J Mol Med (2005) 83: 329–342

DOI 10.1007/s00109-004-0631-3

REVIEW

Kirsty Greenow . Nigel J. Pearce . Dipak P. Ramji

The key role of apolipoprotein E in atherosclerosis

Received: 20 July 2004 / Accepted: 8 November 2004 / Published online: 13 April 2005
# Springer-Verlag 2005

Abstract Apolipoprotein E is a multifunctional protein
that is synthesized by the liver and several peripheral tissues
and cell types, including macrophages. The protein is in-
volved in the efficient hepatic uptake of lipoprotein par-
ticles, stimulation of cholesterol efflux from macrophage
foam cells in the atherosclerotic lesion, and the regulation
of immune and inflammatory responses. Apolipoprotein E
deficiency in mice leads to the development of atheroscle-
rosis and re-expression of the protein reduces the extent of
the disease. This review presents evidence for the potent
anti-atherogenic action of apolipoprotein E and describes
our current understanding of its multiple functions and regu-
lation by factors implicated in the pathogenesis of cardio-
vascular disease.

Keywords Atherosclerosis . Lipid metabolism .

Lipoproteins . Macrophages . Reverse cholesterol
transport

Abbreviations ABCA1: ATP-binding cassette transporter

KIRSTY GREENOW
received her Ph.D. degree from
Cardiff School of Biosciences,
Cardiff University, Wales, UK.
She is currently carrying out
post-doctoral research at the
School of Medicine at Cardiff
University.
K. Greenow . D. P. Ramji (*)
Cardiff School of Biosciences,
Cardiff University, Museum Avenue,
P.O. Box 911 Cardiff, CF10 3US, UK
e-mail: ramji@cardiff.ac.uk
Tel.: +44-29-20876753
Fax: +44-29-20876753
N. J. Pearce
GlaxoSmithKline,
Gunnels Wood Road,
Stevenage, SG1 2NY, UK
DIPAK RAMJI
received his Ph.D. degree from
the School of Biochemistry and
Molecular Biology, University
of Leeds, UK. He is currently a
Senior Lecturer at Cardiff
School of Biosciences, Cardiff
University, Wales, UK. His
laboratory’s interests focus on
understanding the mechanisms
involved in the regulation of
gene expression during inflam-
mation and cardiovascular
disease.
A1 . AD: Alzheimer’s disease . apo: Apolipoprotein .
apoER2: ApoE receptor 2 . HCR: Hepatic control regions .
HDL: High density lipoproteins . HSPG: Heparan-sulphate
proteoglycans . LDL: Low-density lipoproteins . LDL-R:
LDL receptor . LRP: LDL receptor-related protein . ME:
Multi-enhancer . SMC: Smooth muscle cells . VLDL: Very
low density lipoproteins
Introduction
Atherosclerosis, the underlying cause of heart attacks and
stroke, is responsible for about 50% of deaths in Western
countries. The risk factors for this multifactorial disease
include genetic predisposition, age, gender, smoking, hy-
pertension, stress, dietary habits and physical inactivity [1].
Atherosclerosis is often considered to be a form of chronic
inflammation that is triggered by injury to the endothelial
lining of the arterial wall by many such risk factors, which
then cause changes in the permeability of the arterial wall,
increased expression of several cell surface adhesion
molecules, and production of cytokines [1]. These changes
in endothelial function lead to an increased migration of
monocytes and T-lymphocytes from the circulation into the
330
intima of the arterial wall [1]. Once monocytes enter the
intima, they differentiate into macrophages and begin to
accumulate cell debris and oxidatively modified lipopro-
teins, resulting in the development of lipid-laden foam cells
[1]. This early vascular lesion, called a fatty streak, prog-
resses further with the migration and proliferation of smooth
muscle cells (SMC) from the media into the intima. The
most advanced and unstable lesions within the intima con-
sist of a lipid-rich necrotic core surrounded by an extracel-
lular matrix and covered by a fibrous cap containing smooth
muscle cells [1]. The major clinical complications, such as
myocardial infarction and stroke, arise due to plaque rup-
ture and thrombosis.
A major focus of recent research on cardiovascular dis-
ease has been to understand the molecular basis of ath-
erosclerosis in detail and has resulted in the identification of
a key, but complex, role for apolipoprotein (apo) E in the
process. ApoE was first described by Shore and Shore [2] as
a lipoprotein constituent of triglyceride-rich very low den-
sity lipoproteins (VLDL). It has since been found to be a
major component of several classes of plasma lipoproteins
and has been implicated in the maintenance of overall plas-
ma cholesterol homeostasis by facilitating the hepatic uptake
of lipoproteins by binding to their receptors. The protein
also carries out additional functions such as stimulation of
cholesterol efflux from macrophages, prevention of platelet
aggregation, and inhibition of proliferation of T-lympho-
cytes and endothelial cells. ApoE therefore plays a key pro-
tective role in atherosclerosis. A number of polymorphisms
in the apoE gene have been identified, and the functional
consequences of these changes along with their association
with cardiovascular disease have been investigated. This
review describes the molecular and cell biology of apoE, the
polymorphisms that have been identified and their con-
sequences, the multiple functions of the protein, and evi-
dence supporting its key role in atherosclerosis. In addition,
the regulation of apoE expression by factors implicated in
atherosclerosis is addressed.
