Oxidized cholesteryl esters and inflammation

The oxidation hypothesis of atherosclerosis proposes that oxidized LDL is a major causative factor in the development of atherosclerosis. Although this hypothesis has received strong mechanistic support and many animal studies demonstrated profound atheroprotective effects of antioxidants, which reduce LDL oxidation, the results of human clinical trials with antioxidants were mainly negative, except in selected groups of patients with clearly increased systemic oxidative stress. We propose that even if reducing lipoprotein oxidation in humans might be difficult to achieve, deeper understanding of mechanisms by which oxidized LDL promotes atherosclerosis and targeting these specific mechanisms will offer novel approaches to treatment of cardiovascular disease. In this review article, we focus on oxidized cholesteryl esters (OxCE), which are a major component of minimally and extensively oxidized LDL and of human atherosclerotic lesions. OxCE and OxCE-protein covalent adducts induce profound biological effects. Among these effects, OxCE activate macrophages via toll-like receptor-4 (TLR4) and spleen tyrosine kinase and induce macropinocytosis resulting in lipid accumulation, generation of reactive oxygen species and secretion of inflammatory cytokines. Specific inhibition of OxCE-induced TLR4 activation, as well as blocking other inflammatory effects of OxCE, may offer novel treatments of atherosclerosis and cardiovascular disease. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.     

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.     

Flavonoids protect LDL from oxidation and attenuate atherosclerosis

Consumption of some plant-derived flavonoids results in their absorption and appearance in plasma and tissues. The inverse relationship between dietary flavonoids consumption and cardiovascular diseases may be associated with the ability of flavonoids to attenuate LDL oxidation, macrophage foam cell formation and atherosclerosis. The effect of flavonoids on arterial cell-mediated oxidation of LDL is determined by their accumulation in the lipoprotein and in arterial cells, such as macrophages. Flavonoids can reduce LDL lipid peroxidation by scavenging reactive oxygen/nitrogen species, chelation of transition metal ions and sparing of LDL-associated antioxidants. They can also reduce macrophage oxidative stress by inhibition of cellular oxygenases [such as nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase] or by activating cellular antioxidants (such as the glutathione system). Thus, plant flavonoids, as potent natural antioxidants that protect against lipid peroxidation in arterial cells and lipoproteins, significantly attenuate the development of atherosclerosis.     

Intracellular generation of MDA-LYS epitope in foam cells.

Oxidative stress plays a central role in atherogenesis. Antioxidants, such as probucol, inhibit oxidation of LDL, retard secretion of interleukin-1, growth factors and chemoattractants, and thus inhibit progression of atherosclerosis. Other antioxidants with an ability to inhibit LDL oxidation, however, could not prevent progression of atherosclerosis. The inconsistency between antioxidant potencies indicated oxidative events might have occurred at locations other than LDL. MDA-lysine epitope (MDA-lys) is closely associated with atherogenesis and was recognized as marker for oxidation. We traced formation of MDA-lys during oxidation of LDL and formation of foam cells. The results indicated that thiobarbituric acid reactive substance (TBARS) was primarily present in lipid fraction of ox-LDL not associated with protein fraction after Cu2+ oxidation in vitro. Oxidized LDL did not increase significant immunoreactivity of MDA-lys epitope under our experimental conditions. Foam cells, however, showed the presence of MDA-lys epitope suggesting that intracellular oxidation events occurred to internalized lipids. The uptake of non-oxidatively modified LDL (acetylated LDL) was sufficient to generate MDA-lys epitope in foam cells, consistent with the hypothesis that atherosclerosis is associated with oxidative events in addition to LDL oxidation. We hypothesized that MDA-lys may be generated through intracellular lipid metabolism during the formation of foam cells.     

