HDL Species Research Articles

Abstract 170: Deep Phenotyping of HDL Particles: Characterization of Seven HDL Species and Their Relationship to Cardiometabolic Phenotypes in a Multi-Ethnic Population (Dallas Heart Study)

Background: Size-based HDL particle analysis based on small, medium, and large categories has led to inconsistent associations with cardiovascular disease (CVD). A new algorithm expands characterization of HDL-P from three to seven species, but the clinical significance remains unknown. We investigated the relationships between the seven HDL species and traditional risk factors, lipids, and cardiometabolic phenotypes in the Dallas Heart Study, a multiethnic, probability-based, population cohort of Dallas county adults. Methods: This study included 2,996 DHS participants (56% women, 50% Black), excluding those with prior CVD and statin users. HDL species were determined by nuclear magnetic resonance using the LP4 algorithm, with increasing size from H1P to H7P. Insulin resistance was determined by homeostatic model assessment index (HOMA-IR). Visceral fat was measured by MRI. Results: The largest HDL species were most directly associated with HDL cholesterol (HDL-C) (H6P: r = 0.61, p < 0.0001; H7P: r = 0.66, p < 0.0001). H2P was inversely associated with all HDL species including H1P (r = -0.19, p < 0.0001) and HDL-C (r = -0.18, p < 0.0001), but was directly associated with total cholesterol, triglycerides, and LDL-C (p < 0.0001). Female gender and Black ethnicity were associated with lower H2P levels (p < 0.0001). H2P alone was directly associated with diabetes, hypertension, waist circumference, insulin resistance, and visceral fat (Figure, p < 0.0001 for all values) Conclusion: Our study of a novel 7-species designation of HDL particles revealed that the smallest HDL particle species (H1P and H2P) confer differential associations with cardiometabolic phenotypes. These findings suggest further investigation specifically into the role of H2P and CVD.

Role of apolipoproteins, ABCA1 and LCAT in the biogenesis of normal and aberrant high density lipoproteins.

In this review, we focus on the pathway of biogenesis of HDL, the essential role of apoA-I, ATP binding cassette transporter A1 (ABCA1), and lecithin: cholesterol acyltransferase (LCAT) in the formation of plasma HDL; the generation of aberrant forms of HDL containing mutant apoA-I forms and the role of apoA-IV and apoE in the formation of distinct HDL subpopulations. The biogenesis of HDL requires functional interactions of the ABCA1 with apoA-I (and to a lesser extent with apoE and apoA-IV) and subsequent interactions of the nascent HDL species thus formed with LCAT. Mutations in apoA-I, ABCA1 and LCAT either prevent or impair the formation of HDL and may also affect the functionality of the HDL species formed. Emphasis is placed on three categories of apoA-I mutations. The first category describes a unique bio-engineered apoA-I mutation that disrupts interactions between apoA-I and ABCA1 and generates aberrant preβ HDL subpopulations that cannot be converted efficiently to α subpopulations by LCAT. The second category describes natural and bio-engineered apoA-I mutations that generate preβ and small size α4 HDL subpopulations, and are associated with low plasma HDL levels. These phenotypes can be corrected by excess LCAT. The third category describes bio-engineered apoA-I mutations that induce hypertriglyceridemia that can be corrected by excess lipoprotein lipase and also have defective maturation of HDL. The HDL phenotypes described here may serve in the future for diagnosis, prognoses and potential treatment of abnormalities that affect the biogenesis and functionality of HDL.

