Interactions of C-reactive protein with low-density lipoproteins: implications for an active role of modified C-reactive protein in atherosclerosis

The interaction of C-reactive protein with low-density lipoprotein is considered to be one of the key properties that link C-reactive protein with atherosclerosis. However the data obtained to date are controversial, and hence make it difficult to conclude actual physiological or pathological impact of such interaction. The incompatible findings could be ascribed to the different structural state of C-reactive protein and/or low-density lipoprotein. We investigated in detail the interaction of various C-reactive protein isoforms with native and modified low-density lipoprotein. Our data showed "C-reactive protein" could indeed interact with each of native low-density lipoprotein, oxidized or enzymatically modified low-density lipoprotein, but that interaction occurs primarily when C-reactive protein is conformed in a modified form and not pentameric structure. Low level of modified C-reactive protein "contaminant" could confer C-reactive protein obvious low-density lipoprotein binding capacity. Interaction of modified C-reactive protein and low-density lipoprotein was mediated synergistically by both electrostatic association with ApoB and hydrophobic insertion into lipid layer. When complexed with modified C-reactive protein, macrophage binding/uptake of native and oxidized low-density lipoprotein was either increased 150% or decreased 35%, respectively. Thus the interaction of modified C-reactive protein with low-density lipoprotein may contribute to the regulation of low-density lipoprotein metabolism and foam cell formation in arterial wall. These results highlight an active role of modified C-reactive protein in atherosclerotic process.     

Tricarbocyanine cholesteryl laurates labeled LDL: new near infrared fluorescent probes (NIRFs) for monitoring tumors and gene therapy of familial hypercholesterolemia

For monitoring low-density lipoprotein receptors (LDLr) in tumors and in livers of patients with familial hypercholesterolemia (FH) treated with gene therapy, a series of tricarbocyanine cholesteryl laurates were synthesized with the cholesteryl laurate moiety serving as the lipid-chelating anchor for low-density lipoprotein (LDL). One of these conjugates, TCL17, was successfully used to label LDL to give a new NIRF, TCL17-LDL. Ex vivo biological studies on an LDLr overexpressing tumor model, human hepatoblastoma G(2) (HepG(2)), confirmed that this NIRF were internalized selectively by the tumor and detected with high sensitivity by a low-temperature 3-D redox scanner.     

Defective endocytosis of low-density lipoprotein in monensin-resistant mutants of the mouse Balb/3T3 cell line

Two monensin-resistant clones show similar low-density lipoprotein binding activity but less internalization or degradation of low-density lipoprotein than the parental Balb/3T3 or other resistant clone. Sterol synthesis from radioactive acetate in the resistant mutant, MO-5, is inhibited by more than 70% of control in the presence of tenfold higher amounts of low-density lipoprotein than the dose that inhibits the parental Balb/3T3 to similar level. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity of Balb/3T3 and MO-5 is inhibited by 48% and 27% of control, respectively, in the presence of 10 micrograms/ml of low-density lipoprotein. Colloidal silica gradient centrifugation shows that transport of low-density lipoprotein from the surface membrane to the lysosome is much slower in MO-5 cells than in Balb/3T3 cells. Down regulation of low-density lipoprotein receptors on the cell surface in Balb/3T3 is observed by exposing the cells to 5-15 micrograms/ml low-density lipoprotein, whereas only slight if any down regulation is observed when MO-5 cells are treated with low-density lipoprotein. The altered endocytosis of low-density lipoprotein behaves as a dominant trait in hybrids of MO-5 and THO2-2, a derivative of Balb/3T3 resistant to both ouabain and 6-thioguanine.     

