Tangier disease: isolation and characterization of LpA-I,LpA-II,LpA-I:A-II and LpA-IV particles from plasma

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 ± 0.14 and 0.14 ± 0.04 μmol of cholesterol esterified/h/μg of protein, and 7 ± 2.5 and 1.4 ± 0.3 μmol of cholesteryl ester transferred/h/μg of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2–1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 ± 0.16 and 0.01 ± 0.01 μmol of cholesterol esterified/h/μg of protein and, 41 ± 3.7 and 1 ± 0.4 μmol of cholesteryl ester transferred /h/μg of protein). The LpA-II particle in TD represented in fact in LpA-II: A-IV complex (75% mol apoA-II and 22% mol apoA-IV). This particle did not promote cholesterol efflux, but LCAT and CETP activity were present. LpA-IV particles had the capacity to promote cholesterol efflux and had both LCAT and CETP activity. LpA-IV may contribute to maintain the reverse cholesterol transport in TD. Our results indicate the potential importance of apoA-IV in maintaining reverse cholesterol transport in TD. In spite of the low steady state HDL-cholesterol levels in TD, LpA-I, LpA-I: A-II: A-IV complex and LpA-IV appear to be active in reverse cholesterol transport and may help to prevent premature CHD in TD.     

Effects of weight loss,induced by gastric bypass surgery,on HDL remodeling in obese women

Plasma lipoproteins and glucose homeostasis were evaluated after marked weight loss before and over 12 months following Roux-en-Y gastric-bypass (RYGBP) surgery in 19 morbidly obese women. Standard lipids, remnant-lipoprotein cholesterol (RLP-C); HDL-triglyceride (TG); apolipoproteins (apo) A-I, A-II, E, and A-I-containing HDL subpopulations; lecithin-cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) mass and activity; plasma glucose and insulin levels were measured before and at 1, 3, 6, and 12 months after GBP surgery. Baseline concentrations of TG, RLP-C, glucose, and insulin were significantly higher in obese than in normal-weight, age-matched women, whereas HDL cholesterol (HDL-C), apoA-I, apoA-II, α-1 and α-2 levels were significantly lower. Over 1 year, significant decreases of body mass index, glucose, insulin, TG, RLP-C, HDL-TG, and preβ-1 levels were observed with significant increases of HDL-C and α-1 levels (all P < 0.05). Changes of fat mass were correlated with those of LDL cholesterol (P = 0.018) and LCAT mass (P = 0.011), but not with CETP mass (P = 0.265). Changes of fasting plasma glucose concentrations were inversely correlated with those of CETP mass (P = 0.005) and α-1 level (P = 0.004). Changes of fasting plasma insulin concentrations were positively correlated with those of LCAT mass (P = 0.043) and inversely with changes of α-1 (P = 0.03) and α-2 (P = 0.05) concentrations. These results demonstrate beneficial changes in HDL remodeling following substantial weight loss induced by RYGBP surgery and that these changes are associated with improvement of glucose homeostasis in these patients.     

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic,CETP transgenic,LDLr-/- mice

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.     

High density lipoprotein,apolipoprotein A-1, and coronary artery disease

High density lipoproteins (HDL), one of the main lipoprotein particles circulating in plasma, is involved in the reverse cholesterol transport. Several lines of evidence suggest that elevated levels of HDL is protective against coronary heart disease. The role of HDL in the removal of body cholesterol and in the regression of atherosclerosis add to the importance of understanding the molecular-cellular processes that determine plasma levels of HDL. Factors modulating plasma levels of HDL may have influence on the predisposition of an individual to premature coronary artery disease. Apolipoprotein (apo) A-I is the main apolipoprotein component of HDL and, to a large extent, sets the plasma levels of HDL. Thus, understanding the regulation of apoA-I gene expression may provide clues to raise plasma levels of HDL. This review discusses the various pathways that alter plasma levels of HDL. Since apoA-I is the main protein component of HDL and determines the plasma levels of HDL, this review also covers the regulation of apoA-I gene expression.     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

Thematic Review Series: Proteomics: Lipoproteomics: using mass spectrometry-based proteomics to explore the assembly, structure, and function of lipoproteins

