Reviews
This Review is part of a thematic series on ABC Transporters in the Cardiovascular System, which includes the
following articles:
ATP-Binding Cassette Cholesterol Transporters and Cardiovascular Disease
Structure and Function of ABC Transporters
John F. Oram, Guest Editor
ATP-Binding Cassette Cholesterol Transporters and
Cardiovascular Disease
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John F. Oram, Ashley M. Vaughan
Abstract—A hallmark of atherosclerotic cardiovascular disease (CVD) is the accumulation of cholesterol in arterial
macrophages. Factors that modulate circulating and tissue cholesterol levels have major impacts on initiation,
progression, and regression of CVD. Four members of the ATP-binding cassette (ABC) transporter family play
important roles in this modulation. ABCA1 and ABCG1 export excess cellular cholesterol into the HDL pathway and
reduce cholesterol accumulation in macrophages. ABCG5 and ABCG8 form heterodimers that limit absorption of
dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. All
4 transporters are induced by the same sterol-sensing nuclear receptor system. ABCA1 expression and activity are also
highly regulated posttranscriptionally by diverse processes. ABCA1 mutations can cause a severe HDL-deficiency
syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. ABCG5 or
ABCG8 mutations can cause sitosterolemia, in which patients accumulate cholesterol and plant sterols in the circulation
and develop premature CVD. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excess cholesterol in
macrophages, and manipulating mouse macrophage ABCA1 expression affects atherogenesis. Overexpressing ABCG5
and ABCG8 in mice attenuates diet-induced atherosclerosis in association with reduced circulating and liver cholesterol.
Metabolites elevated in individuals with the metabolic syndrome and diabetes destabilize ABCA1 protein and inhibit
transcription of all 4 transporters. Thus, impaired ABC cholesterol transporters might contribute to the enhanced
atherogenesis associated with common inflammatory and metabolic disorders. Their beneficial effects on cholesterol
homeostasis have made these transporters important new therapeutic targets for preventing and reversing CVD. (Circ
Res. 2006;99:1031-1043.)
Key Words: atherosclerosis 䡲 cholesterol homeostasis 䡲 HDL cholesterol 䡲 lipoproteins 䡲 ABC transporters
C
for steroid production. Thus, an inadequate supply of cholesterol would have devastating effects on cell function, tissue
development, and whole-body physiology.
Too much cholesterol, however, can also have pathological
consequences. Cholesterol is a waxy substance that is poorly
soluble in water. An uncontrolled buildup of cholesterol in
cells can disrupt membranes and promote apoptosis. Accumulation of excess cholesterol causes atherosclerotic cardiovascular disease (CVD)3 and might contribute to the early
onset of Alzheimer’s disease4 and renal dysfunction.5,6
holesterol is an abundant metabolite in mammalian
tissues that is essential for several biological systems.
Membrane fluidity of all cells is tightly modulated by the
ordered packing of cholesterol between phospholipid molecules. Cholesterol also concentrates in sphingolipid-rich domains of the plasma membrane called rafts that contain a
variety of important signaling molecules.1 Cholesterol covalently links to sonic hedgehog,2 a cell morphogen required
for normal embryonic development and cell differentiation.
In addition to its structural functions, cholesterol is a substrate
Original received June 20, 2006; resubmission received September 18, 2006; accepted October 3, 2006.
From the Department of Medicine, University of Washington, Seattle.
Correspondence to John F. Oram, Department of Medicine, Box 356426, University of Washington, Seattle, WA 98195-6426. E-mail
[email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000250171.54048.5c
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November 10, 2006
Figure 1. Pathways for cholesterol delivery and production.
Details are described in the text. C indicates free cholesterol;
CE, cholesteryl esters; TG, triglycerides; Rem, remnants; LPL,
lipoprotein lipase; LDLR, LDL receptor.
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Because of this need for strict maintenance of tissue
cholesterol levels, the body relies on a complex homeostatic
network to modulate the availability of cholesterol for cells
and tissues. This network operates within both cells and the
plasma compartment.
Lipoprotein Metabolism
Sources of Cholesterol
Cholesterol is derived from both exogenous dietary sources
and endogenous biosynthetic pathways. Intestinal enterocytes
absorb dietary cholesterol and package most of it into
triglyceride-rich lipoproteins called chylomicrons (Figure
1).7,8 Endothelial-bound lipoprotein lipase hydrolyzes triglycerides in circulating chylomicrons to generate chylomicron
remnants, which are cleared rapidly by the liver (Figure 1).7
The intrahepatic cholesterol can be repackaged and secreted
along with triglycerides in very-low-density lipoproteins
(VLDLs), which are also substrates for lipoprotein lipase.
Most of the cholesterol secreted from the liver is synthesized
by hepatocytes rather than derived from dietary sources
(Figure 1).
The remnants formed by hydrolysis of VLDL triglycerides
are either taken up by the liver or converted to a cholesterol-rich lipoprotein subclass called low-density lipoprotein
(LDL) (Figure 1),7,8 which carries approximately two-thirds
of the cholesterol in human plasma. LDL provides a source of
cholesterol for steroidogenesis and cellular membranes. This
occurs through the interaction of LDL with a cell surface
receptor that mediates internalization and degradation of the
lipoprotein particles.9 The hepatic LDL receptor is responsible for clearing most of the LDL cholesterol from the
circulation. Most cells in the body can also synthesize
cholesterol when needed.
