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Cholesterol efflux capacity: An introduction for clinicians

Article  in  American Heart Journal · July 2016

DOI: 10.1016/j.ahj.2016.07.005

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 Progress in Cardiology

Cholesterol efflux capacity: An introduction

for clinicians
Malcolm Anastasius, MBBS, MMed, FRACP, a Maaike Kockx, PhD, a Wendy Jessup, PhD, a David Sullivan, MBBS, FRACP, b
Kerry-Anne Rye, PhD, c and Leonard Kritharides, MBBS, FRACP, PhD a,d New South Wales, Australia

Epidemiologic studies have shown an inverse correlation between high-density lipoprotein (HDL) cholesterol (HDL-C) levels and
cardiovascular disease outcomes. However, the hypothesis of a causal relationship between HDL-C and cardiovascular disease has
been challenged by genetic and clinical studies. Serum cholesterol efflux capacity (CEC) is an important measure of HDL function in
humans. Recent large clinical studies have shown a correlation between in vitro CEC and cardiovascular disease prevalence and
incidence, which appears to be independent of HDL-C concentration. The present review summarizes recent large clinical studies
and introduces important methodological considerations. Further studies are required to standardize and establish the
reproducibility of this measure of HDL function and clarify whether modulating CEC will emerge as a useful therapeutic target.
(Am Heart J 2016;180:54-63.)

Recent major studies have identified that serum “choles-
terol efflux capacity” (CEC) is associated with both the
prevalence and incidence of coronary artery disease. Because
the concept of CEC may be unfamiliar to most cardiovascular
clinicians, the purposes of this review is to introduce the
scientific rationale of CEC in relation to the removal of
cholesterol from cells (cholesterol efflux) by high-density
lipoproteins (HDLs) and to summarize key recent clinical
literature in this area. The limitations of the CEC model and
likely future developments will also be discussed.
High-density lipoprotein cholesterol
and its relation to cardiovascular disease
The HDL-C hypothesis and associated limitations
Epidemiologic evidence indicates that a low level of
HDL cholesterol (HDL-C) is an independent risk factor for
cardiovascular disease (CVD). 1 More recent studies have
From the aANZAC Research Institute, Concord Repatriation General Hospital, University of
Sydney, Sydney, New South Wales, Australia, bDepartment of Biochemistry, Royal Prince
Alfred Hospital, Sydney, New South Wales, Australia, cSchool of Medical Sciences, The
University of New South Wales, Sydney, New South Wales, Australia, and dCardiology
Department, Concord Repatriation General Hospital, University of Sydney, Sydney, New
South Wales, Australia.
Source of funding: L.K., W.J., and K.A.R. are supported by NHMRC Program Grant
1037903.
Submitted April 27, 2016; accepted July 7, 2016.
Reprint request: Leonard Kritharides, MBBS, FRACP, PhD, Department of Cardiology,
Concord Repatriation General Hospital, 1A Hospital Rd, Concord, New South Wales
2139, Australia.
E-mail: leonard.kritharides@sydney.edu.au
0002-8703
© 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ahj.2016.07.005
confirmed this association, including contemporary
populations with diabetes (UK Prospective Diabetes
Study) or populations taking statins. 2 This association
gave rise to the hypothesis that HDL-C is causally related
to the development of CVD and may be atheroprotective.
In animal models of atherosclerosis, genetic manipulation
of circulating levels of HDLs or its major apolipoprotein
component, apolipoprotein A-I (apoA-I), has been shown
to prevent progression and/or induce regression of
atherosclerosis, 3-5 adding significant plausibility to the
hypothesis that HDL itself is directly antiatherogenic.
However, the notion that circulating HDLs are anti-
atherogenic in humans has been contentious. First, a
cardiovascular risk prediction model including HDL-C
was examined in the Copenhagen General Population
Study. 6 In this study, the addition of HDL-C to cardiovas-
cular risk estimation reduced the sensitivity of the
high-risk threshold for detecting fatal cardiovascular
events and decreased the net reclassification index. 6
Second, patients with low HDL-C commonly have
elevated triglycerides and increased concentrations of
small dense low-density lipoprotein (LDL; the so-called
metabolic syndrome phenotype). 7 Consequently, distin-
guishing between the atheroprotective effects of HDLs
and the harmful effects of other components of this
phenotype has been challenging. Recent studies confirm
that elevated triglycerides and elevated apoC-III (a cause
of increased triglycerides) increase the risk of CVD. 8
Third, in Mendelian randomization studies investigating
the protective effect of high HDL-C, genetic variations
linked to elevated HDL-C were not associated with
reduced risk of myocardial infarction. 9-11
Fourth, and perhaps most importantly, recent random-
ized controlled trials of nicotinic acid and cholesterol ester

