- Email: [email protected]
Oxidative stress: new approaches to diagnosis and prognosis in atherosclerosis
Oxidative Stress: New Approaches to Diagnosis and Prognosis in Atherosclerosis Jay W. Heinecke,
MD
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. 䊚2003 by Excerpta Medica, Inc. Am J Cardiol 2003;91(suppl):12A–16A
levated levels of low-density lipoproteins (LDL) appear to play a central role in the risk for atheroE sclerosis, but studies suggest that LDL itself is not
tive antioxidants can block oxidative stress in vivo.4,5 The long-term goal is to use markers to investigate the role of antioxidants in preventing human disease associated with aging.
atherogenic in vitro.1,2 These observations suggest that LDL must be modified in some way to promote atherogenesis. Many lines of evidence support the hypothesis that oxidative modifications of LDL play a critical role in the pathogenesis of atherosclerosis.2,3 The LDL oxidation hypothesis raises a key point—the need to know and understand the pathways that promote oxidative stress. The dilemma that confronts us as clinical investigators is the identification of the biologically relevant sources of oxidative stress in the human artery wall. The strategy for approaching this problem is reviewed here. To understand oxidative stress, the chemistry that is occurring in the arterial wall must be understood. It is apparent that to rationally design and test antioxidants, it is important to know what the oxidants are, analogous to identifying the infecting bacterium in order to select an antibiotic to eliminate an infection. The approach has been to use oxidized amino acids as markers for oxidative pathways by first studying oxidative chemistry in vitro and then looking for evidence of generation of these markers in vivo.3 Animal models have been used to validate the biochemical relevance of the reaction pathways for generating oxidative stress and to test the idea that putaFrom the Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington School of Medicine, Seattle, Washington, USA. This work was supported by a grant from AtheroGenics, Inc. Address for reprints: Jay W. Heinecke, MD, Box 356426, Metabolism/Endocrinology, University of Washington, Seattle, Washington 98195. E-mail: [email protected].
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©2003 by Excerpta Medica, Inc. All rights reserved.
PATHWAYS OF OXIDATIVE STRESS IN EXPERIMENTAL MODELS
Myeloperoxidase: A key event in atherogenesis is the appearance of proinflammatory macrophages in the artery wall.2,6 In fact, lipid-laden macrophages are the cellular hallmark of the early atherosclerotic lesion, and they are abundant at all stages of the atherosclerotic process. These cells possess specialized biochemical pathways that generate reactive oxygen species. In particular, macrophages use a membraneassociated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to produce superoxide that, in turn, can dismutate to form hydrogen peroxide.7 Another key component of the oxidative pathways in these cells is myeloperoxidase,8,9 a heme protein that is secreted by phagocytic white blood cells when they become activated. Myeloperoxidase uses hydrogen peroxide to generate much more potent cytotoxic oxidants.7,10 An interesting hypothesis over the last 10 years is that myeloperoxidase is a pathway for oxidative damage in the human artery wall. To test this idea, Daugherty et al8 first searched for evidence that the enzyme is expressed in human atherosclerotic lesions and found that myeloperoxidase colocalizes with macrophages in intermediate and advanced lesions. In intermediate lesions, myeloperoxidase was expressed in macrophages in the shoulder region of human atherosclerotic lesions, a region that is particularly prone to rupture that might trigger acute occlusion of the lumen 0002-9149/03/$ – see front matter PII S0002-9149(02)03145-4
of the artery. Immunoreactive protein is also present in high abundance in the core of necrotic lesions, suggesting that myeloperoxidase might play a role in more advanced atherosclerotic vascular disease. A pattern of immunostaining for myeloperoxidase was observed that was remarkably similar to that seen by others for oxidation-specific adducts in intermediate and advanced lesions.6 The similarity of these patterns to that observed for myeloperoxidase suggests that myeloperoxidase might be a pathway for oxidative damage of LDL. After this, our strategy shifted to a chemical approach, to look for molecular fingerprints that could serve as diagnostic indicators of enzymatically active myeloperoxidase. The search for such markers was greatly aided by the fact that myeloperoxidase is the only human enzyme that is known to generate hypochlorous acid under physiologically plausible conditions.7,10 Hypochlorous acid is more familiar to people as bleach. When bleach is used to sterilize water, a molecular pathway that was developed by our white blood cells long ago to kill invading bacterial organisms is recapitulated. Our approach was to search for chlorinated biomolecules as molecular fingerprints for oxidative damage by this pathway. With a long-standing interest in protein oxidation,11–13 the rationale for studying proteins as opposed to lipid oxidation was clear, in that proteins can retain the fingerprint of the initial oxidative insult that mediates damage. This contrasts with lipid peroxidation, where propagation reactions involving the initial lipid oxidation products result in loss of the information that tells us about the initial oxidative insult.14 Thus, examining the ability of myeloperoxidase to oxidatively modify the aromatic amino acid tyrosine, we were able to show in vitro that myeloperoxidase uses hydrogen peroxide and chloride ion to generate 3-chlorotyrosine.15 To investigate the role of this pathway in damaging human tissue, we used a technique called isotope dilution gas chromatography/mass spectrometry (GC/ MS) to quantify these molecules.16,17 The important point is that, in contrast to most methods used to measure oxidative stress in vivo, GC/MS is very sensitive and specific. It permits detection of trace amounts of oxidized amino acids, and because it is very specific, quantification of the analyte of interest can be ensured.16,18 Using isotope dilution GC/MS to measure 3-chlorotyrosine, we observed a remarkable 30-fold increase in the levels of 3-chlorotyrosine in lesion LDL compared with circulating LDL.17 These observations provide compelling evidence that myeloperoxidase is a pathway for oxidatively damaging LDL in the human artery wall. Tyrosyl radical, reactive nitrogen species, and hydroxyl radical: Having shown that 3-chlorotyrosine
