High Density Lipoproteins and Arteriosclerosis

Role of Cholesterol Efflux and Reverse Cholesterol Transport

Arnold von Eckardstein, Jerzy-Roch Nofer, Gerd Assmann

Abstract—High density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease, and HDL
exerts various potentially antiatherogenic properties, including the mediation of reverse transport of cholesterol from
cells of the arterial wall to the liver and steroidogenic organs. Enhancement of cholesterol efflux and of reverse
cholesterol transport (RCT) is considered an important target for antiatherosclerotic drug therapy. Levels and
composition of HDL subclasses in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes,
lipid transfer proteins, receptors, and cellular transporters. In vitro experiments as well as genetic family and population
studies and investigation of transgenic animal models have revealed that HDL cholesterol plasma levels do not
necessarily reflect the efficacy and antiatherogenicity of RCT. Instead, the concentration of HDL subclasses, the
mobilization of cellular lipids for efflux, and the kinetics of HDL metabolism are important determinants of RCT and
the risk of atherosclerosis. (Arterioscler Thromb Vasc Biol. 2001;21:13-27.)
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Key Words: apolipoproteins 䡲 lipid transfer proteins 䡲 lipases 䡲 ABC transporter 䡲 Tangier disease

A low level of HDL cholesterol is an important cardiovas-

cular risk factor.1 Moreover, HDLs exert various poten-
tially antiatherogenic properties.2– 4 As a consequence, thera-
peutic modifications of HDL cholesterol (HDL-C) levels
have attracted considerable interest. Drugs increasing HDL-C
are sought for antiatherogenic therapies, and drugs decreasing
HDL-C are suspected to increase cardiovascular risk.4
Reverse cholesterol transport (RCT) describes the metab-
olism and an important antiatherogenic function of HDL,
namely, the HDL-mediated efflux of cholesterol from non-
hepatic cells and its subsequent delivery to the liver and
steroidogenic organs, in which it is used for the synthesis of
lipoproteins, bile acids, vitamin D, and steroid hormones.2– 4
Approximately 9 mg cholesterol per kilogram body weight is
synthesized by peripheral tissues every day and must be
moved to the liver for effective catabolism.5 Distortion of
RCT can favor the deposition of cholesterol within the
arterial wall and thereby contribute to the development of
arteriosclerosis. This ⬎30-year-old concept of John Glomset6
has been much revived only recently by the identification of
several important players in this metabolic pathway, which is
reviewed in the present article. Because of the limitation of
space, additional potentially antiatherogenic functions of
HDL will be reviewed by us in detail elsewhere.
Epidemiological Background
Numerous clinical and epidemiological studies have demon-
strated the inverse and independent association between
HDL-C and the risk of coronary heart disease (CHD).1 More
than 40% of patients with myocardial infarction have low
HDL-C as a cardiovascular risk factor.7 In the prospective
and multicentric European Concerted Action on Thrombosis
and Disabilities (ECAT) Angina Pectoris Study, we identified
low HDL-C and low apoA-I as the most important biochem-
ical risk factors for coronary events in patients with angio-
graphically assessed CHD.8 By convention, the risk threshold
value of HDL-C has been defined as 35 mg/dL (0.9 mmol/L)
in men and 45 mg/dL (1.15 mmol/L) in women.9 –11 Because
of interaction, the strength of the association between HDL-C
and cardiovascular risk depends on the presence of additional
risk factors. Therefore, threshold values are higher in men
with diabetes mellitus or hypercholesterolemia or in the
presence of multiple risk factors.4 Low HDL-C has been
identified as the most frequent familial dyslipoproteinemia in
patients with premature myocardial infarction.12 Finally, in
the Helsinki Heart Study13 and the High-Density-Lipoprotein
Cholesterol Intervention Trial of the Department of Veterans
Affairs (VA-HIT) study,14 increases of HDL-C on treatment
with gemfibrozil were correlated with the prevention of CHD
events. Thus, HDL-C has become an important component of
algorithms to assess the global cardiovascular risk of patients
and also a target for therapeutic intervention and for the
definition of treatment goals.9,10 However, in contrast to LDL
cholesterol and smoking, it is as yet unknown whether the
epidemiological association between HDL-C and CHD is
causal.
Several arguments against a causal relationship must be
taken into account: First, the association between HDL-C and

Received May 22, 2000; revision accepted October 18, 2000.

From the Institut für Klinische Chemie und Laboratoriumsmedizin (A.v.E., J.-R.N., G.A.), Zentrallaboratorium, Westfälische Wilhelms-Universität
Münster, and the Institut für Arterioskleroseforschung an der Universität Münster (A.v.E., J.-R.N., G.A.), Münster, Germany.
Correspondence to Dr Arnold von Eckardstein, Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische
Wilhelms-Universität Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany. E-mail vonecka@uni-muenster.de
© 2001 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

