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Hyperglycemia and hyperlipidemia are important risk factors for diabetes-accelerated atherosclerosis. Macrophage proliferation has been implicated in the progression of atherosclerosis. We therefore investigated the effects of hyperglycemia and hyperlipidemia on macrophage proliferation in murine atherosclerotic lesions and isolated primary macrophages. Hyperglycemic LDL receptor-deficient mice that were fed a cholesterol-free diet for 12 weeks did not have elevated cholesterol levels compared with nondiabetic mice, and there was no evidence of increased macrophage proliferation in atherosclerotic lesions. Moreover, elevated glucose levels did not increase proliferation of isolated mouse peritoneal macrophages. In contrast, hyperglycemic LDL receptor-deficient mice that were fed a cholesterol-rich diet showed increased cholesterol levels concomitant with macrophage proliferation in atherosclerotic lesions. Glucose promoted lipid and protein oxidation of LDL in vitro. Glucose-oxidized LDL resulted in phosphorylation of extracellular signal-regulated kinase and protein kinase B/Akt and stimulated proliferation of isolated macrophages. The mitogenic effect of glucose-oxidized LDL was mediated by CD36 and by extracellular signal-regulated kinase activation induced by protein kinase C-dependent and phosphatidylinositol 3-kinase-dependent pathways. Thus, hyperglycemia is not sufficient to stimulate macrophage proliferation in lesions of atherosclerosis or in isolated macrophages. A combination of hyperglycemia and hyperlipidemia, however, stimulates macrophage proliferation by a pathway that may involve the glucose-dependent oxidation of LDL.

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.

Oxidative modifications of low-density lipoprotein (LDL) have been proposed to play a critical role in atherogenesis. To test the role of proposed antioxidants in inhibiting LDL oxidation and vascular disease, it is important to identify the biologically relevant sources of oxidative stress in the human arterial wall. Mass spectrometric (MS) quantification of oxidized amino acids in proteins was used as a "molecular fingerprint" to identify the pathways that inflict oxidative damage in vivo. For example, myeloperoxidase is expressed in macrophages in human atherosclerotic lesions, and immunohistochemical studies suggest that it might be a pathway for LDL oxidation. We found that hypochlorous acid, tyrosyl radical, and reactive nitrogen species generated by myeloperoxidase each yielded a unique pattern of protein oxidation products in vitro. MS analysis of human atherosclerotic tissue revealed a similar pattern of oxidation products. This strategy has pinpointed myeloperoxidase as a pathway that promotes LDL oxidation in the human artery wall. It is noteworthy that vitamin E fails to inhibit LDL oxidation by myeloperoxidase in vitro. Because the utility of an antioxidant depends critically on the nature of the oxidant that inflicts tissue damage, interventions that specifically inhibit physiologically relevant pathways would be logical candidates for clinical trials of antioxidants. Such a rational approach to therapy is likely to accelerate progress against oxidative stress and coronary artery disease.

Oxidized low-density lipoprotein (LDL) is implicated in atherogenesis, but the mechanisms that oxidize LDL in the human artery wall have proven difficult to identify. A powerful investigative approach is mass spectrometric quantification of the oxidized amino acids that are left in proteins by specific oxidation reactions. Comparison of these molecular fingerprints in biological samples with those produced in proteins by various in vitro oxidation systems can indicate which biochemical pathway has created damage in vivo. For example, the pattern of oxidized amino acids in proteins isolated from atherosclerotic lesions implicates reactive intermediates generated by myeloperoxidase, a major phagocyte enzyme. These intermediates include hypochlorous acid, tyrosyl radical, and reactive nitrogen species, each of which generates a different pattern of stable end products. Despite this strong evidence that myeloperoxidase promotes LDL oxidation in vivo, the antioxidant that has been tested most extensively in clinical trials, vitamin E, fails to inhibit myeloperoxidase pathways in vitro. Because the utility of an antioxidant depends critically on the nature of the pathway that inflicts tissue damage, interventions that specifically inhibit myeloperoxidase or other physiologically relevant pathways would be more logical candidates for the prevention of cardiovascular disease. Moreover, levels of oxidized amino acids in urine and plasma might reflect those in tissues and therefore identify individuals with high levels of oxidative stress. Trials with such subjects would seem more likely to uncover effective antioxidant therapies than trials involving the general population.