Abstract 13218: Remodeling of High Density Lipoprotein Induced by CSL112: Structure and Function of the Remodeled Lipoproteins

Introduction: CSL112 is a novel formulation of human apolipoprotein A-I (apoA-I) suitable for intravenous infusion, currently in development for reducing the high rate of early recurrent events after acute myocardial infarction. Infusion of CSL112 into healthy volunteers causes HDL remodeling, with an immediate rise in lipid-poor apoA-I and a strong and immediate increase in the ABCA1-dependent cholesterol efflux capacity of plasma. Interaction of CSL112 with whole plasma or purified HDL gives rise to three new remodeled HDL species: large (L-HDL rem ) and small (S-HDL rem ) species, and lipid-poor apoA-I. Here we characterized the composition and morphology of remodeled HDL species and compared their ability to efflux cholesterol via different transporters. Methods and Results: CSL112 and HDL3, fluorescently labeled on protein or lipid, were incubated together for varying periods of time at 37°C. Resultant HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescence imaging. Morphology of the individual purified products of HDL remodeling was examined by negative stain electron microscopy. We show that L-HDL rem is a large, spherical species composed of apoA-I and phospholipid from both native HDL and CSL112. S-HDL rem is a small, disc-shaped species composed of apoA-I from CSL112 that is smaller due to the loss of phospholipids. The smallest species, lipid-poor apoA-I, is composed of apoA-I from HDL and CSL112. Lipid-poor apoA-I and S-HDL rem were found to have the greatest ability to efflux cholesterol via ABCA1. While S-HDL rem and L-HDL rem showed comparable ability to efflux cholesterol via ABCG1, L-HDL rem mediated additional efflux via SR-B1 and by an aqueous diffusion process. Conclusion: The generation of these remodeled HDL species can account for the strong and immediate increase in cholesterol efflux capacity observed upon infusion of CSL112, and make CSL112 a promising approach for rapidly removing cholesterol from atherosclerotic plaque.

Distinct Roles of Apolipoproteins A1 and E in the Modulation of High-Density Lipoprotein Composition and Function.

In addition to high-density lipoprotein cholesterol (HDL-C) levels, HDL quality also appears to be very important for atheroprotection. Analysis of various clinical paradigms suggests that the lipid and apolipoprotein composition of HDL defines its size, shape, and functions and may determine its beneficial effects on human health. Previously, we reported that like apolipoprotein A-I (Apoa1), apolipoprotein E (Apoe) is also capable of promoting the de novo biogenesis of HDL with the participation of ATP binding cassette A lipid transporter member 1 (Abca1) and plasma enzyme lecithin:cholesterol acyltransferase (Lcat), in a manner independent of a functional Apoa1. Here, we performed a comparative analysis of the functions of these HDL subpopulations. Specifically, Apoe and Apoa1 double-deficient (Apoe(-/-) × Apoa1(-/-)) mice were infected with APOA1- or APOE3-expressing adenoviruses, and APOA1-containing HDL (APOA1-HDL) and APOE3-containing HDL (APOE3-HDL), respectively, were isolated and analyzed by biochemical and physicochemical methods. Western blot and lipidomic analyses indicated significant differences in the apolipoprotein and lipid composition of the two HDL species. Moreover APOE3-HDL presented a markedly reduced antioxidant potential and Abcg1-mediated cholesterol efflux capacity. Surprisingly, APOE3-HDL but not APOA1-HDL attenuated LPS-induced production of TNFα in RAW264.7 cells, suggesting that the anti-inflammatory effects of APOA1 are dependent on APOE expression. Taken together, our data indicate that APOA1 and APOE3 recruit different apolipoproteins and lipids on the HDL particle, leading to structurally and functionally distinct HDL subpopulations. The distinct role of these two apolipoproteins in the modulation of HDL functionality may pave the way toward the development of novel pharmaceuticals that aim to improve HDL functionality.

Abstract 28: Adiponectin Stimulates Cholesterol Efflux Efficiently in Human THP-1 Macrophages and Modulates HDL-apoA-I Biogenesis