Cells other than those in steroidogenic tissues and the liver
cannot metabolize cholesterol. Instead they modulate their
membrane cholesterol content by a feedback system that
controls the rate of cholesterol uptake by the LDL receptor
and de novo biosynthesis.10 With most cell types, this system
is sufficient to provide enough cholesterol for membrane
integrity and other functions without overloading them.11
Some cells, particularly macrophages, can ingest cholesterol
Figure 2. Reverse cholesterol transport. Details are described in
the text. C indicates free cholesterol; CE, cholesteryl esters;
LCAT, lysolecithin cholesterol acyltransferase; CETP, cholesteryl
ester transfer protein; LDLR, LDL receptor; SR-B1, scavenger
receptor B1.
by endocytotic and phagocytotic pathways that are not
feedback regulated by cholesterol.12 These cells must either
store this excess cholesterol as esters or secrete it.
Reverse Cholesterol Transport
High-density lipoproteins (HDLs), which carry approximately one-third of the cholesterol in human plasma, are
involved in the removal of excess cholesterol from cells.13,14
HDL is a multifunctional and heterogeneous class of particles
that transports a variety of lipids and lipophilic molecules
between tissues and lipoproteins. One of the major functions
of HDL is to transport cholesterol from peripheral tissues to
the liver for elimination in the bile. This occurs by a pathway
called reverse cholesterol transport, which involves the coordinate action of multiple cellular and plasma proteins.14,15
Although HDL particles contain a variety of proteins,
⬇70% of its total protein cargo is apolipoprotein A-I (apoAI). The liver and intestine synthesize and secrete apoA-I into
the circulation as a lipid-free or poorly lipidated protein
(Figure 2). This apoA-I rapidly acquires phospholipids and
cholesterol from cells to become partially lipidated. Most of
this lipidation appears to involve the interaction of apoA-I
with liver cells, but a fraction of the newly secreted or
partially lipidated apoA-I may circulate to the periphery,
where it interacts with other cholesterol-loaded cells, particularly macrophages. These nascent HDL particles acquire
additional phospholipids and cholesterol through cellular
cholesterol efflux processes (Figure 2) and by transfer from
the surface of other lipoproteins, such as chylomicrons and
VLDL, during lipolysis of their triglycerides (not shown).
Most of these nascent HDL particles are disc-shaped
because their cholesterol is in an unesterified (free) form that
incorporates between phospholipid molecules (Figure 2). In
the circulation, an enzyme (lysolecithin cholesterol acyltransferase [LCAT]) esterifies the free cholesterol in these particles, converting it to cholesteryl ester lipid droplets that
comprise the core of the mature spherical HDL particles
(Figure 2).8
Mature HDL particles are remodeled by plasma proteins
that transfer their core cholesteryl esters and surface phospholipid to other lipoproteins and hydrolyze their phospho-
Oram and Vaughan
Figure 3. Topographic models for ABCA whole transporters (A)
or ABCG half-transporters in their orientations as dimers (B).
NBD indicates nucleotide binding domains.
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lipids. These particles also selectively deliver their core
cholesteryl esters into cells, particularly those of the liver and
steroidogenic tissues, through their interaction with a receptor
called scavenger receptor B1 (SR-B1) (Figure 2).16 These
HDL-remodeling processes are thought to regenerate lipidpoor apoA-I that dissociates from the particles and recycles
through the reverse cholesterol transport pathway (Figures 2
and 4).17
ATP-Binding Cassette
Cholesterol Transporters
Different steps that control the delivery and disposal of
cholesterol are regulated by membrane transporters of the
ATP-binding cassette (ABC) superfamily. The human genome contains 48 distinct ABC transporters that are grouped
into 7 subclasses labeled ABCA through ABCG.18 Mutations
in ABC genes cause a variety of diseases, including cystic
fibrosis, Startgardt’s macular degeneration, and disturbances
in lipid and lipoprotein metabolism. All ABC transporters use
ATP to generate the energy needed to transport metabolites
across membranes. Structurally, ABCs fall into 2 groups:
whole transporters having 2 similar structural units joined
covalently and half-transporters of single structural units that
form active heterodimers or homodimers (Figure 3).18
Although cholesterol has been implicated as a direct or
indirect substrate for at least 7 ABC transporters, 4 members
of this family have been shown to have a major impact on
lipoprotein metabolism and cell cholesterol biology: ABCA1,
a whole transporter that mediates export of cellular cholesterol, phospholipids, and other metabolites to lipid-poor HDL
apolipoproteins; ABCG1, a homodimeric half-transporter that
mediates cellular cholesterol export to lipidated lipoprotein
particles; and ABCG5 and ABCG8, which form heterodimers
that restrict intestinal absorption and promote biliary excretion of sterols. These 4 transporters have common modes of
regulation and act in a coordinated fashion to rid tissues and
the plasma compartment of excess cholesterol.
ATP-Binding Cassette A1
Structure and Function
ABCA1 is a 2261 amino acid integral membrane protein that
comprises 2 halves of similar structure.19 Each half has a
ABC Cholesterol Transporters
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Figure 4. Model for ABCA1- and ABCG1-mediated lipid transport from macrophages. Details are described in the text. C
indicates free cholesterol; CE, cholesteryl esters; PL, phospholipids; ACAT, acyl-CoA:cholesterol acyltransferase; NCEH, neutral cholesteryl ester hydrolase.
transmembrane domain containing 6 helices and a nucleotide
binding domain (NBD) containing 2 conserved peptide motifs known as Walker A and Walker B, which are present in
many proteins that use ATP, and a Walker C signature unique
to ABC transporters (Figure 3A).18 ABCA1 is predicted to
have an N terminus oriented into the cytosol and 2 large
extracellular loops that are highly glycosylated and linked by
1 or more cysteine bonds (Figure 3A).19,20
ABCA1 mediates the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes, where they are removed from cells by lipid-poor HDL
apolipoproteins.21 It is likely that ABCA1 forms a channel in
the membrane that promotes “flopping” of lipids from the
inner to outer membrane leaflet by an ATPase-dependent
process.21 Although still controversial, most data support the
idea that ABCA1 simultaneously translocates cholesterol and
phospholipids to the outer membrane leaflet to generate lipid
domains that are accessible for solubilization by apolipoproteins. ABCA1 localizes to the plasma membrane and intracellular compartments,22–24 where it could potentially facilitate transport of lipids to either cell surface– bound or
internalized apolipoproteins.
