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lipoprotein; RCT, reverse cholesterol transport; LCAT, lecithin:cholesterol acyltransferase; CETP, cholesterol ester transfer protein; EL, endothelial lipase; SRBI, scavenger receptor BI; PPAR, peroxisome proliferator activated receptor; RXR, retinoid X receptor; LXR, liver X receptor; GGPP, geranylgeranyl pyrophosphate; PKC, protein kinase C; PKA, protein kinase A; SCD, stearoyl CoA-desaturase; BMT, bone marrow transplantation; LTR, long terminal repeat; WT, wild-type

Lipid Metabolism and Atherosclerosis

Nearly half of the deaths of persons in the Western world are caused by atherosclerotic processes, the most common of which manifest as coronary artery disease and stroke [1–4]. Aberrant regulation of lipid and lipoprotein homeostasis can lead to atherosclerosis [5–19]. Well-established risk factors include elevated plasma low-density lipoprotein (LDL) and depressed highdensity lipoprotein (HDL) levels [4, 15, 20–27]. Conversely, high levels of HDL protect from atherosclerosis even in the presence of high LDL levels [2, 26, 27]. Statins, drugs that lower LDL levels by inhibiting the rate-limiting step in cholesterol biosynthesis, are widely used to control LDL levels and to reduce the risk of cardiovascular disease [19]. However, the clinical event rate in patients on statins is approximately 70% of the rate in subjects treated with a placebo, suggesting that additional therapeutic strategies are still needed to reduce the burden of cardiovascular disease [19]. Therapeutic approaches that increase the levels of HDL, augment its antiatherogenic properties, and promote reverse cholesterol transport (RCT) are considered particularly desirable [19, 21].

RCT is the process by which excess cholesterol is extracted from peripheral cells and transported to the liver in the form of HDL particles for elimination as bile acids [21, 28, 29]. HDL itself exhibits several antiatherogenic properties including stimulating nitric oxide production, inhibiting endothelial cell adhesion molecule expression, inhibiting the oxidation of LDL, preventing endothelial cell apoptosis, and inhibiting platelet aggregation [19, 21, 27]. HDL is the smallest and densest circulating lipoprotein whose major function is to transfer excess lipids from extrahepatic tissues to the liver. HDL is a heterogenous lipoprotein consisting mainly of phospholipid, cholesterol, and protein. Biogenesis of HDL occurs predominately in the liver and intestine, and requires the action of the ATP-binding cassette transporter ABCA1 to transfer cholesterol and phospholipids onto lipid-free apoAI to form discoidal pre-β-HDL, which then matures into α-migrating spherical HDL particles upon acquisition of additional lipids and esterification of cholesterol into cholesterol esters (CE) by lecithin:cholesterol acyltransferase (LCAT) [4, 26].

Currently used agents that can raise HDL levels include nicotinic acid, fibrates, statins, thiazolidinediones, estrogen, and ethanol, but these typically result in relatively modest increases of HDL [19]. Strategies that target specific

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genes involved in HDL metabolism, including apoAI, cholesterol ester transfer protein (CETP), and endothelial lipase (EL), show promise as potential therapies. Weekly intravenous injections of recombinant apoAI-Milano, a single base-pair variant of apoAI, resulted in significant regression of coronary atherosclerosis over a 6-week treatment period [30]. Additionally, inhibition of CETP-mediated movement of cholesterol from HDL to LDL in rabbits increases plasma HDL and reduces atherosclerosis, and CETP inhibition also increases HDL levels in humans [31–33]. Recently, overexpression of EL in mice was shown to reduce HDL levels by approximately 19%, whereas inactivation of EL increases HDL levels by approximately 50% [34–36].

Although absolute levels of circulating HDL are important determinants of atherosclerotic risk, the ability of the macrophage to efflux accumulated cholesterol within the arterial wall plays a crucial role in the development and progression of atherosclerotic lesions. Strategies to augment the process of macrophage cholesterol efflux, and thus retard foam cell formation and the initiation of inflammatory pathways that exacerbate lesion progression, may also yield novel therapeutic agents that slow the development of advanced atherosclerotic lesions and reduce the incidence of plaque rupture and infarction.

Pathways for Cholesterol Efflux from Macrophages

Although mammalian cells can synthesize cholesterol, they do not have the capacity to degrade the sterol ring. In order to remove excess cholesterol, macrophages and other cell types must export cholesterol to extracellular acceptors that transport excess cholesterol to the liver for eventual excretion in bile. It is generally accepted that there are several pathways by which macrophages are thought to efflux excess cholesterol (reviewed in Refs. [37, 38]). For the purposes of this review, we will classify these into two major subdivisions: passive/facilitated aqueous diffusion and transporter-mediated active efflux.