Myeloperoxidase uses hydrogen peroxide (H2O2) to generate hypochlorous acid (HOCl), a potent cytotoxic oxidant. We demonstrate that HOCl regulates the activity of matrix metalloproteinase-7 (MMP-7, matrilysin) in vitro, suggesting that this oxidant activates MMPs in the artery wall. Indeed, both MMP-7 and myeloperoxidase were colocalized to lipid-laden macrophages in human atherosclerotic lesions. A highly conserved domain called the cysteine switch has been proposed to regulate MMP activity. When we exposed a synthetic peptide that mimicked the cysteine switch to HOCl, HPLC analysis showed that the thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as sulfinic acid, sulfonic acid, and a dimer containing a disulfide bridge. In contrast, the peptide reacted slowly with H2O2, and the only product was the disulfide. Moreover, HOCl markedly activated pro-MMP-7, an MMP expressed at high levels in lipid-laden macrophages in vivo. Tandem mass spectrometric analysis of trypsin digests revealed that the thiol residue of the enzyme's cysteine switch domain had been converted to sulfinic acid. Thiol oxidation was associated with autolytic cleavage of pro-MMP-7, strongly suggesting that oxygenation activates the latent enzyme. In contrast, H2O2 failed to oxidize the thiol residue of the protein or activate the enzyme. Thus, HOCl activates pro-MMP-7 by converting the thiol residue of the cysteine switch to sulfinic acid. This activation mechanism is distinct from the well-studied proteolytic cleavage of MMP pro-enzymes. Our observations raise the possibility that HOCl generated by myeloperoxidase contributes to MMP activation, and therefore to plaque rupture, in the artery wall. HOCl and other oxidants might regulate MMP activity by the same mechanism in a variety of inflammatory conditions.

A wealth of evidence indicates that oxidized low density lipoprotein (LDL) may be of central importance in animal models of atherogenesis. In recent clinical trials, however, dietary vitamin E supplements have not consistently prevented cardiac events in humans with established coronary artery disease. Such mixed results have led many to question the role of LDL oxidation in human atherosclerosis, although this interpretation assumes that the doses of vitamin E used in the studies inhibited lipid oxidation in vivo. In fact, there is remarkably little evidence indicating that those particular regimens effectively inhibit lipid peroxidation in healthy humans. Moreover, evidence of increased oxidative stress was not a criterion for inclusion in the trials; therefore, vitamin E may have benefited only a subset of the participants. These uncertainties raise doubts about the ability of vitamin E to augment antioxidant defense mechanisms in vivo and leave many questions about LDL oxidation and atherosclerosis unanswered.

It has been proposed that elevated levels of tissue iron increase the risk for atherosclerosis, perhaps by favoring the formation of pro-atherogenic oxidized LDL. Working with apoE-deficient (apoE(-/-)) mice, which do not require a high-fat diet to develop atherosclerosis, we compared the effects of standard diet (0.02% iron) or a 2% carbonyl iron diet. After 24 weeks, mice fed the 2% carbonyl iron diet had twice as much iron in their plasma, a ninefold increase in bleomycin-detectable free iron in their plasma, and ten times as much iron in their livers as control mice. Dietary iron overload caused a modest (30%) rise in plasma triglyceride and cholesterol. Nevertheless, this regimen did not exacerbate, but rather reduced the severity of atherosclerosis by 50%, and it failed to elevate hepatic levels of heme oxygenase mRNA, which is induced by many different oxidative insults in vitro. Moreover, hepatic levels of protein-bound dityrosine and ortho-tyrosine, two markers of metal-catalyzed oxidative damage in vitro, failed to rise in iron-overloaded animals. Our observations suggest that elevated serum and tissue levels of iron are not atherogenic in apoE(-/-) mice. Moreover, they call into question the hypothesis that elevated levels of tissue iron promote LDL oxidation and oxidative stress in vivo.

The oxidation hypothesis proposes that oxidative modification of low density lipoprotein (LDL) plays a critical role in atherogenesis. This review critically evaluates the various mechanisms proposed for LDL oxidation, focusing on insights derived from chemical analysis of human artery wall and studies of genetically engineered mice. The implications of recent clinical trials of vitamin E for the oxidation hypothesis are also briefly discussed.