ABCA1 appears to target specific membrane domains for
lipid secretion. These are likely to be regions that are
sensitive to accumulation of cholesterol. This same source of
cholesterol feeds into intracellular compartments that are the
preferred substrate for the esterifying enzyme acyl-coenzyme
A (acyl-CoA):cholesterol acyltransferase (Figure 4).25,26 Because ABCA1 exports only free cholesterol, preformed cholesteryl esters are hydrolyzed by neutral hydrolases before
they are depleted by this pathway (Figure 4). Thus, ABCA1
removes cholesterol that would otherwise accumulate as
cytosolic cholesteryl ester lipid droplets.
Two models have been proposed to account for the ability
of ABCA1 to target lipid domains.21 The first model suggests
that ABCA1 generates lipid domains in the plasma membrane
that are subsequently removed by apolipoproteins that bind to
cell surface ABCA1.27,28 A second model suggests that
ABCA1- and apolipoprotein-containing vesicles endocytose
to intracellular lipid deposits, where ABCA1 pumps lipids
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November 10, 2006
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into the vesicle lumen for release by exocytosis.29,30 There is
evidence that both mechanisms can operate in the same cell.22
ABCA1 might have functions independent of lipid export
into the HDL pathway. There is evidence that ABCA1
promotes engulfment of apoptotic cells by macrophages31 and
generates microparticles that bleb from plasma membranes,32–34 both processes requiring translocation of phosphatidylserine to the cell surface.35
The ABCA1 pathway has broad specificity for multiple
lipid-poor HDL apolipoproteins, including apoA-I, -A-II, -E,
-C-I, -C-II, -C-III, and -A-IV.36 These apolipoproteins contain 11 to 22 amino acid repeats of amphipathic ␣ helices.37
In this type of helix, the charged amino acids align along 1
face of the long axis, whereas the hydrophobic residues align
along the other face. A synthetic 18 amino acid peptide that
is an analog of class A amphipathic ␣ helices, and its dimer
can mimic apoA-I in removing cholesterol and phospholipids
by the ABCA1 pathway.25,38 – 42 These studies imply that the
amphipathic ␣ helix is the major structural motif required for
removing ABCA1-transported lipids. Interestingly, the
D-isomer of the 18-mer ␣ helix is just as active as the
L-isomer,39,41,42 indicating that there are no stereoselective
requirements for these peptides to interact with ABCA1.
Covalent crosslinking studies revealed that apolipoproteins
bind directly to ABCA1 with saturability and high-affinity
(Kd ⬍10⫺7 mol/L).40,43 These ABCA1 binding sites have a
broad specificity for multiple HDL apolipoproteins, including
apoA-I, -A-II, -C-I, -C-II, and -C-III.40,44 These sites also
recognize the 18-mer synthetic amphipathic ␣ helix.40 Thus,
the broad specificity of the ABCA1 binding sites closely
mirrors the ABCA1-dependent lipid transport activity of the
acceptors. These binding sites have not yet been identified,
but their loose specificity for amphipathic ␣ helices suggests
that they might be lipophilic patches of amino acids.
enzymes that are particularly prevalent in the liver and play a
role in bile acid metabolism. One of these enzymes, sterol
27-hydroxylase (Cyp27), is broadly distributed in various
tissues and cell types, including macrophages, suggesting that
27-hydroxycholesterol is a major LXR ligand in macrophages
and other peripheral cells.51
One study showed a high degree of discordance between
ABCA1 mRNA and protein levels in mouse tissues,52 implying that ABCA1 is highly regulated posttranscriptionally.
Most of this regulation appears to occur at the level of protein
stability. Under basal conditions in cultured cells, ABCA1
protein is highly unstable (half-life of 1 to 2 hours).53–55 The
interaction of apolipoproteins with ABCA1-expressing cells
dramatically reduces the rate of ABCA1 protein degradation
by inhibiting ABCA1 proteolysis by calpain54 and by activating other signaling events.56 This regulation acts as a feedback mechanism to sustain ABCA1 levels when acceptors for
cellular lipids are available.
Metabolites associated with common metabolic disorders,
such as inflammation and diabetes, can destabilize ABCA1 in
cultured macrophages. Unsaturated free fatty acids, which are
elevated in diabetes and the metabolic syndrome, directly
destabilize ABCA1 by a signaling pathway involving activation of phospholipase D2 and phosphorylation of ABCA1
serines.55,57–59 The reactive carbonyls glyoxal and glycoaldehyde acutely and severely impair the ABCA1 pathway,
presumably by directly damaging ABCA1 protein.60 These
carbonyls are generated during glucose metabolism and
protein glycoxidation and form advanced glycated end products (AGEs) on tissue proteins.61– 63 AGEs accumulate with
aging, and their formation is enhanced by diabetes. These and
other metabolic factors could promote accumulation of cholesterol in cells by reducing ABCA1 levels.
ABCA1 In Vivo
Regulation
ABCA1 protein levels and activity are highly regulated by
multiple transcriptional and posttranscriptional processes (reviewed previously21). These regulatory steps are likely to
coordinate the overall activity of ABCA1 in a cell-specific
manner in response to different environmental stimuli. As
discussed below, several of these processes are likely to play
important roles in lipoprotein metabolism and atherogenesis.