Passive/Facilitated Aqueous Diffusion

Passive diffusion involves the desorption of membrane cholesterol down a concentration gradient into the aqueous environment followed by absorption onto a phospholipid-containing acceptor [39, 40]. Cholesterol efflux by passive diffusion does not consume metabolic energy and is independent of the cellular growth state and cholesterol content. Rather, the rate of cholesterol transfer is influenced both by the structure and lipid composition of the plasma membrane on the donor cell and properties of the acceptor molecule including concentration, size, and lipid composition. Because the aqueous layer between donor and acceptor represents a significant diffusion barrier, cholesterol efflux by passive diffusion is inefficient and slow.

However, passive diffusion can be facilitated by expression of the scavenger receptor BI (SRBI), which is the first membrane protein shown to function in the bidirectional flux of free cholesterol (FC). SRBI is expressed in many tissues including macrophages, and the highest levels of SRBI are found in the liver and steroidogenic tissues such as adrenals, testis, and ovary. A major function of SRBI is to promote the selective uptake of esterified cholesterol from HDL, a unidirectional process that occurs without endocytosis of the entire HDL particle [41–43]. Expression of SRBI also stimulates the bidirectional flux of FC, with the net direction of transfer dictated by the concentration gradient between donor and acceptor [44, 45]. In a number of cell types, the rate of FC efflux to acceptors correlates well with expression levels of SRBI [46]. Although many acceptors bind to SRBI, including HDL, LDL, oxidized and acetylated LDL, small unilamellar vesicles, and lipid-free apoAI [47, 48], only those acceptors that contain phospholipids will accept cholesterol via SRBI. Furthermore, both phospholipid content and species influence SRBI-medi- ated efflux. For example, it is well established that enrichment of HDL or serum with phosphotidylcholine increases SRBI-mediated efflux, whereas depletion of phosphotidylcholine decreases SRBI-mediated efflux [45, 49]. Enrichment of HDL with sphingomyelin also results in a net increase in SRBI–cholesterol efflux, but this occurs by inhibiting SRBI-mediated FC influx rather than by enhancing FC efflux [45].

SRBI is thought to facilitate passive diffusion by reorganizing membrane microdomains into cholesterol-rich lipid rafts that supply the majority of cholesterol during passive diffusion [48, 50–52]. This mechanism may be particularly important at high HDL concentrations when SRBI receptors are saturated, and increased efflux is independent of HDL binding. However, under conditions of low HDL concentrations, binding of HDL to SRBI influences cholesterol efflux. In this case, it appears that SRBI and the acceptor molecule must be precisely aligned into a productive complex in order to promote efficient FC efflux [53]. For example, SRBI-mediated FC efflux to HDL was found to be severely decreased in the presence of two apoAI mutations even though binding of the mutant HDL particle to SRBI was not affected. Intriguingly, efficient FC efflux to HDL containing the mutated apoAI was rescued in the presence of mutations within SRBI, presumably by restoring the alignment between donor and acceptor [53].

Transporter-Mediated Active Efflux

Several members of the ATP-binding cassette transporter superfamily are key regulators of cholesterol efflux, RCT, and HDL metabolism. There are 48 known human ABC transporters that are grouped into seven classes [54]. Members of the ABCA and ABCG classes are believed to form a functional network of lipid transporters that are critical gatekeepers of cell and sterol homeostasis [54–56]. Two of these transporters, ABCA1 and ABCG1, are highly induced in cholesterol-loaded macrophages and mediate lipid efflux to

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apoAI and HDL, respectively. These observations suggest a prominent role for ABCA1 and ABCG1 in the coordinated regulation of macrophage lipid homeostasis.

ABCA1

ABCA1 is the prototype of the full-sized ABCA class of transporters and contains two membrane spanning regions interspersed with two ATP-bind- ing domains [55, 56]. Deficiency of ABCA1 results in Tangier disease, which is characterized by extremely low plasma HDL levels, tissue deposition of CE, and an increased risk of atherosclerosis [57–60]. ABCA1 catalyses the ATP-dependent transport of cholesterol and phospholipids from the plasma membrane to lipid-free apoAI to form immature, pre-β-HDL, which is considered to be a key step in RCT [57, 59, 61, 62]. Mature, α-HDL is formed by further accumulation of lipids on pre-β-HDL followed by esterification of cholesterol by LCAT [29, 63–65]. Cholesterol from α-HDL is taken up by the liver through a number of pathways including SRBI-mediated selective uptake, holoprotein uptake by apoE receptors, or indirect pathways involving CETP, hepatic lipase, and EL. Within hepatocytes, cholesterol is converted to bile acids and excreted from the body in bile [41, 42, 66]. Because ABCA1 has a singular role in the biogenesis of HDL and the initiation of RCT, it is therefore an attractive candidate molecule on which to base atheroprotective therapeutic strategies. Additional support for ABCA1 as a potential therapeutic target has come from studies demonstrating that overexpression of ABCA1 in mice increases plasma HDL levels [67, 68] and dramatically reduces the progression of atherosclerotic lesions on an apoE-deficient background [69].

