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HDL – a difficult friend
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Vol. 5, No. 3–4 2008
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Cardiology MECHANISMS
HDL – a difficult friend Arnold von Eckardstein Institute of Clinical Chemistry, University Hospital Zurich, Raemistrasse 100, CH 8091 Zurich, Switzerland
High-density lipoprotein (HDL) is an attractive target for antiatherogenic drug therapy because of the inverse association between HDL cholesterol and cardiovascu-
Section Editor: Christian Weber – Institute for Molecular Cardiovascular Research (IMCAR), Aachen University, Germany
lar risk as well as many potentially antiatherogenic functions. However, controversial data from inborn
Introduction
errors of human HDL metabolism and genetic animal
Among the many risk factors of atherosclerotic cardiovascular diseases dyslipidemias play a pivotal pathogenetic role and have hence become a prime target for both prevention and therapy. In several controlled intervention trials encompassing more than 500,000 patient years of follow-up statins, which inhibit cholesterol synthesis and lower LDL cholesterol, have proved as safe and effective in reducing cardiovascular morbidity and mortality [1,2]. However, 60–80% of cardiovascular events could not be prevented in the statin arms of these trials. In addition, intracoronary ultrasound studies revealed that the treatment of hypercholesterolemia to the current LDL cholesterol goal of <100 mg/dL (<2.6 mmol/L) reduces progression but is not sufficient to induce regression of atherosclerosis [3]. As a consequence, lower treatment goals (e.g. LDL cholesterol < 70 mg/dL/ <1.7 mmol/L) and treatment of other risk factors are intensively discussed [4]. Among these other risk factors low HDL cholesterol has turned out as very attractive because both in populationbased studies of healthy individuals (i.e. primary prevention setting) and in patients with pre-existing coronary heart disease (i.e. secondary prevention setting) low concentrations of HDL cholesterol or apolipoprotein (apo) A-I, that is the major protein component of HDL, turned out as independent risk factors for future myocardial infarction [5]. Interestingly, this association was found in old studies, such as the ECAT Angina pectoris study, which were performed in the 1980s before the introduction of statins [6], as well as in very recent studies where LDL cholesterol was very well controlled by statin therapy [7,8].
models as well as the frequent confounding of low HDL cholesterol with other proatherogenic conditions in the population have complicated the proof of a causal relationship between HDL cholesterol and atherosclerosis. Because HDLs form a very heterogenous class of lipoproteins which differ in protein and lipid composition, it is increasingly accepted that the quality rather than quantity of HDL is relevant for its atheroprotective activity. As a consequence, protein or lipid or functional biomarkers are postulated to be better biomarkers than HDL cholesterol to assess and monitor the cardiovascular risk exerted by disturbed HDL metabolism and to estimate the benefit of any therapeutic intervention. In addition, novel therapeutics are searched which either improve HDL metabolism, mimic HDL function or cure the regulatory network underlying disturbed HDL metabolism and function. Beyond better biomarkers of HDL functionality and metabolism early clinical endpoint studies are needed to assess the therapeutic benefit of any novel HDLmodifying therapy.
E-mail address: A. von Eckardstein ([email protected]) 1740-6765/$ ß 2008 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2008.10.004
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Structure, function, metabolism and regulation of HDL Structure HDL is a heterogenous class of lipoproteins which differ by composition, shape, size and density [9,10]. In a recent shotgun proteomic approach more than 50 different HDL-associated proteins have been identified which include apolipoproteins in the proper sense, such as apoA-I and apoA-II, lipocalins such as apoD and apoM, lipid-transfer proteins and lipid-modifying enzymes, serpins and complement regulatory proteins [11]. Also the lipid content is highly
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heterogenous. Traditionally lipids of HDL have been considered as structural components and cargo only. More recently it has become evident that, beyond cholesterol, phosphatidylcholine and sphingomyelin, HDL carries a great variety of biologically quantitatively minor active lipids such as oxysterols, lysophospholipids and lysosphingolipids [12–14].
Functions HDL and its structural compounds exert a broad scope of biological actions which potentially counteract several steps in the pathogenesis of atherosclerosis (Fig. 1): for example,
Figure 1. Structural and functional heterogeneity of HDL. HDLs consist of various subclasses that can be differentiated by shape, size, density, electrophoretic mobility and protein composition. It contains more than 50 different proteins and many lipid classes. Several of them exert biological activities that may protect from atherosclerosis. apo, apolipoprotein; LCAT, lecithin:cholesterol acyl-transferase; CETP, cholesteryl ester transfer protein; PLTP, phospholipid-transfer protein; PON, paraoxonase; LpPLA2, lipoprotein-associated phospholipase A2; glutathionperoxidase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; IL1b, interleukin 1 beta; TNFa, tumor necrosis factor alpha; NO, nitric oxide.
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they correct endothelial dysfunction including monocyte adhesion and disturbed vasoreactivity, stimulate cholesterol efflux and inhibit lipoprotein oxidation, proinflammatory cytokine secretion from macrophages, platelet aggregation and coagulation [9,10,15,16]. Unfortunately it is as yet unknown which of the many functions and hence also components of HDL are most crucial for the protective effects on atherosclerosis. Furthermore, these and additional functions of HDL are also of interest for the treatment and prevention of diseases other than atherosclerosis. For example, HDLs were found to reduce ischemia-reperfusion injury, to preserve survival and function of beta-cells, and to prolong survival of animals infected with viruses, bacteria or protozoa (e.g. malaria or trypanosoma) [9,13,16–18].
Metabolism HDL metabolism is a multistep process which involves the secretion of lipid-free apolipoproteins by the liver or intestine, the acquisition of phospholipids and cholesterol from cells via ATP-binding cassette (ABC) transporters A1 and G1, the maturation by LCAT-mediated cholesterol esterification and PLTP-mediated particle fusion, and the final delivery of lipids to the liver, either directly via receptors or indirectly via CETP-mediated transfer to LDL (Fig. 2) [9,10,15,19]. The investigation of further important steps in HDL metabolism
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has been started only recently, for example, the traffic of HDL between intra- and extravascular compartments which is a prerequisite for the exertion of many antiatherogenic functions within the arterial wall [20]. The incomplete knowledge of HDL metabolism is also highlighted by the findings of genetic linkage analyses and genome-wide association studies which identified more than 30 loci in men and mice which regulate HDL cholesterol levels. For several genes and their products it is not known, how they are related to HDL metabolism [21,22].
Regulation Like other cardiovascular risk factors, HDL cholesterol is regulated by many genetic and nongenetic factors which are only partially resolved and interact with one another [23]. Genetic factors account for about 50% of the variability in HDL cholesterol levels among individuals [22,23]. Both monogenic and polygenic factors are involved. Heterozygosities for private mutations in the genes of apoA-I, ABCA1 and LCAT explain about 10% of HDL cholesterol levels below the 5th percentile [24,25]. Polymorphisms in CETP, LPL, hepatic lipase or apoE explain 1–4 mg/dL mean differences between carriers and noncarriers [23]. Most recently, genome-wide linkage analysis identified most of the genes mentioned above as well as endothelial lipase and four other genes of
Figure 2. Key steps in HDL metabolism and their modulation by factors relevant in metabolic syndrome. HDL precursors may be secreted as nascent HDL by hepatocytes and the intestinal mucosa. They may also dissociate from triglyceride-rich lipoproteins (TGRL, i.e. VLDL and chylomicrons) during lipoprotein lipase-mediated hydrolysis of triglycerides. Alternatively, they may be generated by the interconversion of HDL by cholesteryl ester transfer protein (CETP), phospholipid-transfer protein (PLTP), hepatic lipase (HL), scavenger receptor BI (SR-BI) and endothelial lipase (EL). The interaction of lipidfree apoA-I or lipid-poor particles with the ATP-binding cassette transporter A1 (ABCA1), which is expressed in many cells including hepatocytes, enterocytes and macrophages, leads to efflux of cellular phospholipids and unesterified cholesterol, and thereby to the formation of discoidal HDL precursors. These discoidal HDL precursors become mature, lipid-rich and spherical through the esterfication of cholesterol by the enzyme lecithin:cholesterol acyltransferase (LCAT). They increase in size through the acquisition of additional phospholipids and unesterified cholesterol, from cells (in a process that is mediated by ABCG1 and SR-BI) or apoB-containing lipoproteins (by an at least partially PLTP-mediated process); by the association of additional apolipoproteins; by ongoing LCAT-mediated cholesterol esterification and by PLTP-mediated fusion with other HDL. Lipids of mature HDL are removed from the circulation by at least two pathways, which involve the selective uptake of lipids via SR-BI and by exchanging cholesteryl esters of HDL with triglycerides of apoB-containing lipoproteins, which are then removed via the LDL-receptor (LDLR) pathway. Various genes or their encoded proteins in this pathway are dysregulated by free fatty acids or insulin resistance.