can serve as a marker for protein damage by myeloperoxidase, this same kind of strategy was used to develop markers that could serve as indicators for protein oxidative damage by other reaction pathways. Another pathway that appears to be physiologically relevant involves a species called tyrosyl radical,
which generates another molecular fingerprint called o,o⬘-dityrosine.19,20 Other pathways of interest involve reactive nitrogen species that generate the molecular fingerprint 3-nitrotyrosine, and reduction-oxidation active metal ions and hydroxyl radical that will generate the unnatural tyrosine isomers ortho-tyrosine and meta-tyrosine.21,22 All of these oxidized amino acids have been quantified in LDL isolated from human atherosclerotic lesions and have shown that markers for reactive nitrogen species, tyrosyl radical, and myeloperoxidase are present at greatly elevated levels.12,13,17 Moreover, markers from myeloperoxidase are present at all stages of human atherosclerosis. In striking contrast, metal ion– catalyzed chemistry does not appear to be taking place in human atherosclerosis—at least not in fatty streaks or in intermediate lesions—although there is some evidence that it may be involved in advanced atherosclerotic lesions.13
CLINICAL RELEVANCE OF PATHWAYS FOR OXIDATIVE STRESS Clinical trials of vitamin E for preventing cardiac events in patients with established coronary artery disease have been largely disappointing, and this has led many to question the validity of the oxidation hypothesis of atherogenesis.23–26 Such an interpretation of the clinical studies assumes that vitamin E inhibits LDL oxidation in vivo.26 In contrast, in vitro studies reveal that vitamin E fails to inhibit LDL oxidation initiated by tyrosyl radical generated by myeloperoxidase.27 Moreover, vitamin E actually promotes LDL lipid peroxidation by many oxidation systems when the flux of radicals is low.28 These observations suggest that if myeloperoxidase and tyrosyl radical are biologically relevant, vitamin E would not be an effective mechanism to prevent LDL oxidation in the artery wall. These studies reinforce the proposition that in order to understand how to design antioxidant interventions it is necessary to know which oxidative pathways are operative in the human artery wall.
ROLE OF OXIDANTS IN PLAQUE RUPTURE It is not actually atherosclerosis that triggers acute myocardial infarction in most human beings with this disease. Instead, most clinical events appear to be precipitated by rupture of the atherosclerotic plaque.29,30 This potentially catastrophic event exposes tissue factor and other procoagulants that trigger clot formation and acute obstruction of the arterial lumen. The idea that oxidants might play a role in triggering plaque rupture in the human artery wall led to work showing that in human atherosclerotic lesions that had ruptured, there was intense peroxidase staining in the regions of the plaque that had broken down.31 Using a specific antibody, evidence was found for hypochlorous acid–modified proteins in the region of plaques that had ruptured. These observations suggested that the enzyme was present in regions
A SYMPOSIUM: CLOSED ROUNDTABLE ON ATHEROSCLEROSIS
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of the artery wall that were prone to plaque rupture, and that it was damaging proteins in these regions. Thus, oxidants generated by myeloperoxidase might play a role in triggering plaque rupture. A potential pathway involves a family of enzymes called the matrix metalloproteinases (MMPs), enzymes that are expressed in high levels in human atherosclerotic lesions, particularly in association with macrophages.30,32 It is also known that in vitro these enzymes are capable of degrading collagen and elastin, the major structural elements in the atherosclerotic lesion, suggesting that activation of these enzymes might play a role in degrading matrix and triggering plaque rupture.30,32 There are 2 highly conserved structural elements in MMPs.32 First, the enzymes are secreted in a latent or proenzyme form, and in the region of the propeptide that inactivates the enzyme, there is a highly conserved cysteine residue. In the crystal structure of these enzymes, this cysteine ligates the zinc at the catalytic site of the enzyme, inactivating the enzyme. The conventional notion of activation is that a protease clips this region of the enzyme, releasing the propeptide and exposing the active site of the enzyme. This, of course, begs the question as to where the first protease comes from. In fact, there is little in vivo evidence that this proteolytic activation mechanism is taking place. We were interested in the role of oxidants in regulating the activity of latent MMPs because cysteine is the most reactive amino acid.33 In fact, mixing 1 mol/L of hypochlorous acid with 1 mol/L of cysteine residue will quantitatively oxidize the thiol residue. We hypothesized that oxidants modify the thiol residue, causing it to lose its ability to ligate to zinc and inactivate latent MMPs. To examine this hypothesis, pro–MMP-7, which is expressed by macrophages in atherosclerotic lesions, was incubated with increasing concentrations of either hypochlorous acid or hydrogen peroxide.33 There was a striking increase in activation of the enzyme exposed to hypochlorous acid, but not the enzyme exposed to peroxide. To understand the molecular basis for this activation event, a