Inhibition of low density lipoprotein oxidation by melatonin

The antioxidant activity of the lipophilic hormone melatonin, “an ideal inhibitor of free radicals,” is studied in models of cellular (peritoneal mouse macrophages) and copper-induced oxidation of low density lipoproteins. Oxidative modification of low density lipoproteins is assessed by accumulation of thiobarbituric acid reactive substances and degradation of¹²⁵I-labeled lipoproteins in a fresh culture of macrophages. Melatonin inhibits in a dose-dependent manner cellular and copper-induced oxidation of lipoproteins and production of the superoxide anion radical by macrophages, the mean concentrations of 50% inhibition being 300, 1230, and 900 μM, respectively. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 10, pp. 399–402, October, 1996     

Susceptibility to oxidation of copper-induced plasma lipoproteins from Japanese eel: protective effect of vitellogenin on the oxidation of very low density lipoprotein.

The susceptibility to oxidation of copper-induced plasma lipoproteins from Japanese eel Anguilla japonica was examined with the guidance of thiobarbituric acid-reactive substances (TBARS). The TBARS values of copper-induced plasma lipoproteins increased with increasing the lipid-to-apolipoprotein ratios and very low density lipoprotein (VLDL) exhibited the highest TBARS value. On the other hand, vitellogenin, estrogen-induced precursor of egg yolk proteins, was resistant to copper-induced oxidation and seemed to chelate low concentrations of copper ion. Vitellogenin also protected the copper-induced oxidation of VLDL because of its antioxidant function. Vitellogenin seemed to serve as transition metals-binding lipoprotein by which free-radical reactions in the oocytes were extensively depressed.     

姜黄素抑制ox-LDL诱导HUVECs凋亡

目的研究姜黄素对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)凋亡的保护作用及其可能机制。方法体外培养HUVECs,实验分为:对照组、ox-LDL组、ox-LDL加内质网应激(ERS)抑制剂PBA组、姜黄素组、ox-LDL加姜黄素组和ox-LDL加姜黄素加PI3K抑制剂LY294002组。CCK-8法检测细胞存活率;流式细胞仪检测细胞凋亡;激光共聚焦显微镜观察活化转录因子6(ATF6)转位;Western bolt检测ERS相关蛋白:糖调节蛋白78(GRP78)、蛋白激酶样内质网激酶(PERK)和肌醇激酶-1(IRE-1)以及相关通路蛋白:LOX-1、AKT和p-AKT的表达。结果与对照组相比,ox-LDL可增加细胞凋亡,提高ERS相关蛋白的表达(P0.01),促使ATF6向核内转位,以及提高LOX-1(P0.01)和降低p-AKT的表达(P0.01);与ox-LDL组相比,PBA可抑制ox-LDL诱导的细胞凋亡(P0.01),姜黄素可抑制ox-LDL诱导的ERS相关蛋白和LOX-1的表达(P0.01),ATF6的核转位,内皮细胞的凋亡(P0.01),同时它还可提高ox-LDL引起的p-AKT表达下调(P0.01);LY294002可部分削弱姜黄素抑制ox-LDL诱导ERS相关蛋白表达的作用(P0.05)。结论姜黄素可降低ox-LDL诱导HUVECs的凋亡,其可能机制是通过抑制LOX-1的表达和激活AKT通路减轻细胞ERS来实现的。     

维拉帕米对巨噬细胞氧化低密度脂蛋白的影响

观察Ca^2 在细胞介导的低密度脂蛋白氧化过程中的作用。巨噬细胞分别培养在不加处理因素、添加了20mg／L低密度脂蛋白以及低密度脂蛋白加不同浓度钙离子拮抗剂维拉帕米(0，25，50，100，150μmol/L)的培养基中。测定培养基中的丙二醛、丙二烯、髓过氧化物酶的活性和浓度的变化以及细胞内的髓过氧化物酶的活性及含量的变化。结果显示，维拉帕米显著地抑制巨噬细胞介导的低密度脂蛋白的氧化和巨噬细胞髓过氧化物酶的分泌，且这种抑制作用呈浓度依赖性。这些结果提示维拉帕米引起钙离子向巨噬细胞膜内转移减少，细胞分泌髓过氧化物酶的随之减少，髓过氧化物酶氧化修饰低密度脂蛋白下降。     