13
 14 Arterioscler Thromb Vasc Biol. January 2001
cardiovascular mortality is U-shaped rather than linear; ie,
cardiovascular death rates are higher in individuals with very
high levels of HDL-C (eg, fifth quintile) than in individuals
with intermediate levels of HDL-C (eg, third quintile)15
(Figure I, available online at http://atvb.ahajournals.org).
Second, in ecological studies, considerable differences in
HDL-C between ethnic populations did not explain the
differences in cardiovascular morbidity and mortality (eg,
differences between Germany and Israel).16 Third, in some
but not all populations (eg, in Germans but not in Turks),17
low HDL-C is frequently found as a component of the
metabolic syndrome, ie, together with hypertriglyceridemia,
small dense LDL, glucose intolerance or overt diabetes
mellitus, hypertension, overweight or obesity, and hyperin-
sulinemia. Insulin resistance is considered the common soil of
these cardiovascular risk factors. As a consequence, insulin
resistance and/or the proatherogenic components of the met-
abolic syndrome rather than an antiatherogenic function of
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HDL may underlie the inverse association between HDL-C
and cardiovascular risk.18 The coincidence of low HDL-C
with many additional risk factors puts patients with the
metabolic syndrome at high global risk for coronary events.
This is in contrast to individuals who have low HDL-C as an
isolated risk factor.4,11 Fourth, as a negative acute-phase
reactant, low HDL-C can serve as a surrogate marker for a
systemic inflammation, which causes or aggravates athero-
sclerosis (eg, smoking or chronic infections), or as a disease
marker for local inflammation, especially in unstable
plaques.19 In support of the importance of this association,
low HDL-C is a more powerful risk factor in short-term
follow-ups than in long-term follow-ups of prospective epi-
demiological studies. For example, the relative risk of CHD
events associated with HDL-C ⬍35 mg/dL (⬍0.9 mmol/L)
was 6.1 in a 2-year follow-up of the Prospective Cardiovas-
cular Münster (PROCAM) study but only 2.1 to 2.7 in longer
follow-ups (Figure II, available online at http://atvb.ahajour-
nals.org). In the ECAT Angina Pectoris Study, HDL choles-
terol had no association with coronary events when plasma
levels of C-reactive protein or fibrinogen, which are positive
acute-phase reactants, were low.8 Also, in the PROCAM
study, HDL-C had a significant association with cardiovas-
cular and overall mortality in smokers but not in nonsmokers
(Figure I). Fifth, the elevation of HDL-C with statins,
fibrates, or estrogens was correlated with the prevention of
CHD events in some trials (ie, Helsinki Heart Study and
VA-HIT)13,14 but not in other trials (Bezafibrate Infarction
Prevention [BIP], Scandinavian Simvastatin Survival Study
[4S], Cholesterol and Current Events [CARE], West of
Scotland Pravastatin Trial [WOSCOP], Air Force/Texas Cor-
onary Atherosclerosis Prevention Study [AFCAPS/Tex-
CAPS], and the Heart and Estrogen/Progestin Replacement
Study [HERS]).20 –24 Moreover, most of these interventions
have only moderate effects on HDL-C levels, and none of
them is specific for HDL, but they exert a broad scope of
mostly antiatherogenic but sometimes even proatherogenic
effects on lipid metabolism, the hemostatic system, and
inflammation.
In summary, the data from observational and interventional
studies demonstrate that the increased coronary risk associ-
ated with low HDL-C does not necessarily reflect a defective
antiatherogenic repair system and that therapeutically in-
duced changes in HDL-C may not necessarily lead to ex-
pected changes in clinical outcomes. Despite this skepticism
toward HDL-C as a suitable intermediary phenotype for the
monitoring of the cardiovascular effects of therapeutic inter-
ventions, it is still believed that modulation of HDL metab-
olism and the RCT system might be an important target for
antiatherosclerotic drug therapy.
HDL Subclasses
HDL encompasses a heterogeneous class of lipoproteins,
which have in common a high density (⬎1.063 g/mL) and a
small size (Stoke’s diameter 5 to 17 nm).25 The majority of
the HDL particles contain apoA-I. Differences in the quanti-
tative and qualitative content of lipids, apolipoproteins, en-
zymes, and lipid transfer proteins (Table 1) result in the
presence of various HDL subclasses, which are characterized
by differences in shape, density, size, charge, and antigenic-
ity.25 After agarose gel electrophoresis of plasma and anti–
apoA-I immunoblotting, the majority of apoA-I is present in
a fraction, which migrates with ␣-electrophoretic mobility
and is designated ␣-LpA-I. This fraction contains eventually
all of the cholesterol, which is quantified in the routine
laboratory as HDL-C. About 5% to 15% of apoA-I in human
plasma is associated with particles, which have electro-
phoretic pre-␤ mobility and which can be further distin-
guished by subsequent polyacrylamide gradient gel electro-
phoresis into pre-␤1-LpA-I, pre-␤2-LpA-I, and pre-␤3-LpA-I
(Figure III, available online at http://atvb.ahajournals.org).
Pre-␤1-LpA-I is the smallest particle, discoidal in shape, and
contains apoA-I either as a lipid-free apolipoprotein or in
association with a few molecules of sphingomyelin and
phosphatidylcholine.26,27 Similar lipid-poor particles contain
only apoE (␥-LpE) or apoA-IV (LpA-IV) as their only
apolipoproteins.28 –30 It is also important to note that relative
to the concentration of lipid-rich ␣-HDL, the concentration of
these lipid-poor particles is increased in extravasal compart-
ments including the lymph, where RCT is initiated in
vivo.27,31,32 ␣-HDL can be further differentiated by ultracen-
trifugation according to density (HDL2 and HDL3), by non-
denaturing polyacrylamide gradient gel electrophoresis ac-
cording to size, or by immunoaffinity chromatography
according to apolipoprotein composition (eg, LpA-I/A-II and
LpA-I).25
HDL Metabolism
Lipid-rich ␣-HDLs arise from lipid-poor particles or even
lipid-free apolipoproteins.25–27,33 These lipid-poor HDL pre-
cursors are produced either as nascent HDL by hepatocytes34
and the intestinal mucosa,35 or they dissociate from chylomi-
crons and VLDL during lipoprotein lipase–mediated hydro-
lysis of triglycerides36 or are generated by the interconversion
of HDL2 and HDL3 by cholesteryl ester transfer protein
(CETP),37,38 phospholipid transfer protein (PLTP),39,40 and
hepatic lipase (HL)41 (Figure 1, Table 1). Lipid-free apoli-
poproteins or lipid-poor particles acquire phospholipids and
unesterified cholesterol from hepatic and nonhepatic cells.42
It is not known whether this lipidation occurs intracellularly
or extracellularly or both. On the one hand, lipid-free apoli-
poproteins were shown to induce phospholipid and choles-
terol efflux from various cells, including hepatocytes and
macrophages, which suggests extracellular assembly.43,44 On
 von Eckardstein et al
Figure 1. Pathways involved in the generation and conversion
of HDL. Mature HDL3 and HDL2 are generated from lipid-free
apoA-I or lipid-poor pre-␤1-HDL as the precursors. These pre-
cursors are produced as nascent HDL by the liver or intestine or
are released from lipolysed VLDL and chylomicrons or by inter-
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conversion of HDL3 and HDL2. ABC1-mediated lipid efflux from
cells is important for initial lipidation; LCAT-mediated esterifica-
tion of cholesterol generates spherical particles that continue to
grow on ongoing cholesterol esterification and PLTP-mediated
particle fusion and surface remnant transfer. Larger HDL2 parti-
cles are converted into smaller HDL3 particles on CETP-
mediated export of cholesteryl esters from HDL onto apoB-
containing lipoproteins, on SR-BI–mediated selective uptake of
cholesteryl esters into liver and steroidogenic organs, and on
HL- and EL-mediated hydrolysis of phospholipids. HDL lipids
are catabolized either separately from HDL proteins (ie, by
selective uptake or via CETP transfer) or together with HDL pro-
teins (ie, via uptake through as-yet-unknown HDL receptors or
apoE receptors). The conversion of HDL2 into HDL3 and the
PLTP-mediated conversion of HDL3 into HDL2 liberated lipid-free
or poorly lipidated apoA-I. A part of lipid-free apoA-I undergoes
glomerular filtration in the kidney and tubular readsorption
through cubilin. For further details, see text and Table 1. Blue
arrows represent lipid transfer processes, and red arrows repre-
sent protein transfer processes. TGRL indicates triglyceride-rich
lipoproteins.
the other hand, macrophages, hepatocytes, and fibroblasts
were shown to internalize lipid-free apolipoproteins, HDL,
and chylomicron remnants and to resecrete lipidated apoli-
poproteins.45,46 This process has been termed retroendocyto-
sis and was found to be defective in Tangier disease,47 in
which mutations in the ATP binding cassette transporter 1
(ABC1) interfere with cellular lipid efflux and lead to the
absence of lipid-rich ␣-HDL from plasma.48 –52 Because
ABC1 is expressed in many cells, including hepatocytes and
enterocytes,53,54 this protein probably plays an important role
not only in lipid efflux from peripheral cells but also in the
hepatic and intestinal birth of HDL.
Lipid-poor HDL precursors become mature, lipid-rich, and
spherical ␣-LpA-I by the acquisition of phospholipids and
unesterified cholesterol from either cells or apoB-containing
lipoproteins, by the lecithin:cholesterol acyltransferase
(LCAT)-mediated esterification of cholesterol, and by the
association of additional apolipoproteins.33,55–57 The initial
products are small HDL3 particles, which on esterification of
cholesterol through LCAT and fusion with other HDL3
particles through PLTP and PLTP-mediated acceptance of
surface remnants of triglyceride-rich lipoproteins grow into
larger HDL2 particles33,58 – 60 (Figure 1, Table 1).
Lipids or proteins of ␣-HDL are removed from the circu-
lation by at least 2 direct pathways, which involve the
HDLs and Arteriosclerosis 15
selective uptake of lipids by scavenger receptor B1 (SR-BI)
and the holoparticle uptake by apoE or apoA-I receptors,
respectively, and 2 indirect pathways, which involve the
action of CETP, HL, and endothelial lipase (EL) (Figure 1,
Table 1).61– 67 (For details see below.)
The removal of lipids from HDL2 by SR-BI, CETP, and
HL, the subsequent conversion of HDL2 into HDL3, and the
conversion of HDL3 into HDL2 by PLTP regenerate pre-␤1-
LpA-I or lipid-free apoA-I.26,33,37– 40 These small apolipopro-
teins or particles can leave the plasma into the extravascular
space,25–27 where they serve as acceptors of cellular lipids and
thus again initiate the generation of HDL. In the kidney, these
small particles are filtered and removed from the plasma. The
reuptake of apoA-I from the proximal tubulus lumen is
mediated by cubilin68 –70 (Figure 1, Table 1).
Cholesterol Efflux
Cellular Cholesterol Trafficking
The uptake of modified lipoproteins by macrophages of the
vascular wall plays an important role in the pathogenesis of
atherosclerosis because accumulation of lipids turns them
into activated foam cells, which produce various growth
factors, cytokines, and proteases and thereby influence the
course of atherosclerosis. Macrophages internalize modified
lipoproteins (especially by type A scavenger receptors) and
cell debris (by phagocytosis). Neither process is regulated by
cholesterol. In consequence and in contrast to other cells
(which limit their cholesterol content by a finely tuned,
sterol-regulated interplay of endogenous cholesterol synthesis
and LDL receptor–mediated uptake of exogenous lipopro-
teins), macrophages accumulate lipids. Released from cho-
lesteryl esters by lysosomal acid lipase, unesterified choles-
terol is transferred to the endoplasmic reticulum (ER) either
directly or indirectly via the plasma membrane. In the ER,
cholesterol is esterified by acyl coenzyme A:cholesterol
acyltransferase (ACAT) to protect the cell from the cytotox-
icity of excess unesterified cholesterol.71–73 Cholesteryl esters
formed by ACAT appear as cytosolic lipid droplets, which
give lipid-laden macrophages their foamy appearance. Cyto-
solic cholesteryl esters can be hydrolyzed by neutral cho-
lesteryl ester hydrolase (NCEH), which is activated by a
cAMP-dependent protein kinase A.74 Cholesterol released by
NCEH is transferred to the cell membrane, where it can be
transported to the ER for reesterification by ACAT.71–73 This
cycle of cholesterol and cholesteryl esters between ACAT
and NCEH is interrupted by the presence of extracellular
cholesterol acceptors, including HDL, which cause cholester-
ol efflux.42,71–73,75,76
The plasma membrane, from which cholesterol is ulti-
mately released into the extracellular space, accounts for 60%
to 90% of unesterified cholesterol in mammalian cells, 95%
of which is localized in the cytofacial leaflet of the bilayer
membrane.72,73 The lateral distribution of cholesterol within
the plasma membrane is organized into microdomains.77
Coated pits, ie, clathrin-stabilized plasma membrane invagi-
nations that contain lipoprotein receptors, have less choles-
terol and sphingolipids than the rest of the plasma membrane.
In contrast, caveolae, detergent-resistant plasma membrane
invaginations that are characterized by the presence of caveo-
lins but the absence of clathrin, are enriched in cholesterol
 16 Arterioscler Thromb Vasc Biol. January 2001

