Baohai Shao

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

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 μmol/mol Tyr) than that in circulating HDL (104 ± 11 μmol/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.

Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I.

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, protein-bound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo.

Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I

HDL protects against vascular disease by accepting free cholesterol from macrophage foam cells in the artery wall. This pathway is critically dependent on lecithin:cholesterol acyltransferase (LCAT), which rapidly converts cholesterol to cholesteryl ester. The physiological activator of LCAT is apolipoprotein A-I (apoA-I), the major HDL protein. However, cholesterol removal is compromised if apoA-I is exposed to reactive intermediates. In humans with established cardiovascular disease, myeloperoxidase (MPO) oxidizes HDL, and oxidation by MPO impairs apoA-Is ability to activate LCAT in vitro. Because a single methionine residue in apoA-I, Met-148, resides near the center of the proteins LCAT activation domain, we determined whether its oxidation by MPO could account for the loss of LCAT activity. Mass spectrometric analysis demonstrated that oxidation of Met-148 to methionine sulfoxide associated quantitatively with loss of LCAT activity in both discoidal HDL and HDL3, the enzymes physiological substrates. Reversing oxidation with methionine sulfoxide reductase restored HDLs ability to activate LCAT. Discoidal HDL prepared with apoA-I containing a Met-148→Leu mutation was significantly resistant to inactivation by MPO. Based on structural data in the literature, we propose that oxidation of Met-148 disrupts apoA-Is central loop, which overlaps the LCAT activation domain. These observations implicate oxidation of a single Met in apoA-I in impaired LCAT activation, a critical early step in reverse cholesterol transport.

Myeloperoxidase: An Oxidative Pathway for Generating Dysfunctional High-Density Lipoprotein

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by artery wall macrophages triggers atherosclerosis, the leading cause of cardiovascular disease. Conversely, high-density lipoprotein (HDL) retards atherosclerosis by promoting cholesterol efflux from macrophages by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway. HDL has been proposed to lose its cardioprotective effects in subjects with atherosclerosis, but the underlying mechanisms are poorly understood. One potential pathway involves oxidative damage by myeloperoxidase (MPO), a heme enzyme secreted by human artery wall macrophages. We used mass spectrometry to demonstrate that HDL isolated from patients with established cardiovascular disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of MPO. When apolipoprotein A-I (apoA-I), the major HDL protein, was oxidized by MPO, its ability to promote cellular cholesterol efflux by ABCA1 was impaired. Moreover, oxidized apoA-I was unable to activate lecithin:cholesterol acyltransferase (LCAT), which rapidly converts free cholesterol to cholesteryl ester, a critical step in HDL maturation. Biochemical studies implicated tyrosine chlorination and methionine oxygenation in the loss of ABCA1 and LCAT activity by oxidized apoA-I. Oxidation of specific residues in apoA-I inhibited two key steps in cholesterol efflux from macrophages, raising the possibility that MPO initiates a pathway for generating dysfunctional HDL in humans.

Myeloperoxidase Targets Apolipoprotein A-I, the Major High Density Lipoprotein Protein, for Site-Specific Oxidation in Human Atherosclerotic Lesions

Background: Oxidation of apolipoprotein A-I by myeloperoxidase has been proposed to deprive HDL of its cardioprotective effects. Results: Tyrosine 192 is the major site of chlorination in apoA-I in both plasma and lesion HDL isolated from humans. Conclusion: Chlorination of apolipoprotein A-I by myeloperoxidase may contribute to generation of a dysfunctional form of HDL in vivo. Significance: Quantifying apolipoprotein A-I chlorination might help diagnose and perhaps treat human cardiovascular disease. Oxidative damage by myeloperoxidase (MPO) has been proposed to deprive HDL of its cardioprotective effects. In vitro studies reveal that MPO chlorinates and nitrates specific tyrosine residues of apoA-I, the major HDL protein. After Tyr-192 is chlorinated, apoA-I is less able to promote cholesterol efflux by the ABCA1 pathway. To investigate the potential role of this pathway in vivo, we used tandem mass spectrometry with selected reaction monitoring to quantify the regiospecific oxidation of apoA-I. This approach demonstrated that Tyr-192 is the major chlorination site in apoA-I in both plasma and lesion HDL of humans. We also found that Tyr-192 is the major nitration site in apoA-I of circulating HDL but that Tyr-18 is the major site in lesion HDL. Levels of 3-nitrotyrosine strongly correlated with levels of 3-chlorotyrosine in lesion HDL, and Tyr-18 of apoA-I was the major nitration site in HDL exposed to MPO in vitro, suggesting that MPO is the major pathway for chlorination and nitration of HDL in human atherosclerotic tissue. These observations may have implications for treating cardiovascular disease, because recombinant apoA-I is under investigation as a therapeutic agent and mutant forms of apoA-I that resist oxidation might be more cardioprotective than the native form.

