Relations between particle size of HDL and LDL lipoproteins and cholesterol esterification rate

Particle size of low density (LDL) and high density (HDL) lipoproteins and cholesterol esterification rate in HDL plasma (FER(HDL)) are important independent predictors of coronary artery diseases (CAD). In this study we assessed the interrelations between these indicators and routinely examined plasma lipid parameters and plasma glucose concentrations. In 141 men, healthy volunteers, we examined plasma total cholesterol (TC), triglycerides (TG), HDL and LDL cholesterol (HDL-C, LDL-C) and HDL unesterified cholesterol (HDL-UC). Particle size distribution in HDL and LDL was assessed by gradient gel electrophoresis and FER(HDL) was estimated by radioassay. An effect of particle size and FER(HDL) on atherogenic indexes as the Log(TG/HDL-C) and TC/HDL-C was evaluated. Subjects in the study had plasma concentrations (mean +/- S.D.) of TC 5.2+/-0.9 mmol/l, HDL-C 1.2+/-0.3 mmol/l, TG 2.1+/-1.7 mmol/l, glucose 5+/-0.8 mmol/l. Relative concentration of HDL(2b) was 17.6+/-11.5 % and 14.6+/-11.8 % of HDL(3b,c). The mean diameter of LDL particles was 25.8+/-1.5 nm. The increase in FER(HDL) significantly correlated with the decrease in HDL(2b) and LDL particle size (r = -0.537 and -0.583, respectively, P<0.01) and the increase in HDL(3b,c) (0.473, P<0.01). Strong interrelations among TG and HDL-C or HDL-UC and FER(HDL) and particle size were found, but TC or LDL-C did not have such an effect. Atherogenic indexes Log(TG/HDL-C) and TC/HDL-C correlated with FER(HDL) (0.827 and 0.750, respectively, P<0.0001) and with HDL and LDL particle size.     

HDL and reverse cholesterol transport. Role of cholesterol ester transfer protein]

As most of peripheral cells are not able to catabolize cholesterol, the transport of cholesterol excess from peripheral tissues back to the liver, namely "reverse cholesterol transport", is the only way by which cholesterol homeostasis is maintained in vivo. Reverse cholesterol transport pathway can be divided in three major steps: 1) uptake of cellular cholesterol by the high density lipoproteins (HDL), 2) esterification of HDL cholesterol by the lecithin: cholesterol acyltransferase and 3) captation of HDL cholesteryl esters by the liver where cholesterol can be metabolized and excreted in the bile. In several species, including man, cholesteryl esters in HDL can also follow an alternative pathway which consists in their transfer from HDL to very low density (VLDL) and low density (LDL) lipoproteins. The transfer of cholesteryl esters to LDL, catalyzed by the Cholesteryl Ester Transfer Protein (CETP), might affect either favorably or unfavorably the reverse cholesterol transport pathway, depending on whether LDL are finally taken up by the liver or by peripheral tissues, respectively. In order to understand precisely the implication of CETP in reverse cholesterol transport, it is essential to determine its role in HDL metabolism, to know the potential regulation of its activity and to identify the mechanism by which it interacts with lipoprotein substrates. Results from recent studies have demonstrated that CETP can promote the size redistribution of HDL particles. This may be an important process in the reverse cholesterol transport pathway as HDL particles with various sizes have been shown to differ in their ability to promote cholesterol efflux from peripheral cells and to interact with lecithin: cholesterol acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple QTLs influencing triglyceride and HDL and total cholesterol levels identified in families with atherogenic dyslipidemia

We conducted a genome-wide scan using variance components linkage analysis to localize quantitative-trait loci (QTLs) influencing triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol, and total cholesterol (TC) levels in 3,071 subjects from 459 families with atherogenic dyslipidemia. The most significant evidence for linkage to TG levels was found in a subset of Turkish families at 11q22 [logarithm of the odds ratio (LOD)=3.34] and at 17q12 (LOD=3.44). We performed sequential oligogenic linkage analysis to examine whether multiple QTLs jointly influence TG levels in the Turkish families. These analyses revealed loci at 20q13 that showed strong epistatic effects with 11q22 (conditional LOD=3.15) and at 7q36 that showed strong epistatic effects with 17q12 (conditional LOD=3.21). We also found linkage on the 8p21 region for TG in the entire group of families (LOD=3.08). For HDL-C levels, evidence of linkage was identified on chromosome 15 in the Turkish families (LOD=3.05) and on chromosome 5 in the entire group of families (LOD=2.83). Linkage to QTLs for TC was found at 8p23 in the entire group of families (LOD=4.05) and at 5q13 in a subset of Turkish and Mediterranean families (LOD=3.72). These QTLs provide important clues for the further investigation of genes responsible for these complex lipid phenotypes. These data also indicate that a large proportion of the variance of TG levels in the Turkish population is explained by the interaction of multiple genetic loci.     

