Hugh Sinclair Lecture: The regulation and remodelling of HDL by plasma factors

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1 Atherosclerosis Supplements 3 (2002) 39/47 Hugh Sinclair Lecture: The regulation and remodelling of HDL by plasma factors P.J. Barter Hanson Institute, Adelaide, SA, Australia Abstract High density lipoproteins (HDLs) originate as lipid-free or lipid-poor apolipoproteins that acquire most of their lipid in the extracellular space. They accept phospholipids from cells in a process promoted by the ATP binding cassette A1 transporter to form prebeta-migrating discoidal HDL that are efficient acceptors of cholesterol released from cell membranes. The cholesterol in discoidal HDL is esterified by lecithin:cholesterol acyltransferase (LCAT) in a process that converts the prebeta-migrating disc into an alpha-migrating, spherical HDL. Spherical HDL are further remodelled by cholesteryl ester transfer protein (CETP) that transfers cholesteryl esters from HDL to other lipoproteins and by hepatic lipase that hydrolyses HDL triglyceride in processes that reduce HDL size and lead to the dissociation of prebeta-migrating, lipid-poor apolipoprotein (apo)a-i from the particle. Prebetamigrating, lipid-poor apoa-i is also generated as a product of the remodelling of HDL by phospholipid transfer protein. Thus, apoa-i cycles between lipid-poor and lipid associated forms as part of a highly dynamic metabolism of HDL. The other main HDL apolipoprotein, apoa-ii is incorporated into apoa-i-containing particles in a process of particle fusion mediated by LCAT. Extracellular assembly and remodelling of HDL not only plays a major role in HDL regulation but also provides potential targets for therapeutic intervention. One example of this is the development of inhibitors of CETP. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: High density lipoproteins; HDL remodelling; HDL subpopulations; CETP 1. Introduction The regulation of high density lipoproteins (HDLs) is complex. It involves not only intracellular factors that control the synthesis and cell uptake of the lipoproteins but also factors that promote the assembly and remodelling of HDL in the extracellular space. This paper is concerned with the extracellular assembly and remodelling of HDL, with a special emphasis on two processes: (i) the cycling of apolipoprotein (apo)a-i between prebeta-migrating, lipid-poor particles and alpha-migrating, lipid-rich HDL particles and (ii) the incorporation of apoa-ii into apoa-i-containing HDL to form particles that contain both apoa-i and apoa-ii. The paper will conclude with a discussion of disorders Address: Cardiovascular Investigation Unit, Royal Adelaide Hospital, North Terrace, Adelaide, SA, 5000 Australia. Tel.: / ; fax: / address: pbarter@medicine.adelaide.edu.au (P.J. Barter). related to abnormalities of HDL remodelling and of HDL remodelling factors as potential therapeutic targets. But first, the composition, structure and subpopulation distribution of human HDL will be summarised. 2. Composition and structure of HDL HDL are the smallest (diameter 7.4 /12 nm) and densest (1.063B/d B/1.21 g/ml) of the plasma lipoproteins. In their mature form, they consist of spherical particles with a hydrophobic core (mainly cholesteryl esters plus a small amount of triglyceride) surrounded by a surface molecular monolayer comprising phospholipids, unesterified cholesterol and apolipoproteins. Human HDLs contain two main apolipoproteins: apoa-i and apoa-ii which account, respectively, for 70 and 20% of the HDL protein. There are also several minor apolipoproteins in HDL, including apoa-iv, the C- apolipoproteins, apod, apoe and apoj /02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S ( 0 2 )

2 40 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/47 3. Factors involved in the extracellular assembly and remodelling of HDL 3.1. ATP-binding cassette A1 Fig. 1. Heterogeneity of human HDL. The HDL fraction in human plasma is heterogeneous [1] (Fig. 1). When viewed by electron microscopy, HDL appear either as discoidal or spherical particles, although in normal plasma most are spherical. When isolated on the basis of density by ultracentrifugation, human HDL separate into two major subfractions, which have been designated HDL 2 (1.063B/dB/1.125 g/ml) and HDL 3 (1.125B/dB/1.21 g/ml). Non-denaturing polyacrylamide gradient gel electrophoresis has been used to separate HDL, on the basis of particle size, into five distinct subpopulations [2]. In order of decreasing particle size, these are HDL 2b (mean diameter 10.6 nm), HDL 2a (9.2 nm), HDL 3a (8.4 nm), HDL 3b (8.0 nm) and HDL 3c (7.6 nm). HDL 2a and HDL 2b fall within the HDL 2 density range, while HDL 3a, HDL 3b and HDL 3c are within the HDL 3 density range. HDL may also be divided into two main subpopulations on the basis of their apolipoprotein composition (Fig. 1). One subpopulation comprises HDL that contain apo A-I but no apo A-II (A-I HDL), while another comprises particles that contain both apo A-I and apo A-II (A-I/A-II HDL) [3]. Apo A-I is divided approximately equally between A-I HDL and A-I/A-II HDL in most human subjects [3], while virtually all of the apo A-II is in A-I/A-II HDL [3,4]. Most of the A-I/ A-II HDLs are found in the HDL 3 density range, while A-I HDL are prominent components of both HDL 