As expected for a transporter that mediates secretion of
excess cellular cholesterol, transcription of ABCA1 is markedly induced by overloading cells with cholesterol.45,46 This
occurs exclusively through the nuclear receptors liver X
receptor (LXR␣ and/or LXR␤) and retinoid X receptor
(RXR).47,48 LXR and RXR form obligate heterodimers that
preferentially bind to response elements within the ABCA1
gene promoter and the first intron. LXRs and RXRs bind to
and are activated by oxysterols and retinoic acid, respectively.49 Treatment of cells with either an oxysterol or 9-cisretinoic acid induces ABCA1, but their combined treatment
has synergistic effects.47,48
Excess intracellular cholesterol is converted to a “second
messenger” oxysterol, which regulates ABCA1 and other
cholesterol homeostatic processes through the LXR system.50,51 Most oxysterols are generated by cytochrome P450
ABCA1 is widely expressed throughout animal tissues, where
it may have multiple and diverse functions. In humans and
baboons, ABCA1 mRNA was reported to be most abundant
in liver, placenta, small intestine, and lung.64,65 In mice,
ABCA1 mRNA was reported to be most abundant in liver,
kidney, adrenal, heart, bladder, testis, and brain.52 Because of
extensive posttranscriptional regulation of expression and
function, mRNA levels may not truly reflect the actual
activity of the ABCA1 pathway in vivo.
Based on cell culture studies, macrophage-rich tissues
should have relatively high levels of ABCA1. As scavenger
cells, macrophages ingest modified lipoproteins and damaged
cell membranes and thus can accumulate large amounts of
cholesterol and oxysterols, which are ABCA1 inducers.
ABCA1 is also highly expressed in the liver, where it is
upregulated when mice are fed a high-cholesterol diet.52 It is
therefore expected that hepatic ABCA1 would make an
important contribution to whole-body lipoprotein
metabolism.
Human and animal model studies have confirmed that
ABCA1 is a major determinant of plasma HDL levels and
macrophage cholesterol content. Mutations in human ABCA1
can cause a genetic disorder called Tangier disease,46,66 – 68
which is characterized by very low levels of plasma HDL and
Oram and Vaughan
deposition of sterols in macrophages.69 The low levels of
plasma HDL probably reflect an inability of lipid-poor
apoA-I to acquire lipids, leading to a rapid clearance of
apoA-I from the plasma (Figure 2). Targeted disruption of the
Abca1 gene in mice produces a phenotype similar to that of
Tangier disease,70 –72 and overexpressing ABCA1 in mice
elevates plasma HDL levels.73–76 Tissue-specific expression
or disruption of Abca1 revealed that the hepatic ABCA1
pathway is responsible for generating most of the plasma
HDL in mice (Figure 2),76,77,78 presumably because of the
large flux of cholesterol through the liver when compared
with peripheral tissues.
Genetic Variations
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More than 70 mutations in ABCA1 have been identified from
family studies and population screens for single-nucleotide
polymorphisms (SNPs), nearly a third of which are missense
mutations.79 – 81 Most of the ABCA1 mutations were present
in subjects with low plasma HDL levels, implying that they
play a causative role in lowering HDL levels. Some mutations, however, were more frequent in subjects with the
highest HDL levels, consistent with an enhancement of the
ABCA1 pathway. Although these mutations occur throughout
the gene, they tend to cluster in the extracellular loops, the
NBD domains, and the C-terminal region (Figure 3). SNP
analyses have also identified more than 20 common polymorphisms (⬎1% allelic frequency) in the coding, promoter,
and 5⬘ untranslated regions of ABCA1,79 – 82 many of which
are associated with either low or high plasma HDL levels.
The functional impact of a small subset of the missense
mutations has been studied in cultured cells. When overexpressed in cells, most of these mutants appear in the plasma
membrane but have severely impaired lipid transport and
apolipoprotein binding activities.83,84 ABCA1 proteins with
missense mutations in the first and second extracellular loop
and in the ninth membrane spanning domain have been
reported to impair ABCA1 trafficking to the plasma membrane.85– 88 Only 1 mutant has been described that has near
normal apolipoprotein binding activity but defective lipid
transport.83
ABCA1 and CVD
There is an inverse relationship between plasma HDL levels
and CVD risk,89 implying that HDL protects against atherosclerosis. Although there are multiple mechanisms by which
HDL can be cardioprotective, the relative activity of ABCA1
plays a major role. Because accumulation of sterol-laden
macrophage “foam cells” in the artery wall is an early event
in formation of atherosclerotic lesions, the relative activity of
ABCA1 in arterial macrophages would be expected to have a
major impact on atherogenesis. Studies of human genetic
HDL deficiencies and mouse models support this assumption.
Tangier disease homozygotes and compound heterozygotes who are more than 30 years of age have a 6-fold higher
than normal incidence of CVD.90 The low levels of LDL in
these subjects may partially protect them from atherogenesis.
Studies of heterozygotes, who tend to have more normal
levels of LDL, showed a significant inverse correlation
between ABCA1 activity in their cultured skin fibroblasts and
ABC Cholesterol Transporters
1035
the prevalence and severity of CVD.91 In general, premature
CVD is associated with ABCA1 mutations that impair function.81 Some polymorphisms in ABCA1 are associated with
either increased or decreased CVD.81 Interestingly, many of
these variants show an altered severity of atherosclerosis
without changes in plasma lipid levels.
Mouse studies have also provided support for the cardioprotective effects of macrophage ABCA1. Some but not all
reports showed that overexpression of human ABCA1 in
mice significantly reduced diet-induced atherosclerosis in
association with a favorable change in lipoprotein profile.73,75
Reciprocal bone marrow transplantation studies using wildtype and Abca1-null mice have shown that selectively overor underexpressing ABCA1 in macrophages decreases or
increases atherosclerosis, respectively.92–94 In contrast, selective overexpression of ABCA1 in the liver of mice lacking
LDL receptors increases the plasma levels of more atherogenic apoB-containing particles and enhances atherosclerosis.95 Taken together, these studies clearly show that macrophage ABCA1 is a cardioprotective factor. Hepatic ABCA1
may override some of these protective effects by increasing
atherogenic apoB-containing lipoproteins.