TABLE 1. Characteristics of Pivotal Genes/Proteins Involved in RCT

Regulation

Gene/Protein Locus Structure* Function in RCT Major Sites of Synthesis Up Down

Apolipoproteins
ApoA-I 11q13-qter 243 Major protein component of HDL Liver, small intestine E2, T3, Testosterone,
activation of LCAT, stimulation of glucocorticoids, TGF-␤, cholic
cholesterol efflux, ligand of HDL IL-6, retinoids, acid
binding sites (eg, SR-BI, ABC1) fibrates
ApoA-II 1q21-qter 77 Major protein component of HDL Liver Retinoids, Thyroid hormone,
ligand of HDL binding site, inhibition fibrates TNF-␣
of HL
ApoA-IV 11q13-qter 376 Activation of LCAT, modulation of LPL, Small intestine Unsaturated fatty Leptin
stimulation of cholesterol efflux acids, bile acids,
peptide YY
ApoC-I 19cen-q13.2 57 Activation of LCAT, inhibition of Liver ? ?
hepatic TGRL uptake
ApoC-II 19cen-q13.2 79 Activation of LPL, inhibition of hepatic Liver ? ?
TGRL uptake
ApoC-III 11q13-qter 79 Inhibition of LPL, inhibition of hepatic Liver Retinoids, Insulin, TNF-␣,
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TGRL uptake cytokines fibrates

