Oxidized lipids as mediators of coronary heart disease

PURPOSE OF REVIEW: To summarize the recent evidence on the physiological relevance of the view that LDL lipid oxidation may play a major role in the inflammatory reaction that leads to or amplifies atherogenesis. Oxidation of LDL phospholipids containing arachidonic acid at the sn-2 position occurs when a critical concentration of 'seeding molecules' derived from the lipoxygenase pathway is reached in LDL. This generates a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. RECENT FINDINGS: We have observed that LDL from mice that are genetically predisposed to diet-induced atherosclerosis is highly proinflammatory when the mice are maintained on an atherogenic diet, when they are injected with LDL-derived oxidized phospholipids, or once they are infected with influenza A virus. Patients with coronary atherosclerosis also had highly proinflammatory LDL, despite having normal blood lipid levels or normal plasma HDL levels. SUMMARY: We and others have hypothesized that HDL and LDL-derived oxidized phospholipids may be part of a system of nonspecific innate immunity. We therefore propose that determination of HDL capacity against LDL oxidation and the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.     

Role of oxidatively modified LDL in atherosclerosis

Oxidative modification of LDL is accompanied by a number of compositional and structural changes, including increased electrophoretic mobility, increased density, fragmentation of apolipoprotein B, hydrolysis of phosphatidylcholine, derivatization of lysine amino groups, and generation of fluorescent adducts due to covalent binding of lipid oxidation products to apo B. In addition, oxidation of LDL has been shown to result in numerous changes in its biologic properties that could have pathogenetic importance, including accelerated uptake in macrophages, cytotoxicity, and chemotactic activity for monocytes. The present article summarizes very recent developments related to the mechanism of oxidation of LDL by cells, receptor-mediated uptake of oxidized LDL in macrophages, the mechanism of phosphatidylcholine hydrolysis during LDL oxidation, and other biologic actions of oxidized LDL including cytotoxicity, altered eicosanoid metabolism, and effects on the secretion of growth factors and chemotactic factors. In addition, this review will examine the evidence for the presence of oxidized LDL in vivo and the evidence that oxidized LDL plays a pathogenetic role in atherosclerosis.     

The oxidative modification hypothesis of atherogenesis: an overview

The literature relating lipid and lipoprotein oxidation to atherosclerosis has expanded enormously in recent years. Papers on the “oxidative modification hypothesis” of atherogenesis have ranged from the most basic studies of the chemistry and enzymology of LDL oxidation, through studies of the biological effects of oxidized LDL on cultured cells, and on to in vivo studies of the effects of antioxidants on atherosclerosis in animals and humans. The data in support of this theory are mounting but many key questions remain unanswered. For example, while it is generally agreed that LDL undergoes oxidation and that oxidized LDL is present in arterial lesions, it is still not known how and where LDL gets oxidized in vivo nor which of its many biological effects demonstrable in vitro are relevant to atherogenesis in vivo. This brief review is not intended to be comprehensive but rather to offer a perspective and a context for this Forum. We discuss the strengths and weaknesses of each line of evidence, try to identify areas in which further research is needed, assess the relevance of the hypothesis to the human disease, and point to some of the potential targets for therapy.     

Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement.

The 'oxidation theory' of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although little direct evidence for a causative role of 'oxidized LDL' in atherogenesis exists, several studies show that, in vitro, oxidized LDL exhibits potentially proatherogenic activities and lipoproteins isolated from atherosclerotic lesions are oxidized. As a consequence, the molecular mechanisms of LDL oxidation and the actions of alpha-tocopherol (alpha-TOH, vitamin E), the major lipid-soluble lipoprotein antioxidant, have been studied in detail. Based on the known antioxidant action of alpha-TOH and epidemiological evidence, vitamin E is generally considered to be beneficial in coronary artery disease. However, intervention studies overall show a null effect of vitamin E on atherosclerosis. This confounding outcome can be rationalized by the recently discovered diverse role for alpha-TOH in lipoprotein oxidation; that is, alpha-TOH displays neutral, anti-, or, indeed, pro-oxidant activity under various conditions. This review describes the latter, novel action of alpha-TOH, termed tocopherol-mediated peroxidation, and discusses the benefits of vitamin E supplementation alone or together with other antioxidants that work in concert with alpha-TOH in ameliorating lipoprotein lipid peroxidation in the artery wall and, hence, atherosclerosis.     

