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ANNEMA W et al.
REVIEW
Circulation Journal
Official Journal of the Japanese Circulation Society
http://www. j-circ.or.jp
High-Density Lipoproteins
– Multifunctional but Vulnerable Protections from Atherosclerosis –
Wijtske Annema, PhD; Arnold von Eckardstein, MD
Low plasma levels of high-density lipoprotein (HDL) cholesterol are associated with increased risks of coronary artery
disease (CAD). HDL particles exert many effects in vitro and in vivo that may protect arteries from chemical or biological harm or facilitate repair of injuries. Nevertheless, HDL has not yet been successfully exploited for therapy.
One potential reason for this shortfall is the structural and functional complexity of HDL particles, which carry more
than 80 different proteins and more than 200 lipid species as well as several microRNAs and other potentially bioactive
molecules. This physiological heterogeneity is further increased in several inflammatory conditions that increase cardiovascular risk, including CAD itself but also diabetes mellitus, chronic kidney disease, and rheumatic diseases. The
quantitative and qualitative modifications of the proteome and lipidome, as well as the resulting loss of functions or
gain of dysfunctions, are not recovered by the biomarker HDL-cholesterol. As yet the relative importance of the many
physiological and pathological activities of normal and dysfunctional HDL, respectively, for the pathogenesis of atherosclerosis is unknown. The answer to this question, as well as detailed knowledge of structure-function-relationships
of HDL-associated molecules, is a prerequisite to exploit HDL for the development of anti-atherogenic drugs as well
as of diagnostic biomarkers for the identification, personalized treatment stratification, and monitoring of patients at
increased cardiovascular risk. (Circ J 2013; 77: 2432 – 2448)
Key Words: Cholesterol efflux; Endothelium; HDL functionality; Reverse cholesterol transport
I
n both epidemiological and clinical studies, as well as the
meta-analyses thereof, low plasma levels of high-density
lipoprotein (HDL) cholesterol (HDL-C) identified individuals at increased risk of major coronary events.1,2 In line with
a causally protective effect, HDLs exert a broad spectrum of
potentially anti-atherogenic properties. Moreover, atherosclerosis was decreased or even reverted in several animal models by
transgenic overexpression or exogenous application of apolipoprotein (apoA-I),3–5 the most abundant protein of HDL. However, to date, drugs increasing HDL-C, such as fibrates, niacin,
and inhibitors of cholesteryl ester transfer protein (CETP), have
failed to consistently and significantly reduce the risk of major
cardiovascular events, especially when combined with statins.6–10
Moreover, mutations in several human genes as well as targeting of several murine genes modulate HDL-C levels without
changing cardiovascular risk and atherosclerotic plaque load,
respectively, in the opposite direction as expected from the inverse correlation of HDL-C levels and cardiovascular risk in
epidemiological studies.3–5,11,12 Because of these controversial
data, the pathogenic role and, hence, suitability of HDL as a
therapeutic target has been increasingly questioned. Because
of the frequent confounding of low HDL-C with hypertriglyceridemia, it has been argued that low HDL-C is an innocent
bystander of (postprandial) hypertriglyceridemia or another cul-
prit related to insulin resistance or inflammation.11,12
In this controversy it is important to note that previous interventional and genetic studies targeted HDL-C; that is, the cholesterol measured by clinical laboratories in HDL. By contrast
to the pro-atherogenic and, hence, disease-causing cholesterol in
LDL (ie, LDL-C), which after internalization turns macrophages
of the arterial intima into pro-inflammatory foam cells, the cholesterol in HDL (ie, HDL-C) neither exerts nor reflects any of
the potentially anti-atherogenic activities of HDLs.3,4 In contrast
to LDL-C, HDL-C is only a non-functional surrogate marker for
estimating HDL particle number and size without deciphering
the heterogeneous composition and, hence, functionality of
HDLs. In fact, the MESA study13 and the EPIC study14 previously found a stronger and more robust association of intimamedia thickness and coronary event rates with HDL particle
number than with HDL-C level. Successful exploitation of HDL
for therapy and prevention will depend on improved understanding of the structure-function relationships of HDL in health and
disease. This is an essential prerequisite for the development of
biomarkers that reflect the functionality of HDLs better than the
plasma levels of HDL-C or apoA-I and can guide both the development of anti-atherogenic drugs and the clinical management of patients at increased risk for cardiovascular events. In
this context we here summarize our current knowledge on the
Received August 18, 2013; accepted August 20, 2013; released online September 20, 2013
Institute of Clinical Chemistry, University Hospital Zurich (W.A., A.v.E.); Center of Integrative Human Physiology, University of Zurich
(A.v.E.), Zurich, Switzerland
Mailing address: Arnold von Eckardstein, MD, Institute of Clinical Chemistry, University Hospital Zurich, Rämistrasse 100, CH 8091,
Zurich, Switzerland. E-mail: [email protected]
ISSN-1346-9843 doi: 10.1253/circj.CJ-13-1025
All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]
Circulation Journal Vol.77, October 2013
Multifunctional but Vulnerable HDL
2433
Figure 1. Structural and compositional heterogeneity of high-density lipoprotein (HDL). HDL forms macromolecular complexes
containing >80 proteins and peptides, >200 lipid species, and several microRNAs. Apo, apolipoprotein; AT, antitrypsin; AP, antiplasmin; C3, C4, complement factors 3 and 4, respectively; CETP, cholesteryl ester transfer protein; Coe, ceruloplasmin; Fib, fibrinogen;
GPx, glutathione peroxidase; HRP, haptoglobin-related protein; Hp, haptoglobin; Hb, hemoglobin; hPBP, human phosphatidylethanolamine-binding protein; LCAT, lectin:cholesterol acyltransferase; Lp-PLA2, lipoprotein-associated phospholipase A2; MPO, myeloperoxidase; PI, proteinase inhibitor; PLTP, phospholipid transfer protein; PON, paraoxonase; PSP, parotid secretory protein; SAA,
serum amyloid A; TTR, transthyrein. *microRNAs found at very low copy numbers. The structural complexity gives rise to the formation of HDL subclasses that differ by size, shape, density, and charge.
molecular composition and vascular functions of HDL in health
and disease as well as underlying structure-function relationships.
Structure and Composition of HDL
HDLs form a heterogeneous class of lipoproteins that differ
by protein and lipid composition, shape, size, and density
(Figure 1).3,4,15–17 A prototypic HDL particle contains 2–5 molecules of apoA-I18,19 and approximately 100 molecules of phosphatidylcholine or sphingomyelin. They form an amphipathic
shell that contains several molecules of unesterified cholesterol
and wraps a core of completely water-insoluble cholesteryl
esters and triglycerides.19 Quantitative variation of apoA-I and
the major lipid constituents of HDL (phosphatidylcholine, sphingomyelin, cholesterol, and cholesteryl esters) cause considerable
heterogeneity of HDL in shape, density, size, and charge, which
can be analyzed by electron microscopy, ultracentrifugation, gel
filtration, polyacrylamide gel electrophoresis or nuclear magnetic resonance spectroscopy, and agarose gel electrophoresis,
respectively.15–17 Although these analytical methods yield differentiations into various HDL subclasses that cannot be simply translated into each other, a consensus group has previously
suggested a simplified differentiation into 5 subclasses (verysmall-, small-, medium-, large-, and very-large-sized HDL) to
facilitate their application to clinical studies as well as the com-
munication of results.15 As yet, clinical and epidemiological
studies have come to discrepant and inconsistent conclusions on
the prognostic performance of HDL subclasses differentiated by
size.14,20–24 Sometimes the associations of cardiovascular risk
with the various HDL subclass concentrations were not stronger
than with HDL-C,20,21 and even if so, the associations with size
were discrepant: significant associations with risk of cardiovascular events or stroke were found for small HDL in some
studies,22,23 but for medium-sized HDL21,23 or large HDL14,20,21,24
in others.
