Selenoprotein P protects low-density lipoprotein against oxidation

Selenoprotein P (SeP) is an extracellular glycoprotein with 8-10 selenocysteines per molecule, containing approximately 50% of total selenium in human serum. An antioxidant function of SeP has been postulated. In the present study, we show that SeP protects low-density lipoproteins (LDL) against oxidation in a cell-free in-vitro system. LDL were isolated from human blood plasma and oxidized with CuCl₂, 2,2'-azobis(2-amidinopropane) (AAPH) or peroxynitrite in the presence or absence of SeP, using the formation of conjugated dienes as parameter for lipid peroxidation. SeP delayed the CuCl₂- and AAPH-induced LDL oxidation significantly and more efficiently than bovine serum albumin used as control. In contrast, SeP was not capable of inhibiting peroxynitrite-induced LDL oxidation. The protection of LDL against CuCl₂- and AAPH-induced oxidation provides evidence for the antioxidant capacity of SeP. Because SeP associates with endothelial membranes, it may act in vivo as a protective factor inhibiting the oxidation of LDL by reactive oxygen species.     

Vitamin C protects low-density lipoprotein from homocysteine-mediated oxidation

Homocysteine, an atherogenic amino acid, promotes iron-dependent oxidation of low-density lipoprotein (LDL). We investigated whether vitamin C, a physiological antioxidant, could protect LDL from homocysteine-mediated oxidation. LDL (0.2 mg of protein/ml) was incubated at 37 degrees C with homocysteine (1000 microM) and ferric iron (10-100 microM) in either the absence (control) or presence of vitamin C (5-250 microM). Under these conditions, vitamin C protected LDL from oxidation as evidenced by an increased lag time preceding lipid diene formation (> or = 5 vs. 2.5 h for control), decreased thiobarbituric acid-reactive substances accumulation (< or = 19 +/- 1 nmol/mg when vitamin C > or = 10 microM vs. 32 +/- 3 nmol/mg for control, p <.01), and decreased lipoprotein anodic electrophoretic mobility. Near-maximal protection was observed at vitamin C concentrations similar to those in human blood (50-100 microM); also, some protection was observed even at low concentrations (5-10 microM). This effect resulted neither from altered iron redox chemistry nor enhanced recycling of vitamin E in LDL. Instead, similar to previous reports for copper-dependent LDL oxidation, we found that vitamin C protected LDL from homocysteine-mediated oxidation through covalent lipoprotein modification involving dehydroascorbic acid. Protection of LDL from homocysteine-mediated oxidation by vitamin C may have implications for the prevention of cardiovascular disease.     

Characterization of low-density lipoprotein uptake by murine macrophages exposed to Chlamydia pneumoniae

Exposure to Chlamydia pneumoniae is correlated with atherosclerosis in a variety of clinical and epidemiological studies, but how the organism may initiate and promote the disease is poorly understood. One pathogenic mechanism could involve modulation of macrophage function by C. pneumoniae. We recently demonstrated that C. pneumoniae induces macrophages to accumulate excess cholesterol and develop into foam cells, the hallmark of early atherosclerotic lesions. To determine if C. pneumoniae-induced foam cell formation involved increased uptake of low-density lipoprotein (LDL), the current study examined macrophage association of a fluorescent carbocyanine (DiI)-labeled LDL following infection. C. pneumoniae enhanced the association of DiI-LDL with macrophages in a dose-dependent manner with respect to both C. pneumoniae and DiI-LDL. Interestingly, increased association was inhibited by native LDL and occurred in the absence of oxidation byproducts and in the presence of antioxidants. However, enhanced DiI-LDL association occurred without the participation of the classical Apo B/E native LDL receptor, since C. pneumoniae increased DiI-LDL association and induced foam cell formation in macrophages isolated from LDL-receptor-deficient mice. Surprisingly, DiI-LDL association was inhibited not only by unlabeled native LDL but also by high-density lipoprotein, very low density lipoprotein, and oxidized LDL. These data indicate that exposure of macrophages to C. pneumoniae increases the uptake of LDL and foam cell formation by an LDL-receptor-independent mechanism.     