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unknown role in HDL metabolism as genetic determinants of HDL cholesterol levels [21]. Important life style and environmental factors influencing HDL cholesterol include gender, body fat, physical activity, smoking and alcohol intake as well as several drugs [25]. Clinically most relevant is the frequent association of low HDL cholesterol with overweight or obesity, hypertriglyceridemia, latent or overt type 2 diabetes mellitus and hypertension, that is with components of the metabolic syndrome [26,27]. Therefore, insulin resistance and metabolic sequelae thereof appear to play an important role in the determination of HDL cholesterol levels in the population. In fact, insulin and free fatty acids, which are found at increased concentrations in the metabolic syndrome, regulate several important steps of HDL metabolism at the transcriptional and posttranslational level (Fig. 2) [19,27,28]. Several pivotal genes in HDL metabolism are regulated by nuclear hormone receptors such as the retinoid-X- and A-receptors RXR and RAR, the liver and farnesoid X receptors LXR and FXR as well as the peroxisome proliferator agent receptors PPARa, PPARg and PPARb/d. Because these receptors are activated by endogenous and exogenous lipids or derivatives thereof, they and their cofactors are also strong candidates for contributing to the pathogenesis of low HDL cholesterol and other components of the metabolic syndrome [19,29].
Is HDL a causal risk factor? Despite the pleiotropic potentially antiatherogenic functions of HDL, the causality of the inverse association between HDL cholesterol and cardiovascular risk has not been definitely proven, because several data from epidemiological, clinical as well as human and animal genetic studies are contradictory or can be differently interpreted.
Epidemiology The relationship between HDL cholesterol concentration and cardiovascular risk is not strictly dose-dependent. While the association of low HDL cholesterol with increased cardiovascular risk is a rather constant finding, the association of high HDL cholesterol with reduced risk is not: in several primary and secondary prevention studies HDL cholesterol levels in the highest quartile or quintile were not associated with less risk than intermediate HDL cholesterol levels [5,7,8]. Furthermore, although the epidemiological association of low HDL cholesterol with increased cardiovascular risk is statistically independent of the confounding risk factors constituting the metabolic syndrome, it has been argued that low HDL cholesterol may be an innocent bystander of these proatherogenic situations [5]. Especially fasting and, even more so, postprandial hypertriglyceridemia are found in more than 50% of individuals with HDL cholesterol <1 mmol/L, so some authors suggested that low HDL cholesterol is a nonpathogenic long-term marker of disturbed postprandial lipid e318
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metabolism exactly as glycated hemoglobin A1 is a nonpathogenic long-term marker of disturbed glucose metabolism [30–32].
Human genetics Several inborn errors of metabolism causing low and high HDL cholesterol have been identified in patients with premature atherosclerosis and high age, respectively. For example, several patients with complete apoA-I deficiency because of nonsense mutations in the apoA-I gene suffered from myocardial infarction before the age of 50 years [23,33]. Likewise CETP deficiency, which causes a high HDL cholesterol level was originally thought to contribute to prolonged life expectancy and low myocardial infarction rates in Japan [34]. However, if these single reports on rare homozygous cases were extended to systematic family studies or, most recently even to the population level, the picture became more complicated: for example, heterozygosity for rare mutations in the ABCA1 gene, which decreases HDL cholesterol significantly below the commonly accepted threshold value of 40 mg/dL (1.05 mmol/L), was not associated with increased cardiovascular risk [35]. Likewise data on mutations and polymorphisms in CETP have been controversial [23,36]. However, in a recent meta-analysis of 46 studies with data on 27,196 coronary cases and 55,338 controls showed significant associations of three CETP polymorphisms with decreased CETP activity and mass by 8–10% and with increased HDL cholesterol by 4–5% and with slightly reduced cardiovascular risk [36]. Interestingly several mutations in pivotal genes of HDL metabolism, for example, ABCA1, do not affect HDL cholesterol levels but increase cardiovascular risk [37,38]. This finding, together with findings in ABCA1 animal models (see below), points to antiatherogenic functions of ABCA1 which are not reflected by HDL cholesterol, for example, in macrophages, endothelial and beta cells [15,17,20].
Clinical intervention studies Life style interventions such as smoking cessation, increased physical activity, body weight reduction or moderate alcohol intake as well as drug interventions with statins, fibrates, glitazones, nicotinic acid or rimonabant increase HDL cholesterol by 5–30% [26] (Fig. 3), so more effective regimens are sought. Some of these interventions, for example smoking cessation and drug therapies with statins, fibrates and nicotinic acid were shown to reduce cardiovascular event rates. However, these interventions have profound effects on other cardiovascular risk factors as well and the clinical benefit was not found correlated with changes in HDL cholesterol. Therefore, the clinical benefit of these interventions does not prove the causal role of altered HDL metabolism [26,28,34]. More direct effects can be assessed in studies where reconstituted HDLs were given to patients with acute coronary syndromes. Although intracoronary ultrasound analyses revealed some
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Figure 3. Maximal effects of established therapeutic interventions on HDL cholesterol.
regression of atherosclerotic plaque load in these studies, it is important to note that most of the predefined endpoints in these studies were not reached or that the benefit was not dose-dependent [39,40]. The most effective HDL cholesterol increasing drugs are inhibitors of CETP. The most advanced representative of this class, torcetrapib, doubled HDL cholesterol levels if given at the highest dosage [26,34]. Its clinical efficacy in reducing cardiovascular events was tested in three phase III trials using carotid intima media thickness and coronary plaque volume as intermediate surrogate endpoints or cardiovascular events as clinical endpoints [41–43]. Surprisingly, the combination of atorvastatin with torcetrapib did not prevent progression of carotid or coronary atherosclerosis more effectively than atorvastatin alone despite further reducing LDL cholesterol by about 15% and increasing HDL cholesterol by more than 50% [41,42]. Even worse, the clinical endpoint study had to be stopped prematurely because of an excess in cardiovascular and overall mortality in the torcetrapib + atorvastatin arm [43]. As yet it is not clear whether the failure of torcetrapib has been caused by adverse off-target effects, notably the increase in blood pressure as well as aldosterone and sodium levels, or by the choice of the wrong target, that is CETP inhibition [26,34,43].
Animal studies Also genetic mouse models provided controversial data on the role of HDL in atherosclerosis [5,10,21,33]. However, before going into details, it is important to recall that murine and human lipoprotein metabolism is considerably different: notably wild-type mice lack CETP and partially therefore carry almost all of their plasma cholesterol in HDL. Only data from animals with altered apoA-I expression are consistent with one another and the expectations: the knock-out of apoA-I caused HDL deficiency and increased atherosclerosis in a proatherogenic but not in a normolipidemic background [44,45]. Overexpression of apoA-I was found to increase HDL cholesterol
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and to inhibit progression or, if accomplished by somatic gene transfer, even induce regression of atherosclerosis [10,19,28,34,46]. Systemic or macrophage deletion of ABCA1 increased atherosclerosis at least when ABCG1 also was deleted [47–49]. Systemic overexpression of ABCA1 increased HDL cholesterol but was found to increase atherosclerosis [50,51]. However, macrophage-specific overexpression of ABCA1 was found to decrease atherosclerosis without altering HDL cholesterol [52]. The effects of knocking-out or overexpressing LCAT were modulated by CETP. LCAT-deficient mice had low HDL cholesterol but developed atherosclerosis only if CETP was overexpressed. LCAT-overexpressing mice had high HDL cholesterol but developed atherosclerosis. Atherosclerosis, however, was reduced if both LCAT and CETP were expressed. Also the effects of overexpressed CETP or SR-BI in mice on atherosclerosis were strongly modulated by the parallel modulation of other genes [5,10,21,34].
Consequences for HDL as a therapeutic target Several of the aforementioned inconsistencies on the causal relationship of HDL to atherosclerosis can be resolved if one considers HDL as a heterogenous class of lipoproteins which differ in function and which interact with other risk factors.