ATP-Binding Cassette G1
Structure and Function
ABCG1 belongs to the ABCG family of reverse halftransporters (Figure 3). Because an active transporter needs 2
nucleotide-binding domains and 2 transmembrane bundles,
members of the ABCG family have to form either hetero- or
homodimers to gain function. ABCG1 is believed to act as a
homodimer in macrophages.96 –99 Although earlier studies
suggested a shorter gene, it now appears that the full-length
human gene has 23 exons spanning 98 kb of genome.100
Multiple potential transcripts of mouse and human ABCG1
have been identified that use alternate exons or promoters.101
These are predicted to encode proteins that differ in their
N-terminal end. However, there appears to be only one active
transcript in mice and humans.
As with ABCA1, ABCG1 is highly expressed in cholesterol-loaded macrophages,97,101,102 consistent with it playing a
role in ridding these cells of excess cholesterol. ABCG1 is
expressed both on the cell surface and within intracellular
compartments.98,103 ABCG1 promotes cholesterol efflux from
cells to HDL and other lipoprotein particles but not to
lipid-free apoA-I.96 –98,103,104 Both ABCA1 and ABCG1 appear to have the intrinsic ability to translocate cholesterol to
cell surface domains accessible to extracellular molecules,28,98 suggesting that they directly or indirectly operate as
cholesterol floppases. In contrast to ABCA1-dependent lipid
export, which requires apolipoprotein binding, ABCG1dependent cholesterol efflux does not appear to require the
direct interaction of lipoproteins.99 It is likely that ABCG1
forms cell surface lipid domains that allow cholesterol to
desorb into the surrounding fluid and be picked up by
lipoproteins (Figure 4).
In contrast to ABCA1, which transports phospholipids and
other lipophilic compounds as well as cholesterol, ABCG1 is
largely a cholesterol transporter.98,99 This is partly because
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November 10, 2006
the acceptors that remove ABCG1-transported lipids contain
phospholipids, which can readily incorporate cholesterol. It
has been reported, however, that ABCG1 promotes some
efflux of phospholipids, particularly sphingomyelin.105
ABCA1 and ABCG1 can act synergistically to remove
cholesterol from cells (Figure 4).104,106 ABCA1 converts
lipid-poor apoA-I to partially lipidated “nascent” lipoproteins
that are effective acceptors for cholesterol exported by
ABCG1. This is probably because ABCA1 transports phospholipids to apoA-I. These findings raise the interesting
possibility that ABCA1 and ABCG1 coordinate the removal
of excess cholesterol from macrophages by using diverse
types of lipid acceptor particles (Figure 4).
Regulation
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Additional evidence that ABCA1 and ABCG1 cooperate to
export cholesterol from cells is that they are both regulated by
the LXR/RXR nuclear receptor system.100,101,107,108 Thus the
conversion of cholesterol to the oxysterol ligands for LXRs
drives expression of both ABCA1 and ABCG1 in cholesterol-loaded cells. In macrophages, LXR agonists stimulate the
movement of ABCG1 from intracellular compartments to the
plasma membrane, which enhances the subsequent efflux of
cholesterol to HDL.103 Thus, the LXR system performs the
dual function of increasing ABCG1 expression and its translocation to the plasma membrane.
In Vivo Activity
ABCG1 was shown to be highly expressed in mouse and
human brain, thymus, lung, adrenals, and spleen.109,110
ABCG1 is expressed in both the gray and white matter in
mouse brain.111 In the cerebellar astroglia, which are macrophage-like cells, expression of ABCG1 rather than ABCA1 is
correlated with cholesterol release,112 suggesting that ABCG1
plays a greater role than ABCA1 in exporting lipids from
these cells in the brain. ABCG1 is also expressed in the
intestine, suggesting that it may play a role in transport of
dietary lipids. In CaCo-2 human intestinal cells, induction of
ABCG1 by LXR agonists increased HDL-mediated cholesterol efflux from the basolateral membrane,113 consistent with
the possibility that ABCG1 is involved in formation and
maturation of intestinal HDL.
Studies of ABCG1⫺/⫺ mice have demonstrated the importance of this transporter in macrophage and liver lipid
transport. The targeted disruption of Abcg1 in mice had no
effect on plasma lipid levels but did result in massive
accumulation of both neutral lipids and phospholipids in
hepatocytes and in macrophages within multiple tissues
following administration of a high-fat and -cholesterol diet.97
In contrast, overexpression of human ABCG1 protected
tissues from dietary fat–induced lipid accumulation.97 The
results imply that ABCG1 is essential for cholesterol homeostasis in macrophages and is needed to remove excess
cholesterol from these cells. The elevated triglycerides in
cells from ABCG⫺/⫺ mice also suggest that ABCG1 may
also play a role in modulating metabolism of this class of
lipids in vivo.
The accumulation of lipids in the liver of ABCG1⫺/⫺ mice
raises the possibility that this transporter is involved in
hepatic lipid metabolism or transport. ABCG1 expression is
relatively low in the liver,97 especially when compared with
ABCA1 expression. The lack of change in plasma lipoproteins with ablation or overexpression of ABCG1 implies that
this transporter has little effect on hepatic lipoprotein production or clearance. It is possible, however, that ABCG1 plays
some role in modulating intrahepatic lipid trafficking or
biliary secretion of cholesterol.114
Role in CVD
To date, no genetic variations in human ABCG1 have been
identified that link it to any inheritable disease. The search for
functionally significant rare mutations and polymorphism in
this gene may be complicated by the likelihood that ABCG1
contributes little to plasma lipid or lipoprotein levels, thus
limiting large-scale screening based on abnormal plasma
lipids. If dysfunctional ABCG1 causes CVD in humans, it is
likely to be in a patient population with relatively normal
levels of lipoprotein cholesterol.