Lipoproteins are centrally important in lipid transport, fuel metabolism, and cardiovascular disease. The prototypic lipoprotein has an outer shell of amphipathic lipids and proteins that solubilizes a hydrophobic lipid core. Lipoprotein-associated proteins have classically been viewed as structural elements and factors important in lipid metabolism. Recent mass spectrometric analyses reveal that the protein cargo of lipoproteins is much more diverse than previously appreciated, raising the possibility that lipoproteins play previously unsuspected roles in host defense mechanisms and inflammation. They further suggest that lipoprotein-associated proteins can identify humans at increased risk of cardiovascular disease. Here, we summarize recent developments in lipoproteomics, the proteomic analysis of lipoproteins. We also discuss the promises and challenges this powerful analytical strategy offers for expanding our understanding of the biology and structures of lipoproteins.     

Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process ( approximately 72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid ( approximately 4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.     

Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT

Mutations in ABCA1, APOA1, and LCAT reduce HDL cholesterol (HDLc) in humans. However, the prevalence of these mutations and their relative effects on HDLc reduction and risk of coronary artery disease (CAD) are less clear. Here we searched for ABCA1, APOA1, and LCAT mutations in 178 unrelated probands with HDLc < 10th percentile but no other major lipid abnormalities, including 89 with ≥ 1 first-degree relative with low HDLc (familial probands) and 89 where familial status of low HDLc is uncertain (unknown probands). Mutations were most frequent in LCAT (15.7%), followed by ABCA1 (9.0%) and APOA1 (4.5%), and were found in 42.7% of familial but only 14.6% of unknown probands (p = 2.44 ∗ 10^− 5). Interestingly, only 16 of 24 (66.7%) mutations assessed in families conferred an average HDLc < 10th percentile. Furthermore, only mutation carriers with HDLc < 5th percentile had elevated risk of CAD (odds ratio (OR) = 2.26 for 34 ABCA1 mutation carriers vs. 149 total first-degree relative controls, p = 0.05; OR = 2.50 for 26 APOA1 mutation carriers, p = 0.04; OR = 3.44 for 38 LCAT mutation carriers, p = 1.1 ∗ 10^− 3). These observations show that mutations in ABCA1, APOA1, and LCAT are sufficient to explain > 40% of familial hypoalphalipoproteinemia in this cohort. Moreover, individuals with mutations and large reductions in HDLc have increased risk of CAD. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Description of the torcetrapib series of cholesteryl ester transfer protein inhibitors, including mechanism of action

We have identified a series of potent cholesteryl ester transfer protein (CETP) inhibitors, one member of which, torcetrapib, is undergoing phase 3 clinical trials. In this report, we demonstrate that these inhibitors bind specifically to CETP with 1:1 stoichiometry and block both neutral lipid and phospholipid (PL) transfer activities. CETP preincubated with inhibitor subsequently bound both cholesteryl ester and PL normally; however, binding of triglyceride (TG) appeared partially reduced. Inhibition by torcetrapib could be reversed by titration with both native and synthetic lipid substrates, especially TG-rich substrates, and occurred to an equal extent after long or short preincubations. The reversal of TG transfer inhibition using substrates containing TG as the only neutral lipid was noncompetitive, suggesting that the effect on TG binding was indirect. Analysis of the CETP distribution in plasma demonstrated increased binding to HDL in the presence of inhibitor. Furthermore, the degree to which plasma CETP shifted from a free to an HDL-bound state was tightly correlated to the percentage inhibition of CE transfer activity. The finding by surface plasmon resonance that torcetrapib increases the affinity of CETP for HDL by approximately 5-fold likely represents a shift to a binding state that is nonpermissive for lipid transfer. In summary, these data are consistent with a mechanism whereby this series of inhibitors block all of the major lipid transfer functions of plasma CETP by inducing a nonproductive complex between the transfer protein and HDL.     

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.     