Previous proteomic, lipidomic, and transcriptomic studies
revealed a much greater structural complexity of HDLs: 80 different proteins,25,26 more than 200 lipid species,27,28 and several
microRNAs29,30 have been identified in HDL isolated from plasma by either ultracentrifugation, gel filtration, or immunoaffinity chromatography (Figure 1). Among the proteins, apoA-II is
present in HDLs at the highest concentration after that of apoA-I
and has been investigated for its cardiovascular risk association
by several studies. In contrast to initial findings, large prospective
studies did not find any significant differences in the risk association between apoA-II-containing and apoA-II-free HDL.15–17
Despite their much lower concentrations relative to the 2
major proteins apoA-I and apoA-II and relative to the major
lipids, many quantitatively minor components of HDLs are not
passive cargo but biologically active and thereby contribute to
the potentially anti-atherogenic properties of HDLs beyond the
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ANNEMA W et al.
Figure 2. Concentration ranges of high-density lipoprotein (HDL) particles as well as the different proteins and lipids found in
HDL. Apo, apolipoprotein; CE, cholesteryl esters; CETP, cholesteryl ester transfer protein; LCAT, lectin:cholesterol acyltransferase;
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PON, paraoxonase; PLTP, phospholipid transfer protein; Sphingosine1-P, sphingosine-1-phosphate; SPM, sphingomyelin; TAG, triacylglyceride; UC, unesterified cholesterol.
transport and metabolism of lipids (Figure 1). A general theme
of these functions is host defense from chemical or biological
hazards such as oxidation (eg, paraoxonase-1 [PON1], plateletactivating factor acylhydrolase [PAF-AH], ceruloplasmin) and
infection (eg, lipopolysaccharide binding protein, apoL1, serum
amyloid A [SAA]), respectively, or the repair of resulting homeostatic disturbances (eg, serpins, antithrombin, antiplasmin)
or damages of cells and tissue (eg, sphingosine-1-phosphate
[S1P], clusterin). Some minor subcomponents of HDLs appear
to form well-defined platforms with distinct functionality; for
example, a complex formed by apoA-I, haptoglobin-related protein (HRP), and apoL1 that, despite its very low concentration,
protects humans from sleeping sickness caused by the general
strain of Trypanosoma brucei.26 HRP mediates the binding to
a cell surface receptor and subsequent internalization, whereas
apoL1 causes the lysosomal lysis of these protozoa.31
The various lipids and proteins of HDLs cover a broad concentration range from the submicromolar (S1P: 0.5 µmol/L; apoL1:
0.1 µmol/L) to the supramillimolar (cholesterol >1 mmol/L)
(Figure 2). Assuming that every HDL particle carries 2–5 molecules of apoA-I, which has an average plasma concentration of
50 µmol/L, the average particle concentration of HDL in plasma
ranges between 10 and 25 µmol/L. The much lower concentration of many HDL-associated proteins, lipids, and microRNAs
indicates that they are residing on different HDL particles. This
has been confirmed by proteomics and lipidomics of subfractionated HDL particles,32–34 which identified the biggest number of microcomponents in the smallest HDL particles. However, these HDL3 particles are also present in plasma at higher
concentrations than their microcomponents, which hence appear to be dispersed throughout different HDL3 particles. The
great concentration differences between HDL components and
particles provide indirect evidence that the established biomarkers HDL-C (1–2 mmol/L) and apoA-I (50 µmol/L), but also the
HDL size/density subclasses, lack both sensitivity and specificity to record the complex structure-function relationships of
HDLs, which in various inflammatory diseases is further increased by the molecular modification of canonical HDL proteins and the acquisition of atypical constituents.35–40
An intriguing example for the diagnostic potential of minor
HDL components is provided by the identification of 2 opposing associations of HDL-C with cardiovascular risk depending
on the presence or absence of apoC-III in HDL:41 In two nested
case-control analyses of the Nurses’ Health and the Health
Professionals Follow-Up Studies, the cholesterol concentration
in both total HDL and apoC-III-free HDL showed the expected
inverse association with CAD events, whereas the concentration of cholesterol in apoC-III-containing HDL was positively
associated with the risk of cardiovascular events.41 Interestingly,
the enrichment of HDLs with apoC-III was found as one reason
for the reduced activity of HDL from CAD patients to protect
endothelial cells from apoptosis.37
HDL Functions
Traditionally, the anti-atherogenic potential of HDLs is assigned
to their capacity to remove cholesterol from peripheral cells and,
most importantly, macrophage foam cells in the blood vessel
intima. The efflux of cholesterol from macrophages is the first
step in the reverse cholesterol transport pathway, a process in
which HDLs play a central role by transporting cellular cholesterol back to the liver for final excretion in bile and feces. As
HDL research moves increasingly towards HDL functionality,
more and more novel functions of HDLs are being uncovered.
Some of these functions have been mechanistically linked to the
well-known ability of HDLs to induce the activation of cellular
cholesterol efflux pathways, whereas many other functions of
HDLs are independent of the effects of HDLs on cellular cholesterol homeostasis. Moreover, besides beneficial functions that
provide protection against atherosclerotic plaque development,
HDLs have been found to positively influence other processes
that indirectly affect atherosclerosis such as glucose homeosta-
Circulation Journal Vol.77, October 2013
Multifunctional but Vulnerable HDL
2435
sis. Below, we will discuss the most relevant functions of HDLs
for atherosclerosis and, whenever possible, describe the underlying molecular mechanism(s) and specific agonist(s) in HDLs
responsible for the effects.
Cholesterol Efflux and Reverse Cholesterol Transport
A key process in the formation of an atherosclerotic plaque is
accumulation of cholesterol in macrophage foam cells. HDLs
and their major protein constituent, apoA-I, are efficient acceptors of excess free cholesterol from cells. This ability of HDLs
and apoA-I to promote cellular cholesterol efflux might reverse or prevent the formation of macrophage foam cells. Four
different pathways have consistently been described by which
HDLs and their apolipoproteins remove cholesterol from cells.
First of all, cells can release cholesterol toward HDLs by passive aqueous diffusion, which involves spontaneous desorption of free cholesterol molecules from the plasma membranes
and diffusion through the intervening aqueous face until collision with and incorporation into acceptor particles.42,43 Such
an unmediated bidirectional flux of free cholesterol is rather
inefficient and follows a cholesterol concentration gradient between the plasma membrane and HDL.42 The 3 other pathways
for cholesterol efflux are more specific and depend on interaction with a cellular receptor. Lipid-free or lipid-poor apoA-I
can mediate cellular efflux of both cholesterol and phospholipids via the ATP-binding cassette transporter A1 (ABCA1), resulting in the rapid lipidation of apoA-I to generate nascent HDL
particles.44,45 However, the nature of the molecular interaction
between apoA-I and ABCA1 is still not entirely understood. The
mature HDL particles can then serve as acceptors of cholesterol
provided by ABCG146 or scavenger receptor class B type I
(SR-BI).47 Although it is currently unknown how ABCG1 mediates cholesterol efflux to HDLs, it does not appear to require
direct binding of HDLs to the cells.46 Rather, there is considerable evidence that ABCG1 modulates the flux of sterols from
intracellular organelles to the plasma membrane for efflux.48
SR-BI facilitates a bidirectional flux of free cholesterol between
the cell and HDL, and the net movement of cholesterol is similar to aqueous diffusion determined by the cholesterol concentration gradient.49 In contrast to ABCG1, HDLs have to bind to
SR-BI in order to stimulate net cholesterol efflux.50,51
After efflux, cholesterol in HDLs may be esterified by the
enzymatic activity of lecithin:cholesterol acyltransferase (LCAT)
whereupon HDLs can deliver the excess cholesterol from peripheral cells back to the liver by 3 distinct ways: (1) HDL cholesteryl esters, but not the protein component of HDLs, are selectively taken up into the liver via SR-BI;52 (2) HDL particles
(lipids together with proteins) can undergo holoparticle endocytosis mediated by the interaction of apoA-I with the ectopic
β-chain of F0F1 ATPase and subsequent activation of the nucleotide receptor P2Y13;53 and (3) in humans, CETP exchanges cholesteryl esters in HDLs for triglycerides in apoB-containing lipoproteins, which in turn are removed from the circulation
by the liver via members of the LDL receptor family.54 Ultimately, cholesterol is excreted from the liver into the bile, either
directly as free cholesterol or after conversion into bile acids, and
eliminated from the body via the feces.55,56 This pathway involving cholesterol efflux from cells toward HDLs, uptake of cholesterol from HDLs into the liver, and finally excretion into bile and
feces is termed reverse cholesterol transport. Besides HDLmediated reverse cholesterol transport through the classical
hepatobiliary route, there is also an HDL-independent pathway
for body cholesterol removal, the so-called transintestinal cholesterol excretion, which has been described in mice.57–59
The flux of cholesterol from macrophage foam cells repre-
sents only a very small fraction of the total cholesterol pool and
therefore has no effect on whole body cholesterol homeostasis.
