Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins

Review Article Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins A. R. Tall From the Division of Molecular Medicine, Department of Medicine, Columbia University, NY, USA doi: 10.1111/j.1365-2796.2007.01898.x Abstract. Tall AR (Division of Molecular Medicine, Department of Medicine, Columbia University, New York, USA). Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins (Review). J Intern Med 2008; 263: 256 273. Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. Although HDL has anti-oxidant, anti-inflammatory, vasodilating and antithrombotic properties, the central anti-atherogenic activity of HDL is likely to be its ability to remove cholesterol and oxysterols from macrophage foam cells, smooth muscle cells and endothelial cells in the arterial wall. To some extent, the pleotropic atheroprotective properties of HDL may be related to its ability to promote sterol and oxysterol efflux from arterial wall cells, as well as to detoxify oxidized phospholipids. In cholesterol-loaded macrophages, activation of liver X receptors (LXRs) leads to increased expression of adenosine triphosphate (ATP) binding cassetter transporter (ABCA1), ATP binding cassetter transporter gene (ABCG1) and apoe and promotes cholesterol efflux. ABCA1 stimulates cholesterol efflux to lipid-poor apolipoproteins, whilst ABCG1 promotes efflux of cholesterol and oxysterols to HDL. Despite some recent setbacks in the clinical arena, there is still intense interest in therapeutically targeting HDL and macrophage cholesterol efflux pathways, via treatments with niacin, cholesterol ester transfer protein inhibitors, LXR activators and infusions of apoa-1, phospholipids and peptides. Keywords: ATP binding cassetter transporters A-1, G1, HDL, liver X receptors. Introduction Plasma high density lipoproteins (HDL) levels have a strong inverse relationship to atherosclerotic cardiovascular disease. A major hypothesis to explain the anti-atherogenic properties of HDL is that that HDL promotes a process of reverse cholesterol transport from arteries to the liver [1]. In recent years there has been substantial progress in understanding the biology of HDL and its interaction with cells, and the reverse cholesterol transport hypothesis has been modified and amplified. Progress has been punctuated by the discovery of new molecules involved in the different steps of reverse cholesterol transport or other aspects of HDL biology, development of transgenic mouse models and the beginning of human clinical trials targeting HDL. This review will summarize recent advances in the basic science of HDL, macrophage cholesterol efflux pathways and reverse cholesterol transport. We will describe how the effects of HDL on cellular sterol efflux may be related to a variety of potentially novel anti-atherogenic actions of HDL, we will also describe other novel mechanisms that have been proposed, and we will attempt to integrate this information with recent clinical studies involving HDL therapeutics. 256 ª 2008 Blackwell Publishing Ltd

HDL, cholesterol efflux and reverse cholesterol transport The ability of HDL to stimulate efflux of cholesterol from peripheral tissues, transport in the plasma, uptake in the liver and excretion into the bile is termed reverse cholesterol transport. The specific process involving efflux of cholesterol from macrophage foam cells in the artery wall has been termed macrophage reverse cholesterol transport [2] and is thought to be central to the anti-atherogenic properties of HDL (Fig. 1). The principal molecules involved in efflux of cholesterol from macrophage foam cells are adenosine triphosphate (ATP) binding cassette transporter A1 (ABCA1) and ATP binding cassette transporter gene G1 (ABCG1). ABCA1 promotes efflux of cholesterol and phospholipids onto lipid-poor apoa-1 [3 6]. In addition to cholesterol efflux from arterial wall cells, ABCA1 is primarily responsible for the initiation of HDL formation, principally in the liver and to a lesser extent in the small intestine [7, 8]. ABCG1 promotes cholesterol efflux from macrophage foam cells onto HDL particles but the activity of ABCG1 does not influence overall HDL levels [9, 10]. Fig. 1 Overview of macrophage cholesterol efflux pathways and reverse cholesterol transport to the liver. In macrophages active cholesterol efflux occurs via adenosine triphosphate (ATP) binding cassette transporter A1 (ABCA1) and ATP binding cassette transporter gene G1 (ABCG1). ABCA1 promotes phospholipids and cholesterol efflux to lipid-poor apoa-1 or apoe, whilst ABCG1 promotes efflux of cholesterol and oxysterols modified in the seventh position to HDL particles. ABCA1 is also involved in the initiation of HDL formation by addition of lipids to apoa-1 in the liver and to a lesser extent the small intestine. Within HDL, some of the free cholesterol (FC) is esterified by the lecithin:cholesterol acyltransferase (LCAT) enzyme forming cholesteryl esters (CE). In humans, CE is transferred to triglyceride-rich lipoproteins by CE transfer protein and subsequently removed from the circulation when LDL receptors mediate the hepatic uptake of remnant lipoproteins. Another lipid transfer protein, phospholipid transfer protein mediates transfer of phospholipids from triglyceride-rich lipoproteins into HDL, providing phospholipids for the LCAT reaction. In rodents and possibly also in humans, direct uptake of HDL CE and FC into the liver is mediated by scavenger receptor BI in a process of selective uptake. ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273 257

Lecithin:cholesterol acyltransferase (LCAT) esterifies cholesterol on HDL particles, and this activity may help to drive cholesterol efflux via passive efflux or the ABCG1 pathway [11]. In mice and rats, free and esterified cholesterol is taken up into hepatocytes by scavenger receptor (SR-BI), in a process of selective uptake that does not lead to catabolism of whole HDL particles [12]. Although primary human hepatocytes display active selective uptake of HDL-cholesteryl esters (CE) leading to regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity [13], the quantitative importance of selective uptake in the catabolism of HDL CE in humans is unknown. In humans, cholesterol ester transfer protein (CETP) transfers CE from HDL into VLDL, and chylomicrons by exchanging CE for triglycerides. Remnants formed from the triglyceride-rich lipoproteins are thus enriched with CE and the reverse transport to the liver is completed by uptake of remnants via the LDL receptor or heparin sulphate proteoglycans [14]. Following uptake into the liver, cholesterol can be utilized in a variety of different ways, but there is some evidence for metabolic channelling of HDL cholesterol into