A high-throughput screen for endothelial lipase using HDL as substrate

Endothelial lipase (EL) is a 482-amino-acid protein from the triglyceride lipase gene family that uses a Ser-His-Asp triad for catalysis. Its expression in endothelial cells and preference for phospholipids rather than triglycerides are unique. Animal models in which it is overexpressed or knocked out indicate EL levels are inversely correlated with high-density lipoprotein cholesterol (HDL-C). HDL-C is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, in which excess cholesterol is effluxed from peripheral tissues for excretion or reabsorption. Thus, EL inhibition in humans is expected to lead to increases in HDL levels and possibly a decrease in cardiovascular disease. To discover inhibitors of EL, a coupled assay for EL has been developed, using its native substrate, HDL. Hydrolysis of HDL by EL yields free fatty acids, which are coupled through acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase to produce the fluorescent species resorufin. This assay was developed into a 5-microL, 1536-well assay format, and a high-throughput screen was executed against the GSK collection. In addition to describing the screening results, novel post-HTS mechanism-of-action studies were developed for EL and applied to 1 of the screening hits as an example.     

Metabolic and genetic determinants of HDL metabolism and hepatic lipase activity in normolipidemic females.

The metabolic and genetic determinants of HDL cholesterol (HDL-C) levels and HDL turnover were studied in 36 normolipidemic female subjects on a whole-food low-fat metabolic diet. Lipid, lipoprotein, and apolipoprotein levels, lipoprotein size, and apolipoprotein turnover parameters were determined, as were genetic variation at one site in the hepatic lipase promoter and six sites in the apolipoprotein AI/CIII/AIV gene cluster. Menopause had no significant effect on HDL-C or turnover. Stepwise multiple regression analysis revealed that HDL-C was most strongly correlated with HDL size, apolipoprotein A-II (apoA-II), and apolipoprotein A-I (apoA-I) levels, which together could account for 90% of the variation in HDL-C. HDL size was inversely correlated with triglycerides, body mass index, and hepatic lipase activity, which together accounted for 82% of the variation in HDL size. The hepatic lipase promoter genotype had a strong effect on hepatic lipase activity and could account for 38% of the variation in hepatic lipase activity. The apoA-I transport rate (AI-TR) was the major determinant of apoA-I levels, but AI-TR was not associated with six common genetic polymorphism in the apoAI/CIII/AIV gene cluster.A simplified model of HDL metabolism is proposed, in which A-I and apoA-II levels combined with triglycerides, and hepatic lipase activity could account for 80% of the variation in HDL-C.     

Nascent HDL formation by hepatocytes is reduced by the concerted action of serum amyloid A and endothelial lipase

Inflammation is associated with significant decreases in plasma HDL-cholesterol (HDL-C) and apoA-I levels. Endothelial lipase (EL) is known to be an important determinant of HDL-C in mice and in humans and is upregulated during inflammation. In this study, we investigated whether serum amyloid A (SAA), an HDL apolipoprotein highly induced during inflammation, alters the ability of EL to metabolize HDL. We determined that EL hydrolyzes SAA-enriched HDL in vitro without liberating lipid-free apoA-I. Coexpression of SAA and EL in mice by adenoviral vector produced a significantly greater reduction in HDL-C and apoA-I than a corresponding level of expression of either SAA or EL alone. The loss of HDL occurred without any evidence of HDL remodeling to smaller particles that would be expected to have more rapid turnover. Studies with primary hepatocytes demonstrated that coexpression of SAA and EL markedly impeded ABCA1-mediated lipidation of apoA-I to form nascent HDL. Our findings suggest that a reduction in nascent HDL formation may be partly responsible for reduced HDL-C during inflammation when both EL and SAA are known to be upregulated.     

Endothelial lipase: a new lipase on the block

Endothelial lipase (EL) is a newly described member of the triglyceride lipase gene family. It has a considerable molecular homology with lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%). Unlike LPL and HL, this enzyme is synthesized by endothelial cells and functions at the site where it is synthesized. Furthermore, its tissue distribution is different from that of LPL and HL. As a lipase, EL has primarily phospholipase A1 activity. Animals that overexpress EL showed reduced HDL cholesterol levels. Conversely, animals that are deficient in EL showed a marked elevation in HDL cholesterol levels, suggesting that it plays a physiologic role in HDL metabolism. Unlike LPL and HL, EL is located in the vascular endothelial cells and its expression is highly regulated by cytokines and physical forces, suggesting that it may play a role in the development of atherosclerosis. However, there is only a limited amount of information available about this enzyme. Some of our unpublished data in addition to previously published data support the possibility that the enzyme plays a role in the formation of atherosclerotic lesion.     