However, macrophage-specific reverse cholesterol transport is
highly relevant for maintaining normal macrophage cholesterol
homeostasis, with profound consequences for atherosclerosis
development. Two crucial questions to address regarding the
cholesterol efflux and reverse cholesterol transport functionality of HDLs are (1) how HDLs reach macrophage foam cells in
the subendothelial space, and (2) how HDLs leave the arterial
intima, so that they can transport macrophage-derived cholesterol to the liver for elimination from the body. First, in order to
get access to the cholesterol-loaded macrophages within atherosclerotic lesions, HDLs or their major protein constituent,
apoA-I, need to cross the endothelium. Our group found that
aortic endothelial cells are able to bind, internalize, and transport both HDLs and lipid-free apoA-I in a specific and saturable manner.60,61 Transcytosis of apoA-I through aortic endothelial cells resulted in lipidation of lipid-free apoA-I and was
reduced by knockdown of ABCA1 by small interfering RNA,60,62
providing evidence that ABCA1 is involved in this process.
In contrast, binding, uptake, and transendothelial transport of
mature HDLs were modulated by ABCG1 and SR-BI, not by
ABCA1.61 Further research identified a novel mechanism for
transport of both apoA-I and HDLs through endothelial cells,
in which binding of apoA-I to the β-chain of cell surface F0F1
ATPase expressed on endothelial cells stimulates ATP hydrolysis, and the generated ADP selectively activates P2Y12, leading to internalization and transport of initially lipid-free apoA-I
and HDLs.63 Once apoA-I and HDLs have been loaded with
cholesterol, they need to leave the arterial wall to complete
reverse cholesterol transport. It seems conceivable that HDL
particles can return from the extravascular space to the systemic circulation either by yet again passing through the endothelium or alternatively via the lymphatic system. Although
the first hypothesis has not been disproved, recent data support
a role for the lymphatic vessels in reverse cholesterol transport.64,65 Macrophage-specific reverse cholesterol transport
was substantially reduced in mice with surgical interruption of
lymphatic transport and in Chy mice, which have absent dermal lymphatics because of a mutation in vascular endothelial
growth factor (VEGF) receptor 3 (VEGFR3).64,65 Ameliorating the lymphatic function in apoE knockout mice by VEGF-C
treatment improved the transport of fluorescent cholesterol
from macrophages injected into the footpad to the plasma and
liver.64,65 More importantly, the cholesterol label could be detected in lymph nodes regional to the site of injection and in the
efferent lymph.65 In vitro observations revealed that lymphatic
endothelial cells internalize and transcytose HDLs by a mechanism that involves SR-BI.65 In more complex experiments,
aortic arches from apoE–/– donor mice, which are rich in atherosclerotic lesions, were loaded with D6-cholesterol and transplanted into the abdominal cavity of apoE–/– recipient mice.64
Partial inhibition of reformation of lymphatic connections by
treating the recipient mice with an antibody blocking VEGFR3
was associated with increased retention of D6-cholesterol in
the transplanted aortic segments,64 thereby providing more
direct evidence for the specific involvement of lymphatic vessels draining the wall of the aorta in cholesterol clearance.
Antioxidative Effects
Oxidation of LDL yields a more pro-atherogenic particle, and in
numerous studies HDLs have been found to be capable of impeding oxidative changes in LDL. An inhibitory effect of HDLs
on the oxidation of LDL has been detected both in cocultures of
endothelial cells and vascular smooth muscle cells (VSMCs)66,67
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ANNEMA W et al.
Figure 3. Molecules and receptors mediating endothelial functions of high-density lipoproteins (HDLs). Apo, apolipoprotein; eNOS,
endothelial nitric oxide synthase; EPC, endothelial progenitor cell; NO, nitric oxide; S1P, sphingosine-1-phosphate; SR, scavenger
receptor; VCAM, vascular cell adhesion molecule.
and several cell-free systems.68–70 A major part of the antioxidative activity of HDLs is attributed to apoA-I. In cell culture
experiments, apoA-I removes lipids from LDL and thereby
makes LDL resistant to vascular cell-mediated oxidation and
prevents oxidized LDL-induced monocyte adherence and chemotaxis.67 Similarly, LDL isolated from mice and humans injected with apoA-I can no longer be oxidized by coculture with
human artery wall cells.67 There is also evidence indicating that
apoA-I binds molecules produced by vascular cells that stimulate the formation of biologically active phospholipids in LDL.66
The oxidation of 2 methionine residues in apoA-I to their respective methionine sulfoxide forms is highly important for the
ability of HDLs to reduce lipid hydroperoxides.71
PON1 is another HDL-associated protein that plays an important role in the antioxidative functionality of HDLs. Purified
PON1 offers protection against oxidation of LDL in vitro67,72
by reducing oxidized phospholipids that accumulate in oxidized
LDL.66,73 Notably, HDL isolated from mice with transgenic
overexpression of human PON1 is more potent at inhibiting
oxidation of LDL than HDL from wild-type mice,74 and the lack
of a functional PON1 gene in mice renders the HDLs of those
mice unable to block accumulation of lipid hydroperoxides in
human LDL.75
Another HDL enzyme possessing antioxidative activity is
PAF-AH, which nowadays is termed lipoprotein-associated
phospholipase A2 (Lp-PLA2). Inactivation of HDL-associated
Lp-PLA2 abolishes the inhibitory effect of HDLs against LDL
modification and production of biologically active phospholipids.76 A protective action of Lp-PLA2 against LDL oxidation
has been further supported by mouse studies demonstrating that
adenoviral-mediated overexpression of human Lp-PLA2 in mice
reduced the circulating levels of oxidatively modified LDL.77
In contrast to the potentially anti-atherogenic functions of LpPLA2, plasma levels of Lp-PLA2 positively correlate with cardiovascular risk.78–80 A possible explanation is that the majority
of Lp-PLA2 resides in LDL where it generates pro-inflammatory lysophospholipids.81 Indeed, the findings of a 3-year pro-
spective follow-up study of patients with stable coronary artery
disease (CAD) suggested that a higher total plasma Lp-PLA2
level is associated with increased future cardiovascular mortality, whereas an association in the opposite direction was found
with Lp-PLA2 in apoB-depleted plasma.82
Finally, LCAT might also contribute to the beneficial effects
of HDLs in modulating LDL oxidation. Reports showing that
incubation of LDL with LCAT considerably decreased Cu2+induced formation of lipid hydroperoxides in LDL83 and that
infection of mice with a recombinant adenovirus encoding
human LCAT resulted in lower levels of autoantibodies against
oxidized LDL84 support an antioxidative function for LCAT.