Small dense low-density lipoprotein and its role as an independent predictor of cardiovascular disease

PURPOSE OF REVIEW: This review discusses whether the relationship of small dense low-density lipoprotein to cardiovascular risk is direct, due to the atherogenic properties of the particle, or a reflection of concomitant abnormalities in high-density lipoprotein and plasma triglyceride. RECENT FINDINGS: Recent studies have examined whether low-density lipoprotein size distribution or concentration of small low-density lipoprotein is related more strongly to risk. It appears that the latter is a better predictor in major surveys, although in smaller cohort studies particle size shows a strong association with atherosclerosis burden. While the main causes of the formation of small dense low-density lipoprotein are relatively well understood, novel metabolic factors may also play a role, and pharmacologic interventions such as glitazones may have a direct regulatory impact. SUMMARY: Evidence links abnormalities in low-density lipoprotein structure to cardiovascular risk. The plasma concentration of small dense low-density lipoprotein is likely to be more informative than relative low-density lipoprotein particle size, and although methods are available for quantitation of this subfraction, there is considerable room for improvement. It is not yet clear how knowledge of the small dense low-density lipoprotein concentration may add to risk prediction.     

Low-Density Lipoprotein Receptor on Endothelium of Brain Capillaries

The presence of lipoproteins, apolipoproteins, and their receptors in the brain could provide a system for cholesterol homeostasis, as they do in other tissues. This study was undertaken to determine whether plasma low-density lipoprotein, the major carrier of cholesterol, is involved in the delivery of lipids through the blood-brain barrier. 125I-Labeled low-density lipoprotein bound to a specific receptor on the endothelium of brain capillaries when it was injected immediately postmortem into bovine brain circulation. In contrast, no specific binding of 125I-low density lipoprotein was found when the incubations were performed with isolated capillaries. Incubations of endothelial or basement membranes of brain capillaries with 125I-low density lipoprotein demonstrated a high-affinity association of low-density lipoprotein with the membranes of bovine cerebral endothelial cells. The specificity of the low-density lipoprotein binding was determined in several ways using a dot blot assay. This receptor shows the same characteristics as the low-density lipoprotein receptor on human fibroblasts. The molecular weight of the bovine brain capillary low-density lipoprotein receptor (132,000) was determined by ligand blotting. These results demonstrated the occurrence of a low-density lipoprotein receptor on the endothelial cells of brain capillaries.     

Clinical applications of circulating oxidized low-density lipoprotein biomarkers in cardiovascular disease

PURPOSE OF REVIEW: The aim of this article is to review, analyze and interpret the growing body of evidence on circulating oxidized low-density lipoprotein and its relationship to diagnosis and prognosis of cardiovascular disease. RECENT FINDINGS: Previous studies focused on indirect measures of oxidative stress such as susceptibility of low-density lipoprotein to oxidation and measurement of autoantibodies to oxidized low-density lipoprotein. The generation of monoclonal antibodies recognizing distinct oxidation-specific epitopes has allowed the development of sensitive and specific assays to measure circulating oxidized low-density lipoprotein. Recent work in human populations has demonstrated that circulating oxidized low-density lipoprotein is associated with preclinical atherosclerosis, coronary and peripheral arterial atherosclerosis, acute coronary syndromes and vulnerable plaques. Several studies have also suggested that elevated levels of oxidized low-density lipoprotein are a prognostic indicator of cardiovascular outcomes. In addition, it has been shown that lipoprotein(a) is the primary carrier of oxidized phospholipids in the circulation of humans, suggesting additional mechanisms through which lipoprotein(a) may be pro-atherogenic. SUMMARY: Research on circulating oxidized low-density lipoprotein biomarkers is rapidly accelerating and providing novel insights into the pathophysiology of cardiovascular disease. Future studies will further assess the clinical utility of oxidized low-density lipoprotein biomarkers by determining their prognostic value in the diagnosis and prognosis of cardiovascular disease and will also evaluate the relative merit of specific assays by performing comparative studies.     