The macrophage cholesterol export activity of ABCG1
predicts that this transporter should be cardioprotective.
Surprisingly, 3 studies showed that transplantation of bone
marrow from ABCG1⫺/⫺ mice into atherogenic mouse models caused only a moderate increase or actually decreased
atherosclerotic lesions.115–117 The decreased atherosclerosis
was associated with increased macrophage apoptosis and
enhanced expression of both apoE and ABCA1. Thus, the
potential harmful effects of impaired ABCG1 may be overridden by the beneficial effects on clearing apoptotic cells and
increasing other compensatory cholesterol efflux pathways. It
remains to be determined whether ABCG1 is cardioprotective
in humans.
ABCG5 and ABCG8
Structure and Function
ABCG5 and ABCG8 are half-transporters that form heterodimers to become functional (Figure 3).118 –120 ABCG5 and
ABCG8 each contain 13 exons and are arranged in a headto-head configuration on chromosome 2p21, with only 140
bases separating them.121
In cultured hepatocytes, coexpression of the transporters
caused a translocation of the functional heterodimer from the
endoplasmic reticulum to the canalicular membrane of the
apical surface of the cell.118 –120 Thus, ABCG5 and ABCG8
form an obligate heterodimer that traffics to the surface of the
cell membrane, where it presumably performs its sterol
export function. It has not yet been shown, however, that
ABCG5 and ABCG8 actually promote sterol export from
cultured cells.
Studies of human disease and mouse models have shown
that ABCG5 and ABCG8 regulate the whole-body retention
of plant sterols and biliary secretion of cholesterol (Figure
2).122–128 There is evidence that these transporters promote
flopping of sterols from the inner to outer leaflets of the
plasma membrane.129 Thus, ABCG5 and ABCG8 heterodimers probably function in a similar manner as other
ABC cholesterol exporters.
The observation that patients with mutations in either
ABCG5 or ABCG8 accumulate plant and shellfish sterols in
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their plasma is consistent with the idea that these transporters
are selective for noncholesterol sterols. Mouse model studies,
however, have shown that these transporters also promote
hepatobiliary secretion of cholesterol.130 The major plant
sterols are sitosterol, campesterol, stigmasterol, and avenasterol. ABCG5/8 also transports plant stanols, such as campestanol and sitostanol.131,132 Plant sterols differ from cholesterol
by the addition of methyl or ethyl groups or a double bond in
the carbon-24 acyl side chain. Stanols differ from cholesterol
by the saturation of the ⌬5 double bond. It is unknown
whether ABCG5/8 also transports phospholipids. Because it
pumps sterol into the intestinal lumen, the presumed acceptors of ABCG5/8-transported sterols are complexes of bile
acids and phospholipids.
Regulation
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ABCG5 and ABCG8 belong to the family of ABC transporters that are induced by the LXR/RXR nuclear receptors.133
Both cholesterol feeding and the addition of a LXR agonist to
the mouse diet increased ABCG5 and ABCG8 levels in the
liver and intestine.128,134 Activation of LXRs was associated
with an increased biliary cholesterol secretion, decreased
fractional cholesterol absorption, and increased fecal neutral
sterol excretion.135
In Vivo Activity
Studies of human genetic disorders and mouse models have
shown that intestinal ABCG5/8 functions to limit absorption
of dietary plant sterols. The daily average Western diet
contains 250 to 500 mg of cholesterol and 200 to 400 mg of
noncholesterol sterols, the majority of which are plant sterols.
Typically, 50% to 60% of this cholesterol is absorbed in the
intestine, which is in stark contrast to only 1% to 5% of the
plant sterols.131,136,137 A major entry point for the absorption
of dietary sterols is through the Niemann–Pick C1 like 1
(NPC1L1) protein, which is expressed on the brush border
membrane of enterocytes.138 It is believed that a small
fraction of plant sterols enter the circulation because those
taken up by NPC1L1 are secreted back into the lumen by
enterocyte ABCG5/8 (Figure 2).
These human and mouse studies have shown that
ABCG5/8 is also expressed in hepatocytes, where it mediates
the biliary secretion of sterols (Figure 2). This secretion
appears to require another ABC transporter, MDR2
(ABCB4),130 which mediates biliary secretion of phospholipids and, secondarily, cholesterol.139 These studies also suggested that the decreased intestinal absorption of plant sterols
in mice overexpressing ABCG5 and ABCG8 is primarily
attributable to the increased biliary secretion of cholesterol,
which could compete for the uptake of noncholesterol sterols
by enterocytes.130 This implies that the ability of enterocytes
to secrete sterols through ABCG5/8 plays a minor role in
decreasing fractional absorption of plant sterols.
In humans, the hepatic ABCG5/8 system may play the
biggest role in controlling dietary sterol absorption. This idea
is supported by a study showing that a liver transplant in a
patient with genetically nonfunctional ABCG5/8 normalized
plant sterol levels despite the fact that intestinal ABCG5/8
was still impaired.140 It is possible that the enterocyte
ABC Cholesterol Transporters
1037
ABCG5/8 pathway is the initial defense against an acute
supply of dietary plant sterols but that the hepatic pathway
has a more sustained effect.