Inhibitory effect of flavonoids on low-density lipoprotein peroxidation catalyzed by mammalian 15-lipoxygenase

Lipoxygenase-dependent low-density lipoprotein (LDL) oxidation is believed to be involved in atherogenesis. Inhibition of lipoxygenase-induced lipid peroxidation might, therefore, be an important mode to suppress the development of atherosclerosis. Because dietary antioxidants inhibit LDL oxidation in vitro and their intake is inversely associated with coronary heart diseases, we compared the inhibitory effect of three typical flavonoids-quercetin, epicatechin, and flavone-with alpha-tocopherol and ascorbic acid against human LDL oxidation catalyzed by mammalian 15-lipoxygenase. The oxidative modification of LDL was monitored by measurement of cholesteryl ester hydroperoxide (CE-OOH) formation and consumption of antioxidants by using HLPC. Quercetin and epicatechin were the strongest inhibitors of LDL oxidation catalyzed by 15-lipoxygenase; ascorbic acid was an effective inhibitor in the first 3 h of oxidation; and fivefold alpha-tocopherol-enriched LDL showed a partial inhibition of CE-OOH formation only after 4-6 h of incubation. Flavone had no effect. Quercetin, ascorbic acid, and alpha-tocopherol were consumed in the first 3 h of incubation. Consumption of LDL alpha-tocopherol was partially inhibited by ascorbic acid and quercetin, whereas epicatechin and flavone were without effect. These results emphasize the inhibitory effect of the flavonoids quercetin and epicatechin on 15-lipoxygenase-mediated LDL lipid peroxidation. At similar concentrations, they are stronger antioxidants than ascorbic acid, alpha-tocopherol, and flavone.     

The degree of unsaturation of dietary fatty acids and the development of atherosclerosis (review)

Atherosclerosis is the principal contributor to the pathogenesis of myocardial and cerebral infarction, gangrene and loss of function in the extremities. It results from an excessive inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle of the artery wall. Atherosclerotic lesions develop fundamentally in three stages: dysfunction of the vascular endothelium, fatty streak formation and fibrous cap formation. Each stage is regulated by the action of vasoactive molecules, growth factors and cytokines. This multifactorial etiology can be modulated through the diet. The degree of unsaturation of dietary fatty acids affects lipoprotein composition as well as the expression of adhesion molecules and other pro-inflammatory factors, and the thrombogenicity associated with atherosclerosis development. Thus, the preventive effects of a monounsaturated-fatty acid-rich diet on atherosclerosis may be explained by the enhancement of high-density lipoprotein-cholesterol levels and the impairment of low-density lipoprotein-cholesterol levels, the low-density lipoprotein susceptibility to oxidation, cellular oxidative stress, thrombogenicity and atheroma plaque formation. On the other hand, the increase of high-density lipoprotein cholesterol levels and the reduction of thrombogenicity, atheroma plaque formation and vascular smooth muscle cell proliferation may account for the beneficial effects of polyunsaturated fatty acid on the prevention of atherosclerosis. Thus, the advantages of the Mediterranean diet rich in olive oil and fish on atherosclerosis may be due to the modulation of the cellular oxidative stress/antioxidant status, the modification of lipoproteins and the down-regulation of inflammatory mediators.     

Oxidation of low-density lipoprotein in atherosclerosis from basic biochemistry to clinical studies

Although it has been known for long time that atherosclerosis is associated with lipid deposition, only recently it has been accepted that the plasmatic concentration of cholesterol, especially LDL cholesterol, is a risk factor for atherosclerosis. However, chemically modified LDL, but not native LDL, is able to induce the formation of foam cells, the hallmark of atherosclerosis. LDL oxidation is likely to be the most important form of LDL modification in humans. In biochemical terms, LDL oxidation is a free radical driven chain reaction where polyunsaturated fatty acids are converted to lipid peroxides, which easily decompose to many products, including biologically active aldehydes. The assay of LDL oxidation in biological fluids is problematic; direct assays detect a product of LDL oxidation whereas indirect assays give an indicator of LDL oxidation susceptibility. In general, epidemiological studies support the concept that the level of plasmatic lipophilic antioxidants, tocopherols and carotenoids, is low in populations at increased risk for atherosclerosis. However, clinical trials based on vitamin E as antioxidant showed inconclusive results, suggesting that supplementation with vitamin E is not generically recommended for atherosclerotic patients. These results, however, do not contradict that oxidation of lipoprotein is involved in atherosclerosis; rather, this negative outcome raises a number of considerations such as the need for a reliable marker of lipoprotein oxidation in plasma and a more complete information about the physiological triggers of lipoprotein oxidation.     

