LDL. Representative cultures were then washed with PBS and removed for analysis. After being washed with PBS, the remaining plates were treated for 30 minutes with either 2.25 pg/ml Brefeldin A (dissolved in ethanol) or an equivalent volume of ethanol alone. Finally, these plates were exposed to 75 pg/ml HDL3 for 12 hours. Prior to incubation with acetylated LDL, cells had only 63 pg free cholesterol/mg cell protein and the esterified cholesterol content of these cells was negligible. After 24 hours, total cholesterol increased to 326 pg cholesterol/mg cell protein — a five-fold augmentation over the baseline level. Figure 12 depicts the degree of cholesterol efflux observed in each case. We found that treating acetylated LDL-loaded cells with inhibitors of Golgi transport completely abolished the ability of HDL3 to stimulate cholesterol efflux. This is in agreement with the findings of Mendez et al. (355, 356). It has been suggested that free cholesterol is transported from the endoplasmic reticulum and/or lysosomes to the Golgi apparatus and then assembled in phospholipid-rich vesicles that are subsequently transported to the cell surface. Upon fusion with the plasma membrane, these vesicles may form discrete membrane domains with unique compositions of lipids and proteins, similar to what has been described for caveolae (365, 366). 61 36 33 30" 27 -Degree of Cholesterol 2 4 Efflux (expressed as % of Total Cholesterol) 1 5 12 2 H 18H no inhibitor BFA-treated Figure 12 Comparison of cholesterol efflux in normal and Brefeldin A-treated cells. Each bar represents the percent decrease in total cholesterol after HDL-mediated cholesterol efflux in cells that had been loaded with acetylated LDL. Prior to exposure to HDL, cells were treated for 30 minutes with either 2.25 ug/ml of Brefeldin A (in ethanol) or the equivalent volume of ethanol alone. The acetylated LDL-loaded cells that had not been exposed to Brefeldin A exhibited a significant (p < 0.0001) decrease in total cholesterol upon exposure to HDL 3 . Furthermore, there was a statistically significant (p < 0.05) difference in the degree of cholesterol efflux in the BFA-treated cells compared to the untreated cells. Each value represents the mean percentage difference ± SD from triplicate plates from one of two experiments with similar results. 62 When resident peritoneal macrophages isolated from CD-1 mice were incubated for 24 hours with either 50 pg/ml acetylated LDL or 50 ug/ml oxidized LDL, there was an increase in both esterified cholesterol and free cholesterol compared to control cells which were given no lipoproteins during the same time period. After the initial 24 hours incubation, cells were washed and then exposed to 75 pg/ml HDL3 for 12 hours. This resulted in a significant (* p < 0.0001) decrease in total cholesterol from the acetylated LDL-loaded cells compared to the acetylated LDL-loaded cells that had not been exposed to HDL3. In contrast, there was no significant decrease in total cholesterol in the oxidized LDL-loaded cells compared to the oxidized LDL-loaded cells that had not been exposed to HDL3. Figure 13 shows the results of one representative experiment out of thirty-six. At the end of the first 24 hour incubation in the presence or absence of modified lipoproteins, half of the cells were extracted and assayed for protein and cholesterol content, and the remaining plates were washed and then treated with HDL3 for an additional 12 hours, after which they were processed in the same manner as the first set. As would be expected from the composition of these.modified LDL's (Table 5), the acetylated LDL-loaded cells accumulated large quantities of cholesterol ester, as well as free cholesterol, whereas the oxidized LDL-loaded cells accumulated predominantly free cholesterol. The increase in total cholesterol in the acetylated LDL-loaded macrophages represented a five-fold augmentation over the baseline level. Similarly, the increase in total cholesterol in the oxidized LDL-loaded macrophages represented a greater than two-fold augmentation over the baseline level. In such experiments, we have demonstrated that mouse peritoneal macrophages incubated with either acetylated LDL or oxidized LDL typically exhibit a two-fold to six-fold augmentation of their total cholesterol compared to control (nonloaded) cells. Indeed, many 63 0 Cholesterol Ester Figure 13 Total cholesterol content of cells after lipid-loading and exposure to HDL3. Each bar represents the micrograms of total cholesterol per milligram of cell protein; within each bar the solid area represents the free cholesterol content of the cells, and the hatched area represents the cholesterol ester content of the cells. Panel A depicts macrophages that were loaded for 24 hours with no lipoprotein, acetylated LDL, or oxidized LDL. Panel B represents macrophages that were treated in the same manner as the cells in Panel A, only they underwent a further 12 hour incubation in the presence of HDL 3 . There was a massive accumulation of cholesterol in the macrophages loaded with modified LDL compared to the control cells. The acetylated LDL-loaded macrophages exhibited a significant (* p < 0.0001) decrease in total cholesterol upon exposure to HDL 3 , whereas the decrease in cholesterol in the oxidized LDL-loaded cells was not statistically significant. Each value represents the mean ± S.D. for three determinations from one typical experiment out of thirty-six with similar results. 64 other studies have also reported that acetylated LDL and oxidized LDL stimulate significant (100-600 pg/mg cell protein) cholesterol accumulation in macrophages from many species (11, 300, 335, 367-370). In comparison to acetylated LDL-loaded cells, we have consistently found that oxidized LDL-loaded cells exhibit a significant impairment of HDL-mediated cholesterol efflux. This is in agreement with the findings of Kritharides et al. (300). Because of the precision of the protein and cholesterol assays, the relatively small changes in cholesterol observed in oxidized LDL-loaded cells after treatment with HDL3 often failed to achieve significance in individual experiments. However, when we compared the decrease in total cholesterol in oxidized LDL-loaded cells to the decrease measured in acetylated LDL-loaded cells, the difference was always statistically significant. When we averaged the data from experiments in which there was a nearly identical degree of loading, we found that the cholesterol efflux from oxidized LDL-loaded cells amounted to about 10% of the total cholesterol content of these cells (as depicted in Figure 14) — which is much lower than the approximately 30% decrease in total cholesterol observed in acetylated-LDL loaded cells that had been treated with HDL3. 65 3 5 3 0 ; 2 5 " Degree of Cholesterol 20-Efflux (expressed as % of Total 1 5 -Cholesterol) 10-5-o--acetylated L D L oxidized L D L Figure 14 Decrease in total cholesterol after HDL3-mediated cholesterol efflux. Each bar represents the percent decrease in total cholesterol after HDL-mediated cholesterol efflux in cells that had been loaded with either acetylated LDL or oxidized LDL. Each value represents the mean ± S.E. of triplicate determinations from three experiments with similar results. The design of the experiments used to generate the data in this figure is the same as the experimental design of Figure 13. Macrophages were incubated for 24 hours with no lipoprotein, 50 pg/ml acetylated LDL, or 50 pg/ml oxidized LDL. Half of the plates were removed and the contents were lipid extracted and analyzed for protein and cholesterol content. The remaining plates were washed and then incubated for a further 12 hours with 75 pg/ml HDL 3 . Finally, these cells were also lipid extracted and analyzed for protein and cholesterol content. There was a significant (* p < 0.01) difference between the degree of cholesterol efflux observed in the acetylated LDL-loaded cells compared to the degree of cholesterol efflux observed in the oxidized LDL-loaded cells. 66 3.3 Impairment of cholesterol efflux involves both lipid and protein modification of LDL Oxidized LDL is known to be susceptible to aggregation (371). Like acetylated LDL and oxidized LDL, uptake of vortex-aggregated LDL also leads to cholesterol accumulation within macrophages. In order to rule out aggregation as a cause of impaired cholesterol efflux from macrophages, the efflux of cholesterol from aggregated LDL-loaded macrophages was measured. Figure 15 depicts the results from one such experiment. At the end of the first 24 hour incubation in the presence or absence of modified lipoproteins, half of the cells were extracted and assayed for protein and cholesterol content, and the remaining plates were washed and then treated with HDL3 for an additional 12 hours, after which they were processed in the same manner as the first set. The increase in total cholesterol in the acetylated LDL-loaded macrophages represented a greater than four-fold augmentation over the baseline level. Similarly, the increase in total cholesterol in both the vortex-aggregated LDL-loaded macrophages and the oxidized LDL-loaded macrophages represented a five-fold augmentation over the baseline level. The HDL-mediated efflux of cholesterol from the aggregated LDL-loaded cells was intermediate (31% of total) between that of the acetylated LDL-loaded cells (51%) and the oxidized LDL-loaded cells (5%). This suggests that aggregation of LDL may contribute to the impaired efflux of cholesterol from oxidized LDL-loaded cells. However, it does not appear to be the predominant cause of the impaired efflux. 67 Figure 15 Accumulation and efflux of aggregated LDL-derived cholesterol. Each bar represents the micrograms of total cholesterol per milligram of cell protein. Panel A depicts macrophages that were loaded for 24 hours with no lipoprotein, 50 pg/ml acetylated LDL (acLDL), 50 pg/ml oxidized LDL (oxLDL) or 50 pg/ml vortex-aggregated LDL (aggreg. LDL). Panel B represents macrophages that were treated in the same manner as the cells in Panel A, only they underwent a further 12 hour incubation in the presence of HDL 3. There was a significant accumulation of cholesterol in the macrophages loaded with modified LDL compared to the control cells. Both the acetylated LDL-loaded macrophages and the vortex-aggregated LDL-loaded macrophages exhibited a significant (* p < 0.05) decrease in total cholesterol upon exposure to HDL 3. Each value represents the mean ± S.D. for four determinations from a single experiment. 68 The impairment of efflux of cholesterol from macrophages loaded with oxidized LDL suggests that the metabolism of oxidized LDL is different from that of acetylated LDL. In fact, it has been reported that the protein moiety of oxidized LDL is hydrolyzed at a much slower rate than that of acetylated LDL (129, 295, 304). In order to compare the rate at which macrophages take up and degrade each of these modified lipoproteins, cells were loaded with 125I-labelled modified LDL's, and then we determined the degradation of these lipoproteins as well as the cholesterol mass and radioactive LDL content of both loaded and effluxed cells. It has been debated whether extensive oxidation occurs in vivo. To determine whether mild oxidation could result in impairment of cholesterol efflux, we loaded cells with acetylated LDL that had been either mildly (5 hrs) or extensively (24 hrs) oxidized. It has been reported that the apolipoprotein component of oxidized LDL is resistant to lysosomal cathepsins, whereas the apolipoprotein component of acetylated LDL is hypersensitive to lysosomal cathepsins (129). The oxidation of acetylated LDL provides a molecule that contains an acetylated apolipoprotein component along with an oxidized lipid component. In acetylated LDL that has been subsequently oxidized, the apolipoprotein does get oxidized to some degree, but it is not oxidized nearly as extensively as oxidized LDL. Thus, it can serve as a useful tool because it allows us to deliver oxidized lipids without rendering the protein resistant to cathepsins. Figures 16, 17 and 18 depict the results from one of two experiments. 69 2 5 0 200 | j g of 150 r a d i o a c t i v e L D L p e r m g o f c e l l p r o t e i n 100 50 o CO X o o CO X o LO O CC X o CM o CO _ l _ l _ l Q Q Q _ l _ l _ i X o o o cp cp X X o o _^ _c LO OJ Figure 16 125 I-labelled modified LDL content of loaded and effluxed cells. Each bar represents the micrograms of radiolabeled modified LDL in the cells per milligram of cell protein. Panel A depicts macrophages that were loaded for 24 hours with 50 pg/ml of one of the following radiolabeled lipoproteins: acetylated LDL (acLDL), oxidized LDL (oxLDL), acetylated LDL that had been oxidized for 5 hours (5 hr ox-acLDL), or acetylated LDL that had been oxidized for 24 hours (24 hr ox-acLDL). Panel B represents macrophages that were treated in the same manner as the cells in Panel A, only they underwent a further 12 hour incubation in the presence of 75 pg/ml HDL 3 . The most pronounced increase in radioactivity occurred in the cells loaded with oxidized LDL (oxLDL), followed by the cells loaded with 24 hr ox-acLDL. With respect to HDL-mediated cholesterol efflux, there was a statistically significant (* p < 0.01) decrease in radioactive LDL in two of the conditions: the plates loaded with oxidized LDL and then exposed to HDL 3 exhibited a significant decrease in radioactive LDL compared to the corresponding plates that were not treated with HDL 3; similarly, the plates loaded with 24 hr ox-acLDL and then exposed to HDL 3 exhibited a significant decrease in radioactive LDL compared to the corresponding plates that were not treated with HDL 3. The values are mean ± S.D. for triplicate plates from one of two experiments with similar results. 70 150 125 H Each bar represents the micrograms of radioactive L D L degraded per milligram of cell protein, over the course of 24 hours. Cells loaded with acetylated L D L (acLDL) exhibited the highest degree of L D L degradation, followed by the cells that had been loaded with acetylated L D L that had been oxidized for 5 hours (5 hr ox-acLDL). The cells loaded with acetylated L D L that had been oxidized for 24 hours (24 hr ox-acLDL) degraded one-third less L D L than the cells loaded with acLDL. Finally the cells loaded with oxidized L D L (oxLDL) degraded less than half as much L D L as the cells loaded with acLDL. However, only the oxLDL-loaded cells exhibited a statistically significant difference in L D L degradation compared to acLDL-loaded cells (* p < 0.01). The values are mean ± S.D. for triplicate plates from one of two experiments with similar results. 