Whom shall we treat? The increased cardiovascular risk associated with low HDL cholesterol is modulated by other risk factors (Fig. 4; [5]). This is probably even truer for HDL than for other risk factors because it conveys protection or repair and hence is especially relevant in the presence of other risk factors or pre-existing atherosclerosis. Like a fire brigade that needs conflagration to show its usefulness HDL needs a proatherogenic condition to prove its relevance. Interestingly in most individuals low HDL cholesterol is a component of the metabolic syndrome which frequently coexists because of the common metabolic and regulatory soil with other proatherogenic risk factors such as diabetes, hypertriglyceridemia as well as procoagulatory and proinflammatory states. In these conditions the risk threshold value of HDL cholesterol is 40 mg/dL (1.05 mmol/ L) or even higher (Fig. 4). By contrast in the much rarer inborn errors of HDL metabolism, such as heterozygous apoA-I or ABCA1 deficiency, the coexistence of other risk factors is a chance finding, so frequently in these situations low HDL cholesterol is the only risk factor. In these situations, the risk threshold is much lower, namely 20 mg/dL (Fig. 4). It is hence no surprise that relatively rare isolated low HDL cholesterol conditions because of monogenic defects in HDL metabolism do not confer the same cardiovascular risk as much more frequent polygenic low HDL cholesterol conditions, for example, in patients with the metabolic syndrome, which because of their metabolic origin are obligatorily accompanied by other risk factors [33,35]. Interestingly also in genetic mouse models a proatherogenic condition (e.g. LDL receptor www.drugdiscoverytoday.com
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Figure 4. Incidence of coronary events in men according to HDL-cholesterol and other risk factors in the PROCAM study. Hypertriglyceridemia: triglycerides > 2.1 mmol/L; hypercholesterolemia: LDL cholesterol > 4.1 mmol/L; IFG: impaired fasting glucose (glucose 6.1–7.0 mmol/L) (modified from Ref. [5]).
deficiency) must coexist to see the aggravating effects of apoA-I deficiency or the antiatherogenic effects of apoA-I overexpression [44,45]. Accordingly patients with pre-existing atherosclerosis and/or multiple or severe additional risk factors appear to be the most relevant group for HDL-modifying therapies.
How and how much shall we treat? For a long time and in analogy to the ‘the lower the better concept’ for LDL cholesterol patients, physicians and scientists longed for very high levels of the ‘good’ HDL cholesterol. However, in contrast to the robust epidemiological evidence for low HDL cholesterol being a risk factor of atherosclerosis there is much less observational evidence for the protective effects of very high HDL cholesterol [5,7,8]. Instead of supporting ‘the higher the better’ the epidemiological data rather indicate that a threshold of HDL cholesterol must be passed (e.g. 50–60 mg/dL or 1.3–1.5 mmol/L; see Fig. 4 and data in Refs. [5,7,8]). Depending on the basal level, drugs increasing HDL cholesterol by 20–100% are needed to surpass this threshold level. Among the presently available drugs only nicotinic acid shows an efficacy at the lower end of this range (Fig. 3). Among the CETP inhibitors dalcetrapib (=JTT 705) increases HDL cholesterol by 25–30% while torcetrapib and the structurally related anacetrapib can double HDL cholesterol levels [41–43,53,54]. However, as yet nicotinic acid is the only drug which was shown to reduce cardiovascular endpoint rates as well [55]. Because the risk of low HDL cholesterol is modulated by additional risk factors (Fig. 4), elimination of accompanying risk factors including lowering of LDL cholesterol by statin treatment is still the most important measure of cardiovascular risk reduction in patients with low HDL cholesterol. Moreover, to improve the proven therapeutic benefit of statins, the current philosophy is that any HDL cholesterol-modifying therapy (e.g. nicotinic acid) is combined with statin therapy. Mostly because of ethical e320
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reasons, also the design of clinical endpoint studies on HDL cholesterol increasing treatments such as CETP inhibition follows this paradigm [41–43].
What shall we measure? For the present time HDL cholesterol and apoA-I concentrations are equally well suited to identify patients at risk. However, in no study, changes of HDL cholesterol predicted the outcome of any intervention. This is no surprise because the cholesterol in HDL is neither a protective agent nor a reflection of the antiatherogenic HDL action for example in reverse cholesterol transport. It is simply an indirect estimation of HDL particle number which, however, owing to HDL size heterogeneity varies considerably among individuals with equal HDL cholesterol concentration [5,9,12]. The particle heterogeneity of HDL is incompletely and insufficiently characterized by HDL2 and HDL3 so that measurement of cholesterol in these two subfractions does not much improve the diagnostic performance [9]. More comprehensive data on the size and charge heterogeneity of HDL particles are provided by NMR spectroscopy or refined electrophoretic techniques [56,57]. In fact some of the thereby determined HDL subclasses were found to predict the incidence of cardiovascular events better than HDL cholesterol; however, intellectual property right issues and the complexity, respectively, of the applied technology and difficulties in standardization aggravate the roll out into clinical laboratories [9]. It appears more feasible to measure defined functional protein or lipid components of HDL by immunoassays and mass spectrometry; however, it is as yet unknown which of the many protein and lipid components of HDL and the thereof exerted functions are most important in protecting from atherosclerosis [9,11–13,16]. As yet only apoA-I and apoA-II levels have been evaluated in larger populations. In observational studies these parameters showed no consistent advantage over HDL cholesterol [58]. In some intervention
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studies such as AFCAPS/TexCAPS or IDEAL changes in apoA-I levels unlike HDL cholesterol concentrations were significantly associated with outcome [59,60]. In addition, the compostion and hence functionality of HDL changes in response to inflammatory or metabolic stress, so that physiological protein components, for example apoA-I, are either displaced by acute phase proteins such as serum amyloid protein A (SAA) [61] or modified for example by carbamylation of lysine residues, nitration or chlorination of tyrosine or tryptophan residues or oxidation of methionine residues [62]. Several of these oxidative modifications as well as glycation in diabetes mellitus impair the functionality of HDL or its components, for example the ability of apoA-I to induce ABCA1-mediated cholesterol efflux and activate LCAT [63,64]. Also the lipid moiety of HDL can be modified in the course of inflammation, for example by phospholipases which generate proinflammatory lysophospholipids or by reactive oxygen species which oxidize polyunsaturated fatty acids of phospho- and sphingolipids. Some of these modifications do not only interfere with the antiatherogenic functionality of HDLs but turn them into proinflammatory or proatherogenic particles [61]. In any case at equal levels of HDL cholesterol two individuals may differ considerably in atheroprotection. Proteomic and lipidomic analyses of HDL are hence needed to identify new potential biomarkers of HDL functionality, which then must be validated in clinical studies for their ability to improve differential diagnosis, prognosis and therapy monitoring low HDL cholesterol. Alternatively, functional tests may be used to assay the heterogeneity of HDL, for example measurement of activities to stimulate cholesterol efflux, to inhibit oxidation or adhesion of monocytes to endothelial cells [9]. However, these bioassays are difficult to standardize and automate so that they appear feasible in specialized laboratories and clinical studies only [9]. Finally, instead of HDL components or functions one may measure the effect of any HDL-modifying therapy on the extent and/or severity of atherosclerosis. However, also for this approach we need novel disease biomarkers (similar to tumor markers) since as yet only expensive imaging techniques can do this job.