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American Heart Journal
Volume 180
transfer protein (CETP) inhibitors, which clearly increased
HDL-C, failed to reduce cardiovascular events when added
to optimal medical therapy including statins. 12-14 The
discrepancy between epidemiology and animal studies on
the one hand, and genetic and clinical studies on the other,
have directed attention toward a better understanding of
HDL function and HDL subpopulations.
High-density lipoprotein–mediated
cholesterol efflux
High-density lipoproteins are spherical particles with a
core consisting of nonpolar lipids (triglycerides and
cholesterol esters), encased by a surface coat of
apolipoproteins (mainly apoA-I), phospholipids, and
unesterified cholesterol. High-density lipoproteins are
heterogeneous and comprise subpopulations that vary in
size, as well as apolipoprotein and lipid composition. This
variation in HDL subpopulations results in heterogeneity
in the capacity of HDLs to perform cell cholesterol efflux
(see below).
The mechanisms by which HDLs might confer ather-
oprotection have been extensively investigated. These
include reverse cholesterol transport (RCT); anti-inflammatory
actions on leucocytes and endothelial cells; improved
endothelial function, through stimulating production of nitric
oxide; cytoprotection (prevention of apoptosis); and anti-
thrombotic effects. Of these, RCT is the best studied and is the
process relevant to CEC.
A key element of atherosclerosis development and
progression is the accumulation of cholesterol within
macrophages in the arterial wall and subsequent forma-
tion of foam macrophages. This cholesterol is derived
from circulating lipoproteins, principally LDL. Reverse
cholesterol transport involves the movement of choles-
terol from peripheral sites, such as foam macrophages in
the arterial wall, with subsequent excretion of cholester-
ol from the body through the liver and bile. 15,16
Macrophage-specific RCT has been demonstrated in
animal models which use cholesterol-enriched macro-
phages as the cholesterol donor and track the movement
of radiolabeled cholesterol from the macrophage into
interstitial fluid, blood, thence bile, and feces. 17Reverse
cholesterol transport has also been measured in humans
using infusions of synthetic/reconstituted HDL (apoA-I–
phospholipid disks) and quantifying the excretion of fecal
sterols. 18However, it needs to be kept in mind that
cholesterol efflux from macrophages provides only a
small component of the overall turnover of cholesterol in
the body as a whole. 19
Cholesterol is a highly hydrophobic molecule and cannot
be cleared from cells without a cholesterol acceptor to
transport it in the aqueous milieu of plasma. Cholesterol
efflux describes the movement of cholesterol from a cell
to a cholesterol acceptor and is the first step of RCT. 15 In
the case of macrophage-specific RCT, it represents the
movement of cholesterol from macrophages in the arterial
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Anastasius et al 55
wall (Fig. 1 16) to a cholesterol acceptor such as HDL. The
mechanisms of cholesterol efflux have been extensively
reviewed elsewhere. 20-22
Serum efflux capacity
What is serum efflux capacity and how is
it measured?
Lipoproteins in general, and HDL species in particular,
appear to be highly dynamic. Consequently, assessing the
functional properties of HDLs, as they exist in vivo, is
challenging. The CEC assay evolved from the need for an
assay that would measure the capacity of HDLs to remove
cholesterol from cells in vivo, without modifying or
excluding subpopulations of HDL. It derived from a series
of studies first performed in the Rothblat laboratory in
Philadelphia, which identified that most cholesterol efflux
stimulated by human serum was attributable to the non–
apolipoprotein B (apoB) lipoprotein-containing fraction, 20
and that this could be measured simply and reliably using
total serum from which apoB had been precipitated.
Measurement of cellular cholesterol efflux in vitro
requires 2 components: a donor cell releasing a cholesterol
tracer and a cholesterol acceptor 23 (Fig. 2). The cholesterol
acceptors that have been most commonly examined in
relation to CEC include the following (Fig. 2):
1. Whole serum or plasma
2. ApoB-depleted serum
3. Isolated HDLs
The use of apoB-depleted serum rather than isolated HDLs
has the advantage of avoiding the shedding of apolipoproteins
and small dense particles, which can occur during ultracen-
trifugation. Apolipoprotein B depletion removes LDL, inter-
mediate-density lipoprotein, and very low density lipoprotein
from serum and reduces the exchange of labeled cholesterol
from cells to the cholesterol-rich atherogenic apoB-containing
lipoproteins, and thus provides an indirect measure of HDL
function. Apolipoprotein B–depleted serum is the standard
preparation of serum used in major studies of CEC.
The specific steps involved in measuring in vitro
cellular CEC are outlined in schematic form in Figure 2.
The roles of different pathways in cholesterol efflux from
macrophages
Cholesterol-enriched macrophages can release cholesterol
to human sera using the following pathways 24:
(i) Aqueous diffusion
(ii) ABCA1 (ATP-binding cassette transporter A1)
(iii) ABCG1 (ATP-binding cassette transporter G1)
(iv) SR-B1 (scavenger receptor class B type 1)
The relative importance of these pathways will vary
according to the precise cell systems and the cholesterol
acceptor used. In the setting of cholesterol enrichment,