Higher cholesterol in human LDL is associated with the increase of oxidation susceptibility and the decrease of antioxidant defence: experimental and simulation data

Increased low-density lipoprotein (LDL) cholesterol is a recognized risk factor for atherosclerosis. There is also strong evidence that oxidatively modified LDL initiates the development of this pathological process and the administration of antioxidants might have a protective effect. However, the appropriate trials did not provide completely consistent results. We found in this study that the oxidation kinetics and also the antioxidant effectiveness are different depending on the cholesterol content in LDL. Higher cholesterol in LDL causes an acceleration of its oxidation as well as an increase of resistance to the antioxidative effect of ascorbic acid. In searching for a theoretical background of this dual impact of cholesterol in LDL, computer simulation of LDL oxidation was used. It was found that the pre-existing level of lipid hydroperoxides together with the total amount of oxidizable lipid substrate associated with the cholesterol level in LDL were satisfactory prerequisites for a best fit to the experimental data. In conclusion, this study provides at least a partial explanation for some failures to arrest, by administration of antioxidants, the progression of atherosclerosis in animal and human hypercholesterolemia.     

Protection of low density lipoprotein oxidation by the antioxidant agent IRFI005, a new synthetic hydrophilic vitamin E analogue

The oxidative modification of low density lipoprotein (LDL) is thought to be an important factor in the initiation and development of atherosclerosis. Antioxidants have been shown to protect LDL from oxidation and to inhibit atherosclerosis development in animals. Potent synthetic antioxidants are currently being tested, but they are not necessarily safe for human use. We here characterize the antioxidant activity of IRFI005, the active metabolite of Raxofelast (IRFI0016) that is a novel synthetic analog of vitamin E under clinical development, and demonstrate that it prevents oxidative modification of LDL. IFI005 inhibited the oxidative modification of LDL, measured through the generation of MDA, electrophoretic mobility and apo B100 fluorescence. During the oxidation process IRF1005 was consumed with the formation of the benzoquinone oxidation product. The powerful antioxidant activity of IRFI005 is at least in part mediated by a chain breaking mechanism as it is an efficient peroxyl radical scavenger with a rate constant k(IRFI005 + LOO(o)) of 1.8 X 10(6) M(-1)s(-1). 4. IRFI005 substantially preserved LDL-associated antioxidants, alpha-tocopherol and carotenoids, and when co-incubated with physiologic levels of ascorbate provoked a synergistic inhibition of LDL oxidation. Also the co-incubation of IRFI005 with Trolox caused a synergistic effect, and a lag phase in the formation of the trolox-benzoquinone oxidation product. A synergistic inhibition of lipid peroxidation was also demonstrated by co-incubating IRFI005 and alpha-tocopherol incorporated in linoleic acid micelles. These data strongly suggest that IRFI005 can operate by a recycling mechanism similar to the vitamin E/ascorbate sysem.     

Potential role of oxidized lipids and lipoproteins in antioxidant defense

The atherogenic oxidative modification of low-density lipoprotein is suggested to occur in the aortic intima. There is reasonable evidence to suggest that antioxidants might be beneficial in preventing or retarding the progression of atherosclerosis. Exercise, estrogens, and substitution of polyunsaturated fat for saturated fat are beneficial in the prevention of atherosclerosis. Yet, paradoxically, they are capable of inducing an oxidative stress. To reconcile with this paradox, we postulate that under certain conditions an oxidative stress might be beneficial by inducing antioxidant enzymes in arterial cells. However, those with genetic deficiency in antioxidant enzymes or those who poorly respond to oxidative stress or those with overwhelming plasma oxidative stress might need additional antioxidant protection.     

Potential role of oxidized lipids and lipoproteins in antioxidant defense

The atherogenic oxidative modification of low-density lipoprotein is suggested to occur in the aortic intima. There is reasonable evidence to suggest that antioxidants might be beneficial in preventing or retarding the progression of atherosclerosis. Exercise, estrogens, and substitution of polyunsaturated fat for saturated fat are beneficial in the prevention of atherosclerosis. Yet, paradoxically, they are capable of inducing an oxidative stress. To reconcile with this paradox, we postulate that under certain conditions an oxidative stress might be beneficial by inducing antioxidant enzymes in arterial cells. However, those with genetic deficiency in antioxidant enzymes or those who poorly respond to oxidative stress or those with overwhelming plasma oxidative stress might need additional antioxidant protection.     