What is the right therapeutic target? In view of the structural, functional and metabolic complexity of HDL it is difficult to predict the most attractive target for therapy development. Evidence from animal models and inborn errors of human HDL metabolism points to the stimulation of apoA-I, ABCA1 (at least in macrophages) and LCAT as the most interesting targets. Unfortunately no small molecules have been found to stimulate apoA-I or LCAT production. Because in general stimulators or agonists are notoriously difficult to be developed, efforts have been increased to substitute apoA-I and other potentially antiatherogenic HDL components or mimic their antiathero-
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genic properties. Because of the need of infusion and largescale protein production substitution therapy with isolated or recombinant apoA-I appears to be limited to acute situations such as the acute coronary syndrome. Initial data on shortterm treatment of patients with acute coronary syndrome indicate some regression of lesions [39,40] but the value of this intervention with respect to clinical outcome is not known. Limitations in producing large quantities of (recombinant) protein and the need of intravenous therapy for apoA-I can be circumvented by using small peptides that mimic the structure of apoA-I and that are synthesized of D-amino acids. In animal experiments, these mimetics reduced the progression of atherosclerosis [65]. Data of animal experiments also point to the antiatherogenic potential of lipid mimetics like FTY702 which binds to sphingosine-1phosphate receptors and was found to inhibit atherosclerosis in apoE- or LDL-receptor deficient mice [66–68]. More promising for a longtime therapy are small molecules. LXR agonists are known to increase ABCA1 expression, HDL cholesterol levels and reverse cholesterol transport [18]. However, because they also induce hepatic VLDL production and, hence, hypertriglyceridemia, and because hepatic ABCA1 overexpression may be proatherogenic, macrophage-specific LXR agonists would be needed [18,27,33,69]. Also because inhibitors or antagonists can be more easily developed than activators or stimulators much effort is invested to find small molecules that inhibit the catabolism of HDL. However, although inhibition or deletion of CETP, PLTP or SR-BI increases HDL cholesterol, the effects of such interventions on atherosclerosis are uncertain. Because of the negative outcomes of the torcetrapib trials [41,43] and because of the controversial data of animal models with high HDL cholesterol levels owing to delayed HDL catabolism [5,10,22] the development of these drugs needs biomarkers that assess HDL functionality better than HDL cholesterol or apoA-I levels. Because of the frequent association of low HDL cholesterol with other proatherogenic components of the metabolic syndrome, it appears of special importance to target therapies to a common denominator of this condition, for example obesity or insulin resistance, so that most of, if not all, the components/risk factors of the metabolic syndrome are corrected. Dual PPAR agonists or rimonabant have been developed according to this paradigm but raised safety issues [70,71]. Alternatively, one may consider the metabolic syndrome as the product of several vicious cycles of interconnected metabolic disturbances which can be interrupted by correcting or improving HDL functionality ([27], Fig. 5): insulin resistance directly or indirectly produces metabolic stress factors such as fatty acids, triglycerides and cytokines and interferes with protective or repairing factors such including HDL. The resulting disbalance of metabolic stress and repair systems is not only proatherogenic but also harmful for other organs including www.drugdiscoverytoday.com
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impair the functionality of HDL, notably those which inhibit the catabolism and hence prolong the exposure time of HDL to unwanted enzymatic and nonenzymatic modifications such as oxidation, glycation, apolipoprotein displacement, proteolysis or lipolysis. There is also a strong need of biomarkers that reflect the antiatherogenicity of HDL better than plasma concentrations of HDL cholesterol or apoA-I. In addition any novel HDL therapy must be subjected to clinical imaging and endpoint trials at an early stage to prove that it not only changes concentration and composition of HDL but also reduces progression or even induces regression of atherosclerosis and lowers cardiovascular event rates.
Acknowledgement The author is supported by the 6th Framework of the European Commission (HDLomics, LSHM-CT-2006-037631) and the Swiss National Research Foundation.
References
Figure 5. Mutual relationships between diabetes mellitus, HDL and atherosclerosis.
beta cells and the liver, that is, contributes to the manifestation of diabetes mellitus and nonalcoholic fatty liver disease which in turn aggravate the metabolic disturbances. Interestingly, low HDL cholesterol is also a risk factor of future diabetes mellitus and HDL as well as important HDL regulatory genes like ABCA1 were recently found to inhibit beta cell apoptosis and to secure insulin secretion, respectively [16,27,72]. Hence in a prediabetic state, insulin resistance compromises HDL metabolism and thereby (and other factors) also beta cell protection (Fig. 5). In the metabolic syndrome it may hence also be promising to address HDL as a prime target to interrupt the transition of insulin resistance to diabetes mellitus.
Overall conclusion The independent association of low HDL cholesterol with increased cardiovascular risk, many potentially antiatherogenic properties activities of HDL and its components as well as the beneficial effects of application or overexpression of apoA-I on atherosclerosis in humans and animals point to a causal role of HDL or some of its components in atherosclerosis protection. The antiatherogenic effect of HDL is incompletely recovered by the measurement of HDL cholesterol, especially in clinical situations which alter the composition of HDL, for example in the course of inflammation and diabetes. Of note, also pharmacological interventions may e322
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Brunham, L.R. et al. (2008) Cholesterol in islet dysfunction and type 2 diabetes. J. Clin. Invest. 118, 403–408 18 Gomaraschi, M. et al. (2008) Anti-inflammatory and cardioprotective activities of synthetic high-density lipoprotein containing apolipoprotein A-I mimetic peptides. J. Pharmacol. Exp. Ther. 324, 776–783 19 Rader, D.J. (2006) Molecular regulation of HDL metabolism and function: implications for novel therapies. J. Clin. Invest. 116, 3090–3100 20 Cavelier, C. et al. (2006) ABCA1 modulates ApoA-I transcytosis through aortic endothelial cells. Circ. Res. 99, 1060–1066 21 Kathiresan, S. et al. (2008) Defining the spectrum of alleles that contribute to blood lipid concentrations in humans. Curr. Opin. Lipidol. 19, 122–127 22 Rollins, J. et al. (2006) In search of new targets for plasma high-density lipoprotein cholesterol levels: promise of human-mouse comparative genomics. Trends Cardiovasc. Med. 16, 220–234 23 Holleboom, A.G. et al. (2008) The value of HDL genetics. Curr. Opin. Lipidol. 19, 385–394 24 Frikke-Schmidt, R. et al. (2004) Genetic variation in ABC transporter A1 contributes to HDL cholesterol in the general population. J. Clin. Invest. 114, 1343–1353 25 Cohen, J.C. et al. (2004) Multiple rare alleles contribute to low plasma levels of HDL cholesterol. Science 305, 869–872 26 Singh, I.M. et al. (2007) High-density lipoprotein as a therapeutic target: a systematic review. JAMA 298, 786–798 27 Rohrer, L. et al. (2004) High density lipoproteins in the intersection of diabetes mellitus, inflammation and cardiovascular disease. Curr. Opin. Lipidol. 15, 269–278 28 Linsel-Nitschke, P. and Tall, A.R. (2005) HDL as a target in the treatment of atherosclerotic cardiovascular disease. Nat. Rev. Drug Discov. 4, 193– 205 29 Desvergne, B. et al. (2006) Transcriptional regulation of metabolism. Physiol. Rev. 86, 465–514 30 Nordestgaard, B.G. et al. (2007) Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women. JAMA 298, 299–308 31 Bansal, S. et al. (2007) Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women. JAMA 298, 309–316 32 Zilversmit, D.B. (1979) Atherogenesis: a postprandial phenomenon. Circulation 60, 473–485 33 von Eckardstein, A. (2006) Differential diagnosis of familial high density lipoprotein deficiency syndromes. Atherosclerosis 186, 231–239 34 Joy, T. and Hegele, R.A. (2008) Is raising HDL a futile strategy for atheroprotection? Nat. Rev. Drug Discov. 7, 143–155 35 Frikke-Schmidt, R. et al. (2008) Association of loss-of-function mutations in the ABCA1 gene with high-density lipoprotein cholesterol levels and risk of ischemic heart disease. JAMA 299, 2524–2532 36 Thompson, A. et al. (2008) Association of cholesteryl ester transfer protein genotypes with CETP mass and activity, lipid levels, and coronary risk. JAMA 299, 2777–2788 37 Frikke-Schmidt, R. et al. (2005) Mutation in ABCA1 predicted risk of ischemic heart disease in the Copenhagen City Heart Study Population. J. Am. Coll. Cardiol. 46, 1516–1520 38 Frikke-Schmidt, R. et al. (2008) Genetic variation in ABCA1 predicts ischemic heart disease in the general population. Arterioscler. Thromb. Vasc. Biol. 28, 180–186 39 Nissen, S.E. et al. (2003) Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA 290, 2292–2300 40 Tardif, J.C. et al. (2007) Effect of rHDL on Atherosclerosis-Safety and Efficacy (ERASE) Investigators. Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial. JAMA 297, 1675–1682 41 Barter, P.J. et al. (2007) Effects of torcetrapib in patients at high risk for coronary events. N. Engl. J. Med. 357, 2109–2122 42 Kastelein, J.J. et al. (2007) Effect of torcetrapib on carotid atherosclerosis in familial hypercholesterolemia. N. Engl. J. Med. 356, 1620–1630 43 Nissen, S.E. et al. (2007) Effect of torcetrapib on the progression of coronary atherosclerosis. N. Engl. J. Med. 356, 1304–1316
