Ferritin protects endothelial cells from oxidized low density lipoprotein in vitro.

Low density lipoprotein (LDL), if it becomes oxidized, develops several unique properties including the capacity to provoke endothelial cytotoxicity via metal-catalyzed free radical-mediated mechanisms. As were previously have shown that iron-catalyzed oxidant injury to endothelial cells can be attenuated by the addition of exogenous iron chelators such as the lazaroids and deferoxamine, we have examined whether the endogenous iron chelator, ferritin, might provide protection from oxidized LDL. LDL oxidized by iron-containing hemin and H2O2 is toxic to endothelial cells in a time- and dose-dependent fashion. Endothelial cell ferritin content is increased by pretreatment of cells with iron compounds or by the direct addition of exogenous apoferritin; ferritin-loaded cells are markedly resistant to the toxicity caused by oxidized LDL. Iron inactivation by ferritin depends on its ferroxidase activity. When a recombinant human ferritin heavy chain mutant, 222, which is devoid of ferroxidase activity, is added to endothelial cells, unlike the excellent protection afforded by the wild-type recombinant heavy chain, endothelial cells are not protected from oxidized LDL. To assess the in vivo relevance of our observation, we examined human coronary arteries of cardiac explants taken from patients with end-stage atherosclerosis. Large amounts of immunoreactive ferritin are focally detected in atherosclerotic lesions, specifically in the myofibroblasts, macrophages, and endothelium without a notable increase in Prussian blue-detectable iron. These findings suggest that ferritin may modulate vascular cell injury in vivo.     

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.     

Ox-LDL增加人脐静脉内皮细胞分泌白三烯C4

氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)是动脉粥样硬化(atherosclerosis,AS)最重要的危险因素之一.它可以直接损伤内皮细胞,也可刺激内皮细胞表达大量的黏附分子、趋化因子,促使单核细胞黏附于内皮细胞、趋化单核细胞进入内膜和促进泡沫细胞形成,从而导致AS的发生.     

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     

氧化低密度脂蛋白下调血管内皮细胞表达Rictor

目的探讨氧化低密度脂蛋白对培养血管内皮细胞表达基因Rictor的影响。方法分离并培养人脐静脉内皮细胞,用不同浓度(10、20、40和80 mg/L)氧化低密度脂蛋白作用24 h后,用RT-PCR及Western blot检测Rictor的表达情况,并检测转染Rictor后,蛋白激酶Akt及eNOS磷酸化的变化,用硝酸还原酶还原法测定NO释放量。结果氧化低密度脂蛋白显著降低Rictor的mRNA及蛋白的表达量(P<0.01),使Rictor-mTOR复合物形成减少。转染Rictor后,不仅Rictor表达增加而且使蛋白激酶Akt及eNOS磷酸化增加;内皮细胞NO释放量增多(P<0.05)。结论影响内皮细胞表达Rictor,是氧化低密度脂蛋白诱发血管内皮细胞功能不良的重要机制之一。     

Perturbation of cultured human endothelial cells by atherogenic levels of low density lipoprotein.

Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic levels of low density lipoprotein (LDL) for protracted periods demonstrated no measurable evidence of overt cytotoxicity, but were perturbed as indicated by an increase in prostacyclin (PGI2) production. Confluent EC were incubated with high LDL concentrations (240 or 330 mg/dl cholesterol) for 1 to 12 days. LDL was added to culture media containing 25% human lipoprotein-deficient serum to determine the effects of LDL independent of other lipoproteins. LDL did not injure EC as assessed by cell count, vital dye exclusion, 51chromium release, and lactate dehydrogenase release. Although high concentrations of LDL did not cause EC cytotoxicity, such LDL concentrations did results in increased PGI2 generation. PGI2 accumulation in postincubation media was increased two-to-fivefold in otherwise unstimulated cells as measured by radioimmunoassay of the stable PGI2 breakdown product, 6-keto-PGF1-alpha. This elevation persisted for the entire 12-day exposure to high LDL concentrations. These results indicate that prolonged exposure to atherogenic concentrations of LDL does not effect EC viability, but does cause an endothelial perturbation as demonstrated by an increased PGI2 production.     

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,高脂血清对内皮细胞膜脂流动性的影响

实验用DPH荧光偏振技术测定经人低密度脂蛋白(LDL)及兔高脂血清(HRS)孵育的牛主动脉内皮细胞(EC)的膜脂微粘度。结果表明LDL孵育EC12小时导致细胞膜脂流动性下降,过氧化脂质(Lpo)含量增高;EC经HRS作用36小时后也发生相似的变化。形态观察证明EC胞浆中有大量脂滴样结构。结果提示高胆固醇含量的LDL和HRS可能通过增加膜的胆固醇含量而降低膜的流动性,高浓度Lpo可能也起重要作用。     

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.     