bile [15]. The bile canalicular transporters ABCG5 and ABCG8 are responsible for the excretion of cholesterol into bile [16]. A remarkable feature of the reverse cholesterol transport pathway is that each of the individual steps involve molecules that are directly targeted by LXR retinoid X receptor (RXR) transcription factors, including ABCA1, ABCG1 and apoe in macrophages, CETP, Cyp7a (in mice but not humans) and ABCG5 8 [17, 18]. LXRs are activated by accumulation of certain oxysterols in cells, suggesting that a specific molecular control mechanism has evolved to enable the removal of cholesterol from the body. Macrophage cholesterol efflux pathways: ABCA1 and ABCG1 A major breakthrough in the HDL field was the discovery that mutations in the ABCA1 are responsible for Tangier Disease [4 6], a condition characterized by an almost complete deficiency of plasma HDL, macrophage foam cell accumulation in various tissues, peripheral neuropathy and an apparent moderate increase in atherosclerosis. More than twenty different mutations in ABCA1 have been shown to cause Tangier Disease. Moreover, about 10% of individuals with very low HDL (the bottom first percentile of the population) have a variety of different mutations in ABCA1 [19, 20]. Whilst ABCA1 promotes cholesterol efflux to lipidpoor apoa-1, it only modestly stimulates lipid efflux to smaller HDL-3 particles and does not promote cholesterol efflux to the larger HDL-2 fraction [3, 21]. This suggested the possibility that there might be another LXR-activated cholesterol efflux pathway promoting lipid efflux to HDL particles that led to the discovery that ABCG1 and ABCG4 promote cholesterol efflux from transfected cells to HDL particles but not to lipid-poor apoa-1 [9]. ABCG1 and ABCG4 are half-transporters that are likely to act as homo-dimers. ABCG1 is highly expressed in macrophages and promotes cholesterol efflux from macrophage foam cells to HDL particles [22]. ApoE and LCAT in the HDL particles have an important role in promoting cholesterol efflux via ABCG1, especially in CETP deficiency states (Fig. 2) [11]. Adenosine triphosphate binding cassette transporter A1 and ABCG1 are ATPases that promote unidirectional, net cholesterol efflux to lipid-poor helical apolipoproteins and lipoprotein particles, respectively [22] (T. Pagler and A.R. Tall, unpublished). In transfected cells and probably in macrophages, ABCA1 and ABCG1 can act in a sequential fashion, with ABCA1 generating nascent HDL particles which then promotes cholesterol efflux via ABCG1 [23]. Genetic knock-down studies suggest that ABCA1 and ABCG1 together account for about 60 70% of the net cholesterol efflux to HDL or serum from cholesterol-loaded LXR-activated macrophages [24]. Even though SR-BI can promote the bidirectional exchange of free cholesterol between cells and HDL, SR-BI knock-out macrophages have no change in net cholesterol efflux to HDL, suggesting it does not make a significant contribution to this process. However, transplantation of SR-BI deficient bone marrow into LDL receptor (LDLR) or apoe-deficient mice results in an increase in atherosclerosis [25], and an in vivo role of SR-BI 258 ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273

Fig. 2 Co-ordinate role of adenosine triphosphate (ATP) binding cassette transporter A1 (ABCA1), ATP binding cassette transporter gene G1 ABCG1, ApoE and lecithin:cholesterol acyltransferase (LCAT) in efflux of cholesterol from macrophage foam cells. Accumulation of certain oxysterols in macrophage foam cells leads to activation of liver X receptor transcription factors, which induce expression of ABCA1 and ABCG1. ABCA1 promotes efflux of phospholipid and cholesterol to lipid-poor apoa- 1 or apoe. ABCG1 promotes efflux of cholesterol to HDL particles. These transporters can act sequentially within the same cell, with ABCA1 initiating formation of a nascent HDL particle which then acquires additional lipids via the ABCG1 pathway. ApoE and LCAT act to facilitate cholesterol efflux via the ABCG1 pathway. in cholesterol efflux from bone marrow-derived cells cannot be completely excluded. Other unknown transporters or diffusional (passive) cholesterol efflux could account for the residual cholesterol efflux observed in Abca1) )Abcg1) ) macrophages [24]. Cellular mechanisms mediating cholesterol efflux via ABCA1 and ABCG1 Adenosine triphosphate binding cassette transporter A1 promotes the efflux of cellular phospholipids and cholesterol onto lipid-poor apoa-1, as well as other lipid-poor apolipoproteins, such as apoe and synthetic peptides containing ampipathic helices [26]. ABCA1 directly binds and cross-links apoa-1 [3] and this appears to be an essential step in its ability to promote lipid efflux [27, 28]. There are two components in the binding of apoa-1 to cells overexpressing ABCA1, a high-affinity, low-capacity binding that likely represents a direct interaction, and a distinct high-affinity high-capacity binding that may represent binding to cholesterol-enriched lipid domains in the plasma membrane created by the activity of ABCA1 [29]. ABCA1 is active in the plasma membrane and also trafficks to late endosomes where it promotes efflux of cholesterol deposited in late endosomes by modified forms of LDL [30]. Previous studies showed that apoa-i can be internalized by macrophages and that cholesterol efflux might involve retro-endocytosis of apoa-1 [31]. It is possible that these observations represented trafficking of ABCA1 carrying bound apoa-i. ABCA1 is found in the plasma membrane, as well as in the endosomal system [32, 33]. Mutations in the NPC1 (Niemann Pickel) molecule, involved in the exit of cholesterol from late endosomes result in a severe defect in cholesterol efflux to lipid-poor apoa-1, suggesting that ABCA1 acts downstream of NPC1 in promoting cholesterol efflux from late endosomes [34]. The cytoplasmic domain of ABCA1 contains a proline-glutanic acid-serine-threonine (PEST) sequence, typical of proteins undergoing rapid turnover either ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273 259