Recent evidence argues strongly that the marked increase in risk for atherosclerotic heart disease seen in diabetics cannot be explained by a generalized increase in oxidative stress. Here, we used streptozotocin to induce hyperglycemia in cynomolgus monkeys for 6 months and tested whether high glucose levels promote localized oxidative damage to artery wall proteins. We focused on three potential agents of oxidative damage: hydroxyl radical, tyrosyl radical, and reactive nitrogen species. To determine which pathways operate in vivo, we quantified four stable end products of these reactants -- ortho-tyrosine, meta-tyrosine, o,o'-dityrosine, and 3-nitrotyrosine -- in aortic proteins. Levels of ortho-tyrosine, meta-tyrosine, and o,o'-dityrosine, but not of 3-nitrotyrosine, were significantly higher in aortic tissue of hyperglycemic animals. Of the oxidative agents we tested, only hydroxyl radical mimicked this pattern of oxidized amino acids. Moreover, tissue levels of ortho-tyrosine and meta-tyrosine correlated strongly with serum levels of glycated hemoglobin, a measure of glycemic control. We conclude that short-term hyperglycemia in primates promotes oxidation of artery wall proteins by a species that resembles hydroxyl radical. Our observations suggest that glycoxidation reactions in the arterial microenvironment contribute to early diabetic vascular disease, raising the possibility that antioxidant therapies might interrupt this process.

Inflammation and oxidative stress contribute to the pathogenesis of many human diseases including atherosclerosis. Advanced human atheroma contains high levels of the enzyme myeloperoxidase that produces the pro-oxidant species, hypochlorous acid (HOCl). This study documents increased numbers of myeloperoxidase-expressing macrophages in eroded or ruptured plaques causing acute coronary syndromes. In contrast, macrophages in human fatty streaks contain little or no myeloperoxidase. Granulocyte macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, selectively regulates the ability of macrophages to express myeloperoxidase and produce HOCl in vitro. Moreover, myeloperoxidase-positive macrophages in plaques co-localized with granulocyte macrophage colony-stimulating factor. Pro-inflammatory stimuli known to be present in human atherosclerotic plaque, including CD40 ligand, lysophosphatidylcholine, or cholesterol crystals, could induce release of myeloperoxidase from HOCl production by macrophages in vitro. HOCl-modified proteins accumulated at ruptured or eroded sites of human coronary atheroma. These results identify granulocyte macrophage colony-stimulating factor as an endogenous regulator of macrophage myeloperoxidase expression in human atherosclerosis and support a particular role for the myeloperoxidase-expressing macrophages in atheroma complication and the acute coronary syndromes.

Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, generates an array of oxidants proposed to play critical roles in host defense and local tissue damage. Both MPO and its reaction products are present in human atherosclerotic plaque, and it has been proposed that MPO oxidatively modifies targets in the artery wall. We have now generated MPO-deficient mice, and show here that neutrophils from homozygous mutants lack peroxidase and chlorination activity in vitro and fail to generate chlorotyrosine or to kill Candida albicans in vivo. To examine the potential role of MPO in atherosclerosis, we subjected LDL receptor-deficient mice to lethal irradiation, repopulated their marrow with MPO-deficient or wild-type cells, and provided them a high-fat, high-cholesterol diet for 14 weeks. White cell counts and plasma lipoprotein profiles were similar between the two groups at sacrifice. Cross-sectional analysis of the aorta indicated that lesions in MPO-deficient mice were about 50% larger than controls. Similar results were obtained in a genetic cross with LDL receptor-deficient mice. In contrast to advanced human atherosclerotic lesions, the chlorotyrosine content of aortic lesions from wild-type as well as MPO-deficient mice was essentially undetectable. These data suggest an unexpected, protective role for MPO-generated reactive intermediates in murine atherosclerosis. They also identify an important distinction between murine and human atherosclerosis with regard to the potential involvement of MPO in protein oxidation.

Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein.

