Low density lipoprotein (LDL), atherosclerosis and antioxidants

A crucial and causative role in the pathogenesis of atherosclerosis is believed to be the oxidative modification of low density lipoprotein (LDL). The oxidation of LDL involves released free radical driven lipid peroxidation. Several lines of evidence support the role of oxidized LDL in atherogenesis. Epidemiologic studies have demonstrated an association between an increased intake of dietary antioxidant vitamins, such as vitamin E and vitamin C and reduced morbidity and mortality from coronary artery diseases. It is thus hypothesized that dietary antioxidants may help prevent the development and progression of atherosclerosis. The oxidation of LDL has been shown to be reduced by antioxidants, and, in animal models, improved antioxidants may offer possibilities for the prevention of atherosclerosis. The results of several on going long randomized intervention trials will provide valuahle information on the efficacy and safety of improved antioxidants in the prevention of atherosclerosis. This review a evaluates current literature involving antioxidants and vascular disease, with a particular focus on the potential mechanisms.     

Structure of low density lipoprotein in complexes formed with arterial matrix components

Temperature dependent techniques (differential scanning calorimetry, polarizing light microscopy and n.m.r. spectroscopy) were used to study the physico-chemical state of low density lipoprotein (LDL) in LDL-aorta glycosaminoglycan and LDL aorta proteoglycan complexes. Complex formation between LDL and glycosaminoglycans (GAGs) resulted in a reversible liquid-liquid crystalline reorganization of the core lipids within the LDL molecule. In the proteoglycan-LDL complexes, prepared by the addition of porcine arterial proteoglycans or of human ‘lipoprotein complexing proteoglycan’, the formation of liquid crystals was an irreversible process, suggesting that the protein moiety of the proteoglycans also participates in the interaction with LDL. Arterial specimens were also examined. In atheromatous intima samples, spherulites with the above characteristics could be identified at the sites of lipoprotein deposition. Since proteoglycans are present in the arterial intima media, the phenomena observed in the present model system may also take place in vivo. The formation of proteoglycan-LDL complexes may result in the establishment of a liquid crystalline LDL structure and may thus play a role in the immobilization of LDL and in the development of the atherosclerotic lesion.     

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

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.     

The fatty acid distribution in low density lipoprotein in diabetes

Atherosclerosis is commonly found in diabetes. There is an association between small dense low density lipoprotein (LDL) phenotype, which is more prevalent in the diabetic state, and atherosclerosis. Small dense LDL is more easily oxidised and it is possible that fatty acid compositional changes, particularly an increase in polyunsaturated fatty acids, could underlie this association. However, there is little information about fatty acids in the different LDL phenotypes in the literature. This study examined LDL subfraction composition in 18 non-insulin-dependent diabetic (NIDDM) patients and 11 control subjects. LDL was isolated and fractionated into LDL 1, 2 and 3 by density gradient ultracentrifugation. NIDDM patients had significantly more fatty acids in all LDL subfractions than control subjects (P<0.01). Palmitic and linoleic acid were significantly greater in all subfractions in the diabetic patients compared to control subjects (P<0.01) and palmitoleic and oleic acids were also greater in LDL1 and LDL2 in diabetic patients (P<0.01). We conclude that in NIDDM fatty acids are increased in all LDL subfractions and this may be the reason for the increased atherosclerosis in diabetes irrespective of phenotype.     

Comparative studies of the physical state of the lipid phase of normal and hypercholesterolemic very low density lipoprotein.

Very low density lipoproteins (VLDL) were prepared from the serum of rabbits at various stages of hypercholesterolemia (95--1665 mg cholesterol/100 ml of serum). The most notable chemical change in hypercholesterolemic (hc) VLDL was the greatly increased content of cholesteryl esters and the greatly decreased content of triglycerides, compared to normal (n) VLDL. Structurally, the lipid region of n VLDL possessed a much lower microviscosity than did hc VLDL, when analyzed by fluorescence polarization and pyrene eximer methods. The microviscosity of the redispersed n VLDL lipid extract was considerably greater than the observed in n VLDL; but less than that of hc VLDL. Incorporation of pyrene into the lipid region of n VLDL and hc VLDL allowed assessment of various properties of the surface and hydrocarbon regions of these lipoproteins. Only slight differences were found in the pyrene monomer 3 : 1 fluorescence emission peak ratios, and in the rate constant for quenching of pyrene by O2. However, the quenching rate constant of pyrene by I- and iodoheptane were different for each lipoprotein.     