trypsin digest of the enzyme was subjected to electrospray ionization MS and tandem MS analysis. It was predicted that if the enzyme were being activated by hypochlorous acid, 2 alterations in the protein would be observed. First, there would be oxidation of the thiol residue to an oxygenated product. Second, if oxidation were turning on the enzyme, it would cleave itself autolytically at a site called EY in the propeptide.32 It is important to note that this would generate a proteolytic cleavage site distinct from the site involved in a trypsin digest of the enzyme. Thus, if hypochlorous acid were activating the protein, a modified form of the thiol residue that incorporated oxygen atoms in concert with the appearance of the autolytic clip site in the prodomain would be expected. In contrast, if oxidation of the thiol residue by hypochlorous acid was not involved in activation, the trypsin clip site in the digest of latent MMP-7 should be seen. 14A THE AMERICAN JOURNAL OF CARDIOLOGY姞
FIGURE 1. Proposed mechanism for regulation of pro–matrix metalloproteinase-7 (pro–MMP-7) by myeloperoxidase. (Reprinted with permission from J Biol Chem.33)
Using electrospray ionization MS and tandem MS analysis, the appearance of a unique peptide was observed in pro–MMP-7 exposed to concentrations of hypochlorous acid that activated the enzyme.33 The peptide had the anticipated mass to charge ratio of the prodomain peptide that had been cleaved at EY and that had 2 oxygen atoms incorporated into the cysteine residue. To confirm this observation, the peptide was sequenced using tandem MS analysis, which showed that that cysteine residue was selectively modified by a gain of 32 atomic mass units. Recalling that oxygen has an atomic weight of 16, this strongly suggests that the thiol side chain of the amino acid was converted to a doubly oxygenated product. Looking at loss of the precursor peptide and gain of the oxygenated cleaved switch peptide, a reciprocal relation was observed that was optimal at the concentration of hypochlorous acid that optimally activates the enzyme. In summary, these and other studies suggest that oxidative modifications of latent MMP may be a mechanism for triggering plaque rupture in the human artery wall.32 Fu et al33 propose a mechanism that involves oxygenation of the cysteine by hypochlorous acid to the sulfonic acid derivative (Figure 1). This in turn exposes the zinc atom at the active site of the enzyme, which autolytically cleaves the enzyme, releasing the prodomain peptide and generating active enzyme. This mechanism may be biologically relevant because MMP-7 and myeloperoxidase colocalize in macrophages in the shoulder region of fibrous plaques in human atherosclerotic lesions, a region known to be prone to plaque rupture. We are currently investigating the relevance of this pathway in the human artery wall, using tandem MS analysis to look for the oxygenated, autolytically cleaved prodomain of pro–MMP-7.
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ASSESSING THE IN VIVO EFFECTIVENESS OF ANTIOXIDANT INTERVENTIONS
CONCLUSION
Because a key issue of interest is determining whether antioxidant interventions are effective at blocking oxidative stress in vivo, a noninvasive approach to this might be a powerful strategy for trying to understand the role of reactive oxygen stress in vivo. Our strategy has been to look for evidence of oxidized amino acids in urine or plasma.34 –36 Although this review focuses on studies in urine, similar studies are being conducted that also look at plasma in animal models of inflammation as well as in humans. By using MS to quantify these markers, this might be a noninvasive way to assess oxidative stress in vivo. A pathway of interest involves the conversion of tyrosine by myeloperoxidase into a tyrosyl radical.16,19,20 The tyrosyl radical in turn can undergo a radical–radical recombination reaction to generate o,o⬘-dityrosine. This molecule has been detected at high levels in human atherosclerotic lesions, suggesting that tyrosyl radical is another pathway for damaging LDL in the artery wall.16 To explore the role of o,o⬘-dityrosine as a marker for oxidative stress in vivo, a mouse model of inflammation in the peritoneum was developed and levels of the oxidized amino acid in leukocytes isolated from this peritoneal inflammatory infiltrate were quantified.36 In resident peritoneal leukocytes, there was a baseline protein-bound level of o,o⬘-dityrosine that was increased about 3-fold by activating these cells in situ. This increase was completely blocked in mice made deficient in NADPH oxidase. These results suggest that activation of leukocytes in vivo can generate o,o⬘-dityrosine by a pathway that is completely dependent on NADPH oxidase. To determine whether quantifying o,o⬘-dityrosine in urine could be used as a noninvasive way to assess oxidative stress, urine was collected from these animals after triggering inflammation in the peritoneum, and levels of o,o⬘-dityrosine were measured using isotope dilution GC/MS.36 A marked increase was observed in urinary levels of o,o⬘-dityrosine in animals with leukocytes recruited into the peritoneum and activated in situ. In mice that are deficient in NADPH oxidase, the increase in urinary o,o⬘-dityrosine was completely blocked. These observations suggest that it might be possible to use urine levels of oxidized amino acids as a surrogate marker for monitoring oxidative stress in vivo. They also indicate that NADPH oxidase is involved in forming o,o⬘-dityrosine in this model system of acute inflammation. There is preliminary evidence that these observations may be relevant to human disease. Levels of o,o⬘-dityrosine have been measured in urine from patients who are in the intensive care unit with and without acute sepsis (as assessed by clinical criteria). There was a 2- to 3-fold increase in the levels of o,o⬘-dityrosine in the urine of septic humans.36 These results suggest that acute inflammation mediated by phagocytes may, in fact, be triggering o,o⬘-dityrosine formation in humans.