Molecular and cell biology of apoE
Gene organization, synthesis and expression profile
The human apoE gene has been mapped to chromosome 19,
where it is located at the 5′ end of a 50-kb gene cluster with
apoCI, an apo-CI pseudogene, apoCII and apoCIV [3]. The
apoE gene contains four exons separated by three introns
and specifies for a secretory protein that follows the classi-
cal pathway for secretory protein synthesis and release [4].
The primary translation product is 317 amino acids long and
contains an 18 amino acid extension at the amino-terminus,
which acts as a signal peptide that directs the nascent poly-
peptide chain to the endoplasmic reticulum [4]. The protein
is then transported to the Golgi, where it undergoes O-
linked glycosylation with the addition of carbohydrate
chains containing sialic acid, prior to secretion [5]. The
secretion of apoE is, however, a complex process. For ex-
ample, a significant proportion of newly synthesized apoE
is degraded prior to secretion [6, 7]. In addition, newly
secreted apoE may remain tightly bound to proteoglycans
on the cell surface, and this source of the protein can either
serve as a precursor for the secreted protein or be internal-
ized and then degraded or recycled back to the cell surface/
extracellular medium [8–12]. Furthermore, the secretion of
apoE can be regulated by a number of factors such as
cholesterol loading of the cells [7, 13, 14] (see below).
ApoE is produced by various tissues including the liver,
brain, spleen, lung, ovary, adrenal gland, kidney and mus-
cles [15]. In general, most of the apoE found in the plasma is
synthesized by the liver with about 20–40% derived from
extra-hepatic sources, such as macrophages [15, 16]. The
expression of apoE is also known to be modulated during
physiological and pathophysiological changes by a number
of factors at the transcriptional and post-transcriptional
levels (see below).
Structure of apoE
ApoE contains two independently folded domains that are
linked by a protease-sensitive loop [17]. The amino-ter-
minal domain (residues 1–191) has an ordered structure
consisting of four amphipathic α helix bundle (residues 24–
42, 54–81, 87–122 and 130–164, respectively) arranged in
an anti-parallel fashion with the hydrophobic faces orien-
tated towards the interior of the bundle [18]. A short addi-
tional helix (residues 44–53) connects helices 1 and 2. This
amino-terminal region contains the recognition sites for low-
density lipoprotein (LDL) receptor (LDL-R) and LDL-R
related protein (LRP), with an arginine- and lysine-rich seg-
ment of helix 4 (residues 136–150) being essential for the
interaction with the acidic residues of the receptors [17].
The importance of these lysine or arginine residues for re-
ceptor binding activity has been demonstrated by muta-
genesis and chemical modification [17]. A region spanning
residues 171–183 also contains essential elements for sta-
bilization or alignment of the receptor binding domain [19].
Association with lipids is essential for apoE to recognize
the LDL-R [17]. It has been proposed that upon binding to
lipids the amino-terminal helix bundle opens, thereby in-
creasing exposure of lysines 143 and 146 to the aqueous
phase and triggering helix formation by amino acids 165–
169 [20, 21]. In this confirmation the positive electrostatic
potential in the receptor-binding region of apoE is enhanced,
thus allowing its high affinity binding to the LDL-R [22].
The segment containing helix 4 also has a high-affinity
binding site for heparan-sulphate proteoglycans (HSPG)
with the lysine- and arginine-rich residues interacting with
the negatively charged carboxylate and sulfate groups of
proteoglycans [23, 24]. More recently the amino-terminal
region of apoE has been shown to contain the binding site
for scavenger receptor class B type and, as with LDL-R,
lipidation enhances the interaction [25, 26].
The carboxyl-terminal region (residues 216–299) con-
tains high-affinity lipid-binding sites and is also responsible
for the self-association of apoE in the absence of lipids [17].
The structure of the carboxyl-terminus is currently poorly
 331

understood but is believed to adopt an amphipathic α-helical gene frequencies in different populations see [48]). A
conformation, with a segment that coincides with the puta- second, non-genetically determined form of apoE polymor-
tive lipoprotein binding site having a high propensity to phism arises due to differential glycosylation [28]. Indeed,
form a coiled-coil [27]. Residues 243–272 are also thought several glycosylated isoforms of apoE exist mainly because
to bear a heparin-binding site [23]. of the different extent of sialization on the carbohydrate
chain added to threonine 194 [49]. Although sialated iso-
forms account for about 10–20% of plasma apoE, the func-
ApoE polymorphisms tional relevance of this modification remains currently
unclear. Whilst most apoE is secreted from hepatic cells in
Nature of polymorphisms the glycosylated form, glycolysation is not essential for the
synthesis or the secretion of the protein [50].
ApoE is a polymorphic protein with three common iso-
forms and more than 20 rare variants (Table 1) [28]. The
three most common polymorphisms are E2, E3 and E4, Functional consequences of genetically determined
which are the products of three alleles (ε2, ε3 and ε4) at a apoE polymorphisms
single gene locus [28]. ApoE3 (Cys112, Arg158) is consid-
ered to be the parent form and apoE4 (Arg112, Arg158) and Although the three common apoE isoforms differ from one
apoE2 (Cys112, Cys158) are variants [28]. Overall, six phe- another by only a single amino acid substitution, the changes
notypes are possible with their ranking from most to least have profound effects at the structural and functional level
common being E3/3, E4/3, E3/2, E4/4, E4/2 and E2/2 (for [28]. For example, the three isoforms exhibit different ther-
mal and chemical stabilities (apoE4<apoE3<apoE2) [51,
52]. In addition, the binding affinity of apoE2 for LDL-R is
Table 1 Rare variants of apoE
less than 2% of the affinity of apoE3 and apoE4 [53]. Such
Variant References reduced binding capability of apoE2 is because of the ab-
sence of a positive charge at position 158, which drastically
ApoE1 (Gly127→Asp, Arg158→Cys) 29 changes salt bridges within helix 4 and between helices 3
ApoE1Harrisburg (LyS146→Glu) 28 and 4 that are required to maintain the receptor binding
ApoE1Hammersmith (LyS146→Asn, Arg147→Trp) 30 region of the protein in a conformation that can interact ef-
ApoE1Baden (Arg180→Cys) 31 fectively with the receptor and, additionally, lowers the pos-
ApoE1 (Leu252→Glu) 32 itive ion potential of the receptor binding domain [53, 54].