低密度脂蛋白对血管内皮细胞骨架及粘附能力的影响

目的研究低密度脂蛋白(LDL)诱导的血管内皮细胞脂质及细胞骨架的改变及其对内皮细胞粘附的影响。方法不同浓度LDL(0mg/L,50mg/L,100mg/L,150mg/L)孵育内皮细胞24h。采用高效液相色谱法检测细胞内胆固醇酯;油红O检测细胞内脂质;FITC标记的鬼笔环肽进行F-actin纤维荧光标记;流室系统检测生理剪切流场下(1.5Pa)内皮细胞的粘附。结果不同浓度LDL(50mg/L,100mg/L,150mg/L)均导致细胞内胆固醇酯含量增加,其结果分别为(80.3±1.5)μg/g,(115.5±2.2)μg/g和(160.6±3.2)μg/g,与对照组(61.3±1.6)μg/g相比P<0.05;油红O染色内皮细胞内脂质含量明显增加。荧光染色发现,对照组内皮细胞的F-actin纤维主要富集在相邻细胞膜的周边,细胞质中未见密集的F-actin纤维;随着LDL浓度的增加,内皮细胞内F-actin纤维形态和分布发生明显的改变,出现应力纤维;在高浓度LDL下,大部分细胞周边的F-actin纤维消失,胞质中出现密集的应力纤维。在生理剪切流场下,高浓度LDL降低内皮细胞的粘附与保留,150mg/L组内皮细胞的粘附与保留仅50%±10%,对照组为93%±5%。结论LDL增加内皮细胞中脂质含量,损伤细胞骨架,降低内皮细胞的顺应性,降低内皮细胞的粘附。     

OX-LDL诱导内皮细胞条件培养液对内皮细胞VCAM-1和 ICAM-1的影响

目的探讨受损内皮细胞的自分泌和旁分泌对内皮细胞自身的影响。方法利用正常内皮细胞条件培养液和用氧化型低密度脂蛋白(OX-LDL)诱导内皮细胞的条件培养液分别作用于正常内皮细胞和受损内皮细胞,用酶联免疫细胞化学法检测血管内皮细胞黏附分子-1(VCAM-1)和细胞间黏附分子-1(ICAM-1)表达的变化。结果正常内皮细胞的条件培养液和OX-LDL诱导的内皮细胞条件培养液对正常内皮细胞VCAM-1和ICAM-1的表达作用不明显(P>0.05),而对受损内皮细胞VCAM-1和ICAM-1的表达具有明显的下调作用(P<0.01)。结论正常和受到氧化损伤的内皮细胞的自分泌和旁分泌作用对正常内皮细胞黏附分子没有影响,而对受损内皮细胞黏附分子有下调作用,说明内皮细胞可通过下调黏附分子的表达来实现自身的抗损伤作用。     

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。     

Decreased uptake of low density lipoprotein by LLC-PK cells cultured at low magnesium concentration.

LLC-PK cells grown in tissue culture on medium containing normal or deficient amounts of magnesium were presented with 125I-low density lipoprotein (LDL) and the uptake determined. Total LDL uptake by Mg-deficient cells decreased as the magnesium concentration decreased. The receptor binding of LDL and the internalization of LDL by the Mg-deficient cells were reduced. Degradation of LDL by Mg-deficient cells, when corrected for the reduced uptake, was less affected.     

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     

软骨细胞膜蛋白低密度脂蛋白受体相关蛋白1低表达的意义

BACKGROUND: Tumor necrosis factor α, as a pathogenic factor, induces the inflammatory reaction mainly via the activation of the nuclear factor kappa B signaling pathway. Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the regulation of the inflammatory reaction induced by cytokines.     