2 and HDL 3 [5]. The heterogeneity of HDL extends to variations in surface charge. When subjected to agarose gel electrophoresis, HDL have either alpha, prebeta or gamma migration [6 /9]. The alpha-migrating particles are spherical lipoproteins and account for the major proportion of HDL in plasma. They include the HDL 2 and HDL 3 subfractions as well as A-I HDL and A-I/A-II subpopulations. Prebeta HDL are discoidal particles consisting of one or two molecules of apo A-I complexed with phospholipids and possibly a small amount of unesterified cholesterol [10]. Gamma-HDL are discoidal particles that contain apoe but no apoa-i [7]. As outlined below, much of the heterogeneity of HDL is the consequence of remodelling that takes place in the extracellular space. ATP binding cassette A1 (ABCA1) is a cell membrane transporter that facilitates the delivery of phospholipids from cell membranes to lipid-poor apoa-i in the extracellular space [11 /15]. This process results in the formation of discoidal apoa-i-containing HDL Lecithin:cholesterol acyltransferase Lecithin:cholesterol acyltransferase (LCAT) is a plasma glycoprotein that catalyses the transfer of an acyl group from phosphatidylcholine to cholesterol to form cholesteryl esters and lysophosphatidylcholine. This reaction takes place mainly on the surface of HDL particles and accounts for most of the cholesteryl esters that circulate in human plasma [16]. A-I HDLs are the preferred substrates for LCAT, with discoidal A- I HDL being superior as substrates to spherical A-I HDL [17] Cholesteryl ester transfer protein Cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein of hepatic origin that circulates in plasma bound mainly to HDL [18]. It promotes the redistribution of cholesteryl esters, triglyceride and, to a lesser extent, phospholipids between plasma lipoproteins. CETP picks up lipids from lipoprotein particles and deposits them in other lipoproteins in a process that results in an equilibration of lipids between lipoprotein fractions [19]. The net effect of CETP is a mass transfer of cholesteryl esters from HDL to triglyceride-rich lipoproteins and LDL and of triglyceride from triglyceride-rich lipoproteins to LDL and HDL Hepatic lipase The endothelial enzyme, hepatic lipase (HL), is anchored by glycosaminoglycans to the luminal surface of endothelial cells lining hepatic sinusoids and the capillary beds of steroid hormone synthesising tissues. It hydrolyses the triglycerides and phospholipids of all lipoprotein fractions, although the preferred substrates are the lipids in HDL. Within the HDL fraction, HL has the greatest affinity for HDL that contain apoa-ii, although the maximal rate of the HL-mediated hydrolysis of both triglyceride and phospholipids is greatest when they are components of A-I HDL [20].

3 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/ Lipoprotein lipase Lipoprotein lipase (LPL) catalyses the hydrolysis of triglyceride (and to a lesser extent phospholipids) in chylomicrons and very low density lipoproteins. This hydrolysis results in a reduction in particle size and a consequent redundancy of surface constituents (phospholipids and apolipoproteins) that may be transferred to HDL Phospholipid transfer protein Phospholipid transfer protein (PLTP) is another plasma glycoprotein that promotes transfers of phospholipids between HDL and other plasma lipoproteins. Of particular, importance is the role of PLTP in delivering phospholipids from chylomicrons to HDL [21]. As with LCAT, CETP and HL, PLTP promotes extensive remodelling of HDL. These factors play a fundamental role in the extracellular processes responsible for the assembly and remodelling of both A-I HDL and A-I/A-II HDL. They play a particularly important role in a series of reactions that result in the cycling of apoa-i between prebeta and alpha-migrating pools. 4. Cycling of apoa-i between prebeta- and alphamigrating pools ApoA-I cycles between lipid-poor and lipid-rich forms [22] in a series of reactions that generate the prebeta-migrating, discoidal particles that function as the preferred initial acceptors of cell cholesterol in the first step of the pathway of reverse cholesterol transport [23]. This cycling of apoa-i involves several identifiable steps: (i) formation prebeta-migrating, lipid-poor apoa- I, (ii) the acquisition by the lipid-poor apoa-i of cell phospholipids and cholesterol to form prebeta-migrating, discoidal A-I HDL particles, (iii) the conversion of prebeta-migrating, discoidal A-I HDL into small alphamigrating, spherical A-I HDL, (iv) the conversion of small alpha-migrating, spherical A-I HDL into larger alpha-migrating, spherical A-I HDL and finally (v) remodelling of the larger alpha-migrating, spherical A- I HDL in processes that regenerate prebeta-migrating, lipid-poor apoa-i, thus completing the cycle. Each of these steps is described below. HDL as a product of their remodelling by plasma factors Lipidation of lipid-poor apoa-i to form discoidal A-I HDL (Fig. 2) Prebeta-migrating, lipid-poor apoa-i, whether secreted directly from the liver or generated as a product of the extracellular remodelling of HDL, rapidly acquires phospholipids from cell membranes in the process promoted by ABCA1 [11 /15]. ABCA1 translocates phospholipids from the inner to the outer layer of the cell membrane where it