71 Figure 18 Accumulation and efflux of cholesterol from various modified LDL's. Each bar represents the micrograms of total cholesterol per milligram of cell protein; within each bar the solid area represents the free cholesterol content of the cells, and the hatched area represents the cholesterol ester content of the cells. Panel A depicts macrophages that were loaded for 24 hours with either no lipoprotein or 50 ug/ml of one of the following: acetylated LDL (acLDL), acetylated LDL that had been oxidized for 5 hours (5hr ox-acLDL), acetylated LDL that had been oxidized for 24 hours (24hr ox-acLDL), or oxidized LDL (oxLDL). Panel B represents macrophages that were treated in the same manner as the cells in Panel A, only they underwent a further 12 hour incubation in the presence of 75 ug/ml HDL 3 . Compared to the control cells, there was a statistically significant accumulation of cholesterol in all of the lipid-loaded macrophages. After treatment with HDL 3, there was a decrease in total cholesterol in all of the cells except the cells loaded with oxidized LDL. However, a statistically significant decrease in total cholesterol occurred in only two of the conditions: the plates loaded with acetylated LDL and then exposed to HDL 3 exhibited a significant decrease (** p < 0.001) in total cholesterol compared to the corresponding plates that were not treated with HDL 3; 72 similarly, the plates loaded with 5 hr ox-acLDL and then exposed to HDL 3 exhibited a significant decrease (* p < 0.01) in total cholesterol compared to the corresponding plates that were not treated with HDL 3 . The values are mean ± S.D. for triplicate plates from one of two experiments with similar results. Protein was labelled by radioiodination (311). In the experiment depicted in Figures 16 and 17, we measured the cellular content and degradation of modified apolipoprotein B. In Figure 16, we see a massive accumulation of 125I-labelled oxidized apo B derived from oxidized LDL. In contrast, there was very little accumulation of 125I-labelled acetylated apo B derived from acetylated LDL or acetylated LDL that had been oxidized for 5 hours. However, there was an appreciable accumulation of 125I-labelled acetylated apo B derived from acetylated LDL that had been oxidized for 24 hours. Since we have seen massive accumulation of cholesterol in macrophages loaded with acetylated LDL, the results from this experiment provide further evidence that the protein component of acetylated LDL is metabolized differently from the lipid component; the results also confirm that the protein component of acetylated LDL is hypersensitive to lysosomal degradation and therefore, very little accumulation of acetylated apo B occurs. On the other hand, the large accumulation of oxidized apo B within the cells suggests that the protein component of oxidized LDL is resistant to lysosomal degradation, and that is why it accumulates within the macrophages. Following a 12 hour exposure to HDL3, we saw a statistically significant decrease in radiolabeled modified apo B from the plates loaded with either oxidized LDL or acetylated LDL that had been oxidized for 24 hours. This may seem in opposition to our results which showed that oxidized LDL-derived macrophages exhibit impaired efflux of cholesterol; however, it must be recognized that although three-quarters of the accumulated oxidized apo B was removed from the cells upon treatment with HDL3, the oxidized apo B that remained within the cells was still more than 20-fold higher than the acetylated apo B that remained 73 within the cells. Nevertheless, it is possible that the rapid decrease in cell-associated oxidized apolipoprotein B during the 12 hour "efflux" incubation involves exocytosis of undegraded oxidized LDL. If this were the case, the efflux medium would contain not only free cholesterol but also esterified cholesterol and oxysterols. To test this hypothesis, LDL was labelled with [3H]-cholesteryl linoleate before being oxidized. Macrophages were treated with 50 pg/ml of this radiolabeled oxidized LDL for 24 hours, washed and then treated with 75 pg/ml HDL3 for 12 hours. The efflux medium was removed, lipid extracted, and run on thin layer chromatography. The spots corresponding to free cholesterol, cholesterol oleate, cholesterol linoleate, and 7-ketocholesterol were scraped and counted on a Beckman LS5000CE scintillation spectrometer. The radioactivity of each spot is presented in Table 6 as the mean ± SD of triplicate plates from one single experiment. Table 6 Sterol efflux into the medium from oxidized LDL-loaded macrophages cholesterol 4230 ± 423 dpm's cholesterol oleate 30 ± 18 dpm's cholesterol linoleate 12 ± 12 dpm's 7-ketocholesterol 7 ± 19 dpm's The same experiment was conducted using radiolabeled acetylated LDL, and although a decrease in cellular cholesterol ester content occurred, we found that only free cholesterol appeared in the medium, not cholesterol ester (data not shown). This is consistent with previous reports that any decrease in cholesterol ester content of cells is the result of hydrolysis of the ester rather than efflux of esterified cholesterol (347). Furthermore, the 74 appearance of only free cholesterol in the efflux medium opposes the hypothesis that exocytosis of undegraded oxidized LDL occurs. In Figure 17, we see that the acetylated apo B is efficiently degraded but the oxidized apo B is not. Although the 5 hr ox-acLDL-loaded cells and the 24 hr ox-acLDL-loaded cells degraded less LDL than the cells loaded with acetylated LDL, the difference was not statistically significant. The significantly lower (* p < 0.01)) degradation of oxidized LDL-derived apo B compared to acetylated LDL-derived apo B is consistent with the retention of oxidized apo B within the cells. Thus, compared to apolipoprotein derived from acetylated LDL, apolipoprotein derived from oxidized LDL exhibits impaired degradation within macrophages, which results in accumulation within the cell. This experiment would be easier to interpret if the acetylated LDL degradation was not different from that of the acetylated LDL that had subsequently been oxidized, because that would clearly demonstrate that introducing oxidized lipids has no effect on the rapid metabolism of apolipoprotein from acetylated LDL. However, our data indicate that increasing the degree of oxidation of acetylated LDL from 5 hours of oxidation to 24 hours of oxidation resulted in poorer degradation and greater accumulation within the cells. Nevertheless, the data in Figures 16 and 17 does suggest that oxidized lipid may interfere with the normal degradation of the apolipoprotein component of LDL. In Figure 18, we see the corresponding cholesterol content of the cells. There was an approximately three-fold augmentation of total cholesterol in all of the lipid-loaded cells compared to the control (nonloaded) cells. Here, again we see the characteristic impairment of cholesterol efflux from oxidized LDL-loaded macrophages, as compared to acetylated LDL-loaded macrophages. Introducing oxidized lipids into acetylated LDL resulted in 75 decreased efflux of cholesterol to HDL3. Even mildly oxidized (5 hr) acetylated LDL resulted in impaired efflux of cholesterol compared to unoxidized acetylated LDL. Moreover, increasing the degree of oxidation of acetylated LDL resulted in a decrease in cholesterol efflux in the cells loaded with these modified LDL's. This suggests a prominent causal role for oxidized lipids in the impairment of cholesterol efflux. In order to investigate the role of the oxidized protein component in the impairment of cholesterol efflux, cells were loaded with arachidonic acid-oxidation product LDL (A-OP LDL). This way, we were able to see the effects of delivering LDL that contained an oxidatively modified apolipoprotein component but not an oxidized lipid component. It has been proposed that aldehydic adducts on oxidized LDL that are unreactive at neutral pH become exposed at acidic pH and then covalently bind thiols on neighbouring proteins such as cathepsin B in lysosomes, thereby inducing cross-linking of proteins and consequently, enzyme inactivation (302). In order to test whether inactivation of such proteases by aldehydes in oxidized LDL plays a role in impairment of cholesterol efflux, we loaded macrophages with oxidized LDL that had been treated with sodium borohydride (to reduce the aldehydes to alcohols) and then determined whether the rate of efflux to HDL3 was greatly enhanced. At the end of the first 24 hour incubation in the presence or absence of 50 pg/ml modified LDL's, representative cultures were extracted and assayed for protein and cholesterol content, and the remaining plates were washed and then treated with 75 pg/ml HDL3 for an additional 12 hours, after which they were processed in the same manner as the first set. Prior to incubation, cells contained only 70 pg cholesterol/mg cell protein, nearly all of which was in the form of free (unesterified) cholesterol. After 24 hours, total cholesterol increased to 230 pg cholesterol/mg cell protein in the acetylated LDL-loaded 76 cells, 172 pg cholesterol/mg cell protein in the arachidonic acid-oxidation product LDL-loaded cells, 210 pg cholesterol/mg cell protein in the oxidized LDL-loaded cells, and 211 pg cholesterol/mg cell protein in the cells loaded with oxidized LDL that had been treated with sodium borohydride. The increase in total cholesterol in the A-OP LDL-loaded cells represented a 2.5-fold augmentation compared to the control cells; and there was an approximately three-fold augmentation of total cholesterol in all of the other lipid-loaded cells compared to control. The degree to which cholesterol efflux occurred in each of the conditions is represented in Figure 19. Compared to acetylated LDL-loaded cells, the cells loaded with any of the other three modified LDL's resulted in decreased efflux of cholesterol to HDL3. Only the decrease in total cholesterol from the acetylated LDL-loaded cells can be regarded as a statistically significant (p < 0.05)) decrease in total cholesterol, as assessed by student's t-test. In this experiment, we found that A-OP LDL-loaded cells exhibited a profound impairment of cholesterol efflux to HDL3, which highlights the role of the oxidized apolipoprotein component in this phenomenon. Finally, quenching the aldehydes in oxidized LDL increased the rate of cholesterol efflux, suggesting that inactivation of certain lysosomal enzymes by aldehydes is a contributing factor in impairment of cholesterol efflux from oxidized LDL-loaded macrophages. Taken together, these results indicate that impairment of cholesterol efflux can be attributed to both the oxidized apolipoprotein component of oxidized LDL, as well as the oxidized lipid component. The latter claim is consistent with previous reports (372). It is clear that lipid and protein oxidation products, such as those found in oxidized LDL, can influence lysosomal metabolism of lipoproteins. For example, it has been 77 suggested that 7-ketocholesterol in oxidized LDL inhibits sphingomyelinase leading to a build-up of free cholesterol in lysosomes (92). 501 acLDL A-OP LDL oxLDL aid-free oxLDL Figure 19 Decrease in cholesterol in cells loaded with NaBH4-treated LDL and A-OP LDL. Each bar represents the percent decrease in total cholesterol after HDL-mediated cholesterol efflux in cells that had been loaded with acetylated LDL (acLDL), arachidonic acid-oxidation product LDL (A-OP LDL), oxidized LDL (oxLDL), or aldehyde-free oxidized LDL (aid-free oxLDL). Each value is expressed as mean percentage decrease ± SD from triplicate plates from one of two experiments with similar results. 78 3.4 Cholesterol delivered by oxidized LDL accumulates in lysosomes It has been postulated that degradation of the entire oxidized LDL particle may be arrested in the lysosome, and this would certainly account for the observed impairment of cholesterol efflux (304). If this is the case, one would expect that cholesterol would be sequestered in the lysosomal compartment of oxidized LDL-loaded cells. It has been previously reported that the apolipoprotein B component of oxidized LDL accumulates in the lysosomes of macrophages (305). However, there are conflicting reports regarding the lysosomal accumulation of cholesterol in oxidized LDL-loaded cells (92, 295, 296, 299-301, 335). In order to reconcile the contradictory findings, we investigated the subcellular distribution of cholesterol in both oxidized LDL-loaded cells and acetylated LDL-loaded cells. Since we hypothesized that the oxidized LDL-derived cholesterol would accumulate within the lysosomal fraction, after loading cells with oxidized LDL, we disrupted the plasma membranes, fractionated using a density gradient, and analyzed each fraction for lysosomal marker enzyme activity and [3H]-cholesterol content. As a point of reference, each fraction was also measured for endoplasmic reticulum marker enzyme activity. Before loading the cells with modified lipoproteins, we determined the relative distribution of the marker enzymes in control (nonloaded) macrophages. The results from one representative experiment out of three are presented in Figures 20 through 24. The distribution in control cells is given in Figure 20. 79 00 00 to CM o « — CM C O C O C O C O co C O L O to C O cn C v J q q q q q q q q q q q <-; (NJ < — » - < — « — 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Fraction Number Figure 20 Distribution of ER and lysosome enzyme markers in control cells. Each point represents the percentage of total enzyme activity in the fraction. In order to identify the lysosomal fraction, we assayed each fraction for acetylglucosaminidase. In order to identify the endoplasmic reticulum fraction, we assayed each fraction for neutral ct-glucosidase. The density of the fractions (in grams/milliliter) is written across the top of the graph. Macrophages were incubated in the absence of lipoproteins for 24 hours. Then the plasma membranes were disrupted by nitrogen cavitation, and the nuclei, mitochondria, and intact cells were removed by centrifugation. Finally, the various subcellular fractions were separated by Percoll density gradient centrifugation. 80 In control (nonloaded) cells, we found that the endoplasmic reticulum fraction occurred at a density of approximately 1.040 g/ml, and the lysosomal fraction occurred at a density of approximately 1.100 g/ml. Figure 21 represents the distribution in macrophages that have been loaded for 24 hours with 50 pg/ml radiolabeled oxidized LDL. Figure 21 Distribution in oxidized LDL-loaded cells. Each point represents the percentage of total enzyme activity in the fraction or in the case of radiolabeled cholesterol, each point represents the nanomoles of [3H]-cholesterol in each fraction. Macrophages were incubated with 50 pg/ml radiolabeled oxidized LDL for 24 hours. Other methods are described briefly in the legend for the previous figure, and in detail in Chapter 2. In macrophages loaded with oxidized LDL, we found that the endoplasmic reticulum fraction remained at a density of approximately 1.040 g/ml. However, the lysosomal marker 81 shifted down to approximately 1.050 g/ml. The cholesterol clearly concentrates in the fractions that contain high lysosomal marker activity, suggesting that the cholesterol from oxidized LDL is sequestered in lysosomes after the initial 24 hour incubation. Figure 22 represents the distribution in macrophages that have been loaded for 24 hours with 50 pg/ml radiolabeled oxidized LDL, followed by a 12 hour incubation with 75 pg/ml HDL3. Fraction Number Figure 22 Distribution in oxidized LDL-loaded cells after HDL-mediated efflux. Each point represents the percentage of total enzyme activity in the fraction or in the case of radiolabeled cholesterol, each point represents the nanomoles of [3H]-cholesterol in each fraction. Macrophages were incubated with 50 pg/ml radiolabeled oxidized LDL for 24 hours, washed, and then treated for an additional 12 hours in the presence of 75 pg/ml HDL 3 . Other methods are described briefly in the legend for figure 20, and in detail in Chapter 2. 82 In macrophages loaded with oxidized LDL and then treated with HDL3, we found that the endoplasmic reticulum fraction remained at a density of approximately 1.040 g/ml and the lysosomal marker remained shifted down to approximately 1.050 g/ml. The cholesterol remained highly concentrated in the fractions that contained high lysosomal marker activity, suggesting that the cholesterol from oxidized LDL is sequestered in lysosomes even after incubation with HDL3. From the cholesterol content we can see that less than one-quarter of the radiolabeled cholesterol is effluxed after being treated with HDL3. Figure 23, represents the distribution in macrophages that have been loaded for 24 hours with 50 pg/ml radiolabeled acetylated LDL. In macrophages loaded with acetylated LDL, we found that the endoplasmic reticulum fraction remained at a density of approximately 1.040 g/ml. However, the lysosomal marker shifted down to approximately 1.050 g/ml. Again, the radiolabeled cholesterol concentrated in the fractions that contained high lysosomal marker activity. However, very little cholesterol accumulated within the lysosomes compared to the radiolabeled cholesterol content of lysosomes in oxidized LDL-loaded cells. Figure 24, represents the distribution in macrophages that have been loaded for 24 hours with 50 pg/ml radiolabeled acetylated LDL, followed by a 12 hour incubation with 75 pg/ml HDL3. In macrophages loaded with acetylated LDL and then treated with HDL3, we found that the endoplasmic reticulum fraction remained at a density of approximately 1.040 g/ml and the lysosomal marker remained shifted down to approximately 1.050 g/ml. Again, we found that there was a low level of radiolabeled cholesterol present in the fractions that contained high lysosomal marker activity even after incubation with HDL3. However, from 83 the cholesterol content we can see that nearly two-thirds of the radiolabeled cholesterol was effluxed after being treated with HDL3. Figure 23 Distribution in acetylated LDL-loaded cells. Each point represents the percentage of total enzyme activity in the fraction or in the case of radiolabeled cholesterol, each point represents the nanomoles of [3H]-cholesterol in each fraction. Macrophages were incubated with 50 pg/ml radiolabeled acetylated LDL for 24 hours. Other methods are described briefly in the legend for figure 20, and in detail in Chapter 2. 