Endothelial Chlamydia pneumoniae infection promotes oxidation of LDL

The bacterium Chlamydia pneumoniae chronically infects atheromatous lesions and is linked to atherosclerosis by modifying inflammation, proliferation, and the lipid metabolism of blood monocytes. As continuous LDL modification in the vascular intima is crucial for atherogenesis we investigated the impact of endothelial infection on LDL oxidation. HUVEC were infected with a vascular C. pneumoniae strain. Supernatants of infected cells but not cell lysates increased lipid peroxidation products (6.44 vs 6.14 nmol/ml, p<0.05) as determined by thiobarbituric acid reacting substances assay. Moreover, supernatants rendered human LDL more susceptible to oxidation as shown in a copper-ion catalysed LDL oxidation assay by a 16% reduction of LDL resistance against pro-oxidative stimuli (p<0.05). Chlamydial infection of vascular endothelial cells releases acellular components that convert LDL to its proatherogenic form and reduce its resistance against oxidation. Foci of chronic endothelial chlamydial infection may thus continuously contribute to the dysregulated lipid metabolism that promotes atherogenesis.     

Implication of lipoprotein associated phospholipase A2 activity in oxLDL uptake by macrophages

Recognition and uptake of oxidized LDL (oxLDL) by scavenger receptors of macrophages and foam cell formation are mediated by the oxidatively modified apolipoprotein B (ApoB) and lipid moiety of oxLDL. A great amount of oxidized phosphatidylcholine (oxPC) of oxLDL is hydrolyzed at the sn-2 position by lipoprotein associated phospholipase A₂ (Lp-PLA₂) to lysophosphatidylcholine and small oxidation products. This study examines the involvement of Lp-PLA₂ in the uptake of oxLDL by mouse peritoneal macrophages. LDL with intact Lp-PLA₂ activity [LDL (+)] and LDL with completely inhibited Lp-PLA₂ activity [LDL (-)] were subjected to oxidation with 5 μM CuSO₄ for 6 h [moderately oxLDL (MoxLDL)], or 24 h [heavily oxLDL (HoxLDL)] and peritoneal macrophages were incubated with these preparations. The uptake of MoxLDL(-) was about 30% increased compared with that of MoxLDL(+), and HoxLDL(-) uptake was about 20% increased compared with that of HoxLDL(+). Inhibition of Lp-PLA₂ activity had no effect on the uptake of ApoB-liposomes conjugates with ApoB isolated from MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+). Liposomes prepared from the lipid extract of MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+) exhibited a similar pattern to that observed in the uptake of the corresponding intact lipoproteins. This study suggests that the progressive inactivation of Lp-PLA₂ during LDL oxidation leads to an increased uptake of oxLDL by macrophages, which could be primarily attributed to the increased uptake of the oxidized phospholipids enriched lipid moiety of oxLDL.     

Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products

Modification of low density lipoproteins (LDL) by oxidation has been shown to permit recognition by the acetyl-LDL receptor of macrophages. The extensive oxidation of LDL that is required before interaction occurs with this receptor produces major alterations in both the lipid and protein components of LDL. Several chemical modifications of LDL also lead to recognition by this receptor; all of these involve derivatization of lysine residues of apolipoprotein B by adducts that neutralize the positively charged epsilon-amino group. The present studies show that oxidation also results in derivatization of LDL lysine residues. Analysis of amino acid composition indicated that 32% of lysine residues were modified after oxidation of LDL by exposure to 5 microM CuSO4 for 20 h. About one-half of the derivatized lysines were labile under the conditions of acid hydrolysis. Fluorescence of LDL protein was greatly increased by oxidation, with excitation maximum at 350 nm and emission maximum at 433 nm. When LDL containing phosphatidylcholine with isotopically labeled arachidonic acid in the sn-2 position was oxidized, there was a 5-fold increase in radioactivity bound to protein compared to nonoxidized LDL or oxidized LDL labeled with 2-[1-14C]palmitoyl phosphatidylcholine. Prior methylation of LDL prevented the rapid uptake and degradation by macrophages that normally accompanies oxidation. These findings suggest that oxidation of LDL is accompanied by derivatization of lysine epsilon-amino groups by lipid products and that these adducts may be important in the interaction of oxidized LDL with the acetyl-LDL receptor.     

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu²⁺-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R^−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.     

Formation of monohydroxy derivatives of arachidonic acid, linoleic acid, and oleic acid during oxidation of low density lipoprotein by copper ions and endothelial cells.