ApoE2 (Arg136→Cys) 33 The binding properties of these apoE variants to lipoproteins
ApoE2Christchurch (Arg136→Ser) 34 also differ, with apoE3 and apoE2 preferentially binding to
ApoE2Heidelberg (Arg136→Cys) 35 high-density lipoprotein (HDL) and apoE4 to VLDL. Do-
ApoE2 (Arg142→Leu) 36 main interaction between the amino- and the carboxyl-ter-
ApoE2 (Arg142→Cys) 28 minal regions of apoE is responsible for such a difference
ApoE2 (Arg145→Cys) 28 [55, 56]. The substitution of cysteine 112 by arginine in
ApoE2Sendai (Arg145→Pro) 37 apoE4 causes a reorientation in the side chain of arginine
ApoE2 (Lys146→GIn) 28 61, allowing it to interact with glutamate 255 within the
ApoE2Dunedin (Arg228→Cys) 28 critical lipoprotein-binding region of the carboxyl terminus,
ApoE2 (Val236→Glu) 32 thereby leading to an alteration in conformation [55]. In
ApoE3 (Ala99→Thr, Ala152→Pro) 28 addition, the carboxyl-terminal domain becomes less or-
ApoE3-Leiden (Cys112→Arg; residues 121–127a) 28 ganized, which results in a significantly higher affinity for
ApoE3 (Cys112→Arg, Arg142→Cys) 28 lipids and therefore the preferential association with VLDL
ApoE3 (Cys112→Arg, Arg142→Gly) 29 [56].
ApoE3 (Arg136→Ser) 38 A number of studies investigating the role of apoE on the
ApoE3 (Arg145→His) 39 normal variation of its own expression and that of plasma
ApoE3 (Arg251→Gly) 32 lipids have also revealed isoform-specific differences. Thus,
ApoE3′(Arg136→His) 40 compared to apoE3, apoE2 tends to be associated with de-
ApoE4Philadelphia (Glu13→Lys, Arg145→Cys) 41
creased levels of apoB and cholesterol and increased levels
ApoE4Freiburg (Leu28→Pro) 42
of apoE and triglycerides [57]. On the other hand, apoE4 is
ApoE5 (Glu3→Lys) 43
associated with increased apoB and cholesterol levels and
decreased apoE levels [57]. The mechanisms that are re-
ApoE5 (Glu13→Lys) 44
sponsible for such changes are currently poorly understood
ApoE5 (Pro84→Arg, Cys112→Arg) 43
except for the variation in cholesterol levels, where an ac-
ApoE5 (GIn204→Lys, Cys112→Arg) 45
tion on hepatic LDL-R levels is likely to make a major
ApoE5 (Glu212→Lys) 46
contribution [57]. ApoE4 preferentially distributes to triglyc-
ApoE7 Suita (Glu244, 245→Lys) 47
eride-rich lipoproteins and accelerates its uptake, thereby
a
Seven amino acid insertionthat is a tandem repeat of residues leading to down-regulation of hepatic LDL-R expression
121–127 and increased levels of LDL [57–59]. On the other hand, the
332
lower affinity of apoE2 for LDL-R would decrease cellular
cholesterol influx and lead to lower intracellular levels of
the sterol and up regulation of LDL-R [57–59].
ApoE polymorphisms and pathophysiological
conditions
The association between homozygous ε2 phenotype, in
which the protein is defective in binding to lipoprotein re-
ceptors, and type III hyperlipoproteinaemia is well docu-
mented [57]. This disorder is characterized by increased
triglyceride and cholesterol levels, presence of β-VLDL,
cholesterol-rich remnants of chylomicrons and VLDL,
xanthomas and premature cardiovascular disease [57]. How-
ever, more than 90% of homozygous E2 individuals have
either normal or low cholesterol levels [57]. Thus the action
of other secondary hormonal, environmental and/or genetic
factors is required for overt type III hyperlipoproteinae-
mia [57, 60]. It is interesting that some of the rare apoE
variants (see Table 1) are associated either with dominant
type III hyperlipoproteinaemia or hypertriglyceridaemia
[57, 60]. Such dominant variants of apoE include apoE3-
Leiden, apoE2 (Lys146→Gln), apoE3 (Cys112→Arg,
Arg142→Cys), apoE4 (Glu13→Lys, Arg145→Cys), apoE2
(Arg145→Cys), apoE1 (Lys146→Glu) and apoE1 (Lys146→
Asn, Arg147→Trp) [28, 30, 41]. In most cases the mutations
involve substitution of lysine or arginine in the cluster of
basic amino acids in the receptor/heparin-binding region of
the protein (residues 136–150) by a neutral or acidic amino
acid.
A number of studies have investigated the impact of apoE
polymorphisms on cardiovascular disease, often yielding
conflicting results (for recent reviews see [48, 61, 62]).