Hepatic proprotein convertases modulate HDL metabolism

The risk of atherosclerosis is inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). However, HDL metabolism is incompletely understood, and there are few effective approaches to modulate HDL-C levels. Here we show that inhibition in the liver of the classical proprotein convertases (PCs), but not the atypical PCs S1P and PCSK9, decreases plasma HDL-C levels. This metabolic effect of hepatic PCs is critically dependent on expression of endothelial lipase (EL), an enzyme that directly hydrolyzes HDL phospholipids and promotes its catabolism. Hepatic PCs reduce EL function through direct inactivating cleavage of EL as well as through activating cleavage of angiopoietin-like protein 3 (ANGPTL3), an endogenous inhibitor of EL. Thus, inhibition of hepatic PCs results in increased EL activity, leading to reduced HDL-C as well as impaired reverse cholesterol transport. The hepatic PC-ANGPTL3-EL-HDL pathway is therefore a novel mechanism controlling HDL metabolism and cholesterol homeostasis.     

Long-chain polyunsaturated fatty acids, endothelial lipase and atherosclerosis

Endothelial lipase (EL), a new member of the lipase gene family, was recently cloned and has been shown to have a significant role in modulating the concentrations of plasma high-density lipoprotein levels (HDL). EL is closely related to lipoprotein and hepatic lipases both in structure and function. It is primarily synthesized by endothelial cells, functions at the cell surface, and shows phospholipase A1 activity. Overexpression of EL decreases HDL cholesterol levels whereas blocking its action increases concentrations of HDL cholesterol. Pro-inflammatory cytokines suppress plasma HDL cholesterol concentrations by enhancing the activity of EL. On the other hand, physical exercise and fish oil (a rich source of eicosapentaenoic acid and docosahexaenoic acid) suppress the activity of EL and this, in turn, enhances the plasma concentrations of HDL cholesterol. Thus, EL plays a critical role in the regulation of plasma HDL cholesterol concentrations and thus modulates the development and progression of atherosclerosis. The expression and actions of EL in specific endothelial cells determines the initiation and progression of atherosclerosis locally explaining the patchy nature of atheroma seen, especially, in coronary arteries. Both HDL cholesterol and EPA and DHA enhance endothelial nitric oxide (eNO) and prostacyclin (PGI2) synthesis, which are known to prevent atherosclerosis. On the other hand, pro-inflammatory cytokines augment free radical generation, which are known to inactivate eNO and PGI2. Thus, interactions between EL, pro- and anti-inflammatory cytokines, polyunsaturated fatty acids, and the ability of endothelial cells to generate NO and PGI2 and neutralize the actions of free radicals may play a critical role in atherosclerosis.     

Endothelial lipase modulates susceptibility to atherosclerosis in apolipoprotein-E-deficient mice

Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant approximately 70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.     

Fluorogenic substrates for high-throughput measurements of endothelial lipase activity

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum.     

Design and synthesis of boronic acid inhibitors of endothelial lipase

Endothelial lipase (EL) and lipoprotein lipase (LPL) are homologous lipases that act on plasma lipoproteins. EL is predominantly a phospholipase and appears to be a key regulator of plasma HDL-C. LPL is mainly a triglyceride lipase regulating (V)LDL levels. The existing biological data indicate that inhibitors selective for EL over LPL should have anti-atherogenic activity, mainly through increasing plasma HDL-C levels. We report here the synthesis of alkyl, aryl, or acyl-substituted phenylboronic acids that inhibit EL. Many of the inhibitors evaluated proved to be nearly equally potent against both EL and LPL, but several exhibited moderate to good selectivity for EL.     

A naturally occurring variant of endothelial lipase associated with elevated HDL exhibits impaired synthesis

Human endothelial lipase (EL) is a member of a family of lipases and phospholipases that are involved in the metabolism of plasma lipoproteins. EL displays a preference to hydrolyze lipids in HDL. We report here that a naturally occurring low frequency coding variant in the EL gene (LIPG), glycine-26 to serine (G26S), is significantly more common in African-American individuals with elevated HDL cholesterol (HDL-C) levels. To test the hypothesis that this variant results in reduced EL function, we extensively characterized and compared the catalytic and noncatalytic functions of the G26S variant and wild-type (WT) EL. While the catalytic-specific activity of G26S EL is similar to WT EL, its secretion is markedly reduced. Consistent with this observation, we found that carriers of the G26S variant had significantly reduced plasma levels of EL protein. Thus, this N-terminal variant results in reduced secretion of EL protein, plausibly leading to increased HDL-C levels.     

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.     

Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3-4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 microg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.     