is picked up by prebetamigrating, lipid-poor apoa-i in the extracellular space. The acquisition of cell phospholipids by the lipid-poor apoa-i results in the formation of prebeta-migrating, discoidal complexes that are efficient acceptors of unesterified cholesterol in cell membranes in a process that appears not to require ABCA1 [24]. Regardless of their origin, prebeta-migrating, discoidal HDL readily accept unesterified cholesterol from cell membranes to form particles that are highly reactive with LCAT Conversion of prebeta-migrating, discoidal A-I HDL into small alpha-migrating, spherical HDL (Fig. 3) LCAT catalyses the esterification of cholesterol in discoidal A-I HDL. The cholesteryl esters that are formed are hydrophobic and cannot be accommodated at the water interface on the surface of the HDL disc. Rather, they form a hydrophobic core between the bilayers of the HDL disc in a process that ultimately converts the prebeta-migrating disc into a small, alphamigrating sphere [1]. These small spheres (diameter 7.8 / 8.2 nm) consist of a surface molecular monolayer consisting of two molecules of apoa-i, phospholipids (mainly phosphatidylcholine) and unesterified cholesterol surrounding a hydrophobic core of cholesteryl esters Formation of prebeta-migrating, lipid-poor apoa-i Prebeta-migrating, lipid-poor apoa-i may arise from two major sources. It may be secreted directly from the liver where it is synthesised or, as described below, it may be released from alpha-migrating, spherical A-I Fig. 2. Role of ABCA1 in the lipidation of apoa-i to form discoidal A-I HDL.

4 42 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/47 Fig. 3. Role of LCAT in the conversion of prebeta-migrating, discoidal A-I HDL into small alpha-migrating, spherical HDL Conversion of small spherical A-I HDL into larger spherical A-I HDL The small, alpha-migrating, spherical A-I HDL which are formed by the esterification of cholesterol in discoidal particles are also subject to remodelling by plasma factors in processes that further change their composition and size. Two factors that increase the size of small spherical HDL are LCAT (which retains a degree of reactivity with the small spheres) and PLTP. Each of these factors remodel small, alpha-migrating A- I HDL spheres into larger particles which contain three (or four) rather than two molecules of apoa-i per particle. These larger, alpha-migrating spheres represent the main subpopulations of A-I HDL that circulate in normal human plasma Lecithin:cholesterol acyltransferase Interaction of small spherical A-I HDL with LCAT provides the particle with additional cholesteryl esters that must be accommodated in an expanding particle core. Coincident with the LCAT-mediated increase in the volume of the particle core, the HDL surface acquires additional apoa-i either in the form of lipidpoor apolipoprotein [22] or in a process involving the fusion of HDL particles [25]. LCAT promotes the fusion of small, spherical HDL particles, each of which contains two molecules of apoa-i to form larger particles that contain four molecules of apoa-i. It appears that these larger fusion products are not stable until they shed a molecule of apoa-i to form a particle containing three molecules of apoa-i (Fig. 4). The lipidfree (or lipid poor) apoa-i that dissociates from the unstable intermediate has several potential fates as described in greater detail below. Fig. 4. LCAT-mediated fusion of small, spherical A-I HDL, each of which contains two molecules of apoa-i to form larger spherical A-I HDL that contain three molecules of apoa-i. This remodelling of A-I HDL by PLTP is enhanced by the presence of triglyceride in the particle [27] Generation of prebeta-migrating, lipid-poor apoa-i from large, alpha-migrating, spherical HDL (Fig. 5) Cholesteryl ester transfer protein CETP promotes transfers cholesteryl esters from HDL to other lipoproteins and of triglycerides in the reverse direction in a process that simultaneously depletes the HDL core of cholesteryl esters and enriches it with triglyceride [19]. However, the magnitude of the transfer of cholesteryl esters out of HDL is greater than that of triglyceride into the HDL fraction, such that here is a net reduction in HDL core lipid content [28]. The resulting reduction in HDL particle size is accompanied by the dissociation of prebeta-migrating, lipid-poor apoa-i from the HDL surface [28] Combination of CETP and hepatic lipase The combined activities of CETP and HL in the presence of triglyceride-rich lipoproteins are especially effective in reducing the particle size of HDL and generating lipid-poor apoa-i [29]. CETP promotes the net mass transfer of cholesteryl esters from HDL to triglyceride-rich lipoproteins in exchange for a transfer of triglyceride into the HDL. This exchange results in the formation of HDL that are depleted of cholesteryl esters and enriched in triglyceride. Hydrolysis of this newly acquired triglyceride reduces the volume of the Phospholipid transfer protein PLTP also increases the size of spherical A-I HDL in a process involving particle fusion [26]. As with the LCAT-mediated HDL fusion outlined above, the fusion products formed by PLTP appear not to be stable until they shed one or more molecules of lipid-poor apoa-i. Fig. 5. Cycling of apoa-i between HDL and a lipid-poor pool.