mediated by ubiquitination and proteasomal degradation or proteolysis by calpain proteases [35]. ABCA1 is susceptible to thiol protease-mediated degradation, most likely involving calpains, and this process is dependent on the cytoplasmic PEST sequence [36]. Binding of apoa-1 or apoe to ABCA1 results in stabilization of ABCA1 and this is apparently because of reduced PEST sequence-dependent proteolysis. Deletion of the PEST sequence results in accumulation of ABCA1 in the plasma membrane and also leads to reduced concentrations of ABCA1 in late endosomes. This results in a higher level of cholesterol efflux from cells after loading the plasma membrane with cylodextrin-cholesterol, but impaired cholesterol efflux after loading endosomes with modified LDL [30]. Together with other information [37], this suggests that whilst ABCA1 is active in promoting cholesterol efflux from the plasma membrane, the trafficking of ABCA1 to late endosomes is important in promoting efflux of cholesterol from late endosomes following uptake of modified forms of LDL. In contrast to ABCA1 and SR-BI, overexpression of ABCG1 does not lead to increased cellular binding of apoa-1 or HDL [9, 22]. ABCG1 overexpression causes increased cholesterol efflux to HDL-2, HDL-3, LDL and cyclodextrin, whilst knock-down of ABCG1 in cholesterol-loaded, LXR-activated macrophages decreases cholesterol efflux to these various acceptors. In noncholesterol loaded peritoneal macrophages, ABCG1 is predominantly intracellular and does not contribute significantly to cholesterol efflux to HDL. However, after cholesterol loading and LXR activation, ABCG1 levels are increased and ABCG1 can be readily detected in the macrophage plasma membrane; this correlates with a significant role for ABCG1 in cholesterol efflux to HDL and other lipoprotein acceptors in cholesterol-loaded, LXR-activated macrophages. ABCG1 deficient macrophages show increased cholesterol esterification activity, as well as induction of various LXR target genes and suppression of cholesterol biosynthesis. These observations suggest that ABCG1 acts in the macrophage plasma membrane to increase the availability of cholesterol to a variety of different lipoprotein acceptors, and thereby depletes the regulatory sterol pool in the endoplasmic reticulum. Small [38] has argued that the related transporters, ABCG5 and ABCG8, may utilize ATP to promote the protrusion of cholesterol from the bile canalicular membrane into the aqueous phase, overcoming the large energetic barrier to desorption of sterol from the membrane into water. Subsequently, a transient collision with a micelle could result in the release of cholesterol onto the acceptor particle. By analogy, ABCG1 could act to promote desorption of cholesterol or selected oxysterols from the plasma membrane onto HDL or other lipoprotein particles. It is also possible that the primary action of ABCG1 leads to a re-organization of membrane phospholipids leading to decreased retention of sterol in the plasma membrane. In addition to its apparent role in the plasma membrane, ABCG1 may be active within cells, for example in the Golgi or recycling endosomes [22]. HDL, ABCG1 and efflux of oxysterols The death of macrophages in atherosclerotic lesions by apoptosis or postapoptotic necrosis is thought to contribute to inflammation, necrotic core formation and destabilization of plaques [39 42]. Recently, we have shown that HDL reduces macrophage apoptosis induced by loading with free cholesterol or oxidized LDL [43]. Interestingly, the ability of HDL to protect against apoptosis, especially that induced by oxidized LDL, is abolished in ABCG1) ) macrophages. This reflects the specific ability of HDL and ABCG1 to promote efflux of 7-ketocholesterol and related oxysterols modified at the seventh position from macrophages loaded with oxidized LDL. In contrast, ABCA1 and apoa-1 do not promote efflux of 7-ketocholesterol from cells. 7-Ketocholesterol is the major oxysterol found in oxidized LDL and atherosclerotic plaques. Consistent with an in vivo role of ABCG1 in promoting efflux of oxysterols, ABCG1) ) mice show accumulation of 7-ketocholesterol in macrophages, as well as reduced levels of 7-ketocholesterol in plasma, when fed a high-cholesterol diet. These observations suggest a specific role of ABCG1 in promoting efflux of toxic oxysterols from plaque macrophages to HDL. As ABCG1 also appears to be highly 260 ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273

expressed in arterial endothelium [44], this activity could also potentially protect against oxidized LDLinduced endothelial dysfunction and reduced endothelial nitric oxide synthase (enos) activity [45]. The role of ABC transporters in phagocytes Adenosine triphosphate binding cassette transporter A1 and ABCG1 are both induced in phagocytes following ingestion of apoptotic cells [46], and are involved in promoting cholesterol efflux after ingestion of the cholesterol-rich apoptotic cell, thus protecting the phagocyte from free cholesterol-induced cell death [47]. The induction of ABCA1 involves both LXRs and activation of mitogen-activated protein kinase (MAPK) signalling pathways in phagocytes, specifically NFjB and p38 MAPK signalling, whilst the induction of ABCG1 is mediated via LXRs [46]. The efflux of ingested cholesterol and possibly oxidized phospholipids [48] and sterols from phagocytes likely helps maintain the viability of phagocytes. Thus, in atherosclerotic plaques, the activities of ABCA1 and ABCG1 probably help to get rid of cholesterol, oxysterols and oxidized phospholipids that are taken up during ingestion of modified LDL or apoptotic cells. The efficient and healthy clearance of apoptotic cells is likely to be a key event limiting lesion progression, and the failure of this process may lead to directly to postapoptotic necrosis and inflammatory cell death, promoting plaque destabilization [41]. In vivo measurements of reverse cholesterol transport Whilst there is a general agreement that macrophage cholesterol efflux and reverse cholesterol transport are central players in the anti-atherogenic properties of HDL, the ability to measure these processes in vivo has proven elusive. A compelling demonstration of the ability of HDL to promote net reverse cholesterol transport was the demonstration of a transient rise in HDL levels followed by an increase in faecal neutral sterol and bile acid excretion (approximately 30 35%) in a small number of heterozygous familial hypercholesterolemia (FH) subjects infused with human proapoa-1 [49]. In contrast to these findings, some other approaches to raising HDL, such as treatment with CETP inhibitors do not result in an increased output of faecal neutral sterols [50]. However, from the point of view of atherogenesis, the key issue is the efflux of sterols from the tiny pool of foam cells in the artery wall, and this could be favourably influenced by raising HDL levels or increasing macrophage ABC transporter activity without necessarily increasing the overall process of reverse cholesterol transport. An example of this is a bone marrow transplantation from ABCA1) ) mice into apoe) ) recipients. This results in increased atherosclerosis as a result of defective macrophage cholesterol efflux, but does not lead to overall changes in HDL levels, and is unlikely to affect overall reverse cholesterol transport to the liver. An intermediate measurement that may have some use in predicting the outcome of treatments aimed at HDL has