Brief Reviews |
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{at}uni-muenster.de
| Abstract |
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Key Words: apolipoproteins lipid transfer proteins lipases ABC transporter Tangier disease
| Introduction |
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Reverse cholesterol transport (RCT) describes the metabolism and an important antiatherogenic function of HDL, namely, the HDL-mediated efflux of cholesterol from nonhepatic 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 3 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 |
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Several arguments against a causal relationship must be taken into account: First, the association between HDL-C and 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 hyperinsulinemia. Insulin resistance is considered the common soil of these cardiovascular risk factors. As a consequence, insulin resistance and/or the proatherogenic components of the metabolic syndrome rather than an antiatherogenic function of 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 atherosclerosis (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 epidemiological 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 Cardiovascular Münster (PROCAM) study but only 2.1 to 2.7 in longer follow-ups (Figure II, available online at http://atvb.ahajournals.org). In the ECAT Angina Pectoris Study, HDL cholesterol 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 cardiovascular 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 Coronary Atherosclerosis Prevention Study [AFCAPS/TexCAPS], and the Heart and Estrogen/Progestin Replacement Study [HERS]).20 21 22 23 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 associated with low HDL-C does not necessarily reflect a defective antiatherogenic repair system and that therapeutically induced changes in HDL-C may not necessarily lead to expected changes in clinical outcomes. Despite this skepticism toward HDL-C as a suitable intermediary phenotype for the monitoring of the cardiovascular effects of therapeutic interventions, it is still believed that modulation of HDL metabolism and the RCT system might be an important target for antiatherosclerotic drug therapy.
| HDL Subclasses |
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-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 electrophoretic pre-ß mobility and which
can be further distinguished by subsequent polyacrylamide
gradient gel electrophoresis into
pre-ß1-LpA-I,
pre-ß2-LpA-I, and
pre-ß3-LpA-I (Figure III, available online at
http://atvb.ahajournals.org). Pre-ß1-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 29 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 compartments including the lymph, where RCT is
initiated in
vivo.27 31 32
-HDL can be further differentiated by
ultracentrifugation according to density
(HDL2 and HDL3), by
nondenaturing polyacrylamide gradient gel electrophoresis
according to size, or by immunoaffinity chromatography
according to apolipoprotein composition (eg, LpA-I/A-II and
LpA-I).25
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| HDL Metabolism |
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-HDLs arise from lipid-poor particles or
even lipid-free
apolipoproteins.25 26 27 33
These lipid-poor HDL precursors are produced either as nascent HDL by
hepatocytes34 and
the intestinal mucosa,35 or
they dissociate from chylomicrons and VLDL during lipoprotein
lipasemediated hydrolysis 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
-HDL
from
plasma.48 49 50 51 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.
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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 56 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 59 60
(Figure 1
, Table 1
).
Lipids or proteins of
-HDL are removed from the
circulation by at least 2 direct pathways, which involve the 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 62 63 64 65 66 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-ß1-LpA-I or lipid-free
apoA-I.26 33 37 38 39 40
These small apolipoproteins or particles can leave the plasma into the
extravascular
space,25 26 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 69 70
(Figure 1
, Table 1
).
| Cholesterol Efflux |
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The plasma membrane, from which cholesterol is ultimately 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 invaginations that contain lipoprotein receptors, have less cholesterol and sphingolipids than the rest of the plasma membrane. In contrast, caveolae, detergent-resistant plasma membrane invaginations that are characterized by the presence of caveolins but the absence of clathrin, are enriched in cholesterol and sphingolipids. Relatively small amounts of unesterified cholesterol are found in intracellular organelles except in those that communicate with the plasma membrane (endosomes, 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, lysosomes, and caveolae72 73 78 and subsequently distributes cholesterol and phospholipids either in the form of detergent-resistant and caveolin-containing vesicles (rafts or cytolipoproteins) or as secretory detergent-soluble vesicles.72 73 79 80 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
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Protein-free phospholipid vesicles, synthetic cyclodextrins, albumin, and trypsinized HDL mediate a slow and unsaturable 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 molecules.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, unidirectional,
independent of LCAT, and efficient in reducing the content of cytosolic
cholesteryl
esters.28 42 75 76 84 85 86 87
Phospholipid efflux appears to precede cholesterol
efflux.76 87 As a
result, apolipoprotein-mediated lipid efflux lipidates apolipoproteins
and hence produces HDL-like lipoproteins with electrophoretic pre-ß
and pre-
mobilities.43 85
Apolipoprotein-mediated lipid efflux involves unspecific desorption 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 remove phospholipids and
cholesterol from normal human skin fibroblasts, aortic
smooth muscle cells, and
macrophages28 75 76 83 84 85 86 87 90 91
but not from erythrocytes, rat or porcine smooth muscle cells, or
fibroblasts of patients with Tangier
disease.84 91 92 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 brefeldin
A).83 84 It has
been suggested that lipid-free apolipoproteins and
pre-ß1-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 98 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 translocation of
cholesterol from intracellular compartments to the plasma
membrane.42 75 The
nature of this receptor is unknown. 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 49 50 51 52 84 92 93
In support of this possibility, apoA-Imediated
cholesterol efflux is severely decreased by the inhibition
of ABC1 with either antisense oligonucleotides or
pharmacological compounds 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 example, 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 bidirectional 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 Expression of SR-BI increases HDL-mediated cholesterol efflux.102 However, SR-BImediated 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 cholesterol- and caveolae-rich domains within the plasma membrane.103
HDL-mediated cholesterol efflux also shares some
properties 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 transduction
pathways, which may activate intracellular lipid transfer
processes.95 96 104 105 106 107 108
HDL induces the hydrolysis of phosphatidylcholine and