Low levels of high density lipoproteins in Turks, a population with elevated hepatic lipase. High density lipoprotein characterization and gender-specific effects of apolipoprotein e genotype

Turks have strikingly low levels of high density lipoprotein cholesterol (HDL-C) (10-15 mg/dL lower than those of Americans or Western Europeans) associated with elevated hepatic lipase mass and activity. Here we report that Turks have low levels of high density lipoprotein subclass 2 (HDL(2)), apoA-I-containing lipoproteins (LpA-I), and pre-beta-1 HDL and increased levels of HDL(3) and LpA-I/A-II particles (potentially an atherogenic lipid profile). The frequency distributions of HDL-C and LpA-I levels were skewed toward bimodality in Turkish women but were unimodal in Turkish men. The apoE genotype affected HDL-C and LpA-I levels in women only. In women, but not men, the varepsilon2 allele was strikingly more prevalent in those with the highest levels of HDL-C and LpA-I than in those with the lowest levels. The higher prevalence of the epsilon2 allele in these subgroups of women was not explained by plasma triglyceride levels, total cholesterol levels, age, or body mass index. The modulating effects of apoE isoforms on lipolytic hydrolysis of HDL by hepatic lipase (apoE2 preventing efficient hydrolysis) or on lipoprotein receptor binding (apoE2 interacting poorly with the low density lipoprotein receptors) may account for differences in HDL-C levels in Turkish women (the epsilon2 allele being associated with higher HDL levels). In Turkish men, who have substantially higher levels of hepatic lipase activity than women, the modulating effect of apoE may be overwhelmed. The gender-specific impact of the apoE genotype on HDL-C and LpA-I levels in association with elevated levels of hepatic lipase provides new insights into the metabolism of HDL.     

Testosterone administration to men increases hepatic lipase activity and decreases HDL and LDL size in 3 wk

Testosterone administration to men is known to decrease high-density lipoprotein cholesterol (HDL-C) and the subclasses HDL(2) and HDL(3). It also might increase the number of small, dense, low-density lipoprotein cholesterol (LDL-C) particles in hypogonadal men. The decrease in HDL-C and in LDL-C size is potentially mediated by hepatic lipase activity, which hydrolyzes lipoprotein phospholipids and triacylglycerol. To determine how HDL-C and LDL-C particles are affected by testosterone administration to eugonadal men, testosterone was administered as a supraphysiological dose (600 mg/wk) for 3 wk to elderly, obese, eugonadal men before elective hip or knee surgery, and lipids were measured by routine methods and by density gradient ultracentrifugation. Hepatic lipase activity increased >60% above baseline levels, and HDL-C, HDL(2), and HDL(3) significantly declined in 3 wk. In addition, the LDL-C peak particle density and the amount of LDL-C significantly increased. Testosterone is therefore a potent stimulator of hepatic lipase activity, decreasing HDL-C, HDL(2), and HDL(3) as well as increasing LDL particle density changes, all associated with increased cardiovascular risk.     

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras

The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain.     

Effects of nonlipolytic ligand function of endothelial lipase on high density lipoprotein metabolism in vivo

Endothelial lipase (EL) influences high density lipoprotein (HDL) metabolism in vivo and mediates bridging and uptake of HDL particles independent of its lipolytic activity in vitro. To determine whether EL has a nonlipolytic ligand function in HDL metabolism in vivo, 1 x 1011 particles of a recombinant adenovirus encoding human EL (AdEL), catalytically inactive human EL (AdELS149A), or control (Adnull) were injected into wild-type, apoA-I transgenic, and hepatic lipase knockout mice. ELS149A protein was expressed at higher levels than wild-type EL. EL and ELS149A protein were both substantially increased in the postheparin plasma compared with preheparin, indicating that both the wild-type and mutant EL were bound to cell-surface heparan sulfate proteoglycans. Overexpression of wild-type EL was associated with a significantly increased postheparin-plasma phospholipase activity and dramatically decreased levels of total cholesterol, HDL cholesterol, phospholipids, and apoA-I. Injection of AdELS149A did not result in increased phospholipase activity confirming that ELS149A was catalytically inactive. Expression of ELS149A did not decrease lipid or apoA-I levels in wild-type and apoA-I transgenic mice yet led to an intermediate reduction of total cholesterol, HDL cholesterol, and phospholipids in hepatic lipase-deficient mice compared with control and EL-expressing mice. Our study demonstrates for the first time that EL has both a lipolytic and nonlipolytic function in HDL metabolism in vivo. Lipolytic activity of EL, however, seems to be most important for its effects on systemic HDL metabolism.     