ApoD 3p14.2-qter 169 Formation of pre-␤3-LpA-I Liver, spleen, CNS, Testosterone, Estradiol
small intestine, adrenals, glucocorticoids
placenta
ApoE 19cen-q13.2 299 Ligand of apoE receptors, mobilization Liver, macrophages, Cholesterol, E2 Cytokines
of cellular cholesterol in macrophages CNS
and cholesterol efflux
ApoF 12 326 Inhibition of CETP ? ? ?
Enzymes and lipid transfer proteins
LPL 8p22 475 Hydrolysis of triglycerides in TGRL, Muscle, adipose tissue Vitamin D3, Endothelin-1,
generation of HDL precursors and cAMP, insulin, IL-1, IL-11,
surface remnants glucose, ␣1-AR, TNF-␣, IFN-␥,
␤3-AR, FFAs, LPS, PTH, PGE2
fibrates,
thiazolidinediones
HL 15q15-15q22 499 Hydrolysis of triglycerides and Liver Testosterone E2, growth
phospholipids in HDL, cofactor of hormone,
SR-BI in selective uptake, generation dexamethasone
of lipid-free apoA-I fibrates
EL ? 482 Hydrolysis of phospholipids in HDL Endothelium, liver, TNF-␣, IL-1␤,
macrophages shear stress
LCAT 16q22 440 Cholesterol esterification, maturation Liver, testes, CNS IL-6, TGF-␤
of HDL dexamethasone,
FFAs,
corticotropin
CETP 16q12-16q21 476 Exchange of cholesteryl esters and Liver, small intestine, Cholesterol,
triglycerides between HDL and LpB, spleen, macrophages oxysterols,
conversion of HDL, generation of rexinoids
lipid-free apoA-I
PLTP 20q12-q13.1 493 Transfer of phospholipids (surface Liver, endothelium Fibrates, bile
remnants) from TGRL onto HDL, acids, hypoxia
fusion of small HDL, generation of
lipid-free apoA-I
Cellular receptors and transporters
ABC1 9q31 2201 Mediation of cholesterol and Liver, macrophages, Cholesterol, IFN-␥
phospholipid efflux, maturation of HDL intestine, fetal tissues oxysterols,
(ubiquitous) rexinoids, cAMP
SR-BI 12q24.2-qter 509 Mediation of selective uptake and Liver, adrenals, testes, E2 (Kupffer Cholesterol, E2
cholesterol efflux ovary, macrophages cells), cAMP, (hepatocytes),
gonadotropins, glucocorticoids
corticotrophin,
fibrates, PGF2␣
Cubilin 10p12-10p14 3597 Tubular resorption of HDL and apoA-I Renal tubulus cells, Shear stress
intestinum, yolk sac,
placenta
AR indicates adrenergic receptor; CNS, central nervous system; E2, estradiol; FFAs, free fatty acids; IL, interleukin; IFN-␥, interferon-␥; LPS, lipopolysaccharide,
PGE2, prostaglandin E2; PGF2␣, prostaglandin 2␣; PTH, parathyroid hormone; T3, thyroxin; TGRL, triglyceride-rich lipoproteins; TGF-␤, tissue growth factor-␤; and
TNF-␣, tumor necrosis factor-␣.
*Number of amino acids in the mature protein.
 von Eckardstein et al
Figure 2. Regulation of cholesterol efflux from cells. Aqueous
diffusion of unesterified cholesterol (UC) from the plasma mem-
brane onto lipid-rich lipoproteins, albumin, phospholipid vesi-
cles, or cyclodextrins is slow. Binding of HDL to SR-BI leads to
reorganization of cholesterol within the plasma membrane and
facilitates cholesterol efflux. ABC1-mediated efflux of UC and
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phospholipids (PL) onto lipid-free apolipoproteins or lipid-poor
particles is fast and involves the translocation of cholesterol
from intracellular compartments to the plasma membrane. This
transport process appears to involve signal transduction pro-
cesses, ie, the cAMP-mediated activation of a protein kinase A
(PKA) and the degradation of phosphatidylcholine by phospho-
lipase C (PC-PLC) and phospholipase D (PC-PLD). The prod-
ucts DAG and phosphatidic acid (PA) act as second messen-
gers, which activate intracellular transporters either directly or
indirectly by activation of a PKC.
and sphingolipids. Relatively small amounts of unesterified
cholesterol are found in intracellular organelles except in
those that communicate with the plasma membrane (endo-
somes, lysosomes, and trans-Golgi network [TGN]).72 The
TGN is an acceptor of newly synthesized cholesterol from the
ER and exogenous cholesterol from endocytic vesicles, lyso-
somes, and caveolae72,73,78 and subsequently distributes cho-
lesterol and phospholipids either in the form of detergent-re-
sistant and caveolin-containing vesicles (rafts or
cytolipoproteins) or as secretory detergent-soluble
vesicles.72,73,79 – 81
Diverse Cholesterol Efflux Pathways
Cholesterol efflux from cells is the result of unspecific and
passive as well as specific and active processes (Figure
2).30,42,75,76
Protein-free phospholipid vesicles, synthetic cyclodextrins,
albumin, and trypsinized HDL mediate a slow and unsatur-
able cholesterol efflux from all cell types.76,82 This form of
cholesterol efflux is not prevented by partial proteolysis of
plasma membranes or by inhibition of intracellular vesicular
transport.76,81,83 It does not involve specific interactions with
cell surface receptors or the specific activation of cellular
transport processes but simply reflects aqueous diffusion of
cholesterol out of the plasma membrane onto acceptor mol-
ecules.76 Although the loss in cholesterol from the plasma
membrane can be replenished, this form of cholesterol efflux
has little effect in depleting cells of intracellularly stored
cholesteryl esters.76,83
By contrast, lipid-free apoA-I, apoA-II, apoA-IV, apoC,
and apoE and also amphipathic synthetic peptides without
sequence homology to these apolipoproteins cause an efflux
of phospholipids and cholesterol that is fast, saturable, uni-
directional, independent of LCAT, and efficient in reducing
HDLs and Arteriosclerosis 17
the content of cytosolic cholesteryl esters.28,42,75,76,84 – 87 Phos-
pholipid efflux appears to precede cholesterol efflux.76,87 As
a result, apolipoprotein-mediated lipid efflux lipidates apoli-
poproteins and hence produces HDL-like lipoproteins with
electrophoretic pre- ␤ and pre- ␣ mobilities. 43,85
Apolipoprotein-mediated lipid efflux involves unspecific de-
sorption of plasma membrane lipids, ie, microsolubilization,
and specific interactions with plasma membrane proteins.76,88
It is cell specific and takes place only in growth-arrested and
cholesterol-enriched cells.51,89 Lipid-free apolipoproteins re-
move phospholipids and cholesterol from normal human skin
fibroblasts, aortic smooth muscle cells, and macro-
phages28,75,76,83– 87,90,91 but not from erythrocytes, rat or por-
cine smooth muscle cells, or fibroblasts of patients with
Tangier disease.84,91–93 Moreover, lipid-free apolipoprotein-
mediated cholesterol efflux is suppressed by low temperature,
by partial proteolysis of cell membranes, or by interference
with the regular function of the Golgi apparatus and with
intracellular vesicular transport (eg, with monensin or brefel-
din A).83,84 It has been suggested that lipid-free apolipopro-
teins and pre-␤1-LpA-I specifically release cholesterol that is
located in the caveolae.72,94 Activation of protein kinase C
(PKC) enhances and inhibition of PKC suppresses
apolipoprotein-mediated cholesterol efflux.90,95,96 cAMP was
also found to increase apolipoprotein-induced cholesterol
efflux from macrophage cell lines.45,97–99 In RAW264 cells,
this was associated with internalization and resecretion of
apoA-I45 as well as with secretion of apoE.98 It has been
hypothesized that apoA-I binds to a signal-transducing cell-
surface receptor and that this binding facilitates the translo-
cation of cholesterol from intracellular compartments to the
plasma membrane.42,75 The nature of this receptor is un-
known. Because cholesterol efflux from cells and ABC1 are
defective in Tangier disease, it is likely that ABC1 plays an
important role in apolipoprotein-mediated efflux.48 –52,84,92,93