糖基化低密度脂蛋白对巨噬细胞及单核细胞的作用

我们观察了体外制备的非酶法糖基化低密度脂蛋白(glcLDL)在小鼠腹腔巨噬细胞(Mφ)及人单核细胞(U_(937)株)的降解代谢特点,发现小鼠腹腔Mφ在glcLDL的作用下未见胆固醇蓄积增加,对glcLDL的降解亦未见增加。U_(937)细胞对glcLDL的降解与glcLDL加样浓度成正比,用肉豆蔻酰乙酰佛波酯(TPA)处理细胞后其对glcLDL的降解较未处理者显著增加,且仍与加样浓度成正比。本文对上述现象与糖尿病时加速发生的动脉粥样硬化(AS)之间的关系作了初步分析。     

The expression and down stream effect of lectin like-oxidized low density lipoprotein 1 (LOX-1) in hyperglycemic state

The lesions of atherosclerosis represent a series of highly specific cellular and molecular responses. Low density lipoprotein (LDL), which may be modified by oxidation, glycation, aggregation, association with proteoglycans, or incorporation into immune complexes, is a major cause of injury to the endothelium and vascular smooth muscle cells (VSMC).The major major cell types involved in atherogenesis, macrophages and VSMC, are activated by pro-inflammatory stimuli including modified LDL. Modified LDL induces inflammatory responses in macrophages, migration and proliferation of SMC, and triggers foam cell formation. Scavenger receptors, including LOX-1, play a key role in foam cell formation by mediating the uptake of modified LDL. LOX-1 expression is detected in endothelial cells of early atherosclerosis lesions of human carotid arteries. Advanced lesions showed LOX-1 expression not only in endothelial cells but also in macrophages and more frequently in VSMC, and may be involved in foam cell transformation in macrophages and VSMC. The metabolic abnormalities that characterize diabetes, particularly hyperglycemia, free fatty acids, and insulin resistance, provoke molecular mechanisms that alter the function and structure of blood vessels. These include increased oxidative stress, intracellular signal transduction disturbances, and activation of the receptor for advanced glycation end products (R-AGE). Data showed that LOX-1 expression is enhanced by proatherogenic factors relevant to human diabetes, including high glucose, oxLDL, advance glycation end products, and C-reactive protein. LOX-1 expression increased also through oxygen species (ROS), endothelin-1 (ET-1), tumor necrosis factor-alpha (TNF-alpha), shear stress, activation of protein kinase-C (PKC), angiotensin-II (ANG-II), and through inflammatory pathways.     

In vitro isolation of circulating endothelial progenitor cells is related to the high density lipoprotein plasma levels

Circulating endothelial progenitor cells (EPCs) play an important role in post natal neovascularization. High density lipoproteins (HDL) protect the vascular wall from atherosclerosis. The role exerted by HDL on EPCs physiology is unknown. In this study we investigated whether the levels of plasma HDL can modulate the number of EPCs. The number of EPCs was evaluated in 24 subjects as the number of endothelial colony-forming unit (e-CFU) growth in culture. The number of AC133 positive progenitor cells present in the gate of the CD34 bright positive lymphocytes was also evaluated. Plasma levels of HDL, triglycerides and total cholesterol/HDL cholesterol ratio correlated with the number of e-CFU (r=0.62, P=0.006; r=-0.54, P=0.019, and r=-0.61, P=0.007 respectively), but not with the number of CD34/AC133 positive progenitor cells. In vitro, the incubation of the mononuclear cellular fraction with HDL did not increase the number of e-CFU in culture, whereas LDL and VLDL reduced the number of e-CFU. Our results indicate that human HDL plasma levels directly relate to the number of circulating endothelial progenitor cells that can be isolated in vitro, as determined by the number of e-CFU.     

THP-1单核细胞氧化低密度脂蛋白过程中基因表达谱的改变

目的检测与巨噬细胞氧化低密度脂蛋白可能相关的信号传导相关蛋白基因。方法利用低密度脂蛋白作用人单核细胞系THP-1,通过基因芯片分析人信号相关蛋白的表达谱,期望获得阐明氧化机制的有价值的线索。结果在被检测的1 651个基因中,13个基因的表达变化超过了2倍以上,其中9个基因表达增加,4个基因表达减少。在表达增加的基因中,有2个基因已在新近的报道中被认为可能与动脉粥样硬化相关。结论我们的发现可能为揭示巨噬细胞氧化低密度脂蛋白的机制提供全新的思路。     

Interaction of the apolipoprotein E receptors low density lipoprotein receptor-related protein and sorLA/LR11

In this study, we examined protein–protein interactions between two neuronal receptors, low density lipoprotein receptor-related protein (LRP) and sorLA/LR11, and found that these receptors interact, as indicated by three independent lines of evidence: co-immunoprecipitation experiments on mouse brain extracts and mouse neuronal cells, surface plasmon resonance analysis with purified human LRP and sorLA, and fluorescence lifetime imaging microscopy (FLIM) on rat primary cortical neurons. Immunocytochemistry experiments revealed widespread co-localization of LRP and sorLA within perinuclear compartments of rat primary neurons, while FLIM analysis showed that LRP-sorLA interactions take place within a subset of these compartments.