84 ~l—1—I—1—I— 2 4 6 8 10 12 14 16 18 20 22 24 Fraction Number Figure 24 Distribution in acetylated LDL-loaded cells after HDL-mediated efflux. Each point represents the percentage of total enzyme activity in the fraction or in the case of radiolabeled cholesterol, each, point represents the nanomoles of [3H]-cholesterol in each fraction. Macrophages were incubated with 50 pg/ml radiolabeled acetylated LDL for 24 hours, washed, and then treated for an additional 12 hours in the presence of 75 pg/ml HDL 3 . Other methods are described briefly in the legend for figure 20, and in detail in Chapter 2. In this experiment, we found that loading cells with either oxidized or acetylated LDL resulted in a shift in the density of the lysosomal fraction, compared to control cells. In these instances, the lysosomal fraction became less dense after taking up so much lipoprotein-derived cholesterol. Even after the 12 hour incubation with HDL3, we found that the lysosomal marker remained shifted down to a density of approximately 1.050 g/ml. In the case of oxidized LDL-loaded cells this is not surprising since less than one-quarter of the 85 radiolabelled cholesterol was effluxed during the incubation with HDL3. However, in the case of acetylated LDL-loaded cells, the lysosomal content of cholesterol decreased significantly from 0.57 nanomoles to 0.22 nanomoles, but the lysosomal marker density did not shift up to a higher density. The only explanation that we can provide for this is that during the processing of acetylated LDL-derived cholesterol, lighter endosomes may have fused with the lysosomal compartment containing lipid and the lysosomal marker. Nevertheless, these results provide further evidence that macrophages loaded with oxidized LDL exhibit impaired efflux of cholesterol compared to macrophages loaded with acetylated LDL. Moreover, the cholesterol delivered by oxidized LDL is sequestered in a lysosomal compartment. However, this is in sharp contrast to the findings of Kritharides et al. (300), who reported that there was equilibration of cholesterol to all membrane pools in murine macrophages loaded with oxidized LDL. The contrast between this and our observations may be based on differences in the analytical methods employed to fractionate the cells and detect cholesterol. Kritharides et al. arbitrarily combined the collected fractions into five major fractions representing large ranges in density, in which the density range of 1.035 g/ml to 1.050 g/ml was labelled the endosomal fraction and the density range of 1.050 g/ml to 1.095 g/m was labelled the lysosomal fraction. In our experience, the fractions containing lysosomal enzyme activity in oxidized LDL-loaded cells form a sharp peak between the density range of 1.045 g/ml and 1.055 g/ml. They reported a large proportion of free cholesterol and 7-ketocholesterol in both their endosomal and lysosomal fractions; this is in agreement with our data which demonstrates that most of the oxidized LDL-derived cholesterol is found in the 1.050 g/ml density fraction. Unlike, Kritharides et al., we did not find any accumulation of cholesterol in the density range 1.020 g/ml to 1.035 g/ml. This may 86 be explained by the fact that they measured the distribution of all cholesterol in the cell, whereas we measured only the radiolabelled cholesterol delivered by oxidized LDL. Our results also differ from those of Yancey et al., who found that mouse peritoneal macrophages stored most of their oxidized LDL-derived cholesterol (71 %) within cytoplasmic inclusions (301, 335). In contrast to mouse peritoneal macrophages, Yancey et al. reported that pigeon and THP-1 macrophages stored most (60%-90%) of their oxidized LDL-derived cholesterol in lysosomes, whereas the bulk (64%-88%) of acetylated LDL cholesterol was stored in cytoplasmic inclusions (301, 335). It is difficult to reconcile the findings of Yancey et al. with our data because we used extensively oxidized LDL for our work, whereas they used only mildly oxidized LDL. Our identification of the lysosomal accumulation of cholesterol in oxidized LDL-loaded macrophages is in agreement with the findings of Maor et al. (92, 299), who studied this phenomenon in the J-774 A.l murine macrophage-like cell line. Moreover, immunolocalization and ultrastructural studies also suggest that oxidized LDL accumulates in the lysosomes of both cultured macrophages and human atherosclerotic tissues (295, 296). Because acetylated LDL does not exist in vivo, there have been few reports on the subcellular distribution of cholesterol in acetylated LDL-loaded cells. In disagreement with the findings of Yancey et al., we found that there was a moderate accumulation of radiolabelled cholesterol in the lysosomes of acetylated LDL-loaded cells (301, 335). It is difficult to surmise an explanation for the contrast between their work and ours. However, it may be based on differences in the analytical methods or culture conditions used. We used subcellular fractionation followed by assays for lysosomal and endoplasmic reticulum enzyme markers to determine the subcellular distribution of acetylated LDL-derived [3H]-87 cholesterol. They, on the other hand, used electron microscopy and cytochemistry to determine the subcellular distribution of lipid; specifically, they stained cells for the lysosomal enzyme marker, acid phosphatase, and then analyzed electron micrographs to determine whether lipid associated with acid phosphatase-positive lysosomes (301). Alternatively, it is possible that the conditions under which they cultured the cells may have had an effect. They loaded their cells with medium containing 100 pg/ml acetylated LDL and 1% FBS, for 48 hours; we loaded our cells for only 24 hours, with medium containing 50 pg/ml acetylated LDL and 0.5 mg/ml BSA. It is possible that the longer incubation time in the presence of FBS redistributed the lipid within the cell as it was being effluxed from the cell by FBS. This would also explain their very low levels of lipid accumulation compared to the levels that we achieve in 24 hours. There are no reports of the subcellular distribution of cholesterol in modified LDL-loaded cells that have been treated with a cholesterol acceptor such as HDL. We found that subcellular distribution of cholesterol in oxidized LDL-loaded macrophages that had been treated with HDL3 was identical to the distribution of cholesterol in untreated oxidized LDL-loaded macrophages. This is in keeping with the observed impairment of cholesterol efflux from these cell. Surprisingly, when the same analysis was done on cells that had been loaded with acetylated LDL and then treated with HDL3, it was found that the distribution was again similar to that of untreated acetylated LDL-loaded cells. We would have expected the lysosomal compartment in acetylated LDL-loaded macrophages to become denser as cholesterol was released to HDL3. Since this did not occur, we surmise that lighter endosomes may have fused with the lysosomal compartment containing lipid and the lysosomal marker, during the efflux of cholesterol from the cell. Despite the lack of change 88 in the subcellular distribution of cholesterol after HDL-mediated cholesterol efflux, it can be seen that the acetylated LDL-loaded cells released nearly two-thirds of their radiolabelled cholesterol to HDL3, whereas the oxidized LDL-loaded cells released less than one-quarter of their radiolabelled cholesterol to HDL3. This provides further evidence that macrophages loaded with oxidized; LDL exhibit impaired efflux of cholesterol compared to macrophages loaded with acetylated LDL. To seek morphologic confirmation of the cell fractionation results indicating that oxidized LDL accumulates within macrophages and oxidized LDL-derived lipids are inefficiently effluxed in the presence of HDL3, we loaded cells with fluorescently-labelled oxidized LDL and then observed the decrease in fluorescence, if any, after treating them with HDL3. Figure 25 depicts a typical example of macrophages loaded for 24 hours with 50 pg/ml NBD-C6-sphingomyelin-labelled oxidized LDL. Figure 26 depicts an example of macrophages that were loaded under the same conditions as the cells in Figure 25, only the cells in Figure 26 underwent a further 12 hour incubation with 75 pg/ml HDL3. These pictures were taken from one of three experiments. The caption for Figure 25 is provided below,-but the photographs corresponding to this caption are on page 89. Figure 25 Macrophages loaded with NBD-Q-sphingomyelin-labelled oxidized LDL. Both pictures were taken under 400 X magnification. Cells were loaded for 24 hours with 50 ug/ml NBD-Q-sphingomyelin-labelled oxidized LDL, washed, and then fixed onto microscope slides. The photograph on the top of the next page was taken in the phase mode of the microscope, and the photograph on the bottom is the corresponding fluorescent micrograph. The NBD-C6-sphingomyelin appears green. 89 90 The caption for Figure 26 is provided below, but the photographs corresponding to this caption are on page 91. Figure 26 Macrophages treated with fluorescent oxidized LDL followed by HDL3. Both pictures were taken under 400 X magnification. Cells were loaded for 24 hours with 50 pg/ml NBD-C 6-sphingomyelin-labelled oxidized LDL, washed, treated for an additional 12 hours with 75 pg/ml HDL 3 , washed again, and then fixed onto microscope slides. The photograph on the left was taken in the phase mode of the microscope, and the photograph on the right is the corresponding fluorescent micrograph. The NBD-C 6-sphingomyelin appears green. 91 92 Comparing photographs of fluorescence in cells is highly subjective because the degree of exposure affects the apparent fluorescence intensity of the cells. Furthermore, photobleaching of the fluorescent probe does occur the longer it is exposed to light. It is for this reason that both the amount of time that each slide was exposed to light and the exposure time of the film were kept constant for each photograph in order to ensure comparable photographs were taken. After examining numerous slides under the microscope, we are confident that there appeared to be little or no decrease in fluorescence after HDL-mediated cholesterol efflux. However, in cells loaded for 24 hours with NBD-C6-sphingomyelin-labelled acetylated LDL, very little NBD-C6-sphingomyelin accumulated within the cells because unlike oxidized LDL derived sphingomyelin, acetylated LDL-derived sphingomyelin is rapidly hydrolyzed within macrophages and the water-soluble fluorescent label is also expelled from the cell. Figure 27 depicts a typical example of macrophages loaded for 24 hours with 50 pg/ml NBD-C6-sphingomyelin-labelled acetylated LDL. Cells that were loaded with fluorescently-labelled acetylated LDL and subsequently treated with HDL3 contained little or no NBD-C6-sphingomyelin, as we would expect (photographs not shown). These results suggest that, in addition to cholesterol, sphingolipids delivered by oxidized LDL are also poorly degraded and consequently accumulate within oxidized LDL-loaded cells. 93 *4r Figure 27 Macrophages loaded with NBD-C6-sphingomyelin-labelled acetylated LDL. Both pictures were taken under 400 X magnification. Cells were loaded for 24 hours with 50 fig/ml NBD-Q-sphingomyelin-labelled acetylated LDL, washed, and then fixed onto microscope slides. The photograph on the top of the next page was taken in the phase mode of the microscope, and the photograph on the bottom is the corresponding fluorescent micrograph. The NBD-C6-sphingomyelin appears green. 3.5 H D L - m e d i a t e d cholesterol efflux Because interstitial fluid contains a greater concentration than plasma of lipoprotein-unassociated apolipoprotein A-I (373) and because cholesterol-laden fibroblasts release cholesterol to an apo A-I-only-containing particle (374), it has been proposed that apo A-I may be involved in the initial steps of cholesterol efflux. Moreover, purified apolipoprotein A-I has been shown to promote cholesterol efflux in vitro (375). Very little efflux of cholesterol from oxidized LDL-loaded cells occurs, compared to cells loaded with acetylated 94 LDL. In order to determine whether the residual cholesterol efflux from oxidized LDL-loaded cells is apolipoprotein-dependent, lipid-loaded cells were treated with either HDL3 or purified apo A-I. In this experiment, control cells contained only 80 pg total cholesterol/mg cell protein. After 24 hours, total cholesterol increased to 276 pg cholesterol/mg cell protein in the acetylated LDL-loaded cells, and 335 pg cholesterol/mg cell protein in the oxidized LDL-loaded cells. As would be expected from the composition of these modified LDL's (Table 5), the acetylated LDL-loaded cells accumulated large quantities of cholesterol ester, as well as free cholesterol, whereas the oxidized LDL-loaded cells accumulated predominantly free cholesterol. The increase in total cholesterol in the acetylated LDL-loaded macrophages represented an approximately 3.5-fold augmentation over the baseline level. Similarly, the increase in total cholesterol in the oxidized LDL-loaded macrophages represented an approximately four-fold augmentation over the baseline level. In such experiments, the degree of cholesterol accumulation varied from two-fold to six-fold, but in all cases, the acetylated LDL-loaded cells effluxed significant quantities of cholesterol, whereas the oxidized LDL-loaded cells exhibited impaired efflux of cholesterol. Some efflux of cholesterol from the acetylated LDL-loaded cells occurred in the absence of apo A-I or HDL3. In this case, the BSA in the medium acted as the cholesterol acceptor and cholesterol efflux occurred by passive diffusion from the cells to the BSA. Figure 28 depicts the degree of cholesterol efflux observed in this experiment. 