been developed by Rader and colleagues who have developed a method for measuring macrophage reverse cholesterol transport in animal models. This involves the injection of macrophages containing radiolabelled cholesterol into the peritoneal cavity followed by measurement of the appearance of cholesterol radioactivity in plasma, liver and faeces. Increased movement of 3H-cholesterol into all three compartments is observed following the treatment of animals with LXR activators [51], or following overexpression of apoa-1 [52]. A shortcoming of this method is that it does not distinguish isotope exchange from net cholesterol movement, and in both of these previous examples, increased HDL levels may have led to greater exchange of macrophage 3Hcholesterol with the enlarged HDL pool. Recently, this technique has been used to estimate the contribution of different transporters to macrophage reverse cholesterol transport. In these studies, plasma HDL levels would not be appreciably altered as a result of macrophage deficiency of ABCA1 and or ABCG1. As noted above, ABCA1 and ABCG1 are unidirectional cholesterol transporters and thus changes in 3H-cholesterol movement into plasma from macrophages deficient in these transporters should represent net cholesterol movement. These studies have shown that ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273 261

both transporters contribute to macrophage reverse cholesterol transport in an additive fashion, whilst similar studies with SR-BI ) ) macrophages show no role for SR-BI [53]. Recent studies from Tall s laboratory have shown a dramatic increase in atherosclerosis in LDLR-deficient mice transplanted with ABCA1) )ABCG1) ) mice compared to mice receiving bone marrow with individual deficiencies of transporters or wild-type bone marrow (Yvan-Charvet et al., submitted). This would appear to correlate well with the in vivo reverse cholesterol transport (RCT) measurements and emphasizes the importance of these two transporters in the athero-protective effects of HDL and its apolipoproteins. Atherosclerosis studies In animal models and probably also in humans (see below), over-expression or infusion of apoa-1 reduces progression or mediates regression of atherosclerosis. There appears to be premature atherosclerosis in a substantial proportion of human families with genetic deficiency of apoa-1. Although initial studies of apoa-i deficiency in mice indicated no major effect on atherosclerosis [54], subsequent studies of apoa-1 gene knock-out in apob transgenic [55] or LDLR) ) [56] mice have shown an increase in atherosclerosis. Long-term studies (22 months) of chow-fed LDLR) )apoa1) ) mice have indicated a significant increase in atherosclerosis, associated with increased indices of inflammation and oxidation [56]. Surprisingly, this occurred without substantial reductions of HDL cholesterol in the apoa-1) ) mice, suggesting specific anti-atherogenic properties of apoa-1. Whether these observations are related to direct anti-oxidant and anti-inflammatory properties of apoa-1, or are secondary to the ability of apoa-1 and HDL to mediate efflux of cholesterol and oxysterols via ABCA1 and ABCG1 is presently unknown. Mice with deficiency of SR-BI have increased HDL levels but are markedly more susceptible to atherosclerosis in apoe- or LDLR-deficient backgrounds [57]. Conversely, overexpression of SR-BI leads lower HDL levels and reduced atherosclerosis [58]. This is perhaps the clearest example of discordance between HDL levels and effects on atherosclerosis. However, in addition to regulating selective uptake of HDL CE and HDL levels, SR-BI also has a back-up role in the clearance of remnant lipoproteins in mice with LDLR or apoe deficiency [59], and in some studies altered expression of SR-BI also appeared to affect the levels of apob-lipoproteins [58]. HDL from SR-BI-deficient mice appears to be capable of promoting cholesterol efflux from macrophage foam cells [60] and as noted above, SR-BI-deficient macrophages do not have any change in cholesterol efflux to HDL or in macrophage reverse cholesterol transport. Thus, it remains unclear to what extent effects of SR-BI under- and over-expression on atherosclerosis reflect changes in levels of remnant lipoproteins versus defects in reverse cholesterol transport. Patients with Tangier disease probably have a moderate increase in atherosclerosis risk, but nothing like that seen in genetic disorders causing increased LDL such as familial hypercholesterolemia. The explanation for this has always been that ABCA1 deficiency in addition to causing low HDL also reduced VLDL and LDL cholesterol levels, and thus the atherogenic stimulus may be diminished in Tangier disease patients. A clever way around this issue was to study individuals with heterozygous mutations in ABCA1, who have isolated low HDL without significant changes in LDL or VLDL cholesterol levels [61]. These individuals have a moderate increase in carotid intima-media thickness (a measure of atherosclerosis volume) compared to age-matched control subjects, but the effects are not very dramatic despite about 40% decreases in HDL levels. The role of ABCA1 in atherosclerosis has also been extensively studied in mouse models. Bone marrow transplantation from ABCA1) ) mice into apoe- or LDLR-deficient recipients resulted in two- to three-fold increases in atherosclerosis compared to mice receiving control bone marrow [62, 63] without any major change in the plasma lipoprotein profiles. This indicates that the expression of ABCA1 in macrophages has an antiatherogenic role. Administration of LXR activators to ApoE- or LDLRdeficient mice results in a decrease in atherosclerosis 262 ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273

[64]. Based on bone marrow transplantation studies, there is evidence that macrophage LXRs have antiatherogenic functions [65], and are specifically responsible for the anti-atherogenic effects of systemically administered synthetic LXR agonists [66]. LXR activators reproducibly cause fatty liver as result of activation of the sterol regulatory element-binding protein-1c gene expression as well as direct targeting of genes involved in fatty acid synthesis [67]. However, in LXRa, apoe double knock-out mice effects of LXR activators on fatty liver and hypertriglyceridemia are minimal, whilst anti-atherogenic effects of these drugs are retained [68]. This suggests that small molecule LXRb selective activators or LXR activators that are relatively inactive in the liver might be ideal anti-atherogenic drugs. To date the identification of such selective activators has proven challenging. The discovery of LXRs and ABCA1 as key molecules involved in the regulation and mediation of reverse cholesterol transport has provided compelling