5 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/47 43 HDL core in a process accompanied by a decrease in HDL particle size and the dissociation of lipid-poor apoa-i from the particle [29] Phospholipid transfer protein The fusion of HDL particles promoted by PLTP is accompanied by the dissociation of lipid-poor apoa-i from the particle. The importance of this process is uncertain but in species such as rats and mice that lack activity of CETP, PLTP may provide most of the prebeta-migrating apoa-i for initiating reverse cholesterol transport Lecithin:cholesterol acyltransferase As outlined above, LCAT converts small, spherical HDL containing two molecules of apoa-i per particle into larger spherical particles containing three molecules of apoa-i. This is achieved by the fusion of two of the smaller particles to form an unstable intermediate with four molecules of apoa-i [25]. Dissociation of a molecule of lipid-poor apoa-i restores stability to a particle that now contains three molecules of apoa-i. One fate of this lipid-poor apoa-i is to be incorporated into small spherical HDL to provide an alternate mechanism for increasing the number of molecules of apoa-i from two to three [22]. However, as outlined below, there are several other potential fates for lipidpoor apoa-i. 5. Metabolic fate of prebeta-migrating, lipid-poor apoa-i The prebeta-migrating, lipid-poor apoa-i that is generated as a product of the remodelling of alphamigrating, spherical A-I HDL has several potential fates (Fig. 5). (i) It may act as the recipient of phospholipids released from cells by ABCA1 to form discoidal HDL complexes that are efficient acceptors of cell cholesterol, thus completing the cycle outlined above. (ii) Lipid-poor apoa-i may also be incorporated into new discoidal HDL particles within the plasma by accepting phospholipids transferred from other plasma lipoproteins in a process linked to the hydrolysis of triglyceride-rich lipoproteins [30]. (iii) Lipid-poor apoa-i may be reincorporated directly into pre-existing HDL particles to provide an alternate means by which, as described above, the number of molecules of apoa-i increases from two to three during interactions with LCAT. (iv) And finally, lipid-poor apoa-i may be excreted through the kidney and thus be lost irreversibly from the plasma [31]. 6. Assembly of A-I/A-II HDL All of the discussion of HDL remodelling outlined above has been concerned with A-I HDL that contain apoa-i but no apoa-ii. However, about 50% of the apoa-i in human HDL exists in particles that also contain apoa-ii. Until recently, the origin of these A-I/ A-II HDL was not known. It is probable that apoa-i and apoa-ii enter the plasma separately and are assembled into A-I/A-II HDL within the plasma. As with apoa-i, apoa-ii is synthesised in the liver and is most likely secreted in a lipidpoor form. Once in the extracellular space, this hydrophobic protein acquires phospholipids from cell membranes, probably in an ABCA1-dependant process, to form discoidal apoa-ii/phospholipid complexes that function as acceptors of cell cholesterol to form discoidal A-II HDL. However, unlike discoidal A-I HDL, discoidal A-II HDL are non-reactive with LCAT [32] and are not converted into spherical particles. Rather, the presence of apoa-ii in spherical A-I/A-II HDL is the result of a process in which discoidal A-II HDL fuse with A-I HDL in a reaction catalysed by LCAT [33]. The postulated mechanism by which LCAT promotes the formation of spherical A-I/A-II HDL is shown schematically in Fig. 6. According to this scheme, the initial product of the interaction of LCAT with discoidal A-I HDL is a small spherical A-I HDL particle that contains the same number of apoa-i molecules (two) as the precursor discoidal particles. Continuing activity of LCAT increases the cholesteryl ester content of the small, spherical A-I HDL. To accommodate this increase in cholesteryl ester content of the particle core, the expanding HDL needs to acquire additional apolipoproteins in the surface monolayer. This may be achieved in two ways. One mechanism involves the fusion of the expanding particle with discoidal A-I HDL to form a larger spherical A-I HDL in which the number of molecules of apoa-i is also increased [25]. A second mechanism involves the expanding spherical A-I HDL fusing with discoidal A-II HDL to form spherical A-I/ A-II HDL [33]. Fig. 6. Mechanism by which LCAT promotes the incorporation of apoa-ii into A-I HDL to form spherical A-I/A-II HDL.