Oxidized amino acids in urine and in plasma may be useful surrogate markers for studying the pathways that promote oxidative stress in vivo.33–36 The advantage of looking at oxidized amino acids is that it permits us to study specific reaction pathways, such as myeloperoxidase, reactive nitrogen species, or tyrosyl radical. Preliminary evidence in humans suggests that o,o⬘-dityrosine, a marker for tyrosyl radical, increases in urine during acute inflammation. Mouse studies suggest that the pathway is dependent on the phagocyte NADPH oxidase. It will be of interest to extend studies in humans, with the long-term goals of investigating the role of reactive oxygen species in the pathogenesis of human disease. It may also be possible to use oxidized amino acids as surrogate markers to establish the efficacy of proposed antioxidant interventions. In concert with other markers of oxidative stress, such as products of lipid peroxidation, the quantification of oxidized amino acids may represent a powerful strategy for evaluating the effectiveness of antioxidant interventions. 1. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34 –47. 2. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991;88:1785–1790. 3. Heinecke JW. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis 1998;141:1–15. 4. Gaut JP, Byun J, Tran HD, Lauber WM, Carroll JA, Hotchkiss RS, Belaaouaj A, Heinecke JW. Myeloperoxidase produces nitrating oxidants in vivo. J Clin Invest 2002;109:1311–1319. 5. Gaut JP, Yeh GC, Tran HD, Byun J, Henderson JP, Richter GM, Belaaoua A, Hotchkiss RS, Heinecke JW. Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis. Proc Natl Acad Sci U S A 2001;98:11961–11966. 6. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein-B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis 1990; 10:336 –349. 7. Hurst JK, Barette WC Jr. Leukocyte oxygen activation and microbicidal oxidative toxins. Crit Rev Biochem Mol Biol 1989;24:271–328. 8. Daugherty A, Rateri DL, Dunn JL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest 1994;94:437–444. 9. Brown KE, Brunt EM, Heinecke JW. Immunohistochemical detection of myeloperoxidase and its oxidation products in Kupffer cells of human liver. Am J Pathol 2001;159:2081–2088. 10. Harrison JE, Schultz J. Studies on the chlorinating activity of myeloperoxidase. J Biol Chem 1976;251:1371–1376. 11. Heinecke JW, Li W, Francis GA, Goldstein JA. Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative crosslinking of proteins. J Clin Invest 1993;91:2866 –2872. 12. Leeuwenburgh C, Hardy MM, Hazen SL, Wagner P, Oh-ishi S, Steinbrecher UP, Heinecke JW. Reactive nitrogen intermediates promote low density lipoprotein oxidation in human atherosclerotic intima. J Biol Chem 1997;272:1433– 1436. 13. Leeuwenburgh C, Rasmussen JE, Hsu FF, Mueller DM, Pennathur S, Heinecke JW. Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J Biol Chem 1997;272:3520 –3526. 14. Morrow JD, Roberts LJ. The isoprostanes. Current knowledge and directions for future research. Biochem Pharmacol 1996;51:1–9. 15. Hazen SL, Hsu FF, Mueller DM, Crowley JR, Heinecke JW. Human neutrophils employ chlorine gas as an oxidant during phagocytosis. J Clin Invest 1996;98:1283–1289. 16. Heinecke JW, Hsu FF, Crowley JR, Hazen SL, Leeuwenburgh C, Mueller DM, Rasmussen JE, Turk J. Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: sensitive and quantitative assays for oxidized amino acids in proteins and tissues. Methods Enzymol 1998;300:124 – 144.
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17. Hazen SL, Heinecke JW. 3-Chlorotyrosine, a specific marker of myeloper-
oxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J Clin Invest 1997;99:2075–2081. 18. McCloskey JA. Mass spectrometry. San Diego, Academic Press, 1990. 19. Heinecke JW, Li W, Daehnke HL, Goldstein JA. o,o⬘-Dityrosine, a specific marker of oxidation, is synthesized by the myeloperoxidase-hydrogen peroxide system of human neutrophils and macrophages. J Biol Chem 1993;268:4069 –4077. 20. McCormick ML, Gaut JP, Lin T, Britigan BE, Buettner GR, Heinecke JW. Electron paramagnetic resonance detection of free tyrosyl radical generated by myeloperoxidase, lactoperoxidase, and horseradish peroxidase. J Biol Chem 1998;273:32030 –32037. 21. Beckman JS, Chen J, Ischiropoulos H, Crow JP. Oxidative chemistry of peroxynitrite. Methods Enzymol 1994;233:229 –240. 22. Huggins TG, Wells-Knecht MC, Detorie NA, Baynes JW, Thorpe SR. Formation of o-tyrosine and o,o⬘-dityrosine in proteins during radiolytic and metal-catalyzed oxidation. J Biol Chem 1993;268:12341–12347. 23. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico. Lancet 1999;354: 447–455. 24. Yusuf S, Dagenais G, Pogue J, Bosch J, Sleight P. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med 2000;342:154 –160. 25. de Gaetano G. Low-dose aspirin and vitamin E in people at cardiovascular risk: a randomised trial in general practice. Collaborative Group of the Primary Prevention Project. Lancet 2001;357:89 –95. 26. Heinecke JW. Is the emperor wearing clothes? Clinical trials of vitamin E and the LDL oxidation hypothesis. Arterioscler Thromb Vasc Biol 2001;21:1261– 1264. 27. Savenkova MI, Mueller DM, Heinecke JW. Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation in low density lipoprotein. J Biol Chem 1994;269:20394 –20400.