95 70-60" D e g r e e o f a 501 C h o l e s t e r o l E f f l u x 4 0 1 ( e x p r e s s e d 3 o - | a s % o f T o t a l C h o l e s t e r o l ) 20 10 0 B S A H D L a p o A - I acetylated LDL-loaded cells B S A H D L a p o A - I oxidized LDL-loaded cells Figure 28 Decrease in total cholesterol after treatment with apo A-I or HDL3. Each bar represents the percent decrease in total cholesterol after cholesterol efflux to BSA, HDL 3, or apo A - l , in cells that had been loaded with either acetylated LDL (Panel A) or oxidized LDL (Panel B). Each value represents the percentage difference of the means ± SD from triplicate plates from one of two experiments with similar results. Once again, we see the striking difference in cholesterol efflux from cells loaded with oxidized LDL compared to cells loaded with acetylated LDL. It has been shown previously that using apolipoproteins such as apo A-I to mediated cholesterol efflux from cultured cells results in the formation of HDL-like particles in the culture medium (75, 78, 375-383). Our data indicated that HDL3 was equally as effective as apolipoprotein A-I at bringing about cholesterol efflux from macrophages loaded with either acetylated LDL or oxidized LDL. This raises the question of the role of the lipid component of HDL3 in cholesterol efflux. As discussed in Chapter 1 (Section 1.2), there are two proposed mechanisms of efflux: one involving passive diffusion of cholesterol from the cell and the other involving apolipoprotein binding to the cell surface. In order to determine the importance of HDL 96 lipids in bringing about cholesterol efflux, loaded cells were treated with either HDL, or an equivalent amount of trypsinized HDL3. The equivalent amount of trypsinized HDL was calculated by determining the phosphorus content of both native HDL and the trypsinized HDL preparation. To ensure that the apolipoprotein component had been adequately digested, the trypsinized HDL sample was run on SDS-polyacrylamide gel electrophoresis (Figure 29). MW Standard native HDL trypsinized HDL Figure 29 SDS-PAGE of native HDL, and trypsinized HDL,. Each band in the gel represents a protein of a particular molecular weight; the molecular weights are labelled on the gel. Each lane is labelled on the bottom of the gel. The molecular weight of apolipoprotein E is 34 kDa. The molecular weight of apolipoprotein A-I is 28 kDa. It can be seen from the lanes containing trypsinized HDL, that there is a nearly complete absence of intact apolipoprotein A-I. 97 In Figure 29, we see that the trypsinized HDL3 preparation contained small amounts of intact apolipoprotein E. However, it was a suitable preparation to use because nearly all of the apolipoprotein A-I was degraded. Macrophages loaded with either oxidized LDL or acetylated were treated with either native HDL or trypsinized HDL, and the degree of cholesterol efflux was measured. Control cells contained only 77 pg total cholesterol/mg cell protein. After 24 hours, total cholesterol increased to 147 pg cholesterol/mg cell protein in the acetylated LDL-loaded cells, and 144 pg cholesterol/mg cell protein in the oxidized LDL-loaded cells. The increase in total cholesterol in both the acetylated LDL-loaded cells and the oxidized LDL-loaded cells represented an approximately two-fold augmentation over the baseline level. There was a slight decrease in total cholesterol in all of the cells treated with trypsinized HDL3, but it was not statistically significant. Figure 30 depicts the degree of cholesterol efflux observed in this experiment. Once again, we see the striking difference in cholesterol efflux from cells loaded with oxidized LDL compared to cells loaded with acetylated LDL. The data from Figure 30 suggests that HDL lipids are responsible for less than one-third of the efflux observed when lipid-loaded cells are treated with native HDL3. This is in agreement with previous reports that cholesterol efflux from plasma membranes is mediated by HDL lipids, whereas removal of excess intracellular^ accumulated cholesterol is mediated by HDL apolipoproteins (77). Furthermore, our results are consistent with the hypothesis that HDL apolipoproteins must bind to a cell surface receptor in order to stimulate translocation of excess intracellular cholesterol to the cell surface where it becomes accessible for removal by HDL (53, 65, 67, 78). 98 Degree of Cholesterol Efflux (expressed as % of Total Cholesterol) % efflux with trypsinized H D L % efflux with native H D L AcLDL Load OxLDL Load Figure 30 Decrease in total cholesterol after treatment with trypsinized HDL3. Each bar represents the percent decrease in total cholesterol after cholesterol efflux to trypsinized HDL 3 or native HDL 3 , in cells that had been loaded with either acetylated LDL (acLDL) or oxidized LDL (oxLDL). Each value represents the mean percentage difference ± SD from triplicate plates from one of three experiments with similar results. Some of our early experiments showed substantial efflux of cholesterol from oxidized LDL-loaded cells. Subsequently, we were not able to verify these results. Nevertheless, these unexpected results prompted us to investigate possible mechanisms whereby oxidized 99 LDL-derived cholesterol could be effluxed to HDL-containing medium. In addition to the two mechanisms of HDL-mediated cholesterol efflux discussion in Chapter 1 (Section 1.2), another pathway has been reported in differentiated enterocytes (384, 385) and rat peritoneal macrophages (386). These reports demonstrated that HDL3 was bound specifically to these cells, leading to a subsequent intracellular passage and resecretion through a process of retroendocytosis effecting the efflux of cellular cholesterol. To investigate whether retroendocytosis was involved in HDL-mediated cholesterol efflux from cultured mouse peritoneal macrophages, lipid-loaded cells were treated with fluorescently-labelled HDL3. This experiment allowed us to determine whether the mechanism of cholesterol efflux involves uptake and resecretion of HDL3. Figure 31 depicts the control for this experiment: macrophages were treated for 24 hours with BSA-containing medium only, followed by a 12 hour incubation with 75 pg/ml Texas Red HDL3. Figure 32 depicts an example of macrophages that were loaded for 24 hours with 50 pg/ml NBD-C6-sphingomyelin-labelled oxidized LDL, washed, and then treated with 75 pg/ml Texas Red HDL3 for an additional 12 hours. These pictures were taken from one of three experiments. The caption for Figure 31 is provided below, but the photographs corresponding to this caption are on page 100. Figure 31 Control macrophages treated with Texas Red HDL. Both pictures were taken under 100 X magnification. Cells were incubated for 24 hours with medium in the absence of lipoproteins, washed, and then treated with 75 pg/ml Texas Red HDL 3 for 12 hours. Finally, the cells were washed and fixed onto microscope slides. The photograph on the top of the next page was taken in the phase mode of the microscope, and the photograph on the bottom is the corresponding fluorescent micrograph. Texas Red HDL appears red. 100 Figure 32 OxLDL-loaded macrophages treated with Texas Red HDL. All three pictures were taken under 400 X magnification. Cells were loaded for 24 hours with 50 pg/ml NBD-C6-sphingomyelin-labelled oxidized LDL, washed, treated for an additional 12 hours with 75 pg/ml Texas Red HDL,, washed again, and then fixed onto microscope slides. The photograph below was taken in the phase mode of the microscope. The photographs on the next page are the corresponding fluorescent micrographs taken using the fluorescein filter set (top) and the rhodamine filter set (bottom). The NBD-C6-sphingomyelin appears green. Texas Red HDL appears red 102 103 We found that control (non-loaded) cells took up very little HDL3, whereas cells loaded with either acetylated LDL (photographs not shown) or oxidized LDL took up a substantial amount of HDL3. In some cases, lipid loaded cells that had been exposed to Texas Red HDL were washed and incubated in the absence of lipoproteins for an additional 12 hours. It appeared that there was minimal decrease in Texas Red HDL fluorescence in the cells that were treated with no lipoproteins during this "chase" period; however, it was impossible to come to any meaningful conclusions about this "chase" incubation because there was a significant decrease in cell viability during this additional incubation (photographs not shown). Since HDL3 was taken up by lipid-loaded cells but not by control (nonloaded) cells, retroendocytosis could not be ruled out as a possible mechanism of cholesterol efflux in these cells. To quantify this phenomenon, we measured the retention and degradation of HDL3 in lipid-loaded and control (nonloaded) cells. Macrophages were incubated for 24 hours with no lipoprotein (control), 50 pg/ml acetylated LDL (acLDL) or 50 pg/ml oxidized LDL (oxLDL). Then, they were washed and treated for an additional 12 hours with 125I-labelled native HDL3. Finally, we measured both the HDL retained within the cells as well as the HDL that had been degraded during the 12 hour incubation. Figure 33 depicts the results of a representative experiment out of three. Iodine labels proteins by associating with tyrosine rings. In the experiment depicted in Figure 33, we measured the cellular content and degradation of HDL. We found that after 12 hours of incubation with 75 pg/ml HDL3, both the lipid-loaded and control (nonloaded) macrophages retained approximately 0.76 pg of HDL3 per mg of cell protein. Rogler et al. reported a lower (0.40 pg/mg of cell protein) retention of HDL3 (385). However, they used 104 enterocytes (Caco-2) and they incubated their cells with HDL3 for only three hours, so this may account for the conflicting results. 3.0n u.g of HDL 2 0 retained or degraded per mg of cell protein HDL retained HDL degraded control acLDL oxLDL Figure 33 HDL retention and degradation by macrophages. Each bar represents the micrograms of radioactive HDL retained (solid bar) or degraded (hatched bar) per milligram of cell protein at the end of a 12 hours incubation with 1 2 5I-HDL 3. Before treating the macrophages for 12 hours with 75 ug/ml radiolabelled HDL, the cells were incubated for 24 hours with no lipoprotein (control), 50 pg/ml acetylated LDL (acLDL), or 50 pg/ml oxidized LDL (oxLDL). There was no statistically significant difference in the retention of HDL in the lipid-loaded cells compared to the control cells. There was a statistically significant difference in the degradation of HDL in the in the lipid-loaded cells compared to the control cells (* p < 0.05). However, there was no statistically significant difference in the degradation of HDL in the acLDL-loaded cells compared to the oxLDL-loaded cells. The values are mean ± S.D. for triplicate plates from one of three experiments with similar results. 105 The pathway of retroendocytosis described by Rogler et al. does not involve degradation of HDL (385); in fact, intact HDL particles are a requirement for this pathway. In contrast to previous reports (385, 386), we found that degradation of HDL3 by nonloaded cells was not negligible. Again, the discrepant results may be attributed to the difference in the cell type used as well as the different incubation times. However, it was clear from our findings that HDL3 retroendocytosis did not occur in our experiments. The most interesting findings came from the lipid-loaded cells, which exhibited a nearly four-fold increase in the degradation of HDL3 compared to control cells (* p < 0.05). This suggests that uptake of either oxidized LDL or acetylated LDL results in upregulation of an internalizing receptor for HDL. Han et al. have reported that exposure to either oxidized LDL or acetylated LDL resulted in marked induction of CD36 mRNA expression in both J774 cells and mouse peritoneal macrophages (177). However, they also found that exposure to native LDL increased the expression of CD36 mRNA (177). As outlined in Table 3, CD36 is capable of binding to and internalizing oxidized LDL. The scavenger receptor class B type I (SR-BI) is the only known HDL receptor which can mediate selective uptake of cholesteryl esters from HDL (without apolipoprotein uptake). Fluiter et al. reported that rats that were fed a high-cholesterol diet for two weeks exhibited decreased SR-BI expression in parenchymal cells, while the expression in Kupffer cells increased (387). Our finding that lipid-loaded cells exhibit increased degradation of HDL may represent upregulation of SR-BI expression, and it is plausible (although untested) that under these culture conditions, this receptor could mediate uptake of the entire HDL particle by macrophages, not just selective uptake of cholesteryl esters from HDL as in liver and adrenal cells. 106 In summary, the results of the present study demonstrate that oxidized LDL-loaded macrophages exhibit impaired efflux of cholesterol. This impairment of HDL-mediated cholesterol efflux involves both the oxidized lipid component and the oxidized apolipoprotein B moiety of oxidized LDL. Furthermore, cholesterol delivered by oxidized LDL is sequestered in lysosomes. With regards to the mechanism of HDL-mediated cholesterol efflux in modified LDL-loaded mouse peritoneal macrophages, our data suggest that exocytosis of undegraded lipoproteins does not occur, nor does HDL3 retroendocytosis. It is possible that the impairment of cholesterol efflux from oxidized LDL loaded cells influences the formation and persistence of the foam cell phenotype in vivo and may therefore contribute to the atherogenicity of oxidized LDL. 107 Chapter 4 Conclusions The data in this study present compelling evidence that macrophages loaded with oxidized LDL have a reduced ability to release cholesterol to HDL, compared to cells loaded with acetylated LDL. Oxidation of LDL results in profound alterations of both the lipid and protein compositions of LDL, including fragmentation of apolipoprotein B, hydrolysis of phosphatidylcholine and loss of esterified cholesterol. We have found that both the oxidized lipid portion and the oxidized apolipoprotein component are involved in the observed impairment of HDL-mediated cholesterol efflux from macrophages loaded with oxidized LDL. In addition, the data also provide evidence that oxidized LDL-derived cholesterol is sequestered within lysosomes. This implicates a possible defect in cholesterol metabolism and transport in foam cells of atherosclerotic lesions. Finally, our work has demonstrated that HDL-mediated cholesterol efflux from lipid-loaded mouse peritoneal macrophages involves neither HDL3 retroendocytosis, nor exocytosis of undegraded oxidized LDL. These findings not only increase our understanding of the role of oxidized LDL in lipid accumulation, but they also give insight into the physiologically-relevant process of HDL-mediated cholesterol efflux. To fully understand how oxidized LDL causes impairment of cholesterol efflux from macrophages, further investigations are required. Future studies should focus on the identification of the specific oxidized protein and oxysterol components responsible for the impaired metabolism of cholesterol. Whether oxidized LDL is responsible for cholesterol accumulation in atherosclerotic lesions is unknown and will be a challenge to determine. However, the present findings provide impetus to look at the 108 metabolism and transport of cholesterol within cells isolated from atheromas, and also to further characterize the mechanism of HDL-mediated cholesterol efflux. 109 References 1. Brown MS, Goldstein JL. Receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34-47. 2. Small DM. Progression and regression of atherosclerotic lesions. Insights from lipid physical biochemistry. Arteriosclerosis 1988;8:103-29. 3. Yokoyama S. Apolipoprotein-mediated cellular cholesterol efflux. Biochim. Biophys. Acta 1998;1392(1):1-15. 4. Vance DE, Vance J, editors. Biochemistry of Lipids, Lipoproteins and Membranes. 2nd ed. Amsterdam: Elselvier Science Publishers B.V.; 1991. 5. Schneider W. The low density lipoprotein receptor. Biochim. Biophys. Acta 1989;988:303-17. 6. Goldstein J, Dana S, Faust J, Beaudet A, Brown M. Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease. J. Biol. Chem. 1975;250(21):8487-95. 7. de Duve C. Lysosomes revisited. Eur. J. Biochem. 1983;137(3):391-7. 8. Goldstein J, Brunschede G, Brown M. Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339. J. Biol. Chem. 1975;250(19):7854-62. 9. de Duve C, de Barsy T, Poole B, Trouet A, Tulkens P, van Hoof F. Commentary. Lysosomotropic agents. Biochem. Pharmacol. 1974;23(18):2495-531. 10. Brown M, Ho Y, Goldstein J. The cholesteryl ester cycle in macrophage foam cells. J. Cell. Biol. 1980;255:9344-52. 11. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Ann. Rev. Biochem. 1983;52:223-61. 12. Brown M, Goldstein J. Receptor-mediated endocytosis: Insights from the lipoprotein receptor system. Proc. Natl. Acad. Sci. U.S.A. 1979;76:3330-7. 13. Goldstein J, Dana S, Brown M. Esterification of low density lipoprotein cholesterol in human fibroblasts and its absence in homozygous familial hypercholesterolemia. Proc. Natl. Acad. Sci. U.S.A. 1974;71(ll):4288-92. 