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Moore, R.E. et al. (2005) Increased atherosclerosis in mice lacking apolipoprotein A-I attributable to both impaired reverse cholesterol transport and increased inflammation. Circ. Res. 97, 763–771 Li, H. et al. (1993) Lack of apoA-I is not associated with increased susceptibility to atherosclerosis in mice. Arterioscler. Thromb. 13, 1814– 1821 Lebherz, C. et al. (2007) Gene transfer of wild-type apoA-I and apoA-I Milano reduce atherosclerosis to a similar extent. Cardiovasc. Diabetol. 6, 15 Out, R. et al. (2008) Coexistence of foam cells and hypocholesterolemia in mice lacking the ABC transporters A1 and G1. Circ. Res. 102, 113–120 Yvan-Charvet, L. et al. (2007) Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice. J. Clin. Invest. 117, 3900–3908 Out, R. et al. (2008) Combined deletion of macrophage ABCA1 and ABCG1 leads to massive lipid accumulation in tissue macrophages and distinct atherosclerosis at relatively low plasma cholesterol levels. Arterioscler. Thromb. Vasc. Biol. 28, 258–264 Joyce, C.W. et al. (2002) The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice. Proc. Natl. Acad. Sci. U. S. A. 99, 407–412 Joyce, C.W. et al. (2006) ABCA1 overexpression in the liver of LDLr-KO mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis. J. Biol. Chem. 281, 33053–33065 Van Eck, M. et al. (2006) Macrophage ATP-binding cassette transporter A1 overexpression inhibits atherosclerotic lesion progression in low-density lipoprotein receptor knockout mice. Arterioscler. Thromb. Vasc. Biol. 26, 929–934 de Grooth, G.J. et al. (2002) Efficacy and safety of a novel cholesteryl ester transfer protein inhibitor, JTT-705, in humans: a randomized phase II dose-response study. Circulation 105, 2159–2165 Krishna, R. et al. (2007) Effect of the cholesteryl ester transfer protein inhibitor, anacetrapib, on lipoproteins in patients with dyslipidaemia and on 24-h ambulatory blood pressure in healthy individuals: two doubleblind, randomised placebo-controlled phase I studies. Lancet 370, 1907– 1914 Canner, P.L. et al. (1986) Fifteen year mortality in Coronary Drug Project patients: long-term benefit with niacin. J. Am. Coll. Cardiol. 8, 1245–1255 Lamon-Fava, S. et al. (2008) Plasma levels of HDL subpopulations and remnant lipoproteins predict the extent of angiographically-defined coronary artery disease in postmenopausal women. Arterioscler. Thromb. Vasc. Biol. 28, 575–579 Ala-Korpela, M. (2008) Critical evaluation of 1H NMR metabonomics of serum as a methodology for disease risk assessment and diagnostics. Clin. Chem. Lab. Med. 46, 27–42 Holme, I. et al. (2008) Relationships between lipoprotein components and risk of myocardial infarction: age, gender and short versus longer followup periods in the Apolipoprotein MOrtality RISk study (AMORIS). J. Intern. Med. 264, 30–38 Gotto, A.M., Jr et al. (2000) Relation between baseline and on-treatment lipid parameters and first acute major coronary events in the Air Force/ Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS). Circulation 101, 477–484 van der Steeg, W.A. et al. (2008) High-density lipoprotein cholesterol, high-density lipoprotein particle size, and apolipoprotein A-I: significance for cardiovascular risk: the IDEAL and EPIC-Norfolk studies. J. Am. Coll. Cardiol. 51, 634–642 Kontush, A. and Chapman, M.J. (2006) Functionally defective highdensity lipoprotein: a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol. Rev. 58, 342– 374 Ansell, B.J. et al. (2007) The paradox of dysfunctional high-density lipoprotein. Curr. Opin. Lipidol. 18, 427–434 Shao, B. et al. (2008) Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I. Proc. Natl. Acad. Sci. U. S. A. 105, 12224– 12229 [Epub 2008 August 21] Shao, B. et al. (2006) Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine
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chlorination of apolipoprotein A-I. J. Biol. Chem. 281, 9001–9004 [Epub 2006 February 22] Navab, M. et al. (2006) Apolipoprotein A-I mimetic peptides and their role in atherosclerosis prevention. Nat. Clin. Pract. Cardiovasc. Med. 3, 540–547 Keul, P. et al. (2007) The sphingosine-1-phosphate analogue FTY720 reduces atherosclerosis in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 27, 607–613 Nofer, J.R. et al. (2007) FTY720, a synthetic sphingosine 1 phosphate analogue, inhibits development of atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation 115, 501–508 Klingenberg, R. et al. (2007) Sphingosine-1-phosphate analogue FTY720 causes lymphocyte redistribution and hypercholesterolemia in ApoE-
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deficient mice. Arterioscler. Thromb. Vasc. Biol. 27, 2392–2399 [Epub 2007 August 30] Teupser, D. et al. (2008) Effect of macrophage overexpression of murine liver X receptor-{alpha} (LXR-{alpha}) on atherosclerosis in LDL-receptordeficient mice. Arterioscler. Thromb. Vasc. Biol. [Epub ahead of print] Scheen, A.J. (2008) CB1 receptor blockade and its impact on cardiometabolic risk factors: overview of the RIO programme with rimonabant. J. Neuroendocrinol. 20 (Suppl. 1), 139–146 Chang, F. et al. (2007) Evolution of peroxisome proliferator-activated receptor agonists. Ann. Pharmacother. 41, 973–983 Roehrich, M.E. et al. (2003) Insulin-secreting beta-cell dysfunction induced by human lipoproteins. J. Biol. Chem. 278, 18368–18375
DRUG DISCOVERY
TODAY
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Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Cardiology MECHANISMS
HDL – a difficult friend Arnold von Eckardstein Institute of Clinical Chemistry, University Hospital Zurich, Raemistrasse 100, CH 8091 Zurich, Switzerland
High-density lipoprotein (HDL) is an attractive target for antiatherogenic drug therapy because of the inverse association between HDL cholesterol and cardiovascu-
Section Editor: Christian Weber – Institute for Molecular Cardiovascular Research (IMCAR), Aachen University, Germany
lar risk as well as many potentially antiatherogenic functions. However, controversial data from inborn
Introduction
errors of human HDL metabolism and genetic animal
Among the many risk factors of atherosclerotic cardiovascular diseases dyslipidemias play a pivotal pathogenetic role and have hence become a prime target for both prevention and therapy. In several controlled intervention trials encompassing more than 500,000 patient years of follow-up statins, which inhibit cholesterol synthesis and lower LDL cholesterol, have proved as safe and effective in reducing cardiovascular morbidity and mortality [1,2]. However, 60–80% of cardiovascular events could not be prevented in the statin arms of these trials. In addition, intracoronary ultrasound studies revealed that the treatment of hypercholesterolemia to the current LDL cholesterol goal of <100 mg/dL (<2.6 mmol/L) reduces progression but is not sufficient to induce regression of atherosclerosis [3]. As a consequence, lower treatment goals (e.g. LDL cholesterol < 70 mg/dL/ <1.7 mmol/L) and treatment of other risk factors are intensively discussed [4]. Among these other risk factors low HDL cholesterol has turned out as very attractive because both in populationbased studies of healthy individuals (i.e. primary prevention setting) and in patients with pre-existing coronary heart disease (i.e. secondary prevention setting) low concentrations of HDL cholesterol or apolipoprotein (apo) A-I, that is the major protein component of HDL, turned out as independent risk factors for future myocardial infarction [5]. Interestingly, this association was found in old studies, such as the ECAT Angina pectoris study, which were performed in the 1980s before the introduction of statins [6], as well as in very recent studies where LDL cholesterol was very well controlled by statin therapy [7,8].
models as well as the frequent confounding of low HDL cholesterol with other proatherogenic conditions in the population have complicated the proof of a causal relationship between HDL cholesterol and atherosclerosis. Because HDLs form a very heterogenous class of lipoproteins which differ in protein and lipid composition, it is increasingly accepted that the quality rather than quantity of HDL is relevant for its atheroprotective activity. As a consequence, protein or lipid or functional biomarkers are postulated to be better biomarkers than HDL cholesterol to assess and monitor the cardiovascular risk exerted by disturbed HDL metabolism and to estimate the benefit of any therapeutic intervention. In addition, novel therapeutics are searched which either improve HDL metabolism, mimic HDL function or cure the regulatory network underlying disturbed HDL metabolism and function. Beyond better biomarkers of HDL functionality and metabolism early clinical endpoint studies are needed to assess the therapeutic benefit of any novel HDLmodifying therapy.
E-mail address: A. von Eckardstein ([email protected]) 1740-6765/$ ß 2008 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2008.10.004
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Structure, function, metabolism and regulation of HDL Structure HDL is a heterogenous class of lipoproteins which differ by composition, shape, size and density [9,10]. In a recent shotgun proteomic approach more than 50 different HDL-associated proteins have been identified which include apolipoproteins in the proper sense, such as apoA-I and apoA-II, lipocalins such as apoD and apoM, lipid-transfer proteins and lipid-modifying enzymes, serpins and complement regulatory proteins [11]. Also the lipid content is highly
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heterogenous. Traditionally lipids of HDL have been considered as structural components and cargo only. More recently it has become evident that, beyond cholesterol, phosphatidylcholine and sphingomyelin, HDL carries a great variety of biologically quantitatively minor active lipids such as oxysterols, lysophospholipids and lysosphingolipids [12–14].