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28. Thomas SR, Stocker R. Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis. Free Radic Biol Med 2000;28:1795–1805. 29. Cho A, Graves J, Reidy MA. Mitogen-activated protein kinases mediate
matrix metalloproteinase-9 expression in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2000;20:2527–2532. 30. Aikawa M, Rabkin E, Sugiyama S, Voglic SJ, Fukumoto Y, Furukawa Y, Shiomi M, Schoen FJ, Libby P. An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro. Circulation 2001;103:276 –283. 31. Sugiyama S, Okada Y, Sukhova GK, Virmani R, Heinecke JW, Libby P. Macrophage myeloperoxidase regulation by granulocyte macrophage-colony stimulating factor in human atherosclerosis and implications in acute coronary syndromes. Am J Pathol 2001;158:879 –891. 32. Woessner JF, Nagase H. Matrix Metalloproteinases and TIMPs. New York: Oxford University Press, 2000. 33. Fu X, Kassim SY, Parks WC, Heinecke JW. Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase. J Biol Chem 2001;276:41279 –41287. 34. Leeuwenburgh C, Hansen PA, Holloszy JO, Heinecke JW. Oxidized amino acids in the urine of aging rats: potential markers for assessing oxidative stress in vivo. Am J Physiol 1999;276:R128 –R135. 35. Gaut JP, Byun J, Tran HD, Heinecke JW. Artifact-free quantification of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma by electron capture-negative chemical ionization gas chromatography/mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry. Anal Biochem 2002;300:252–259. 36. Bhattacharjee S, Pennathur S, Byun J, Crowley J, Mueller D, Gischler J, Hotchkiss RS, Heinecke JW. The NADPH oxidase of neutrophils elevates o,o⬘dityrosine cross-links in proteins and urine during inflammation. Arch Biochem Biophys 2001;395:69 –77.
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MD
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. 䊚2003 by Excerpta Medica, Inc. Am J Cardiol 2003;91(suppl):12A–16A
levated levels of low-density lipoproteins (LDL) appear to play a central role in the risk for atheroE sclerosis, but studies suggest that LDL itself is not
tive antioxidants can block oxidative stress in vivo.4,5 The long-term goal is to use markers to investigate the role of antioxidants in preventing human disease associated with aging.
atherogenic in vitro.1,2 These observations suggest that LDL must be modified in some way to promote atherogenesis. Many lines of evidence support the hypothesis that oxidative modifications of LDL play a critical role in the pathogenesis of atherosclerosis.2,3 The LDL oxidation hypothesis raises a key point—the need to know and understand the pathways that promote oxidative stress. The dilemma that confronts us as clinical investigators is the identification of the biologically relevant sources of oxidative stress in the human artery wall. The strategy for approaching this problem is reviewed here. To understand oxidative stress, the chemistry that is occurring in the arterial wall must be understood. It is apparent that to rationally design and test antioxidants, it is important to know what the oxidants are, analogous to identifying the infecting bacterium in order to select an antibiotic to eliminate an infection. The approach has been to use oxidized amino acids as markers for oxidative pathways by first studying oxidative chemistry in vitro and then looking for evidence of generation of these markers in vivo.3 Animal models have been used to validate the biochemical relevance of the reaction pathways for generating oxidative stress and to test the idea that putaFrom the Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington School of Medicine, Seattle, Washington, USA. This work was supported by a grant from AtheroGenics, Inc. Address for reprints: Jay W. Heinecke, MD, Box 356426, Metabolism/Endocrinology, University of Washington, Seattle, Washington 98195. E-mail: [email protected].
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©2003 by Excerpta Medica, Inc. All rights reserved.
PATHWAYS OF OXIDATIVE STRESS IN EXPERIMENTAL MODELS
Myeloperoxidase: A key event in atherogenesis is the appearance of proinflammatory macrophages in the artery wall.2,6 In fact, lipid-laden macrophages are the cellular hallmark of the early atherosclerotic lesion, and they are abundant at all stages of the atherosclerotic process. These cells possess specialized biochemical pathways that generate reactive oxygen species. In particular, macrophages use a membraneassociated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to produce superoxide that, in turn, can dismutate to form hydrogen peroxide.7 Another key component of the oxidative pathways in these cells is myeloperoxidase,8,9 a heme protein that is secreted by phagocytic white blood cells when they become activated. Myeloperoxidase uses hydrogen peroxide to generate much more potent cytotoxic oxidants.7,10 An interesting hypothesis over the last 10 years is that myeloperoxidase is a pathway for oxidative damage in the human artery wall. To test this idea, Daugherty et al8 first searched for evidence that the enzyme is expressed in human atherosclerotic lesions and found that myeloperoxidase colocalizes with macrophages in intermediate and advanced lesions. In intermediate lesions, myeloperoxidase was expressed in macrophages in the shoulder region of human atherosclerotic lesions, a region that is particularly prone to rupture that might trigger acute occlusion of the lumen 0002-9149/03/$ – see front matter PII S0002-9149(02)03145-4