110 14. Luskey K, Faust J, Chin D, Brown M, Goldstein J. Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells. J. Biol. Chem. 1983;258(13):8462-9. 15. Gil G, Faust J, Chin D, Goldstein J, Brown M. Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme. Cell 1985;41(l):249-58. 16. Brown M, Goldstein J. Regulation of the activity of the low density lipoprotein receptor in human fibroblasts. Cell 1975;6(3):307-16. 17. Russell D, Yamamoto T, Schneider W, Slaughter C, Brown M, Goldstein J. cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA. Proc. Natl. Acad. Sci. U.S.A. 1983;80(24):7501-5. 18. Goldstein JL, Brown MS. The low density lipoprotein pathway and its relation to atherosclerosis. Annu. Rev. Biochem. 1977;46:897-930. 19. Hakamata H, Miyazaki A, Sakai M, Suginohasa Y, Sakamoto Y-I, Fforiuchi S. Species difference in cholesterol ester cycle and HDL-induced cholesterol efflux from macrophage foam cells. Arterioscler. Thromb. 1994;14:1860-5. 20. Reue K, Cohen R, Schotz M. Evidence for hormone-sensitive lipase mRNA expression in human monocyte/macrophages. Arterioscler. Thromb. Vase. Biol. 1997;17(12):3428-32. 21. Bjbrkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu R, Duan C, et al. Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages. Proc. Natl. Acad. Sci. USA 1994;91:8592-6. 22. Babiker A, Andersson O, Lund E, Xiu R, Deeb S, Reshef A, et al. Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-hydroxylase mechanism: comparison iwth high density lipoprotein-mediated reverse cholesterol transport. J. Biol. Chem. 1997;272:26253-61. 23. Miller G, Miller N. Plasma high density lipoprotein concentration and development of ischaemic heart disease. Lancet 1974;1:16-20. 24. Fielding C, Fielding P. Molecular physiology of reverse cholesterol transport. J. Lipid Res. 1995;36:211-28. 25. Badimon J, Badimon L, Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J. Clin. Invest. 1990;85:1234-41. I l l 26. Glomset J. The plasma lecithimcholesterol acyltransferase reaction. J. Lipid Res. 1968;9:155-67. 27. Gordon D, Knoke J, Probstfield J, Superko R, Tyroler H. High-density lipoprotein cholesterol and coronary artery disease in hypercholesterolemic men: the Lipid Research Clinic's Coronary Primary Prevention Trial. Circulation 1986;73:1217-25. 28. Gordon D, Rifkind B. High density lipoprotein — The clinical implications of recent studies. N. Engl. J. Med. 1989;321:1311-6. 29. Stampfer M, Sacks F, Salvini S, Willett W, Hennekens C. A prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction. N. Engl. J. Med. 1991;325:373-81. 30. von Eckardstein A. Cholesterol Efflux from macrophages and other cells. Curr. Opin. Lipidol. 1996;7(5):308-19. 31. Phillips M, Johnson W, Rothblat G. Mechanism and consequence of cellular cholesterol exchange and transfer. Biochim. Biophys. Acta 1987;906:223-76. 32. Johnson W, Mahlberg F, Rothblat G, Phillips M. Cholesterol transport between cells and high density lipoproteins. Biochim. Biophys. Acta 1991;1085:273-98. 33. Daniels R, Guertler L, Parker T, Steinberg D. Studies on the rate of efflux of cholesterol from cultured human skin fibroblasts. J. Biol. Chem. 1981;256:4978-83. 34. Johnson W, Bamberger M, Latta R, Rapp P, Phillips M, Rothblat G. The bidirectional flux of cholesterol between cells and lipoproteins: effects of phospholipid depletion of high density lipoprotein. J. Biol. Chem. 1986;261:5766-76. 35. Johnson W, Mahlberg F, Chacko G, Phillips M, Rothblat G. The influence of cellular and lipoprotein cholesterol contents on the efflux of cholesterol between fibroblasts and high density lipoprotein. J. Biol. Chem. 1988;263:14099-106. 36. Fielding C, Fielding P. Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and its relationship to lecithin:cholesterol acyltransferase. Proc. Natl. Acad. Sci. U.S.A. 1981;78:3911-4. 37. Ko K, Ohnishi T, Yokoyama S. Trigylceride transfer is required for net cholesteryl ester transfer between lipoproteins in plasma by lipid transfer protein. J. Biol. Chem. 1994;269:28206-13. 38. Huang Y, von Eckerdstein A, Wu S, Assmann G. Cholesterol efflux, cholesterol esterification, and cholesterol ester transfer by LpA-I and LpA-I/A-II in native plasma. Arterioscler. Thromb. Vase. Biol. 1995;15:1412-8. 112 39. Czarnecka H, Yokoyama S. Regulation of cellular cholesterol efflux by lecithimcholesterol acyltransferase reaction through nonspecific lipid exchange. J. Biol. Chem. 1996;271:2023-8. 40. Fielding CJ. The origin and properties of free cholesterol potential gradients in plasma and their relation to atherogenesis. J. Lipid Res. 1984;25:1624-8. 41. Rothblat G, Mahlberg F, Johnson W, Phillips M. Apolipoproteins, membrane cholesterol domains and the regulation of cholesterol efflux. J. Lipid Res. 1992;33:1091-7. 42. Rothblat G, Phillips M. Mechanism of cholesterol efflux from cells: effects of acceptor structure and concentration. J. Biol. Chem. 1982;257:4775-82. 43. Gold J, Phillips M. Effects of membrane lipid composition on the kinetics of cholesterol exchange between lipoproteins and different species of red blood cells. Biochem. Biophys. Acta 1990;1027:85-92. 44. Kilsdonk E, Dorsman A, van Gent T, van Tol A. Effect of phospholipid fatty acid composition of endothelial cells on cholesterol efflux rates. J. Lipid Res. 1992;33:1373-82. 45. Davidson W, Lund-Katz S, Johnson W, Anantharamaiah G, Palgunachari M, Segrest J, et al. The influence of apolipoprotein structure on the efflux of cellular free cholesterol to high density lipoprotein. J. Biol. Chem. 1994;269:22975-82. 46. Lagrost L, Dengremont C, Athias A, de Geitere C, Fruchart J, Lallemant C, et al. Modulation of cholesterol efflux from Fu5AH hepatoma cells by the apolipoprotein content of high density lipoprotein particles: particles containing various proportions of apoA-I and apoA-II. J. Biol. Chem. 1995;270:13004-9. 47. Davidson W, Gillote K, Lund-Katz S, Johnson W, Rothblat G, Phillips M. The effect of high-density lipoprotein phospholipid acyl chain composition on the efflux of cellular free cholesterol. J. Biol. Chem. 1995;270:5882-90. 48. Davidson W, Rodrigueza W, Lund-Katz S, Johnson W, Rothblat G, Phillips M. Effects of acceptor particle size on the efflux of cellular free cholesterol. J. Biol. Chem. 1995;270:17106-13. 49. Gillotte K, Davidson W, Lund-Katz S, Rothblat G, Phillips M. Apolipoprotein A-I structural modification and the functionality of reconstituted high density lipoprotein particles in cellular cholesterol efflux. J. Biol. Chem. 1996;270:17106-13. 50. Brinton E, Oram J, Chen C, Albers J, Bierman E. Binding of high density lipoprotein to cultured fibroblasts after chemical alteration of apoprotein amino acid residues. J. Biol. Chem. 1986;261:495-503. 113 51. Oram J, Johnson C, Brown T. Interaction of high density lipoprotein with its receptor on cultured fibroblasts and macrophages: evidence for reversible binding at the cell surface without internalization. J. Biol. Chem. 1987;262:2405-10. 52. Duel B, Oram J, Bierman E. Nonenzymatic glycosylation of HDL and impaired HDL-receptor-mediated cholesterol efflux. Diabetes 1991;40:377-84. 53. Mendez AJ, Oram JF. Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux. Biochim. Biophys. Acta 1997;1346(3):285-99. 54. Graham D, Oram J. Identification and characterization of a high density lipoprotein-binding protein in cell membranes by ligand blotting. J. Biol. Chem. 1987;262:7439-42. 55. McKnight G, Reasoner J, Gilbert T, Sundquist K, Hoklund B, McKernan P, et al. Cloning and expression of a cellular high density lipoprotein-binding protein that is up-regulated by cholesterol loading of cells. J. Biol. Chem. 1992;267:12131-41. 56. Hokland B, Mendez AJ, Oram JF. Cellular localization and characterization of proteins that bind high density lipoprotein. J. Lipid Res. 1992;33(9): 1335-42. 57. Temel RE, Trigatti B, DeMattos RB, Azhar S, Krieger M, Williams DL. Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells. Proc. Natl. Acad. Sci. USA 1997;94(25): 13600-5. 58. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism. Proc. Natl. Acad. Sci. USA 1997;94(23): 12610-5. 59. Murao K, Terpstra V, Green SR, Kondratenko N, Steinberg D, Quehenberger O. Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes. J. Biol. Chem. 1997;272(28): 17551-7. 60. Acton S, Rigotti A, Landschulz K, Xu S, Hobbs H, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 1996;271:518-20. 61. Ji Y, Jian B, Wang N, Sun Y, de la Llera-Moya M, Phillips MC, et al. Scavenger receptor BI promotes high density lipoprotein-mediated cellular cholesterol efflux. J. Biol. Chem. 1997;272(34):20982-5. 114 62. Jian B, de la Llera-Moya M, Ji Y, Wang N, Phillips M, Swaney J, et al. Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors. J. Biol. Chem. 1998;273(10):5599-606. 63. Slotte J, Oram J, Bierman E. Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface. J. Biol. Chem. 1987;262:12904-7. 64. Aviram M, Bierman E, Oram J. High density lipoprotein stimulates sterol translocation between intracellular plasma membrane pools in human monocyte-derived macrophages. J. Lipid Res. 1989;30:65-76. 65. Oram JF, Mendez AJ, Slotte JP, Johnson TF. High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts. Arterioscler. Thromb. 1991;11(403-414). 66. Johnson W, Fischer R, Phillips M, Rothblat G. Efflux of newly synthesized cholesterol and biosynthetic sterol intermediates from cells: evidence on acceptor type and on enrichment of cells with cholesterol. J. Biol. Chem. 1995;270:25037-46. 67. Mendez AJ, Oram JF, Bierman EL. Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol. J. Biol. Chem. 1991;266(16):10T04-11. 68. Theret N, Delbart C, Aguie G, Fruchart J, Vassaux G, Ailhaud G. Cholesterol efflux from adipose cells is coupled to diacylglycerol production and protein kinase C activation. Biochem. Biophys. Res. Commun. 1990;173:1361-8. 69. Li Q, Tsujita M, Yokoyama S. Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cellular cholesterol efflux in macrophages. Biochemistry 1997;36(40): 12045-52. 70. Darbon J, Tourneir J, Tauber J, Bayard F. Possible role of protein phosphorylation in the mitogenic effect of high density lipoproteins on cultured vascular endothelial cells. J. Biol. Chem. 1986;261:8002-8. 71. Voyno-Yasenetskaya T, Dobbs L, Erickson S, Hamilton R. Low density lipoprotein-and high density lipoprotein-mediated signal transduction and exocytosis in alveolar type II cells. Proc. Natl. Acad. Sci. U.S.A. 1993;90:4256-60. 72. Walter M, Reinecke H, Nofer J, Seedorf U, Assmann G. HDL3 stimulates multiple signaling pathways in human skin fibroblasts. Arterioscler. Thromb. Vase. Biol. 1995;15:1975-86. 115 73. Oram J, Brinton E, Bierman E. Regulation of HDL receptor activity in cultured human skin fibroblasts and human arterial smooth muscle cells. J. Clin. Invest. 1983;72:1611-21. 74. Schmitz G, Niemann R, Brennhausen B, Krauss R, Assmann G. Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl CoA: cholesterol acyltransferase. EMBO J. 1985;4:2773-9. 75. Bielicki J, Johnson W, Weinberg R, Glick J, Rothblat G. Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol. J. Lipid. Res. 1992;33:1699-709. 76. Mazzone T, Pustelnikas L. Growth-related modulation of human skin fibroblast cholesterol distribution and metabolism. Biochem. Biophys. Acta 1990;1047:180-6. 77. Oram JF, Yokoyama S. Apolipoprotein-mediated removal of cellular cholesterol and phospholipids. J. Lipid Res. 1996;37(12):2473-91. 78. Francis GA, Knopp RH, Oram JF. Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier Disease. J. Clin. Invest. 1995;96(l):78-87. 79. Smith E, Ashall C, Walker J. High density lipoprotein (HDL) subfractions in interstitial fluid from human aortic intima and atherosclerotic lesions. Biochem. Soc. Trans. 1984;12:843-4. 80. Oram J. Effects of high density lipoprotein subfractions ort cholesterol homeostasis in human fibroblasts and arterial smooth muscle cells. Arteriosclerosis 1983;3:420-32. 81. Daerr W, Gianturco S, Patsch J, Smith L, Gotto A. Stimulation and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in normal fibroblasts by high density lipoprotein subclasses. Biochim. Biophys. Acta 1980;619:287-301. 82. de la Llera Moya M, Atger V, Paul J, Fournier N, Moatti N, Giral P, et al. A cell culture system for screening human serum for ability to promote cellular cholesterol efflux. Arterioscler. Thromb. 1994; 14(7): 1056-65. 83. Fuster V, Lewis A. Conner Memorial Lecture. Mechanisms leading to myocardial infarction: insights from studies of vascular biology. Circulation 1994;90:2126-46. 84. Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico (GISSI). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet 1986;1:397-402. 116 V 85. Nivelstein-Post P, Mottino G, Fogelman A, Frank J. An ultra-structural study of lipoprotein accumulation in cardiac valves of the rabbit. Arterioscler. Thromb. 1994;14:1151-61. 86. Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc. Natl. Acad. Sci. U.S.A. 1984;81(12):3883-7. 87. Steinbrecher UP, Zhang H, Lougheed M. Role of oxidatively modified LDL in atherosclerosis. Free Rad. Biol. Med. 1990;9(2): 155-68. 88. Steinbrecher U. Role of lipoprotein peroxidation in the pathogenesis of atherosclerosis. Clin. Cardiol. 1991; 14(11):865-7. 89. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J. Clin. Invest. 1989;84(4): 1086-95. 90. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, et al. Low density lipoprotein undergoes oxidative modification in vivo. Proc. Natl. Acad. Sci. USA 1989;86:1372-6. 91. Hoff HF, O'Neil J, Pepin JM, Cole TB. Macrophage uptake of cholesterol-containing particles derived from LDL and isolated from atherosclerotic lesions. Eur Heart J 1990. 92. Maor I, Mandel H, Aviram M. Macrophage uptake of oxidized LDL inhibits lysosomal sphingomyelinase, thus causing the accumulation of unesterified cholesterol-sphingomyelin-rich particles in the lysosomes. A possible role for 7-ketocholesterol. Arterioscler. Thromb. Vase. Biol. 1995;15:1378-87. 93. Diaz MN, Frei B, Vita JA, Keaney JF, Jr. Antioxidants and atherosclerotic heart disease. N. Engl. J. Med. 1997;337:408-16. 94. Klinkner AM, Waites CR, Kerns WD, Bugelski PJ. Evidence of foam cell and cholesterol crystal formation in macrophages incubated with oxidized LDL by fluorescence and electron microscopy. Journal of Histochemistry & Cytochemistry 1995;43(10): 1071-8. 95. Greenspan P, Yu H, Mao F, Gutman R. Cholesterol deposition in macrophages: foam cell formation mediated by cholesterol-enriched oxidized low density lipoprotein. J. Lipid Res. 1997;38:101-9. 117 96. Liu SX, Zhou M, Chen Y, Wen WY, Sun MJ. Lipoperoxidative injury to macrophages by oxidatively modified low density lipoprotein may play an important role in foam cell formation. Atherosclerosis 1996;121(1):55-61. 97. Berliner J, Navab M, Fogelman A, Frank J, Demer L, Edwards P, et al. Atherosclerosis: Basic Mechanisms. Circulation 1995;91:2488-96. 98. Williams K, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler. Thromb. Vase. Biol. 1995;15:551-61. 99. Hajjar D, Haberland M. Lipoprotein Trafficking in Vascular Cells. J. Biol. Chem. 1997;272(37):22975-8. 100. Wolfbauer G, Glick JM, Minor LK, Rothblat GH. Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions. Proc. Natl. Acad. Sci. U.S.A. 1986;83:7760-4. 101. Nikkari ST, Jarvelainen HT, Wight TN, Ferguson M, Clowes AW. Smooth muscle cell expression of extracellular matrix genes after arterial injury. American Journal of Pathology 1994;144(6):1348-56. 102. Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemori EN, Lark MW, et al. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion. Circulation Research 1994;75(1): 181-9. 103. Galis ZS, Kranzhofer R, Fenton JWn, Libby P. Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells. Arteriosclerosis, Thrombosis & Vascular Biology 1997;17(3):483-9. 104. Breslow JL. Cardiovascular disease burden increases, NIH funding decreases. Nature 1997;3:600-1. 