Platelet activation by low density lipoprotein and high density lipoprotein

Cardiovascular disease is the main cause of death and disability in the Western society. Lipoproteins are important in the development of cardiovascular disease since they change the properties of different cells involved in atherosclerosis and thrombosis. The interaction of platelets with lipoproteins has been under intense investigation. Particularly the initiation of platelet signaling pathways by low density lipoprotein (LDL) has been studied thoroughly, since platelets of hypercholesterolemic patients, whose plasma contains elevated LDL levels due to absent or defective LDL receptors, show hyperaggregability in vitro and enhanced activity in vivo. These observations suggest that LDL enhances platelet responsiveness. Several signaling pathways induced by LDL have been revealed in vitro, such as signaling via p38 mitogen-activated protein kinase and p125 focal adhesion kinase. High density lipoprotein (HDL) consists of two subtypes, HDL(2) and HDL(3), which have opposing effects on platelet activation. This review provides a summary of the activation of signaling pathways after platelet-LDL and platelet-HDL interaction, with special emphasis on their role in the development of thrombosis and atherosclerosis.     

内源性高甘油三酯血症患者血浆VLDL、LDL及HDL对血凝的影响

目的：研究内源性高甘油三酯血症(HTG)患血浆极低密度脂蛋白(VLDL)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)是否发生了氧化修饰及其对血凝的影响。方法：对2l例内源性高甘油三酯血症患与2l例年龄性别相近的正常人的血脂、脂质过氧化物进行了分析。用一次性密度梯度超速离心法分离血浆VLDL、LDL及HDL，测定这三种脂蛋白的234nm光吸收、相对电泳迁移率(REM)和硫代巴比妥酸反应物质(TBARS)，分别将这三种脂蛋白加入由正常人新鲜混合血浆构成的反应系统中，按试剂盒分别测定凝血酶原时间(PT)及活化部分凝血酶原时间(APIT)。结果：内源性HTG患血浆TG含量平均升高2．73倍，HDLC下降l.7l倍，同时LPO升高1．22倍;HTG组VLDL、LDL及HDL的REM、234nm光吸收值、TBARS含量均较对照组显增加(P＜0．01)，表明内源性HTG患血浆VLDL、LDL及LDL均发生了氧化修饰生成Ox—VLDL、Ox-LDL.PT及APTT在分别加入HTG组的VLDL、LDL及HDL后均比加入相应正常组脂蛋白明显缩短(P均＜0．05)。相关分析表明，HTG组血浆VLDL及HDL相对电泳迁移率(REM)与PT呈负相关(P＜0．01)。结论：HTG患血浆VLDL、LDL及HDL发生了氧化修饰，并使PT及APTT明显缩短。     

Genetics of the low density lipoprotein receptor:

Six indices of low density lipoprotein (LDL) receptor activity were assayed in cultured fibroblasts from seven subjects with familial hypercholesterolemia (HC) and six subjects without HC (non-HCs). Four non-HCs, three HC heterozygotes and one HC homozygous proband belonged to one kindred (kindred A). The proband's fibroblast 125I-LDL processing values fell within or were slightly above the range defined by fibroblasts from three "receptor-negative" HC homozygotes. Thus, the plasma membrane receptor defect in this kindred is probably of the "receptor-negative" category. LDL receptor-dependent 125I-LDL processing was about twice as high in fibroblasts from non-HCs as in those from HC heterozygotes belonging to kindred A. The segregation pattern of LDL receptor activity in this kindred was compatible with control by a single gene locus. 125I-LDL processing values from non-HCs, HC heterozygotes and HC homozygotes differed significantly from one another, but non-HCs and HC heterozygotes showed some overlap. LDL receptor-dependent 125I-LDL association (plasma membrane binding plus intracellular accumulation) data for 6 HC heterozygous and 13 non-HC fibroblast strains clustered into two and into three groups, respectively. Median 125I-LDL association levels in these groups appeared to be in agreement with hypothesis that two different geno-types in HC heterozygotes and three in non-HCs determined LDL receptor activity. These findings suggest the possibility that 125I-LDL processing studies may reveal "normal" alleles at the LDL receptor locus.     