6 44 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/47 This postulated scheme has some interesting implications. For example, if the probability of small spherical A-I HDL fusing with discoidal A-I HDL rather than discoidal A-II HDL is a function of the relative concentrations of the two discoidal particles, it follows that the distribution of apoa-i between spherical A-I HDL and spherical A-I/A-II HDL in plasma will be determined by the relative rates of formation of discoidal A-I HDL and A-II HDL. Such a proposition is consistent with the observation that apoa-ii production rate determines the distribution of apoa-i between the two subpopulations in human plasma in vivo [34]. 7. Disorders of HDL related to remodelling Genetic abnormalities that impact on HDL assembly and remodelling include mutations of the genes encoding ABCA1 [11], LCAT [35], CETP [36], LPL [37] and HL [38]. Low levels of HDL-C related to HDL remodelling are also a well-recognised component of a variety of dyslipidaemic states. These include genetic conditions such as familial hypertriglyceridaemia, familial combined hyperlipidaemia and metabolic disorders such as non-insulin-dependent diabetes mellitus and the metabolic syndrome. Several of these conditions are summarised below, with particular attention given to CETP deficiency because of the development of novel CETP inhibitors as potential anti-atherogenic agents ATP binding cassette A1 deficiency The rate of formation of HDL is determined partly by the rate secretion of the main apolipoproteins into plasma and partly by the activity of ABCA1 that promotes lipidation of the apoa-i (and possibly apoa- II) in a process that retains the apolipoproteins in the plasma. A genetic deficiency of ABCA1 has been identified as the basic abnormality in Tangier disease [11 /13,15] in which, in the homozygous state, there is virtually no circulating HDL. In this condition, apoa-i and apoa-ii are synthesised and secreted into plasma as in normal subjects. However, a failure to lipidate the apolipoproteins results in their rapid disappearance from the plasma, possibly by excretion through the kidney. It is interesting that patients with Tangier disease do not necessarily develop premature atherosclerosis despite their absent HDL. This may be because such patients tend also to have a low level of LDL cholesterol, suggesting that a substantial proportion of the cholesterol in LDL originates in HDL from which it is transferred to LDL by CETP Lecithin:cholesterol acyltransferase deficiency The effects on HDL of a genetic deficiency of LCAT are predictable from the known function of LCAT. The HDL lacks a cholesteryl ester core and circulate predominantly as prebeta-migrating, discoidal particles [39,40]. The concentrations of all HDL lipids (not only cholesteryl esters) and all apolipoproteins are markedly reduced. The inability to esterify HDL cholesterol in this condition is associated with an obvious impairment of cholesterol efflux from cells as evidenced by the accumulation of unesterified cholesterol in cell membranes in many tissues in this condition. It is interesting that subjects with LCAT deficiency appear not to be at particularly high risk of developing atherosclerosis Cholesteryl ester transfer protein deficiency The effects of a deficiency of CETP are predictable from what is known of the function of this protein. When CETP deficiency is complete, the resulting failure to transfer LCAT-derived cholesteryl esters from HDL to other lipoprotein fractions leads to a retention of cholesteryl esters in HDL. The associated reduction in transfer of triglyceride into HDL translates into a triglyceride-poor HDL core. Overall, however, the total HDL core lipid content and the HDL particle size are increased. This results in a retention of additional surface components (apoa-i, phospholipids, unesterified cholesterol) that are required to accommodate the increased amount of core lipids. The net effect of these changes is reflected by substantial increases in the concentration of HDL cholesterol and apoa-i that are accommodated in large HDL particles. The HDL fraction in subjects with CETP deficiency comprises mainly larger, less dense HDL 2 particles that are enriched in cholesteryl ester and apoe [41]. The concentrations of both apoa-i and apoa-ii are increased due to a reduction in the rate of their catabolism [42]. In contrast, the synthesis of both apoa-i and apoa-ii is similar to control subjects [42] Hepatic lipase deficiency The HDL in subjects with HL deficiency are again as predicted from the known action of HL on HDL [38,43]. The HDL tend to be larger than normal and are considerably enriched in triglyceride. The pathophysiological implications of these HDL changes are uncertain and may be less important than the accumulation of triglyceride-rich lipoproteins that depend on activity of HL for their normal catabolism.