Lipophorin receptor of Bombyx mori: cDNA cloning, genomic structure, alternative splicing, and isolation of a new isoform

The cDNA and genomic structure of a putative lipophorin receptor from the silkworm, Bombyx mori (BmLpR), indicated the presence of four isoforms, designated LpR1, LpR2, LpR3, and LpR4. The deduced amino acid sequence of each isoform showed five functional domains that are homologous to vertebrate very low density lipoprotein receptor (VLDLR). All four isoforms seem to have originated from a single gene by alternative splicing and were differentially expressed in a tissue- and stage-specific manner. BmLpR1 harbored an additional 27 amino acids in the O-linked sugar domain, resulting in an extra exon. The silkworm BmLpR gene consisted of 16 exons separated by 15 introns spanning >122 kb and was at least three times larger than the human VLDLR gene. Surprisingly, one of the isoforms, LpR4, was expressed specifically in the brain and central nervous system. Additionally, it had a unique cytoplasmic tail, leading to the proposition that it represents a new candidate LpR for possible brain-related function(s). This is the first report on the genomic characterization of an arthropod lipoprotein receptor gene and the identification of a brain-specific receptor variant from a core member of the low density lipoprotein receptor family in invertebrates.     

Sortilin: an unusual suspect in cholesterol metabolism: from GWAS identification to in vivo biochemical analyses, sortilin has been identified as a novel mediator of human lipoprotein metabolism

The concentration of low-density lipoprotein (LDL) cholesterol (C) in plasma is a key determinant of cardiovascular disease risk and human genetic studies have long endeavoured to elucidate the pathways that regulate LDL metabolism. Massive genome-wide association studies (GWASs) of common genetic variation associated with LDL-C in the population have implicated SORT1 in LDL metabolism. Using experimental paradigms and standards appropriate for understanding the mechanisms by which common variants alter phenotypic expression, three recent publications have presented divergent and even contradictory findings. Interestingly, although these reports each linked SORT1 to LDL metabolism, they did not agree on a mechanism to explain the association. Here, we review recent mechanistic studies of SORT1 - the first gene identified by GWAS as a determinant of plasma LDL-C to be evaluated mechanistically.     

Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function

L-4F, an apolipoprotein A-I (apoA-I) mimetic peptide (also known as APL180), was administered daily by either intravenous (IV) infusion for 7 days or by subcutaneous (SC) injection for 28 days in patients with coronary heart disease in two distinct clinical studies. L-4F was well tolerated at all doses tested. Despite achieving plasma levels (mean maximal plasma concentration of 2,907 ng/ml and 395 ng/ml, following IV infusion and SC injection, respectively), that were effective in previously published animal models, treatment with L-4F, as assessed by biomarkers of HDL function such as HDL-inflammatory index (HII), and paraoxonase activity, did not improve. Paradoxically, there was a 49% increase in high-sensitivity C-reactive protein (hs-CRP) levels after seven IV infusions of 30 mg L-4F (P < 0.05; compared with placebo) and a trend for hs-CRP increase in subjects receiving 30 mg SC injection for 28 days. In a subsequent, ex vivo study, addition of L-4F at concentrations of 150, 375, or 1,000 ng/ml to plasma from subjects prior to L-4F treatment resulted in significant dose-dependent HII improvement. In conclusion, in vivo L-4F treatment, delivered by either SC injection or IV infusion, did not improve HDL functional biomarkers despite achieving plasma levels that improved identical biomarkers ex vivo and in animal models.     

HDL3, but not HDL2, stimulates plasminogen activator inhibitor-1 release from adipocytes: the role of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that regulates numerous key cardiovascular functions. High-density lipoproteins (HDLs) are the major plasma lipoprotein carriers of S1P. Fibrinolysis is a physiological process that allows fibrin clot dissolution, and decreased fibrinolytic capacity may result from increased circulating levels of plasminogen activator inhibitor-1 (PAI-1). We examined the effect of S1P associated with HDL subfractions on PAI-1 secretion from 3T3 adipocytes. S1P concentration in HDL3 averaged twice that in HDL2. Incubation of adipocytes with increasing concentrations of S1P in HDL3, but not HDL2, or with S1P complexed to albumin stimulated PAI-I secretion in a concentration-dependent manner. Quantitative RT-PCR revealed that S1P_1–3 are expressed in 3T3 adipocytes, with S1P₂ expressed in the greatest amount. Treatment of adipocytes with the S1P₁ and S1P₃ antagonist VPC23019 did not block PAI-1 secretion. Inhibiting S1P₂ with JTE-013 or reducing the expression of the gene coding for S1P₂ using silencing RNA (siRNA) technology blocked PAI-1 secretion, suggesting that the S1P₂ receptor mediates PAI-1 secretion from adipocytes exposed to HDL3 or S1P. Treatment with the phospholipase C (PLC) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, the protein kinase C (PKC) inhibitor RO-318425, or the Rho-associated protein kinase (ROCK) inhibitor Y27632 all significantly inhibited HDL3- and S1P-mediated PAI-1 release, suggesting that HDL3- and/or S1P-stimulated PAI-1 secretion from 3T3 cells is mediated by activation of multiple, downstream signaling pathways of S1P₂.     