Excessive centrifugal fields damage high density lipoprotein [S]

HDL is typically isolated ultracentrifugally at 40,000 rpm or greater, however, such high centrifugal forces are responsible for altering the recovered HDL particle. We demonstrate that this damage to HDL begins at approximately 30,000 rpm and the magnitude of loss increases in a rotor speed-dependent manner. The HDL is affected by elevated ultracentrifugal fields resulting in a lower particle density due to the shedding of associated proteins. To circumvent the alteration of the recovered HDL, we utilize a KBr-containing density gradient and a lowered rotor speed of 15,000 rpm to separate the lipoproteins using a single 96 h centrifugation step. This recovers the HDL at two density ranges; the bulk of the material has a density of about 1.115 g/ml, while lessor amounts of material are recovered at >1.2 g/ml. Thus, demonstrating the isolation of intact HDL is possible utilizing lower centrifuge rotor speeds.     

氧化性低密度脂蛋白与细胞自噬

氧化性低密度脂蛋白(oxygenized low density lipoprotein,oxLDL)水平的升高不仅引发动脉粥样硬化,还与癌症等疾病的发生有密切关系。研究发现,高水平的oxLDL在引发细胞凋亡的同时,也诱导多种细胞自噬。本文归纳了oxLDL与血管内皮细胞、乳腺上皮细胞、结肠癌细胞、颗粒细胞和神经细胞自噬关系的最新研究进展。     

HDL is redundant for adrenal steroidogenesis in LDLR knockout mice with a human-like lipoprotein profile

The contribution of HDL to adrenal steroidogenesis appears to be different between mice and humans. In the current study, we tested the hypothesis that a difference in lipoprotein profile may be the underlying cause. Hereto, we determined the impact of HDL deficiency on the adrenal glucocorticoid output in genetically modified mice with a human-like lipoprotein profile. Genetic deletion of APOA1 in LDL receptor (LDLR) knockout mice was associated with HDL deficiency and a parallel increase in the level of cholesterol associated with nonHDL fractions. Despite a compensatory increase in the adrenal relative mRNA expression levels of the cholesterol synthesis gene, HMG-CoA reductase, adrenals from APOA1/LDLR double knockout mice were severely depleted of neutral lipids, as compared with those of control LDLR knockout mice. However, basal corticosterone levels and the adrenal glucocorticoid response to stress were not different between the two types of mice. In conclusion, we have shown that HDL is not critical for proper adrenal glucocorticoid function when mice are provided with a human-like lipoprotein profile. Our findings provide the first experimental evidence that APOB-containing lipoproteins may facilitate adrenal steroidogenesis, in an LDLR-independent manner, in vivo in mice.     

A comparison of anion-exchange and steric-exclusion HPLC assays of mouse plasma lipoproteins

We compared two HPLC methods (anion exchange [AE] and steric exclusion [SE]) for analysis of mouse lipoprotein profiles by determining coefficients of variability (CVs) under varying conditions. CVs for AE and SE were comparable on fresh samples. There was an inverse relationship between subfraction curve area and CV [r = -0.65 (AE) and -0.50 (SE)], consistent with the interpretation that as curve area decreased, error variance increased and signal-to-noise ratio decreased. Sample storage did not affect SE. In contrast, with AE, alterations in measured lipoproteins were apparent after storage, including a decrease in the HDL subfraction [66.8% (baseline) vs. 15.9% (1 week); P < 0.01] and an increase in areas under LDL and VLDL peaks. Concomitant with decreasing HDL area, reproducibility deteriorated with the duration of storage. Analysis of the effects of decreasing sample injectate volume to <25 microl on SE lipoprotein subfractions revealed that areas under LDL and VLDL peaks decreased and persisted as volume was decreased further. Areas under all lipoprotein subfractions measured with either AE or SE were linearly correlated with the amount of cholesterol [r = 0.69 (AE) and 0.87 (SE)]. Both AE and SE yield reproducible, accurate, and rapid measurements of lipoproteins from small amounts of serum. AE yields more sensitive, high-amplitude, well-defined peaks that can be easily distinguished and necessitates the use of smaller sample volumes compared with SE, but sample storage causes alterations in the chromatogram. SE appears better suited to serial analyses involving stored samples.     

Quantitative trait loci influencing low density lipoprotein particle size in African Americans

Genomic regions that influence LDL particle size in African Americans are not known. We performed family-based linkage analyses to identify genomic regions that influence LDL particle size and also exert pleiotropic effects on two closely related lipid traits, high density lipoprotein cholesterol (HDL-C) and triglycerides, in African Americans. Subjects (n = 1,318, 63.0 +/- 9.5 years, 70% women, 79% hypertensive) were ascertained through sibships with two or more individuals diagnosed with essential hypertension before age 60. LDL particle size was measured by polyacrylamide gel electrophoresis, and triglyceride levels were log-transformed to reduce skewness. Genotypes were measured at 366 microsatellite marker loci distributed across the 22 autosomes. Univariate and bivariate linkage analyses were performed using a variance components approach. LDL particle size was highly heritable (h(2) = 0.78) and significantly (P < 0.0001) genetically correlated with HDL-C (rho(G) = 0.32) and log triglycerides (rho(G) = -0.43). Significant evidence of linkage for LDL particle size was present on chromosome 19 [85.3 centimorgan (cM), log of the odds (LOD) = 3.07, P = 0.0001], and suggestive evidence of linkage was present on chromosome 12 (90.8 cM, LOD = 2.02, P = 0.0011). Bivariate linkage analyses revealed tentative evidence for a region with pleiotropic effects on LDL particle size and HDL-C on chromosome 4 (52.9 cM, LOD = 2.06, P = 0.0069). These genomic regions may contain genes that influence interindividual variation in LDL particle size and potentially coronary heart disease susceptibility in African Americans.     