7 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/ Lipoprotein lipase deficiency Subjects with a deficiency of LPL have very high concentrations of triglyceride-rich lipoproteins and low concentrations of HDL cholesterol and apolipoproteins [37]. The HDL in these people are small and enriched in triglyceride. There are two potential mechanisms accounting for the low HDL: (i) the impaired catabolism of triglyceride-rich lipoproteins limits the availability of phospholipids for transfer to HDL by PLTP and (ii) the expanded pool of triglyceride-rich lipoproteins favours an exaggerated transfer of cholesteryl esters out of HDL. 8. Factors that remodel HDL as potential therapeutic targets Several factors that remodel HDL are potential targets for therapies designed to reduce atherosclerosis. For example, an up-regulation of ABCA1 in macrophages would be predicted to promote the efflux of cholesterol from these cells and inhibit their conversion into foam cells. An increase in activity of LCAT may be anti-atherogenic by virtue of its ability to increase the concentration of HDL cholesterol, a possibility that is supported by studies of rabbits genetically engineered to over-express LCAT [44]. However, comparable overexpression of LCAT in transgenic mice has been reported to be pro- rather than anti-atherogenic [45]. LPL has the potential to be anti-atherogenic for several reasons, including its ability to increase the concentration of HDL. HL, on the other hand, could be argued to be either pro-atherogenic by decreasing the concentration of HDL or anti-atherogenic by virtue of generating prebeta-migrating apoa-i and by accelerating the catabolism of the atherogenic remnants of triglyceride-rich lipoproteins. However, interventions that target these factors are still the subject of basic research and are probably many years from possible clinical development. Inhibitors of CETP, however, are in an advanced stage of clinical development, with at least one CETP inhibitor currently undergoing human phase 3 trials CETP deficiency and atherosclerosis There has been considerable debate about the effects of CETP deficiency on the development of atherosclerosis, with evidence of both pro- and anti-atherogenic effects [46]. For example, inhibition of CETP in rabbits (a species with naturally high CETP activity) results in substantial increases in the concentration of HDL cholesterol and is powerfully and consistently antiatherogenic [47 /49]. In one study of cholesterol-fed rabbits, the inhibition of CETP by injection of antisense oligodeoxynucleotides (ODNs) against CETP resulted in a reduction in CETP mrna and mass in the liver, a reduction in plasma total cholesterol and an increased concentration of HDL cholesterol [47]. There was also an increase in LDL receptor mrna associated with the antisense ODNs. These changes were accompanied by a marked reduction in aortic cholesterol content as a marker of the extent of atherosclerosis. A vaccine approach has been used to generate autoantibodies against CETP in vivo in rabbits [48]. In a study of cholesterol-fed rabbits, animals that were immunized against CETP had a reduced plasma activity of CETP and a substantial increase in the concentration of HDL cholesterol, a modest decrease in LDL cholesterol concentration and a significant reduction in aortic atherosclerotic lesions. A chemical inhibitor of CETP has been used in another study of cholesterol-fed rabbits [49]. This inhibitor reduced CETP activity in rabbits by more than 90%, almost doubled the level of HDL cholesterol and decreased the non-hdl cholesterol by about 50%. There was an accompanying 70% reduction in atherosclerotic lesions in the aortas of these animals. It was not possible to determine the relative importance of the increased HDL versus the decreased LDL in the reduction of atherosclerosis observed in these rabbit studies. It was stated in this paper that short-term treatment of human subjects with the same CETP inhibitor resulted in a 40 /45% increase in HDL cholesterol and a 15/20% decrease in LDL cholesterol. In contrast to the highly consistent results of CETP inhibition in rabbits, the results of over-expressing CETP in mice have been more difficult to interpret. Mice are naturally deficient in CETP, an observation that explains why most of the cholesteryl esters in murine plasma is transported as a component of HDL. Expression of CETP in mice results in a redistribution of cholesteryl esters from HDL to LDL and the development of atherosclerosis [50,51]. However, in studies of LCAT transgenic mice [52] and of mice genetically engineered to over-express apoc-iii [53], expression of CETP has been reported to be antiatherogenic. It seems that the relationship between CETP activity and atherosclerosis may differ in different metabolic settings. The situation in humans remains uncertain, with studies of humans with natural CETP deficiency being interpreted as supporting a view that CETP may be both pro-atherogenic [54] and antiatherogenic [55]. It is still premature to predict the effects of therapeutic CETP inhibition in humans. More information is required about how CETP inhibition impacts on cholesterol transport in vivo under a range of metabolic conditions. On balance, however, it is probable that the response to CETP inhibition in humans will resemble that in a high CETP expressing animal such as the