Introduction: Adiponectin (APN) is an anti-inflammatory and anti-atherogenic adipokine that is strongly correlated with circulating HDL levels. However, its role in macrophage lipid metabolism, a crucial process in atherogenesis, remains poorly investigated. We examined the effect of APN on cholesterol efflux from human THP-1 macrophages, elucidated its kinetics, and investigated its role in HDL biogenesis. Methods: APN dose-dependent (0.1 to 60 μM) and time-dependent (0.5 to 24 hours) cholesterol efflux studies were performed in 3 [H]-cholesterol labeled human THP-1 macrophages in the presence of apoA-I. Following efflux studies, the HDL fractions within media were concentrated (10kDa cut-off filter) and subjected to analytical FPLC and 2D-PAGGE technique to reveal HDL species. Results: APN stimulated ABCA1-mediated cholesterol efflux in a dose-dependent and time-dependent manner. Kinetics analysis revealed that increased molar doses of APN and apoA-I had similar Km efficiency of cholesterol efflux but greater velocity ( Km =3.24±0.71 μM, Vmax =4.90±0.07 efflux/6h) when compared to apoA-I alone ( Km =3.33±0.57 μM, Vmax =3.83±0.24 efflux/6h). Importantly, once APN was tested against a fixed dose of apoA-I (10 μg/mL), it promoted cholesterol efflux with Km = 0.17±0.06 μM. This was associated with a 75.7% decrease in intracellular free cholesterol in THP-1 cells in the presence of APN and apoA-I when compared to apoA-I alone (P<0.01). APN alone had no effect on the level of residual efflux (reached a level of 1%). The FPLC cholesterol profiles demonstrated that in the presence of APN and apoA-I there was increased lipidated nascent HDL (nHDL) during the process of cholesterol efflux, compared to apoA-I alone. This was associated with increased size of nHDL-apoA-1 pre-β and α species via 2D-PAGGE analyses. By immunoblotting for apoA-I and APN, APN oligomers exhibited a molecular weight range of 9 to 20 nm, appearing within the size range of nHDL-apoA-I. Conclusion: In addition to promoting macrophage cholesterol efflux in vitro , APN can modulate HDL-apoA-I biogenesis, by increasing the generation of nHDL particles. These findings suggest that APN may be of potential therapeutic value in the modulation of HDL’s protective role in atherosclerosis.

Speciated High-Density Lipoprotein Biogenesis and Functionality.

Plasma high-density lipoprotein cholesterol (HDL-C) concentration is a negative risk factor for atherosclerotic cardiovascular disease (CVD). Despite this, most attempts to raise plasma HDL-C concentrations in a cardioprotective way have failed. Recently, hypotheses about the atheroprotective effects of HDL have shifted away from quantity to quality, mostly HDL function in reverse cholesterol transport. Plasma HDL from CVD patients is a poorer acceptor of cellular cholesterol than plasma from healthy controls, independent of plasma HDL-C concentrations. The function of HDL is likely determined by two other factors, stability and composition. The kinetic instability of HDL, which varies according to subclass, is a likely determinant of its reactivity in response to many HDL-modifying activities. HDL composition is also heterogeneous and variable; all HDL particles contain apo AI but only about two-thirds contain apo AII. This occurs despite the fact that apo AI and apo AII are hepatically secreted on separate HDL that later fuse in plasma. HDL also contains traces of other proteins, some of which have not yet been associated with HDL function. One minor HDL species are those that are secreted with intact signal peptides, which enhances their binding to HDL; these HDL have special properties that are independent of cholesterol transport. Here, we review and provide a perspective about what is currently known about speciated HDL biogenesis in the context of health and disease.

Amphipathic polyproline peptides stimulate cholesterol efflux by the ABCA1 transporter

ApoA-I mimetics are short synthetic peptides that contain an amphipathic α-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenyl group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop-Prog-Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-helical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20–35% (p < 0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides.

Abstract 17107: Distribution of Anti-inflammatory Activity During Remodeling of High Density Lipoprotein: Studies With CSL112