In support of this possibility, apoA-I–mediated cholesterol
efflux is severely decreased by the inhibition of ABC1 with
either antisense oligonucleotides or pharmacological com-
pounds and is increased by the overexpression of ABC1.51,100
In addition to ABC1, other ABC transporters appear to be
involved in apolipoprotein-mediated lipid efflux. For exam-
ple, inhibition of the sterol-regulated half-transporter ABC8
(ABCG1) impairs cholesterol efflux from cells.55
Native and reconstituted lipid-rich HDLs induce specific
and unspecific forms of cholesterol efflux. Partial proteolysis
of either HDL or cells does not fully prevent HDL-mediated
cholesterol efflux, which is slow, unsaturable, and bidirec-
tional and thus appears to occur by aqueous diffusion.28,42,75,76
Esterification of the released cholesterol by LCAT prevents
the rediffusion of cholesterol from HDL back to the plasma
membrane and thus enhances net cholesterol efflux.101 Ex-
pression of SR-BI increases HDL-mediated cholesterol ef-
flux.102 However, SR-BI–mediated cholesterol efflux does
not simply reflect the binding of HDL to cells, inasmuch as
binding of HDL to CD36, which is another HDL binding
scavenger receptor, does not facilitate cholesterol efflux.103
Therefore, it has been suggested that binding of HDL to
SR-BI facilitates the bidirectional flux between HDL and
plasma membrane by reorganization of lipids within choles-
terol- and caveolae-rich domains within the plasma
membrane.103
 18 Arterioscler Thromb Vasc Biol. January 2001
HDL-mediated cholesterol efflux also shares some proper-
ties of lipid-free apolipoprotein-mediated cholesterol efflux.
In the presence of HDL, intracellularly stored cholesteryl
esters are removed.75,76 After incubation of lipid-enriched
cells with brefeldin A or the PKC inhibitor sphingosine,
HDL-mediated cholesterol efflux is reduced by ⬇50%.84,96
Native and reconstituted HDL elicit various signal transduc-
tion pathways, which may activate intracellular lipid transfer
processes.95,96,104 –108 HDL induces the hydrolysis of phos-
phatidylcholine and phosphatidylinositol biphosphates by
phospholipases C and D and thereby the generation of
diacylglycerol (DAG), phosphatidic acid, and inositol phos-
phates. DAG activates PKC, which has been shown to
stimulate the translocation of newly synthesized cholesterol
to the plasma membrane as well as cholesterol ef-
flux.95,96,104,108 By contrast, HDL-mediated breakdown of
phosphatidylinositol biphosphate and the subsequent mobili-
zation of intracellular calcium106,109 does not stimulate cho-
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lesterol efflux but rather mediates the mitogenic effects of
HDL.110 In macrophages, HDL has been shown to increase
the concentration of cAMP, resulting in the activation of
protein kinase A, which may activate the hydrolysis of
cytosolic cholesteryl esters by NCEH and the mobilization
of cholesterol by ABC1.45,74,97–99,111 Another mechanism of
HDL-mediated cholesterol efflux is retroendocytosis, ie, the
uptake of HDL into clathrin-coated endosomes, which is
followed by intracellular enrichment with lipids and
resecretion.45– 47
ABC1 Is a Pivotal Regulator of Cholesterol Efflux
As a complete ABC transporter, ABC1 has 2 highly con-
served cytoplasmic ATP binding cassettes and 2 transmem-
brane domains, each of which consists of 6 transmembrane-
spanning segments.53,112 In analogy with the multidrug
resistance (MDR) protein 1, whose low-resolution structure is
known, it has been suggested that the transmembrane seg-
ments form the wall of an aqueous chamber within the plasma
membrane. This chamber is opened to the extracellular space
and to the lipid phase of the plasma membrane but not to the
cytosol. Substrates of ABC1, ie, cholesterol, phospholipids,
vitamins A, E, and K, anions, and interleukin-1␤, may be
transported through this channel.113–116
ABC1 is expressed in many organs. The highest expression
was found in fetal tissues, placenta, liver, lung, and adrenal
glands.53,54 In cell culture, ABC1 was expressed in confluent
but not in growing fibroblasts, indicating that ABC1-
mediated cholesterol efflux is important in cell quiescence
and other conditions of low need for cholesterol.51
The promotor of the ABC1 gene has been analyzed.112,117
It contains binding motifs for several transcription factors,
including the sterol regulatory binding protein, the liver X
receptor/retinoid X receptor, and activator proteins. In agree-
ment with a regulatory role of these transcription factors,
ABC1 expression and lipid efflux are upregulated by choles-
terol,51,53 oxysterols,117 rexinoids,118 and cAMP ana-
logues.51,99 The linker segment of ABC1 has several serine
and threonine residues that can be phosphorylated and
thereby modulate ABC1 activity posttranslationally.53
In analogy with MDRs, it has been suggested that ABC1
forms a channel within the plasma membrane through which
phospholipids (sphingomyelin, phosphatidylserine, and phos-
phatidylinositol?) and cholesterol are transferred (“flopped”)
from the inner leaflet to the outer leaflet of the plasma bilayer
membrane.100,115 There they may be picked up by lipid-free
apolipoproteins or lipid-poor particles, which may even bind
to ABC1.119,120
HDL and apoA-I have been previously found to be
internalized by macrophages into an endosomal compart-
ment, from which they are resecreted together with lip-
ids.45– 47 Tangier macrophages appear to have a defect in
resecretion and erroneously target internalized HDL to lyso-
somes for degradation.47 For this reason and because of the
presence of hyperplastic Golgi structures within lipid-laden
Tangier macrophages,121 it has been suggested that ABC1
serves as a protein component of vesicles or rafts that target
lipids and proteins between lipid-rich intracellular organelles
(lysosomes and TGN) and the plasma membrane. In fact,
several ABC transporters, such as cystic fibrosis transduc-
tance receptor, Rim, and MDRs, are important for the
trafficking of proteins and lipids between intracellular or-
ganelles and the plasma membrane.122,123
In addition to ABC1, there are several other sterol-
regulated ABC transporters.54,124 One of them, ABC8, also
appears to be involved in cholesterol efflux from macro-
phages.54 It is a so-called half-transporter, inasmuch as it
consists of only 1 ATP binding cassette and 1
transmembrane-spanning domain. To be operative, this half-
transporter needs another half-transporter, which has not yet
been identified.
Role of Endogenous ApoE for Cholesterol Efflux
From Macrophages
Macrophages produce apoE, although to a much lesser extent
than do hepatocytes.125 Transplantation of wild-type bone
marrow126,127 or the selective expression of a human apoE
transgene in macrophages127,128 decreased atherosclerosis in
apoE-deficient mice. Conversely, on a western diet, athero-
sclerosis was promoted in wild-type mice receiving bone
marrow from apoE-deficient mice.129 Because expression of a
human apoE transgene in macrophages of apoE-deficient
mice128 and transplantation of apoE-deficient macrophage
stem cells into wild-type mice129 produced little changes in
plasma lipid levels, macrophage-derived apoE exerts anti-
atherogenic properties largely independent of its effects on
plasma lipoproteins.
One possible explanation for the antiatherogenicity of
macrophage-derived apoE is its contribution to cholesterol
efflux from these cells.125 In vitro, lipid loading and activa-
tion of protein kinase A with 8-bromo-cAMP stimulates the