An important event in the formation of atherosclerotic lesions is the uptake of modified low density lipoprotein (LDL) by macrophages via scavenger receptors. Modification of LDL, which results in its recognition by these receptors, can be initiated by peroxidation of LDL lipids. The first step in this process is the formation of monohydroperoxy derivatives of fatty acids, which are subsequently degraded to the corresponding monohydroxy compounds, or to a variety of secondary oxidation products. In order to understand this process more completely, we have developed a mass spectrometric procedure to measure the amounts of specific hydroperoxy/hydroxy fatty acids formed by oxidation of the major unsaturated fatty acids in human LDL, oleic acid, linoleic acid, and arachidonic acid. Oxidation of human LDL in the presence of a relatively strong stimulus (20 microM CuSO4) resulted in very large increases in the amounts of the major monohydroxy derivatives of linoleic acid (9- and 13-hydroxy derivatives) and arachidonic acid (5-, 8-, 9-, 11-, 12-, and 15-hydroxy derivatives) in LDL lipids in the early stages of the reaction. After 20 h, the amounts of these products declined due to substrate depletion, but large amounts of monohydroxy derivatives of oleic acid (8-, 10-, and 11-hydroxy derivatives) were detected. Although thiobarbituric acid-reactive substances clearly increased under these conditions, the changes were not nearly so dramatic as those observed for monohydroxy fatty acids. Oxidation of LDL in the presence of endothelial cells, a much milder stimulus, resulted in 2.5- to 3-fold increases in the amounts of monohydroxy derivatives of linoleic and arachidonic acids, as well as thiobarbituric acid-reactive substances, with more modest increases in the amounts of hydroxylated derivatives of oleic acid. There was little positional specificity in the oxidation of the above fatty acids in the presence of either stimulus, suggesting that the formation of these products proceeds primarily by lipid peroxidation, rather than by catalysis by lipoxygenases. However, an important role for lipoxygenases in the initiation of these reactions cannot be excluded. In conclusion, oxidation of LDL in the presence of copper ions or endothelial cells results in the formation of a large number of monohydroxy derivatives of oleic, linoleic, and arachidonic acids. The relative amounts of products formed from each of these fatty acids depends on the strength of the stimulus as well as the incubation time.     

Esterified lipid hydroperoxide-derived modification of protein: formation of a carboxyalkylamide-type lysine adduct in human atherosclerotic lesions

We have recently identified Nepsilon-azelayllysine (AZL) as a carboxyalkylamide-type novel lysine adduct in the reaction of linoleic acid hydroperoxides with the lysine derivative. To examine the formation of AZL in vivo, a novel monoclonal antibody (mAb19D5) specific to AZL moiety was prepared. The mAb19D5 scarcely recognized oxidized low-density lipoprotein (oxLDL), whereas the treatment of oxLDL with alkali or phospholipase A2 significantly increased the immunoreactivity. Similarly, the immunopositive materials were detected in alkali- or phospholipase A2-treated sections from human atherosclerotic aorta but not in untreated sections. These results suggest that esterified lipid hydroperoxide-derived modification of protein may serve as one mechanism for the oxidative modification of LDL and subsequent formation of atherosclerotic lesions in vivo.     

Inflammation-related gene expression by lipid oxidation-derived products in the progression of atherosclerosis

Vascular areas of atherosclerotic development persist in a state of inflammation, and any further inflammatory stimulus in the subintimal area elicits a proatherogenic response; this alters the behavior of the artery wall cells and recruits further inflammatory cells. In association with the inflammatory response, oxidative events are also involved in the development of atherosclerotic plaques. It is now unanimously recognized that lipid oxidation-derived products are key players in the initiation and progression of atherosclerotic lesions. Oxidized lipids, derived from oxidatively modified low-density lipoproteins (LDLs), which accumulate in the intima, strongly modulate inflammation-related gene expression, through involvement of various signaling pathways. In addition, considerable evidence supports a proatherogenic role of a large group of potent bioactive lipids called eicosanoids, which derive from oxidation of arachidonic acid, a component of membrane phospholipids. Of note, LDL lipid oxidation products might regulate eicosanoid production, modulating the enzymatic degradation of arachidonic acid by cyclooxygenases and lipoxygenases; these enzymes might also directly contribute to LDL oxidation. This review provides a comprehensive overview of current knowledge on signal transduction pathways and inflammatory gene expression, modulated by lipid oxidation-derived products, in the progression of atherosclerosis.     