Elevated levels of circulating inflammatory markers predict an unfavorable cardiovascular outcome in acute coronary syndrome patients. CSL112 is human apolipoprotein A-I (apoA-I), reconstituted with phosphatidylcholine to form HDL particles suitable for infusion. Addition of CSL112 to stimulated human whole blood ex vivo strongly reduces pro-inflammatory cytokine production. Infusion of CSL112 into human subjects or addition to human plasma ex vivo causes remodeling of endogenous HDL. Similar remodeling occurs upon incubation of CSL112 with purified HDL3 and results in accumulation of three HDL species: enlarged HDL (HDL2), a smaller, dense species (HDL3c), and lipid-poor apoA-I (pre-β1 HDL). Study aim was to determine the anti-inflammatory activity of remodeled HDL species. CSL112 was incubated with HDL3 and the products of particle remodeling were purified by ultracentrifugation. The inhibitory effects on pro-inflammatory cytokine production were examined using human peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin-M (PHA-M) in vitro. Lipid-poor apoA-I, HDL3c as well as parent CSL112 exerted powerful inhibitory effects on secretion of pro-inflammatory mediators (&gt; 89.2% + 4.0% inhibition of TNF-α, IL-1β, IL-6 and Mip-1β); HDL3 and HDL2 were much less effective (&lt; 54.1% + 3.9% inhibition). The extent of inhibition correlated positively with induction of the transcription repressor ATF3, a negative regulator of pro-inflammatory cytokine production, with lipid-poor apoA-I and HDL3c inducing higher protein levels of ATF3 in PHA-stimulated PBMC compared to control medium, HDL3 or HDL2. Anti-inflammatory activity of the remodeled species also correlated with their ability to support cellular cholesterol efflux via the ABCA1 transporter: lipid-poor apoA-I and HDL3c were potent acceptors of cholesterol; HDL2 was inactive. The ability to generate HDL species with high cholesterol efflux and anti-inflammatory activity makes CSL112 a promising candidate for removing cholesterol and reducing inflammation in atherosclerotic plaque, thus reducing the high risk of early recurrent atherothrombotic events following acute MI (AMI). A Phase IIb trial (AEGIS-I; NCT02108262) of CSL112 in AMI patients is ongoing.

Abstract 17135: Distribution of Anti-oxidative Activity During Remodeling of High Density Lipoprotein: Studies With CSL112

We have previously shown that reconstituted HDL has the ability to inactivate phospholipid hydroperoxides (PLOOH) in oxidized LDL by virtue of redox-active Met residues present in apolipoprotein A-I (apoA-I), with the level of activity being proportional to the amount of apoA-I protein added. CSL112 is human apoA-I, reconstituted with phosphatidylcholine to form HDL particles suitable for infusion. Infusing CSL112 into human subjects or adding it to human plasma ex vivo, causes remodeling of endogenous HDL. Similar remodeling occurs upon incubation of CSL112 with purified HDL3 and results in accumulation of three HDL species: enlarged HDL (HDL2), a smaller, dense species (HDL3c-like), and lipid-poor apoA-I (pre-β1 HDL). Here we examine the distribution of anti-oxidant activity upon remodeling of native plasma HDL induced by CSL112. We incubated CSL112 with human plasma HDL3, isolated the three populations of remodeled HDL particles by density gradient ultracentrifugation and compared (on a total protein basis) their ability to inactivate PLOOH derived from oxidized LDL as well as their apoA-I content of unoxidized and oxidized Met residues. Both parent CSL112 and HDL3 displayed anti-oxidative activity, inactivating 22±4 and 47±9% of PLOOH, respectively (n=4). Upon remodeling, this activity was mainly found in enlarged HDL2 and smaller HDL3c-like species (PLOOH inactivation, 46±3 and 47±9%, respectively) and was low (23±17%) in lipid-poor apoA-I. In parallel, apoA-I Met residues were oxidized in HDL; oxidation was several fold lower in the lipid-poor apoA-I (12±3%) compared to larger species (62±1% and 69±2% in HDL2 and HDL3c-like particles), consistent with a limited accessibility of apoA-I Met to PLOOH molecules in the lipid-poor particles. These studies further confirm the unequal distribution of HDL function among HDL subclasses and emphasize the important role of HDL remodeling in the production and maintenance of HDL functionality. The antioxidant properties of CSL112 add to its potential as a promising therapy for reducing the high risk of early recurrent atherothrombotic events following acute MI (AMI). A Phase IIb trial (AEGIS-I; NCT02108262) of CSL112 in AMI patients is ongoing.