Superoxide, the reduced form of molecular oxygen, has been implicated in the genesis of vascular disease. One potential mechanism involves oxidation of low density lipoprotein into an atherogenic particle. A second involves reaction with nitric oxide to generate peroxynitrite, a highly oxidizing intermediate. A third involves regulation of signal transduction in artery wall cells. One well-characterized pathway for superoxide production resides in macrophages, the cellular hallmark of the early atherosclerotic lesion. Macrophages contain a membrane-bound NADPH oxidase that reduces oxygen to superoxide. In the current studies, we used mice that are deficient in the gp91-phox subunit of the NADPH oxidase-a model of chronic granulomatous disease (CGD)-to explore the role of superoxide in atherosclerotic vascular disease. Wild-type and CGD mice on the C57BL/6 background received a high-fat diet for 20 weeks to induce hypercholesterolemia. At the end of this period, the 2 strains of mice had comparable plasma lipid levels, and their atherosclerotic lesions were similar in size. We also crossed CGD mice with apolipoprotein E-deficient (apoE-/-) mice to generate spontaneously hypercholesterolemic animals that lacked functional NADPH oxidase. After 24 weeks, the CGD-apoE-/- animals had lower plasma cholesterol and triglyceride levels than did the apoE-/- animals, but there was no difference in the extent of atherosclerotic plaque. Our findings suggest that superoxide generated by the NADPH oxidase of phagocytes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia.

Oxidation of low density lipoprotein (LDL) may be of critical importance in the pathogenesis of atherosclerosis. Recent studies suggest that oxidized phospholipids render LDL atherogenic. However, both the structures and the physiologically relevant pathways for the formation of modified phospholipids in oxidized LDL remain poorly understood. We previously showed that p-hydroxyphenylacetaldehyde (pHA) is the major product of L-tyrosine oxidation by the myeloperoxidase/hydrogen peroxide/chloride system of phagocytes. In the current studies, we demonstrate that this reactive aldehyde targets the aminophospholipids of LDL in vitro and in vivo. Activated human neutrophils generated pHA-ethanolamine, the reduced adduct of pHA with the amino group of phosphatidylethanolamine, on LDL phospholipids by a reaction that required myeloperoxidase, H(2)O(2), and L-tyrosine. The cellular system could be replaced by HOCl and L-tyrosine but not by a wide variety of other oxidation systems, indicating that pHA-ethanolamine is a specific marker for covalent modification of aminophospholipids by myeloperoxidase. To determine whether aldehydes modify aminophospholipids in vivo, we quantified levels of pHA-ethanolamine in acid hydrolysates of reduced lipid extracts through isotope dilution gas chromatography/mass spectrometry. Circulating LDL contained undetectable levels of pHA-modified phospholipid (<0.1 mmol/mol). In contrast, the concentration of pHA-ethanolamine in LDL isolated from human atherosclerotic lesions was strikingly elevated (4.5 mmol/mol). Collectively, these results demonstrate a novel, myeloperoxidase-based mechanism for modifying the amino group of LDL phospholipids. They also offer the first evidence that myeloperoxidase may damage LDL lipids in vivo, raising the possibility that aldehyde-modified aminophospholipids play a role in inflammation and vascular disease.

The oxidation hypothesis proposes that low density lipoprotein must be oxidatively modified to trigger the pathological events of atherosclerosis. In this article, we evaluate recent studies addressing the pathways that promote low density lipoprotein oxidation in vivo and the impact of antioxidants on atherogenesis in animals, paying particular attention to the clinical implications of these studies for the oxidation hypothesis.

Oxidatively damaged LDL may play a critical role in the pathogenesis of atherosclerotic vascular disease. Several pathways promote LDL oxidation in vitro but the physiologically relevant mechanisms have proven difficult to identify. Detection of stable compounds that result from specific reaction pathways has provided the first insights into the mechanism of oxidative damage in the human artery wall. Mass spectrometric analysis of protein oxidation products isolated from atherosclerotic tissue implicate tyrosyl radical, reactive nitrogen intermediates and hypochlorous acid in LDL oxidation and lesion formation in vivo. Hypochlorous acid is only generated by the phagocytic enzyme myeloperoxidase, which can also generate tyrosyl radical and reactive nitrogen intermediates. Chiral phase high-pressure liquid chromatography analysis of lipid oxidation products suggests that cellular lipoxygenases may also play a role at certain stages. In contrast, LDL isolated from atherosclerotic tissue is not enriched in protein oxidation products characteristic of free metal ions, which are the most widely studied in vitro model of LDL oxidation. These observations provide the first direct chemical evidence for reaction pathways that promote LDL oxidation in human atherosclerosis.