HDL and triglyceride as therapeutic targets

PURPOSE OF REVIEW: Epidemiological studies have shown that plasma HDL-cholesterol is inversely related to coronary artery disease and that there is an inverse relationship between HDL-cholesterol and triglyceride levels, but it is now demonstrated that hypertriglyceridemia is an independent risk factor for coronary heart disease (CHD). The goal of this review is to discuss if triglycerides and HDL-cholesterol could be therapeutic targets to reduce cardiovascular risk. RECENT FINDINGS: Triglyceride measurement is not informative on the specificity of the triglyceride-rich lipoproteins present in the plasma because some of these are not atherogenic (chylomicrons, large VLDLs) while others are highly atherogenic (small VLDLs, remnants, IDL...). Statins, in addition to reducing LDL-cholesterol, significantly reduced atherogenic remnant lipoprotein cholesterol levels. 4S, CARE+LIPID, and AFCAPS/TexCAPS studies, suggested enhanced therapeutic potential of statins for improving triglyceride and HDL-cholesterol levels in patients with CHD. A fibrate (gemfibrozil) was shown to reduce death from CHD and non-fatal myocardial infarction in secondary prevention of CHD in men with low levels of HDL-cholesterol (VA-HIT); during the treatment these levels predicted the magnitude of reduction in risk for CHD events. SUMMARY: ATP III recommendations state, on triglycerides and HDL-cholesterol as targets to reduce cardiovascular risk: (1) that lowering LDL-cholesterol levels is the primary target of therapy, (2) a secondary target is to achieve a triglyceride level < 150 mg/dL and (3) clinical trial data are considered to be insufficient to support recommended a specific HDL-cholesterol goal even if HDL-cholesterol < 40 mg/dL is considered to be a major risk factor of CHD.     

HDL2 and HDL3 cholesterol in hepatic cirrhosis

In order to further investigate plasma lipoproteins abnormalities secondary to serious liver damage, we studied plasma lipids and lipoproteins, and in particular HDL subfractions (HDL2, HDL3), in 12 patients with cirrhosis of the liver and in 12 sex, age and weight matched healthy volunteers. Enzymatic methods were used to determine total cholesterol and triglycerides, while the extractive method of Abell et al. was used for the determination of HDL-cholesterol levels after LDL and VLDL precipitation with polyanions (MnCl2 and Na-heparin) and of HDL3-cholesterol values after HDL2 precipitation with dextran-sulphate 15,000 m.w. Total cholesterol and HDL-cholesterol levels were significantly lower in cirrhotic patients compared to normal subjects. We must emphasize that only HDL3-cholesterol was decreased in cirrhotics, whereas HDL2-cholesterol values were normal or high. We suggest that a diminished activity of hepatic triglyceride lipase might account for the decrease in HDL3-cholesterol in liver cirrhosis.     

ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins

Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.     

Apolipoprotein-lipid association in oxidatively modified HDL and LDL

We investigated the effect of Cu²⁺ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation of HDL with Cu²⁺ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (λ_max). LDL incubated with Cu²⁺ also showed a rapid decrease in Trp fluorescence intensity with time, but with no associated increase in λ_max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in λ_max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in λ_max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in λ_max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.     

A gene score of nine LDL and HDL regulating genes is associated with fluvastatin-induced cholesterol changes in women

While conventional pharmacogenetic studies have considered single gene effects, we tested if a genetic score of nine LDL- and HDL-associated single nucleotide polymorphisms, previously shown to predict cardiovascular disease, is related to fluvastatin-induced lipid change. In patients with asymptomatic plaque in the right carotid artery, thus candidates for statin therapy, we related score LDL [APOB(rs693), APOE(rs4420638), HMGCR(rs12654264), LDLR(rs1529729), and PCSK9(rs11591147)] and score HDL [ABCA1(rs3890182), CETP(rs1800775), LIPC(rs1800588), and LPL(rs328)] as well as the combined score LDL+HDL to fluvastatin-induced LDL reduction (± metoprolol) (n = 395) and HDL increase (n = 187) following 1 year of fluvastatin treatment. In women, an increasing number of unfavorable alleles (i.e., alleles conferring higher LDL and lower HDL) of score LDL+HDL (P = 0.037) and of score LDL (P = 0.023) was associated with less pronounced fluvastatin-induced LDL reduction. Furthermore, in women, both score LDL+HDL (P = 0.001) and score HDL (P = 0.022) were directly correlated with more pronounced fluvastatin-induced HDL increase, explaining 5.9–11.6% of the variance in treatment response in women. There were no such associations in men. This suggests that a gene score based on variation in nine different LDL- and HDL-associated genes is of importance for the magnitude of fluvastatin HDL increase in women with asymptomatic plaque in the carotid artery.     