105. Braunwald E. Shattuck Lecture — Cardiovascular Medicine at the turn of the millennium: triumphs, concerns, and opportunities. N. Engl. J. of Med. 1997;337:1360-9. 106. Ross R. Atherosclerosis — An Inflammatory Disease. N. Engl. J. of Med. 1999;340(2): 115-26. 107. Daugherty A, Zwiefel BS, Sobel BE, Schonfeld G isolation of low density lipoprotein from atherosclerotic vascular tissue of Watanabe heritable hyperlipidemic rabbits. Arteriosclerosis 1988;8:768-77. 108. Hollander W, Colombo MA, Kirkpatrick BJ, Paddock J. Soluble proteins in the human atherosclerotic plaque: with special reference to immunoglobulins, C3 118 complement component, oc-1 antitrypsin and oc-2 macroglobulin. Atherosclerosis 1979;34:391-405. 109. Steinberg D. Lipoproteins and atherosclerosis. A look back and a look ahead. Arteriosclerosis 1983;3:283-301. 110. Steinberg D. Arterial metabolism of lipoproteins in relation to atherogenesis. Annals of the New York Academy of Sciences 1990;598(125): 125-35. 111. Steinberg D, Witztum JL. Lipoproteins and atherogenesis. Current concepts. JAMA 1990;264:3047-52. 112. Hoff HF, Morton RE. Lipoproteins containing apoB extracted from human aortas. Ann. N.Y. Acad. Sci. 1985;454:183-94. 113. Hoff HF, Gaubatz JW. Isolation, purification and characterization of a lipoprotein containing apo B from the human aorta. Atherosclerosis 1982;42:273-97. 114. Mendez AJ. Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion. J. Lipid Res. 1997;38(9): 1807-21. 115. Bernini F, Calabresi L, Bonfadini G, Franceschini G. The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux. Biochim. Biophys. Acta 1996;1299(1): 103-9. 116. Aqel NM, Ball RY, Waldman H, Mitchinson MJ. Monocytic origin of foam cells in human atherosclerotic plaques. Atherosclerosis 1984;53:265-71. 117. Fowler S, Shio 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;4:372-8. 118. Faggiotto A, Ross R. Studies of hypercholesterolemia in the nonhuman primate, II. Fatty streak conversion to fibrous plaque. Arteriosclerosis 1984;4:341-56. 119. Gerrity RG. The role of monocyte in atherogenesis. I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am. J. Pathol. 1981;103:181-90. 120. Gerrity RG. The role of the monocyte in atherogenesis. II. Migration of foam cells from atherosclerotic lesions. Am. J. Pathol. 1981;103:191-200. 121. Rosenfeld ME, Tsukada T, Gown AM, Ross R. Fatty streak initiation in Watanabe Heritable Hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis 1987;7:9-23. 119 122. Schaffner T, Taylor K, Bartucci EJ, Fischer-Dzoga K, Beenson JH, Glagov S, et al. Arterial foam cells with distinctive immunomorphologic and histochemical features of macrophages. Am. J. Pathol. 1980;100: 57-80. 123. 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. U.S.A. 1979;76:333-7. 124. Goldstein JL, Kita T, Brown MS. Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia. N. Engl. J. Med. 1983;309:288-96. 125. Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira P, Kreiger M. Type I macrophage scavenger receptor contains a-helical and collagen-like coiled coils. Nature 1990;343:532-5. 126. Mahley RW, Innerarity TL, Weisgraber KH, Oh SY. Altered metabolism (in vivo and in vitro) of plasma lipoproteins after selective chemical modification of lysine residues of the apoproteins. J. Clin. Invest. 1979;64:743-50. 127. Fogelman AM, Schechter I, Seager J, Hodom M, Child JS, Edwards PA. Malondialdehyde alteration of low density lipoproteins leads to choleteryl ester accumulation in human moonocyte macrophages. Proc. Natl. Acad. Sci. U.S.A. 1980;77:2214-8. 128. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J. Biol. Chem. 1997;272(34):20963-6. 129. Lougheed M, Zhang H, Steinbrecher U. Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages. J. Biol. Chem. 1991;266:14519-25. 130. Navab M, Berliner J, Watson A, Hama S, Territo M, Lusis A, et al. The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture. Arterioscler. Thromb. Vase. Biol. 1996;16:831-42. 131. Lopes-Virella MF, Klein RL, Lyons TJ, Stevenson HC, Witztum JL. Glycosylation of low-density lipoprotein enhances cholesteryl ester synthesis in human monocyte-derived macrophages. Diabetes 1988;37:550-7. 132. Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N. Engl. J. Med. 1988;318:1315-21. 120 133. Steinbrecher U, Witztum J. Glucosylation of low-density lipoproteins to an extent comparable to that seen in diabetes slows their catabolism. Diabetes 1984;33(2):130-4. 134. Khoo JC, Miller E, McLoughlin P, Steinberg D. Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis 1988;8:348-58. 135. Polacek D, Byrne RE, Scanu AM. Modification of Low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway. J. Lipid Res. 1988;29:797-808. 136. Yla-Herttuala S, Jaakkola O, Solakivi T, Kuivaniemi H, Nikkari T. T h e e f f e c t o f proteoglycans, collagen, and lysyl oxidase on the metabolism of low density lipoprotein by macrophages. Atherosclerosis 1986;62-73-80 137. Hurt E, Camejo G. Effect of arterial proteoglycans on the interaction of LDL with human monocyte-derived macrophages. Atherosclerosis 1987;67:115-26. 138. Hurt E, Bondjers G, Camejo G. Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages. J. Lipid Res. 1990;31:443-54. 139. Hollander W, Lazzari A, Franzblau C. Biochemical and immunological evidence for the presence of lipoprotein-antilipoprotein immune complexes in human atherosclerotic plaque. In: Glagov S, Newman, W.P., and Schaffer, S.A., editor. Pathobiology of the human atherosclerotic plaque. New York: Springer Verlag; 1990. p. 209-34. 140. Tertov VV, Sobenin IA, Orekhov AN, Jaakkola O, Solakivi T, Nikkari T. Characteristics of low density lipoprotein isolated from circulating immune complexes. Atherosclerosis 1996;122(2):191-9. 141. Tertov VV, Orekhov AN, Kacharava AG, Sobenin IA, Perova NV, Smirnov VN. Low density lipoprotein-containing circulating immune complexes and coronary atherosclerosis. Exp. Mol. Pathol. 1990;52:300-8. 142. Szondy E, Lengyel E, Mezey Z, Fust G, Gero S. Occurrence of anti-low density lipoprotein antibodies and circulating immune complexes in aged subjects. Mech. Ageing Dev. 1985;29(2): 117-23. 143. Szondy E, Horvath M, Mezey Z, Szekely J, Lengyel E, Fust G, et al. Free and complexed anti-lipoprotein antibodies in vascular diseases. Atherosclerosis 1983;49(l):69-77. 121 144. Witztum JL, Steinbrecher UP, Fisher M, Kesaniemi A. Nonenzymatic glucosylation of homologous low density lipoprotein and albumin renders them immunogenic in the guinea pig. Proc. Natl. Acad. Sci. U.S.A. 1983;80(9):2757-61. 145. Witztum JL, Steinbrecher UP, Kesaniemi YA, Fisher M. Autoantibodies to glucosylated proteins in the plasma of patients with diabetes mellitus. Proc. Natl. Acad. Sci. U.S.A. 1984;81(10):3204-8. 146. Henriksen T, Mahoney E, 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;78:6499-503. 147. Henriksen T, Mahoney E, Steinberg D. Interactions of plasma lipoproteins with endothelial cells. Ann NY Acad Sci 1982;401:102-16. 148. Henriksen T, Mahoney EM, Steinberg D. Enhanced macrophage degradation of biologically modified low density lipoprotein. Arteriosclerosis 1983;3:149-59. 149. Heinecke JW, Rosen H, Chait A. Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J. Clin. Invest. 1984;74:1890-4. 150. Cathcart MK, Morel DW, Chisolm III GM. Monocytes and neutrophils oxidize low density lipoproteins making it cytotoxic. J. Leukocyte Biol. 1985;38:341-50. 151. Lamb DJ, Wilkins GM, Leake DS. The oxidative modification of low density lipoprotein by human lymphocytes. Atherosclerosis 1992;92:187-92. 152. Morel DW, DiCorleto PE, Chisolm GM. Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation. Arteriosclerosis 1984;4:357-64. 153. Parthasarathy S, Printz DJ, Boyd D, Joy L, Steinberg D. Macrophage oxidation of low density lipoprotein generates a form recognized by the scavenger receptor. Arteriosclerosis 1986;6:505-10. 154. Lougheed M, Ming Lum C, Ling W, Suzuki H, Kodama T, Steinbrecher UP. High-affinity saturable uptake of oxidized low density lipoprotein by macrophages from mice lacking the scavenger receptor class A type I/II. J. Biol. Chem. 1997;272(20): 12938-44. 155. Dhaliwal B, Steinbrecher U. Scavenger Receptors and Oxidized LDL. Clinica Chimica Acta 1999:m press. 122 156. Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: Macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu. Rev. Biochem. 1994;63:601-37. 157. Emi M, Asaoka H, Matsumoto A, Itakura H, Kurihara Y, Wada Y, et al. Structure, organization, and chromosomal mapping of the human macrophage scavenger receptor gene. J. Biol. Chem. 1993;268:2120-5. 158. Freeman M, Ashkenas J, Rees DJG, Kingsley DM, Copeland NG, Jenkins NA, et al. An ancient, highly conserved family of cysteine-rich protein domains revealed by cloning type I and type II murine macrophage scavenger receptors. Proc. Natl. Acad. Sci. USA 1990;87:8810-4. 159. Penman M, Lux A, Freedman NJ, Rohrer L, Ekkel Y, McKinstry H, et al. The type I and type II bovine scavenger receptors expressed in Chinese hamster ovary cells are trimeric proteins with collagenous triple helical domains comprising noncovalently associated monomers and Cys83-disulfide-linked dimers. J. Biol. Chem. 1991;266(35):23985-93. 160. Doi T, Higashino K, Kurihara Y, Wada Y, Miyazaki T, Nakamura H, et al. Charged collagen structure mediates the recognition of negatively charged macromolecules by macrophage scavenger receptors. J. Biol. Chem. 1993;268:2126-33. 161. Rohrer L, Freeman M, Kodama T, Penman M, Krieger M. Coiled-coil fibrous domains mediate ligand binding by macrophage scavenger receptor type II. Nature 1990;343:570-2. 162. Gough P, Greaves D, Gordon S. A naturally occurring isoform of the human scavenger receptor (SR-A) gene generated by alternative splicing blocks modified LDL uptake. J. Lipid Res. 1998;39:531-43. 163. Pearson AM, Rich A, Krieger M. Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-strand helices. J. Biol. Chem. 1993;268:3546-54. 164. Nishikawa K, Arai H, Inoue K. Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages. J. Biol. Chem. 1990;265:5226-31. 165. Hampton RY, Golenbock DT, Penman M, Krieger M, Raetz CR. Recognition and plasma clearance of endotoxin by scavenger receptors. Nature 1991;352(6333):342-4. 166. Piatt N, Suzuki H, Kurihara Y, Kodama T, Gordon S. Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro. Proc. Natl. Acad. Sci. USA 1996;93:12456-60. 123 167. Terpstra V, Kondratenko N, Steinberg D. Macrophages lacking scavenger receptor A show a decrease in binding and uptake of acetylated low-density lipoprotein and of apoptotic thymocytes, but not of oxidatively damaged red blood cells. Proc. Natl. Acad. Sci. USA 1997;94(15):8127-31. 168. Lee KD, Pitas RE, Papahadjopoulos D. Evidence that the scavenger receptor is not involved in the uptake of negatively charged liposomes by cells. Biochim. Biophys. Acta 1992;llll(l):l-6. 169. Steinbrecher UP, Lougheed M, Kwan WC, Dirks M. Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation. J. Biol. Chem. 1989;264(26): 15216-23. 170. Zhang H, Yang Y, Steinbrecher UP. Structural requirements for the binding of modified proteins to the scavenger receptor of macrophages. J. Biol. Chem. 1993;268(8):5535-42. 171. Haberland M, Fogelman A. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte-macrophages. Proc. Natl. Acad. Sci. USA 1985;82:2693-7. 172. Elomaa O, Kangas M, Sahlberg C, Tuukkanen J, Sormunen R, Liakka A, et al. Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages. Cell 1995;80(4):603-9. 173. van der Laan LJW, Kangas M, Dopp EA, Brougholub E, Elomaa O, Tryggvason K, et al. Macrophage scavenger receptor MARCO - in vitro and in vivo regulation and involvement in the anti-bacterial host defense. Immunol. Lett. 1997;57(l-3):203-8. 174. Fearon DT, Locksley RM. The instructive role of innate immunity in the acquired immune response. Science 1996;272(5258):50-3. 175. Greenwalt D, Lipsky R, Ockenhouse C, Ikeda H, Tandon N, Jamieson G. Membrane glycoprotein CD36: A Review of its Roles in Adherance, Signal Transduction, and Transfusion Medicine. Blood 1992;80:1105-15. 176. Abumrad N, El-Maghrabi M, Amri E, Lopez E, Grimaldi P. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. J. Biol. Chem. 1993;268:17665-8. 177. Han JH, Hajjar DP, Febbraio M, Nicholson AC. Native and modified low density lipoproteins increase the functional expression of the macrophage class B scavenger receptor, CD36. J. Biol. Chem. 1997;272(34):21654-9. 124 178. Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RG, Xu S, et al. Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J. Biol. Chem. 1997;272:13242-9. 179. Armesilla AL, Calvo D, Vega MA. Structural and functional characterization of the human CD36 gene promoter: identification of a proximal PEBP2/CBF site. J. Biol. Chem. 1996;271(13):7781-7. 180. Calvo D, Gomez-Coronado D, Suarez Y, Lasuncion M, Vega M. Human CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, and VLDL. J. Lipid Res. 1998;39:777-88. 181. Acton S, Scherer P, Lodish H, Krieger M. Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J. Biol. Chem. 1994;269:21003-9. 182. Puente Navazo MD, Daviet L, Ninio E, McGregor J. Identification on human CD36 of a domain (155-183) implicated in binding oxidized low-density lipoproteins (Ox-LDL). Arterioscler. Thromb. Vase. Biol. 1996;16:1033-9. 183. Ryeom SW, Sparrow JR, Silverstein RL. CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium. J. Cell Sci. 1996;109(Pt 2):387-95. 184. Ryeom SW, Silverstein RL, Scotto A, Sparrow JR. Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36. J. Biol. Chem. 1996;271(34):20536-9. 185. Endemann G, Stanton LW, Madden KS, Bryant CM, White RT, Protter AA. CD36 is a receptor for oxidized low density lipoprotein. J. Biol. Chem. 1993;268:11811-6. 186. Nicholson A, Frieda S, Pierce A, Silverstein R. Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines. Arterioscler. Thromb. Vase. Biol. 1995;15:269-75. 187. Nozaki S, Kashiwagi H, Yamashita S, Nakagawa T, Kostner B, Tomiyami Y, et al. Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects. J. Clin. Invest. 1995;96:1859-65. 188. Calvo D, Vega MA. Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J. Biol. Chem. 1993;268:18929-35. 189. Rigotti A, Acton S, Krieger M. The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids. J. Biol. Chem. 1995;270:16221-4. 190. Calvo D, Gomez-Coronado D, Lasuncion M, Vega M. CLA-1 in an 85-kD plasma membrane glycoprotein that acts as a high-affinity receptor for both native (HDL, 125 LDL, and VLDL) and modified (OxLDL and AcLDL) lipoproteins. Arterioscler. Thromb. Vase. Biol. 1997;17:2341-9. 191. Xu SZ, Laccotripe M, Huang XW, Rigotti A, Zannis VI, Krieger M. Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake. J. Lipid Res. 1997;38:1289-98. 192. Kozarsky KF, Donahee MH, Rigotti A, Iqbal SN, Edelman ER, Krieger M. Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels. Nature 1997;387(6631):414-7. 193. Pearson A, Lux A, Krieger M. Expression cloning of dSR-CI, a class C macrophage -specific scavenger receptor from Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 1995;92(9):4056-60. 194. Abrams JM, Lux A, Steller H, Krieger M. Macrophages in Drosophila embryos and L2 cells exhibit scavenger receptor-mediated endocytosis. Proc. Natl. Acad. Sci. USA 1992;89(21): 10375-9. 195. da Silva RP, Piatt N, de Villiers JS, Gordon S. Membrane molecules and macrophage endocytosis: scavenger receptor and macrosialin as markers of plasma-membrane and vacuolar functions. Biochem. Soc. Trans. 1996;24(l):220-4. 196. Martinez-Pomares L, Piatt N, McKnight AJ, da Silva RP, Gordon S. Macrophage membrane molecules: markers of tissue differentiation and heterogeneity. Immunobiology 1996; 195(4-5) :407-16. 197. Holness CL, da Silva RP, Fawcett J, Gordon S, Simmons DL. Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family. J. Biol. Chem. 1993;268(13):9661-6. 198. Ramprasad M, Terpstra V, Kondratenko N, Quehenberger O, Steinberg D. Cell surface expression of mouse macrosialin and human CD68 and their roles as macrophage receptors for oxidized low density lipoprotein. Proc. Natl. Acad. Sci. USA 1996;93(25):14833-8. 