Endothelial Function and Integrity
HDLs exert several direct protective effects on the vascular endothelium (Figure 3). First, HDLs are presumed endotheliumdependent vasodilators because the addition of HDLs to precontracted aortic segments leads to vasorelaxation.85 Additional
human studies have revealed that heterozygotes for loss-offunction mutations in ABCA1 with low levels of HDL-C display impaired endothelium-dependent vasodilation, which can
be restored by a single infusion of apoA-I/phosphatidylcholine
disks.86 Likewise, administration of reconstituted HDLs resulted
in normalized endothelial vasodilator function in hypercholesterolemic men.87 The effect of HDLs on vascular tone is mechanistically explained by the ability of HDLs to induce the phosphorylation of endothelial nitric oxide synthase (eNOS) and
thereby stimulate nitric oxide (NO) production. This vasoprotective activity involves the interaction of HDLs with SR-BI and
the lysophospholipid receptor S1P3, which leads to the parallel
activation of phosphatidylinositol 3-kinase (PI3K)/Akt and
mitogen-activated protein-kinase signaling.85,88,89 By the same
cellular signaling pathways, HDLs also lead to a significant
increase in eNOS protein abundance in endothelial cells.90 Three
bioactive lysosphingolipids in HDLs (ie, sphingosylphosphorylcholine, S1P, and lysosulfatide) were attributed to arterial
vasodilation by HDLs.85 Evidence has accumulated that also
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Multifunctional but Vulnerable HDL
2437
the cholesterol efflux capacity of HDLs via ABCG1 has some
effect in the stimulatory activity of HDLs on eNOS activity. In
mice lacking ABCG1 the reduction in endothelium-dependent
vasorelaxation is associated with increased aortic levels of
7-oxysterols and decreased levels of active eNOS dimer.91 Moreover, the inhibitory interaction of eNOS with caveolin-1 in response to cholesterol loading can be reversed by HDLs in an
ABCG1-dependent fashion.92
HDLs also beneficially affect the vasculature by promoting
endothelial cell survival. HDLs suppress apoptosis in endothelial cells induced by several stimuli, including oxidized LDL,93–95
tumor necrosis factor-α (TNF-α),96 and growth factor withdrawal.37,95 Studies of the molecular mechanism indicate that
HDLs potentially can maintain mitochondrial integrity, thereby
preventing the release of cytochrome c and apoptosis-inducing
factor from mitochondria94,97 and subsequent activation of the
caspase cascade.94,96,97 In addition, HDLs are negative regulators
of the pro-apoptotic effector protein, Bid,94 and positively modulate the expression of the anti-apoptotic protein Bcl-xl.37 Stimulation of the PI3K/Akt/eNOS signaling pathway again seems
to play a role in this endothelial cell protection afforded by
HDLs.37,97 The anti-apoptotic capacity of HDLs in endothelial
cells has been attributed to their protein components apoA-I 98
and clusterin (or apoJ),37 as well as to lysophospholipids carried by HDLs.95,97
Loss of the structural integrity of blood vessels facilitates the
development of atherosclerotic lesions, and electrical cell substrate impedance sensing assays have revealed that HDLs can
promote endothelial barrier function.99 In this context, HDLassociated S1P acts on its receptor S1P1 to stimulate signaling
primarily through the Akt/eNOS pathway, an effect that has been
shown to be partially dependent on the hydrolysis of HDLassociated phospholipids by endothelial lipase and ultimately
leads to endothelial barrier enhancement.99–101 Recent studies
have identified apoM as the binding molecule of S1P in HDLs.102
Notably, apoM-deficient mice, which lack S1P in their HDL
fraction, exhibit significantly impaired endothelial barrier function in the lungs.102
Finally, HDLs may contribute to repair processes after injury.
Carotid artery re-endothelialization after perivascular electric
injury is blunted in apoA-I knockout mice with very low HDL-C
levels, but can be restored through liver-directed gene transfer.103
In vitro studies indicate that HDLs may in part accelerate reendothelialization by driving endothelial cell migration through
Rac-mediated formation of actin-based lamellipodia in an SRBI-dependent manner.103 Moreover, blockage of the SR-BI
adaptor protein molecule, PDZK1, prevented HDL-mediated
activation of eNOS and endothelial cell migration.104 In PDZK1
knockout mice, re-endothelialization after perivascular electric
injury does not occur.104 These data suggest that PDZK1 is required for re-endothelialization events activated by HDL/SRBI. Although somewhat controversial, endothelial progenitor
cells (EPCs) are thought to participate in vascular repair. EPCs
cultured in the presence of reconstituted HDLs show increased
proliferation, differentiation, and cell survival as well as enhanced adhesion to endothelial cells and transendothelial migration.105–107 The beneficial effects of HDLs on EPCs can be
abolished by either blocking Akt or PI3K in the cells.106,107 In a
study using mice, the administration of reconstituted HDLs increased the number of EPCs and stimulated the re-endothelialization of injured carotid arteries.105 In that same study, treatment
with reconstituted HDLs improved blood flow and capillary
density following hindlimb ischemia, conceivably by enhanced
incorporation of bone marrow-derived endothelial cells in the
ischemic tissue.107 The potential relevance for humans is indi-
cated by the positive correlation between HDL-C levels and
circulating EPC numbers in CAD patients,105 as well as by the
increase of EPC numbers in the blood of patients with type 2
diabetes mellitus (T2DM) who received infusions of reconstituted HDLs.108 Furthermore, the administration of HDLs and
their constituent, S1P, diminished myocardial damage in a murine model of myocardial ischemia-reperfusion. These protective activities of HDLs require NO as well as the S1P3 receptor
and entail the ability of the lipoprotein to suppress the recruitment of inflammatory neutrophils and apoptosis of cardiomyocytes.109
Anti-Inflammatory Effects
An early event in the pathogenesis of atherosclerosis is upregulated expression of adhesion molecules on the endothelium,
leading to the recruitment and infiltration of monocytes. It is
generally well established that native110,111 as well as reconstituted HDLs112,113 have the capacity to inhibit cytokine-induced
expression of adhesion molecules on endothelial cells in vitro.
Several lines of evidence confirm that HDL-mediated suppression of adhesion molecule expression also occurs in vivo. Injection of reconstituted HDLs reduced the expression of E-selectin
in interleukin 1α-induced skin lesions in a pig model.114 Moreover, infusions of apoA-I either in the free form or as reconstituted HDL mitigated the vascular inflammation caused by implantation of a periarterial collar in rabbits, characterized among
other effects by decreased endothelial expression of various adhesion molecules.115,116 Additional human studies have shown
that infusions of reconstituted HDLs lower the expression level
of vascular cell adhesion molecule 1 (VCAM-1) in atherosclerotic lesions.117 Lysosphingolipids present in HDLs, such as S1P,
have been proposed as mediating the HDL-induced inhibition
of VCAM-1 expression on endothelial cells.118 However, despite
considerable research in this area, the mechanisms underlying
this anti-inflammatory effect of HDLs are still not completely
understood. TNF-α enhances sphingosine kinase activity and the
generation of S1P in endothelial cells, and this can be blocked
by pre-incubation with HDL3.119 Moreover, it has been postulated that HDLs impair VCAM-1 expression on endothelial cells
via SR-BI- and S1P1-mediated activation of PI3K and eNOS.120
Another mechanism may involve repression of TNF-α-induced
IκB kinase activity in response to HDLs, thereby preventing
nuclear factor-κB (NF-κB) inhibitor degradation, and subsequently reducing nuclear translocation, DNA binding, and the
transcriptional activity of NF-κB.119,121,122 More recent studies
indicate that the interaction of HDL with SR-BI on endothelial cells increases the expression of heme oxygenase-1 by
activation of PI3K/Akt through 3β-hydroxysteroid-∆24 and
that this may play an important role in the suppression of endothelial inflammation by HDLs.122,123
Although interactions between endothelial cells and monocytes require the expression of adhesion molecules on both cell
types, the effect of HDLs on monocytic adhesion molecules has
been less extensively investigated. There is one study showing
that treatment of human monocytes with native HDL, reconstituted HDL, or apoA-I reduced the expression and activation of
the monocyte adhesion molecule CD11b.124 This inhibition of
monocyte activation by HDLs or apoA-I was associated in vitro
with decreased monocyte adhesion to endothelial cells, monocyte spreading, and monocyte migration in response to monocyte chemoattractant protein-1 (MCP-1).124 ApoA-I was found
to prevent monocyte activation through efflux of cholesterol
via ABCA1, whereas for HDLs this occurred via an ABCA1independent mechanism.124
Besides reducing the number of adhesion molecules, HDLs
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ANNEMA W et al.