Genetics of the low density lipoprotein receptor:

Fibroblast low density lipoprotein (LDL) plasma membrane receptor activity, measured as 125I-LDL association (plasma membrane binding plus intracellular accumulation) and degradation was determined in cell strains from 14 monozygotic (MZ) and 21 like-sexed dizygotic (DZ) normolipidemic twin pairs. The twins were between 57 and 62 years old and had liver apart for an average of 38 years (range 0-60). The intrapair differences were significantly smaller in MZ than in DZ twin pairs in fibroblast 125I-LDL association as well as degradation assays (P less than 0.05). These findings suggest a genetic influence on normal variation in LDL receptor activity in vitro. In two MZ pairs discordant for psoriasis, the psoriatic twin had markedly lower LDL receptor activity than the cotwin.     

Genetics of the low density lipoprotein receptor:

Fibroblast association (plasma membrane binding plus intracellular accumulation) and degradation of radioiodinated low density lipoprotein (125I-LDL) index plasma membrane LDL receptor activity. Cultured fibroblasts from 23 subjects affected with familial hypercholesterolemia (HC) and from 95 subjects without HC (non-HCs) were tested for 125I-LDL association and degradation. Both LDL receptor activity indices were twice as high in non-HC and HC heterozygous cell strains. This is compatible with a major gene effect on LDL receptor activity. However, a considerable overlap between non-HC and HC heterozygous values was found in the 125I-LDL association assay [median (range) 970 (330-2500), and 450 (250-490), respectively] and in the degradation assay [median (range) 810 (280-2020), and 470 (160-790), respectively]. The values are expressed as ng 125I-LDL X mg cell protein-1 X 4.5 h-1. These great overlaps in the LDL receptor activity indices support the view that the influence of LDL receptor activity on the HC phenotype may be smaller than believed previously. Furthermore, for the diagnosis of HC, these LDL receptor activity assays are far more expensive and have less sensitivity and specificity than simple serum cholesterol determination. The LDL receptor-dependent 125I-LDL association values for the HC heterozygous individuals clustered into four groups. Family data supported the hypothesis that this variation could be due to four different LDL receptor variants, each coded for by different alleles at the LDL receptor locus. If confirmed, this finding may have implications for the understanding of the variable expression of HC and also of the genetic impact on lipoprotein metabolism and susceptibility to atherosclerosis in non-HCs.     

Very low density lipoprotein and low density lipoprotein isolated from patients with hepatitis C infection induce altered cellular lipid metabolism

Several abnormalities of lipid metabolism, including hypo-beta-lipoproteinemia and liver steatosis are associated with infection by hepatitis C virus (HCV). The aim of this study was to determine whether circulating lipoproteins of patients with HCV infection could directly cause alterations of lipid cellular metabolism. To this end the metabolic response of human monocyte-derived macrophages (HMDM) to very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), measuring the cholesteryl ester (CE) and triglyceride (TG) production was analyzed. Lipoproteins were isolated from 18 patients infected with hepatitis C virus (HCV-VLDL and HCV-LDL) and from normal healthy donors (ct-VLDL and ct-LDL). In comparison to ct-lipoproteins, HCV-lipoproteins induced significant differences in HMDM CE and TG production. HCV-VLDL decreased CE and TG production; while HCV-LDL induced an increased TG synthesis. The present findings suggest that HCV infection modifies VLDL and LDL molecular composition, affecting cellular lipid metabolism, thus promoting intracellular lipid accumulation and hypo-beta-lipoproteinemia.     