Differences in study design, geographic and ethnic back-
ground, allele frequency, sex, and potential gene-gene and
gene-environment interactions are likely to have contrib-
uted to such conflicting results [61]. In addition, apoE
genotypes have also been found to potentially affect life
expectancy [62]. Although several, but not all, studies link
the ε4 allele with greater risk for cardiovascular disease, the
relationship with respect to the ε2 allele remains in com-
plete [48, 57, 61, 62]. For example, a study of middle-aged
men from nine populations estimated a 40% greater risk for
mortality from coronary heart disease for carriers of the ε4
allele than ε2 carriers or individuals with the ε3/ε3 geno-
type [63]. In addition, populations with higher cholesterol
levels and higher coronary heart disease mortality rates
have a greater risk of the ε4 allele [48, 57, 63–65]. A meta-
analysis of 14 published studies in 1996 reported that
compared with carriers of the ε3/3 genotype those with ε4/4
had a higher risk for developing coronary heart disease, and
that the ε2 allele was not associated with such a risk [66].
However, numerous other studies have been performed
thereafter with conflicting results (see [61] and references
therein). A comprehensive recent meta-analysis of 48 pub-
lished studies comprising 15,492 case patients and 32,965
controls concluded that compared to the ε3/3 genotype
carriers of the ε4 allele had a 42% higher risk for coronary
heart disease whereas the ε2 allele had no significant asso-
ciation [61]. Several other studies have demonstrated a
context dependency of apoE genotype on cardiovascular
disease risk. An emerging pattern is that the association of
the ε4 allele with risk is present in individuals who smoke
[67–69].
In addition to cardiovascular disease, apoE polymor-
phisms have been investigated as a risk factor for other
pathophysiological conditions, including Alzheimer’s dis-
ease (AD), Parkinson’s disease, diabetes, renal disease, stroke,
schizophrenia, and cancer [48, 57, 61, 62, 70, 71]. From
these, the action of apoE and its polymorphisms on neuro-
nal function and AD has been the subject of intense research
in a number of laboratories. A detailed description of the
current literature on this topic is beyond the scope of this
review, where the focus is on the role of apoE in athero-
sclerosis. The readers are therefore directed to recent re-
views [70, 71]. Briefly, apoE is expressed at high levels in
the brain, where it acts as a principal lipid transport protein
in the cerebrospinal fluid [57, 70, 71]. Furthermore, the
expression of apoE is induced during peripheral nerve in-
jury, where it is involved in the repair process by redis-
tributing lipids to regenerating neurons and Schwann cells,
and additionally it modulates neurite extension and cy-
toskeletal function [57, 70, 71]. Both clinical and epide-
miological studies have uncovered a strong correlation
between apoE variants and the development of AD, with
the ε4 allele accounting for over one-half of AD cases in
the United States and the rarity of the disease in carriers
of the ε2 allele [57, 70, 71]. Such an isoform-specific ac-
tion has also been shown in a mouse model of AD [72].
A number of potential mechanisms have been proposed
for the contribution of apoE4 to the pathogenesis of AD,
including the regulation of deposition and clearance of
amyloid β peptides and formation of plaques, altered phos-
phorylation of the microtubule-associated protein tau and
the formation of neurofibrillary tangles, modulation of neu-
ronal signalling pathways, disruption of cytoskeletal struc-
ture and function, and impairment of the anti-oxidative
defence system [57, 70–73].
Interaction of apoE polymorphisms with other genes
and environmental factors
Several studies have also investigated the interaction be-
tween apoE polymorphisms and diet, smoking, gender, al-
cohol consumption, obesity, weight gain, physical inactivity,
cholesterol lowering drugs and other genetic factors [48, 61,
62, 67–69, 74–84]. A large number of such association
studies have also often produced conflicting findings [48,
57, 61, 62]. For example, in relation to lipid-lowering drugs
Gerdes et al. [78] reported that ε4 carriers had a greater
reduction in mortality in response to simvastatin than non-ε4
carriers. On the other hand, a meta-analysis study in 1995
[79] showed that apoE genetic variations had a significant
impact on the lowering of LDL cholesterol by 3-hydroxy-3-
methylgluaryl coenzyme A reductase inhibitors, with ε2
carriers being more responsive than ε4 carriers or ε3/ε3

subjects. A number of other studies, however, have failed to
find such an association between apoE genotype and lipid-
lowering agents [80–82].
Role of apoE in atherosclerosis
Several lines of evidence have implicated a potent anti-
atherogenic role of apoE. Thus depressed expression of
apoE in humans has been found to be associated with a pro-
atherogenic lipoprotein profile and diffuse atherosclerotic
disease [85, 86]. In addition, apoE knockout (apoE−/−) mice
are severely hypercholesterolaemic compared to the wild-
type counterparts, and the increase in cholesterol is largely
distributed in lower density lipoproteins due to their im-
paired clearance [87–89]. The accumulation of remnant
lipoproteins in such cases leads to the development of com-
plex atherosclerotic lesions, which develop even when the
mice are fed a low fat diet [87, 88, 89]. Atherosclerosis
in apoE-null mice has been shown to be prevented by in-
creasing circulating apoE levels through either recombinant
adenovirus-mediated gene transfer to the liver [90, 91], ex-
pression of apoE transgenes [92], or injection of synthetic
apoE peptide mimics [93].