phosphatidylinositol biphosphates by phospholipases C and D and thereby
the generation of diacylglycerol (DAG), phosphatidic acid, and inositol
phosphates. DAG activates PKC, which has been shown to
stimulate the translocation of newly synthesized
cholesterol to the plasma membrane as well as
cholesterol
efflux.95 96 104 108
By contrast, HDL-mediated breakdown of phosphatidylinositol biphosphate
and the subsequent mobilization of intracellular
calcium106 109
does not stimulate cholesterol 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 98 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 46 47
ABC1 Is a Pivotal Regulator of
Cholesterol Efflux
As a complete ABC transporter, ABC1 has 2 highly
conserved cytoplasmic ATP binding cassettes and 2 transmembrane
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 segments 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-1ß, may be transported
through this
channel.113 114 115 116
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 agreement with a regulatory role of these transcription factors, ABC1 expression and lipid efflux are upregulated by cholesterol,51 53 oxysterols,117 rexinoids,118 and cAMP analogues.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 phosphatidylinositol?) 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 compartment, from which they are resecreted together with lipids.45 46 47 Tangier macrophages appear to have a defect in resecretion and erroneously target internalized HDL to lysosomes 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 transductance receptor, Rim, and MDRs, are important for the trafficking of proteins and lipids between intracellular organelles 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 macrophages.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, atherosclerosis 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 antiatherogenic 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 activation 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.98 125 126 127 128 129 130 131 132 133 134 ApoE secretion is also stimulated in the presence of exogenous 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.125 130 131 132 However, at least human monocytederived 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 macrophages).134 Moreover, in the presence of excess exogenous apolipoproteins, including apoE cholesterol, efflux from apoE-producing mouse peritoneal macrophages or J774 macrophages transfected with human apoE was more effective than cholesterol efflux from apoE-deficient mouse macrophages and untransfected J774 macrophages, respectively.137 138 This suggests that endogenous apoE modulates cholesterol efflux and cholesteryl ester hydrolysis in macrophages by the regulation of intracellular transport processes.137 138
| HDL-Mediated Delivery of Cholesterol to the Liver and Steroidogenic Organs |
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-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
Selective Cholesterol Uptake by
SR-BI
SR-BI mediates the selective uptake of cholesteryl
esters from HDL and also LDL into hepatocytes and steroid
hormoneproducing cells without internalizing HDL
proteins.61 62 140
In contrast to holoparticle receptors (eg, members 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 predominantly 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 layers 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, suggesting 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 cholesterol 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 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 knockout 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 catabolism 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 triglycerides 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-ß1-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 modulated 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.156 157 158 Responsiveness of CETP to sterols is probably the basis of reduced cholesteryl ester transfer activity in plasmas of patients treated with statins.159 Cholesteryl ester transfer activity is increased in hypertriglyceridemic individuals independently of changes in CETP mass. This is considered 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
associated with an increased risk of coronary
events.163 Likewise,
nonsynonymous alleles of the polymorphic CETP gene increased
the risk of CHD events despite being associated with increased
levels of
HDL-C.164 165 166
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
).169 170 171 172 173
Thus, whether inhibition of CETP prevents or enhances
atherosclerosis appears to depend on the presence or
absence of other dyslipidemias. CETP has been suggested to
be proatherogenic in hypercholesterolemia
(especially when the LDL receptor pathway is ineffective) and
antiatherogenic in hypertriglyceridemia and
hyperalphalipoproteinemia.162
|
HL and EL
HL hydrolyzes phospholipids and
triglycerides in all lipoprotein
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-ß-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 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 between 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 atherosclerosis.175 183 The controversial data from genetic animal models of HL (and SR-BI) demonstrate the difficulty of interpreting 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 phospholipids 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 catabolism 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 69 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 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 69 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 internalization, the ligands of cubilin (HDL, apoA-I, and the vitamin B12intrinsic 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 receptorrelated 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 internalized 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 presence 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 |
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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-Ideficient mouse model.194 195 Plasmas of apoA-Ideficient 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.198 199 200 201 202 203 Somatic overexpression of apoA-I even induces the regression of preexisting lesions.202 203 In line with improved HDL function and accelerated 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 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 transgenic mice show features of enhanced RCT, namely, increased 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
structural 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 increased 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 165 166
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
antiatherogenic effects in different mouse
models.169 170 171 172 173
Likewise, human HL deficiency as well as transgenic overexpression or
knockout of HL or apoA-II in mice unraveled the antiatherogenic and
proatherogenic effects of HL and apoA-II,
respectively.174 175 182 183 213 214 215 216
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
metabolism of atherogenic apoB-containing lipoproteins.
Thus, LCAT-deficient men, ABC1-deficient Tangier patients, and
SR-BIoverexpressing 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, atherogenic 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 |
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| Acknowledgments |
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Received May 22, 2000; accepted October 18, 2000.
| References |
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