can also affect the infiltration of monocytes in the arterial intima by regulating the expression of chemokines and their receptors.125 The beneficial influence of HDLs on the expression of
various chemokines is mediated through direct inhibition of the
NF-κB-pathway.125 On the other hand, the capacity of HDLs to
reduce the chemokine receptor, CX3CR1, on monocytes is dependent on peroxisome proliferator-activated protein γ.125 In
addition, diminished production of the chemokine MCP-1 after
incubation of VSMCs or isolated rat aortas with HDLs has been
attributed to HDL-associated lysosphingolipids and requires signaling through the S1P3 receptor and SR-BI.126
Apart from modulating the functionality of monocytes and
macrophages, HDLs were recently found to control adaptive
immunity.127,128 A defect in cholesterol efflux after combined
knockout of Abca1 and Abcg1 in mice was accompanied by
excessive expansion of both myelopoietic stem and multipotential progenitor cells in bone marrow as well as by leukocytosis
in the peripheral blood.127 Moreover, introduction of a human
apoA-I transgene into heterozygous LDL receptor knockout
mice transplanted with bone marrow of Abca1–/–Abcg1–/– mice
resulted into almost complete normalization of the myeloproliferative disorder.127 In fact, HDLs were shown to suppress the
proliferation of myelopoietic stem cells ex vivo.127 Subsequent
research by the same group revealed that transplantation of bone
marrow from Abca1/Abcg1 double knockout mice into wildtype recipients led to interleukin 23-induced secretion of granulocyte colony-stimulating factor, which in turn increased the
mobilization of hematopoietic stem and progenitor cells as well
as extramedullary hematopoiesis.128 Raising the HDL levels in
these mice via a human apoA-I transgene could attenuate the
number of circulating hematopoietic stem and progenitor cells.128
Findings from other recent work suggest that apart from innate immunity, adaptive immunity is also modulated by apoA-I
and HDLs.129 LDL-receptor knockout mice lacking apoA-I show
signs of autoimmunity in response to a cholesterol-enriched diet,
typically reflected by enlarged cholesterol-rich lymph nodes as
well as increased T cell activation, T cell proliferation, production of autoantibodies, and skin inflammation.129,130 Administration of human lipid-free apoA-I partly reversed the autoimmunity phenotype of these mice, conceivably by increasing the
amount of regulatory T cells in the lymph nodes.129
Antithrombotic Effects
Platelet aggregation and thrombus formation directly contribute
to the pathogenesis and complications of atherosclerotic cardiovascular disease. There are supporting data from several studies
that HDLs are potent inhibitors of platelet activation and aggregation. HDL3 exerts positive regulatory effects on the Na+/H+
antiport system in human platelets by binding to glycoprotein
IIb/IIIa and activation of protein kinase C and phospholipase
C.131 Infusion of reconstituted HDLs into patients with T2DM
attenuated platelet aggregation ex vivo.132 Moreover, native
HDL3 interfered with thrombin-induced platelet activation in
vitro, as evidenced by decreased platelet aggregation, fibrinogen
binding, and P-selectin expression,133,134 which was attributed to
the inhibitory effect of HDLs on thrombin-induced formation of
1,2-diacylglycerol and inositol 1,4,5-tris phosphate as well as to
intracellular calcium mobilization and the ensuing increase in
the phosphorylation and activation of protein kinase C.133,134
The capacity of HDLs to inhibit platelet aggregation appears to
be dependent on SR-BI,134 although this observation could not
be confirmed by another group.132 Cholesterol depletion from
platelet membranes by incubation with either cyclodextrin or
HDLs was accompanied by reduced platelet aggregation,132
indicating that HDLs may modulate platelet function by cho-
lesterol efflux. In addition, HDL-bound phospholipids blunted
platelet activation to a similar extent as native HDL or reconstituted HDL.132,134
Recent investigations have identified a link between HDLmediated cholesterol efflux via ABCG4 and platelet production.135 When Ldlr –/– mice were transplanted with bone marrow
from Abcg4–/– mice, the total platelet count and percentage of
reticulated platelets were significantly increased,135 consistent
with enhanced platelet production. The expression of ABCG4
was found to be specific for bone marrow-derived megakaryocyte progenitors, and deficiency of this transporter promoted the
proliferation and expansion of these platelet progenitors.135 In
megakaryocyte progenitors that lack ABCG4, cholesterol efflux to HDLs was defective.135 Treatment with reconstituted
HDLs reduced platelet numbers and megakaryocyte progenitor
proliferation in Ldlr –/– mice but not in Abcg4–/–Ldlr –/– mice.135
Taken together, these data suggest that HDLs modulate megakaryocyte progenitor cell proliferation and, hence, platelet production by regulating the cholesterol content of megakaryocyte
progenitors via ABCG4. The relevance of this process for human
physiology is indicated by the finding of moderately but significantly reduced platelet numbers in patients with peripheral
vascular disease who were treated with reconstituted HDLs.135
HDLs might also work in an anticoagulatory fashion by inhibiting steps in the extrinsic coagulation cascade. Already in the
1980 s it was documented that HDLs impair the activity of tissue
factor and subsequent activation of factor X.136 Interestingly,
the discovery that HDLs potentiate the anticoagulatory activities of activated protein C and protein S in mediating proteasomal degradation of factor Va was later assigned to contamination of the HDL fraction isolated by ultracentrifugation
with anionic phospholipid membranes.137,138 Other modes of
action that could explain the beneficial effects of HDLs on
coagulation include a reduction in platelet-activating factor production by endothelial cells,139 inhibition of thrombin-induced
endothelial tissue factor expression,140 suppression of the procoagulant activity of erythrocytes,141 and inactivation of anionic
phospholipids that are required to accommodate the prothrombinase complex.142
Consistent with these findings proposing a protective function
of HDLs against thrombosis, Naqvi et al observed an independent inverse correlation between HDL-C levels and ex vivo
thrombus formation in humans.143 Furthermore, flow-induced
thrombus formation was diminished by reconstituted HDLs as
determined by flow-chamber based assays.132 Intravenous injection of apoA-I Milano into rats resulted in significantly delayed
time to thrombus formation and decreased thrombus weight.144
Smooth Muscle Cells
VSMCs play an important role in the stabilization of atherosclerotic plaques. HDLs promote proliferation and cell cycle
entry of VSMCs by inducing cyclin D1 expression and retinoblastoma protein phosphorylation.145 Proliferative effects of
HDLs on VSMCs are dependent on the Raf-1/MEK-1/ERK1/2
mitogen-activated protein kinase cascade, but cannot be mimicked by lipid-free apoA-I.145 Moreover, HDLs have been shown
to inhibit platelet-derived growth factor-induced migration of
VSMCs through their S1P component.146 HDLs also counteract
the pro-inflammatory events mediated by oxidized LDL in rabbit
VSMCs by reducing the generation of intracellular ROS and
thereby activation of NF-κB.147 Additional in vitro experimental data indicate that HDLs might regulate the secretory function of VSMCs. On the one hand, HDLs can facilitate the release
of the cyclooxygenase-2-derived vasodilator prostacyclin in rat
VSMCs via a mitogen-activated protein kinase-dependent mech-
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Multifunctional but Vulnerable HDL
2439
anism.148,149 On the other hand, HDLs decrease the production
and release of basic fibroblast growth factor from bovine aortic
smooth muscle cells.150
HDL Dysfunction
Nearly 20 years ago, it was reported that upon an acute-phase
response, HDL of both patients and animals loses its inhibitory
effect on LDL oxidation and enhances rather than inhibits the
expression of MCP-1 by endothelial cells.151 Since then, many
laboratories worldwide have provided examples of the loss of
atheroprotective functions or even gain of paradoxically proatherogenic functions by HDLs (Table S1). Such dysfunctional
HDL has been isolated from patients with both acute and stable
CAD, T2DM as well as type 1 diabetes mellitus (T1DM), metabolic syndrome (MetS), end-stage (ESRD) as well as mild-tomoderate chronic kidney disease (CKD), rheumatic diseases, or
sepsis (Table S1). Dysfunctions include reduced activities or
capacities of HDLs to induce cholesterol efflux from macrophages and other cells, to inhibit oxidation of lipids in LDL and
cell membranes, and to inhibit cytokine release and expression
of cell surface activation markers by macrophages or dendritic
cells (Table S1). A broad spectrum of dysfunction has been seen
in the interactions of HDLs with endothelial cells: HDL of patients with different disease conditions failed to stimulate NO
production and to inhibit superoxide production and thereby
modulate downstream activities such as vasorelaxation, expression of VCAM-1 and MCP-1 as well as leukocyte adhesion
(Table S1). HDL of CAD, T2DM, or CKD patients was also
found to lack the normal activity to promote endothelial integrity by inhibiting apoptosis and stimulating migration of endothelial cells, as well as the repopulation of denuded arteries by
EPCs (Table S1).