ApoE expression is absent in normal vessels but high in
atherosclerotic plaques, and although the protein can enter
the artery wall from the periphery, it is believed to be mainly
synthesized locally by resident macrophages [94]. As
monocytes/macrophages are the only haematopoietic cells
that express apoE, bone marrow transplantation has been
used largely to investigate the role of macrophage-derived
apoE on atherosclerosis in vivo. Such studies have shown
that macrophage-derived apoE exerts anti-atherogenic prop-
erties largely independently of its effects on plasma lipid
levels [95–97]. Approaches other than bone marrow trans-
plantation have also confirmed the anti-atherogenic role of
apoE expressed in the vessel wall [98–101]. Thus, trans-
genic mice expressing human apoE in the vessel wall show
reduced atherosclerotic lesions in the absence of any changes
in plasma cholesterol and lipoprotein profile [98]. In ad-
dition, expression of low levels of apoE in the artery wall of
Fig. 1 The anti-atherogenic Reverse
role of apoE. See text for details. cholesterol
CETP Cholesteryl ester transfer transport
protein; HL hepatic lipase;
LCAT lecithin:cholesterol acyl-
transferase; LDL low density
lipoproteins; SMC smooth mus- Inhibition of
LDL
cle cells; VLDL very low density
lipoproteins oxidation
Inhibition of
T-lymphocyte
activation and
proliferation
Inhibition of
platelet
aggregation
333
apoE-deficient mice using a retroviral transduction system
leads to reduced early foam cell lesion formation [100]. It
should, however, be noted that there are studies which have
seen normalization of cholesterol levels by expression of
macrophage-derived apoE [102, 103]. In conclusion, both
hepatic- and macrophage-derived apoE can have anti-ath-
erogenic effects through beneficial changes in plasma lipids
and direct action on and from within the artery wall.
Anti-atherogenic role of apoE through the regulation
of lipoprotein metabolism and transport
Figure 1 summarizes the various anti-atherogenic actions of
apoE. Its central role in the regulation of overall lipoprotein
metabolism and transport is clearly a major contributor for
the anti-atherogenic function. Firstly, apoE mediates the
uptake and degradation of lipoproteins through its ability to
bind with high affinity to the LDL-R on parenchymal liver
cells [57]. As detailed above, the importance of such an
interaction is underscored by the massive accumulation of
lipoproteins and lipoprotein remnants in the plasma of pa-
tients with type III hyperlipoproteinaemia, where apoE2
which is defective in LDL-R binding, is present [28]. Sec-
ondly, apoE also binds to another hepatic receptor, LRP,
and this has been implicated in the clearance of chylomicron
remnants [104, 105]. The clearance of chylomicron rem-
nants also involves the binding of apoE to HSPG, as demon-
strated by studies using heparinase treated cells and infusion
of heparinase in mice [104]. Thirdly, apoE is able to directly
stimulate hepatic VLDL and triglyceride production, and
studies on apoE-deficient mouse hepatocytes have demon-
strated impaired secretion of VLDL triglycerides [106].
Consistent with this finding, increased VLDL production is
seen in hepatocytes stably transfected with human apoE or
in apoE3 transgenic mice [107]. Fourthly, apoE stimulates
reverse cholesterol transport, in which excess cholesterol
in peripheral tissues is transported back to the liver, via a
process involving HDL, for excretion through bile forma-
tion [57]. Finally, apoE activates enzymes involved in lipo-
protein metabolism such as hepatic lipase, cholesteryl ester
Hepatic uptake of Influence on
lipoprotein particles hepatic VLDL
secretion and
processing
Activation of
enzymes involved
in lipoprotein
ApoE metabolism (e.g.
HL, CETP, LCAT)
Inhibition of
SMC
proliferation
Anti-inflammatory Inhibition of
activity endothelial cell
proliferation
334
transfer protein and lecithin:cholesterol acyltransferase
[108–110].
The process of cholesterol efflux is particularly important
in maintaining cholesterol homeostasis in cells that are in-
capable of limiting their uptake of lipids, such as macro-
phages, and is vital in the prevention of foam cell formation
and atherogenesis [111]. A key role of apoE in stimulating
macrophage cholesterol efflux has emerged from a number
of studies. For example, the capacity of apoE-depleted HDL
from humans or mice to promote cholesterol efflux from
mouse peritoneal macrophages is decreased and can be
restored to normal by the addition of exogenous apoE [112].
In addition, the apoE genotype has a profound effect on the
efficiency of cholesterol efflux [113]. The expression of en-
dogenous apoE also enhances cholesterol efflux [114, 115],
and the experimental characteristics of cholesterol efflux in
such cases can be distinguished from those produced by the
exogenous addition of lipid free apoE [116]. The precise
mechanisms by which endogenous apoE stimulates cho-
lesterol efflux currently remains unclear, although its reten-
tion at the cell surface via association with proteoglycans
makes a contribution [117]. ApoE arrives at the cell surface
in a relatively lipid-poor state, and conformation changes
attained during lipidation may cause its release [117]. In
addition, apoE held in the proximity to the plasma mem-
brane by proteoglycan binding could facilitate passive de-
sorption of lipid from the plasma membrane, a process that
may involve scavenger receptor class B type I or ATP-
binding cassette transporter A1 (ABCA1) [118, 119]. In-
deed, it has been shown that apoE is an acceptor for free
cholesterol and phospholipid released from macrophages
by ABCA1, and that lipid-free apoE is able to interact with
ABCA1 in vitro [119].