8 46 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/47 rabbit. If so, CETP inhibitors may provide a useful therapeutic approach to raising HDL cholesterol, lowering LDL cholesterol and reducing the development of atherosclerosis in humans. 9. Conclusion Factors operating in the extracellular space not only play a major role in the regulation of HDL concentration, composition and subpopulation distribution but have also emerged as potential targets for therapies designed to inhibit the development of atherosclerosis. The ultimate place of such therapeutic agents is awaited with great interest. References [1] Rye K-A, Clay MA, Barter PJ. Remodelling of high density lipoproteins by plasma factors. Atherosclerosis 1999;145:227/38. [2] Blanche PJ, Gong EL, Forte TM, Nichols AV. Characterization of human high-density lipoproteins by gradient gel electrophoresis. Biochim Biophys Acta 1981;665:408/19. [3] Cheung MC, Albers JJ. Characterization of lipoprotein particles by immunoaffinity chromatography. J Biol Chem 1984;259:12201/9. [4] Bekaert ED, Alaupovic P, Knight-Gibson C, Norum RA, Laux MJ, Ayrault-Jarrier M. Isolation and partial characterization of lipoprotein A-II (LP-A-II) particles of human plasma. Biochim Biophys Acta 1992;1126:105/13. [5] Cheung MC, Albers JJ. Distribution of high density lipoprotein particles with different apoprotein composition: particles with A-I and A-II and particles with A-I but no A-II. J Lipid Res 1982;23:747/53. [6] Asztalos BF, Sloop CH, Wong L, Roheim PS. Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A- I-containing subpopulations. Biochim Biophys Acta 1993;1169:291 /300. [7] Huang Y, von Eckardstein A, Wu S, Assmann G. Effects of the apolipoprotein E polymorphism on uptake and transfer of cellderived cholesterol in plasma. J Clin Invest 1995;96:2693/701. [8] Kunitake ST, La Sala KJ, Kane JP. Apolipoprotein A-I-containing lipoproteins with pre-beta electrophoretic mobility. J Lipid Res 1985;26:549/55. [9] Sparks DL, Lund-Katz S, Phillips MC. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. J Biol Chem 1992;267:25839/47. [10] Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res 1995;36:211/28. [11] Lawn RM, Wade DP, Garvin MR, Wang X, Schwartz K, Porter JG, et al. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. J Clin Invest 1999;104:R25/31. [12] Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, et al. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease. Nat Genet 1999;22:347/51. [13] Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, et al. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency. Nat Genet 1999;22:336/45. [14] Orso E, Broccardo C, Kaminski WE, Bottcher A, Liebisch G, Drobnik W, et al. Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and Abc1- deficient mice. Nat Genet 2000;24:192/6. [15] Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, et al. Tangier disease is caused by mutations in the gene encoding ATPbinding cassette transporter 1. Nat Genet 1999;22:352/5. [16] Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res 1968;9:155/67. [17] Jonas A. Lecithin-cholesterol acyltransferase in the metabolism of high-density lipoproteins. Biochim Biophys Acta 1991;1084:205/ 20. [18] Tall AR. Plasma cholesteryl ester transfer protein. J Lipid Res 1993;34:1255/74. [19] Barter PJ, Hopkins CJ, Calvert GD. Transfers and exchanges of esterified cholesterol between plasma lipoproteins. Biochem J 1982;208:1 /7. [20] Hime NJ, Barter PJ, Rye K-A. The influence of apolipoprotein on the hepatic lipase-mediated hydrolysis of high density lipoprotein phospholipid and triacylglycerol. J Biol Chem 1998;273:27191/9. [21] Qin S, Kawano K, Bruce C, Lin M, Bisgaier C, Tall AR, et al. Phospholipid transfer protein gene knock-out mice have low high density lipoprotein levels, due to hypercatabolism, and accumulate apoa-iv-rich lamellar lipoproteins. J Lipid Res 2000;41:269/ 76. [22] Liang H-Q, Rye K-A, Barter PJ. Cycling of apolipoprotein A-I between lipid-associated and lipid-free pools. Biochim Biophys Acta 1995;1257:31/7. [23] Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein. Biochemistry 1998;27:25 /9. [24] Johnson WJ, Mahlberg FH, Rothblat GH, Phillips MC. Cholesterol transport between cells and high-density lipoproteins. Biochim Biophys Acta 1991;1085:273/98. [25] Liang H-Q, Rye K-A, Barter PJ. Remodelling of reconstituted high density lipoproteins by lecithin: cholesterol acyltransferase. J Lipid Res 1996;37:1962/70. [26] Korhonen A, Jauhiainen M, Ehnholm C, Kovanen PT, Ala- Korpela M. Remodeling of HDL by phospholipid transfer protein: demonstration of particle fusion by 1H NMR spectroscopy. Biochem Biophys Res Commun 1998;249:910/6. [27] Rye KA, Jauhiainen M, Barter PJ, Ehnholm C. Triglycerideenrichment of high density lipoproteins enhances their remodelling by phospholipid transfer protein. J Lipid Res 1998;39:613/ 22. [28] Rye K-A, Hime HJ, Barter PJ. The influence of cholesteryl ester transfer protein on the composition, size and structure of spherical, reconstituted high density lipoproteins. J Biol Chem 1995;270:189/96. [29] Clay MA, Newnham HH, Forte TM, Barter PJ. Cholesteryl ester transfer protein and hepatic lipase activity promote shedding of apo A-I from HDL and subsequent formation of discoidal HDL. Biochim Biophys Acta 1992;1124:52/8. [30] Clay MA, Barter PJ. Formation of new HDL particles from lipidfree apolipoprotein A-I. J Lipid Res 1996;7:1722/32. [31] Horowitz BS, Goldberg IJ, Merab J, Vanni TM, Ramakrishnan R, Ginsberg HN. Increased plasma and renal clearance of an exchangeable pool of apolipoprotein A-I in subjects with low levels of high density lipoprotein cholesterol. J Clin Invest 1993;91:1743/52. [32] Jonas A. Synthetic substrates of lecithin:cholesterol acyltransferase. J Lipid Res 1986;27:689/98. [33] Clay MA, Pyle DH, Rye KA, Barter PJ. Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase. J Biol Chem 2000;275:9019/25.