Iron and atherosclerosis

Iron is a vital element in life. However, it may participate in diverse pathological processes by catalyzing the formation of reactive oxygen free radicals. During the past decade, considerable evidence has supported the role of oxidative stress in the development of atherosclerosis and related cardiovascular diseases. The oxidation of low-density lipoprotein (LDL) and lipid is believed to be one of the crucial events leading to plaque formation in vasculature. It has been hypothesized that iron-mediated oxidation is involved in this process. In favor of this idea, several epidemiological studies have shown that the level of body iron stores is positively correlated with the incidence of coronary heart disease in human populations. However, some studies have yielded conflicting results. Recently, studies conducted in our laboratory and others have demonstrated that iron deposition is prominent in human atherosclerotic lesions. The iron deposits appear to colocalize with ceroid, which is an end product of extensively oxidized lipid and protein complex, in lesions, providing histological evidence to support the iron hypothesis. Additional experiments in animals have further revealed that the severity of atherosclerosis can be markedly influenced by iron overload or deficiency. Collectively, these data provide a strong pathological basis to support the detrimental role of iron in vascular damage and progression of the disease.     

Implications of Lag Time Concept in the Oxidation of LDL

Oxidation of low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. The most common technique for measuring the oxidation of lipoproteins is the continuos measurement of the formation of conjugated diene at OD 234 nm. The concept of “lag time”, derived from such measurements, has been used to test the efficacy of various antioxidants for their ability to inhibit the oxidation of LDL. This review will elaborate on some of the factors that might affect the lag time.     

<Emphasis Type="Italic">N</Emphasis>-acetyl cysteine suppresses the foam cell formation that is induced by oxidized low density lipoprotein via regulation of gene expression

Foam cells derived from macrophages have been implicated as markers of early stage atherosclerosis development. In this study, we found that N-acetyl cysteine (NAC), a well-known inhibitor of reactive oxygen species (ROS), decreased the generation of ROS and suppressed foam cell formation in the presence of oxidized low density lipoprotein through down-regulation of cluster of differentiation 36 expression. We investigated gene expression profiles in order to determine the effects of NAC on foam cell formation using a microarray analysis. The level of apolipoprotein E, which is involved in lipid efflux, was increased and the levels of the antioxidant genes glutathione peroxidase 1 and 3 were also increased. The expression levels of the oxidative stress response and the DNA repair genes were decreased. These results were confirmed using quantitative real-time PCR. Our results indicate that oxidative stress plays an important role in foam cell formation, and that regulation of oxidation using antioxidants is a potential therapeutic method for blocking atherosclerosis development.     

Free and peptide-bound DOPA can inhibit initiation of low density lipoprotein oxidation

Hydroxyl radicals have been shown to convert free tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA) which has reducing properties. During protein or peptide oxidation such reducing species are also formed from tyrosine residues. Free DOPA or peptide-bound DOPA (PB-DOPA) is able to promote radical-generating events, facilitating the damage of biomolecules such as nucleic acids. Radical induced lipid oxidation in low density lipoprotein (LDL) transforms the lipoprotein into an atherogenic particle. As PB-DOPA has been found in atherosclerotic plaques, we tested the ability of free and PB-DOPA to influence LDL oxidation. Free DOPA, in contrast to tyrosine had strong inhibitory action on both, the copper-ion initiated and metal ion independent (AAPH-induced) lipid oxidation. Free DOPA also inhibited LDL oxidation induced by the copper transport protein ceruloplasmin. To test if PB-DOPA was also able to inhibit LDL oxidation, DOPA residues were generated enzymatically in the model peptides insulin and tyr-tyr-tyr, respectively. PB-DOPA formation substantially increased the ability of both molecules to inhibit LDL oxidation by copper or AAPH. We hypothesize that DOPA-peptides and -proteins may have the potential to act as efficacious antioxidants in the atherosclerotic plaque.     