Acetylated low-density lipoprotein is endocytosed through coated pits by rat peritoneal macrophages

The surface distribution of the scavenger receptors for acetylated low-density lipoprotein (acetyl-LDL) and their endocytic behavior were studied by the direct immunoperoxidase method using monomeric conjugates of horseradish peroxidase with Fab' antibody raised against LDL. The receptors were demonstrated to be distributed diffusely on the surface membrane of cultured peritoneal macrophages, with preferential localization in coated pit regions. With temperature shift from 4 degrees C to 37 degrees C, acetyl-LDL bound to the surface membrane rapidly disappeared, but became detectable in coated vesicles or lysosomes. Further incubation in the presence of acetyl-LDL revealed lipid vacuoles devoid of a limiting membrane in the cytoplasm, transforming macrophages into typical foam cells. These data suggest that the binding of acetyl-LDL to its receptors triggers the clustering of the receptors into the coated pit regions through which acetyl-LDL is endocytosed by coated vesicles to be degraded in lysosomes with subsequent intracellular accumulation of cholesterol esters.     

Electronegative low-density lipoprotein

PURPOSE OF REVIEW: The occurrence in blood of an electronegatively charged LDL was described in 1988. During the 1990s reports studying electronegative LDL (LDL(-)) were scant and its atherogenic role controversial. Nevertheless, recent reports have provided new evidence on a putative atherogenic role of LDL(-). This review focuses on and discusses these new findings. RECENT FINDINGS: In recent years, LDL(-) has been found to be involved in several atherogenic features through its action on cultured endothelial cells. LDL(-) induces the production of chemokines, such as IL-8 and monocyte chemotactic protein 1, and increases tumor necrosis factor-alpha-induced production of vascular cell adhesion molecule 1, with these molecules being involved in early phases of leukocyte recruitment. LDL(-) from familial hypercholesterolemic patients also decreases DNA synthesis and intracellular fibroblast growth factor 2 production, which may contribute to impaired angiogenesis and increased apoptosis. In addition, the preferential association of platelet-activating factor acetylhydrolase with LDL(-) has been reported, suggesting a proinflammatory role of this enzyme in LDL(-). SUMMARY: Recent findings suggest that LDL(-) could contribute to atherogenesis via several mechanisms, including proinflammatory, proapoptotic and anti-angiogenesis properties. Further studies are required to define the role of LDL(-) in atherogenesis more precisely and to clarify mechanisms involved in endothelial cell activation.     

Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages

The effects of low-density lipoprotein (LDL) and chylomicron remnants on lipid accumulation in human monocyte-derived macrophages (HMDMs) and in macrophages derived from the human monocyte cell line THP-1 were compared. The HMDMs or THP-1 macrophages were incubated with LDL, oxidized LDL (oxLDL), chylomicron remnant-like particles (CMR-LPs), or oxidized CMR-LPs (oxCMR-LPs), and the amount and type of lipid accumulated were determined. As expected, the lipid content of both cell types was increased markedly by oxLDL but not LDL, and this was due to a rise in cholesterol, cholesteryl ester (CE), and triacylglycerol (TG) levels. In contrast, both CMR-LPs and oxCMR-LPs caused a considerable increase in cellular lipid in HMDMs and THP-1 macrophages, but in this case there was a greater rise in the TG than in the cholesterol or CE content. Lipid accumulation in response to oxLDL, CMR-LPs, and oxCMR-LPs was prevented by the ACAT inhibitor CI976 in HMDMs but not in THP-1 macrophages, where TG levels remained markedly elevated. The rate of incorporation of [(3)H]oleate into CE and TG in THP-1 macrophages was increased by oxLDL, CMR-LPs, and oxCMR-LPs, but incorporation into TG was increased to a greater extent with CMR-LPs and oxCMR-LPs compared with oxLDL. These results demonstrate that both CMR-LPs and oxCMR-LPs cause lipid accumulation in human macrophages comparable to that seen with oxLDL and that oxidation of the remnant particles does not enhance this effect. They also demonstrate that a greater proportion of the lipid accumulated in response to CMR-LPs compared with oxLDL is TG rather than cholesterol or CE and that this is associated with a higher rate of TG synthesis. This study, therefore, provides further evidence to suggest that chylomicron remnants have a role in foam cell formation that is distinct from that of oxLDL.     