Bovine lactoferrin inhibits Japanese encephalitis virus by binding to heparan sulfate and receptor for low density lipoprotein

Lactoferrin is a natural anti-microbial protein which affects Japanese encephalitis virus (JEV) activity. Binding of lactoferrin to cell surface expressed heparan sulfate (HS), one possible receptor for JEV, has been postulated to be the possible mechanism of anti-JEV antiviral activity. In this study, we evaluate the effects of bovine lactoferrin (bLF) against JEV infection in vitro, using both wild-type (WT) and laboratory-adapted strains. bLF inhibited the infectivity of all the JEV strains tested. In particular the infectivity of the HS-adapted JEV strains was strongly reduced, whereas the non HS-adapted JEV strains were inhibited to lesser extent. Using both HS-adapted CJN-S1 and non HS-adapted CJN-L1 viruses, the results showed that bLF inhibited the early events essential to initiate JEV infection, which includes blocking virus attachment to cellular membranes and reducing viral penetration. This anti-JEV activity was the highest using HS-adapted CJN-S1 strain on HS-expressed CHO-K1 cells. Also, binding of bLF to heparin-sepharose blocked JEV binding; and soluble HS attenuated the anti-JEV activity of bLF. The results support the premise that the interaction of bLF with cell surface expressed glycosaminoglycans, in particular the highly sulfated HS, plays an essential role in the antiviral activity of bLF. However, bLF was functional in inhibiting CJN-S1 entry into HS-deficient CHO-pgsA745 cells, and bLF-treated CHO-K1 and -pgsA745 cells also prevented non HS-adapted CJN-L1 virus entry, indicating that a non-HS pathway may be involved in bLF inhibition of JEV entry. The low-density lipoprotein receptor (LDLR), possibly involved in the entry of several RNA viruses, also binds to bLF. We found that both rLDLR and anti-LDLR antibodies reduced the effectiveness of bLF inhibition of JEV infection. This finding provided evidence to suggest that cell surface-expressed LDLR may play a role in JEV infection, especially for non HS-adapted strains.     

Immunochemistry of an allotypic low density lipoprotein in man

This paper presents data on a human allotypic polymorphism carried on a low density lipoprotein (As-LDL). The As polymorphism was investigated by a rabbit antiserum which was firstly used to analyze the serum level and the isoelectric point of the As lipoprotein in sera of different phenotype. This antiserum was also used to purify As-LDL by immunochromatography. Afterwards the peptidic maps of the APO proteins from different As-LDL were compared. While no correlation did result between the serum level and the serological phenotype of the As lipoprotein, the peptidic distribution of the APO parts from different As-LDL showed differences correlated to the allotypic phenotype.     

Metabolic fate of low density lipoprotein and high density lipoprotein labeled with an ether analogue of cholesteryl ester

Summary Low density lipoprotein (LDL) metabolism by human skin fibroblasts was studied using LDL labeled with a nonhydrolyzable cholesteryl ether analogue,³H-cholesteryl linoleyl ether (CLE). The³H-CLE-LDL was taken up by the apo-B, E receptor mediated endocytosis similar to¹²⁵I-labeled LDL. This was shown by saturation kinetics of uptake with respect to³H-CLE-LDL concentration and very low uptake of³H-CLE-LDL by receptor negative cell strains. When injected into rats,³H-CLE-LDL and¹⁴C- cholesteryl ester (CE)-LDL were cleared at equal rates and about 30% of the injected LDL was recovered in the liver. Treatment with ethinyl estradiol resulted in a three-fold increase in³H-CLE-LDL uptake by the liver. The liver is also the major site of uptake of³H-CLE-high density lipoprotein (HDL) (40%–45% of the injected dose) but its uptake by the liver increased only by 20% with estradiol treatment. As³H-CLE-HDL was cleared from the circulation at a somewhat faster rate than¹²⁵I-HDL it appeared that some dissociation in the tissue uptake of the protein and CE moieties occurs.Abbreviations LDL low density lipoprotein - HDL high density lipoprotein - CLE cholesteryl linoleyl ether - CE cholesteryl ester Presented at the 42nd European Atherosclerosis Group Meeting, Munich, 5–6 March, 1984     