Targeted deletion of endothelial lipase increases HDL particles with anti-inflammatory properties both in vitro and in vivo

Previous studies have shown that targeted deletion of endothelial lipase (EL) markedly increases the plasma high density lipoprotein cholesterol (HDL-C) level in mice. However, little is known about the functional quality of HDL particles after EL inhibition. Therefore, the present study assessed the functional quality of HDL isolated from EL(-/-) and wild-type (WT) mice. Anti-inflammatory functions of HDL from EL(-/-) and WT mice were evaluated by in vitro assays. The HDL functions such as PON-1 or PAF-AH activities, inhibition of cytokine-induced vascular cell adhesion molecule-1 expression, inhibition of LDL oxidation, and the ability of cholesterol efflux were similar in HDL isolated from WT and EL(-/-) mice. In contrast, the lipopolysaccharide-neutralizing capacity of HDL was significantly higher in EL(-/-) mice than that in WT mice. To evaluate the anti-inflammatory actions of HDL in vivo, lipopolysaccharide-induced systemic inflammation was generated in these mice. EL(-/-) mice showed higher survival rate and lower expression of inflammatory markers than WT mice. Intravenous administration of HDL isolated from EL(-/-) mice significantly improved the mortality after lipopolysaccharide injection in WT mice. In conclusion, targeted disruption of EL increased HDL particles with preserved anti-inflammatory and anti-atherosclerotic functions. Thus, EL inhibition would be a useful strategy to raise 'good' cholesterol in the plasma.     

Endothelial lipase modulates HDL but has no effect on atherosclerosis development in apoE-/- and LDLR-/- mice

Endothelial lipase (EL) is a determinant of high density lipoprotein-cholesterol (HDL-C) level, which is negatively correlated with atherosclerosis susceptibility. We found no difference in aortic atherosclerotic lesion areas between 26-week-old EL+/+ apolipoprotein E-deficient (apoE-/-) and EL-/- apoE-/- mice. To more firmly establish the role of EL in atherosclerosis, we extended our study to EL-/- and EL+/+ low density lipoprotein receptor-deficient (LDLR-/-) mice that were fed a Western diet. Morphometric analysis again revealed no difference in atherosclerosis lesion area between the two groups. Compared with EL+/+ mice, we found increased HDL-C in EL-/- mice with apoE-/- or LDLR-/- background but no difference in macrophage content between lesions of EL-/- and EL+/+ mice in apoE-/- or LDLR-/- background. EL inactivation had no effect on hepatic mRNAs of proteins involved in reverse cholesterol transport. A survey of lipid homeostasis in EL+/+ and EL-/- macrophages revealed that oxidized LDL-induced ABCA1 was attenuated in EL-/- macrophages. This potentially proatherogenic change may have nullified any minor protective increase of HDL in EL-/- mice. Thus, although EL modulated lipoprotein profile in mice, there was no effect of EL inactivation on atherosclerosis development in two hyperlipidemic atherosclerosis-prone mouse models.     

Protective effect of an endothelial lipase gene variant on coronary artery disease in a Chinese population

The aim of the present study was to assess the influence of the endothelial lipase (EL) gene 584C/T variant, which results in a change at codon 111 of the EL gene from threonine to isoleucine, on the risk of coronary artery disease (CAD) in a Chinese population. The study population consisted of 265 CAD patients and 265 age- and sex-matched control subjects. The T allele frequency was significantly lower among CAD patients than among control subjects (18.3% vs. 29.8%; P < 0.001). In both the CAD and control groups, the T allele carriers had higher high density lipoprotein cholesterol (HDL-C) levels than homozygote C allele carriers. In a multiple logistic regression model adjusted for age, sex, body mass index, smoking, hypertension, diabetes, hyperlipidemia, and low density lipoprotein cholesterol, a significantly decreased risk of developing CAD was found in subjects carrying a variant CT or TT genotype (odds ratio = 0.496, 95% confidence interval = 0.341-0.723; P < 0.001), and the significance persisted after further adjustment for HDL-C. In conclusion, our observation that the EL 584T allele was associated with protection from CAD in this Chinese population replicates the findings in a Japanese study, which found a similar association of this allele with acute myocardial infarction, independent of HDL-C levels.     

Genetic causes of high and low serum HDL-cholesterol

Plasma levels of HDL cholesterol (HDL-C) have a strong inherited basis with heritability estimates of 40-60%. The well-established inverse relationship between plasma HDL-C levels and the risk of coronary artery disease (CAD) has led to an extensive search for genetic factors influencing HDL-C concentrations. Over the past 30 years, candidate gene, genome-wide linkage, and most recently genome-wide association (GWA) studies have identified several genetic variations for plasma HDL-C levels. However, the functional role of several of these variants remains unknown, and they do not always correlate with CAD. In this review, we will first summarize what is known about HDL metabolism, monogenic disorders associated with both low and high HDL-C levels, and candidate gene studies. Then we will focus this review on recent genetic findings from the GWA studies and future strategies to elucidate the remaining substantial proportion of HDL-C heritability. Comprehensive investigation of the genetic factors conferring to low and high HDL-C levels using integrative approaches is important to unravel novel pathways and their relations to CAD, so that more effective means of diagnosis, treatment, and prevention will be identified.