Incubation of acetylated low-density lipoprotein with cholesterol-rich dispersions enhances cholesterol uptake by macrophages

Incubation of J774 macrophages with mixtures of acetylated low-density lipoprotein (acLDL) and free cholesterol-rich phospholipid dispersions increases cellular cholesterol deposition 2-4-fold over that achieved with either acLDL or dispersions alone. Both free and esterified cholesterol accumulate in cells incubated with the mixture of acLDL and dispersions. A similar result is observed when acLDL is replaced by malondialdehyde-LDL. The enhanced deposition of cholesterol is not unique to J774 macrophages, as P388D1 macrophages also accumulate more cholesterol when incubated with the mixture of acLDL and dispersions than either particle alone. A preincubation of the particles for at least 6 h prior to incubation with cells is required in order to observe maximal cholesterol delivery. Both dispersion free cholesterol and phospholipid accumulate in J774 cells, suggesting that a complex is formed between acLDL and dispersions which results in a cholesterol-rich acLDL/dispersion particle. Partial purification of the acLDL-dispersion complex revealed increases in the size distribution of the particles compared to acLDL and increases in free cholesterol and phospholipid contents. Cholesterol uptake from the mixture of acLDL and dispersions was saturable and the enhanced cellular uptake of both cholesterol and phospholipid from the complex could be abolished by inhibitors of the scavenger receptor pathway. In addition to the receptor-mediated uptake of cholesterol from the acLDL-dispersion complex, it was observed that approx. 30% of the total cholesterol uptake from the complex was via non-specific components, including surface transfer.     

Lysophosphatidic acid-induced oxidized low-density lipoprotein uptake is class A scavenger receptor-dependent in macrophages

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoprotein (OxLDL). It is suggested that LPA is involved in atherosclerosis, and our previous studies showed that LPA regulates inflammation in multiple cell types. The main aim of this study was to investigate the effects of LPA on the uptake of OxLDL by mouse J774A.1 macrophages. We observed that LPA upregulated fluorescence-labeled DiI-OxLDL uptake in J774A.1 cells. Meanwhile, expression of the class A scavenger receptor (SR-A), a receptor for modified LDL, was also enhanced. Furthermore, pertussis toxin (PTx) or Ki16425 significantly abolished LPA's effects, indicating that G(i) and LPA(3) are involved in OxLDL uptake and SR-A expression. Of most importance, the LPA-induced OxLDL uptake could be inhibited when cells were incubated with a functional blocking antibody of SR-A. Our results suggest that LPA-enhanced OxLDL uptake is mediated via LPA(3)-G(i) activation and subsequent SR-A expression.     

Lysophosphatidic acid-induced oxidized low-density lipoprotein uptake is class A scavenger receptor-dependent in macrophages

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoprotein (OxLDL). It is suggested that LPA is involved in atherosclerosis, and our previous studies showed that LPA regulates inflammation in multiple cell types. The main aim of this study was to investigate the effects of LPA on the uptake of OxLDL by mouse J774A.1 macrophages. We observed that LPA upregulated fluorescence-labeled DiI-OxLDL uptake in J774A.1 cells. Meanwhile, expression of the class A scavenger receptor (SR-A), a receptor for modified LDL, was also enhanced. Furthermore, pertussis toxin (PTx) or Ki16425 significantly abolished LPA's effects, indicating that G_i and LPA₃ are involved in OxLDL uptake and SR-A expression. Of most importance, the LPA-induced OxLDL uptake could be inhibited when cells were incubated with a functional blocking antibody of SR-A. Our results suggest that LPA-enhanced OxLDL uptake is mediated via LPA₃-G_i activation and subsequent SR-A expression.     

C-reactive protein-bound enzymatically modified low-density lipoprotein does not transform macrophages into foam cells