evidence to support the idea that macrophage cholesterol efflux and reverse cholesterol transport are key processes underlying the anti-atherogenic effects of HDL. Although ABCG1 promotes efflux of cholesterol and oxysterols to HDL, its role in atherogenesis appears complex. When placed on a high-fat and high-cholesterol diet, ABCG1) ) mice accumulate macrophage foam cells in the lungs and also store CE and other neutral lipids in hepatocytes [10]. Surprisingly, however, bone marrow transplantation from ABCG1) ) mice into LDLR) ) recipients resulted in either no change, a small increase [69] or a decrease in atherosclerosis [70, 71]. Studies from two laboratories documented reductions in atherosclerosis in LDLR) ) or apoe) ) mice transplanted with ABCG1) ) bone marrow. In one study, the decrease in atherosclerosis was attributed to an increase in apoptosis of ABCG1) ) macrophages in lesions [70], whilst in the other study, ABCG1) ) macrophages showed up-regulation of some LXR target genes, notably ABCA1, and, an increase in apoe secretion, independent of changes in apoe mrna [71]. Knock-down of ABCA1 in ABCG1) ) macrophages verified that the increase in apoe secretion was secondary to induction of ABCA1. The increase in macrophage apoe secretion was reflected by an increased content of apoe in plasma and a small reduction in VLDL and LDL cholesterol levels in chow-fed mice transplanted with ABCG1) ) bone marrow. As noted above, ABCG1 has a particular role in promoting efflux of oxysterols from macrophages and this may explain induction of ABCA1 and other LXR target genes as well as increased apoptosis in ABCG1) ) cells [43]. Bone marrow transplantation from ABCA1) )ABCG1) ) mice into LDLR-deficient recipients has confirmed that the up-regulation of ABCA1 in ABCG1 deficient macrophages masked the underlying anti-atherogenic functions of ABCG1. Together, this information strongly supports the concept that apoa-1, apoe, HDL, ABCA1, ABCG1 and LXRs act together to promote macrophage cholesterol efflux and to protect against atherosclerosis. Thus, many molecular details of the reverse cholesterol transport pathway have been elucidated, and the hypothesis that cholesterol efflux and RCT are key anti-atherogenic properties of HDL has been sustained. Pleiotropic anti-atherogenic properties of HDL In addition to its cholesterol efflux properties, HDL has proposed to have anti-inflammatory, anti-oxidant, anti-apoptotic, anti-thrombotic and vasodilating properties that could all be potentially relevant to its association with reduced levels of atherosclerotic cardiovascular disease (Table 1). Table 1 Pleotropic anti-atherogenic properties of HDL Cholesterol efflux and reverse cholesterol transport Anti-oxidant and anti-inflammatory effects Anti-apoptotic properties Vasodilation (increased enos activity) HDL proteomics: anti-thrombosis, complement activation enos, endothelial cell nitric oxide synthase. Occam s Razor One should not increase beyond what is necessary the number of entities required to explain anything. Perhaps several different anti-atherogenic qualities of HDL are secondary to effects on cellular sterol efflux? ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273 263

Anti-oxidant and anti-inflammatory properties of HDL High density lipoproteins has been shown to have anti-inflammatory effects in both cell culture and in vivo models. These properties may be in part related to its content of lipoprotein-associated phospholipase A2 [also known as platelet-activation factor (PAF) acetylhydrolase (PAFAH)] [72] and paraoxonase [73]. In addition to its ability to scavenge or break down oxidized phospholipids, some of the beneficial anti-inflammatory properties of HDL may relate to the ability of HDL and its apolipoproteins to remove cholesterol and oxysterols from arterial cell wall. Freshly isolated macrophages from ABCG1) ) mice have increased expression and secretion of inflammatory cytokines, and show an exaggerated inflammatory response when treated with lipopolysaccharide (unpublished). This suggests that cholesterol or oxysterol accumulation secondary to transporter deficiencies somehow leads to enhanced signalling via toll-like Receptor 4 and increased expression of inflammatory genes. High density lipoproteins has the ability to repress tumour necrosis factor-mediated induction of the cell adhesion molecules vascular cell adhesion molecule- 1(VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in cultured aortic endothelial cells, in a mechanism involving suppression of sphingosine kinase activity [74, 75]. Infusion of HDL led to reduced expression of reactive oxygen species (ROS) and ICAM-1 and decreased infiltration of neutrophils in a Collared Carotid Artery Model [76]. It was proposed that HDL interrupted a vicious cycle in which infiltrating neutrophils generated ROS, inducing ICAM-1 in endothelium and thus causing further neutrophil recruitment. However, in another set of studies in a chronic atherosclerosis model, the apoe) ) mouse overexpression of apoa-i did not change arterial cell VCAM-1 or ICAM-1 expression even though it reduced atherosclerosis [77]. Although chronic atherosclerosis does not obviously involve neutrophils, neutrophils are likely to be involved in acute coronary syndromes and stroke. The ability of HDL to suppress neutrophil infiltration could be relevant in these situations. HDL has recently been shown to reduce the area of myocardial infarction in an ischemia-reperfusion model [78]. In this acute injury model, neutrophil degranulation increases the extent of tissue injury. The protective effect was attributed to the presence of sphingosine-1-phosphate (S-1-P) in HDL, and was dependent on the presence of the S-1-P receptor and the generation of nitric oxide (NO) in the heart. However, the role of this receptor in NO release is controversial (see below). These studies suggest that therapeutic elevation of HDL in the setting of acute coronary syndromes or perhaps other models of acute tissue injury involving neutrophils could have therapeutic benefits. A possible role of HDL in the stimulation of rapid emigration of foam cells from lesions? In a series of novel studies, transplantation of atherosclerotic aortic segments from apoe) ) mice into WT recipients but not apoe) ) recipients was associated with rapid regression of atherosclerosis and migration of a population of dendritic-like foam cells to regional lymph nodes [72, 79, 80]. Laser capture microdissection analysis of lesional foam cells indicated up-regulation of LXR and ABCA1 expression, and antibody experiments suggested an essential role of CC chemokine receptor 7 in this process. In an analogous experiment, emigration of dendritic cells from the skin to regional lymph nodes was found to be impaired in western-type diet (WTD)-fed apoe) ) mice, and associated with skin inflammation and deposition of foam cell [72]. Dendritic cell migration was enhanced by the presence of the apoa-1 transgene, or injections of human HDL. Dendritic cell migration was impaired by activity of the PAF receptor, which may be activated by PAF-like oxidized phospholipids in oxidized LDL. Injections of HDL from subjects with genetic deficiency of PAFAH failed to induce mobilization of dendritic cells. Thus, ability of HDL to restore dendritic cell emigration may be related to its content of PAFAH. If similar mechanisms are operative in atherosclerosis, it would imply that the migration of foam cells out of regressing lesions might be stimulated by PAFAH in HDL. However, this has not yet been directly demonstrated. In addition, it is not clear how such observations 264 ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273