Functions HDL and its structural compounds exert a broad scope of biological actions which potentially counteract several steps in the pathogenesis of atherosclerosis (Fig. 1): for example,
Figure 1. Structural and functional heterogeneity of HDL. HDLs consist of various subclasses that can be differentiated by shape, size, density, electrophoretic mobility and protein composition. It contains more than 50 different proteins and many lipid classes. Several of them exert biological activities that may protect from atherosclerosis. apo, apolipoprotein; LCAT, lecithin:cholesterol acyl-transferase; CETP, cholesteryl ester transfer protein; PLTP, phospholipid-transfer protein; PON, paraoxonase; LpPLA2, lipoprotein-associated phospholipase A2; glutathionperoxidase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; IL1b, interleukin 1 beta; TNFa, tumor necrosis factor alpha; NO, nitric oxide.
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they correct endothelial dysfunction including monocyte adhesion and disturbed vasoreactivity, stimulate cholesterol efflux and inhibit lipoprotein oxidation, proinflammatory cytokine secretion from macrophages, platelet aggregation and coagulation [9,10,15,16]. Unfortunately it is as yet unknown which of the many functions and hence also components of HDL are most crucial for the protective effects on atherosclerosis. Furthermore, these and additional functions of HDL are also of interest for the treatment and prevention of diseases other than atherosclerosis. For example, HDLs were found to reduce ischemia-reperfusion injury, to preserve survival and function of beta-cells, and to prolong survival of animals infected with viruses, bacteria or protozoa (e.g. malaria or trypanosoma) [9,13,16–18].
Metabolism HDL metabolism is a multistep process which involves the secretion of lipid-free apolipoproteins by the liver or intestine, the acquisition of phospholipids and cholesterol from cells via ATP-binding cassette (ABC) transporters A1 and G1, the maturation by LCAT-mediated cholesterol esterification and PLTP-mediated particle fusion, and the final delivery of lipids to the liver, either directly via receptors or indirectly via CETP-mediated transfer to LDL (Fig. 2) [9,10,15,19]. The investigation of further important steps in HDL metabolism
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has been started only recently, for example, the traffic of HDL between intra- and extravascular compartments which is a prerequisite for the exertion of many antiatherogenic functions within the arterial wall [20]. The incomplete knowledge of HDL metabolism is also highlighted by the findings of genetic linkage analyses and genome-wide association studies which identified more than 30 loci in men and mice which regulate HDL cholesterol levels. For several genes and their products it is not known, how they are related to HDL metabolism [21,22].
Regulation Like other cardiovascular risk factors, HDL cholesterol is regulated by many genetic and nongenetic factors which are only partially resolved and interact with one another [23]. Genetic factors account for about 50% of the variability in HDL cholesterol levels among individuals [22,23]. Both monogenic and polygenic factors are involved. Heterozygosities for private mutations in the genes of apoA-I, ABCA1 and LCAT explain about 10% of HDL cholesterol levels below the 5th percentile [24,25]. Polymorphisms in CETP, LPL, hepatic lipase or apoE explain 1–4 mg/dL mean differences between carriers and noncarriers [23]. Most recently, genome-wide linkage analysis identified most of the genes mentioned above as well as endothelial lipase and four other genes of
Figure 2. Key steps in HDL metabolism and their modulation by factors relevant in metabolic syndrome. HDL precursors may be secreted as nascent HDL by hepatocytes and the intestinal mucosa. They may also dissociate from triglyceride-rich lipoproteins (TGRL, i.e. VLDL and chylomicrons) during lipoprotein lipase-mediated hydrolysis of triglycerides. Alternatively, they may be generated by the interconversion of HDL by cholesteryl ester transfer protein (CETP), phospholipid-transfer protein (PLTP), hepatic lipase (HL), scavenger receptor BI (SR-BI) and endothelial lipase (EL). The interaction of lipidfree apoA-I or lipid-poor particles with the ATP-binding cassette transporter A1 (ABCA1), which is expressed in many cells including hepatocytes, enterocytes and macrophages, leads to efflux of cellular phospholipids and unesterified cholesterol, and thereby to the formation of discoidal HDL precursors. These discoidal HDL precursors become mature, lipid-rich and spherical through the esterfication of cholesterol by the enzyme lecithin:cholesterol acyltransferase (LCAT). They increase in size through the acquisition of additional phospholipids and unesterified cholesterol, from cells (in a process that is mediated by ABCG1 and SR-BI) or apoB-containing lipoproteins (by an at least partially PLTP-mediated process); by the association of additional apolipoproteins; by ongoing LCAT-mediated cholesterol esterification and by PLTP-mediated fusion with other HDL. Lipids of mature HDL are removed from the circulation by at least two pathways, which involve the selective uptake of lipids via SR-BI and by exchanging cholesteryl esters of HDL with triglycerides of apoB-containing lipoproteins, which are then removed via the LDL-receptor (LDLR) pathway. Various genes or their encoded proteins in this pathway are dysregulated by free fatty acids or insulin resistance.
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unknown role in HDL metabolism as genetic determinants of HDL cholesterol levels [21]. Important life style and environmental factors influencing HDL cholesterol include gender, body fat, physical activity, smoking and alcohol intake as well as several drugs [25]. Clinically most relevant is the frequent association of low HDL cholesterol with overweight or obesity, hypertriglyceridemia, latent or overt type 2 diabetes mellitus and hypertension, that is with components of the metabolic syndrome [26,27]. Therefore, insulin resistance and metabolic sequelae thereof appear to play an important role in the determination of HDL cholesterol levels in the population. In fact, insulin and free fatty acids, which are found at increased concentrations in the metabolic syndrome, regulate several important steps of HDL metabolism at the transcriptional and posttranslational level (Fig. 2) [19,27,28]. Several pivotal genes in HDL metabolism are regulated by nuclear hormone receptors such as the retinoid-X- and A-receptors RXR and RAR, the liver and farnesoid X receptors LXR and FXR as well as the peroxisome proliferator agent receptors PPARa, PPARg and PPARb/d. Because these receptors are activated by endogenous and exogenous lipids or derivatives thereof, they and their cofactors are also strong candidates for contributing to the pathogenesis of low HDL cholesterol and other components of the metabolic syndrome [19,29].
Is HDL a causal risk factor? Despite the pleiotropic potentially antiatherogenic functions of HDL, the causality of the inverse association between HDL cholesterol and cardiovascular risk has not been definitely proven, because several data from epidemiological, clinical as well as human and animal genetic studies are contradictory or can be differently interpreted.
Epidemiology The relationship between HDL cholesterol concentration and cardiovascular risk is not strictly dose-dependent. While the association of low HDL cholesterol with increased cardiovascular risk is a rather constant finding, the association of high HDL cholesterol with reduced risk is not: in several primary and secondary prevention studies HDL cholesterol levels in the highest quartile or quintile were not associated with less risk than intermediate HDL cholesterol levels [5,7,8]. Furthermore, although the epidemiological association of low HDL cholesterol with increased cardiovascular risk is statistically independent of the confounding risk factors constituting the metabolic syndrome, it has been argued that low HDL cholesterol may be an innocent bystander of these proatherogenic situations [5]. Especially fasting and, even more so, postprandial hypertriglyceridemia are found in more than 50% of individuals with HDL cholesterol <1 mmol/L, so some authors suggested that low HDL cholesterol is a nonpathogenic long-term marker of disturbed postprandial lipid e318
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metabolism exactly as glycated hemoglobin A1 is a nonpathogenic long-term marker of disturbed glucose metabolism [30–32].