Detection of distinct receptors for native and acetylated low-density lipoproteins in human placental microvilli by ligand-immunoblotting

Ligand-immunoblotting was used to detect distinct receptors for native low-density lipoprotein and for acetylated low-density lipoprotein on microvillous membranes from human term placentas. Antisera directed against native and modified low-density lipoproteins were prepared in rabbits and their specificities were assessed by immunodiffusion and immunoelectrophoresis. The receptor for low-density lipoprotein was detected as a 160 kDa protein and that for acetylated low-density lipoprotein as a 200 kDa protein. These receptors were compared with their counterparts in cultured human skin fibroblasts, bovine adrenal cortex and J774 macrophage-like cells. This is the first investigation that visualizes the presence of receptors for both native and modified low-density lipoproteins in a steroidogenic tissue.     

From low-density lipoprotein to platelet activation

There is a strong correlation between the level of plasma low-density lipoprotein (LDL) and death by cardiovascular disease (CVD). As a main carrier of cholesterol, a high low-density lipoprotein concentration stimulates atherogenesis by its capacity to become oxidized and to become endocytosed by macrophages in the vessel wall forming cholesterol-rich plaques that are sites for arterial occlusion. New evidence points at a second role of low-density lipoprotein in increasing cardiovascular disease-risk. Contact with low-density lipoprotein induces platelet hypersensitivity to agonists that initiate platelet functions thereby enhancing adhesion, aggregation and secretion of granule contents. The signalling pathways that mediate the priming of platelets by native and oxidized low-density lipoprotein have now been characterized.     

Radioimmunological study of the surface protein of the human serum low-density lipoprotein: comparison of the native particle and the products obtained by tryptic treatment.

The present report describes radioimmunological studies of the native low-density lipoprotein from human serum, and of the products obtained by limited tryptic treatment, i.e.a protein-depleted particle lacking some 20% of the original protein moiety and a peptide fraction of low molecular weight (<5000). The liberated peptides were highly immunogenic and elicited antibodies which reacted with both the native and protein-deficient lipoprotein particles. Moreover these peptides exhibited competitive reactivity with [¹²⁵I]-labelled low-density lipoprotein in binding with homologous antisera, and with antisera to the native and trypsin-treated lipoproteins. These findings suggest that the peptides liberated from low-density lipoprotein by tryptic digestion contain the major antigenic site(s) of the molecule. Consideration of the nature of the competitive displacement of radiolabelled low-density lipoprotein from antisera to low-density lipoprotein, to the trypsinised lipoprotein and to the peptide fraction indicate that a marked repetition of the antigenic site(s) occurs in the structure of the protein moiety, a possibility consistent with the recurrence of similar subunits in the apoprotein of low-density lipoprotein.     

Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.     

Oxidation of low-density lipoprotein by Cu2+ and lipoxygenase: an electron spin resonance study

The aim of this work was to obtain spectroscopic evidence for free radicals formed during copper ion- and lipoxygenase-catalyzed oxidation of the low-density lipoprotein. During the initial oxidation phase, a free-radical metabolite derived from the endogenous alpha-tocopherol present in the low-density lipoprotein was detected by the electron spin resonance technique. The divalent copper ions were bound to the residual EDTA present in the low-density lipoprotein and to the protein. Production of the alpha-tocopherol radical was suppressed in the presence of spin traps. Evidence for the low-density lipoprotein-lipid derived radicals was obtained by ESR-spin trapping methods. Implications of these findings in the oxidative modification of the low-density lipoprotein are discussed.     

Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophages

Nonenzymatic covalent binding (glycation) of reactive aldehydes (from glucose or metabolic processes) to low-density lipoproteins has been previously shown to result in lipid accumulation in a murine macrophage cell line. The formation of such lipid-laden cells is a hallmark of atherosclerosis. In this study, we characterize lipid accumulation in primary human monocyte-derived macrophages, which are cells of immediate relevance to human atherosclerosis, on exposure to low-density lipoprotein glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of low-density lipoprotein-derived lipids and protein has been characterized, together with the subsequent turnover of the modified apolipoprotein B-100 (apoB) protein. Cholesterol and cholesteryl ester accumulation occurs within 24 h of exposure to glycated low-density lipoprotein, and increases in a time-dependent manner. Higher cellular cholesteryl ester levels were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein. Uptake was significantly decreased by fucoidin (an inhibitor of scavenger receptor SR-A) and a mAb to CD36. Human monocyte-derived macrophages endocytosed and degraded significantly more (125)I-labeled apoB from glycolaldehyde-modified than from methylglyoxal-modified, or control, low-density lipoprotein. Differences in the endocytic and degradation rates resulted in net intracellular accumulation of modified apoB from glycolaldehyde-modified low-density lipoprotein. Accumulation of lipid therefore parallels increased endocytosis and, to a lesser extent, degradation of apoB in human macrophages exposed to glycolaldehyde-modified low-density lipoprotein. This accumulation of cholesteryl esters and modified protein from glycated low-density lipoprotein may contribute to cellular dysfunction and the increased atherosclerosis observed in people with diabetes, and other pathologies linked to exposure to reactive carbonyls.     

Toxicity of enzymically-oxidized low-density lipoprotein

Intravenous injection of cholesterol oxidase into hyperlipidemic rabbits in which aortic atheromatous lesions have been induced by dietary means is lethal within hours, whereas injection of the same enzyme into normal rabbits has no visible adverse effect. The lethal effect of the enzyme is explicable by the finding that injection of cholesterol-oxidase treated low-density lipoprotein kills normal rabbits, in contrast to untreated low-density lipoprotein which does not. Enzymically oxidized low-density lipoprotein was also found to be cytotoxic for two human cell lines and for cultured bovine aortic endothelial cells. We suggest that in vivo enzymic conversion of low-density lipoprotein cholesterol to low-density lipoprotein cholestenone may possibly play a role in the initiation of atheromatous lesions in humans.     

Defective catabolism of low-density lipoprotein by fibroblasts from patients with I-cell disease.

Skin fibroblast cultures from patients with I-cell disease (mucolipidosis II) are characterized by multiple lysosomal enzyme deficiencies The present studies deal with the consequences of these deficiencies with respect to the metabolism of plasma low-density lipoproteins. Degradation of the protein moiety was defective in I-cells compared with control cells, but the binding and internalization of low density lipoprotein were much less affected. Measurements of low-density lipoprotein degradation in homogenates demonstrated directly for the first time a deficiency of acid proteinase activity in I-cell fibroblasts. Comparison of results in 6-h incubations with those in 24-h incubations showed accumulation of intracellular low-density lipoprotein in I-cell fibroblasts and an accelerating rate of degradation, possibly attributable to intracellular accumulation of low-density lipoprotein substrate. The significance of these findings with respect to low-density lipoprotein metabolism in vivo is discussed.     

Sulfhydryl-selective fluorescence labeling of lipoprotein(a) reveals evidence for one single disulfide linkage between apoproteins(a) and B-100.

Human lipoprotein(a) and low-density lipoprotein were labeled with two different sulfhydryl-selective fluorescence markers. The hydrophilic fluorophore lucifer yellow iodoacetamide and the apolar compound 6-acryloyl-2-(dimethylamino)naphthalene were used to derivatize free -SH groups in the lipoproteins. Three sulfhydryls could be detected in low-density lipoprotein, whereas only two cysteines were available in lipoprotein(a). One of the three -SH groups in low-density lipoprotein was shown to be located in close proximity to the particle surface. We suggest that this surface-exposed cysteine of apoprotein B-100 serves as a component for the disulfide linkage to apoprotein(a) in lipoprotein(a).     

Effects of cytochalasin B on low-density lipoproteins metabolism by cultured human fibroblasts.

The role of cytoplasmic microfilaments in the metabolism of low-density lipoprotein by human fibroblasts was studied with the aid of cytochalasin B. At concentrations of 5--40 nmol/ml cytochalasin increased the surface binding but decreased the endocytosis of 125I-labelled low-density lipoprotein. Subsequent studies indicated that these changes reflected a reduction of the rate of internalisation of low-density lipoprotein receptors. Independent inhibitory effects were also observed on low-density lipoprotein degradation and on the cellular release of the trichloroacetic acid-soluble degradation products.     