56 Anastasius et al
Figure 1
Diagram illustrating the relationship between macrophage cholesterol efflux and reverse cholesterol transport. Pre–β-HDL, lipid-poor, small HDL particle.
Cholesterol transporters: ABCA1, ABCG1, and SR-B1. Adapted with permission from Macmillan Publishers Ltd: Nature Medicine, 16 copyright 2012.
which is relevant to foam cell macrophages in athero-
sclerotic plaque, cholesterol efflux is mediated mainly by
pathways involving diffusion, ABCA1 and ABCG1, with a
lesser contribution from SR-B1. 24 In the absence of cholesterol
enrichment (and no ABCA1 up-regulation), the pathway used
for cholesterol efflux to human sera is predominantly aqueous
diffusion, with a minor contribution from the SR-B1 pathway.
Because the relative contribution of these pathways will vary
according to the precise cell systems used, extremely
consistent laboratory processes are mandatory for assessing
CEC. Such variation makes the direct comparison of studies
performed in different laboratories challenging.
Of the pathways described above, the most important
contributor to efflux from foam cell macrophages,
especially human foam cell macrophages, is ABCA1. 25-27
Genetic mutations in ABCA1 cause Tangier disease, and
the absence of ABCA1 in macrophages or fibroblasts
impairs cholesterol efflux to HDL. 25,28 Cholesterol efflux from
macrophages, which occurs within the arterial wall, is
predominantly mediated through the ABCA1 transporter.25
Carriers of a loss of function ABCA1 gene mutation
demonstrate impaired HDL CEC and greater carotid or
femoral artery intima-media thickness compared with con-
trols, consistent with accelerated and early atherosclerosis. 29
Comparing the capacity of HDL subpopulations to mediate
cholesterol efflux shows that ABCA1-mediated cholesterol
efflux varies inversely with HDL subpopulation size. Choles-
terol efflux is most efficient with the smaller HDL subpopu-
lations and least efficient with larger HDL particles. 25
Standard system for measuring efflux capacity
The prototype in vitro assay to measure CEC uses a
mouse macrophage cell line (J774) as the cholesterol
donor cell and radiolabeled cholesterol or fluorescently
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American Heart Journal
October 2016
labeled cholesterol (boron-dipyrromethene cholesterol]
as the cholesterol tracer. The cholesterol acceptor is
typically apoB-depleted serum diluted to a final concen-
tration of 1% to 2% (Fig. 2).
In CEC studies using J774 macrophages, cAMP is used to
up-regulate ABCA1 expression. Under conditions of
cholesterol enrichment and cAMP induction, ABCA1
mediates 40% of total cholesterol efflux that occurs during
CEC measurements with J774. 30 Studies vary in the
parameters that they report. Some report total cholesterol
efflux, whereas others report ABCA1-specific or
cAMP-inducible efflux. Although beyond the scope of the
present article to expand on this issue in detail, it is
important for clinicians to be aware that such apparently
minor technicalities could change the relative importance
of individual transporter pathways in the assay and cause
inconsistent results between clinical studies.
Increasingly, CEC assays are also performed using different
cell systems which explore the role of individual cholesterol
transporters very precisely, often using cell lines expressing
a single cholesterol transporter (eg,Chinese hamster ovary
[CHO] cells expressing ABCA1 [ABCA1-CHO] or ABCG1
[ABCG1-CHO]). Each cellular system may be giving different
emphasis to different elements of the cholesterol efflux
process, including the relative contribution of large,
medium, or small HDL particles.
Cellular cholesterol efflux models and
correlation with CVD
Four large clinical studies (Table 1) correlating in vitro
CEC with hard CVD study end points are discussed.
Khera et al 31 established the association between CEC and
prevalent CVD. This study demonstrated a significant
American Heart Journal
Volume 180
Figure 2
Sequential steps to measure in vitro CEC. Cholesterol transporters: ABCA1,
ABCG1, and THP-1 macrophages (human macrophage cell line). Cells
are grown in culture medium and incubated with labeled cholesterol
(radiolabeled or fluorescence-labeled cholesterol) resulting in enrichment
of the cell culture with a cholesterol tracer. Depending on the transporter(s)
being studied, there can be selective up-regulation of relevant transporter
pathways before exposure of cells to a cholesterol acceptor (1-3).
negative association between CEC and subclinical athero-