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.     

beta-Carotene inhibits the oxidative modification of low-density lipoprotein

Several lines of evidence indicate that oxidized LDL (Ox-LDL) may promote atherogenesis. Hence, the role of antioxidants in the prevention of LDL oxidation needs to be determined. beta-Carotene, in addition to being an efficient quencher of singlet oxygen, can also function as a radical-trapping antioxidant. Since previous studies have failed to show that beta-carotene inhibits LDL oxidation, we re-examined its effect on the oxidative modification of LDL. For these studies, LDL was oxidized in both a cell-free (2.5 microM Cu2+ in PBS) and a cellular system (human monocyte macrophages in Ham's F-10 medium). beta-Carotene inhibited the oxidative modification of LDL in both systems as evidenced by a decrease in the lipid peroxide content (thiobarbituric-acid-reacting substances activity), the negative charge of LDL (electrophoretic mobility) and the formation of conjugated dienes. By inhibiting LDL oxidation, beta-carotene substantially decreased its degradation by macrophages. beta-Carotene (2 microM) was more potent than alpha-tocopherol (40 microM) in inhibiting LDL oxidation. Thus, beta-carotene, like ascorbate and alpha-tocopherol, inhibits LDL oxidation and might have an important role in the prevention of atherosclerosis.     

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.     

Oxidation of low-density lipoprotein by iron at lysosomal pH: implications for atherosclerosis

Low-density lipoprotein (LDL) has recently been shown to be oxidized by iron within the lysosomes of macrophages, and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterize the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α-tocopherol on this process. LDL was oxidized by iron at pH 4.5 and 37 °C and its oxidation monitored by spectrophotometry and high-performance liquid chromatography. LDL was oxidized effectively by FeSO(4) (5-50 μM) and became highly aggregated at pH 4.5, but not at pH 7.4. The level of cholesteryl esters decreased, and after a pronounced lag, the level of 7-ketocholesterol increased greatly. The total level of hydroperoxides (measured by the triiodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.5 and 37 °C was similar to that of LDL oxidized by copper at pH 7.4 and 4 °C, i.e., rich in hydroperoxides but low in oxysterols. Previously oxidized LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous iron was much more effective than ferric iron at oxidizing LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepentaacetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages but were unable to prevent aggregation of LDL after it had been partially oxidized. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the rate of oxidation of LDL but decreased it later. The presence of oxidized and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis.     

Baseline diene conjugation in LDL lipids: an indicator of circulating oxidized LDL

The wide acceptance of the diene conjugation-method in monitoring low-density lipoprotein (LDL) oxidation ex vivo has led to development of an assay, which measures the amount of baseline diene conjugation (BDC) in circulating LDL, and is an indicator of oxidized LDL in vivo. The LDL-BDC assay is based on precipitation of serum LDL with buffered heparin, and spectrophotometric determination of baseline level of conjugated dienes in lipids extracted from LDL. Compared to existing methods for oxidized LDL, LDL-BDC is fast and simple to perform. Chemical studies by HPLC and NMR have verified that LDL-BDC is a specific indicator of circulating mildly oxidized LDL. Validity of the assay is further indicated by strong correlation with the titer of autoantibodies against oxidized LDL. Clinical studies have shown that LDL-BDC is closely related to coronary, carotid, and brachial atherosclerosis. Moreover, several independent studies have demonstrated surprisingly strong associations between LDL-BDC and known atherosclerosis risk factors (obesity, physical inactivity, hypertension, diabetes, and arterial functions). Indeed, these studies seem to indicate that as an indicator of the risk of atherosclerosis LDL-BDC clearly exceeds sensitivity and specificity of the common lipid markers of atherosclerosis. It is concluded that LDL-BDC is a promising candidate in search for methods for the evaluation of in vivo LDL oxidation and the risk of atherosclerosis.     

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.     

Neopterin and 7,8-Dihydroneopterin Interfere With Low Density Lipoprotein Oxidation Mediated by Peroxynitrite and/or Copper

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- &#110 generate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO &#109 ). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO &#109 - as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.     

Neopterin and 7,8-dihydroneopterin interfere with low density lipoprotein oxidation mediated by peroxynitrite and/or copper

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- γgenerate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO -). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO -- as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.     