Other anti-atherogenic functions of apoE
As described above, a number of studies have shown that
reduced expression of apoE in the vessel wall can accelerate
atherosclerosis, whereas low expression of the protein in
apoE knockout mice is able to limit the development of the
disease in the absence of any detectable changes in plasma
cholesterol levels or lipoprotein profile [95–101]. This
suggests an additional anti-atherogenic role of apoE that is
independent of its action on cholesterol metabolism and
transport. More recently further support to this proposition
has been provided by the studies of Thorngate et al. [120]
who demonstrated that low-level of apoE expression is able
to inhibit atherogenesis using a mechanism independent of
stimulation of cholesterol efflux. In addition, low levels of
extrahepatic, non-macrophage apoE was found to inhibit
atherosclerosis in transgenic apoE-deficient mice without
correcting hypercholesterolaemia [121]. A number of anti-
atherogenic properties of apoE that are independent of
direct involvement in lipid metabolism and transport have
been identified, and are discussed below in detail.
The apoE expressed by macrophages has been found
to exhibit local cytokine- and hormonal-like effects on the
surrounding cells of the vessel wall, which then contribute
to its atheroprotective role. For example, apoE inhibits
platelet aggregation by interacting with a specific cell sur-
face receptor, the apoE receptor 2 (apoER2) [122, 123].
This interaction initiates a signalling cascade leading to the
activation of nitric oxide synthase [123]. ApoE also inhibits
the expression of vascular cell adhesion molecule 1 on en-
dothelial cells [124] again via interaction with apoER2 and
stimulation of endothelial NO production [125]. It has been
proposed that tyrosine phosphorylation of apoER2 in this
case initiates phosphoinositide-3-kinase signalling leading
to activation of NO synthase [125]. ApoE can also suppress
the activation and proliferation of T lymphocytes [126–
128]. Such an anti-proliferative effect of apoE is thought to
be due to inhibition of critical signalling events such as
intracellular calcium accumulation and phosphatidylinosi-
tol turnover [127]. This inhibition of early signalling is
manifested downstream by a reduction in interleukin 2 ex-
pression [128], which consequently arrests the lymphocytes
at the G1A/G1B boundary in the cell cycle [129]. ApoE also
inhibits SMC migration and proliferation induced by agents
such as serum, platelet-derived growth factor and oxidized
LDL [130–132]. It was reported that the inhibition of SMC
proliferation is mediated through the activation of NO
synthase triggered by the interaction of apoE with HSPG
[130, 131]. More recently, however, Kothapalli et al. [132]
showed that the anti-mitogenic effect maps to the amino-
terminal receptor-binding domain of the protein and in-
volves stimulation of cyclooxygenase-2 expression leading
to prostacyclin synthesis and inhibition of cyclin A gene
expression. The precise reason(s) for the different mecha-
nisms identified remains unclear but may be related to the
nature of the mitogen employed [132]. In contrast to pro-
liferation, apoE inhibition of SMC migration appears to be
mediated by LRP-1, is independent of NO synthase, and
requires activation of the cAMP-dependent protein kinase
A [130–133]. ApoE is also able to inhibit lipid oxidation
[134], which may thereby prevent the accumulation of
oxidized LDL. Indeed, evidence of enhanced oxidative
stress has been found in apoE-deficient mice along with
LDL oxidation specific epitopes in the aortic lesions [135].
In addition, Hayek et al. [112] have shown that circulating
lipoproteins in apoE-deficient mice are more oxidized and
more susceptible to oxidation in vitro than lipoproteins from
the wild-type animals. Although the mechanism is not com-
pletely understood, it has been shown that apoE can bind
metal ions (copper and iron), possibly sequestering them
and preventing their participation in the oxidation process
[134]. Finally, a fraction of macrophage-secreted apoE be-
comes sequestered within the pericellular proteoglycan
matrix where, in addition to influencing the uptake and
retention of atherogenic lipoproteins into the vessel wall,
it can modulate the availability of cytokines and growth
factors retained within it [136]. Such an action of apoE is
likely to make a major contribution to its ability to inhibit
endothelial cell proliferation [136].
 335

Regulation of apoE gene expression and factors that are involved in such regulation have been
the focus of research in a number of laboratories. The
The human apoE, apoCI, apoCIV and apoCII genes are proximal promoter region of apoE has been implicated
closely linked and are located on the long arm of chromo- in the induction of gene expression during macrophage
some 19, spanning a region of 45 kb. ApoE gene expression differentiation and regulation by cAMP [138, 158]. Thus
is extremely complex with regulation in a tissue-specific Basheeruddin et al. [158] showed that the −623 to −447
manner and in response to cellular changes and extracel- promoter region is necessary for transcriptional activation
lular/intracellular factors (Table 2). For example, the differ- during induced differentiation of human THP-1 monocytes
entiation of monocytes into macrophages is accompanied into macrophages. This study suggested that activator pro-
by increased apoE expression [145]. In addition, cholesterol tein 1-like transcription factors were involved, possibly due
loading of macrophages leads to a marked increase in apoE to 12-O-tetradecanoylphorbol-13-acetate activating the pro-
gene transcription [13, 143]. Oxidized forms of cholesterol tein kinase C pathway which is known to regulate these
such as those present in foam cells (e.g. oxysterol deriva- DNA binding proteins [158]. Andreani-Mangeney et al.