199. Stanton LW, White RT, Bryant CM, Protter AA, Endemann G. A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein. J. Biol. Chem. 1992;267:22446-51. 200. Morganelli P, Groveman D, Pfeiffer J. Evidence that human Fey receptor IIA (CD32) subtypes are not receptors for oxidized LDL. Arterioscler. Thromb. Vase. Biol. 1997;17:3248-54. 126 201. Yla-Herttuala S, Palinski W, Butler S, Picard S, Steinberg D, Witztum J. Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL. Arterioscler. Thromb. 1994;14:32-40. 202. Sawamura T, Kume N, Aoyama T, Moriwaki H, Hoshikawa H, Aiba Y, et al. An endothelial receptor for oxidized low-density lipoprotein. Nature 1997;386:73-7. 203. Adachi H, Tsujimoto M, Arai H, Inoue K. Expression cloning of a novel scavenger receptor from human endothelial cells. J. Biol. Chem. 1997;272:31217-20. 204. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radical Biol. Med. 1992;13(4):341-90. 205. Ku G, Thomas CE, Akeson AL, Jackson RL. Induction of interleukin 1(3 expression from human peripheral blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic acid. J. Biol. Chem. 1992;267:14183-8. 206. Lenz ML, Hughes H, Mitchell JR, Via DP, Guyton JR, Taylor AA, et al. Lipid hydroxy and hydroperoxy derivatives in copper-catalyzed oxidation of low density lipoprotein.!. Lipid Res. 1990;31:1043-50. 207. Wang T, Yu W, Powell W. Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells. J. Lipid Res. 1992;33:525-37. 208. Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Rad. Res. Commun. 1989;6(1):67-75. 209. Esterbauer H, Jurgens G, Quehenberger O, Koller E. Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J. Lipid Res. 1987;28:495-509. 210. Steinbrecher UP. Oxidation of human low density lipoproteins results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J. Biol. Chem. 1987;262(8):3603-8. 211. Steinbrecher UP, Witztum JL, Parthasarathy S, Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism. Arteriosclerosis 1987;7(2): 135-43. 212. Fruebis J, Parthasarathy S, Steinberg D. Evidence for a concerted reaction between lipid hydroperoxides and polypeptides. Proc. Natl. Acad. Sci. U.S.A. 1992;89(22): 10588-92. 127 213. Steinbrecher UP, Pritchard PH. Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase. J. Lipid Res. 1989;30(3):305-15. 214. Heinecke JW, Baker L, Rosen L, Chait A. Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells. J. Clin. Invest. 1986;77:757-61. 215. Sparrow C, Olszewski J. Cellular oxidation of low density lipoprotein is caused by thiol production in media containing transition metal ions. J. Lipid Res. 1993;34:1219-28. 216. Parthasarathy S, Wieland E, Steinberg D. A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein. Proc. Natl. Acad. Sci. USA 1989;86:1046-50. 217. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 1989;320(14):915-24. 218. van Hinsbergh V, Scheffer M, Havekes L, Kempen H. Role of endothelial cells and their oxidation products in the modification of low-density lipoproteins. Biochim. Biophys. Acta 1986;878:49-64. 219. Halliwell B. Albumin — an important extracellular antioxidant? Biochem. Pharmacol. 1988;37:569-71. 220. Zawadski Z, Milne RW, Marcel YL. Cu2+-mediated oxidation of dialyzed plasma: effects on low and high density lipoproteins and cholesteryl ester transfer protein. J. Lipid Res. 1991;32:243-50. 221. Kalant N, McCormick S. Inhibition by serum components of oxidation and collagen-binding of low-density lipoprotein. Biochim. Biophys. Acta 1992; 1128(2-3):211-9. 222. Thomas C. The influence of medium components on Cu(2+)-dependent oxidation of low-density lipoprotein and its sensitivity to superoxide dismutase. Biochim. Biophys. Acta 1992;1128:50-7. 223. Dobrian A, Mora R, Simonescu M, Simonescu N. In vitro formation of oxidatively modified and reassembled human low-density lipoproteins: antioxidant effect of albumin. Biochim. Biophys. Acta 1993;1169:12-24. 224. Wagner J, Motchnik P, Stocker R, Sies H, Ames B. The oxidation of blood plasma and low density lipoprotein components by chemically generated singlet oxygen. J. Biol. Chem. 1993;268:18502-6. 128 225. van Berkel TJC, de Rijke YB, Kruijt JK. Different fate in vivo of oxidatively modified-LDL and acetylated-LDL in rats. Recognition by various scavenger receptors on Kupffer cells and endothelial liver cells. J. Biol. Chem. 1991;266:2282-9. 226. de Rijke Y, Jurgens G, Hessels E, Hermann A, van Berkel T. In vivo fate and scavenger receptor recognition of oxidized lipoprotein [a] isoforms in rats. J Lipid Res 1992;33(9):1315-25. 227. Chang M, Lees A, Lees R. Low-density lipoprotein modification and arterial wall accumulation in a rabbit model of atherosclerosis. Biochemistry 1993;32:8518-24. 228. Smith C, Mitchinson M, Aruoma O, Halliwell B. Stimulation of lipid peroxidation and hydroxy radical generation by the contents of human atherosclerotic lesions. Biochem. J. 1992;286:901-5. 229. Haberland M, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science 1988;241:215-8. 230. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis 1990;10(3):336-49. 231. Bjorkhem I, Henriksson-Freyschuss A, Breuer O, Diczfalusy U, Berglund L, Henriksson P. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Arterioscler. Thromb. 1991 ;11(1): 15-22. 232. Carew TE, Schwenke DC, Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc. Natl. Acad. Sci. USA 1987;84:7725-9. 233. Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, et al. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc. Natl. Acad. Sci. USA 1987;84:5928-31. 234. Sparrow CP, Doebber TW, Olszewski J, Wu MS, Ventre J, Stevens KA, et al. Low density lipoprotein is protected from oxidation and the progression of atherosclerosis is slowed in cholesterol-fed rabbits by the antioxidant N,N'-diphenyl-phenylenediamine. J. Clin. Invest. 1992;89:1885-91. 235. Daugherty A, Zweifel BS, Schonfeld G. Probucol attenuates the development of atherosclerosis in cholesterol-fed rabbits. Br. J. Pharmacol. 1989;98:612-8. 129 236. Stein Y, Stein O, Delplanque B, Fesmire JD, Lee DM, Alaupovic P. Lack of effect of probucol on atheroma formation in cholesterol-fed rabbits kept at comparable plasma cholesterol levels. Atherosclerosis 1989;75:145-66. 237. Sasahara M, Raines E, Chait A, Carew T, Steinberg D, Wahl P, et al. Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol. Is the extent of atherosclerosis related to resistance of LDL to oxidation? J. Clin. Invest. 1994;94:155-64. 238. Steinberg D. Lewis A. Conner Memorial Lecture. Oxidative modification of LDL and atherogenesis. Circulation 1997;95(4):1062-71. 239. Heinecke J. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis 1998;141(1):1-15. 240. Regnstrom J, Nilsson J, Tornvall P, Landou C, Hamsten A. Susceptibility to low-density lipoprotein oxidation and coronary atherosclerosis in man. Lancet 1992;339(8803): 1183-6. 241. Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, et al. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc. Natl. Acad. Sci. USA 1990;87:5134-8. 242. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, et al. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J. Clin. Invest. 1990;85:1260-6. 243. Rajavashisth TB, Andalibi A, Territo MC, Berliner JA, Navab M, Fogelman AM, et al. Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins. Nature 1990;344(6263):254-7. 244. Liao F, Berliner JA, Mehrabian M, Navab M, Demer LL, Lusis AJ, et al. Minimally modified low density lipoprotein is biologically active in vivo in mice. J. Clin. Invest. 1991;87:2253-7. 245. Schwartz D, Andalibi A, Chaverri-Almada L, Berliner J, Kirchgessner T, Fang Z-T, et al. Role of the GRO family of chemokines in monocyte adhesion to MM-LDL-stimulated endothelium. J. Clin. Invest. 1994;94:1968-73. 246. Steinbrecher UP. Role of superoxide in endothelial-cell modification of low-density lipoproteins. Biochim. Biophys. Acta 1988;959(l):20-30. 130 247. 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 negatively charged LDL by macrophages. J. Supramol. Struct. 1980;13:67-81. 248. Morel DW, Hessler JR, Chisolm GM. Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid. J. Lipid Res. 1983;24:1070-6. 249. Hessler JR, Robertson Jr. AL, Chisolm GM. LDL-induced cytotoxicity and its inhibition by HDL in human vascular smooth muscle and endothelial cells in culture. Atherosclerosis 1979;32:213-29. 250. Hessler JR, Morel DW, Lewis LJ, Chisolm GM. Lipoprotein oxidation and lipoprotein-induced cytotoxicity. Arteriosclerosis 1983;3:215-22. 251. Henriksen T, Evensen SA, Carlander B. Injury to human endothelial cells in culture induced by low density lipoproteins. Scand. J. Clin. Lab. Invest. 1979;39:361-8. 252. Chisolm G, Ma G, Irwin K, Martin L, Gunderson K, Lindberg L, et al. 70-Hydroperoxycholest-5-en-3(3-ol, a component of human atherosclerotic plaque, is the primary cytotoxin of oxidized human low density lipoprotein. Proc. Natl. Acad. Sci. USA 1994;91:11452-6. 253. Hughes H, Mathews B, Lenz M, Guyton J. Cytotoxicity of oxidized LDL to porcine aortic smooth muscle cells is associated with the oxysterols 7-ketocholesterol and 7-hydroxycholesterol. Arterioscler. Thromb. 1994;14:1177-85. 254. Buttke T, Sandstrom P. Oxidative stress as a mediator of apoptosis. Immunol. Today 1994;15:7-10. 255. Escargueil-Blanc I, Salvayre R, Negre-Salvayre A. Necrosis and apoptosis induced by oxidized low density lipoproteins occur through two calcium-dependent pathways in lymphoblastoid cells. FASEB J. 1994;8:1075-80. 256. Bjorkerud B, Bjorkerud S. Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibroblasts. Arterioscl. Thromb. Vase. Biol. 1996;16:416-24. 257. Hardwick SJ, Hegyi L, Clare K, Law NS, Carpenter KL, Mitchinson MJ, et al. Apoptosis in human monocyte-macrophages exposed to oxidized low density lipoprotein. J. Pathol. 1996;179(3):294-302. 258. Nishio E, Arimura S, Watanabe Y. Oxidized LDL induces apoptosis in cultured smooth muscle cells: a possible role for 7-ketocholesterol. Biochem. Biophys. Res. Comm. 1996;223(2):413-8. 131 259. Papassotiropoulos A, Ludwig M, Naib-Majani W, Rao GS. Induction of apoptosis and secondary necrosis in rat dorsal root ganglion cell cultures by oxidized low density lipoprotein. Neurosci. Lett. 1996;209(l):33-6. 260. Dimmeler S, Haendeler J, Galle J, Zeiher AM. Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the 'response to injury' hypothesis. Circulation 1997;95(7):1760-3. 261. Escargueil-Blanc I, Meilhac O, Pieraggi M-T, Arnal J-F, Salvayre R, Negre-Salvayre A. Oxidized LDLs induce massive apoptosis of cultured human endothelial cells through a calcium-dependent pathway. Prevention by aurintricarboxylic acid. Arterioscler. Thromb. Vase. Biol. 1997;17:331-9. 262. Hoff HF, Whitaker TE, O'Neil J. Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition. J. Biol. Chem. 1992;267(l):602-9. 263. Kalant N, McCormick S, Parniak MA. Effects of copper and histidine on oxidative modification of low density lipoprotein and its subsequent binding to collagen. Arterioscler. Thromb. 1991;ll(5):1322-9. 264. Quinn MT, Parthasarathy S, Steinberg D. Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein. Proc Natl Acad Sci USA 1985;82:5949-53. 265. Quinn MT, Parthasarathy S, Steinberg D. Oxidatively modified low density lipoproteins: A potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc. Natl. Acad. Sci. USA 1987;84:2995-8. 266. Quinn MT, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis. Proc. Natl. Acad. Sci. USA 1988;85:2805-9. 267. Lehr HA, Hiibner C, Finckh B, Angermuller S, Nolte D, Beisiegel U, et al. Role of leukotrienes in leukocyte adhesion following systemic administration of oxidatively modified human low density lipoprotein in hamsters. J. Clin. Invest. 1991;88(1):9-14. 268. Lehr HA, Krober M, Hubner C, Vajkoczy P, Menger MD, Nolte D, et al. Stimulation of leukocyte/endothelium interaction by oxidized low-density lipoprotein in hairless mice. Involvement of CDllb/CD18 adhesion receptor complex. Lab. Invest. 1993;68(4):388-95. 269. Yui S, Sasaki T, Miyazaki A, Horiuchi S, Yamazaki M. Induction of murine macrophage growth by modified LDLs. Arterioscler. Thromb. 1993; 13(3):331-7. 132 270. Martens J, Reiner N, Herrera-Velit P, Steinbrecher U. Phosphatidylinositol 3-kinase is involved in the induction of macrophage growth by oxidized low density lipoprotein. J. Biol. Chem. 1998;273(9):4915-20. 271. Frostegard J, Wu R, Giscombe R, Holm G, Lefvert AK, Nilsson J. Induction of T-cell activation by oxidized low density lipoprotein. Arterioscler. Thromb. 1992;12(4):461-7. 272. Frostegard J, Huang Y, Ronnelid J, Schafer-Elinder L. Platelet activating factor and oxidized LDL induce immune activation by a common mechanism. Arterioscler. Thromb. Vase. Biol. 1997;17:963-8. 273. Terkeltaub R, Banka C, Solan J, Santoro D, Brand K, Curtiss L. Oxidized LDL induces monocytic cell expression of interleukin-8, a chemokine with T-lymphocyte chemotactic activity. Arterioscler. Thromb. 1994;14:47-53. 274. Thomas C, Jackson R, Ohlweiler D, Ku G. Multiple lipid oxidation products in low density lipoproteins induce interleukin-1 beta release from human blood mononuclear cells. J. Lipid Res. 1994;35:417-27. 275. Kume N, Cybulsky M, Gimbrone MA. Lysophosphatidylcholine, a component of atherosclerotic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J. Clin. Invest. 1992;90:1138-44. 276. Khan B, Parthasarathy S, Alexander R, Medford R. Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J. Clin. Invest. 1995;95:1262-70. 277. Fox PL, Chisolm GM, DiCorleto PE. Lipoprotein-mediated inhibition of endothelial cell production of platelet-derived growth factor-like protein depends on free radical lipid peroxidation. J. Biol. Chem. 1987;262:6046-54. 278. Maiden LT, Chait A, Raines EW, Ross R. The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages. J. Biol. Chem. 1991;266:13901-7. 279. Hamilton TA, Ma GP, Chisolm GM. Oxidized low density lipoprotein suppresses the expression of tumor necrosis factor-alpha mRNA in stimulated murine peritoneal macrophages. J. Immunol. 1990;144(6):2343-50. 280. Fong LG, Fong TA, Cooper AD. Inhibition of lipopolysaccharide-induced interleukin-1 beta mRNA expression in mouse macrophages by oxidized low density lipoprotein. J. Lipid Res. 1991;32(12): 1899-910. 133 281. Kume N, Gimbrone M. Lysophosphatidycholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J. Clin. Invest. 1994;93:907-11. 282. Stiko A, Regnstrom J, Shah P, Cercek B, Nilsson J. Active oxygen species and lysophosphatidylcholine are involved in oxidized low density lipoprotein activation of smooth muscle cell DNA synthesis. Arterioscler. Thromb. Vase. Biol. 1996;16:194-200. 283. Chai YC, Howe PH, DiCorleto PE, Chisholm GM. Oxidized low density lipoproteins and lysophosphatidylcholine stimulate cell cycle entry in vascular smooth muscle cells. Evidence for release of fibroblast growth factor-2. J. Biol. Chem. 1996;271:17791-7. 284. Lafont A, Chai Y-C, Cornhill J, Whitlow P, Howe P, Chisolm G. Effect of alpha-tocopherol on restenosis after angioplasty in a model of experimental atherosclerosis. J. Clin. Invest. 1995;95:1018-25. 285. Heery JM, Kozak M, Stafforini DM, Jones DA, Zimmerman GA, Mclntyre TM, et al. Oxidatively modified LDL contains phospholipids with platelet activating factor-like activity and stimulates the growth of smooth muscle cells. J. Clin. Invest. 1995;96:2322-30. 286. Shio H, Haley N, Fowler S. Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. II. Morphometric analysis of lipid-filled lysosomes and lipid droplets in aortic cell populations. Lab. Invest. 1978;1978:390-7. 287. Shio H, Haley N, Fowler S. Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester. Lab. Invest. 1979;41:160-7. 288. Jerome W, Lewis J. Early atherogenesis in White Carneau pigeons. II. Ultrastructural and cytochemical observations. Am. J. Pathol. 1985;119:210-22. 289. Lupu F, Danaricu I, Simionescu N. Development of intracellular deposits in the lipid-laden cells of atherosclerotic lesions. Atherosclerosis 1987;67:127-42. 290. Fowler S, Berberian P, Shio H, Goldfischer S, Wolinsky H. Characterization of cell populations isolated from aortas of rhesus monkeys with experimental atherosclerosis. Circ. Res. 1980;46:520-30. 