Mapping oxidations of apolipoprotein B-100 in human low-density lipoprotein by liquid chromatography-tandem mass spectrometry

Human low-density lipoprotein (LDL) is a major cholesterol carrier in blood. Elevated concentration of low-density lipoprotein, especially when oxidized, is a risk factor for atherosclerosis and other cardiac inflammatory diseases. Past research has connected free radical initiated oxidations of LDL with the formation of atherosclerotic lesions and plaque in the arterial wall. The role of LDL protein in the associated diseases is still poorly understood, partially due to a lack of structural information. In this study, LDL was oxidized by hydroxyl radical. The oxidized protein was then delipidated and subjected to trypsin digestion. Peptides derived from trypsin digestion were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identification of modified peptide sequences was achieved by a database search against apo B-100 protein sequences using the SEQUEST algorithm. At different hydroxyl radical concentrations, oxidation products of tyrosine, tryptophan, phenylalanine, proline, and lysine were identified. Oxidized amino acid residues are likely located on the exterior of the LDL particle in contact with the aqueous environment or directly bound to the free radical permeable lipid layer. These modifications provided insight for understanding the native conformation of apo B-100 in LDL particles. The presence of some natural variants at the protein level was also confirmed in our study.     

A modification of apolipoprotein B accounts for most of the induction of macrophage growth by oxidized low density lipoprotein

It has recently been shown that macrophage proliferation occurs during the progression of atherosclerotic lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this include the interaction of oxidized LDL with integral plasma membrane proteins coupled to signaling pathways, the release of growth factors and autocrine activation of growth factor receptors, or the potentiation of mitogenic signal transduction by a component of oxidized LDL after internalization. The present study was undertaken to further elucidate the mechanisms involved in the growth-stimulating effect of oxidized LDL in macrophages. Only extensively oxidized LDL caused significant growth stimulation, whereas mildly oxidized LDL, native LDL, and acetyl LDL were ineffective. LDL that had been methylated before oxidation (to block lysine derivatization by oxidation products and thereby prevent the formation of a scavenger receptor ligand) did not promote growth, even though extensive lipid peroxidation had occurred. The growth stimulation could not be attributed to lysophosphatidylcholine (lyso-PC) because incubation of oxidized LDL with fatty acid-free bovine serum albumin resulted in a 97% decrease in lyso-PC content but only a 20% decrease in mitogenic activity. Similarly, treatment of acetyl LDL with phospholipase A2 converted more than 90% of the initial content of phosphatidylcholine (PC) to lyso-PC, but the phospholipase A2-treated acetyl LDL was nearly 10-fold less potent than oxidized LDL at stimulating growth. Platelet-activating factor receptor antagonists partly inhibited growth stimulation by oxidized LDL, but platelet-activating factor itself did not induce growth. Digestion of oxidized LDL with phospholipase A2 resulted in the hydrolysis of PC and oxidized PC but did not attenuate growth induction. Native LDL, treated with autoxidized arachidonic acid under conditions that caused extensive modification of lysine residues by lipid peroxidation products but did not result in oxidation of LDL lipids, was equal to oxidized LDL in potency at stimulating macrophage growth. Albumin modified by arachidonic acid peroxidation products also stimulated growth, demonstrating that LDL lipids are not essential for this effect. These findings suggest that oxidatively modified apolipoprotein B is the main growth-stimulating component of oxidized LDL, but that oxidized phospholipids may play a secondary role.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1, a key cardioprotective function of HDL

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine - two characteristic products of MPO - are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Cytotoxicity of lipid-free apolipoprotein B

To investigate the effect of apolipoprotein B (apoB) on cell viability, we used lipid-free apoB as a model for denatured apoB. Lipid-free apoB had cytotoxicity to J774 macrophages, CHO cells and HepG2 cells, whereas apoB bound to low density lipoprotein (LDL) and lipid-free apolipoprotein A-I had no effect on cell viability. Lipid-free apoB induced apoptosis in J774 macrophages assessed by caspase-3 activation and annexin V binding. LDL receptor, heparan sulfate proteoglycans, and class A scavenger receptor were involved in the binding/uptake of lipid-free apoB, but lipid-free apoB binding/uptake by the cells did not correlate with cytotoxicity. Lipid-free apoB disrupted the lipid bilayer of large unilamellar vesicles containing calcein. We evaluated the interaction between apoB and cellular membrane by monitoring the change in intracellular Ca²⁺ concentration using Fura-2, and found that lipid-free apoB rapidly disrupted the cellular membrane in the absence or presence of the inhibitors for cellular binding/uptake mediated by the receptors. Therefore, it is suggested that lipid-free apoB induces cell death by disturbance of the plasma membrane. In addition to other lipid component in modified LDL, apoB itself has an ability to induce apoptosis and plays a crucial role in the development of atherosclerotic lesions.     