synthesis and secretion of apoE in macrophages, which in
turn facilitates cholesterol efflux from these cells.98,125,126 –134
ApoE secretion is also stimulated in the presence of exoge-
nous HDL and apoA-I.130,132,135,136 It was originally suggested
that macrophage-derived apoE facilitates cholesterol efflux
by improving the cholesterol acceptor properties of
HDL.125,130 –132 However, at least human monocyte– derived
macrophages secrete apoE together with cellular lipids also
independent of exogenous HDL.133,134 The efficacy of this
pathway depends on the apoE genotype (ie, it is least efficient
in apoE4 macrophages and most efficient in apoE2 macro-
phages).134 Moreover, in the presence of excess exogenous
apolipoproteins, including apoE cholesterol, efflux from
 von Eckardstein et al
apoE-producing mouse peritoneal macrophages or J774 mac-
rophages transfected with human apoE was more effective
than cholesterol efflux from apoE-deficient mouse macro-
phages and untransfected J774 macrophages, respective-
ly.137,138 This suggests that endogenous apoE modulates
cholesterol efflux and cholesteryl ester hydrolysis in macro-
phages by the regulation of intracellular transport
processes.137,138
HDL-Mediated Delivery of Cholesterol to the
Liver and Steroidogenic Organs
Lipids and proteins of mature ␣-HDL are removed from the
circulation by direct pathways, which involve the selective
lipid uptake by SR-BI61,62 and the holoparticle uptake by
apoE receptors, such as cubilin and probably also other
as-yet-unknown HDL receptors,63,139 as well as by indirect
pathways that involve the action of CETP,64 HL,65,66 and
EL.67
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Selective Cholesterol Uptake by SR-BI
SR-BI mediates the selective uptake of cholesteryl esters
from HDL and also LDL into hepatocytes and steroid
hormone–producing cells without internalizing HDL pro-
teins.61,62,140 In contrast to holoparticle receptors (eg, mem-
bers of the LDL receptor gene family or class A scavenger
receptors), which reside in clathrin-coated pits of the plasma
membrane and which direct internalized lipoproteins via
endosomes to lysosomes for degradation, SR-BI is predomi-
nantly found in caveolae and targets internalized lipids into
nonendosomal and nonlysosomal compartments.141 Epitopes
recognized by SR-BI appear to include phospholipids and
apolipoproteins. The mechanism of how SR-BI achieves the
dissociation of lipids and proteins and the incorporation of
cholesteryl esters into the plasma membrane is not yet
understood. Binding of HDL appears to be a necessary but
not sufficient prerequisite, because binding of HDL to CD36,
another member of the SR-B gene family, does not result in
selective lipid uptake.142,143 Thus, it has been suggested that
SR-BI exerts a lipid transfer activity, eg, by forming a
hydrophobic channel through which cholesterol diffuses from
the HDL particle into the plasma membrane.144 Alternatively
or in addition, selective uptake by SR-BI may depend on the
presence of cofactors. Thus, selective uptake of lipids by
SR-BI is facilitated in the presence of HL. HL and other
phospholipases may hydrolyze phospholipids of surface lay-
ers from HDL and plasma membrane and thereby enable the
flux of cholesteryl esters from the lipoprotein core into the
plasma membrane.145,146 Remodeling of HDLs by CETP also
makes HDLs more viable for selective uptake.146 Finally,
selective uptake is reduced in apoE-deficient mice, suggest-
ing that apoE is also involved in the interaction of HDL with
SR-BI.147
As discussed before, SR-BI also mediates cholesterol
efflux from cells.102,103 The net movement of cholesterol into
or out of the cell may depend on the relative activities of
extracellular lipid transfer enzymes (LCAT, PLTP, and
CETP) and intracellular enzymes, which metabolize choles-
terol to cholesteryl esters, bile acids, lipoproteins, or steroid
hormones.
Turnover studies have suggested that selective uptake
accounts for 90% of cholesteryl ester elimination from HDLs
HDLs and Arteriosclerosis 19
of rodents like mice, which do not express CETP, and 20% of
cholesteryl ester elimination from HDLs of species that
express CETP.61,62 In agreement with the important role of
SR-BI in the regulation of HDL metabolism are the results of
studies in genetically modified mice. Knockout of the SR-BI
gene resulted in a 2-fold elevation of HDL-C but an increase
in atherosclerosis.148 Overexpression of SR-BI enhanced
selective cholesteryl ester uptake and HDL catabolism and
severely decreased HDL-C levels.149 In LDL receptor knock-
out mice or apoE knockout mice, overexpression of SR-BI
also led to the disappearance of atherogenic lipoproteins150,151
and inhibited atherosclerosis. Hence, upregulation of SR-BI
in the liver may be antiatherogenic by increasing the catab-
olism of lipids from HDL and atherogenic lipoproteins.
SR-BI is downregulated by cholesterol and estradiol and
upregulated by polyunsaturated fatty acids in parenchymal
liver cells, by adrenocorticotropic hormone in adrenocortical
cells, by gonadotropins in theca interna and interstitial cells of
the preovulatory ovary, in luteinized granulosa cells, and in
the corpus luteum as well as in Leydig cells and less so in
Sertoli cells of the testis.61,62 Interestingly, at least in rats,
in contrast to their effects in hepatocytes, cholesterol and
estradiol stimulate SR-BI in macrophage-like Kupffer
cells.152
Cholesteryl Ester Transfer Protein
CETP exchanges cholesteryl esters of HDL2 with triglycer-
ides of VLDL, IDL, and LDL. The HDL-derived cholesteryl
esters are removed from the circulation via the LDL receptor
pathway.64,153 Triglycerides in HDL are hydrolyzed by HL.
The concerted action of CETP and HL converts larger HDL2
into smaller HDL3 and releases lipid-free apoA-I and/or
pre-␤1-LpA-I.33 The metabolic importance of this pathway in
humans is emphasized by the finding of retarded HDL
catabolism and elevated HDL-C levels in individuals with
CETP deficiency.154 CETP also exchanges cholesteryl esters
between LDL and VLDL. The direction of transfer is mod-
ulated by apoF, which inhibits the transfer between LDL and
VLDL but activates the transfer from HDL to VLDL.153,155
CETP gene expression is upregulated by cholesterol via
activation of the sterol regulatory binding protein and through
oxysterols via binding to the liver X receptor/retinoid X
receptor.156 –158 Responsiveness of CETP to sterols is proba-
bly the basis of reduced cholesteryl ester transfer activity in
plasmas of patients treated with statins.159 Cholesteryl ester
transfer activity is increased in hypertriglyceridemic individ-
uals independently of changes in CETP mass. This is consid-
ered to be an important mechanism that causes low HDL-C in
patients with (postprandial) hypertriglyceridemia and thus in
patients with the metabolic syndrome because of insulin
resistance.18,160
Because CETP enriches LDL with cholesterol and depletes
HDL of cholesterol and because of the initial finding of
CETP deficiency in Japanese families with longevity, CETP
was originally considered to be a proatherogenic modulator
of HDL metabolism. However, the generation of lipid-free
apoA-I,33,37,38 the enhancement of RCT,158 and the transfer of
oxidized lipids161 also indicate antiatherogenic properties.
Data of genetic studies in men and mice are in agreement with
this more complex scenario.162 Despite high HDL-C, CETP
deficiency in hypertriglyceridemic Japanese has been associ-
 20 Arterioscler Thromb Vasc Biol. January 2001