Human genetics Several inborn errors of metabolism causing low and high HDL cholesterol have been identified in patients with premature atherosclerosis and high age, respectively. For example, several patients with complete apoA-I deficiency because of nonsense mutations in the apoA-I gene suffered from myocardial infarction before the age of 50 years [23,33]. Likewise CETP deficiency, which causes a high HDL cholesterol level was originally thought to contribute to prolonged life expectancy and low myocardial infarction rates in Japan [34]. However, if these single reports on rare homozygous cases were extended to systematic family studies or, most recently even to the population level, the picture became more complicated: for example, heterozygosity for rare mutations in the ABCA1 gene, which decreases HDL cholesterol significantly below the commonly accepted threshold value of 40 mg/dL (1.05 mmol/L), was not associated with increased cardiovascular risk [35]. Likewise data on mutations and polymorphisms in CETP have been controversial [23,36]. However, in a recent meta-analysis of 46 studies with data on 27,196 coronary cases and 55,338 controls showed significant associations of three CETP polymorphisms with decreased CETP activity and mass by 8–10% and with increased HDL cholesterol by 4–5% and with slightly reduced cardiovascular risk [36]. Interestingly several mutations in pivotal genes of HDL metabolism, for example, ABCA1, do not affect HDL cholesterol levels but increase cardiovascular risk [37,38]. This finding, together with findings in ABCA1 animal models (see below), points to antiatherogenic functions of ABCA1 which are not reflected by HDL cholesterol, for example, in macrophages, endothelial and beta cells [15,17,20].
Clinical intervention studies Life style interventions such as smoking cessation, increased physical activity, body weight reduction or moderate alcohol intake as well as drug interventions with statins, fibrates, glitazones, nicotinic acid or rimonabant increase HDL cholesterol by 5–30% [26] (Fig. 3), so more effective regimens are sought. Some of these interventions, for example smoking cessation and drug therapies with statins, fibrates and nicotinic acid were shown to reduce cardiovascular event rates. However, these interventions have profound effects on other cardiovascular risk factors as well and the clinical benefit was not found correlated with changes in HDL cholesterol. Therefore, the clinical benefit of these interventions does not prove the causal role of altered HDL metabolism [26,28,34]. More direct effects can be assessed in studies where reconstituted HDLs were given to patients with acute coronary syndromes. Although intracoronary ultrasound analyses revealed some
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Figure 3. Maximal effects of established therapeutic interventions on HDL cholesterol.
regression of atherosclerotic plaque load in these studies, it is important to note that most of the predefined endpoints in these studies were not reached or that the benefit was not dose-dependent [39,40]. The most effective HDL cholesterol increasing drugs are inhibitors of CETP. The most advanced representative of this class, torcetrapib, doubled HDL cholesterol levels if given at the highest dosage [26,34]. Its clinical efficacy in reducing cardiovascular events was tested in three phase III trials using carotid intima media thickness and coronary plaque volume as intermediate surrogate endpoints or cardiovascular events as clinical endpoints [41–43]. Surprisingly, the combination of atorvastatin with torcetrapib did not prevent progression of carotid or coronary atherosclerosis more effectively than atorvastatin alone despite further reducing LDL cholesterol by about 15% and increasing HDL cholesterol by more than 50% [41,42]. Even worse, the clinical endpoint study had to be stopped prematurely because of an excess in cardiovascular and overall mortality in the torcetrapib + atorvastatin arm [43]. As yet it is not clear whether the failure of torcetrapib has been caused by adverse off-target effects, notably the increase in blood pressure as well as aldosterone and sodium levels, or by the choice of the wrong target, that is CETP inhibition [26,34,43].
Animal studies Also genetic mouse models provided controversial data on the role of HDL in atherosclerosis [5,10,21,33]. However, before going into details, it is important to recall that murine and human lipoprotein metabolism is considerably different: notably wild-type mice lack CETP and partially therefore carry almost all of their plasma cholesterol in HDL. Only data from animals with altered apoA-I expression are consistent with one another and the expectations: the knock-out of apoA-I caused HDL deficiency and increased atherosclerosis in a proatherogenic but not in a normolipidemic background [44,45]. Overexpression of apoA-I was found to increase HDL cholesterol
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and to inhibit progression or, if accomplished by somatic gene transfer, even induce regression of atherosclerosis [10,19,28,34,46]. Systemic or macrophage deletion of ABCA1 increased atherosclerosis at least when ABCG1 also was deleted [47–49]. Systemic overexpression of ABCA1 increased HDL cholesterol but was found to increase atherosclerosis [50,51]. However, macrophage-specific overexpression of ABCA1 was found to decrease atherosclerosis without altering HDL cholesterol [52]. The effects of knocking-out or overexpressing LCAT were modulated by CETP. LCAT-deficient mice had low HDL cholesterol but developed atherosclerosis only if CETP was overexpressed. LCAT-overexpressing mice had high HDL cholesterol but developed atherosclerosis. Atherosclerosis, however, was reduced if both LCAT and CETP were expressed. Also the effects of overexpressed CETP or SR-BI in mice on atherosclerosis were strongly modulated by the parallel modulation of other genes [5,10,21,34].
Consequences for HDL as a therapeutic target Several of the aforementioned inconsistencies on the causal relationship of HDL to atherosclerosis can be resolved if one considers HDL as a heterogenous class of lipoproteins which differ in function and which interact with other risk factors.
Whom shall we treat? The increased cardiovascular risk associated with low HDL cholesterol is modulated by other risk factors (Fig. 4; [5]). This is probably even truer for HDL than for other risk factors because it conveys protection or repair and hence is especially relevant in the presence of other risk factors or pre-existing atherosclerosis. Like a fire brigade that needs conflagration to show its usefulness HDL needs a proatherogenic condition to prove its relevance. Interestingly in most individuals low HDL cholesterol is a component of the metabolic syndrome which frequently coexists because of the common metabolic and regulatory soil with other proatherogenic risk factors such as diabetes, hypertriglyceridemia as well as procoagulatory and proinflammatory states. In these conditions the risk threshold value of HDL cholesterol is 40 mg/dL (1.05 mmol/ L) or even higher (Fig. 4). By contrast in the much rarer inborn errors of HDL metabolism, such as heterozygous apoA-I or ABCA1 deficiency, the coexistence of other risk factors is a chance finding, so frequently in these situations low HDL cholesterol is the only risk factor. In these situations, the risk threshold is much lower, namely 20 mg/dL (Fig. 4). It is hence no surprise that relatively rare isolated low HDL cholesterol conditions because of monogenic defects in HDL metabolism do not confer the same cardiovascular risk as much more frequent polygenic low HDL cholesterol conditions, for example, in patients with the metabolic syndrome, which because of their metabolic origin are obligatorily accompanied by other risk factors [33,35]. Interestingly also in genetic mouse models a proatherogenic condition (e.g. LDL receptor www.drugdiscoverytoday.com
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Figure 4. Incidence of coronary events in men according to HDL-cholesterol and other risk factors in the PROCAM study. Hypertriglyceridemia: triglycerides > 2.1 mmol/L; hypercholesterolemia: LDL cholesterol > 4.1 mmol/L; IFG: impaired fasting glucose (glucose 6.1–7.0 mmol/L) (modified from Ref. [5]).
deficiency) must coexist to see the aggravating effects of apoA-I deficiency or the antiatherogenic effects of apoA-I overexpression [44,45]. Accordingly patients with pre-existing atherosclerosis and/or multiple or severe additional risk factors appear to be the most relevant group for HDL-modifying therapies.
How and how much shall we treat? For a long time and in analogy to the ‘the lower the better concept’ for LDL cholesterol patients, physicians and scientists longed for very high levels of the ‘good’ HDL cholesterol. However, in contrast to the robust epidemiological evidence for low HDL cholesterol being a risk factor of atherosclerosis there is much less observational evidence for the protective effects of very high HDL cholesterol [5,7,8]. Instead of supporting ‘the higher the better’ the epidemiological data rather indicate that a threshold of HDL cholesterol must be passed (e.g. 50–60 mg/dL or 1.3–1.5 mmol/L; see Fig. 4 and data in Refs. [5,7,8]). Depending on the basal level, drugs increasing HDL cholesterol by 20–100% are needed to surpass this threshold level. Among the presently available drugs only nicotinic acid shows an efficacy at the lower end of this range (Fig. 3). Among the CETP inhibitors dalcetrapib (=JTT 705) increases HDL cholesterol by 25–30% while torcetrapib and the structurally related anacetrapib can double HDL cholesterol levels [41–43,53,54]. However, as yet nicotinic acid is the only drug which was shown to reduce cardiovascular endpoint rates as well [55]. Because the risk of low HDL cholesterol is modulated by additional risk factors (Fig. 4), elimination of accompanying risk factors including lowering of LDL cholesterol by statin treatment is still the most important measure of cardiovascular risk reduction in patients with low HDL cholesterol. Moreover, to improve the proven therapeutic benefit of statins, the current philosophy is that any HDL cholesterol-modifying therapy (e.g. nicotinic acid) is combined with statin therapy. Mostly because of ethical e320
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reasons, also the design of clinical endpoint studies on HDL cholesterol increasing treatments such as CETP inhibition follows this paradigm [41–43].