Studies in vitro have shown that copper-zinc superoxide dismutase (CuZn-SOD) inhibits a number of events putatively involved in atherogenesis, including cell-mediated oxidation of LDL. To investigate whether increased activity of CuZn-SOD reduces atherogenesis in vivo, we examined diet-induced fatty streak formation in CuZn-SOD transgenic mice (n = 24) as compared with their nontransgenic littermates (n = 28). Transgenic animals were originally created by introduction of an EcoRI-BamHI human genomic DNA fragment containing the CuZn-SOD gene and its regulatory elements into B6SJL zygotes. For the current studies, the transgene was bred for 12 generations into the atherosclerosis-susceptible C57BL/6 background. Animals were fed atherogenic diets (15% fat, 1.25% cholesterol, 0.5% Na cholate) starting at 100 weeks of age and extending for 18 weeks. At the end of the diet period, aortic SOD activity was two-fold higher in transgenics than nontransgenics (mean +/- SE: 46.7 +/- 5.8 versus 20.1 +/- 2.4 units/mg of protein, P < .001). Levels of protein-bound amino acid oxidation products (meta-, ortho-, and dityrosine) were either similar or lower in aorta and heart from transgenics as compared with nontransgenics, suggesting that amplification of CuZn-SOD activity above the normal complement had modest inhibitory effects on basal oxidative stress in these tissues. CuZn-SOD overexpression did not reduce the extent of lesion development as analyzed by quantitative lipid staining of serial sections of the proximal aorta; mean lesion areas (+/- SE) were 997 +/- 478 and 943 +/- 221 mu 2 in transgenics and nontransgenics, respectively. Notably, the range of values for lesion area was 2.2-fold greater in transgenics (0-8403 versus 0-3868 mu 2 in nontransgenics). Moreover, within this group, lesion area showed a significant positive correlation with SOD activity (r = .611, P < .03). These results do not support an antiatherogenic effect of Cu-Zn-SOD over expression and raise the possibility that high tissue SOD activity may potentiate atherogenesis in fat-fed atherosclerosis-susceptible mice [corrected].

Oxidation of LDL may be of pivotal importance in atherogenesis, but the mechanisms that promote oxidation in vivo remain poorly understood. We have explored the possibility that one pathway involves myeloperoxidase, a heme protein secreted by phagocytes. Myeloperoxidase is the only human enzyme known to generate hypochlorous acid (HOCl), a potent oxidizing agent, at physiological halide concentrations. LDL exposed to the complete myeloperoxidase-H2O2-Cl- system underwent chlorination of its protein tyrosyl residues. Treatment of LDL with reagent HOCl resulted in 3-chlorotyrosine formation, implicating HOCl as an intermediate in the enzymatic reaction pathway. In contrast, 3-chlorotyrosine was undetectable in LDL oxidized by hydroxyl radical, copper, iron, hemin, glucose, peroxynitrite, horseradish peroxidase, lactoperoxidase, or lipoxygenase. These results indicate that 3-chlorotyrosine is a specific marker for LDL oxidation by myeloperoxidase. To address the role of myeloperoxidase in promoting LDL oxidation in vivo, we used stable isotope dilution gas chromatography-mass spectrometry to quantify 3-chlorotyrosine in human aortic tissue and in LDL isolated from atherosclerotic lesions. The level of 3-chlorotyrosine in atherosclerotic tissue obtained during vascular surgery was sixfold higher than that of normal aortic intima. Moreover, the level of 3-chlorotyrosine was 30-fold higher in LDL isolated from atherosclerotic intima compared with circulating LDL. The detection of 3-chlorotyrosine in human atherosclerotic lesions indicates that halogenation reactions catalyzed by the myeloperoxidase system of phagocytes constitute one pathway for protein oxidation in vivo. These findings raise the possibility that the myeloperoxidase-H2O2-Cl- system plays a critical role in converting LDL into an atherogenic form.