Cell-mediated oxidation of LDL: Comparison of different cell types of the atherosclerotic lesion

The three major cell types of the human atherosclerotic lesion — macrophages (Mø), smooth muscle cells (SMC) and endothelial cells (EC) — were compared for their ability to oxidise low density lipoprotein (LDL) in vitro under identical conditions. Near-confluent cultures were incubated for up to 48 h with 50 μg protein/ml LDL in Ham's F10 medium supplemented with 7 μM Fe²⁺. All three cell types oxidised LDL readily using our culture conditions. After 24 and 48 h, the degree of LDL oxidation was in the order: Mø > SMC > EC when based on cell growth area and EC > SMC > Mø when based on cellular DNA content. However, LDL oxidation in vitro progressed more slowly between 24 and 48 h, probably due to increasing toxicity to the cells and/or depletion of polyunsaturated fatty acids. We therefore compared the time of onset of LDL oxidation. The earliest increase in LDL oxidation was always apparent with SMC. Gas chromatography revealed that LDL oxidation by all three cell types followed a similar pattern. The polyunsaturated fatty acids linoleic acid (18:2) and arachidonic acid (20:4) were depleted (to 10.3–18.1% and 4.5–24.7% respectively, compared to native LDL), whereas the content of stearic acid (18:0) and oleic acid (18:1) remained unchanged. Cholesterol was depleted (to 54.1–75.6% of native LDL) with a concomitant rise in 7β-hydroxycholesterol (to 60.6–128.1 μg/mg LDL). This corresponds to a conversion of 4.9, 9.5 and 10.4% of LDL cholesterol in EC-, SMC- and Mø-modified LDL respectively. All three cell types showed significant toxicity in the oxidising culture after 24 h. The possible relevance to LDL oxidation in atherosclerosis is discussed.     

Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis

Oxidized low-density lipoprotein (oxLDL) is thought to promote atherosclerosis through complex inflammatory and immunologic mechanisms that lead to lipid dysregulation and foam cell formation. Recent findings suggested that oxLDL forms complexes with β₂-glycoprotein I (β₂GPI) and/or C-reactive protein (CRP) in the intima of atherosclerotic lesions. Autoantibodies against oxLDL/β₂GPI complexes occur in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid syndrome (APS) and significantly correlate with arterial thrombosis. IgG autoantibodies having similar specificity emerged spontaneously in non-immunized NZW × BXSB F1 mice, an animal model of APS, and a monoclonal autoantibody (WB-CAL-1; IgG2a) against complexed β₂GPI (oxLDL/β₂GPI complexes) was derived from the same mice. WB-CAL-1 significantly increased the in vitro uptake of oxLDL/β₂GPI complexes by macrophages. This observation strongly suggests that such IgG autoantibodies are pro-atherogenic. In contrast, IgM anti-oxLDL natural antibodies found in the atherosclerosis-prone mice (ApoE^-/- and LDL-R^-/- mice) have been proposed to be anti-atherogenic (protective). The presence of IgG anti-oxLDL antibodies in humans has been documented in many publications but their exact clinical significance remains unclear. In this article, we review recent progress in our understanding of the mechanisms involved in oxidation of LDL, formation of oxLDL complexes, and antibody mediated-immune regulation of atherogenesis.     

Aldehyde adducts generated during lipid peroxidation modification of proteins

Abstract

Various lines of evidence indicate that an important part in the pathogenesis of atherosclerosis is the modification of the plasma low-density lipoproteins (LDLs). A large number of pro-inflammatory and pro-atherogenic properties have been ascribed to the oxidatively modified LDLs and their components. There is considerable evidence to support the role of lipid peroxidation products, reactive aldehydes in particular, originating from the oxidized LDL as important signaling molecules in the context of the atherosclerotic lesion. These aldehydes generated during the peroxidation of LDL exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts on the apolipoprotein B-100 particle in LDL. Characterization of the aldehyde adducts generated in the protein is therefore critical in understanding the nature of the oxidized LDL. However, the majority of adducts generated during the oxidative modification of LDL have not yet been chemically characterized. In this review, the current status of aldehyde adducts quantitatively analyzed in the Cu²⁺-oxidized LDL is reviewed.     