of the artery. Immunoreactive protein is also present in high abundance in the core of necrotic lesions, suggesting that myeloperoxidase might play a role in more advanced atherosclerotic vascular disease. A pattern of immunostaining for myeloperoxidase was observed that was remarkably similar to that seen by others for oxidation-specific adducts in intermediate and advanced lesions.6 The similarity of these patterns to that observed for myeloperoxidase suggests that myeloperoxidase might be a pathway for oxidative damage of LDL. After this, our strategy shifted to a chemical approach, to look for molecular fingerprints that could serve as diagnostic indicators of enzymatically active myeloperoxidase. The search for such markers was greatly aided by the fact that myeloperoxidase is the only human enzyme that is known to generate hypochlorous acid under physiologically plausible conditions.7,10 Hypochlorous acid is more familiar to people as bleach. When bleach is used to sterilize water, a molecular pathway that was developed by our white blood cells long ago to kill invading bacterial organisms is recapitulated. Our approach was to search for chlorinated biomolecules as molecular fingerprints for oxidative damage by this pathway. With a long-standing interest in protein oxidation,11–13 the rationale for studying proteins as opposed to lipid oxidation was clear, in that proteins can retain the fingerprint of the initial oxidative insult that mediates damage. This contrasts with lipid peroxidation, where propagation reactions involving the initial lipid oxidation products result in loss of the information that tells us about the initial oxidative insult.14 Thus, examining the ability of myeloperoxidase to oxidatively modify the aromatic amino acid tyrosine, we were able to show in vitro that myeloperoxidase uses hydrogen peroxide and chloride ion to generate 3-chlorotyrosine.15 To investigate the role of this pathway in damaging human tissue, we used a technique called isotope dilution gas chromatography/mass spectrometry (GC/ MS) to quantify these molecules.16,17 The important point is that, in contrast to most methods used to measure oxidative stress in vivo, GC/MS is very sensitive and specific. It permits detection of trace amounts of oxidized amino acids, and because it is very specific, quantification of the analyte of interest can be ensured.16,18 Using isotope dilution GC/MS to measure 3-chlorotyrosine, we observed a remarkable 30-fold increase in the levels of 3-chlorotyrosine in lesion LDL compared with circulating LDL.17 These observations provide compelling evidence that myeloperoxidase is a pathway for oxidatively damaging LDL in the human artery wall. Tyrosyl radical, reactive nitrogen species, and hydroxyl radical: Having shown that 3-chlorotyrosine
can serve as a marker for protein damage by myeloperoxidase, this same kind of strategy was used to develop markers that could serve as indicators for protein oxidative damage by other reaction pathways. Another pathway that appears to be physiologically relevant involves a species called tyrosyl radical,
which generates another molecular fingerprint called o,o⬘-dityrosine.19,20 Other pathways of interest involve reactive nitrogen species that generate the molecular fingerprint 3-nitrotyrosine, and reduction-oxidation active metal ions and hydroxyl radical that will generate the unnatural tyrosine isomers ortho-tyrosine and meta-tyrosine.21,22 All of these oxidized amino acids have been quantified in LDL isolated from human atherosclerotic lesions and have shown that markers for reactive nitrogen species, tyrosyl radical, and myeloperoxidase are present at greatly elevated levels.12,13,17 Moreover, markers from myeloperoxidase are present at all stages of human atherosclerosis. In striking contrast, metal ion– catalyzed chemistry does not appear to be taking place in human atherosclerosis—at least not in fatty streaks or in intermediate lesions—although there is some evidence that it may be involved in advanced atherosclerotic lesions.13
CLINICAL RELEVANCE OF PATHWAYS FOR OXIDATIVE STRESS Clinical trials of vitamin E for preventing cardiac events in patients with established coronary artery disease have been largely disappointing, and this has led many to question the validity of the oxidation hypothesis of atherogenesis.23–26 Such an interpretation of the clinical studies assumes that vitamin E inhibits LDL oxidation in vivo.26 In contrast, in vitro studies reveal that vitamin E fails to inhibit LDL oxidation initiated by tyrosyl radical generated by myeloperoxidase.27 Moreover, vitamin E actually promotes LDL lipid peroxidation by many oxidation systems when the flux of radicals is low.28 These observations suggest that if myeloperoxidase and tyrosyl radical are biologically relevant, vitamin E would not be an effective mechanism to prevent LDL oxidation in the artery wall. These studies reinforce the proposition that in order to understand how to design antioxidant interventions it is necessary to know which oxidative pathways are operative in the human artery wall.