sclerotic disease as measured by carotid-intima media
thickness. In a separate independent case-control cohort,
subjects with a luminal stenosis of N50% in a major coronary
artery based on coronary angiography were recruited. In this
cohort, CEC was negatively associated with prevalent
coronary artery disease (CAD). In both study cohorts, the
significant negative correlation between CEC and CVD was
independent of HDL-C and other traditional cardiovascular
risk factors. Furthermore, HDL-C variation accounted for
only a small component of the relationship between CEC
and atherosclerotic disease. Importantly, almost all of the
CEC was attributable to the HDL fraction of plasma, serum,
suggesting that this is a measure of HDL function.
However, there has been conflicting evidence regarding
the relationship between CEC and CVD as shown by Li et al
32
that used 2 case-control cohorts. This study showed that
heightened cholesterol efflux to apoB-depleted serum was
associated with a reduced risk of prevalent CAD in an
outpatient cohort, but was paradoxically associated with
increased risk of future myocardial infarction, stroke, and
death. This raises concerns that CEC may be both favorable
and unfavorable depending on the outcome measured. It is
possible that index bias contributed to the paradoxical
relationship, which may occur when assessing recurrent
events in subjects with established disease. 35
In a study of a healthy cohort of patients from the
Multiethnic Dallas Heart Study, 33 aged between 30 and 65
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Anastasius et al 57
years and with no history of CVD, subjects had CEC
measured at baseline. At a median follow-up period of
9.4 years, increasing CEC correlated negatively with the
primary end point of incident atherosclerotic CVD (first
nonfatal myocardial infarction, nonfatal stroke, or coro-
nary revascularization or death from cardiovascular
causes). Interestingly, heightened CEC only weakly
correlated with traditional CVD risk factors, except for
lipid levels. For instance, weekly exercise activity and
alcohol consumption only explained 3% of the variance in
CEC. There was only modest correlation between CEC,
markers of inflammation, and insulin resistance. A
limitation of this study is the use of a young cohort of
patients with low risk and only a modest number of
incident atherosclerotic events.
A more recent prospective study 34 of subjects from the
EPIC-Norfolk study also found a significant negative
association between CEC and incident coronary heart
disease, independent of HDL-C and other traditional
cardiovascular risk factors.
This study comprises the largest patient population
studied to date for CEC and is powered with the greatest
number of incident cardiovascular events. In addition, it
is notable for the older average age of its subjects
compared with the Dallas study.
It is important to establish the incremental value of CEC
in cardiovascular risk prediction models if this is to have
any clinical application. Rohatgi et al 33 identified that the
addition of CEC to traditional cardiovascular risk factors
improved the c statistic (c = 0.827-0.841, P = .02) and net
reclassification index (0.37, 95% CI 0.18-0.56) for the
prediction of risk in developing the primary end points of
atherosclerotic CVD. Thus, CEC has the potential to
improve risk stratification. This has been shown in
another study that CEC improves cardiovascular risk
prediction independent of prevalent coronary artery
calcium and a family history of myocardial infarction. 36
Is CEC explained by small dense
HDL particles?
It is now apparent that the size of the HDL particle is a key
determinant of its capacity to promote cellular cholesterol
efflux. In particular, small HDL particles (HDL3) are
considered to have greater efficiency in promoting cellular
cholesterol efflux compared with more mature larger
spherical HDL particles (HDL2). 37 Small-scale studies have
shown that serum efflux capacity correlates with increased
proportions of smaller HDL species. It would be of interest
to examine how well HDL particle size distribution is
predictive of CVD. However, it is difficult and
time-consuming to accurately measure HDL particle size
distribution in large numbers of individual subjects.
Newer techniques such as nuclear magnetic resonance
spectroscopy and ion mobility that directly measure HDL
particle size 38,39 may provide new avenues to standardize the
 American Heart Journal
58 Anastasius et al October 2016