The formation of low-density lipoprotein (LDL) cholesterol-loaded macrophage foam cells contributes to the development of atherosclerosis. C-reactive protein (CRP) binds to atherogenic forms of LDL, but the role of CRP in foam cell formation is unclear. In this study, we first explored the binding site on CRP for enzymatically modified LDL (E-LDL), a model of atherogenic LDL to which CRP binds. As reported previously, phosphocholine (PCh) inhibited CRP-E-LDL interaction, indicating the involvement of the PCh-binding site of CRP in binding to E-LDL. However, the amino acids Phe66 and Glu81 in CRP that participate in CRP-PCh interaction were not required for CRP-E-LDL interaction. Surprisingly, blocking of the PCh-binding site with phosphoethanolamine (PEt) dramatically increased the binding of CRP to E-LDL. The PEt-mediated enhancement in the binding of CRP to E-LDL was selective for E-LDL because PEt inhibited the binding of CRP to another PCh-binding site-ligand pneumococcal C-polysaccharide. Next, we investigated foam cell formation by CRP-bound E-LDL. We found that, unlike free E-LDL, CRP-bound E-LDL was inactive because it did not transform macrophages into foam cells. The function of CRP in eliminating the activity of E-LDL to form foam cells was not impaired by the presence of PEt. Combined data lead us to two conclusions. First, PEt is a useful compound because it potentiates the binding of CRP to E-LDL and, therefore, increases the efficiency of CRP to prevent transformation of macrophages into E-LDL-loaded foam cells. Second, the function of CRP to prevent formation of foam cells may influence the process of atherogenesis.     

Microchip-based human serum atherogenic lipoprotein profile analysis

Owing to the mounting evidence of serum lipid changes in atherosclerosis, there has been increasing interest in developing new methods for analyzing atherogenic lipoprotein profiles. The separation of lipoprotein and lipoprotein subclasses has been demonstrated using a microchip capillary electrophoresis (CE) system [Chromatographia 74 (2011) 799–805]. In contrast to this previous study, the current report demonstrates that sdLDL peak efficiencies can be improved dramatically by adding gold nanoparticles (AuNPs) to the sample. Moreover, NBD C₆-ceramide was identified as a satisfactory dye for specific labeling and quantitation of individual serum lipoproteins. The accuracy of the method was evaluated by comparison with ultracentrifuge separated small, dense, low-density lipoprotein (sdLDL). A high correlation was observed between these two methods for sdLDL cholesterol. Lipid levels were investigated between atherosclerotic patients and healthy controls. The variation of serum atherogenic lipoprotein profiles for atherosclerotic patients pre- and post-treatment was assessed by microchip CE. This method has potential for the rapid and sensitive detection of different lipoprotein classes as well as their subclasses and, therefore, is suitable for routine clinical applications. Microchip-based atherogenic lipoprotein profile assays will greatly improve the analysis of risk factors in atherosclerosis and will provide useful information for monitoring the effect of therapies on atherosclerotic disease.     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.     

Chromium and human low-density lipoprotein oxidation

Chromium is a catalytic metal able to foster oxidant damage, albeit its capacity to induce human LDL oxidation is to date unkown. Thus, we have investigated whether trivalent and hexavalent chromium, namely Cr(III) and Cr(VI), can induce human LDL oxidation. Cr(III) as CrCl₃ is incapable of inducing LDL oxidation at pH 7.4 or 4.5. However, Cr(III), specifically at physiological pH of 7.4 and in the presence of phosphates, causes an absorbance increase at 234 resembling a spectrophotometric kinetics of LDL oxidation with a lag- and propagation-like phase. In this regard, it is conceivable that peculiar Cr(III) forms such as Cr(III) hydroxide and, especially, Cr(III) polynuclear hydroxocomplexes formed at pH 7.4 interact with phosphates generating species with an intrinsic absorbance at 234 nm, which increases over time resembling a spectrophotometric kinetics of LDL oxidation. Cr(VI), as K₂Cr₂O₇, can instead induce substantial human LDL oxidation at acidic pH such as 4.5, which is typical of the intracellular lysosomal compartment. LDL oxidation is related to binding of Cr(VI) to LDL particles with quenching of the LDL tryptophan fluorescence, and it is inhibited by the metal chelators EDTA and deferoxamine, as well as by the chain-breaking antioxidants butylated hydroxytoluene and probucol. Moreover, Cr(VI)-induced LDL oxidation is inhibited by mannitol conceivably by binding Cr(V) formed from LDL-dependent Cr(VI) reduction and not by scavenging hydroxyl radicals (OH); indeed, the OH scavengers sodium formate and ethanol are ineffective against Cr(VI)-induced LDL oxidation. Notably, heightened LDL lipid hydroperoxide levels and decreased LDL tryptophan fluorescence occur in Cr plating workers, indicating Cr-induced human LDL oxidation in vivo. The biochemical, pathophysiological and clinical implications of these novel findings on chromium and human LDL oxidation are discussed.     