9 P.J. Barter / Atherosclerosis Supplements 3 (2002) 39/47 47 [34] Ikewaki K, Zech LA, Kindt M, Brewer HB, Jr., Rader DJ. Apolipoprotein A-II production rate is a major factor regulating the distribution of apolipoprotein A-I among HDL subclasses LpA-I and LpA-I:A-II in normolipidemic humans. Arterioscler Thromb Vasc Biol 1995;15:306/12. [35] Funke H, von Eckardstein A, Pritchard PH, Hornby AE, Wiebusch H, Motti C, et al. Genetic and phenotypic heterogeneity in familial lecithin: cholesterol acyltransferase (LCAT) deficiency. Six newly identified defective alleles further contribute to the structural heterogeneity in this disease. J Clin Invest 1993;91:677/ 83. [36] Koizumi J, Inazu A, Yagi K, Koizumi I, Uno Y, Kajinami K, et al. Serum lipoprotein lipid concentration and composition in homozygous and heterozygous patients with cholesteryl ester transfer protein deficiency. Atherosclerosis 1991;90:189/96. [37] Reymer PW, Gagne E, Groenemeyer BE, Zhang H, Forsyth I, Jansen H, et al. A lipoprotein lipase mutation (Asn291Ser) is associated with reduced HDL cholesterol levels in premature atherosclerosis. Nat Genet 1995;10:28/34. [38] Breckenridge WC, Little JA, Alaupovic P, Wang CS, Kuksis A, Kakis G, et al. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase. Atherosclerosis 1982;45:161/ 79. [39] Glomset JA, Norum KR, Gjone E. Familial lecithin:cholesterol acyltransferase deficiency. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS, editors. Metabolic basis for inherited disease, 5th ed.. New York: McGraw-Hill Book Company, 1983:643/54. [40] Mitchell CD, King WC, Applegate KR, Forte T, Glomset JA, Norum KR, et al. Characterization of apolipoprotein E-rich density lipoproteins in familial lecithin:cholesterol acyltransferase deficiency. J Lipid Res 1980;21:625/40. [41] Yamashita S, Sprecher DL, Sakai N, Matsuzawa Y, Tarui S, et al. Accumulation of apolipoprotein E-rich high density lipoproteins in hyperalphalipoproteinemic human subjects with deficiency of cholesteryl ester transfer protein. J Clin Invest 1990;86:688/95. [42] Ikewaki K, Rader DJ, Sakamoto T, Nishiwaki M, Wakimoto N, et al. Delayed catabolism of high density lipoprotein apolipoprotein A-I and A-II in human cholesteryl ester transfer protein deficiency. J Clin Invest 1993;92:1650/8. [43] Hegele RA, Little JA, Vezina C, Maguire GF, Tu L, Wolever TS, et al. Hepatic lipase deficiency. Clinical, biochemical, and molecular genetic characteristics. Arterioscler Thromb Vasc Biol 1993;13:720/8. [44] Hoeg JM, Santamarina-Fojo S, Berard AM, et al. Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis. Proc Natl Acad Sci USA 1996;93:11448 /53. [45] Berard AM, Remaley AT, Vaisman BL, et al. High plasma HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin-cholesteryl acyltransferase. Nat Med 1997;3:744 /9. [46] Barter PJ, Rye KA. Cholesteryl ester transfer protein, high density lipoprotein and arterial disease. Curr Opin Lipidol 2001;12:377/82. [47] Sugano M, Makino N, Sawada S, Otsuka S, Watanabe M, Okamoto H, et al. Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits. J Biol Chem 1998;273:5033/6. [48] Rittershaus CW, Miller DP, Thomas LJ, Picard MD, Honan CM, Emmett CD, et al. Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:2106/12. [49] Okamoto H, Yonemori F, Wakitani K, Minowa T, Maeda K, Shinkai H. A cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits. Nature 2000;406:203/7. [50] Marotti KR, Castle CK, Boyle TP, Lin AH, Murray RW, Melchior GW. Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein. Nature 1993;364:73/5. [51] Plump AS, Masucci-Magoulas L, Bruce C, Bisgaier CL, Breslow JL, Tall AR. Increased atherosclerosis in apoe and LDL receptor gene knock-out mice as a result of human cholesteryl ester transfer protein transgene expression. Arterioscler Thromb Vasc Biol 1999;19:1105/10. [52] Foger B, Chase M, Amar MJ, Vaisman BL, Shamburek RD, Paigen B, et al. Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice. J Biol Chem 1999;274:36912/20. [53] Hayek T, Masucci-Magoulas L, Jiang X, Walsh A, Rubin E, Breslow JL, et al. Decreased early atherosclerotic lesions in hypertriglyceridemic mice expressing cholesteryl ester transfer protein transgene. J Clin Invest 1995;96:2071/4. [54] Moriyama Y, Okamura T, Inazu A, Doi M, Iso H, Mouri Y, et al. A low prevalence of coronary heart disease among subjects with increased high-density lipoprotein cholesterol levels, including those with plasma cholesteryl ester transfer protein deficiency. Prev Med 1998;27:659/67. [55] Hirano K, Yamashita S, Kuga Y, Sakai N, Nozaki S, Kihara S, et al. Atherosclerotic disease in marked hyperalphalipoproteinemia. Combined reduction of cholesteryl ester transfer protein and hepatic triglyceride lipase. Arterioscler Thromb Vasc Biol 1995;15:1849/56.