TABLE 2. Genetic Animal Models of HDL Metabolism and Atherosclerosis

Animal Crossed Other
Gene Species Expression With/Expressed in HDL Lipoproteins Arteriosclerosis References
ApoA-I Mouse ApoA-I deficiency A 2 N 194
Mouse ApoA-I deficiency ApoB overexpression 2 1 195
Mouse Human apoA-I A 1 2 198
Rabbit Human apoA-I A 1 2 199
Mouse Human apoA-I ApoE deficiency 1 2 200, 201
Mouse Human apoA-I A 1 2 202
Somatic expression
Mouse Human apoA-I ApoE deficiency 1 2 202
Somatic expression
Mouse Human apoA-I LDL-R deficiency 1 2 203
Somatic expression
ApoA-II Mouse Murine apoA-II A 1 1 213
Mouse Murine apoA-II Murine apoA-I 1 1 214
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overexpression
Mouse Human apoA-II A 1 215
Mouse Human apoA-II A 1 Postprandial 2 216
remnants
ApoA-IV Mouse Human apoA-IV A 1 2 206, 207
Mouse Human apoA-IV ApoE deficiency 1 2 206
LCAT Mouse LCAT deficiency A 2 N 210
Mouse Human LCAT A 1 TG 1 211
Rabbit Human LCAT A 1 TG 2 212
Mouse Human LCAT Human CETP 1 TG N 172
overexpression
CETP Mouse Simian CETP A 2 VLDL⫹LDL1 1 169
Mouse Human CETP Human apoC-III 2 2 170
Mouse Human CETP Human 2 2 170
apoA-I/Human
apoC-III
Mouse Human CETP ApoE deficiency 2 TG1 1 171
Mouse Human CETP ApoE 1 TG1 2 171
deficiency/Human
apoA-I
overexpression
Mouse Human CETP Human LCAT 1 TG2 N 171
overexpression
Rat Human CETP Dahl/salt-sensitive 2 1 173
hypertension
Mouse Human CETP LDL-R deficiency N 1 171
HL Mouse HL deficiency ApoE deficiency 1 ␤-VLDL1 2 182
Mouse Human HL A 2 2 183
SR-BI Mouse SR-BI deficiency ApoE deficiency 1 VLDL2N 1 148
Mouse Murine SR-BI LDL-R deficiency 2 LDL2 2 150, 151
LDL-R indicates LDL receptor; TG, triglycerides; N, normal.

ated with an increased risk of coronary events.163 Likewise,
nonsynonymous alleles of the polymorphic CETP gene in-
creased the risk of CHD events despite being associated with
increased levels of HDL-C.164 –166 In contrast, a Taq1
polymorphism of the CETP gene has repeatedly been
associated with increased HDL-C and decreased risk of
CHD.167 Immunization of hypercholesterolemic rabbits
with CETP decreased plasma CETP activity, increased
HDL-C, and reduced atherosclerosis.168 Overexpression of
a human or a simian CETP transgene decreased HDL-C
and resulted in increased atherosclerosis in some but not
all mouse models (Table 2).169 –173 Thus, whether inhibi-
tion of CETP prevents or enhances atherosclerosis appears
to depend on the presence or absence of other dyslipid-
emias. CETP has been suggested to be proatherogenic in
hypercholesterolemia (especially when the LDL receptor
pathway is ineffective) and antiatherogenic in hypertri-
glyceridemia and hyperalphalipoproteinemia.162
 von Eckardstein et al
HL and EL
HL hydrolyzes phospholipids and triglycerides in all lipopro-
tein classes.65,66 In agreement with the broad substrate and
particle specificity of this enzyme, HL deficiency in men and
knockout of the HL gene in mice result in the accumulation
of apoB-containing remnant-like lipoproteins and
phospholipid- and apoE-rich HDLs.174,175 ApoA-II inhibits
HL so that HDLs devoid of apoA-II are the preferred HDL
particle of HL.176,177 The concerted action of CETP-mediated
cholesteryl ester transfer and the HL-mediated hydrolysis of
triglycerides and phospholipids depletes the core of large
HDL2 and helps to form smaller HDL3 as well as lipid-free
apoA-I and/or pre-␤-HDL.33 Independent of its lipolytic
activity, HL appears to serve as a cofactor in the selective
uptake of HDL lipids, which is mediated by SR-BI145,146 but
also by an additional putative HDL binding site in
hepatocytes.178
In addition to regulation of SR-BI, regulation of HL is an
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important mechanism by which sex steroids affect HDL-C
levels. HL activity is suppressed by estradiol and increased by
testosterone.179,180 Experiences with HL deficiency in men
and genetic animal models suggest that HL exerts both
proatherogenic and antiatherogenic activities.66,175 Several
HL-deficient men have suffered from premature CHD despite
high HDL-C levels.174 Despite the inverse correlation be-
tween HL activity and HDL-C, a low HL activity has been
associated with the presence of atherosclerosis.181 Knockout
of HL led to the occurrence of an atherogenic lipoprotein
phenotype but reduced atherosclerosis in apoE-deficient
mice.182 Transgenic overexpression of HL in either mice or
rabbits decreased HDL-C levels but did not cause atheroscle-
rosis.175,183 The controversial data from genetic animal mod-
els of HL (and SR-BI) demonstrate the difficulty of interpret-
ing the estrogen-induced increase of HDL-C and the
testosterone-induced decrease of HDL-C toward the effects
on atherosclerosis.
By contrast to lipoprotein lipase and HL, EL uses phos-
pholipids as its exclusive substrate.67,184,185 By hydrolysis of
phospholipids in HDL, EL generates free fatty acids, which
are taken up by endothelial cells. In addition, removal of
phospholipids eventually generates smaller HDL particles,
which thereby may become viable for the passage through the
endothelial layer into the extravascular space.67 Transient
overexpression of EL lowers HDL-C.184 Animal models are
needed to evaluate proatherogenic or antiatherogenic effects
of EL.
Catabolism of HDL Apolipoproteins
The removal of the protein constituents of HDL from the
circulation is less well understood than the removal of
HDL-associated lipids. Potential mechanisms for the catabo-
lism of HDL holoparticles and lipid-free apolipoproteins are
endocytosis into liver and kidney cells as well as into placenta
and yolk sac during pregnancy.68
The receptor for the intrinsic factor/vitamin B12, cubilin,
has recently been identified as an HDL/apoA-I binding site in
epithelial cells of the proximal tubulus of the kidney and of
the yolk sac.68 –70 Small HDL particles with a size of ⬍8 nm
and lipid-free apoA-I are filtrated by renal glomeruli into the
primary urine. Because cubilin-deficient men or dogs have
increased excretion of apoA-I in their urine, cubilin has been
HDLs and Arteriosclerosis 21
suggested to mediate the uptake of apoA-I into proximal
tubulus cells.68,69 However, cubilin has no transmembrane
domain and can therefore not mediate the internalization of
its ligands without coreceptors.68 –70 Megalin, a member of
the LDL receptor gene family, has been suggested to play this
role in the kidney and the yolk sac.68,69,186 After internaliza-
tion, the ligands of cubilin (HDL, apoA-I, and the vitamin
B12–intrinsic factor complex) are targeted to lysosomes for
degradation. Thus, it is unlikely that apoA-I of the primary
urine taken up by proximal tubulus cells reenters the plasma
compartment and that cubilin is an important determinant of
HDL-C plasma concentration.68
ApoE-containing HDLs, which constitute the minority of
HDLs, are internalized by hepatic apoE receptors (LDL
receptor and LDL receptor–related protein).63,187 There is
considerable evidence of the presence of additional HDL
receptors on liver cells, which mediate the catabolism of
apoE-free HDL. Ligand blotting studies have identified HDL
binding sites of different size in liver cells, which are
candidates for receptors mediating hepatic HDL holoparticle
uptake.139,188 Morphological studies provided evidence for
the binding and endocytosis of HDL as well. Some authors
have demonstrated resecretion of HDL, which was internal-
ized into liver cells.189,190 Interestingly, hepatic binding,
uptake, degradation, and resecretion of HDL is disturbed in
ob/ob mice, which are deficient in leptin.191 Because ob/ob
mice have elevated HDL-C levels because of retarded HDL
catabolism,192 Tall and colleagues have suggested the pres-
ence of a hepatic leptin-regulated HDL receptor, which
regulates HDL-C levels by mediating holoparticle uptake into
liver cells.191
Genetic Disturbances of HDL Metabolism in Men
and Animal Models
The pivotal role of apoA-I, ABC1, LCAT, PLTP, CETP,
SR-BI, HL, and EL in the regulation of HDL metabolism is
highlighted by the eventually tremendous changes of HDL-C
levels in men with inborn errors of HDL metabolism or in
genetically modified animal models (Tables 2 and 3). How-
ever, changes in HDL-C were not always associated with the
expected inverse changes in atherosclerosis, especially when
the catabolism of HDL was modified. This indicates that
HDL-C levels lack both specificity and sensitivity in assess-
ing the effect of an intervention on atherosclerosis. Rather,
the concentration of various HDL subclasses and thereby the
function of HDL as well as the kinetics of HDL metabolism
appear to influence the course of atherosclerosis.
Many patients with apoA-I null alleles have suffered from
CHD very early in their life.193 Increased atherosclerosis was
also found in one but not in another apoA-I– deficient mouse
model.194,195 Plasmas of apoA-I– deficient men and mice have
half-normal cholesterol efflux capacity.196,197 In mice and
rabbits, transgenic overexpression of the apoA-I gene yielded
the expected result, namely, increased HDL-C levels and
reduced atherosclerosis.198 –203 Somatic overexpression of
apoA-I even induces the regression of preexisting le-
sions.202,203 In line with improved HDL function and accel-
erated RCT, expression of apoA-I transgenes in mice resulted
in an increased cholesterol efflux capacity of plasma.204 In
men, infusion of apoA-I increased fecal sterol excretion.205
Likewise, overexpression of a human apoA-IV transgene
 22 Arterioscler Thromb Vasc Biol. January 2001