Pathological structural changes in HDLs were previously
found to diminish or eliminate physiological interactions, for
example, with ABCA1152–154 or SR-BI,155,156 resulting in defective cholesterol efflux, or in pathophysiological interactions, for
example with the lectin-oxidized lipoprotein receptor LOX-138
or the toll-like receptor TLR2,39 both of which result in inhibition rather than activation of eNOS and, hence, abnormal suppression of NO production.38,39 As yet, the relative importance
of the many physiological and pathological activities of normal
and dysfunctional HDL, respectively, in the pathogenesis of
atherosclerosis or T2DM is unknown. The elucidation of this
problem, however, is the major limiting factor in the clinical
exploitation of HDL for treatment and prevention of cardiovascular and possibly other diseases. The identification of the
most relevant biological activities of HDLs and the mediating
molecules within HDLs as well as their cellular interaction partners are pivotal for the development of anti-atherogenic drugs
as well as of diagnostic biomarkers for the identification, personalized treatment stratification, and monitoring of patients at
increased cardiovascular risk.
Three principle types of molecular changes appear to underlie
HDL dysfunction: (1) compositional changes of the proteome,
(2) post-translational modifications of proteins, and (3) alterations of the lipid moiety and other cargo molecules.
These phenomena can be interrelated. For example, the displacement of apoA-I, PON1, and PAF-AH by acute-phase proteins reduces the capacity of HDL to protect its own lipids and
proteins from oxidation. Oxidized lipids or post-translational
modification of proteins by thereof derived advanced lipoxidation endproducts (ALE) can directly interfere with the functionality of HDLs and/or lead to the formation of antigenic neoepitopes; for example in apoA-I, which can bind neutralizing
autoantibodies.
Compositional Changes of the Proteome
Traditionally, the loss of HDL function has been attributed to
replacement of apoA-I by other proteins, notably acute-phase
proteins such as SAA, which in inflammation can represent
more than 80% of HDL proteins, but also haptoglobin, ceruloplasmin, and fibrinogen.151,157 An effect of SAA on HDL’s cholesterol efflux capacity is controversial. The laboratory of van
der Westhuyzen and de Beer showed in several publications
that SAA-enriched HDLs elicit normal cholesterol efflux via
ABCG1- and SR-BI-mediated pathways.158,159 The SAA-induced remodeling of HDLs liberates apoA-I,160 so that ABCA1mediated cholesterol efflux was also found to be normal in the
presence of SAA.161 By contrast, Malle’s group found reduced
cholesterol efflux capacity by both SAA-enriched HDLs and
lipid-free SAA.162,163 Interestingly, adenovirus-mediated expression of SAA reduced the fecal excretion of macrophage-derived
cholesterol in mice,164 indicating that SAA interferes with macrophage reverse cholesterol transport, possibly by mechanisms
other than cholesterol efflux. In addition, SAA was found to reduce the antioxidative capacity of HDLs by displacing PON1
and PAF-AH.151 SAA also increases the binding of HDLs to
vascular proteoglycans and thereby the retention in the arterial
intima as well as exposition to oxidative modifications.165 Moreover, SAA mimicked the stimulatory activity of HDL from CKD
patients on the production of inflammatory cytokines and chemokines by monocytes and VSMCs.166
Other hypothesis-driven analyses of dysfunctional HDL have
identified atypical abundance of myeloperoxidase (MPO) in
HDL of patients with T2DM,40 which is considered an important
causal factor for the oxidative protein modification of apoA-I
(see later).35 Vice versa, the activity of the antioxidative PON1
was found to be decreased in either sera or HDL of patients with
CAD, T2DM, T1DM, CKD, rheumatoid arthritis, or cardiac
surgery (Table S1). The decreased PON1 activity showed very
plausible associations with reduced antioxidative activities of
HDLs in various disease conditions (Table S1). In addition, low
PON1 activity was found as a causal factor in the inhibition of
NO production in endothelial cells, probably by allowing the
formation of malondialdehyde and subsequent lysine residue
modifications in HDL-associated proteins.38
Much more compositional changes have been found by recent small but comprehensive proteomic case-control studies of
patients with CAD, T2DM, CKD, rheumatoid arthritis, or psoriasis (Table S1). Some findings of previous hypothesis-driven studies were confirmed by this hypothesis-free approach; for
example, the enrichment of HDL from patients with rheumatoid arthritis, psoriasis, CAD, or CKD with SAA (Table S1).
Enrichment with apoC-III in the HDL of patients with stable
or acute CAD, T2DM, or CKD is another very robust observation in proteomic case-control studies from different laboratories.33,37,167,168 This finding has been confirmed in 2 nested
prospective studies by the opposite associations of apoC-IIIcontaining HDL and apoC-III-free HDL with cardiovascular
risk.41 However, proteomics studies have also generated controversial findings; for example, the enrichment of HDL from
CAD patients with PON1 and apoE in one study,167 but deprivation of the same proteins in HDL from CAD and T2DM
patients in other studies.33,37 Differences in patients and methods of HDL isolation may be the reason, in addition to chance
findings in the as yet small sample numbers. Quantitative proteomic studies using single-reaction-monitoring mass spectrometry or arrayed immunoassays in larger sample sizes are hence
needed to firmly establish proteomic footprints, which differen-
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2440
ANNEMA W et al.
tiate the HDL of healthy and diseased individuals. In addition,
the (dys)functional relevance of many differentially expressed
proteins in HDLs needs to be shown, especially if this cannot be
inferred from previous investigations of candidate proteins such
as SAA or PON1. For many newly detected differences in HDL
proteomes, the functional relevance needs to be established.
For example, enriched apoC-III but decreased clusterin content
was found to account for the reduced anti-apoptotic activity of
HDL from CAD patients towards endothelial cells.37
Post-Translational Modifications of Proteins
A broad range of enzymatic and oxidative modifications has
been reported for both the proteins and lipids of HDLs. Notably,
amino acid residues of apoA-I have been found modified by
oxidation through MPO and carbonylation through advanced
glycation endproducts (AGE) and ALE.35,169 These modifications have been found at increased concentrations in the HDL of
patients with inflammatory diseases, including CAD, T2DM,
and CKD, and have been associated with dysfunction. However,
it must be emphasized that the concentration of these modifications is in the low or even submicromolar range and hence lower
than the concentration of apoA-I or HDL particles (Figure 2).
It hence appears that defined and functionally highly relevant
subfractions of HDL rather than the entire HDL fraction are
critically hit by these modifications.
