The effects of apolipoprotein B depletion on HDL subspecies composition and function

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl₂), heparin sodium/manganese chloride (MnCl₂), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl₂, heparin sodium/MnCl₂, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.     

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

apoA-I plays important structural and functional roles in reverse cholesterol transport. We have described the molecular structure of the N-terminal domain, Δ(185-243) by X-ray crystallography. To understand the role of the C-terminal domain, constructs with sequential elongation of Δ(185-243), by increments of 11-residue sequence repeats were studied and compared with Δ(185-243) and WT apoA-I. Constructs up to residue 230 showed progressively decreased percent α-helix with similar numbers of helical residues, similar detergent and lipid binding affinity, and exposed hydrophobic surface. These observations suggest that the C-terminal domain is unstructured with the exception of the last 11-residue repeat (H10B). Similar monomer-dimer equilibrium suggests that the H10B region is responsible for nonspecific aggregation. Cholesterol efflux progressively increased with elongation up to ∼60% of full-length apoA-I in the absence of the H10B. In summary, the sequential repeats in the C-terminal domain are probably unstructured with the exception of H10B. This segment appears to be responsible for initiation of lipid binding and aggregation, as well as cholesterol efflux, and thus plays a vital role during HDL formation. Based on these observations and the Δ(185-243) crystal structure, we propose a lipid-free apoA-I structural model in solution and update the mechanism of HDL biogenesis.     

ABCA1 single nucleotide polymorphisms on high-density lipoprotein-cholesterol and overweight: the D.E.S.I.R. study

The adenosine triphosphate-binding cassette A1 (ABCA1) gene plays a key role in reverse cholesterol transport. Some ABCA1 gene polymorphisms have been associated with high-density lipoprotein-cholesterol (HDL-C) concentrations. The aim of this study was to assess the effect of three polymorphisms, C69T, G378C, and G1051A (R219K), on HDL-C levels and their interaction with BMI in more than 5000 French whites from the D.E.S.I.R. (Data from an Epidemiological Study on the Insulin Resistance syndrome) cohort study. The T allele of the C69T single nucleotide polymorphism (SNP) was associated with higher HDL-C levels in normal-weight men (BMI <25 kg/m(2)). The C allele of the G378C SNP was associated with lower HDL-C in overweight subjects (BMI > or =25 kg/m(2)). For the G1051A SNP, in the normal-weight group, the minor A allele was significantly associated with higher HDL-C levels. In contrast, in overweight people, the minor allele was associated with lower HDL-C levels. After accounting for multiple testing, empiric p values remained significant for the associations between G378C SNP and HDL-C in the overweight group and between G1051A SNP and HDL-C in the normal-weight group. This study suggests that ABCA1 gene polymorphisms modulate HDL-C concentrations, in interaction with BMI, and, thus, they might influence cardiovascular risk in the general population.     

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT

The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I^−/− mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-β-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I^−/− × apoE^−/− mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-β-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1^−/− or LCAT^−/− mice. Coexpression of apoA-IV and LCAT in apoA-I^−/− mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.     

The central type Y amphipathic alpha-helices of apolipoprotein AI are involved in the mobilization of intracellular cholesterol depots

We studied the role of a central domain of human apolipoprotein AI (apoAI) in cholesterol mobilization and removal from cells. In order to check different protein conformations, we tested different sized and cholesterol-content reconstituted apoAI particles (rHDL). Meanwhile cholesterol-free discs were active to induce mobilization, only small cholesterol-containing rHDL were active. To test the influence of a central domain in such events, we used two apoAI variants: one, with its central Y helix pair replaced by the C-terminal domain, and the other having a lysine deleted in central region. The helix-swapping variant decrease the cholesterol pool available to acyl-CoA cholesterol acyl transferase and increase mobilization of newly synthesized cholesterol. Instead, the deletion mutant had no effect on both events. We conclude that the central domain of apoAI is involved in cholesterol cell traffic and solubilization, and that a Y-type charge distribution in polar face may be required, as well as a correct helices-polar face orientation.     