Cell density dependent uptake of LDL in cultured rat hepatocytes

An inverse relationship between low-density lipoprotein uptake and cell density was observed in rat hepatocyte monolayers incubated with lipoprotein-deficient serum. This was also true for cell association, binding and degradation of low-density lipoproteins. Compactin stimulated cell association and degradation of low-density lipoproteins both at low and high concentrations. Insulin, on the other hand, had no consistent effect on low-density lipoprotein cell association or degradation.     

No evidence of interaction between known lipid-associated genetic variants and smoking in the multi-ethnic PAGE population

Genome-wide association studies (GWAS) have identified many variants that influence high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and/or triglycerides. However, environmental modifiers, such as smoking, of these known genotype–phenotype associations are just recently emerging in the literature. We have tested for interactions between smoking and 49 GWAS-identified variants in over 41,000 racially/ethnically diverse samples with lipid levels from the Population Architecture Using Genomics and Epidemiology (PAGE) study. Despite their biological plausibility, we were unable to detect significant SNP × smoking interactions.     

杨梅素对高脂喂养小鼠代谢及自发活动节律的影响研究

摘要 目的：探讨杨梅素对高脂喂养小鼠代谢情况及自发活动节律的影响。方法：6周龄清洁级C57BL/6雄性小鼠15只,随机分为普通饲料组（CON）、高脂饲料组（HFD）、高脂饲料+杨梅素组100 mg /（kg?d）组（HFD+MYR）。从干预第10周开始使用Clocklab生物节律采集分析系统记录三组小鼠自发活动数据。干预第13周结束,检测三组小鼠体重、血脂数据。结果：与CON组相比,HFD组体重、甘油三酯、总胆固醇、低密度脂蛋白均显著升高（P<0.001）,高密度脂蛋白显著降低（P<0.001）,活动峰值时相（Activity phase）显著后移（P<0.001）,自发活动量中值（Activity mesor）和总自发活动量（Total counts）明显增加（P<0.05）,HFD+MYR组体重和低密度脂蛋白无明显变化（P>0.05）,甘油三酯、总胆固醇均显著升高（P< 0.01）,高密度脂蛋白显著降低（P<0.01）,活动峰值时相显著后移（P<0.001）,自发活动量中值和总自发活动量无明显变化（P>0.05）。与HFD组相比,HFD+MYR组体重、甘油三酯、总胆固醇、低密度脂蛋白明显降低（P<0.05）,高密度脂蛋白明显升高（P<0.05）,活动峰值时相明显前移（P<0.05）,自发活动量中值和总自发活动量明显减少（P<0.05）。结论：杨梅素可改善高脂喂养小鼠的代谢状态及减轻小鼠自发活动节律紊乱。     

Rapid transformation of [3H]cholesteryl ester in rat high-density lipoprotein: in vivo and in vitro studies

[24,25-3H]Cholesteryl ester-labeled rat high-density and low-density lipoproteins were administered to recipient rats. Following death of the rats, a major portion of the radioactivity in administered [3H]cholesteryl ester-high-density lipoprotein rapidly appeared in less dense [3H]cholesteryl ester-lipoproteins and was isolated with the low-density lipoprotein fraction. The specific activity of the esterified cholesterol in the product lipoproteins found with the low-density lipoproteins exceeded that of the precursor high-density lipoproteins. In vitro, the addition of [3H]cholesteryl ester-high-density lipoprotein to plasma resulted in a five- to six-fold increase in radioactivity recovered in the low-density lipoprotein. These results demonstrate that, under a variety of experimental conditions, isolated high-density lipoprotein particles (both in vitro and in vivo) tend to become larger and less dense. Rapid changes in the density of lipoproteins labeled with [3H]cholesteryl ester must be considered when interpreting physiologic studies using this label.