would relate to the epidemiological observations concerning HDL levels and atherosclerosis, as PAFAH is found in both LDL and HDL and it has not been shown that acitivty of PAFAH or other components of HDL like paraoxonase are correlated with overall HDL levels. HDL and vascular reactivity High density lipoproteins has been shown to increase enos activity and protein levels in cultured endothelial cells [81], and to reverse the oxidized LDL-mediated decrease in NO production in endothelial cells [82]. HDL increases NO-mediated vascular relaxation in aortic ring preparations [83]. In humans, elevated HDL levels were less likely to be associated with abnormal vasoconstrictor responses in response to acetylcholine, over diseased segments of coronary arteries [84]. Tangier disease heterozygotes show impaired flow-mediated vasodilation in the brachial artery, and infusion of recombinant HDL particles containing apoa-1 and phospholipids into Tangier disease heterozygotes or hyperlipidemic subjects leads to acute improvements in flow-mediated vasodilation in the brachial artery [85]. Although there seems to be widespread agreement that HDL improves vascular reactivity by increasing NO bioavailability, there is no consensus on the underlying mechanisms. The ability of HDL to cause relaxation of vascular rings is markedly impaired in SR-BI) ) mice, and cell transfection of SR-BI enables HDL to increase enos activity in cell culture [83], in a mechanism that apparently depends on the cholesterol efflux properties of HDL [86]. Shaul et al. have suggested that HDL by influencing the pool of cholesterol in caveolae may acutely induce signalling via AKT, MAPK and Src pathways that leads to enos phosphorylation and activation [81]. However, the effect of HDL in aortic rings was very rapid (within few minutes), saturated at low HDL concentrations (10 lg ml) and was reportedly variable in different preparations [83]. Thus, it is unclear if mechanisms defined in cell culture are directly relevant to these very rapid effects that saturate at low HDL concentrations. It also remains unclear whether the defect in vascular relaxation in SR-BI) ) mice is a direct effect of SR-BI in endothelium or reflects perturbations in sterol metabolism in endothelial cells secondary to altered lipoprotein levels in vivo. Alternative explanations for the ability of HDL to increase enos activity relate to the presence of minor components in the HDL. Thus, in one study, HDL from premenopausal and oestrogen-treated postmenopausal women, but not HDL from nontreated postmenopausal women was found to be effective in inducing vascular relaxation, and the vascular relaxing properties of HDL were attributed to its content of oestrogen [87]. However, others have questioned whether the oestrogen content in HDL is sufficiently high to induce enos activity [81], and whether there is a difference between male and female HDL in this regard [88]. In another study, the increase in enos protein and activity was attributed to lysopholipids in HDL, particularly S-1-P, and is shown to depend on the S-1-P receptor in endothelial cells [88]. However, again it has been questioned whether the content of S-1-P in HDL is high enough to activate this receptor [81]. Moreover, in another study, the ability of HDL to increase coronary blood flow was found to be independent of the S-1-P3 receptor but dependent on enos expression [89]. Thus, at this time, it is unclear whether the ability of HDL to increase enos in model studies is because of minor bioactive components of HDL, or relates to the more established properties of HDL to induce sterol efflux from cells. It is worth noting that the ability of recombinant HDL consisting of phospholipids and apoa-1 to induce improvements in vascular reactivity in vivo [85] is likely to be independent of such minor components as they would not be included in the infused preparation and more likely to be related to sterol efflux. Anti-thrombotic properties of HDL High density lipoprotein levels are inversely related to athero-thrombotic disease but it is difficult to know whether this reflects primarily effects on atherosclerosis or thrombosis. A recent report suggests that HDL levels are inversely related to risk of recurrent venous thrombo-embolism [90]. A number of anti-thrombotic properties of HDL have been described. HDL stimulates endothelial cell prostacyclin synthesis both by ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273 265

providing substrate (arachidonic acid) [91, 92] and inducing cyclooxygenase-2 expression [93]. In addition, large HDL particles can act as a surface on which the ability of activated protein C or S to cleave factor Va is enhanced [81]. Finally, as result of its content of S-I-P and activation of the S-1-P receptor, as well as stimulation of NO synthesis, HDL can oppose apoptosis of endothelial cells [81, 94]. Although all of these mechanisms could be acting to oppose athero-thrombosis and possibly venous thrombosis, there is still only limited in vivo evidence for such activity in humans. The proteome of HDL: potential roles in complement regulation, inflammation and coagulation A recent study used a proteomics approach to catalogue the proteome of human HDL. This revealed quite unexpectedly that HDL is enriched in several proteins involved in the complement cascade, as well as in a variety of protease inhibitors, supporting the concept that HDL plays a role in innate immunity and in the regulation of proteolytic cascades involved in inflammatory and coagulation processes. Many acute phase response proteins were also detected, supporting the proposal that HDL is of central importance in the regulation of inflammation. Indeed, HDL binds to and modulates the actions of endotoxin and other bacterial antigens, provides a platform for assembly of innate immune complexes, acts as an