Copper-induced LDL peroxidation: interrelated dependencies of the kinetics on the concentrations of copper, hydroperoxides and tocopherol

Excessive uptake of oxidized low density lipoprotein plays a role in the onset of atherosclerosis. Lipid-associated antioxidants, the most abundant of which is tocopherol (vitamin E), are therefore believed to have anti-atherogenic properties. By contrast, hydroperoxides enhance the peroxidation of low density lipoprotein. We demonstrate that none of these compounds markedly affect the maximal rate of oxidation of low density lipoprotein, whereas the lag preceding rapid oxidation is prolonged by tocopherol but shortened by hydroperoxides. The corresponding 'prolongation' and 'shortening' can be compensated by each other in low density lipoprotein preparations enriched with both these compounds. The dependence of the balance between the effects of tocopherol and hydroperoxides on the copper concentration indicates that the antioxidative effect of vitamin E increases with the oxidative stress.     

Evidence for inhibition of low density lipoprotein oxidation and cholesterol accumulation by apolipoprotein H (beta2-glycoprotein I)

Low density lipoprotein (LDL) oxidation and lipid accumulation are thought to enhance the progression of atherosclerosis. Apolipoprotein H (apoH) has been implicated in the development of human atherosclerosis. However, the roles of apoH in the oxidative modification of LDL and cellular accumulation of lipid constituents remained uncharacterized. In this study, the level of plasma apoH was found to be significantly associated with the oxidative susceptibility of LDL in human subjects. Plasma levels of apoH were positively correlated with the lag time but negatively correlated with LDL oxidation rate in conjugated diene formation. By using a J774 A.1 macrophage culture system, we found that apoH could not only inhibit the formation of conjugated diene and thiobarbituric acid-reactive substances, but also reduce the electrophoretic mobility of oxidized LDL. Furthermore, apoH decreased cellular accumulation of cholesterol via a reduction in cholesterol influx and an increase in cholesterol efflux. This is the first demonstration that apoH appears to have "antioxidant"-like effects on LDL oxidation. The results also suggest that apoH can inhibit the translocation of cholesterol from extracellular pools to macrophages, suggesting that apoH may play an important role in the prevention of atherosclerosis.     

Oxidation of lipids and membranes I: In vitro formation of peroxidative lipid polymers

Fluorescent polymers were obtained by oxidizing partly emulsified linolenic acid with different oxidants. The speed of formation of polymers differed for the various oxidants, and the difference was not a simple function of the oxidation potential. The speed of polymerization also depended on the nature of the emulsion. The presence of egg albumen in the emulsion enhanced polymer formation with all oxidants. When the oxidants used are arranged in the order of decreasing speed of polymer formation, the order is different in the presence of albumen from what it is in the absence of albumen. With different oxidation catalysts most antioxidants and amino acids tested enhanced polymerization. In oxidation with ferric ions, with K-dichromate, and without added oxidants the only antioxidants which delayed polymerization were “inhibitors”. “Retarders” enhanced polymerization. With KMnO₄ slight delay was caused by some retarders. The findings indicate that not only oxidation catalysts, but also proteins, amino acids, and antioxidants enhance polymerization. The possibility is suggested that in animal cells lipid pigment formation might represent a mechanism for neutralizing free radicals.     

Interactions of nitric oxide and peroxynitrite with low-density lipoprotein

Nitric oxide (*NO) is a free radical species that diffuses and concentrates in the hydrophobic core of low-density lipoprotein (LDL) to serve as a potent inhibitor of lipid oxidation processes. Peroxynitrite (PN), the product of the diffusion-limited reaction between *NO and superoxide (O2*-) represents a relevant mediator of oxidative modifications in LDL. The focus of this review is the analysis of interactions between *NO and PN and its secondary reactions with oxygen radicals on LDL oxidation, which are relevant in the development of the early steps as well as progression of atherosclerosis. We propose that the balance between rates of PN and *NO production, which greatly depends on oxidative stress processes within the vascular wall, will critically determine the final extent of oxidative LDL modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.