291. Lewis J, Taylor R, Ohta T. Lysosomal alterations during coronary atherosclerosis in the pigeon: correlative cytochemical and three-dimensional HVEM/IVEM observations. Exp. Mol. Pathol. 1988;48:103-15. 134 292. Balis J, Haust M, More R. Electron-microscopic studies in human atherosclerosis. Cellular elements in aortic fatty streaks. Exp. Mol. Pathol. 1964;3:511-25. 293. Marshall J, Adams J, O'Neal R, DeBakey M. The ultrastructure of uncomplicated human atheroma in surgically resected aortas. J. Atheroscler. Res. 1966;6:120-31. 294. Guyton JR, Klemp KF. The lipid-rich core region of human atherosclerotic fibrous plaques. Prevalence of small droplets and vesicles by electron microscopy. Am. J. Pathol. 1989;134:705-17. 295. Roma P, Bernini F, Fogliatto R, Bertulli SM, Negri S, Fumagalli R, et al. Defective catabolism of oxidized LDL by J774 murine macrophages. J. Lipid Res. 1992;33(6):819-29. 296. Kaesberg B, Harrach B, Dieplinger H, Robeneck H. In situ immunolocalization of lipoproteins in human arteriosclerotic tissue. Arterioscler. Thromb. 1993;13:133-46. 297. Mahlberg F, Glick J, Jerome W, Rothblat G. Metabolism of cholesterol ester lipid droplets in a J774 macrophage foam cell model. Biochim. Biophys. Acta 1990;1045:291-8. 298. Tangirala R, Mahlberg F, Glick J, Jerome W, Rothblat G. Lysosomal accumulation of unesterified cholesterol in model macrophage foam cells. J. Biol. Chem. 1993;268:9653-60. 299. Maor I, Aviram M. Oxidized low density lipoprotein leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesterol ester. J. Lipid Res. 1994;35:803-9. 300. Kritharides L, Jessup W, Mander E, Dean R. Apolipoprotein A-I-mediated efflux of sterols from oxidized LDL-loaded macrophages. Arterioscler. Thromb. Vase. Biol. 1995;15:276-89. 301. Yancey P, Jerome W. Lysosomal sequestration of free and esterified cholesterol from oxidized low density lipoprotein in macrophages of different species. J. Lipid Res. 1998;39:1349-61. 302. O'Neil J, Hoppe G, Sayre L, Hoff H. Inactivation of cathepsin B by oxidized LDL involves complex formation induced by binding of putative reactive sites exposed at low pH to thiols on the enzyme. Free Radical Biol. Med. 1997;23:215-25. 303. Hoppe G, O'Neil J, Hoff H. Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages. J. Clin. Invest. 1994;94:1506-12. 135 304. Jessup W, Mander EL, Dean RT. The intracellular storage and turnover of apolipoprotein B of oxidized LDL in macrophages. Biochim. Biophys. Acta 1992; 1126(2): 167-77. 305. Mander E, Jessup W, Dean R. Apolipoprotein B of oxidized LDL accumulates in the lysosomes of macrophages. Biochim. Biophys. Acta 1994;1212:80-92. 306. Rosenfeld M, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts. J. Clin. Invest. 1991;87:90-9. 307. Araki N, Horiuchi S, Rahim A, Takata K, Morino Y. Microquantification of cholesterol and cholesteryl esters in rat peritoneal macrophages by reverse-phase high-performance liquid chromatography. Anal. Biochem. 1990;1990(185). 308. Zhang H, Basra HJK, Steinbrecher UP. Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages. J. Lipid Res. 1990;31(8): 1361-9. 309. Jialal I, Chait A. Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein. J. Lipid Res. 1989;30:1561-8. 310. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 1955;43:1345-53. 311. McFarlane A. Efficient trace-labelling of proteins with iodine. Nature 1958;182:53. 312. Bilheimer DW, Eisenberg S, Levy RL The metabolism of very low density lipoproteins. Biochim. Biophys. Acta 1972;260:212-21. 313. Levine R, Garland D, Oliver C, Amici A, Clement I, Lenz A, et al., editors. Determination of carbonyl content in oxidatively mofified lipoproteins. New York: Academic Press; 1990. 314. Khoo JC, Miller E, McLoughlin P, Steinberg D. Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I. J. Lipid Res. 1990;31(4):645-52. 315. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265-75. 136 316. Tabas I, Tall A. Mechanism of the association of HDL3 with endothelial cells, smooth muscle cells, and fibroblasts. Evidence against the role of specific ligand and receptor proteins. J. Biol. Chem. 1984;259(22): 13897-905. 317. Morrison J, McPherson G, Fidge N. Evidence for two sites on rat liver plasma membranes which interact with high density lipoprotein3. J. Biol. Chem. 1992;267(19): 13205-9. 318. Scanu A. Forms of human serum high density lipoprotein protein. J. Lipid Res. 1966;7:295-306. 319. Gwynne J, Brewer BJ, Edelhoch H. The molecular properties of Apo A-I from human high density lipoprotein. J. Biol. Chem. 1974;249(8):2411-6. 320. Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(259):680-5. 321. Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 1969;244(16):4406-12. 322. Bligh E, Dyer W. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37(8):911-7. 323. Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. clin. Biochem. 1969;6:24-7. 324. Trinder P, Webster D. Determination of HDL-cholesterol using 2,4,6-tribromo-3-hydroxybenzoic acid with a commercial CHOD-PAP reagent. Annals of Clinical Biochemistry 1984;21(Pt 5):430-3. 325. Amundson D, Zhou M. Fluorometric Method for the Enzymatic Determination of Cholesterol. J. Biochem. Biophys. Meth. 1999;38(43):32334. 326. Rouser G, Fleischer S, Yamamoto A. Two-dimensional thin-layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 1970;5:494-6. 327. Seymour CA, Peters TJ. Enzyme activities in human liver biopsies: assay methods and activities of some lysosomal and membrane-bound enzymes in control tissue and serum. Clin. Sci. MQI. Med. 1977;52(3):229-39. 328. Vaes G. Subcellular localization of glycosidases in lysosomes. Methods Enzymol. 1966;8:509-14. 329. Dean M, Martin J. Intracellular localization of B-glucuronidase in fibroblasts after direct transfer from macrophages. Biochem. J. 1988;256(2):335-41. 137 330. Kritharides L, Jessup W, Dean R. A method for staging the degree of LDL oxidation by the separation of cholesterol and cholesteryl ester oxidation products by HPLC. Anal. Biochem. 1993;213:79-89. 331. Steinbrecher UP, Lougheed M. Scavenger receptor-independent stimulation of cholesterol esterification in macrophages by low density lipoprotein extracted from human aortic intima. Arterioscler. Thromb. 1992;12:608-25. 332. Jialal I, Freeman DA, Grundy SM. Varying susceptibility of different low density lipoproteins to oxidative modification. Arterioscler Thromb 1991;ll(3):482-8. 333. Bhadra S, Arshad MAQ, Rymaszewski Z, Norman E, Wherley R, Subbiah MTR. Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or C u + + ions: identification of major products and their effects. Biochem. Biophys. Res. Commun. 1991;176:431-40. 334. Reaven P, Parthasarathy S, Grasse B, Miller E, Steinberg D, Witztum J. Effect of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects. J. Clin. Invest. 1993;91:668-76. 335. Jerome W, Cash C, Webber R, Horton R, Yancey P. Lysosomal lipid accumulation from oxidized low density lipoprotein is correlated with hypertrophy of the Golgi apparatus and trans-Golgi network. J. Lipid Res. 1998;39:1362-71. 336. Chiesa G, Parolini C, Canavesi M, Colombo N, Sirtori C, Fumagalli R, et al. Human apolipoproteins A-I and A-II in cell cholesterol efflux: studies with transgenic mice. Arterioscler. Thromb. Vase. Biol. 1998; 18(9): 1417-23. 337. Barr D, Russ E, Eder H. Protein-lipid relationship in human plasma II. In atherosclerosis and related conditions. Am. J. Med. 1951;11:480-7. 338. Gordon D, Probstfield J, Garrison R, Neaton J, Castelli W, Knoke J, et al. High-density lipoprotein cholesterol and cardiovascular disease. Circulation 1989;79:8-15. 339. Miller N, Forde O, Thelle S. The Tromso hear study: high density lipoproteins and coronary heart disease; a prospective case-control study. Lancet 1977;1:965-8. 340. Rhoads G, Gulbrandsen C, Kagen A. Serum lipoproteins and coronary heart disease in a population study of Hawaii Japanese men. N. Engl. J. Med. 1976:294-8. 341. Gordon T, Castelli W, Hjortland M. High density lipoproteins as a prospective factor against coronary heart disease. The Framingham Study. Am. J. Med. 1977;62:707-14. 138 342. Gordon T, Kannel W, Castelli W, Dawber T. Lipoproteins, cardiovascular disease and death. The Framingham study. Arch. Intern. Med. 1981; 14:1128-35. 343. Jackson R, Gotto A, Stein O, Stein V. A comparative study on the removal of cellular lipids from Landschut ascites cell by human plasma lipoproteins. J. Biol. Chem. 1971;250:7204-9. 344. Stein O, Vanderhoek J, Stein Y. Cholesterol-ester accumulation in cultured aortic muscle cells. Atherosclerosis 1977;26:465-82. 345. Stein O, Stein Y. High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture. Biochim. Biophys. Acta 1976;431:363-8. 346. Bates S. Effect of HDL on the interaction of hyperlipidemic LDL with monkey smooth muscle cells. Artery 1980;7:303-8. 347. Ho Y, Brown M, Goldstein J. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoproteins and other agents. J. Lipid Res. 1980;21:391-8. 348. Reichl O, Rudura O, Myant N, Pflig J. Further evidence for the role of high density lipoproteins in the removal of tissue cholesterol in vivo. Atherosclerosis 1982;44:73-82. 349. Badimon J, Badimon L, Galvez A, Fuster V. High density lipoprotein plasma fractions inhibit aortic fatty streak lesions in cholesterol-fed rabbits. Lab. Invest. 1989;60:455-61. 350. Lange Y, Steck J. Cholesterol-rich intracellular membranes: a precursor to the plasma membrane. J. Biol. Chem. 1985;260:15592-6. 351. Lange Y. Disposition of intracellular cholesterol in human fibroblasts. J. Lipid Res. 1991;32:329-39. 352. Lange Y. Cholesterol movement from plasma membrane to rough endoplasmic reticulum. Inhibition by progesterone. J. Biol. Chem. 1994;269:3411-4. 353. Liscum L, Dahl N. Intracellular cholesterol transport. J. Lipid. Res. 1992;33:1239-54. 354. Liscum L, Underwood K. Intracellular cholesterol transport and compartmentation. J. Biol. Chem. 1995;270:15443-6. 355. Mendez A. Monensin and Brefeldin A inhibit high density lipoprotein-mediated cholesterol efflux from cholesterol-enriched cells. Implications for intracellular cholesterol transport. J. Biol. Chem. 1995;270:5891-900. 139 356. Mendez A, Uint L. Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus. J. Lipid Res. 1996;37:2510-24. 357. Oda K, Hirose S, Takami N, Misumi Y, Takatsuki A, Ikehara Y. Brefeldin A arrests the intracellular transport of a precursor of complement C3 before its conversion site in rat hepatocytes. FEBS Lett. 1987;214(135-138). 358. Takatsuki A, Tamura G. Brefeldin A, a specific inhibitor of intracellular translocation of vesicular stomatitis virus G protein: intracellular accumulation of high-mannose type G protein and inhibition of its cell surface expression. Agric. Biol. Chem. 1985;49:899-902. 359. Misumi Y, Miki A, Takatsuki A, Tamura G, Ikehara Y. Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes. J. Biol. Chem. 1986;261:11398-403. 360. Klausner R, Donaldson J, Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell. Biol. 1992;116(5): 1071-80. 361. Lippincott-Schwartz J, Donaldson J, Schweizer A, Berger E, Hauri H, Yuan L, et al. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 1990;60:821-36. 362. Lippincott-Schwartz J, Yuan L, Tipper C, Amherdt M, Orci L, Klausner R. Brefeldin A's effects on endosomes, lysosomes and TGN suggest a general mechanism for regulating organelle structure and membrane trafficking. Cell 1991;67(3):601-16. 363. Wood S, Park J, Brown W. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell 1991;67(3):591-600. 364. Hunziker W, Whitney J, Mellman I. Selective inhibition of transcytosis by brefeldin A in MDCK cell. Cell 1991;67:617-28. 365. Conrad P, Smart E, Ying Y, Anderson R, Bloom G. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps. J. Cell Biol. 1995;131:1421-33. 366. Fielding C, Bist A, Fielding P. Caveolin mRNA levels are up-regulated by free cholesterol and down-regulated by oxysterols in fibroblast monolayers. Proc. Natl. Acad. Sci. USA 1997;94:3753-8. 367. Adelman SJ, St. Clair RW. Lipoprotein metabolism by macrophages from atherosclerosis-susceptible White Carneau pigeons and resistant Show Racer pigeons. J. Lipid. Res. 1988;29:643-56. 140 368. Bernard D, Rodriques A, Rothblat G, Glick J. Influence of high density lipoprotein on esterified cholesterol stores in macrophages and hepatoma cells. Arteriosclerosis 1990;10:135-44. 369. Yancey P, St. Clair R. Cholesterol efflux is defective in macrophages from atherosclerosis-susceptible White Carneau pigeons relative to resistant Show Racer pigeons. Arterioscler. Thromb. 1992;12:1291-304. 370. Kritharides L, Christian A, Stoudt G, Morel D, Rothblat G. Cholesterol metabolism and efflux in Human THP-1 Macrophages. Arterioscler. Thromb. Vase. Biol. 1998;18:1589-99. 371. Lougheed M, Steinbrecher U. Mechanism of uptake of copper-oxidized low density lipoprotein in macrophages is dependent on its extent of oxidation. J. Biol. Chem. 1996;271:11798-805. 372. Kilsdonk E, Morel D, Johnson W, Rothblat G. Inhibition of cellular cholesterol efflux by 25-hydroxycholesterol. J. Lipid Res. 1995;36(3):505-16. 373. Lefevre M, Sloop C, Roheim P. Characterization of dog prenodal peripheral lymph lipoproteins: evidence for the peripheral formation of lipoprotein-unassociated apo A-I with slow pre-|3 electrophoretic mobility. J. Lipid Res. 1988;29:1139-48. 374. Castro G, Fielding C. Early incorporation of cell-derived cholesterol into pre-p-migrating high-density lipoprotein. Biochemistry 1988;27:25-9. 375. Hara H, Yokoyama S. Interaction of free apolipoproteins with macrophages: formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol. J. Biol. Chem. 1991;266:3080-6. 376. Komaba A, Li Q, Hara H, Yokoyama S. Resistance of smooth muscle cells to assembly of high density lipoproteins with extracellular free apolipoproteins and to reduction of intracellularly accumulated cholesterol. J. Biol. Chem. 1992;267:17560-6. 377. Hara H, Yokoyama S. Role of apolipoproteins in cholesterol efflux from macrophages to lipid microemulsion: proposal of a putative model for the pre-P high density lipoprotein pathway. Biochemistry 1992;31(2040-2046). 378. Forte T, Goth-Goldstein R, Nordhausen R, McCall M. Apolipoprotein A-I cell membrane interaction: extracellular assembly of heterogeneous nascent HDL particles. J. Lipid Res. 1993;34:317-24. 141 379. Forte T, Bielicki J, Knoff L, McCall M. Structural relationships between nascent apoA-I-containing particles that are extracellularly assembled in cell culture. J. Lipid Res. 1996;37:1076-85. 380. Bielicki J, Johnson W, Glick J, Rothblat G. Efflux of phospholipid from fibroblasts with normal and elevated levels of cholesterol. Biochim. Biophys. Acta 1991 ;1085:7-14. 381. Ffara H, Komaba A, Yokoyama S. a-Helical requirements for free apolipoproteins to generate HDL and to induce cellular lipid efflux. Lipids 1992;27:302-4. 382. Li Q, Yokoyama S. Independent regulation of cholesterol incorporation into free apolipoprotein-mediated cellular lipid efflux in rat vascular smooth muscle cells. J. Biol. Chem. 1995;270:26216-23. 383. Forte T, Bielicki J, Goth-Goldstein R, Selmek J, McCall M. Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT. J. Lipid. Res. 1995;36:148-57. 384. Pinto M, Robine-Leon S, Appay M, Kedinger M, Triadou N, Dussaulx E, et al. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line CaCo-2 in culture. Biol. Cell 1983;47:323-30. 385. Rogler G, Herold G, Fahr C, Fahr M, Rogler D, Reimann F, et al. High-density lipoprotein 3 retroendocytosis: a new lipoprotein pathway in the enterocyte (Caco-2). Gastroenterology 1992; 103(2):469-80. 386. Takahashi K, Fukuda S, Naito M, Horiuchi S, Takata K, Morino Y. Endocytic pathway of high density lipoprotein via trans-Golgi system in rat resident peritoneal macrophages. Lab. Invest. 1989;61(3):270-7. 387. Fluiter K, van der Westhuijzen D, van Berkel T. In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells. J. Biol. Chem 1998;273(14):8434-8. 142 

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