Oxidized LDL autoantibodies are related to apolipoprotein E phenotype,independently of postprandial change in plasma triglycerides and LDL size,among patients with angiographically verified coronary artery disease and healthy controls

OBJECTIVE: Oxidized low-density lipoprotein (LDL) autoantibodies (oxLDLab), apolipoprotein E (apoE) phenotype, postprandial triglyceride changes and LDL size are suggested to be risk factors for coronary artery disease (CAD). Our aim was to study the interaction between these new risk factors among patients with CAD and healthy controls. METHODS: oxLDLab from 31 men with angiographically verified CAD and 31 healthy men were analyzed by enzyme-linked immunosorbent assay. Isoelectric focusing and immunoblotting were used for apoE phenotyping. Triglyceride level was measured after 12 h of fasting and 3, 5 and 7 h after a high-fat meal. Nondenaturing gradient gel electrophoresis was used to separate LDL particles according to size. RESULTS: oxLD- Lab levels increased according to apoE phenotype in the following order: E2 < E3 < E4 (p = 0.004, ANOVA). The postprandial response of triglycerides, the size of LDL particles and the concentration of LDL and high-density lipoprotein (HDL) cholesterol did not differ between apoE phenotypes, and the use of these variables as covariates did not change the statistically significant difference in oxLDLab levels between apoE phenotypes (p = 0.01, ANCOVA). oxLDLab levels did not differ between the patient and control groups. CONCLUSION: We found an association between apoE allele epsilon2 and decreased levels of oxLDLab, which was independent of the postprandial response of triglycerides, the size of LDL particles and plasma LDL and HDL cholesterol levels. The mechanism by which apoE affects oxidation of LDL remains unknown.     

Obesity favors apolipoprotein E- and C-III-containing high density lipoprotein subfractions associated with risk of heart disease

Human HDLs have highly heterogeneous composition. Plasma concentrations of HDL with apoC-III and of apoE in HDL predict higher incidence of coronary heart disease (CHD). The concentrations of HDL-apoA-I containing apoE, apoC-III, or both and their distribution across HDL sizes are unknown. We studied 20 normal weight and 20 obese subjects matched by age, gender, and race. Plasma HDL was separated by sequential immunoaffinity chromatography (anti-apoA-I, anti-apoC-III, anti-apoE), followed by nondenaturing-gel electrophoresis. Mean HDL-cholesterol concentrations in normal weight and obese subjects were 65 and 50 mg/dl (P = 0.009), and total apoA-I concentrations were 119 and 118 mg/dl, respectively. HDL without apoE or apoC-III was the most prevalent HDL type representing 89% of apoA-I concentration in normal weight and 77% in obese (P = 0.01) individuals; HDL with apoE-only was 5% versus 8% (P = 0.1); HDL with apoC-III-only was 4% versus 10% (P = 0.009); and HDL with apoE and apoC-III was 1.5% versus 4.6% (P = 0.004). Concentrations of apoE and apoC-III in HDL were 1.5–2× higher in obese subjects (P ≤ 0.004). HDL with apoE or apoC-III occurred in all sizes among groups. Obese subjects had higher prevalence of HDL containing apoE or apoC-III, subfractions associated with CHD, whereas normal weight subjects had higher prevalence of HDL without apoE or apoC-III, subfractions with protective association against CHD.