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1⁻/⁻) mice were used. Apoa1⁻/⁻ mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1⁻/⁻ mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1⁻/⁻ mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.

Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4

We have used adenovirus-mediated gene transfer in apolipoprotein (apo)E(-/-) mice to elucidate the molecular etiology of a dominant form of type III hyperlipoproteinemia (HLP) caused by the R142C substitution in apoE4. It was found that low doses of adenovirus expressing apoE4 cleared cholesterol, whereas comparable doses of apoE4[R142C] greatly increased plasma cholesterol, triglyceride, and apoE levels, caused accumulation of apoE in VLDL/IDL/LDL region, and promoted the formation of discoidal HDL. Co-expression of apoE4[R142C] with lecithin cholesterol acyltransferase (LCAT) or lipoprotein lipase (LPL) in apoE(-/-) mice partially corrected the apoE4[R142C]-induced dyslipidemia. High doses of C-terminally truncated apoE4[R142C]-202 partially cleared cholesterol in apoE(-/-) mice and promoted formation of discoidal HDL. The findings establish that apoE4[R142C] causes accumulation of apoE in VLDL/IDL/LDL region and affects in vivo the activity of LCAT and LPL, the maturation of HDL, and the clearance of triglyceride-rich lipoproteins. The prevention of apoE4[R142C]-induced dyslipidemia by deletion of the 203-299 residues suggests that, in the full-length protein, the R142C substitution may have altered the conformation of apoE bound to VLDL/IDL/LDL in ways that prevent triglyceride hydrolysis, cholesterol esterification, and receptor-mediated clearance in vivo.

Effects of Annurca Apple Fruit, a Southern Italy Cultivar, on Lipid Metabolism in Wistar Rats

Annurca apple fruit is commonly cultivated in southern Italy and undergoes a typical reddening treatment and, is notable for its high firmness and its characteristic flavour. Apples may have selective effects on abnormalities associated with the plurimetabolic syndrome. Therefore, the effects of 20% lyophilized Annurca apple supplementation on plasma and tissue lipids were investigated in Wistar rats. The experimental diets were equilibrated for sugar supply, containing 0.25g/100g cholesterol and provided only a third of the vitamin E requirement. The apple diet lowered plasma FFA and in parallel reduced triglyceride in heart and liver. Plasma leptin was also suppressed by the apple diet. In conclusion, despite their moderate fiber content, apples improve substantially the lipid status in Wistar rats, suggesting that other plant constituents such as the pectins are involved in these effects. Keywords: Wistar rats, apples, free fatty acids, leptin, lipoprotein, Annurca, Apple Fruit, Lipid Metabolism, plurimetabolic syndrome, tissue lipids, vitamin E, HDL species, triglyceride-lowering effect, insulinemia, glycemia, Leptin synthesis, physiological conditions, obesity, FFA-leptin interaction, ursane triterpen, Antioxidant activities, annurcoic acid, DPPH, MeLo, Heart lipids, Glycaemia

Apolipoprotein Modulation of Streptococcal Serum Opacity Factor Activity against Human Plasma High-Density Lipoproteins

Human plasma HDL are the target of streptococcal serum opacity factor (SOF), a virulence factor that clouds human plasma. Recombinant (r) SOF transfers cholesteryl esters (CE) from approximately 400,000 HDL particles to a CE-rich microemulsion (CERM), forms a cholesterol-poor HDL-like particle (neo HDL), and releases lipid-free (LF) apo A-I. Whereas the rSOF reaction requires labile apo A-I, the modulation effects of other apos are not known. We compared the products and rates of the rSOF reaction against human HDL and HDL from mice overexpressing apos A-I and A-II. Kinetic studies showed that the reactivity of various HDL species is apo-specific. LpA-I reacts faster than LpA-I/A-II. Adding apos A-I and A-II inhibited the SOF reaction, an effect that was more profound for apo A-II. The rate of SOF-mediated CERM formation was slower against HDL from mice expressing human apos A-I and A-II than against WT mice HDL and slowest against HDL from apo A-II overexpressing mice. The lower reactivity of SOF against HDL containing human apos is due to the higher hydropathy of human apo A-I, particularly its C-terminus relative to mouse apo A-I, and the higher lipophilicity of human apo A-II. The SOF-catalyzed reaction is the first to target HDL rather than its transporters and receptors in a way that enhances reverse cholesterol transport (RCT). Thus, effects of apos on the SOF reaction are highly relevant. Our studies show that the "humanized" apo A-I-expressing mouse is a good animal model for studies of rSOF effects on RCT in vivo.