Table I. Summary of major studies examining relationship between CEC and CVD
Cholesterol Cholesterol
Study Donor cell line tracer acceptor Study cohort CVD outcomes

Kheraet al 31 J774 [ 3H]-cholesterol 2.8% apoB-depleted 2 study cohorts: Negative

macrophages serum correlation between
(total HDL Cross-sectional cohort: CEC, prevalent
protein) healthy subjects undergoing carotid-intima
investigation for media thickness,
subclinical atherosclerosis and prevalent CAD
(carotid-intima media thickness)

Case-control cohort:
recruited from subjects undergoing
invasive coronary angiography:
cases with luminal stenosis N50%
matched with controls

Li et al 32 J774 [ 14C]-cholesterol 2.8% apoB 2 study groups: Group A:

macrophages depleted serum
[ 3H]-cholesterol Case-control group A: patients
RAW 264.7 with stable CAD undergoing
macrophages invasive coronary angiography
or computed tomographic
coronary angiography
Case-control group B: patient
population from a cardiology
prevention clinic (subjects with
stable CAD and a matched
control group)
The negative association
between CEC and
prevalent CAD was
attenuated when adjusted
for traditional CVD risk factors.
However, in this group, CEC
was associated with increased
prospective risk for
myocardial infarction,
stroke, and death
over a 3-y period.
Group B: CEC was
negatively associated
with prevalent CAD.

Rohatgi et al 33 J774 BODIPY-cholesterol 2.8% apoB-depleted serum Prospective study of subjects Negative association
macrophages derived from the Dallas between CEC and
Heart Study population primary end point of
incident atherosclerotic
CVD (first nonfatal
myocardial infarction,
nonfatal stroke,
or coronary revascularization
or death from
cardiovascular causes)

Saleheen et al 34 J774 [ 3H]-cholesterol 2.8% Prospective case-control study Negative association between
macrophages apoB-depleted of subjects derived from the CEC and incident fatal and
serum EPIC-Norfolk population nonfatal coronary heart disease
based study

Abbreviations: BODIPY-cholesterol, boron-dipyrromethene (fluorescently labeled cholesterol); J774 macrophages, mouse macrophage cell line; [3H]-cholesterol/[14C]-cholesterol,
radiolabeled cholesterol; RAW 264.7 macrophages, Abelson leukemia virus transformed mouse cell line.

assessment of HDL particle size distribution and to determine
if this is a more accurate predictor of atherosclerotic risk than
measurement of CEC.
Cholesterol efflux capacity and small
HDL particle dysfunction in CAD and risk
factors for CVD
In addition to differences between the efflux activity of
large and small HDL particles, changes in particle compo-
sition are associated with reduced efflux activity. There is
growing evidence for a correlation between small HDL
particle dysfunction and both established CAD and medical
conditions associated with an increased risk of CVD.
Acute coronary syndrome
Specific HDL subpopulations (larger HDL2a and smaller
3a and 3b) from subjects with ST-segment elevation acute
coronary syndromes (STEACSs) were associated with
impaired function shown by a significant reduction in