acceptor for oxidized phospholipids, and blocks oxidation of apob lipoproteins [6, 7, 17, 18]. HDL levels fall during acute inflammation, perhaps to achieve conditions permissive for acute inflammation. The recovery of HDL levels following the acute inflammatory response could play an important role in suppressing ongoing inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with coronary artery disease (CAD) was selectively enriched in apoe, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. However, this might simply reflect a paucity of apoe in larger HDL particles and could thus be a marker of low levels of HDL-2. Collectively, these observations suggested that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its anti-inflammatory and anti-atherogenic properties. However, many of these preliminary observations will need to be confirmed and their functional significance worked out. Recent clinical studies involving drugs targeting HDL Whilst LDL lowering with diet and drugs such as statins remains the cornerstone for treating atherosclerotic cardiovascular disease, statin therapy only reduces cardiovascular events by about one-third. This has led to the suggestion that the large residual burden of atherosclerotic disease might be effectively treated by interventions to raise HDL levels. Despite some recent disappointments in the clinical arena with HDL raising therapies, it is still possible that therapeutic targeting of HDL could represent the next major breakthrough in atherosclerosis research. In the last 5 years, several clinical studies have been completed involving therapies directed primarily or partly at HDL. Together, these studies suggest that raising HDL levels by a variety of approaches may lead to regression of coronary atherosclerosis but also indicate the complexity of further extending this finding into demonstrable clinical benefit. Infusion of HDL-like complexes containing ApoA-I Milano or ApoA-1Wild type In a landmark study, 5-week infusions of apoa-1(milano) phospholipid complexes into patients with acute coronary syndromes were associated with a striking and significant regression of coronary atherosclerosis compared with baseline as assessed by intravascular ultrasound studies (IVUS) measurements [95]. The difference was not statistically significant between the groups. Some authors have questioned whether subjects with the ApoA-1 (Milano) mutation are protected from atherosclerosis [96], and there is some evidence that ApoA-1(Milano) is not superior to wild type ApoA-1 in terms of its anti-atherogenic properties [97, 98]. Nonetheless, the ApoA-I (Milano) infusion study is consistent with a large body of evidence in animals and humans indicating that infusion of apoa-1 or HDL is athero-protective. 266 ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273

A recent much larger study called ERASE involved the infusion of wild type apoa-1 in complexes with phospholipids into humans. This involved two different doses of the complexes or saline control. The study was discontinued in the group receiving the higher dose because of significant hepatoxicity, and this was also seen to some extent in the lower-dose group [99]. The findings could indicate mild hepatotoxicity and haemolysis related to rapid extraction of cholesterol from liver and erythrocytes, respectively. Alternatively, toxicity may have been because of residual cholate in the HDL preparation made by a cholate dialysis technique. Compared to baseline, the group receiving the apoa-1(wild type) phospholipid complexes showed significant regression of coronary atherosclerosis whilst the control group did not, but again the difference was not significant between the groups. The magnitude of regression of coronary atherosclerosis was less than seen in the apoa-1 (Milano) study and this could have simply been because the study was larger or because the infused preparation was sub-optimal. Importantly, this study tended to confirm the major finding of the apoa-1milano study that there was significant regression of atherosclerosis in the group receiving apoa-1 phospholipid complexes. The limited power of fibrates to raise HDL and their variable benefits Whilst these two studies provided grounds for optimism concerning HDL-directed therapies, two other large clinical outcome studies involving HDL-directed therapies, the FIELD study and the ILLUMINATE study were substantial disappointments. The Helsinki Heart and Veterans Affairs-HDL Intervention Trial (VA-HIT) studies using the fibric acid derivative gemofibrozil had indicated significant reductions of coronary artery disease, especially in high risk patients with metabolic syndrome or Type 2 diabetes [100], and part of the benefit of this ppar-alpha activating drug appeared to be mediated through increased HDL levels [101]. The FIELD study was a large, 5-year study carried out using fenofibrate in high risk patients with Type 2 diabetes. This showed a nonsignificant 11% reduction in the primary end-point of coronary artery disease in the group receiving fenofibrate compared with placebo, and mixed findings in secondary end-points [102]. Results were in part vitiated by drop-in of statin therapy in the control group but this is unlikely to have masked a major benefit of fenofibrate. Moreover, HDL elevation by fenofibrate appeared to be limited and in subjects receiving fenofibrate, increases in HDL cholesterol levels were minimal by the end of the study [100]. Whilst there still may be a place for use of fibrates in the treatment of low HDL patients with the metabolic syndrome, statins have already been shown to be useful in such patients. The ACCORD study that is ongoing will assess the efficacy of statin + fenofibrate versus statin alone in patients with Type 2 diabetes [103]. However, in view of the limited efficacy of fenofibrate in the FIELD study, it could be questioned whether fenofibrate is the optimal drug to be used in this context. CETP inhibitors: ILLUMINATE, ILLUSTRATE and RADIANCE The phenotype of very high HDL and moderately reduced LDL levels in subjects with genetic deficiency of CETP [104, 105] spurred the development of small molecule inhibitors that markedly raise HDL and incrementally reduce LDL when added to statin therapy [50]. Unfortunately, a large clinical study involving the CETP inhibitor, torcetrapib was stopped prematurely because of an excess of deaths and adverse myocardial events in the group receiving torcetrapib (60 mg) + atorvastatin versus atorvastatin alone [106, 107]. At this time, it is hard to judge to what extent the adverse outcome reflected mechanism-related toxicity versus off-target toxicity peculiar to torcetrapib. Interestingly, studies by coronary IVUS showed no significant change in the primary endpoint (% atheroma volume) whilst modest regression of atherosclerosis was suggested in the secondary endpoints, (atheroma volume and diameter of most diseased arterial segment) [108]. Studies of carotidintima media thickness in subjects with heterozygous familial hypercholesterolemia or Type 2b hyperlipdemia did not show a significant change in the primary end-point measurement of carotid intima-media ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273 267