Most patients with diabetes die from macrovascular complications. Little is known about the pathogenesis of diabetic vascular disease, but recent advances in molecular genetics and oxidation chemistry provide clues to the mystery of diabetes and atherosclerosis. Genetic variants of well-known proteins such as lipoprotein lipase and apolipoprotein E are common. These proteins are suitable candidates for mediating diabetic vascular risk because their variants can produce hypertriglyceridemia, a risk factor for atherosclerosis in diabetes. However, mutations could have different effects on lipoprotein flux across arteries depending on whether expression is dominant in the vascular space or the vascular wall. Lipoproteins retained in the arterial wall are subject to oxidative modification, which could be dependent on glycoxidation, the enzyme myeloperoxidase, or reactive nitrogen species derived from nitric oxide. Accelerated vascular disease in diabetes is likely the result of complex interactions between metabolic derangements such as hyperglycemia, mutations in genes controlling lipid metabolism, and antioxidant defense mechanisms.

Lipoprotein oxidation has been implicated in the pathogenesis of atherosclerosis. However, the physiologically relevant pathways mediating oxidative damage have not yet been identified. Three potential mechanisms are tyrosyl radical, hydroxyl radical, and redox active metal ions. Tyrosyl radical forms o,o'-dityrosine cross-links in proteins. The highly reactive hydroxyl radical oxidizes phenylalanine residues to o-tyrosine and m-tyrosine. Metal ions oxidize low density lipoprotein (LDL) by poorly understood pathways. To explore the involvement of tyrosyl radical, hydroxyl radical, and metal ions in atherosclerosis, we developed a highly sensitive and quantitative method for measuring levels of o, o'-dityrosine, o-tyrosine, and m-tyrosine in proteins, lipoproteins, and tissue, using stable isotope dilution gas chromatography-mass spectrometry. We showed that o,o'-dityrosine was selectively produced in LDL oxidized with tyrosyl radical. Both o-tyrosine and o, o'-dityrosine were major products when LDL was oxidized with hydroxyl radical. Only o-tyrosine was formed in LDL oxidized with copper. Similar profiles of oxidation products were observed in bovine serum albumin oxidized with the three different systems. Applying these findings to LDL isolated from human atherosclerotic lesions, we detected a 100-fold increase in o,o'-dityrosine levels compared to those in circulating LDL. In striking contrast, levels of o-tyrosine and m-tyrosine were not elevated in LDL isolated from atherosclerotic tissue. Analysis of fatty streaks revealed a similar pattern of oxidation products; compared with normal aortic tissue, there was a selective increase in o,o'-dityrosine with no change in o-tyrosine. The detection of a selective increase of o,o'-dityrosine in LDL isolated from vascular lesions is consistent with the hypothesis that oxidative damage in human atherosclerosis is mediated in part by tyrosyl radical. In contrast, these observations do not support a role for free metal ions as catalysts of LDL oxidation in the artery wall.

Oxidized low density lipoprotein (LDL) may be of central importance in triggering atherosclerosis. One potential pathway involves the production of nitric oxide (NO) by vascular wall endothelial cells and macrophages. NO reacts with superoxide to form peroxynitrite (ONOO-), a potent agent of LDL oxidation in vitro. ONOO- nitrates the aromatic ring of free tyrosine to produce 3-nitrotyrosine, a stable product. To explore the role of reactive nitrogen species such as ONOO- in the pathogenesis of vascular disease, we developed a highly sensitive and specific method involving gas chromatography and mass spectrometry to quantify 3-nitrotyrosine levels in proteins. In vitro studies demonstrated that 3-nitrotyrosine was a highly specific marker for LDL oxidized by ONOO-. LDL isolated from the plasma of healthy subjects had very low levels of 3-nitrotyrosine (9 +/- 7 micromol/mol of tyrosine). In striking contrast, LDL isolated from aortic atherosclerotic intima had 90-fold higher levels (840 +/- 140 micromol/mol of tyrosine). These observations strongly support the hypothesis that reactive nitrogen species such as ONOO- form in the human artery wall and provide direct evidence for a specific reaction pathway that promotes LDL oxidation in vivo. The detection of 3-nitrotyrosine in LDL isolated from vascular lesions raises the possibility that NO, by virtue of its ability to form reactive nitrogen intermediates, may promote atherogenesis, counteracting the well-established anti-atherogenic effects of NO.