Oral nicotine induces an atherogenic lipoprotein profile

Male squirrel monkeys were used to evaluate the effect of chronic oral nicotine intake on lipoprotein composition and metabolism. Eighteen yearling monkeys were divided into two groups: 1) Controls fed isocaloric liquid diet; and 2) Nicotine primates given liquid diet supplemented with nicotine at 6 mg/kg body wt/day. Animals were weighed biweekly, plasma lipid, glucose, and lipoprotein parameters were measured monthly, and detailed lipoprotein composition, along with postheparin plasma lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activity, was assessed after 24 months of treatment. Although nicotine had no effect on plasma triglyceride or high density lipoproteins (HDL), the alkaloid caused a significant increase in plasma glucose, cholesterol, and low density lipoprotein (LDL) cholesterol plus protein while simultaneously reducing the HDL cholesterol/plasma cholesterol ratio and animal body weight. Levels of LDL precursors, very low density (VLDL) and intermediate density (IDL) lipoproteins, were also lower in nicotine-treated primates while total postheparin lipase (LPL + HTGL) activity was significantly elevated. Our data indicate that long-term consumption of oral nicotine induces an atherogenic lipoprotein profile (increases LDL, decreases HDL/total cholesterol ratio) by enhancing lipolytic conversion of VLDL to LDL. These results have important health implications for humans who use smokeless tobacco products or chew nicotine gum for prolonged periods.     

Enhancement of oxidised low-density lipoprotein uptake by macrophages in response to macrophage migration inhibitory factor

We examined the expression of macrophage migration inhibitory factor (MIF) mRNA in murine macrophage cell line (RAW 264.7 cells) in response to oxidized low-density lipoprotein (oxLDL), and investigated the influence of MIF on the uptake and degradation of oxLDL by RAW 264.7 cells. MIF mRNA expression was markedly upregulated in the presence of oxLDL. Consistent with this, the MIF level of the culture medium was increased by stimulation with oxLDL in dose- and time-dependent manners. Next, we added recombinant rat MIF to the culture medium and examined its effects on the uptake of(125)I-labelled oxLDL. Pretreatment with MIF enhanced both the uptake and degradation of(125)I-oxLDL. Taken together, these results suggest that MIF released from macrophages in response to oxLDL stimulates oxLDL uptake and degradation in an autocrine and paracrine fashion, which potentially results in atherosclerosis.     

Measurement of receptor-independent metabolism of low-density lipoprotein. An application of glycosylated low-density lipoprotein

Incubation of human low-density lipoprotein (LDL) with glucose results in a nonenzymatic formation of a Schiff base between the monosaccharide and lysyl residues of apolipoprotein B. Increasing the percentage of lysyl residues of apolipoprotein B modified by glycosylation decreases the fractional catabolic rate of the glycosylated LDL, and decreases the metabolism of the glycosylated LDL by human skin fibroblasts. The glycosylated LDL, containing 20-40% of total lysyl residues of apoprotein B modified, was metabolized at a slow rate by both human skin fibroblasts and mouse peritoneal macrophages. These results led to the suggestion that glycosylated LDL is primarily catabolized via a receptor-independent process. Assuming LDL catabolism occurs via receptor-dependent and receptor-independent processes, the ratio of (fractional catabolic rate of glycosylated LDL)/(fractional catabolic rate of native LDL) should be an estimate of the percentage of LDL catabolism via the receptor-independent process. From the fractional catabolic rates of glucose-LDL (20-40% of lysyl residues modified) and galactose-LDL (30-60% of lysyl residues modified) 41% and 30% respectively, of LDL catabolism occurred by a receptor-independent process.     

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.     

Induced expression of adipophilin mRNA in human macrophages stimulated with oxidized low-density lipoprotein and in atherosclerotic lesions

The RNase activity of barnase mutants obtained by the permutation of modules or secondary structure units was investigated. Four of the 45 mutants had weak but distinct RNase activity, and they had unique optimum pHs and temperatures like natural enzymes. One of the active mutants had an ordered conformation, but the others did not. An active mutant having disordered conformation formed an ordered conformation in the presence of GMP, which is an inhibitor of this mutant. These results indicate that the amino acid sequences derived from barnase have sufficient plasticity to be rearranged into different proteins with basal enzymatic properties.