What shall we measure? For the present time HDL cholesterol and apoA-I concentrations are equally well suited to identify patients at risk. However, in no study, changes of HDL cholesterol predicted the outcome of any intervention. This is no surprise because the cholesterol in HDL is neither a protective agent nor a reflection of the antiatherogenic HDL action for example in reverse cholesterol transport. It is simply an indirect estimation of HDL particle number which, however, owing to HDL size heterogeneity varies considerably among individuals with equal HDL cholesterol concentration [5,9,12]. The particle heterogeneity of HDL is incompletely and insufficiently characterized by HDL2 and HDL3 so that measurement of cholesterol in these two subfractions does not much improve the diagnostic performance [9]. More comprehensive data on the size and charge heterogeneity of HDL particles are provided by NMR spectroscopy or refined electrophoretic techniques [56,57]. In fact some of the thereby determined HDL subclasses were found to predict the incidence of cardiovascular events better than HDL cholesterol; however, intellectual property right issues and the complexity, respectively, of the applied technology and difficulties in standardization aggravate the roll out into clinical laboratories [9]. It appears more feasible to measure defined functional protein or lipid components of HDL by immunoassays and mass spectrometry; however, it is as yet unknown which of the many protein and lipid components of HDL and the thereof exerted functions are most important in protecting from atherosclerosis [9,11–13,16]. As yet only apoA-I and apoA-II levels have been evaluated in larger populations. In observational studies these parameters showed no consistent advantage over HDL cholesterol [58]. In some intervention
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studies such as AFCAPS/TexCAPS or IDEAL changes in apoA-I levels unlike HDL cholesterol concentrations were significantly associated with outcome [59,60]. In addition, the compostion and hence functionality of HDL changes in response to inflammatory or metabolic stress, so that physiological protein components, for example apoA-I, are either displaced by acute phase proteins such as serum amyloid protein A (SAA) [61] or modified for example by carbamylation of lysine residues, nitration or chlorination of tyrosine or tryptophan residues or oxidation of methionine residues [62]. Several of these oxidative modifications as well as glycation in diabetes mellitus impair the functionality of HDL or its components, for example the ability of apoA-I to induce ABCA1-mediated cholesterol efflux and activate LCAT [63,64]. Also the lipid moiety of HDL can be modified in the course of inflammation, for example by phospholipases which generate proinflammatory lysophospholipids or by reactive oxygen species which oxidize polyunsaturated fatty acids of phospho- and sphingolipids. Some of these modifications do not only interfere with the antiatherogenic functionality of HDLs but turn them into proinflammatory or proatherogenic particles [61]. In any case at equal levels of HDL cholesterol two individuals may differ considerably in atheroprotection. Proteomic and lipidomic analyses of HDL are hence needed to identify new potential biomarkers of HDL functionality, which then must be validated in clinical studies for their ability to improve differential diagnosis, prognosis and therapy monitoring low HDL cholesterol. Alternatively, functional tests may be used to assay the heterogeneity of HDL, for example measurement of activities to stimulate cholesterol efflux, to inhibit oxidation or adhesion of monocytes to endothelial cells [9]. However, these bioassays are difficult to standardize and automate so that they appear feasible in specialized laboratories and clinical studies only [9]. Finally, instead of HDL components or functions one may measure the effect of any HDL-modifying therapy on the extent and/or severity of atherosclerosis. However, also for this approach we need novel disease biomarkers (similar to tumor markers) since as yet only expensive imaging techniques can do this job.
What is the right therapeutic target? In view of the structural, functional and metabolic complexity of HDL it is difficult to predict the most attractive target for therapy development. Evidence from animal models and inborn errors of human HDL metabolism points to the stimulation of apoA-I, ABCA1 (at least in macrophages) and LCAT as the most interesting targets. Unfortunately no small molecules have been found to stimulate apoA-I or LCAT production. Because in general stimulators or agonists are notoriously difficult to be developed, efforts have been increased to substitute apoA-I and other potentially antiatherogenic HDL components or mimic their antiathero-
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genic properties. Because of the need of infusion and largescale protein production substitution therapy with isolated or recombinant apoA-I appears to be limited to acute situations such as the acute coronary syndrome. Initial data on shortterm treatment of patients with acute coronary syndrome indicate some regression of lesions [39,40] but the value of this intervention with respect to clinical outcome is not known. Limitations in producing large quantities of (recombinant) protein and the need of intravenous therapy for apoA-I can be circumvented by using small peptides that mimic the structure of apoA-I and that are synthesized of D-amino acids. In animal experiments, these mimetics reduced the progression of atherosclerosis [65]. Data of animal experiments also point to the antiatherogenic potential of lipid mimetics like FTY702 which binds to sphingosine-1phosphate receptors and was found to inhibit atherosclerosis in apoE- or LDL-receptor deficient mice [66–68]. More promising for a longtime therapy are small molecules. LXR agonists are known to increase ABCA1 expression, HDL cholesterol levels and reverse cholesterol transport [18]. However, because they also induce hepatic VLDL production and, hence, hypertriglyceridemia, and because hepatic ABCA1 overexpression may be proatherogenic, macrophage-specific LXR agonists would be needed [18,27,33,69]. Also because inhibitors or antagonists can be more easily developed than activators or stimulators much effort is invested to find small molecules that inhibit the catabolism of HDL. However, although inhibition or deletion of CETP, PLTP or SR-BI increases HDL cholesterol, the effects of such interventions on atherosclerosis are uncertain. Because of the negative outcomes of the torcetrapib trials [41,43] and because of the controversial data of animal models with high HDL cholesterol levels owing to delayed HDL catabolism [5,10,22] the development of these drugs needs biomarkers that assess HDL functionality better than HDL cholesterol or apoA-I levels. Because of the frequent association of low HDL cholesterol with other proatherogenic components of the metabolic syndrome, it appears of special importance to target therapies to a common denominator of this condition, for example obesity or insulin resistance, so that most of, if not all, the components/risk factors of the metabolic syndrome are corrected. Dual PPAR agonists or rimonabant have been developed according to this paradigm but raised safety issues [70,71]. Alternatively, one may consider the metabolic syndrome as the product of several vicious cycles of interconnected metabolic disturbances which can be interrupted by correcting or improving HDL functionality ([27], Fig. 5): insulin resistance directly or indirectly produces metabolic stress factors such as fatty acids, triglycerides and cytokines and interferes with protective or repairing factors such including HDL. The resulting disbalance of metabolic stress and repair systems is not only proatherogenic but also harmful for other organs including www.drugdiscoverytoday.com
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impair the functionality of HDL, notably those which inhibit the catabolism and hence prolong the exposure time of HDL to unwanted enzymatic and nonenzymatic modifications such as oxidation, glycation, apolipoprotein displacement, proteolysis or lipolysis. There is also a strong need of biomarkers that reflect the antiatherogenicity of HDL better than plasma concentrations of HDL cholesterol or apoA-I. In addition any novel HDL therapy must be subjected to clinical imaging and endpoint trials at an early stage to prove that it not only changes concentration and composition of HDL but also reduces progression or even induces regression of atherosclerosis and lowers cardiovascular event rates.
Acknowledgement The author is supported by the 6th Framework of the European Commission (HDLomics, LSHM-CT-2006-037631) and the Swiss National Research Foundation.
References
Figure 5. Mutual relationships between diabetes mellitus, HDL and atherosclerosis.
beta cells and the liver, that is, contributes to the manifestation of diabetes mellitus and nonalcoholic fatty liver disease which in turn aggravate the metabolic disturbances. Interestingly, low HDL cholesterol is also a risk factor of future diabetes mellitus and HDL as well as important HDL regulatory genes like ABCA1 were recently found to inhibit beta cell apoptosis and to secure insulin secretion, respectively [16,27,72]. Hence in a prediabetic state, insulin resistance compromises HDL metabolism and thereby (and other factors) also beta cell protection (Fig. 5). In the metabolic syndrome it may hence also be promising to address HDL as a prime target to interrupt the transition of insulin resistance to diabetes mellitus.
Overall conclusion The independent association of low HDL cholesterol with increased cardiovascular risk, many potentially antiatherogenic properties activities of HDL and its components as well as the beneficial effects of application or overexpression of apoA-I on atherosclerosis in humans and animals point to a causal role of HDL or some of its components in atherosclerosis protection. The antiatherogenic effect of HDL is incompletely recovered by the measurement of HDL cholesterol, especially in clinical situations which alter the composition of HDL, for example in the course of inflammation and diabetes. Of note, also pharmacological interventions may e322
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