The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages

Platelet-derived growth factor (PDGF) is secreted by several cells that participate in the process of atherogenesis, including arterial wall monocyte-derived macrophages. Macrophages in human and non-human primate lesions have recently been demonstrated to contain PDGF-B chain protein in situ. In developing lesions of atherosclerosis, macrophages take up and metabolize modified lipoproteins, leading to lipid accumulation and foam cell formation. Oxidatively modified low density lipoproteins (LDL) have been implicated in atherogenesis and have been demonstrated in atherosclerotic lesions. The effects of the uptake of various forms of modified LDL on PDGF gene expression, synthesis, and secretion in adherent cultures of human blood monocyte-derived macrophages were examined. LDL oxidized in a cell-free system in the presence of air and copper inhibited the constitutive expression of PDGF-B mRNA and secretion of PDGF in a dose-dependent fashion. Oxidatively modified LDL also attenuated lipopolysaccharide-induced PDGF-B mRNA expression. These changes were unrelated to the mechanism of lipid uptake and the degree of lipid loading and were detectable within 2 h of exposure to oxidized LDL. The degree of inhibition of both basal and lipopolysaccharide-induced PDGF-B-chain expression increased with the extent of LDL oxidation. Monocyte-derived macrophages exposed to acetylated LDL or LDL aggregates accumulated more cholesterol than cells treated with oxidized LDL, but PDGF expression was not consistently altered. Thus, uptake of a product or products of LDL oxidation modulates the expression and secretion of one of the principal macrophage-derived growth factors, PDGF. This modulation may influence chemotaxis and mitogenesis of smooth muscle cells locally in the artery wall during atherogenesis.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

Scavenging of Lipid Peroxidation Products from Oxidizing LDL by Albumin Alters the Plasma Half-Life of a Fraction of Oxidized LDL Particles

We analyse LDL oxidation in vitro in the presence of copper (II) ions and differentiate a lag phase and a rapid peroxidation phase. We demonstrate that a physiological concentration of albumin does not alter the kinetics of the dienes in the oxidizing LDL but reduces the fluorescence of the oxidizing LDL and alters the biological properties of oxidized LDL. We find in rats after intravenous administration of oxidized LDL, that it is rapidly cleared from the circulating blood. The presence of albumin during the peroxidation phase, however, reduces the fraction of oxidized LDL with rapid blood clearance. We propose that some lipid peroxidation products formed in oxidizing LDL are hydrophilic enough to diffuse into the aqueous buffer from where they react either with the s-amino-groups of apolipoprotein B or albumin. Effective scavengers for these hydrophilic endproducts of the LDL oxidation pathways such as albumin might reduce modification of the LDL and might be useful to reduce its atherogenicity.     

Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized low density lipoprotein

Oxidation of low density lipoprotein (LDL) occurs in vivo and significantly contributes to the development of atherosclerosis. An important mechanism of LDL oxidation in vivo is its modification with 12/15-lipoxygenase (LO). We have developed a model of minimally oxidized LDL (mmLDL) in which native LDL is modified by cells expressing 12/15LO. This mmLDL activates macrophages inducing membrane ruffling and cell spreading, activation of ERK1/2 and Akt signaling, and secretion of proinflammatory cytokines. In this study, we found that many of the biological activities of mmLDL were associated with cholesteryl ester (CE) hydroperoxides and were diminished by ebselen, a reducing agent. Liquid chromatography coupled with mass spectroscopy demonstrated the presence of many mono- and polyoxygenated CE species in mmLDL but not in native LDL. Nonpolar lipid extracts of mmLDL activated macrophages, although to a lesser degree than intact mmLDL. The macrophage responses were also induced by LDL directly modified with immobilized 12/15LO, and the nonpolar lipids extracted from 12/15LO-modified LDL contained a similar set of oxidized CE. Cholesteryl arachidonate modified with 12/15LO also activated macrophages and contained a similar collection of oxidized CE molecules. Remarkably, many of these oxidized CE were found in the extracts of atherosclerotic lesions isolated from hyperlipidemic apoE(-/-) mice. These results suggest that CE hydroperoxides constitute a class of biologically active components of mmLDL that may be relevant to proinflammatory activation of macrophages in atherosclerotic lesions.