The HDL proteome: a marker–and perhaps mediator–of coronary artery disease

One important cardioprotective function of HDL is to remove cholesterol from lipid-laden macrophages in the artery wall. HDL also exerts anti-inflammatory effects that might inhibit atherogenesis. However, HDL has been proposed to be dysfunctional in humans with established coronary artery disease (CAD), though the underlying mechanisms are unclear. Therefore, we used mass spectrometry to investigate the roles of HDL proteins in inflammation and cardiovascular disease. Shotgun proteomic analysis identified multiple complement regulatory proteins, protease inhibitors, and acute-phase response proteins in HDL, strongly implicating the lipoprotein in inflammation and the innate immune system. Moreover, mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with clinically significant CAD was selectively enriched in apolipoprotein E, suggesting that it carries a distinctive protein cargo in humans with atherosclerosis. HDL from CAD subjects also contained markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, indicating that oxidative damage might generate dysfunctional HDL. Aggressive lipid therapy with a statin and niacin remodeled the HDL proteome to resemble that of apparently healthy subjects. Collectively, our observations indicate that quantifying the HDL proteome by mass spectrometry should help identify novel anti-inflammatory and cardioprotective actions of HDL and provide insights into lipid therapy.

Abstract 5526: Genetic Variation at PCSK5 Influences HDL-C Levels

Low plasma HDL-C is a well-established risk factor for coronary artery disease. The role of endothelial lipase (EL) in HDL metabolism has been recently characterized. The proprotein convertase subtilisin/kexin 5 (PC5/6 or PCSK5) is known to inactivate both EL and lipoprotein lipase (LPL) ex vivo , enzymes critical in modulating plasma levels of HDL-C. In the present study, we investigated the role of human PCSK5 genetic variants on HDL-C; in addition, we characterized lipoprotein fractions in Pcsk5 +/− mice, compared with wild type. Sequencing of the PCSK5 gene was performed in 12 probands with low HDL-C (&lt;5 th percentile) and 7 novel non-coding genetic variants were found. We genotyped these SNPs along with 163 tag SNPs and 12 additional SNPs (n=182 total) selected from the HapMap Project, in 457 individuals with documented coronary artery disease. We identified 10 SNPs associated with HDL-C (p&lt;0.05), with the strongest result being rs11144782 (minor allele frequency: 0.164, p=0.002). In an analysis of HDL-C as a dichotomous trait, 3 of the 10 SNPs were also associated with low HDL-C at either the 5 th or the 10 th percentile (p&lt;0.05). The rare allele of rs111447782 decreased HDL by 0.076 mmol/L in a gene dosage-dependent fashion. We further investigated the effect of this SNP on other lipoprotein levels and identified an association with very low density lipoprotein (p=0.039), triglycerides (p=0.049) and total ApoB (p=0.022) levels. Furthermore, in conditional regression analysis, we identified 3 additional SNPs contributing to HDL-C (p&lt;0.05), all independent of the effect of rs11144782. In addition, we characterized serum from Pcsk5 KO mice. Serum from Pcsk5 +/− mice ( Pcsk5 −/− is embryonic lethal) showed reduction in HDL-C by gel permeation chromatography on HPLC and size redistribution of HDL species by 1D and 2D PAGGE. These results, and the rs11144782 SNP result presented above, are consistent with the concept that PCSK5 modulates HDL, likely upstream of EL and LPL. We conclude that variability at the PCSK5 gene locus influences HDL-C levels and consequently, atherosclerotic cardiovascular disease risk.