Sitosterolemia
Mutations in either ABCG5 or ABCG8 can cause a rare
genetic disorder called sitosterolemia.127,128 The disease is
characterized by markedly elevated plasma levels of plant
sterols and modest increases in plasma cholesterol, which is
attributable to the hyperabsorption of both cholesterol and
plant sterols from the intestine and a low level of excretion
into the bile.131,132 Because of these abnormal sterol levels,
patients with sitosterolemia develop tuberous xanthomas and
premature CVD.131,141 Ezetimibe, a drug that prevents
NPC1L1 from absorbing dietary cholesterol, prevents the
accumulation of plant sterols in patients with sitosterolemia142 and in mice lacking ABCG5 and ABCG8,143 demonstrating the importance of NPC1L1 in both cholesterol and
plant sterol uptake from the intestinal lumen.
Multiple mutations in ABCG5 and ABCG8 have been
identified that cause sitosterolemia.144 SNP analyses have
also uncovered multiple polymorphisms, most of which are in
ABCG8. Many of these polymorphisms are in highly conserved regions, suggesting that they have functional impacts.
A lack of an in vitro lipid export assay has limited the study
of the functional effects of these genetic variations. All of the
missense mutations in either ABCG5 or ABCG8 studied to
date either prevent formation of the obligate heterodimer or
block the efficient trafficking of the heterodimer to the
plasma membrane.120
Role in CVD
Sitosterolemic patients are highly sensitive to dietary cholesterol and become markedly hypercholesterolemic when fed a
high-cholesterol diet.131,145–147 The high plasma cholesterol
levels are associated with severe premature atherosclerosis.141,146,148 The elevated plant sterols may also contribute to
CVD by disrupting normal cholesterol homeostasis. These
observations suggest that interventions to enhance the
ABCG5/8 pathway could protect against CVD in hypercholesterolemic subjects by eliminating more cholesterol in the
bile and reducing plasma cholesterol levels. This concept was
supported by a study showing that overexpressing ABCG5
and ABCG8 in an atherogenic mouse model attenuates
diet-induced atherosclerosis in association with reduced liver
and plasma cholesterol levels.122 Therefore, the role of
ABCG5 and ABCG8 in CVD relates to their abilities to
modulate plasma cholesterol levels.
ABC Cholesterol Transporters and
Atherogenic Complications of Diabetes
The prevalence of diabetes has risen dramatically in the last
20 years worldwide. Patients with a constellation of CVD risk
factors called the metabolic syndrome are predisposed to both
diabetes and CVD.149 As defined by the Adult Treatment
Panel III,149 the criteria for the metabolic syndrome are 3 of
the following risk factors: obesity, hypertriglyceridemia, low
HDL levels, hypertension, and hyperglycemia.
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CVD is the major cause of morbidity and mortality in both
types 1 and 2 diabetes.150 –153 There is a large body of
evidence suggesting that both the hyperglycemia and dyslipidemia accompanying diabetes and the metabolic syndrome
contribute to this enhanced atherogenesis.153–155 Low plasma
HDL is among the CVD risk factors associated with type 2
diabetes and the metabolic syndrome.151,154 –157 Moreover, the
HDL particle distribution is abnormal in both types 1 and 2
diabetes, with decreases in the relative fraction of the large
HDL particles believed to be cardioprotective.151,158 These
results raise the possibility that abnormal HDL metabolism
contributes to the increased CVD caused by diabetes and the
metabolic syndrome.
There is emerging evidence that impaired ABC cholesterol
transporters contribute to the dyslipidemia and enhanced
CVD in these metabolic disorders. Factors that destabilize
ABCA1, such as reactive carbonyls and free fatty acids, are
elevated in diabetes.60,62,159 –162 Hyperglycemia was shown to
reduce ABCA1 expression in leukocytes,163 and unsaturated
fatty acids were shown to suppress ABCA1 and ABCG1
transcription by antagonizing the effects of sterols on LXR
activation.164,165 Peritoneal macrophages isolated from type 2
diabetic mice were reported to have above normal levels of
cholesteryl esters in association with suppressed expression
of ABCG1.166 Transcription of this transporter was severely
reduced by chronic exposure of cultured mouse macrophages
to high-glucose levels.166 In rats, inducing type 1 diabetes led
to a decreased expression of hepatic and intestinal ABCA5
and ABCG8 associated with altered sterol fluxes.167 Humans
with type 2 diabetes were shown to have higher NPC1L1 and
lower ABCG5 and ABCG8 mRNA levels than nondiabetic
subjects.168
The metabolic syndrome and diabetes are associated with
increased chronic inflammation. Several studies have shown
that the inflammatory enzyme myeloperoxidase chlorinates
and oxidizes amino acids in apoA-I, which severely reduce its
ability to interact with ABCA1.169,170 Thus, diabetes and
inflammation can impair these ABC pathways by reducing
the cell content of transporters and damaging the apoA-I
needed to remove cellular cholesterol and generate HDL
particles. Taken together, these studies implicate the disruption of ABC transport pathways as playing a role in the
abnormal HDL and increased CVD associated with diabetes,
the metabolic syndrome, and other inflammatory disorders.
Additional studies with animal models are needed to confirm
that these processes are occurring in vivo.
ABC Cholesterol Transporters As
Therapeutic Targets
The ability of ABCA1, ABCG1, ABCG5, and ABCG8 to
coordinate the depletion of excess tissue cholesterol and its
elimination from the body has made these transporters important new therapeutic targets for treating CVD. The assumption is that increased ABCA1 and ABCG1 activities in
arterial macrophages would prevent foam-cell atherosclerotic
lesion formation, that increased liver ABCA1 activity would
elevate plasma HDL levels and thus enhance the diverse
cardioprotective functions of this lipoprotein, and that increased ABCG5 and ABCG8 in the liver and intestines would
decrease dietary sterol absorption and increase hepatobiliary
cholesterol secretion. The possibility of producing these
beneficial changes in cholesterol homeostasis has launched
multiple programs in the pharmaceutical industry designed to
produce agents that enhance these cholesterol transporters.