MPO, which is strongly expressed by neutrophil granulocytes
and other myeloid cells, plays a pivotal role in the oxidative
chlorination and nitration of free hydroxyl groups in tyrosine
and tryptophan residues, and the sulfoxidation of methionine
residues via formation of hypochlorous acid (HOCl), as well as
the carbamylation of free amino groups, for example, in lysine
residues via the formation of cyanate from thiocyanate.35,169,170
Patients with CAD were found to have increased concentrations
of MPO171,172 as well as its products chlorotyrosine and nitrotyrosine in both plasma and HDL.173 Interestingly, the association of chlorinated or nitrosylated apoA-I with the presence of
CAD was found to be stronger than the association of chlorinated or nitrosylated plasma proteins in general, perhaps reflecting the presence of an MPO binding site in apoA-I that allows
enrichment of this enzyme and its products in HDLs.173 Moreover, as compared with plasma apoA-I, apoA-I isolated from
atherosclerotic plaques was more strongly modified by MPO.152
This indicates that either the modification of apoA-I occurs in
the extravascular rather than intravascular space or that MPOmodified apoA-I and HDLs are trapped within the vascular
wall.174–176 Also, carbamylated HDL was found at increased
concentrations in advanced atherosclerotic plaques, especially of
smokers and CKD patients,155,177 probably because cyanate is
also released from cigarette smoke and formed from urea independently of MPO.155,177 The oxidation of HDLs and apoA-I
by MPO or its immediate product, HOCl, severely compromises
their ability to induce ABCA1-mediated cholesterol efflux152,154
and to activate LCAT,178,179 whereas carbamylated HDL has a
reduced ability to induce SR-BI mediated cholesterol efflux.155
Moreover, treatment of HDLs with MPO or HOCl interferes
with their ability to inhibit apoptosis and TNF-α-induced
VCAM-1 expression, as well as to stimulate NO production,
possibly by reducing the binding of HDL to SR-BI.180 The
observation of decreased macrophage cholesterol transport in
mice after injection of MPO brought in vivo evidence for the
pathophysiological relevance of the previous in vitro findings.164
In addition to the loss of protective activities on cholesterol efflux and esterification as well as endothelial function and survival, MPO-modified HDL gains pro-inflammatory activities as
it induces NF-κB activation and VCAM-1 expression in endo-
thelial cells by binding to an as yet unknown receptor.180 Several amino acid residues are oxidatively modified by MPO or
HOCl in vitro and have been identified as modified in HDL
isolated from patients. However, despite systematic in vitro
mutagenesis studies to generate dysfunctional mimetics of
MPO-modified apoA-I or MPO-resistant apoA-I mutants, the
identity of the pivotal amino acid residues modified by MPO is
not unequivocally resolved. As yet, Tyr166, Tyr192, and
Met146 have been proposed as being critical.154,178,179,181
Another group of protein modifications in HDLs with potential functional relevance is caused by carbonylation of arginine, lysine, and tryptophan residues in their proteins with
either AGE including glyoxal, methylglyoxal, and glycoaldehyde or with ALE including malondialdehyde (MDA), 4-hydroxynonenal (HNE), and acrolein.169,170,182 Both AGE (notably methylglyoxal) and ALE (notably MDA) have been found
at increased concentrations in HDL of patients with CAD or
T2DM.38,183 Methylglyoxal interferes with the ability of HDLs
to bind phospholipids and to activate LCAT.184,185 Exposure of
lipid-free apoA-I, native or reconstituted HDL to methylglyoxal as well as to MDA or acrolein, was found to abrogate their
capacity to induce cholesterol efflux in some186–190 but not all
studies.38,184 In a recent study, HDL and sera of diabetic subjects were found to display increased rather than decreased
cholesterol efflux capacity, which correlated with CETP activity but not with AGE levels.191 Neither showed AGE levels any
association with macrophage reverse cholesterol transport in
vivo of db/db mice or uremic mice.191 HDLs enriched with either AGE in T2DM patients or with MDA in CAD patients
failed to inhibit monocyte or neutrophil diapedesis through
endothelial cells by suppressing VCAM-1 and intracellular adhesion molecule-1 expression.38,192 Both the HDL of CAD patients and that of healthy subjects enriched with MDA bind to
the scavenger receptor LOX-1 of endothelial cells. The resulting activation of protein kinase C βII causes the inhibition instead of activation of phosphorylation of eNOS at residues
Thr495 and Ser1177, respectively, so that MDA-modified HDL,
like the HDL of CAD patients, inhibits rather than stimulates
NO production in endothelial cells.38 The same phenotype and
dysfunction were observed in the HDL of PON1 knockout mice
and could be corrected by addition of recombinant PON1.
Therefore, and because PON1 activity is low in the HDL of
CAD patients, PON1 appears to be crucial to protect HDLs
from lipidoxidation and hence MDA formation and the resulting dysfunction.38 In this regard, it is noteworthy that antioxidative activity and paraxonase itself were found to be modified by AGE.156,193
Symmetric dimethylarginine (SDMA) is a natural protein
degradation product, which was recently identified as the cause
of endothelial dysfunctionality of HDLs in CKD.39 SDMA is
generated intracellularly by the methylation of arginine residues
through a protein arginine methyltransferase, secreted by cells,
transported by plasma proteins, and mostly excreted by the kidney.194 Observational studies identified elevated SDMA concentrations as a risk factor for cardiovascular events.194 As yet,
it is not known why the water-soluble SDMA is enriched in the
HDL of CKD patients. HDL-associated proteins may serve as
SDMA-binding proteins, which are not properly removed from
the circulation in CKD. In fact, complementation of HDLs, reconstituted HDLs, or lipid-free apoA-I with SDMA mimics the
dysfunction of HDL from CKD patients.39 Enrichment with
SDMA transforms HDLs into agonists of TLR2, which via
atypical signaling inhibit eNOS and stimulate the translocation
of NF-κB into the nuclei of endothelial cells.39 In mice, this
translates into disturbed vasodilation and increased blood pres-
Circulation Journal Vol.77, October 2013
Multifunctional but Vulnerable HDL
2441
sure as well as impaired healing of denuded arteries by EPCs.39
In addition, VCAM-1 expression and monocyte diapedesis by
cultivated endothelial are enhanced.39
In addition to being direct causes of HDL dysfunction, structural modifications may generate neo-epitopes in apoA-I that
break self-tolerance and allow the appearance of anti-apoA-I
antibodies.169 Autoantibodies to apoA-I are found in the plasma of the normal population at a prevalence of 1–2% but at a
prevalence of 10–30% in the plasma of patients with systemic
lupus erythematosus (SLE), primary antiphospholipid syndrome
(APLS), or rheumatoid arthritis, as well as in the plasma of
patients with acute coronary syndrome (ACS) or carotid stenosis.195–203 In patients with ACS or rheumatoid arthritis high
titers of anti-apoA-I-autoantibodies increase the risk of cardiovascular events or death.199,204 In patients with SLE or APLS
the presence of anti-apoA-I-autoantibodies interferes with the
antioxidant properties of HDLs by decreasing PON1 activity.196,205 Moreover, the autoantibodies interfere with the negative
chronotropic effects of HDLs on cultivated cardiomyocytes.206
Finally, there is some evidence that anti-apoA-I-autoantibodies
have anti-idiotypic properties by which they bind to the TLR2/
CD14 complex and elicit pro-inflammatory signals.207
Alterations of the Lipid Moiety and Other Cargo Molecules
Modern mass spectrometry has enabled differentiation of more
than 200 lipid species from 5 major lipid classes (glycerophospholipids, sphingolipids, sterols, acylglycerols, and fatty acids)
in the HDL of healthy subjects.28
The content of cholesteryl esters and triglycerides in HDLs is
shifted towards enrichment with triglycerides in many clinical
conditions characterized by hypertriglyceridemia and increased
CETP activity, such as MetS, T2DM, CAD, and the acutephase response.28 By adversely affecting both cholesterol efflux
from macrophages and selective uptake of cholesteryl esters by
hepatocytes the increased triglyceride content may compromise
reverse cholesterol transport.168,208–210 However, it is important
to note that several laboratories reported positive rather than
inverse correlations between plasma concentrations of triglycerides and cholesterol efflux capacity.211–213 In addition, the increased triglyceride content of HDL from patients with T2DM
or familial hypercholesterolemia was associated with decreased
antioxidative,214,215 anti-inflammatory,216 and vasodilatory activities.217 Of note, all these observations reflect associations
among disease, altered triglyceride content, and dysfunction.
Because of confounding with other changes in the lipidome and
proteome of HDLs in these conditions, the causal contribution
of triglycerides is as yet not proven.