ROLE OF OXIDANTS IN PLAQUE RUPTURE It is not actually atherosclerosis that triggers acute myocardial infarction in most human beings with this disease. Instead, most clinical events appear to be precipitated by rupture of the atherosclerotic plaque.29,30 This potentially catastrophic event exposes tissue factor and other procoagulants that trigger clot formation and acute obstruction of the arterial lumen. The idea that oxidants might play a role in triggering plaque rupture in the human artery wall led to work showing that in human atherosclerotic lesions that had ruptured, there was intense peroxidase staining in the regions of the plaque that had broken down.31 Using a specific antibody, evidence was found for hypochlorous acid–modified proteins in the region of plaques that had ruptured. These observations suggested that the enzyme was present in regions
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of the artery wall that were prone to plaque rupture, and that it was damaging proteins in these regions. Thus, oxidants generated by myeloperoxidase might play a role in triggering plaque rupture. A potential pathway involves a family of enzymes called the matrix metalloproteinases (MMPs), enzymes that are expressed in high levels in human atherosclerotic lesions, particularly in association with macrophages.30,32 It is also known that in vitro these enzymes are capable of degrading collagen and elastin, the major structural elements in the atherosclerotic lesion, suggesting that activation of these enzymes might play a role in degrading matrix and triggering plaque rupture.30,32 There are 2 highly conserved structural elements in MMPs.32 First, the enzymes are secreted in a latent or proenzyme form, and in the region of the propeptide that inactivates the enzyme, there is a highly conserved cysteine residue. In the crystal structure of these enzymes, this cysteine ligates the zinc at the catalytic site of the enzyme, inactivating the enzyme. The conventional notion of activation is that a protease clips this region of the enzyme, releasing the propeptide and exposing the active site of the enzyme. This, of course, begs the question as to where the first protease comes from. In fact, there is little in vivo evidence that this proteolytic activation mechanism is taking place. We were interested in the role of oxidants in regulating the activity of latent MMPs because cysteine is the most reactive amino acid.33 In fact, mixing 1 mol/L of hypochlorous acid with 1 mol/L of cysteine residue will quantitatively oxidize the thiol residue. We hypothesized that oxidants modify the thiol residue, causing it to lose its ability to ligate to zinc and inactivate latent MMPs. To examine this hypothesis, pro–MMP-7, which is expressed by macrophages in atherosclerotic lesions, was incubated with increasing concentrations of either hypochlorous acid or hydrogen peroxide.33 There was a striking increase in activation of the enzyme exposed to hypochlorous acid, but not the enzyme exposed to peroxide. To understand the molecular basis for this activation event, a trypsin digest of the enzyme was subjected to electrospray ionization MS and tandem MS analysis. It was predicted that if the enzyme were being activated by hypochlorous acid, 2 alterations in the protein would be observed. First, there would be oxidation of the thiol residue to an oxygenated product. Second, if oxidation were turning on the enzyme, it would cleave itself autolytically at a site called EY in the propeptide.32 It is important to note that this would generate a proteolytic cleavage site distinct from the site involved in a trypsin digest of the enzyme. Thus, if hypochlorous acid were activating the protein, a modified form of the thiol residue that incorporated oxygen atoms in concert with the appearance of the autolytic clip site in the prodomain would be expected. In contrast, if oxidation of the thiol residue by hypochlorous acid was not involved in activation, the trypsin clip site in the digest of latent MMP-7 should be seen. 14A THE AMERICAN JOURNAL OF CARDIOLOGY姞
FIGURE 1. Proposed mechanism for regulation of pro–matrix metalloproteinase-7 (pro–MMP-7) by myeloperoxidase. (Reprinted with permission from J Biol Chem.33)
Using electrospray ionization MS and tandem MS analysis, the appearance of a unique peptide was observed in pro–MMP-7 exposed to concentrations of hypochlorous acid that activated the enzyme.33 The peptide had the anticipated mass to charge ratio of the prodomain peptide that had been cleaved at EY and that had 2 oxygen atoms incorporated into the cysteine residue. To confirm this observation, the peptide was sequenced using tandem MS analysis, which showed that that cysteine residue was selectively modified by a gain of 32 atomic mass units. Recalling that oxygen has an atomic weight of 16, this strongly suggests that the thiol side chain of the amino acid was converted to a doubly oxygenated product. Looking at loss of the precursor peptide and gain of the oxygenated cleaved switch peptide, a reciprocal relation was observed that was optimal at the concentration of hypochlorous acid that optimally activates the enzyme. In summary, these and other studies suggest that oxidative modifications of latent MMP may be a mechanism for triggering plaque rupture in the human artery wall.32 Fu et al33 propose a mechanism that involves oxygenation of the cysteine by hypochlorous acid to the sulfonic acid derivative (Figure 1). This in turn exposes the zinc atom at the active site of the enzyme, which autolytically cleaves the enzyme, releasing the prodomain peptide and generating active enzyme. This mechanism may be biologically relevant because MMP-7 and myeloperoxidase colocalize in macrophages in the shoulder region of fibrous plaques in human atherosclerotic lesions, a region known to be prone to plaque rupture. We are currently investigating the relevance of this pathway in the human artery wall, using tandem MS analysis to look for the oxygenated, autolytically cleaved prodomain of pro–MMP-7.