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American Heart Journal
Volume 180
capacity to promote cholesterol efflux from an in vitro cell
line, compared with age and sex matched normolipidae-
mic individuals. 40
In those with STEACS, alterations in HDL protein and
lipid composition were mostly prominent in the small HDL
particle subpopulation (HDL3b). This included enrichment
with proinflammatory proteins and lipids, and depletion of
apoA-I. 40 Enrichment with proinflammatory proteins and
lipid was negatively correlated with HDL-mediated cell
cholesterol efflux. 40 This suggests that the remodeling
(change in physiochemical properties) of HDL in STEACS
translates to small-particle HDL dysfunction. 40
Diabetes mellitus
In a study of overweight, poorly controlled type 2 diabetes
mellitus (DM) subjects without established CVD, nuclear
magnetic resonance spectroscopic analysis of the HDL
fraction showed that most of the particles were small (59%),
with medium particles making up 25% and large particles
16% of the total HDL fraction. 41 Medium-size HDL particles
significantly correlated with CEC, independent of HDL-C and
apoA-I. However, despite the greater abundance of small
HDL particles, there was no correlation between the
concentration of small HDL particles and CEC. 41 This
could be explained by small-particle HDL dysfunction in
subjects with DM. Other studies have demonstrated
reducedc cell cholesterol efflux by small HDL particles
from DM subjects relative to that of nondiabetic control
particles. 42,43
3Reduced CEC in DM subjects may be related to a
change in HDL composition and nonenzymatic glycosyl-
ation of apoA-I. 44High-density lipoprotein composition is
modified in DM, with triglyceride enrichment and
depletion of cholesterol esters which results in altered
binding of apoA-I to the HDL surface and an unstable HDL
particle that is susceptible to rapid clearance through the
kidney. 45 This reduces the total concentration of HDL
particles available for cell cholesterol efflux. However,
not all studies report reduced CEC in diabetes. At least 2
studies have reported increased CEC in patients
with DM. 46,47
Autoimmune disease
It is established that autoimmune rheumatic diseases,
including rheumatoid arthritis (RA) and systemic lupus
erythematosus (SLE) are associated with an increased risk
of atherosclerosis and cardiovascular mortality, indepen-
dent of traditional cardiovascular risk factors. 48-50 It is
also known that HDL function is altered in the setting of
autoimmune inflammatory disease.
ApoB-depleted serum from subjects with SLE and RA
has reduced capacity to promote cellular cholesterol
efflux particularly via ABCA1 and ABCG1, respectively. 51
It has been previously shown that there is a shift toward
larger HDL particle size in SLE 52,53 which may be
relevant if smaller HDLs are functionally more important.
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Anastasius et al 59
Summary
Further knowledge of the role of specific HDL
subpopulations in promoting cell cholesterol efflux and
how they change in disease states is important to guide
the development of therapeutic agents or novel HDL
analogues to improve HDL function. Further studies will
be needed to see if alternative study end points such as
HDL subpopulations will be able to replace the need for
HDL functional assays.
Cholesterol efflux capacity and
relationship to HDL composition
The composition of the HDL particle is also an
important determinant of CEC. Investigation of asymp-
tomatic hypercholesterolemic male subjects showed that
the CEC of subjects with both high and normal HDL-C
levels was positively correlated with HDL-phospholipid
(HDL-PL) concentration. 54 Multiple regression analysis
examining the association between lipid parameters and
CEC demonstrated that the only parameter to remain
significantly associated with CEC was HDL-PL. 54 These
findings suggest that HDL-PL best explains the capacity of
human serum to stimulate cellular cholesterol efflux. 54
Interestingly, in a study of subjects with high HDL-C and
CAD, HDL-PL concentration and CEC were significantly
reduced in those with CAD compared with healthy
controls. 55 In addition, HDL-PL significantly predicted
CEC. 55 Given these findings, it is plausible that the reduced
HDL-PL levels contribute to the impaired CEC of HDL in this
patient group. 55
Cholesterol efflux capacity in familial
hypercholesterolemia
Familial hypercholesterolemia (FH) is an important cause
of premature CVD. 56It results from a mutation of the LDL
receptor and a subsequent reduction in the number of
functioning receptors. 57 This leads to diminished clearance
of LDL cholesterol from the circulation and increased
deposition in arterial walls triggering accelerated athero-
sclerosis. Familial hypercholesterolemia subjects showed a
significant reduction in HDL2 (large particle) and HDL3
(small particle) concentration when compared with age-
and sex-matched normolipidemic subjects. 58Compared
with controls, HDL2 and HDL3 from FH patients demon-
strated impaired CEC via the SRB1 transporter, and HDL2
alone showed diminished ability to stimulate cholesterol
efflux via the ABCG1 transporter. 58 Furthermore, a significant
negative correlation was found between HDL2-mediated
cholesterol efflux via the SRB1 pathway and premature
atherosclerosis as measured by carotid-intima media
thickness. 58 The impaired capacity of HDL to remove
excess cholesterol from macrophages in the arterial wall
may contribute to elevated cardiovascular risk in FH
subjects. 58