thickness comparing torcetrapib atorvastatin recipients versus atorvastatin recipients [109, 110]. Thus, the adverse clinical outcome, though apparently related to cardiac events was not associated with an obvious worsening of coronary or carotid atherosclerosis. Whilst it is clear that torcetrapib caused significant, nonmechanism-related elevations in (BP), in the range of 3 5 mm SBP, it is uncertain if this degree of BP elevation was sufficient to explain the adverse clinical events. Possibly the BP change was indicative of a serious underlying adverse event such as activation of the renin angiotensin system or vasospasm [107]. Another much discussed possibility is that inhibiting CETP produces increases in HDL particles that are dysfunctional. For example, the HDL could be defective in its ability to promote efflux of cholesterol from arterial wall cells, or could have pro-inflammatory properties or adverse effects on endothelial function. Our studies suggest that HDL from subjects with homozygous CETP deficiency or subjects treated with torcetrapib [11, 24] matched for HDL cholesterol or protein concentration is at least as effective as normal HDL in terms of its ability to promote efflux of cholesterol from cholesterol-loaded macrophages. The HDL that accumulates in homozygous CETP deficiency is large and enriched in apoe and LCAT and has enhanced ability to promote cholesterol efflux, in part is dependent on the ABCG1 pathway of macrophage cholesterol efflux (Fig. 2). As suggested above, the ABCG1 pathway is important in removing both cholesterol and toxic oxysterols from macrophages, and thus the findings suggest that elevation of HDL by CETP inhibition is likely to be beneficial. However, improvements in HDL functionality on a per particle basis were only clearly seen at higher levels of CETP inhibition in association with an increase in the content of apoe and LCAT in the HDL [11, 24]. Thus, it is possible that most of the benefit from CETP inhibition could occur at high levels of inhibition resulting in marked elevations of HDL and enrichment of particles with apoe and LCAT. Further analysis of the ILLUMINATE and ILLUSTRATE data, as well as ongoing studies with CETP inhibitors that do not cause significant hypertension will probably help to clarify these issues. Niacin, HDL and Flushing (Fig. 3) There are some interesting similarities between the effects of niacin and those of CETP inhibitors Fig. 3. Niacin causes increases in larger, HDL-2 like particles in association with a decrease in catabolism of apoa- 1 [111, 112]. Niacin binds a G-protein coupled receptor (GPCR) in adipose tissue, called GPRIDSA (human) resulting in decreased release of free fatty acids (FFA) and decreased VLDL secretion [113]. Niacin does not raise HDL in wild-type mice, but does so in CETP transgenic mice, suggesting that part of its HDL raising effects may be mediated by creating a functional CETP deficiency state i.e. the decrease in VLDL effected by niacin might decrease CETP-mediated transfer of CE from HDL to VLDL [114]. The same GPCR that is responsible for modulation of FFA release from adipocytes is also responsible for niacin-mediated flushing [115]. Activation of this receptor in dendritic cells in the skin leads to synthesis of prostaglandin E2 and prostaglandin D2, which then activate receptors on smooth muscle cells in skin blood vessels leading to vasodilation. The major effect is mediated by the prostaglandin D2 (PGD2) receptor, dorsal protein 1 (DP1), and antagonists of DP1 reduced flushing in subjects treated with niacin [116]. This has led to the idea that combining extended release niacin with a DP1 receptor antagonist could lead to increased tolerability, allowing more subjects to successfully attain a dosage of 2 g niacin, and providing optimal changes in plasma lipoproteins such as maximal HDL elevation. As suggested above, it is possible that more marked elevations of HDL associated with substantial changes in HDL properties and functionality may be desirable. It is interesting to note that the addition of niacin therapy in patients with adequately treated LDL levels has been associated with an improvement in vascular reactivity in forearm blood flow measurements [117]. Is there a future for HDL therapies? Drugs that work by blocking the catabolism of HDL i.e. CETP inhibitors and niacin cause accumulation of large HDL-2 type particles. There is epidemiological evidence that increases in larger HDL-2 type particles 268 ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273

Fig. 3 Mechanisms responsible for niacin-mediated flushing. See section Niacin, HDL and Flushing for further details. Reproduced, with permission, from Pike N. Flushing out the role of GPR109A (HM74A) in the clinical efficacy of nicotinic acid. J Clin Invest 2005; 115: 3400 3. may be most strongly associated with cardiovascular benefit [118]. We have argued that higher levels of HDL, associated with higher level CETP inhibition, and perhaps with higher doses of niacin, may more optimally capture changes in HDL particles functionality [24, 107]. We have emphasized that these type of particles may be particularly effective at enhancing efflux of cholesterol and toxic oxysterols via ABCG1 in macrophages and possibly also in endothelial cells. A potential limitation to this approach may be that efflux of some oxysterols via ABCG1 may lead to lower levels of LXR activation and reduced expression of ABCA1, tending to offset some of the benefit in terms of cholesterol efflux. Theoretically, increasing production of apoa-1, infusing apoa-1 or infusing apoa-1-like peptides might be a more ideal way to increase HDL levels, as it might lead to promotion of cholesterol efflux both by ABCA1 and ABCG1. Similarly, upregulation of ABCA1 and ABCG1 by treatment with LXR activators could also have optimal results. However, whether increasing apoa-1 production or infusions can be carried out safely and effectively remains a challenge. Similarly, it still needs to be seen if LXR activators can be used without causing fatty liver in humans. It appears likely that multiple different therapies directed at HDL will emerge over the next decade, and that clearer goals for HDL elevation in the clinic will be attained. ª 2008 Blackwell Publishing Ltd Journal of Internal Medicine 263; 256 273 269