Transcription of ABCA1 is subject to several layers of control (recently reviewed in Refs. [70–74]). In many cell types including macrophages, the most effective direct transcriptional modulators of ABCA1 include members of the retinoid X receptor (RXR) and liver X receptor (LXR) nuclear orphan receptors, which act as obligate heterodimers upon binding their respective agonists [75–78]. Ligands for RXRs include 9-cis-retinoic acid, and ligands for LXRs are oxysterols such as 22-hydroxycholesterol, 20-hydroxycholesterol, 24-hydroxycholesterol, 24,25-epoxycholesterol, and 27-hydroxycholesterol. Although LXR and RXR agonists can each induce ABCA1 expression independently, the most potent increase in ABCA1 mRNA levels occurs when LXR–RXR agonists act synergistically. It is generally believed that the induction of ABCA1 expression in lipid-loaded macrophages occurs primarily through the LXR–RXR pathway, which is activated upon conversion of accumulated sterols to endogenous oxysterols such as 27-hydroxycholesterol. In J774 and primary murine macrophages, ABCA1 mRNA is also induced by cAMP treatment [79]. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that are activated by fatty acid derivatives and heterodimerize with RXRs to regulate lipid and glucose metabolism [80]. PPARs (PPARα, PPARBβ/δ, and PPARγ) have been shown to upregulate ABCA1 as

well as ABCG1 expression in macrophages [81, 82]. Although this has been thought to employ an indirect mechanism via induction of LXRα expression by PPAR–RXR heterodimers [81], recent studies have shown that ABCA1 expression and apoAI-dependent cholesterol efflux can be induced by the selective PPARδ agonist GW50516 in an LXRα-independent manner in THP- 1 cells, intestinal cells, and fibroblasts [83]. Furthermore, conditional inactivation of PPARγ impaired expression of ABCA1, ABCG1, and apoE in macrophages and led to reduced cholesterol efflux [84]. Recently, PPARα and PPARγ agonists were found to strongly inhibit atherosclerosis in the LDLR− /− model, which was mediated through ABCG1 rather than ABCA1 expression [85]. These observations further support a role for PPARs in macrophage lipid homeostasis and regulation of ABC transporter expression.

ABCA1 mRNA synthesis can be repressed by geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway that reduces the transactivation potential of LXRs [86]. In addition, lipopolysaccharide has recently been shown to downregulate ABCA1 expression in an NFκB- dependent manner [87, 88], and the proinflammatory cytokines IL-1, INFγ, and TNFα each suppress ABCA1 mRNA levels in macrophages [89–91]. These observations suggest that the ability of inflammatory mediators to promote atherosclerosis lesion formation may in part be mediated by suppressing ABCA1 expression and thereby reducing cholesterol efflux. Thyroid hormone (T3) can also inhibit ABCA1 transcription, which occurs when thyroid hormone–RXR complexes compete for LXR–RXR heterodimers at the ABCA1 promoter [92].

ABCA1 protein levels are also subject to regulation through posttranscriptional mechanisms. ABCA1 contains a PEST sequence from amino acids 1283–1306 that modulates protein degradation by calpain [93]. Residues T1286 and T1305 within the PEST sequence are normally constitutively phosphorylated, which results in a rapid turnover (half-life of 1–2 h) of ABCA1 in the absence of apoAI [93, 94]. However, these residues become dephosphorylated in the presence of apoAI, which inhibits calpain-mediated degradation of ABCA1 [93, 94]. This may occur through activation of the protein kinase Cα (PKCα) signaling pathway by lipid-poor apoAI [95]. In contrast, protein kinase A (PKA) appears not to be involved in the stabilization of ABCA1 protein [94], but may promote cholesterol efflux by phosphorylating residues S1044 and S2054 on ABCA1 [96]. In cAMP-stimulated J774 macrophage cells, ABCA1 protein levels are also destabilized by unsaturated but not by saturated fatty acids [97]. In contrast, ABCA1 is destabilized by both saturated and unsaturated fatty acids in LXR–RXR-treated cells [98]. The differential effect of fatty acids on ABCA1 stability is thought to be due to the induction of stearoyl CoA-desaturase (SCD) specifically in LXR–RXR-stimulated cells, resulting in the formation of unsaturated fatty acids from their saturated precursors thereby triggering ABCA1 degradation [98]. ABCA1 is also degraded by the ubiquitin-mediated proteasomal pathway in macrophages loaded with FC [99]. This pathway may be