For the same reason, the causal role of cholesteryl ester enrichment, which is found in CETP deficiency and after CETP
inhibition, for cholesterol efflux capacity is unclear.28 Unesterified cholesterol is enriched in the HDL of patients with LCAT
deficiency and various inflammatory states, but without any
known functional consequence.28 In addition to the millimolar
concentrations of esterified and unesterified cholesterols, HDLs
carry micromolar or lower concentrations of oxysterols,218,219
bile acids,220 and steroid hormones,221–223 many of which are
agonists of nuclear hormone receptors. However, except for
some reports on the effect of 7-ketocholesterol, estradiol, and
dehydroepiandrosterone on cholesterol efflux capacity,221,224
eNOS expression, and endothelium-dependent vasoreactivity,222,223 their contribution to the physiological and pathological
functions of HDLs in health and disease, respectively, are as yet
unknown. By investigating plasma for inborn errors of HDL
metabolism, we have previously found that alterations in the
activity of ABCA1, LCAT, or CETP affect the concentrations
of 27-hydroxycholesterol in HDLs beyond the concentration
of cholesterol.219
The phospholipid content and composition have a strong effect on the size, shape, fluidity, and surface charge of HDLs.28
Their contribution to the functionality of HDLs has been tested
experimentally by reconstituting artificial HDL particles with
defined phosphatidylcholine species at different concentrations.
Increased concentrations of phosphatidylcholine increase the
ability of HDLs to induce SR-BI-mediated cholesterol efflux.225
The negatively charged phosphatidylinositol and phosphatidylserine as well as cardiolipin contribute to the inhibitory effects
of HDLs on platelet activation and coagulation.134,226 A higher
content of unsaturated fatty acids in phosphatidylcholines increases the fluidity of HDLs and thereby also their ability to
accept cholesterol102,225 and lipid hydroperoxides227 from cell
membranes and the anti-inflammatory activity on VCAM-1
expression.228 Surprisingly, however, intervention with a diet
enriched with unsaturated fatty acids was not found to alter the
cholesterol efflux capacity of HDLs.229–231 Phospholipids containing polyunsaturated fatty acids are easily oxidized.28 Some
of the oxidized phospholipids are bioactive and exert inhibitory effects, for example, on eNOS activity.232 Moreover, oxidized phospholipids generate ALE, which modify the proteins
of HDLs. It is generally believed that the high antioxidative
activity of apoA-I, PON1, and Lp-PLA2 (=PAF-AH) as well
as LCAT-mediated transacylation prevents the accumulation
of oxidized phospholipids in HDLs.28 However, as explained
earlier, these antioxidative activities are compromised in several clinical conditions. Lipolysis of glycerophospholipids by
phospholipases such as endothelial lipase and secretory phospholipase A2,233,234 which are activated by inflammation as well
as the LCAT action,235 generate lysophospholipids that in turn
are bioactive and can exert pro-inflammatory activities.233–237
Changes in the glycerophospholipid composition may hence
affect the functionality of HDLs and serve as a biomarker. As
yet, however, no data from clinical studies are available that
show associations and correlations of phospholipid composition with disease and functionality of HDLs, respectively.
Likewise, the clinical relevance of sphingolipids carried by
HDLs needs to be established. By decreasing the fluidity of
HDLs but enhancing the affinity for cholesterol, increases in the
sphingomyelin content of HDLs have a complex effect on cholesterol efflux capacity.28,238 One study reported an inverse association between sphingomyelin in HDL and the presence of
CAD in women.239 Because of their cytoprotective activities,
S1P and other lysosphingolipids are especially interesting candidates to explain HDL dysfunction and to be used as underlying biomarkers. In fact, S1P levels were found by 2 studies to
be lowered in HDL or apoB-depleted plasma of CAD patients.240,241 Interestingly, in inborn errors of HDL metabolism
the concentration of S1P and its binding protein, apoM, do not
simply vary with HDL-C or apoA-I levels. Of note, HDL-C
increasing mutations in CETP, SCARB1 (coding for SR-BI),
LIPC, and LIPG (coding for hepatic lipase and endothelial lipase, respectively) did not increase the S1P levels.242 This
underlines the potential of S1P to serve as a biomarker beyond
HDL-C or apoA-I.
The amphiphilic character and the presence of several specific binding proteins allow HDLs to transport not only lipids but
also vitamins and other micronutrients, drugs, xenobiotics, and
toxins as well as nucleic acids.243 In fact, its broad binding capacity has motivated the generation of reconstituted HDLs for
the detoxification of lipopolysaccharides in sepsis as well as for
the delivery of drugs for therapy or diagnostic imaging.244,245
However, the binding of some exogenous molecules or endog-
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2442
ANNEMA W et al.
enous metabolites may interfere with the normal functionality
of HDLs or convey atypical adverse activities. However, this
concept has not yet been tested experimentally. Likewise, the
role of miRNA transport for physiological and pathological
functions of HDLs in health and disease, respectively, is unknown. One study did not find any effect of disease-associated
alterations in HDL-associated miRNAs on endothelial functionality.30
Overall, quantitative and qualitative changes in the lipidome
of HDLs are interesting candidates for explaining interindividual differences in the functionality of HDLs and to be therefore
exploited as biomarkers. However, comprehensive lipidomic/
metabolomic case-control studies of HDL under different clinical conditions are in their infancy. Ideally, these studies will also
record HDL functions to increase the probability of finding
functionally relevant lipid species. These candidates then need
to be validated by testing reconstituted HDLs with or without
this lipid in functional assays to exclude confounding with other
alterations in HDLs.
Conclusions
For more than 50 years, a low level of HDL-C has been known
as an independent marker of increased cardiovascular risk. Nevertheless and despite the tremendous increase of knowledge
about the structure, function, and metabolism of HDL, it has not
yet been successfully targeted for the prevention or cure of atherosclerosis.
In part this failure originates from the previous targeting of
drug development to the clinical biomarker “HDL-C”, which
reflects neither the functionality of HDLs nor the intensity of
reverse cholesterol transport. The identification of the most relevant biological activities of HDLs (ie, cholesterol efflux and
reverse cholesterol transport or anti-inflammatory or vasoprotective activities) as well as their mediating molecules within HDLs
are important bottlenecks in the development of anti-atherogenic drugs as well as of diagnostic biomarkers for the identification,
personalized treatment stratification, and monitoring of patients
at increased cardiovascular risk. As yet, it is unclear whether
such markers will be native proteins or protein modifications or
lipids or other cargo molecules. To solve this problem, a systems medicine approach is needed to collect comprehensive data
sets on the proteomes, lipidomes, and functionality of HDLs
in different disease conditions for subsequent bioinformatic
integration. Proteins, lipids and/or modifications that show the
closest correlations with dysfunction and disease status will be
the most interesting candidates for subsequent experimental
and epidemiological validation in animal and cell culture models as well as prospective population and patient studies.
Another obstacle is the dynamics of HDL function and dysfunction. HDL and apoA-I of healthy humans and animals exert
innate host defense against many biological and chemical hazards. However, like temporary and local expansion of acute infections compromise the innate defense of the organism by
leukocytes and their humoral mediators and escalate into the
damaging and threatening systemic inflammatory response syndrome, chronic inflammatory conditions may compromise the
functionality of HDLs and even pervert HDLs into hazardous
particles. In this situation it may be harmful rather than helpful
to increase the half-life of HDLs, for example by CETP inhibition. The increase in C-reactive protein concentrations observed
in the failing torcetrapib and dalcetrapib trials6,7 may be indirect
indications of this harm rather than off-target effects. Drugs
that increase the elimination of existing HDLs and generation
of novel HDL particles by increasing de novo synthesis of HDLs
or infusion of reconstituted HDLs may be more promising.
Acknowledgments
Wijtske Annema was supported by funding from the Ter Meulen Fund,
Royal Netherlands Academy of Arts and Sciences. Arnold von Eckardstein
received grants from the Swiss National Science Foundation, the Swiss
COST office at Staatssekretariat für Bildung, Forschung und Innovation
as well as the 7th Framework Program of the European Commission
(“RESOLVE”, Project number 305707). Arnold von Eckardstein is also a
member of the European Cooperation in Science and Technology (COST)
Action “HDLnet” (BM904).
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Supplementary Files
Supplementary File 1
Table S1. Dysfunction of high-density lipoprotein (HDL) in disease
Please find supplementary file(s);
http://dx.doi.org/10.1253/circj.CJ-13-1025
Circulation Journal Vol.77, October 2013