Oxidants and antioxidants in atherosclerosis

The oxidative theory suggests that LDL oxidation contributes to atherogenesis, implying that attenuation of this process by antioxidants should decrease atherosclerosis. However, a causative link between LDL oxidation and atherogenesis is not firmly established. It requires the identification of the oxidants that are responsible for the initiation of LDL oxidation, and an understanding of the modified moieties that are responsible for the proatherogenic activities of oxidized LDL. The present review summarizes recent data on potential biological oxidants for LDL in the vessel wall, and discusses the antiatherogenic role(s) of selected antioxidants.     

Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.     

Antioxidation of human low density lipoprotein by horin hydrate

Oxidative modification of low density lipoprotein (LDL) has been suggested to be a risk factor for the development of atherosclerosis. Agents which can protect LDL from oxidation may be useful in preventing atherogenesis. Here, we found that morin hydrate, at 100 μM concentration, effectively inhibits Cu²⁺- induced oxidation of LDL. The oxidation of LDL was assessed by agarose gel electrophoresis. This was further studied by measuring the increased values of the malondialdehyde equivalents and the decreased numbers of reactive amino groups on oxidized LDL. Trolox, at equimolar concentrations, exhibit similar effects in preventing oxidation of LDL.     

The Pla surface protease/adhesin of Yersinia pestis mediates bacterial invasion into human endothelial cells

Oxidation of low density lipoprotein (LDL) induced by hypochlorous acid (HOCl) leading to LDL(-), a minimally oxidized subspecies of LDL, was investigated. LDL(-) is characterized by its greater electronegativity and oxidative status, and is found in plasma in vivo. Its concentration was found to be elevated under conditions that predispose humans to atherosclerosis. We found that HOCl also converts LDL rapidly to an even more oxidized state, identified as LDL(2-), which is more electronegative than LDL(-). After milder oxidation for short durations, formation of LDL(-) takes place while less LDL(2-) is formed. Under these conditions, addition of methionine not only suppressed further oxidation of LDL but also favored the formation of LDL(-) over LDL(2-), possibly by removing chloramines at lysyl residues of LDL. The presence of lipoprotein-deficient plasma did not prevent HOCl-mediated conversion of LDL to more electronegative species. It is concluded that the HOCl-mediated conversion of LDL into more electronegative species might be physiologically relevant.     

Effects of oolonghomobisflavan A on oxidation of low-density lipoprotein

Oxidation of low-density lipoprotein (LDL) by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been suggested to be involved in the onset of atherosclerosis. Oolong tea contains unique polyphenols including oolonghomobisflavan A (OFA). In this study, the effects of OFA on LDL oxidation by ROS and RNS were investigated in vitro. OFA suppressed formation of cholesterol ester hydroperoxides in LDL oxidized by peroxyl radical and peroxynitrite, and formation of thiobarbituric acid reactive substances in LDL oxidized by Cu²⁺. In addition, OFA inhibited fragmentation, carbonylation, and nitration of apolipoprotein B-100 (apo B-100) in the oxidized LDL, in which heparin-binding activity of apo B-100 was protected by OFA. Our results suggest that OFA exhibits antioxidant activity against both lipid peroxidation and oxidative modification of apo B-100 in LDL oxidized by ROS and RNS. Polyphenols in oolong tea may prevent atherosclerosis by reducing oxidative stress.     

Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

Summary It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3--hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.     

Effects of antioxidants against atherosclerosis

It is generally accepted that the oxidative modification of low density lipoprotein (LDL) plays a pivotal role in the progression of atherosclerosis. This suggests that the antioxidants which suppress the oxidative modification of LDL should be effective in preventing atherogenesis. This brief article reviews the role and potency of antioxidants against the oxidation of LDL. It is emphasized that the LDL can be oxidized by different oxidants by different mechanisms and the efficacy of antioxidants depends on the type of oxidants.     

Effects of antioxidants against atherosclerosis

It is generally accepted that the oxidative modification of low density lipoprotein (LDL) plays a pivotal role in the progression of atherosclerosis. This suggests that the antioxidants which suppress the oxidative modification of LDL should be effective in preventing atherogenesis. This brief article reviews the role and potency of antioxidants against the oxidation of LDL. It is emphasized that the LDL can be oxidized by different oxidants by different mechanisms and the efficacy of antioxidants depends on the type of oxidants.