tives) also modulate apoE expression [143, 153, 154]. The [138] have shown that cAMP inhibits apoE gene transcrip-
inflammatory response also has a profound effect on the tion in HepG2 cells, which is mediated via elements located
expression of apoE. For example, bacterial lipopolysac- between −614 to −804 in the promoter region. Interestingly,
charide decreases apoE expression in mouse macrophages this study also showed that a promoter region containing the
when added exogenously or injected in vivo [141]. ApoE −201/−1 region was activated only by cAMP, thereby in-
production by macrophages is also inhibited by interferon γ dicating a more complex regulation by this factor. Garcia
[144], interleukin 1 [146] and granulocyte colony-stimulat- et al. [140] have identified the transcription factor activator
ing factor [141, 142] and activated by tumour necrosis fac- protein 2, which interacts with the −48 to −74 and −107 to
tor α [157] and transforming growth factor β ([142] and −135 regions of human apoE, as a mediator of synergistic
our unpublished observations). Other agents that modulate activation by cAMP and retinoic acid. It was suggested that
apoE expression include cAMP [138–140], thyroid hor- protein kinase A mediated phosphorylation of activator
mone [156], insulin [148], and oestrogen [147]. protein 2 is likely to play an important role [140]. Trans-
Transcriptional regulation plays a key role in the mod- fection assays using manipulated promoter-reporter gene
ulation of apoE gene expression, and the sequence elements constructs, DNase I and in vivo footprinting, cell-free tran-
scription assays, and yeast one-hybrid system have iden-
Table 2 Regulators of apoE gene expression tified several other transcription factors that interact with
the proximal promoter region (SP1, HNF-3, HNF-4, TF-
Activators LF2, GATA-1, CCAAT/enhancer binding protein, BEF-1,
Apolipoprotein AI 137 Aa, B1b, B2b, Zic1 and Zic2) [159].
cAMP (macrophages, astrocytoma) 139, 140 Polymorphisms in the proximal promoter region of the
Cholesterol/stero loading 9, 13, 143 apoE gene have been described at positions −491 A/T, −427
Differentiation of monocytes to macrophages 145 T/C and −219 G/T [160]. A C/G polymorphism has also
Oestrogen 147 been identified at position +113, which corresponds to an
Expression of ATP-binding cassette transporters A1 139 enhancer element in intron 1 [161]. Such polymorphisms
and 8 are associated with variations in the transcriptional activity.
Glucocorticoids 150 For example, −219G allele shows higher transcriptional
High-density lipoprotein 111 activity than −219T, and the −491T allele shows lower
Oleic acid 152 transcriptional activity than −491A in transfected HepG2
Oxysterol ligands 153 cells and astrocytomas [160, 162]. It is believed that dif-
Oxidized low-density lipoprotein 154 ferential binding of nuclear proteins is responsible for the
Peroxisome proliferator activated receptor γ 155 altered transcriptional activity produced by these polymor-
activators phisms [160, 163]. Indeed, a European population study has
Retinoic acid 140 found an association between the −219G/T polymorphism
Transforming growth factor β 142 with differential plasma apoE levels [164]. The −219T allele
Thyroid hormone 156 was associated with an increased risk of myocardial in-
Tumour necrosis factor α 157 farction and premature coronary heart disease [164, 165]. In
Inhibitors addition, the −219G/T polymorphism has been found to
cAMP (hepatocytes) 138 influence triacylglycerol-rich lipoprotein metabolism dur-
Granulocyte-macrophage colony-stimulating factor 141, 142
ing the postprandial period, thereby prolonging postpran-
Interferon γ 144
dial lipaemia in individuals with the TT genotype [166].
Interleukin 1 146
The −491A/T polymorphism has also been shown to mod-
ulate the lipid-lowering efficiency of atorvastatin and beza-
Insulin 148
fibrate in combined hyperlipidaemia treatment [167]. Finally,
Lipoprotein lipase 149
a relationship between the polymorphisms and risk for AD
Lipopolysaccharide 141
has also been identified [71, 162, 163].
Statins 151
336
Despite the identification of the regulatory sequences and
the polymorphisms detailed above, it has been found that
the proximal promoter region of the apoE gene lacks the
ability to direct gene transcription in vivo in any cell of
transgenic mice in the absence of the distal enhancers [168].
Hepatocyte expression of apoE is controlled by two cell-
specific enhancers, hepatic control regions (HCR) 1 and 2,
which are located 15 kb (HCR.1) and 26 kb (HCR.2) 3′ to
the apoE gene [168]. HCR.1 and HCR.2 share 87% se-
quence identity, thereby suggesting that they have probably
arisen as a result of an evolutionary gene duplication that
yielded the apoCI and apoCI′ genes [168]. More recently,
downstream enhancer sequences have been identified that
direct apoE expression in astrocytes, skin, macrophages and
adipocytes [169–171]. In the case of adipocytes and macro-
phages, two homologous enhancers, designated multi-en-
hancer (ME) 1 and 2, are essential [171]. These are located
3.3 kb (ME1) and 15.9 kb (ME2) downstream of the apoE
gene and contain 620 and 619 nucleotides, respectively [171].
Several binding motifs for the glucocorticoid receptor and
CCAAT/enhancer binding proteins α and β has been lo-
cated in these enhancer regions although their precise roles
remain to be fully investigated [171]. Interestingly, the ME1
and ME2 enhancers along with the promoter also contain a
conserved liver–X receptor responsive elements that medi-
ate activation of gene transcription in response to oxysterol
ligands in macrophages, but not monocytes [153]. Finally,
Galetto et al. [155] has identified a peroxisome proliferator
activated receptor γ response element within the apoE/
apoCI intergenic sequence, the mutation of which abolishes
the induction of expression by the activator ciglitazone.
Overall, therefore, it is clear from all these studies that sub-
stantially more research needs to be carried out to precisely
delineate the role of each of the sequence elements iden-
tified in the proximal and distal regions. In addition, the
signalling pathways associated with changes in apoE ex-
pression are poorly understood and need to be elucidated in
detail.