This article reviews our current understanding of the mechanisms of low-density lipoprotein (LDL) oxidation and the potential role of oxidized lipoproteins in atherosclerosis. Studies in hypercholesterolemic animal models indicate that oxidation of LDL is likely to play an important role in atherogenesis. Epidemiological investigations further suggest that the dietary intake of antioxidants is inversely associated with the risk of vascular disease, suggesting that oxidized LDL may be important in human atherosclerosis. By activating inflammatory events, oxidized lipoproteins may contribute to all stages of the atherosclerotic process. Lipoprotein oxidation is promoted by several different systems in vitro, including free and protein-bound metal ions, thiols, reactive oxygen intermediates, lipoxygenase, peroxynitrite, and myeloperoxidase. Intracellular proteins that bind iron or regulate iron metabolism might also play an important role. The physiologically relevant pathways have yet to be identified, however. We assess recent findings on the effects of antioxidants in vivo and suggest potential strategies for inhibiting oxidation in the vessel wall.

Oxidatively damaged LDL may be of central importance in atherogenesis. Epidemiological evidence suggests that high dietary intakes of beta-carotene and vitamin E decreases the risk for atherosclerotic vascular disease, raising the possibility that lipid-soluble antioxidants slow vascular disease by protecting LDL from oxidation. To test this hypothesis, we fed male New Zealand White rabbits a high-cholesterol diet or the same diet supplemented with either 1% probucol, 0.01% vitamin E, 0.01% all-trans beta-carotene, or 0.01% 9-cis beta-carotene; then we assessed both the susceptibility of LDL to oxidation ex vivo and the extent of aortic atherosclerosis. As in earlier studies, probucol protected LDL from oxidation and inhibited lesion formation. In contrast, vitamin E modestly inhibited LDL oxidation but did not prevent atherosclerosis. While beta-carotene had no effect on LDL oxidation ex vivo, the all-trans isomer inhibited lesion formation to the same degree as probucol. Moreover, all-trans beta-carotene was undetectable in LDL isolated from rabbits fed the compound, although tissue levels of retinyl palmitate were increased. The effect of all-trans beta-carotene on atherogenesis can thus be separated from the resistance of LDL to oxidation, indicating that other mechanisms may account for the ability of this compound to prevent vascular disease. Our results suggest that metabolites derived from all-trans beta-carotene inhibit atherosclerosis in hypercholesterolemic rabbits, possibly via stereospecific interactions with retinoic acid receptors in the artery wall.

Oxidatively modified lipoproteins have been implicated in atherogenesis, but the mechanisms that promote oxidation in vivo have not been identified. Myeloperoxidase, a heme protein secreted by activated macrophages, generates reactive intermediates that oxidize lipoproteins in vitro. To explore the potential role of myeloperoxidase in the development of atherosclerosis, we determined whether the enzyme was present in surgically excised human vascular tissue. In detergent extracts of atherosclerotic arteries subjected to Western blotting, a rabbit polyclonal antibody monospecific for myeloperoxidase detected a 56-kD protein, the predicted molecular mass of the heavy subunit. Both the immunoreactive protein and authentic myeloperoxidase bound to a lectin-affinity column; after elution with methyl mannoside their apparent molecular masses were indistinguishable by nondenaturing size-exclusion chromatography. Peroxidase activity in detergent extracts of atherosclerotic lesions likewise bound to a lectin column and eluted with methyl mannoside. Moreover, eluted peroxidase generated the cytotoxic oxidant hypochlorous acid (HOCl), indicating that enzymatically active myeloperoxidase was present in lesions. Patterns of immunostaining of arterial tissue with antihuman myeloperoxidase antibodies were similar to those produced by an antimacrophage antibody, and were especially prominent in the shoulder region of transitional lesions. Intense foci of myeloperoxidase immunostaining also appeared adjacent to cholesterol clefts in lipid-rich regions of advanced atherosclerotic lesions. These findings identify myeloperoxidase as a component of human vascular lesions. Because this heme protein can generate reactive species that damage lipids and proteins, myeloperoxidase may contribute to atherogenesis by catalyzing oxidative reactions in the vascular wall.