TABLE 3. Inborn Errors of HDL Metabolism and Atherosclerosis in Men

Gene Kind of Defect Zygosity HDL Cholesterol CHD Risk References
ApoA-I Null alleles Homozygosity Virtually zero Increased 2, 193
Heterozygosity Half normal Normal 2, 193
Some structural variants Homozygosity Virtually zero Normal/increased 2, 193
Heterozygosity Half normal Normal 2, 193
LCAT LCAT deficiency Homozygosity Virtually zero Normal/increased 193, 208
Heterozygosity Half normal Normal 193, 208
Fisheye disease Homozygosity Virtually zero Normal/increased 193, 208
Heterozygosity Half normal Normal 193, 208
CETP CETP deficiency Homozygosity Strongly elevated Decreased/normal/under some 2, 162, 163, 193
circumstances increased
Heterozygosity Elevated Normal 2, 162, 163, 193
Ile405Val polymorphism Slightly elevated Increased 164, 166
Asp442Gly Elevated Increased 165
polymorphism
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HL HL deficiency Homozygosity Elevated Increased 65, 174

ABC1 Tangier disease Homozygosity Virtually zero Moderately increased 188, 209
Heterozygosity Half normal Normal/increased? 188, 209

reduced atherosclerosis in mice.206,207 These data suggest that
stimulated production of HDL precursors enhances RCT and
protects from atherosclerosis.
Although they have reduced HDL-C levels, SR-BI trans-
genic mice show features of enhanced RCT, namely, in-
creased biliary cholesterol excretion, and are protected from
atherosclerosis.150,151 Vice versa, knockout of SR-BI causes
atherosclerosis despite doubling the HDL-C levels in mice.148
Other inborn errors and genetic manipulation of HDL
metabolism in men and animal models, respectively, showed
less conclusive relationships between HDL metabolism and
atherosclerosis. Defective HDL maturation because of struc-
tural apoA-I variants, Tangier disease, LCAT deficiency, or
fisheye disease caused premature atherosclerosis in some but
not all patients.188,193,208,209 Heterozygotes for the underlying
defects in the genes of apoA-I, LCAT, and ABC1 have
half-normal levels of HDL-C, ie, usually below the cutoff of
35 mg/dL (0.9 mmol/L) but no increased risk of CHD.
Likewise, knockout of LCAT caused HDL deficiency but not
atherosclerosis in mice.210 Overexpression of LCAT in-
creased HDL-C in mice and rabbits but was antiatherogenic
in rabbits and proatherogenic in mice.211,212 High HDL-C
levels in CETP deficiency were originally found to be
associated with reduced CHD risk but were later found to be
associated with increased CHD risk in hypertriglyceridemic
subpopulations.163 Likewise, some genetic variants of
CETP164 –166 were associated with increases in HDL-C and
CHD risk, whereas others showed the expected inverse
association between HDL-C and CHD risk (Table 3). In
agreement with the controversial data in men, overexpression
of CETP decreased HDL-C but had proatherogenic or anti-
atherogenic effects in different mouse models.169 –173 Like-
wise, human HL deficiency as well as transgenic overexpres-
sion or knockout of HL or apoA-II in mice unraveled the
antiatherogenic and proatherogenic effects of HL and apoA-
II, respectively.174,175,182,183,213–216 Thus, whether LCAT,
CETP, or HL is proatherogenic or antiatherogenic appears to
be strongly influenced by additional factors. In addition,
modification of HDL metabolism can influence the metabo-
lism of atherogenic apoB-containing lipoproteins. Thus,
LCAT-deficient men, ABC1-deficient Tangier patients, and
SR-BI– overexpressing mice, all of which do not develop
atherosclerosis despite HDL deficiency, have low levels of
apoB and LDL cholesterol.150,151,188,208 Vice versa, athero-
genic apoB-containing lipoproteins accumulate in men and
mice with HL deficiency and also in LCAT transgenic mice,
which develop atherosclerosis despite high levels of
HDL-C.174,175,211
Concluding Remarks
Enhancement of RCT has a great potential for antiatheroscle-
rotic drug therapy. However, it is not the increase or decrease
of plasma HDL-C concentration per se but rather the concen-
tration of various HDL subclasses, the cellular mobilization
and transport of lipids, and the kinetics of HDL metabolism
that determine the efficacy of RCT and thus the risk of
atherosclerosis. This challenging concept is also applicable to
other antiatherogenic properties of HDL, such as antioxida-
tive, anti-inflammatory, antiadhesive, antiaggregatory, and
profibrinolytic effects. Importantly, the interpretation of data
from recent trials on statins, fibrates, and estrogens should be
cautious in view of the diverse functionality of HDL and the
pleiotropic effects of these drugs. Moreover, functional and
controlled interventional clinical outcome studies are needed
at an early stage to prove the antiatherogenic effects of
HDL-elevating drugs that are currently being developed by
various pharmaceutical companies.
Acknowledgment
Dr Arnold von Eckardstein is supported by a grant from the
European Union on “The Antiatherogenicity of HDL” (grant No.
BIOMED2 BMH4-CT98-3699).
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High Density Lipoproteins and Arteriosclerosis: Role of Cholesterol Efflux and Reverse
Cholesterol Transport
Arnold von Eckardstein, Jerzy-Roch Nofer and Gerd Assmann

Arterioscler Thromb Vasc Biol. 2001;21:13-27

doi: 10.1161/01.ATV.21.1.13
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