Post-transcriptional mechanisms have also been found
to be involved in the regulation of apoE expression. For
example, Basheeruddin et al. [172] have shown that apoE
mRNA is degraded more slowly in macrophages than in
monocytes due to inhibition of a specific nuclease. In ad-
dition, a substantial amount of newly synthesized apoE
in macrophages is degraded prior to secretion due to either
direct targetting of apoE to lysosomal pathways [173] or
ubiquitinated-mediated proteasomal degradation [174]. Fur-
thermore, the segregation of cell-surface apoE between
secretion and degradation pathways is modulated by the
presence of extracellular lipids, for example, phospholipid
vesicles or lipoproteins [6]. Duan et al. [13] have also dem-
onstrated that pre-incubation of macrophages with sterol
increases secretion of apoE to the medium in part due to the
suppression of apoE degradation, a process that requires the
carboxyl-terminal domain of the protein. A number of other
agents have also been shown to stimulate apoE secretion,
although the molecular mechanisms remain to be deci-
phered in detail. For example, enrichment of cells with cho-
lesterol and activation of protein kinase A with the cAMP
analogue 8-bromo-cAMP stimulates the secretion of apoE
[113, 139]. ApoE secretion is also increased by acceptors of
cholesterol in the efflux process (e.g. apoAI, HDL) at the
post-transcriptional level [111, 137]. On the other hand,
apoE secretion from macrophages is inhibited by interferon
γ due to increased intracellular degradation of newly syn-
thesized protein [144]. ABCA1 and to a lesser extent ATP-
binding cassette transporter 8, both of which are important
regulators of cholesterol efflux, have also been shown to
modulate apoE secretion from human monocyte-derived
macrophages [139]. As cholesterol loading and cAMP treat-
ment up-regulate ABCA1 expression in macrophages [139,
175], it is likely that this plays a role in the intracellular
trafficking of apoE. Indeed, inhibition of ABCA1 leads to
the disappearance of apoE-containing granular structures
from the plasma membrane [139]. More recently, oleic acid
has been shown to induce apoE secretion in macrophages
by modulating the post-translational glycosylation of the
protein [152].
There is an optimal concentration of plasma apoE that is
required for lipoprotein clearance, and too little or too much
of the protein can be detrimental. For instance, over expres-
sion of human apoE3 in apoE-deficient mice at concentra-
tions greater than 30 mg/ml leads to hypertriglyceridaemia
because of elevation in hepatic VLDL triglyceride produc-
tion and interference with VLDL lipolysis [176]. The body
is able to regulate and maintain optimal levels of apoE, and
recent studies have provided novel insights into the recy-
cling of apoE. When radiolabelled triglyceride-rich lipopro-
teins were employed to evaluate intracellular catabolism,
more than one-half of the labelled apoE was released back
to the medium, where it again became part of lipoproteins
[10]. Thus a substantial amount of the internalized lipo-
protein-associated apoE is retained inside the cell and then
reappears in the secretion medium. Such recycling has been
seen in macrophages, hepatocytes and fibroblasts [10–12,
177]. Internalized apoE has been shown to escape the lyso-
somal degradation pathway [177, 178], thereby suggest-
ing the existence of an active cellular mechanism that
maintains apoE levels by dissociating the ligand from the
lipoprotein particle and carry apoE back to the secretory
apparatus. The precise molecular mechanisms responsible
for apoE recycling remains to be deciphered, but the pres-
ence of internalized apoE has been demonstrated in Golgi
apparatus rich fractions isolated from mouse liver [178,
179]. More recently Farkas et al. [180] have investigated the
mechanism in detail and shown that apoE recycling can
occur in the absence of LDL-R or under conditions of dras-
tically reduced LRP expression and also in the absence of an
intact Golgi apparatus. Additionally, recycling occurs even
when apoE lacks its lipoprotein binding domain at the car-
boxyl terminus. It was therefore concluded that apoE recy-
cling occurs through multiple redundant pathways [180].
Interestingly, HDL not only acts as an extracellular acceptor
for recycled apoE but also stimulates the recycling of inter-
nalized triglyceride-rich lipoprotein-derived apoE [12].

Conclusions
The evidence presented in this review demonstrates that
apoE plays an important anti-atherogenic role. Such an
action involves multiple functions of the protein, including
receptor-mediated uptake and metabolism of lipoproteins,
reverse cholesterol transport, and modulation of the in-
flammatory response. The protein therefore represents an
important target for therapeutic intervention of atheroscle-
rosis. Gene therapy, bone marrow transplantation and the
use of recombinant protein represent potential means for
increasing apoE activity. The use of agents which increase
apoE expression is another approach. Each of these strat-
egies currently has a number of limitations, and refinements
will be necessary by further research before use in clinical
trials. It is also clear that a detailed understanding is required
on a number of aspects of apoE function and regulation. For
example, the full molecular interactions of apoE in reverse
cholesterol transport and uptake and metabolism of lipopro-
teins along with the mechanisms of its actions that are
independent of lipid metabolism and transport needs to be
fully understood. In addition, the transcriptional and post-
transcriptional control mechanisms involved in the diverse
regulation of apoE need to be delineated. The findings from
such studies may provide opportunities to exploit the anti-
atherogenic actions of apoE in the prevention and/or treat-
ment of cardiovascular disease.
Acknowledgements We thank the British Heart Foundation for
financial support, and apologize to all those authors whose work
could not be cited because of space limitations.
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