Naturally occurring variant of mouse apolipoprotein A-I alters the lipid and HDL association properties of the protein

Plasma HDL levels are inversely associated with atherosclerosis. Inbred mouse strains differ in plasma HDL levels and susceptibility to atherosclerosis. Atherosclerosis-susceptible C57BL/6J mice possess plasma HDL levels 2-fold lower than atherosclerosis-resistant FVB/NJ mice. Polymorphisms have been previously identified between the two mouse strains in the major HDL apolipoproteins, ApoA-I and ApoA-II, which may affect their function on HDL. To begin to understand the HDL differences, we here report on a detailed comparison of the lipid-associated functions of the two mouse ApoA-I proteins. We demonstrate that these polymorphisms significantly alter the protein self-association properties, the ability of the proteins to clear lipid micelles from solution, and their binding affinity for mature mouse HDL. The changes in lipid binding do not appear to alter the ability of the protein to promote cholesterol efflux from cells or the formation of nascent HDL from primary hepatocytes. These apolipoprotein polymorphisms do not change the rate at which HDL protein or cholesterol are catabolized in vivo. Although the presence of the polymorphisms in ApoA-I alters important factors in HDL formation, the basis for the differences in the HDL plasma levels observed in the various mouse strains is more complex and requires additional investigation.     

Homeostasis of free cholesterol in the blood: a preliminary evaluation and modeling of its passive transport

The rate of noncatalyzed transfer of cholesterol (Chol) among lipoproteins and cells in the blood is of fundamental importance as a baseline to assess the role of active transport mechanisms, but remains unknown. Here we address this gap by characterizing the associa­tion of the Chol analog, ergosta-5,7,9(11),22-tetraen-3β-ol (DHE), with the lipoproteins VLDL, LDL, HDL₂, and HDL₃. Combining these results with data for the association of DHE with liposomes, we elaborated a kinetic model for the noncatalyzed exchange of free Chol among blood compartments. The computational results are in good agreement with experimental values. The small deviations are explained by the nonequilibrium distribution of unesterified Chol in vivo, due to esterification and entry of new unesterified Chol, and eventual effects introduced by incubations at low temperatures. The kinetic profile of the homeostasis of unesterified Chol in the blood predicted by the model developed in this work is in good agreement with the observations in vivo, highlighting the importance of passive processes.     

术前血脂异常和非转移性乳腺癌患者临床病理参数的相关性和临床意义

目的:分析术前血脂(总胆固醇[Total cholesterol,TC]、甘油三酯[Triglyceride,TG]、高/低密度脂蛋白胆固醇[High/Low density lipoprotein cholesterol,HDL-C/LDL])异常和乳腺癌患者临床病理参数的相关性。方法:根据纳入排除标准收集我院自2013年1月-2017年1月经病理证实的乳腺病病例共224例,其中浸润性乳腺癌88例(病例组),乳腺良性疾病136例(对照组),收集患者年龄、肿瘤位置、肿瘤细胞分化程度、肿瘤标记物(癌胚抗原、CA153、CA125)、TNM分期、肿瘤体积、阳性/活检淋巴结数、Luminal分型、初潮年龄、绝经状态、生育子女数、体重指数(BMI)、体表面积(BSA)等临床病理参数,同时收集术前1月内患者血脂检测结果进行统计分析。结果:①病例组术前TC值(p=0.02)、HDL-C/LDL值(p=0.04,p=0.00)与对照组相比差异具有统计学差异;②术前TC取值为4.62 mmol/L、TG取值为0.92 mmol/L、LDL取值为2.86 mmol/L时预测乳腺癌的灵敏度分别为54.5%、69.3%和61.6%,特异度分别为32.4%、45.9%和33%,曲线下面积分别为0.62(95%CI:0.54-0.69,p=0.00)、0.64(95%CI:0.57-0.71,p=0.00)和0.64(95%CI:0.56-0.73,p=0.00);③以上述取值为分割点分析,在病例组中TG和LDL升高更多见于BMI(分别为:21.66±3.09 vs 25.11±3.37,p=0.00;22.99±3.70 vs 24.91±3.48,p=0.03)偏高的患者,而TC和TG升高更多见于已绝经患者(分别为p=0.01;p=0.00)的患者。结论:乳腺癌患者术前存在一定程度血脂异常,该异常对协助诊断乳腺癌具有一定意义,术前BMI偏高和已绝经的患者存在TG和LDL值异常升高的可能性更大。     

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function

HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo. Male mice had significantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol efflux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially (∼50%) protected from MPO-mediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice.     