Very low density lipoprotein apolipoprotein B metabolism in humans

Summary The human plasma lipoproteins encompass a broad spectrum of particles of widely varying physical and chemical properties whose metabolism is directed by their protein components. Apolipoprotein B₁₀₀ (apo B₁₀₀) is the major structural protein resident in particles within the Svedberg flotation range 0–400. The largest of these, the very low density lipoprotein (VLDL), rich in triglyceride, are metabolised by sequential delipidation through a transient intermediate density lipoprotein (IDL) to cholesterol-rich low density lipoproteins (LDL). Several components contribute to the regulation of this process, including (a) the lipolytic enzymes lipoprotein lipase and hepatic lipase (b), apolipoproteins B, CII, CIII and E, and (c) the apolipoprotein B/E or LDL receptor. Lipoprotein lipase acts primarily on large VLDL of Sf 60–400. Hepatic lipase on the other hand seems to be critical for the conversion of smaller particles (Sf 12–60) to LDL (Sf 0–12). Although most apo B₁₀₀ flux is directed to the production of the delipidation end product LDL, along the length of the cascade there is potential for direct removal of particles from the system, probably via the actions of cell membrane receptors. This alternative pathway is particularly evident in hypertriglyceridaemic subjects, in whom the delipidation process is retarded.VLDL metabolism shows inter subject variability even in normal individuals. In this regard, apolipoprotein E plays an important role. Normolipidaemic individuals homozygous for the apo E₂ variant exhibit gross disturbances in the transit of B protein through the VLDL-IDL-LDL chain.Abbreviations apo B, C, E Apolipoprotein B, C, E - CETP Cholesteryl ester transfer protein - FCH Familial combined hyperlipidaemia - FH Familial hypercholesterolaemia - FHTG Familial hypertriglyceridaemia - HDL High density lipoprotein - HL Hepatic lipase - IDL Intermediate density lipoprotein - LDL Low density lipoprotein - LpL Lipoprotein lipase - RFLP Restriction fragment length polymorphism - Sf Svedberg flotation coefficient - VLDL Very low density lipoprotein - WHHL Watanabe heritable hyperlipidemic     

Modulation of endotoxin-induced endothelial cell toxicity by low density lipoprotein

Bacterial endotoxins (lipopolysaccharides (LPS] have been reported to the toxic to endothelial cells in vivo. In vitro they have been shown to be toxic to bovine endothelial cells but not to human endothelial cells. In this report we demonstrate that the presence of plasma low density lipoprotein (LDL) protected bovine endothelial cells from LPS-induced toxicity whereas the presence of LDL actually promoted LPS-induced toxicity to human endothelial cells. These effects of LPS were independent of its source or method of preparation. High density lipoprotein also inhibited LPS-induced toxicity to bovine endothelial cells but unlike LDL, did not enhance LPS-induced toxicity to human cells. The toxicity of LPS to human endothelial cells in the presence of LDL required the oxidation of LDL by free radicals produced by the endothelial cells. LDL modified by acetylation enhanced LPS-induced toxicity to both human and bovine endothelial cells. The toxicity to human endothelial cells of LPS plus either LDL (after endothelial cell-mediated oxidation) or acetyl-LDL was inhibited by fucoidin and polyinosinic acid, blockers of the acetyl-LDL (scavenger) receptor. Polymyxin B, a specific LPS antagonist, inhibited the toxicity of LPS to bovine endothelial cells but not the toxicity of LPS plus LDL to human endothelial cells. These results are consistent with our hypothesis that LDL prevents the toxicity of LPS to bovine endothelial cells by binding the LPS and making it less accessible to the cells. Human endothelial cells are not directly susceptible to LPS-induced toxicity but, unlike bovine cells, produce oxygen free radicals in sufficient quantity to oxidize LDL and render the LDL-LPS complex recognizable for uptake by a scavenger receptor-like process similar to that for acetyl-LDL. LPS thus enters the human endothelial cells via this complex and kills the cells. These findings may have important implications for the study of LPS-induced toxicity to endothelial cells in vitro and for understanding the phenomenon in vivo.     