Anti-Atherosclerotic Effect of a New Synthetic Functional Oil Containing Mono- and Diacylglycerol from Corn Oil

Synthetic oil containing diacylglycerol and monoacylglycerol, called ‘functional oil’ (FO), was newly produced and evaluated for its putative anti-atherosclerotic potential by in vitro assays and in vivo test using hypercholesterolemic mice (C57BL/6). The FO revealed good inhibitory activities against both liver acyl-CoA:cholesterol acyltransferase and serum lipoprotein-associated phospholipase A₂. The FO showed enhanced activities on lipoprotein interaction such as HDL particle rearrangement to produce different sizes of HDL species. In control mice, hypercholesterolemia was induced by consumption of high-cholesterol, high-fat (HCHF) diet that contained 1.25% cholesterol/15% fat/0.5% Na-cholate with or without 5% of corn oil. In experimental mice, 5% of the FO + HCHF diet was fed during the same period. After the 4-week administration of the diet, serum total cholesterol concentration of the FO-fed group decreased by 38 or 20% when compared to the HCHF diet control group or corn oil (99.9% of triacylglycerol) diet group, respectively. The percentage of HDL cholesterol to total cholesterol was 36% of HDL cholesterol in the FO-fed group, while the HCHF control group and corn oil-fed group showed 21 and 25%, respectively. These results indicate that the FO possesses a blood cholesterol-lowering effect in mouse model and inhibition effects against the atherogenic enzymes.

Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages

The initial steps of reverse cholesterol transport involve export of cholesterol from peripheral cells to plasma lipoproteins for subsequent delivery to the liver. The review discusses recent developments in our understanding of how these steps occur, with particular emphasis on the macrophage, the major site of cellular cholesterol accumulation in atherosclerosis. ATP binding cassette transporter (ABC) A1 exports cholesterol and phospholipid to lipid-free apolipoproteins, while ATP binding cassette transporter G1 and scavenger receptor BI export cholesterol to phospholipid-containing acceptors. ABCA1-dependent cholesterol export involves an initial interaction of apolipoprotein AI with lipid raft membrane domains, although ABCA1 and most exported cholesterol are not raft associated. ABCG1 exports cholesterol to HDL and other phospholipid-containing acceptors. These include particles generated during lipidation of apoAI by ABCA1, suggesting that the two transporters cooperate in cholesterol export. Scavenger receptor BI is atheroprotective, mediating clearance of HDL cholesterol by the liver. The relative contributions of scavenger receptor BI and ABCG to cholesterol export to HDL from macrophages is unclear and may depend on cellular cholesterol status and the cholesterol gradient between cell and acceptor. The presence of distinct pathways for cholesterol efflux to lipid-free apolipoprotein AI and phospholipid-containing HDL species clarifies our understanding of reverse cholesterol transport, and provides new opportunities for its therapeutic manipulation.

Type 2 Diabetes as a Lipid Disorder

Diabetic dyslipidemia is a cluster of plasma lipid and lipoprotein abnormalities that are metabolically interrelated. The recognition that the elevation of large VLDL 1 particles initiates a sequence of events that leads to the formation of small dense LDL and HDL species has focused the assembly of VLDL particles on the spotlight as a potential culprit of dyslipidemia. Notably dyslipidemia is associated with insulin resistance, visceral obesity and liver fat content. Insulin resistance is associated with excessive flux of substrates for VLDL assembly to the liver as well as the upregulation of the machinery generating large VLDL particles in excess. The regulation of different molecular steps in this cascade of events are complex and so far poorly understood. The disordered crosstalk between adipose tissue and the liver results in an imbalance of the machinery that orchestrates the regulation of VLDL production. A number of studies indicates that adipocytokines in particular adiponectin may be a seminal player in the regulation of fat metabolism in the liver. Future discoveries hopefully will delineate the regulatory steps to allow more targeted treatment of diabetic dyslipidemia.