Disruption of the murine procollagen C-proteinase enhancer 2 gene causes accumulation of pro-apoA-I and increased HDL levels

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations that impact risk factors associated with the development of this disease continues. Multiple genetic association studies demonstrate that procollagen C-proteinase enhancer 2 (PCPE2) modulates HDL levels. Recent studies revealed an unexpected role for this protein in the proteolytic processing of pro-apolipoprotein (apo) A-I by enhancing the cleavage of the hexapeptide extension present at the N-terminus of apoA-I. To investigate the role of the PCPE2 protein in an in vivo model, PCPE2-deficient (PCPE2 KO) mice were examined, and a detailed characterization of plasma lipid profiles, apoA-I, HDL speciation, and function was done. Results of isoelectric focusing (IEF) electrophoresis together with the identification of the amino terminal peptides DEPQSQWDK and WHVWQQDEPQSQWDVK, representing mature apoA-I and pro-apoA-I, respectively, in serum from PCPE2 KO mice confirmed that PCPE2 has a role in apoA-I maturation. Lipid profiles showed a marked increase in plasma apoA-I and HDL-cholesterol (HDL-C) levels in PCPE2 KO mice compared with wild-type littermates, regardless of gender or diet. Changes in HDL particle size and electrophoretic mobility observed in PCPE2 KO mice suggest that the presence of pro-apoA-I impairs the maturation of HDL. ABCA1-dependent cholesterol efflux is defective in PCPE2 KO mice, suggesting that the functionality of HDL is altered.     

HDL3 reduces the association and modulates the metabolism of oxidized LDL by osteoblastic cells: a protection against cell death

Oxidized low density lipoproteins (OxLDL) are known to promote atherosclerosis, but it is only recently that OxLDL have been associated with alterations of the functions of bone-forming osteoblasts and osteoporosis. Although high density lipoproteins (HDL) are recognized for their anti-atherogenic action, there is less information about their ability to protect against osteoporosis. Therefore, we investigated the capacity of HDL3 to prevent the cell death induced by OxLDL in human osteoblastic cells. Simultaneous exposure of the cells to HDL3 and OxLDL abolished the reduction of cell viability monitored by MTT activity measurement and the induction of apoptosis determined by annexin V staining indicating that HDL3 prevent the apoptosis of osteoblasts induced by OxLDL. This protection correlated with the displacement by HDL3 of OxLDL association to osteoblasts, signifying that OxLDL binding and/or internalization are/is necessary for their cytotoxic effects. We also found that exposition of osteoblastic cells to HDL3 prior to incubation with OxLDL reduced cell death and preserved the lysosomal integrity. This protection was correlated with an increase of SR-BI expression, a modification of OxLDL metabolism with less global uptake of OxLDL and greater selective uptake of cholesterol from OxLDL. These results strongly suggest that, as for atherosclerosis, HDL may exert beneficial actions on bone metabolism.     

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.     

In vitro modification of the chemical composition of human plasma low density lipoproteins: effects of morphology and thermal properties

The effects of enzymatic action on human low density lipoproteins (LDL) occurring during in vitro incubation of plasma have been studied by chemical analysis, analytical ultracentrifugation, negative stain electron microscopy and X-ray small angle scattering. Chemically, the action of cholesteryl ester exchange and transfer proteins(s) (CEPT) leads to a relative increase in trigylcerides at the expense of cholesteryl esters. Morphologically, the particles maintain their characteristic features detectable by X-ray small angel scattering. Additional action of lecithin/cholesterol acyl transferase (LCAT) causes mainly a decrease in polar lipid contents and a reduction in particle size. The associated changes in the thermotropic transition were found to be strongly correlated to the triglyceride/cholesteryl ester ratio.