60 Anastasius et al
Cholesterol efflux capacity and
coronary artery plaque burden
measured by coronary computed
tomographic angiography
Patients with psoriasis are at increased risk for
accelerated atherosclerosis and CVD events. 59,60 Among
patients with psoriasis, there is evidence for a correlation
between impaired HDL function and coronary athero-
sclerosis. 61 A cohort of 101 subjects with psoriasis
underwent coronary computed tomographic angiogra-
phy to assess coronary artery plaque burden. Subjects
with low CEC had higher total burden of coronary plaque
and noncalcified plaque burden compared with those
with high CEC. 61Cholesterol efflux capacity correlated
inversely with noncalcified plaque burden, independent
of traditional cardiovascular risk factors and HDL-C. 61
Thus, there is emerging evidence to support the
correlation between CEC and coronary computed
tomographic angiography–detected CAD.
Cholesterol efflux capacity and
relationship to exercise, diet, and weight loss
There is evolving evidence for modulation of CEC with
level of exercise, dietary modification, and weight loss
interventions. It has been shown that CEC is significantly
enhanced after increased physical activity compared with
control subjects with a sedentary lifestyle, who were
matched for age and anthropometric parameters. 62In
addition to this, engagement in cardiac rehabilitation for
6 months after hospital admission for acute coronary
syndromes resulted in significantly increased CEC,
compared with those who failed to complete the
rehabilitation program. 63
Diets enriched with monounsaturated and polyunsatu-
rated fat result in enhanced CEC. 64,65 Also, in a
randomized controlled trial, diet supplemented with
polyphenol-rich olive oil as opposed to low-polyphenol--
content olive oil lead to enhanced capacity of HDL to
perform cell cholesterol efflux. 66
Obesity surgery with Roux-en-Y gastric bypass pro-
duces sustained weight loss. Obese subjects 12 weeks
after undergoing this procedure demonstrated signifi-
cantly enhanced CEC compared with body mass index–
matched control subjects. 67
Further studies in larger patient populations are
required to corroborate the findings of these studies
that are limited by relatively small sample sizes.
Limitations of current assays of CEC and
future directions
Cholesterol efflux capacity as a measure of HDL
function may help our understanding of the relationship
between HDL function and atherosclerotic CVD events.
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American Heart Journal
October 2016
However, there are limitations to this in vitro measure of
HDL function. First, CEC does not precisely reflect the
complex dynamic in vivo processes of HDL metabolism.
Second, the assay only measures the capacity of serum to
accept cholesterol and does not assess the ability of
macrophages from an individual human subject to release
cholesterol. For instance, changes of in vivo macrophage
cellular function resulting from conditions associated
with increased cardiovascular risk, such as DM 68 and
autoimmune disease, would not be accounted for in the
in vitro CEC assay. Third, the measurement of CEC is
simplified by assuming that movement of radiolabeled
cholesterol is equivalent to the movement of mass
cholesterol and that it is unidirectional. However, as in
any equilibrium, movement of cholesterol between a cell
and a cholesterol acceptor is bidirectional. This bidirec-
tional movement is not accounted for in the currently
used standard cholesterol efflux assay. However, the
effect of cholesterol influx on CEC measurement can be
minimized by limiting the duration of the assay. 69
Perhaps the most important limitation is that choles-
terol efflux assays may provide discrepant results
depending on individual assay conditions. For example,
CEC measurement using different forms of labeled
cholesterol (radiolabel vs fluorescence label) can yield
differing findings. 70 Very few conventional clinically
useful assays routinely rely on cell culture techniques,
and achieving consistency of biological effect can be