低密度脂蛋白诱导巨噬细胞髓过氧化物酶活性的研究

目的：研究低密度脂蛋白（LDL）与巨噬细胞髓过氧化物酶（MPO）活性的关系。方法：LDL诱导培养的小鼠腹水巨噬细胞，用MPO催化底物邻联二茴香胺氧化的方法测定MPO活性；以逆转录多聚酶链反应（RT－PCR）的方法检测MPO基因的表达，借此观察LDL和MPO活性的关系。结果：LDL可以促进巨噬细胞内及分泌的髓过氧化物酶活性升高，但远比细菌脂多糖（LPS）的作用小；氧化型低密度脂蛋白（OX－LDL）没有此作用；随着LDL作用时间的延长巨噬细胞内及分泌的髓过氧化物酶活性逐渐升高，但随着LDL浓度的增加髓过氧化物酶基因的表达达到最大值后便维持该水平而不再随LDL浓度的变化而变化。结论：LDL非特异性地诱导巨噬细胞MPO活性升高以及分泌增强，加强自身的氧化吸收；MPO活性升高的原因可能主要为巨噬细胞内酶活性的激活及分泌增强而非基因表达的增强。     

Intracellular oxidative modification of low density lipoprotein by endothelial cells

We investigated intracellular oxidative modification of low density lipoprotein (IOM-LDL) by endothelial cells (ECs) and the role of ferritin in this process. IOM-LDL was examined by immunocytochemistry with an anti-oxidized phosphatidylcholine antibody and by lipid peroxidation assay. Incubation of LDL-treated ECs (human umbilical vein endothelial cells, passage 3) with ferritin produced cytoplasmic immunostain with the antibody, especially in large or giant ECs, and the formation of thiobarbituric acid-reactive substance (TBARS) in these cells. These observations suggest that ECs can perform IOM-LDL. Incubation with the iron chelator deferoxamine or pretreatment of LDL-treated ECs with deferoxamine suppressed ferritin-induced IOM-LDL by greater than 60%. Antioxidants dimethylsulphoxide and butylated hydroxytoluene markedly inhibited IOM-LDL, but mannitol did so only mildly. Catalase and superoxide dismutase had little or no effect on IOM-LDL. Apoferritin substituted for ferritin did not induce IOM-LDL. Our data suggest that IOM-LDL is mediated by intracellular hydroxyl radical formation, which is catalyzed mainly by free iron released from ferritin, and that ECs contribute to the development of atherosclerosis via IOM-LDL.     

Genetic association of low density lipoprotein receptor and Alzheimer's disease

The low density lipoprotein receptor (LDLR) is an attractive candidate gene for genetic association with Alzheimer's disease (AD) because: (i) the LDLR is an apolipoprotein E (apoE) receptor, alleles of which have been associated with AD, (ii) LDLR resides at chromosome 19p13.3 within a region linked to AD, and (iii) LDLR modulates the homeostasis of cholesterol, which itself appears associated with AD. Therefore, we evaluated whether LDLR haplotypes alter the odds of AD by performing an association study examining three LDLR single nucleotide polymorphisms (SNPs) in 118 AD patients and 133 non-AD subjects. LDLR genotypes were obtained by TaqMan allelic discrimination assays. Although individual LDLR SNPs were not associated with AD, analyses of unambiguous haplotypes suggested the hypothesis that the 211 LDLR haplotype was associated with reduced odds of AD. We then evaluated this hypothesis in a second study cohort, i.e., the Religious Orders Study. These results supported the hypothesis that the 211 LDLR haplotype is associated with reduced odds of AD. Moreover, these data suggested further associations between LDLR variants and AD. Thus, LDLR variants appear significantly associated with AD and merit additional study.     

Immunomodulating effect of low density lipoprotein on human monocytes.

Low density lipoprotein (LDL) isolated from sera of healthy volunteers in 50 micrograms protein/ml concentration induced an early adenylate cyclase activation in human monocytes followed by elevation of cGMP level. In addition, a rapid 45Ca2+ influx was also detected on addition of 25-100 micrograms protein/ml concentrations. The monocyte activating effect of LDL under in vitro circumstances was characterized by an enhanced O2 consumption, H2O2 generation and by the increased release of lysosomal enzymes such as beta-glucuronidase and elastase like protease (ELP). On the other hand, LDL diminished markedly the Fc gamma receptor (Fc gamma R) mediated rosette formation, phagocytosis and the antibody dependent cellular cytotoxicity (ADCC) of monocytes without a significant decrease in the IgG binding capability of cells. High levels of serum LDL may play a significant role in the arterial wall injury by elastase like protease as well as biologically active oxygen species released from monocytes of patients suffering from arteriosclerosis.