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ASSESSING THE IN VIVO EFFECTIVENESS OF ANTIOXIDANT INTERVENTIONS
CONCLUSION
Because a key issue of interest is determining whether antioxidant interventions are effective at blocking oxidative stress in vivo, a noninvasive approach to this might be a powerful strategy for trying to understand the role of reactive oxygen stress in vivo. Our strategy has been to look for evidence of oxidized amino acids in urine or plasma.34 –36 Although this review focuses on studies in urine, similar studies are being conducted that also look at plasma in animal models of inflammation as well as in humans. By using MS to quantify these markers, this might be a noninvasive way to assess oxidative stress in vivo. A pathway of interest involves the conversion of tyrosine by myeloperoxidase into a tyrosyl radical.16,19,20 The tyrosyl radical in turn can undergo a radical–radical recombination reaction to generate o,o⬘-dityrosine. This molecule has been detected at high levels in human atherosclerotic lesions, suggesting that tyrosyl radical is another pathway for damaging LDL in the artery wall.16 To explore the role of o,o⬘-dityrosine as a marker for oxidative stress in vivo, a mouse model of inflammation in the peritoneum was developed and levels of the oxidized amino acid in leukocytes isolated from this peritoneal inflammatory infiltrate were quantified.36 In resident peritoneal leukocytes, there was a baseline protein-bound level of o,o⬘-dityrosine that was increased about 3-fold by activating these cells in situ. This increase was completely blocked in mice made deficient in NADPH oxidase. These results suggest that activation of leukocytes in vivo can generate o,o⬘-dityrosine by a pathway that is completely dependent on NADPH oxidase. To determine whether quantifying o,o⬘-dityrosine in urine could be used as a noninvasive way to assess oxidative stress, urine was collected from these animals after triggering inflammation in the peritoneum, and levels of o,o⬘-dityrosine were measured using isotope dilution GC/MS.36 A marked increase was observed in urinary levels of o,o⬘-dityrosine in animals with leukocytes recruited into the peritoneum and activated in situ. In mice that are deficient in NADPH oxidase, the increase in urinary o,o⬘-dityrosine was completely blocked. These observations suggest that it might be possible to use urine levels of oxidized amino acids as a surrogate marker for monitoring oxidative stress in vivo. They also indicate that NADPH oxidase is involved in forming o,o⬘-dityrosine in this model system of acute inflammation. There is preliminary evidence that these observations may be relevant to human disease. Levels of o,o⬘-dityrosine have been measured in urine from patients who are in the intensive care unit with and without acute sepsis (as assessed by clinical criteria). There was a 2- to 3-fold increase in the levels of o,o⬘-dityrosine in the urine of septic humans.36 These results suggest that acute inflammation mediated by phagocytes may, in fact, be triggering o,o⬘-dityrosine formation in humans.
Oxidized amino acids in urine and in plasma may be useful surrogate markers for studying the pathways that promote oxidative stress in vivo.33–36 The advantage of looking at oxidized amino acids is that it permits us to study specific reaction pathways, such as myeloperoxidase, reactive nitrogen species, or tyrosyl radical. Preliminary evidence in humans suggests that o,o⬘-dityrosine, a marker for tyrosyl radical, increases in urine during acute inflammation. Mouse studies suggest that the pathway is dependent on the phagocyte NADPH oxidase. It will be of interest to extend studies in humans, with the long-term goals of investigating the role of reactive oxygen species in the pathogenesis of human disease. It may also be possible to use oxidized amino acids as surrogate markers to establish the efficacy of proposed antioxidant interventions. In concert with other markers of oxidative stress, such as products of lipid peroxidation, the quantification of oxidized amino acids may represent a powerful strategy for evaluating the effectiveness of antioxidant interventions. 1. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 1986;232:34 –47. 2. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991;88:1785–1790. 3. Heinecke JW. Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis 1998;141:1–15. 4. Gaut JP, Byun J, Tran HD, Lauber WM, Carroll JA, Hotchkiss RS, Belaaouaj A, Heinecke JW. Myeloperoxidase produces nitrating oxidants in vivo. J Clin Invest 2002;109:1311–1319. 5. Gaut JP, Yeh GC, Tran HD, Byun J, Henderson JP, Richter GM, Belaaoua A, Hotchkiss RS, Heinecke JW. Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis. Proc Natl Acad Sci U S A 2001;98:11961–11966. 6. Rosenfeld ME, Palinski W, Yla-Herttuala S, Butler S, Witztum JL. Distribution of oxidation specific lipid-protein adducts and apolipoprotein-B in atherosclerotic lesions of varying severity from WHHL rabbits. Arteriosclerosis 1990; 10:336 –349. 7. Hurst JK, Barette WC Jr. Leukocyte oxygen activation and microbicidal oxidative toxins. Crit Rev Biochem Mol Biol 1989;24:271–328. 8. Daugherty A, Rateri DL, Dunn JL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest 1994;94:437–444. 9. Brown KE, Brunt EM, Heinecke JW. Immunohistochemical detection of myeloperoxidase and its oxidation products in Kupffer cells of human liver. Am J Pathol 2001;159:2081–2088. 10. Harrison JE, Schultz J. Studies on the chlorinating activity of myeloperoxidase. J Biol Chem 1976;251:1371–1376. 11. Heinecke JW, Li W, Francis GA, Goldstein JA. Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative crosslinking of proteins. J Clin Invest 1993;91:2866 –2872. 12. Leeuwenburgh C, Hardy MM, Hazen SL, Wagner P, Oh-ishi S, Steinbrecher UP, Heinecke JW. Reactive nitrogen intermediates promote low density lipoprotein oxidation in human atherosclerotic intima. J Biol Chem 1997;272:1433– 1436. 13. Leeuwenburgh C, Rasmussen JE, Hsu FF, Mueller DM, Pennathur S, Heinecke JW. Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J Biol Chem 1997;272:3520 –3526. 14. Morrow JD, Roberts LJ. The isoprostanes. Current knowledge and directions for future research. Biochem Pharmacol 1996;51:1–9. 15. Hazen SL, Hsu FF, Mueller DM, Crowley JR, Heinecke JW. Human neutrophils employ chlorine gas as an oxidant during phagocytosis. J Clin Invest 1996;98:1283–1289. 16. Heinecke JW, Hsu FF, Crowley JR, Hazen SL, Leeuwenburgh C, Mueller DM, Rasmussen JE, Turk J. Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: sensitive and quantitative assays for oxidized amino acids in proteins and tissues. Methods Enzymol 1998;300:124 – 144.
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