challenging even in experienced hands. Furthermore, at
present, the cholesterol efflux assay is not readily
applicable as a high-throughput assay for use in routine
clinical practice.
How does CEC relate to the effects of
pharmacologic therapies?
Pharmacologic agents have been shown to affect CEC.
Stimulation of a class of nuclear receptors (LXR) leads
to up-regulation of cholesterol transport pathways
(ABCA1 and ABCG1) and enhanced macrophage choles-
terol efflux in vitro 71 and atherosclerosis regression in
mice. 72However, application of LXR agonism in vivo has
been limited by hepatic steatosis and increased levels of
plasma triglycerides. 73
Agonists of the peroxisome proliferator–activated recep-
tor (PPAR) have been shown to modulate CEC. Pioglitazone,
a PPAR-γ agonist, up-regulates the ABCA1 cholesterol
transporter 74 and increases cholesterol efflux from macro-
phages. 75 In a randomized placebo-controlled study, pioglit-
azone treatment for subjects with the metabolic syndrome
for 12 weeks significantly enhanced CEC. 31 Treatment with
fenofibrate, a PPAR-α agonist, in a randomized double-blind
parallel group study for 8 weeks resulted in significantly
enhanced ABCA1-mediated cholesterol efflux. 76 Recently,
treatment for metabolic syndrome subjects with a more
potent and specific PPAR-α agonist (LY518674) for 8 weeks
American Heart Journal
Volume 180
in a randomized placebo controlled study led to significantly
increased cholesterol efflux from macrophages despite no
change in HDL-C levels. 77
There is limited literature addressing change in CEC
after treatment of underlying cardiovascular risk factors
associated with impaired CEC. Comparison of 70 RA
subjects before and after 6 months of treatment with
methotrexate (disease-modifying antirheumatic drug),
adalimumab (anti–tumor necrosis factor α monoclonal
antibody), or tocilizumab (monoclonal antibody against
interleukin-6 receptor) in an uncontrolled longitudinal
study showed significant reduction in RA disease activity;
however, there was no significant change in CEC. 78 In a
subgroup of RA subjects with CEC below the mean at
baseline, however, treatment with tocilizumab was
associated with an improvement in CEC. 78
At present, there is no convincing evidence that a
specific pharmaceutical strategy to promote cellular
cholesterol efflux leads to an improvement in CVD
outcomes. Treatment for a high-risk CVD population
for 12 weeks with a CETP inhibitor, evacetrapib,
either as monotherapy or in combination with a statin
compared with placebo, led to significant improve-
ment in CEC. 79 However, a phase 3 randomized
double-blind, placebo-controlled trial of evacetrapib
treatment in this high-risk CVD population did not lead
to improvements in CVD outcomes, and the study was
prematurely discontinued. 80 Treatment with a novel
CETP inhibitor (TA-8995) has also shown significant
improvement in CEC 81 . These recent studies showing
increased CEC in serum of patients receiving a CETP
inhibitor raise uncertainty about whether increased
CEC will consistently track with improved cardiovas-
cular risk given negative trials to date with CETP
inhibitors. 12,14,79,80
Conclusion
Cholesterol efflux capacity as a measure of HDL
function has been correlated with the prevalence and
incidence of CVD, independent of HDL-C concentra-
tion. This measure of HDL function has been shown to
add incremental value in CVD risk prediction models.
Further validation for the use of this biomarker will
require standardizing cholesterol efflux measurements
across laboratories and establishing the reproducibility of
CEC. The growing understanding of CEC and its
application to specific populations may help risk
stratification and also lead to development of novel
therapeutic approaches for CVD.
Acknowledgements
None.
The authors are solely responsible for the design and
conduct of this manuscript, including the drafting and
editing of the manuscript, and its final contents.
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Anastasius et al 61
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