Low density lipoprotein stimulation of human macrophage proteoglycan secretion

Lipoprotein trapping in arterial intima increases the risk for lipoprotein oxidation, foam cell formation, and inflammatory response in surrounding cells. Modified lipoproteins increase smooth muscle cell production of proteoglycans likely to retain lipoproteins in intimal extracellular matrix. We hypothesized that macrophage proteoglycan production is regulated in a similar manner, and characterized glycosaminoglycan side chains of secreted proteoglycans. Incubation with native low density lipoproteins (LDL) strongly stimulates total proteoglycan secretion in a time and concentration dependent manner. The main secretion product is small-sized (120 kDa) with unusually long galactosaminoglycan chains, predominantly chondroitin-6-O-sulfated. The effect appears specific for native LDL; oxidized LDL, very low density lipoproteins or phospholipid liposomes have only minor effects compared to control. These observations suggest that native LDL stimulate macrophages to secrete a chondroitin sulfate-rich proteoglycan moiety likely to have high capacity for vascular extracellular trapping of apolipoprotein B-containing lipoproteins.     

Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has gained attention as a key regulator of serum low density lipoprotein cholesterol (LDL-C) levels. This novel protease causes the degradation of hepatic low density lipoprotein receptors. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDL-C levels and cardiovascular risk. Previous studies have demonstrated that statins upregulate PCSK9 mRNA expression in cultured cells and animal models. In light of these observations, we studied the effect of atorvastatin on circulating PCSK9 protein levels in humans using a sandwich ELISA to quantitate serum PCSK9 levels in patients treated with atorvastatin or placebo for 16 weeks. We observed that atorvastatin (40 mg/day) significantly increased circulating PCSK9 levels by 34% compared with baseline and placebo and decreased LDL-C levels by 42%. These results suggest that the addition of a PCSK9 inhibitor to statin therapy may result in even further LDL-C decreases.     

Interaction of very low density lipoprotein with chicken oocyte membranes

The interaction of hen 125I-VLDL (very low density lipoprotein) with chicken oocyte membranes was characterized using a rapid sedimentation assay. Equilibrium and kinetic studies showed an apparent dissociation constant (Kd) 8.7-9.1 x 10(-8) M or 43.5-45.5 micrograms VLDL protein/ml. Binding capacity was 2.0 micrograms VLDL protein/mg membrane homogenate protein. The apparent rate constants were k1 = 2.4 x 10(5) M-1 min-1 and k2 = 2.1 x 10(-2) min-1. Specific binding required the presence of divalent cations. Whereas binding was completely restored after treatment with EDTA by the addition of MN++, only 60% of binding was restored using Ca++.     

Tanshinone II-A inhibits low density lipoprotein oxidation in vitro

Tanshinone II-A (TSII-A) is a major component of Salvia miltorrhiza Bunge which has long been used for preventing and ameliorating anginal pain in China. However the effect of TSII-A on low density lipoprotein (LDL) oxidation has not been studied. The present study was performed to investigate the effects of TSII-A on LDL oxidation using four oxidizing systems, including copper-, peroxyl radical- and peroxynitriteinitiated and macrophage-mediated LDL oxidation. LDL oxidation was measured in terms of formation of thiobarbituric acid-reactive substances (TBARS), relative electrophoretic mobility (REM) on agarose gel and lag time. In all four systems, TSII-A has apparent antioxidative effects against LDL oxidation, as evidenced by its dose-dependent inhibition of TBARS formation, prolongation of lag time and suppression of increased REM.

Regarding the mechanism underlying its antioxidative effect, TSII-A neither scavenged superoxide nor peroxynitrite. It also did not chelate copper. But it has mild peroxyl radical scavenging activity. The direct binding to LDL particles and conformational change of LDL structure by TSII-A were suggested, because it increased negative charge of LDL which was shown by increased REM on agarose gel. In conclusion, TSII-A is an effective antioxidant against LDL oxidation in vitro. The underlying mechanism appears to be related to its peroxyl radical scavenging and LDL binding activity.     

Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR-/-) mice. Collectively, although VLDLR deficiency in LRP- and LDLR-/- mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.