Overexpression of apolipoprotein AV in the liver reduces plasma triglyceride and cholesterol but not HDL in ApoE deficient mice

It has been shown that adenovirus-mediated overexpression of human ApoAV (hApoAV) in C57BL/6 mice results in decreased plasma triglyceride (TG) and total cholesterol (TC) levels with a major reduction occurring in the HDL fraction. In order to study the effect of ApoAV on hypercholesterolemic mice, an adenoviral vector expressing hApoAV was constructed and injected into ApoE deficient mice. High levels of hApoAV mRNA in the liver and ApoAV proteins in the liver and plasma were detected. The treatment reduced plasma TG levels by 50% and 75%, and TC levels by 45% and 58% at day 3 and 7, respectively, after treatment as compared with a control group treated with Ad-hAP (human alkaline phosphatase). Plasma HDL-C levels remained unaltered, which were different from normolipidemic mice. These findings suggest that ApoAV might serve as a therapeutic agent for hyperlipidemic disorder.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

Enhancing reverse cholesterol transport/raising HDL cholesterol: new options for prevention and treatment of cardiovascular disease

High-density lipoprotein cholesterol (HDL-c) plays a crucial role in the concept of reverse cholesterol transport and has many other beneficial properties which may interfere with atherogenesis and plaque rupture. Low HDL-c levels are currently considered to be an important risk factor for the development of cardiovascular disease. However until recently no effective and safe treatment for powerfully increasing HDL-c selectively was available. This short overview describes possible new therapeutic approaches that may be able to raise HDL-c levels or improve HDL-c metabolism/reverse cholesterol transport. Today, the most important targets to be evaluated are inhibition of cholesteryl ester transfer protein (CETP) and increasing the HDL-c level by infusion of engineered HDL particles. Trials to prove clinical benefit of new HDL-c raising approaches are underway and may well be a new starting point for an optimised prevention and treatment of atherosclerotic cardiovascular disease.     

A rapid single-step centrifugation method for determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of predominant LDL subclass

Determination of the circulating levels of plasma lipoproteins HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. More recently it has become apparent that the LDL subclass pattern is a further important diagnostic parameter. The reference method for separation of plasma lipoproteins is ultracentrifugation. However, current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoprotein structure and must be removed before further analysis. To overcome these problems we have now investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. A protocol is presented in which HDL, LDL, and VLDL, characterized by electron microscopy and agarose gel electophoresis, separate in three bands in a 2.5 h centrifugation step. Recoveries of cholesterol and TG from the gradients were close to 100%. The distribution profiles of cholesterol and TG in the gradient were used to calculate the concentrations of individual lipoprotein classes. The values correlated with those obtained using commercial kits for HDL and LDL cholesterol. The position of the LDL peak in the gradient and its shape varied between plasma samples and was indicative of the density of the predominant LDL class. The novel protocol offers a rapid, reproducible and accurate single-step centrifugation method for the determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of LDL subclass pattern.     

Apolipoprotein E derived HDL mimetic peptide ATI-5261 promotes nascent HDL formation and reverse cholesterol transport in vitro

Modulation of the reverse cholesterol transport (RCT) pathway may provide a therapeutic target for the prevention and treatment of atherosclerotic cardiovascular disease (CVD). In the present study, we evaluated a novel 26-amino acid apolipoprotein mimetic peptide (ATI-5261) designed from the carboxyl terminal of apoE, in its ability to mimic apoA-I functionality in RCT in vitro. Our data shows that nascent HDL-like (nHDL) particles generated by incubating cells over-expressing ABCA1 with ATI-5261 increase the rate of specific ABCA1 dependent lipid efflux, with high affinity interactions with ABCA1. We also show that these nHDL particles interact with membrane micro-domains in a manner similar to nHDL apoA-I. These nHDL particles then interact with the ABCG1 transporter and are remodeled by plasma HDL-modulating enzymes. Finally, we show that these mature HDL-like particles are taken up by SR-BI for cholesterol delivery to liver cells. This ATI-5621-mediated process mimics apoA-I and may provide a means to prevent cholesterol accumulation in the artery wall. In this study, we propose an integrative physiology approach of HDL biogenesis with the synthetic peptide ATI-5261. These experiments provide new insights for potential therapeutic use of apolipoprotein mimetic peptides.     

HDL and electronegative LDL exchange anti- and pro-inflammatory properties

Electronegative LDL [LDL(–)] is a minor modified LDL subfraction present in blood with inflammatory effects. One of the antiatherogenic properties of HDL is the inhibition of the deleterious effects of in vitro modified LDL. However, the effect of HDL on the inflammatory activity of LDL(–) isolated from plasma is unknown. We aimed to assess the putative protective role of HDL against the cytokine released induced in monocytes by LDL(–). Our results showed that LDL(–) cytokine release was inhibited when LDL(–) was coincubated with HDL and human monocytes and also when LDL(–) was preincubated with HDL and reisolated prior to cell incubation. The addition of apoliprotein (apo)AI instead of HDL reproduced the protective behavior of HDL. HDL preincubated with LDL(–) promoted greater cytokine release than native HDL. Incubation of LDL(–) with HDL decreased the electronegative charge, phospholipase C-like activity, susceptibility to aggregation and nonesterified fatty acid (NEFA) content of LDL(–), whereas these properties increased in HDL. NEFA content in LDL appeared to be related to cytokine production because NEFA-enriched LDL induced cytokine release. HDL, at least in part through apoAI, inhibits phospholipase-C activity and cytokine release in monocytes, thereby counteracting the inflammatory effect of LDL(–). In turn, HDL acquires these properties and becomes inflammatory.