Most of these programs to date have focused on developing
agonists to increase transcription of these transporters. This
approach is made feasible by the fact that each ABC is
induced robustly by the same nuclear receptor ligands, thus
providing an opportunity to simultaneously increase the
expression levels all 4 of them. The first generation drugs
shown to be effective for this endpoint have been LXR
agonists. An additional advantage of these agonists is that
they induce other macrophage proteins that help coordinate
cholesterol export, including apolipoproteins that can act as
cholesterol acceptors171 and enzymes and transfer proteins
that can remodel extracellular HDL particles to regenerate
lipid-free apolipoproteins.17 Two nonsteroidal synthetic LXR
agonists (TO901317 and GW3965) have been shown to
increase plasma HDL levels and reduce atherosclerosis in
mouse models.172–174
The problem with these LXR agonists is that they also
increase hepatic fatty acid synthesis and esterification and
thus generate fatty livers and hypertriglyceridemia when
administered to animals.175–177 LXR agonists induce fatty
acid synthase and a transcriptional factor named sterol regulatory binding element protein-1c (SREBP-1c) that activates
genes for several enzymes in the fatty acid biosynthetic
pathway.11,176 Moreover, SREBP-1c induces stearoyl-CoA
desaturase, which converts saturated fatty acids to unsaturated fatty acids that destabilize ABCA1 protein.58,178
Several studies have provided proof-in-principle that LXR
agonists can be developed that are more selective for the
cholesterol transport pathway than for lipogenesis. It has been
reported that GW3965 can be administered to mice at a
concentration that elevates plasma HDL levels without causing hypertriglyceridemia,173 although this agonist activates
SREBP-1c at high concentrations in vitro.179 A synthetic
oxysterol ligand for the LXR receptor was shown to stimulate
ABCA1 synthesis with only a minor increase in hepatic
SREBP-1c.179 A plant sterol derivative was shown to be a
potent LXR agonist that selectively activates intestinal ABC
transporters with only limited induction of lipogenic enzymes.180 A major challenge is to produce marketable LXR
agonists that selectively induce cholesterol transporters.
The possibility of targeting posttranscriptional regulation
of these ABCs has received less attention. It is clear from
studies of ABCA1 that this is a highly unstable protein that
may be regulated in vivo by metabolic factors associated with
common disorders, such as diabetes and inflammation. Under
these conditions, stimulating transcription may have only a
limited ability to enhance the overall activity of this cholesterol export pathway. It is also possible that transcription of
ABCA1 and ABCG1 in highly cholesterol-loaded cells may
already be maximally stimulated. Additional studies with
animal models are needed to assess the relative importance of
transcriptional and posttranscriptional regulation of these
ABCs under various atherogenic conditions.
Oram and Vaughan
ABC Cholesterol Transporters
1039
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Conclusions
References
Studies of human disorders, mouse models, and cultured cells
have shown that four ABC cholesterol transporters have a
major impact on whole-body cholesterol homeostasis and
CVD. ABCA1 and ABCG1 work independently and synergistically to remove excess cholesterol from cells, particularly
macrophages. Hepatic ABCA1 is largely responsible for
generating cardioprotective HDL particles. ABCG5 and
ABCG8 form heterodimers that limit sterol absorption by the
intestine and promote hepatobiliary secretion of circulating
cholesterol. Transcription of these 4 transporters is regulated
by the same LXR/RXR nuclear receptor system, which is
induced robustly by intracellular sterols and nonsterol
agonists.
These beneficial effects on cholesterol homeostasis have
made these transporters important new therapeutic targets for
preventing and reversing atherosclerotic CVD. Firstgeneration protocols have focused on developing LXR agonists that stimulate transcription of ABCA1 and other genes
involved in cholesterol transport. Administering LXR agonists to atherogenic mouse models markedly reduces their
atherosclerotic lesions. The potential benefits of these agents
have been overshadowed by their broad specificity for lipogenic genes. Several studies, however, have offered proof-inprinciple that drugs selective for cholesterol transport genes
can be developed in the near future.
The most common atherogenic disorders in humans appear
to damage ABC transporters or suppress their expression.
Metabolites that are elevated in both diabetes and in the
metabolic syndrome reduce expression of ABC transporters
by several mechanisms that might contribute to the increased
CVD common to these disorders. These patients could be
relatively resistant to interventions that use transcriptional
regulation to elevate their ABC cholesterol transporters.
Thus, with many individuals, it may be necessary to direct
therapeutic approaches at the underlying mechanisms that
impair ABC transporters or at the actual steps in their cellular
pathways that are affected.
In summary, we have gained a great deal of knowledge
about the biology and pathobiology of the ABC cholesterol
transporters ABCA1, ABCG1, ABCG5, and ABCG8. These
transporters, however, have complex cellular and regulatory
pathways that are far from being completely understood.
Additional studies are needed to identify the molecular
players in these pathways, to determine the array of substrates
targeted for transport, and to characterize the diverse processes that regulate their expression and activity. These
studies will not only provide insight into the role of ABC
cholesterol transporters in health and disease, but will uncover novel therapeutic targets for treating these diseases.
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Sources of Funding
J.F.O. and A.M.V. were supported by NIH grants for work described
in this review (HL18645, HL075340, HL55362, and DK02456).
Disclosures
None.
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ATP-Binding Cassette Cholesterol Transporters and Cardiovascular Disease
John F. Oram and Ashley M. Vaughan
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Circ Res. 2006;99:1031-1043
doi: 10.1161/01.RES.0000250171.54048.5c
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