Serum Lipid Concentrations and Mean Life Span Are Modulated by Dietary Polyunsaturated Fatty Acids in the Senescence-Accelerated Mouse

The senescence-accelerated mouse (SAMP8) is an animal model used in studies of aging. This study was undertaken to investigate the effects of dietary PUFA on longevity (Experiment 1) and serum lipid concentrations (Experiment 2) in SAMP8 mice. Male mice were fed either an (n-3) PUFA-rich (9 g/100 g perilla oil) or an (n-6) PUFA-rich (9 g/100 g safflower oil) diet beginning at 6 wk of age. Experiment 1: The groups did not differ in body weight gain, but those fed perilla oil had significantly lower scores of senescence relative to those fed safflower oil (P<0.05). The mean life span of mice fed perilla oil was 357+/-21 d and of those fed safflower oil, 426+/-24 d (P<0.05). Pathological studies revealed that the incidence of tumors was significantly lower in the perilla oil group than in the safflower oil group (P<0.05). Approximately half the mice fed perilla oil had died after 10 mo, and the direct causes closely connected with death could not be specified. Experiment 2: The serum total cholesterol, HDL cholesterol, triglyceride and phospholipid concentrations were significantly lower in the perilla oil group than in the safflower oil group (P<0.01). A marked decrease of serum HDL cholesterol and apolipoprotein A-II (ApoA-II)concentrations in advanced age were observed in the mice fed perilla oil (P<0.01). Ten-month-old mice fed perilla oil had a significantly greater ratio of apolipoprotein A-I (ApoA-I) to ApoA-II than those fed safflower oil. Separation of HDL subfractions revealed that the smaller HDL species were much more abundant than the larger HDL species in both dietary oil groups. These findings suggest that dietary (n-3) and (n-6) PUFA differ in their effects on serum lipid metabolism which may modulate the mean life span of SAMP8 mice fed each dietary oil.

Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma lipoproteins, age, and gender

Prebeta-1 HDL is a molecular species of plasma HDL of approximately 67 kDa mass that contains apolipoprotein A-I, phospholipids, and unesterified cholesterol. It participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. In this cycle, unesterified cholesterol from cells is incorporated into prebeta-1 HDL, providing a substrate for esterification of cholesterol by lecithin:cholesterol acyltransferase. Prebeta-1 HDL then becomes incorporated into larger HDL species of alpha mobility as esterification proceeds and is regenerated during the transfer of cholesteryl esters from alpha HDL particles to acceptor lipoproteins. Thus the steady state level of prebeta-1 HDL in plasma reflects the relative efficiencies of the major metabolic processes involved in its generation and removal. We have used an isotope dilution technique to measure prebeta-1 HDL levels in the plasmas of 136 normolipidemic individuals (46 M, 90 F). The mean absolute concentration of prebeta-1 HDL as apolipoprotein A-I was 68 ± 40 μg/ml for women, and 84 ± 49 m/ml for men. Prebeta-1 HDL represented 5.5 ± 3.3% of total apolipoprotein A-I in women, and 7.2 ± 4.0% in men. The distributions of both absolute and percent prebeta-1 HDL are highly asymmetric, with skew toward higher values. However, the skew appears not to be attributable to either plasma cholesterol or triglyceride levels which are also skewed in population samples. The percent prebeta-1 HDL was negatively correlated with HDL cholesterol levels (P < 0.0001), whereas absolute levels of prebeta-1 HDL were positively correlated with apolipoprotein A-I and negatively correlated with HDL cholesterol (P, for both, < 0.0001). Multiple linear regression analysis revealed effects of age and gender, but no association with lipoprotein fractions other than HDL. Lower levels of prebeta-1 HDL were associated with female gender in all models.—O'Connor, P. M., B. R. Zysow, S. A. Schoenhaus, B. Y. Ishida, S. T. Kunitake, J. M. Naya-Vigne, P. N. Duchateau, R. F. Redberg, S. J. Spencer, S. Mark, M. Mazur, D. C. Heilbron, R. B. Jaffe, M. J. Malloy, and J. P. Kane. Prebeta-1 HDL in plasma of normolipidemic individuals: influences of plasma, age, and gender.