Modifying Apolipoprotein A-I by Malondialdehyde, but Not by an Array of Other Reactive Carbonyls, Blocks Cholesterol Efflux by the ABCA1 Pathway

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.

Myeloperoxidase Inactivates TIMP-1 by Oxidizing Its N-terminal Cysteine Residue AN OXIDATIVE MECHANISM FOR REGULATING PROTEOLYSIS DURING INFLAMMATION

An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation.

HDL, lipid peroxidation, and atherosclerosis

Genetic, clinical, and pharmacological studies implicate elevated levels of LDL in the pathogenesis of atherosclerotic vascular disease, the leading cause of death in industrialized societies (1). Paradoxically, native LDL fails to exert potentially atherogenic effects in vitro, suggesting that it must be modified to promote vascular disease. Indeed, many lines of evidence support the LDL oxidation hypothesis, which suggests that oxidative damage to LDL is one important mechanism for rendering lipoproteins atherogenic (as reviewed in Refs. 2, 3).

The Interplay between Size, Morphology, Stability, and Functionality of High-Density Lipoprotein Subclasses

High-density lipoprotein (HDL) mediates reverse cholesterol transport (RCT), wherein excess cholesterol is conveyed from peripheral tissues to the liver and steroidogenic organs. During this process HDL continually transitions between subclass sizes, each with unique biological activities. For instance, RCT is initiated by the interaction of lipid-free/lipid-poor apolipoprotein A-I (apoA-I) with ABCA1, a membrane-associated lipid transporter, to form nascent HDL. Because nearly all circulating apoA-I is lipid-bound, the source of lipid-free/lipid-poor apoA-I is unclear. Lecithin:cholesterol acyltransferase (LCAT) then drives the conversion of nascent HDL to spherical HDL by catalyzing cholesterol esterification, an essential step in RCT. To investigate the relationship between HDL particle size and events critical to RCT such as LCAT activation and lipid-free apoA-I production for ABCA1 interaction, we reconstituted five subclasses of HDL particles (rHDL of 7.8, 8.4, 9.6, 12.2, and 17.0 nm in diameter, respectively) using various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, free cholesterol, and apoA-I. Kinetic analyses of this comprehensive array of rHDL particles suggest that apoA-I stoichiometry in rHDL is a critical factor governing LCAT activation. Electron microscopy revealed specific morphological differences in the HDL subclasses that may affect functionality. Furthermore, stability measurements demonstrated that the previously uncharacterized 8.4 nm rHDL particles rapidly convert to 7.8 nm particles, concomitant with the dissociation of lipid-free/lipid-poor apoA-I. Thus, lipid-free/lipid-poor apoA-I generated by the remodeling of HDL may be an essential intermediate in RCT and HDLs in vivo maturation.

Humans With Atherosclerosis Have Impaired ABCA1 Cholesterol Efflux and Enhanced High-Density Lipoprotein Oxidation by Myeloperoxidase

Rationale: The efflux capacity of high-density lipoprotein (HDL) with cultured macrophages associates strongly and negatively with coronary artery disease status, indicating that impaired sterol efflux capacity might be a marker—and perhaps mediator—of atherosclerotic burden. However, the mechanisms that contribute to impaired sterol efflux capacity remain poorly understood. Objective: Our aim was to determine the relationship between myeloperoxidase-mediated oxidative damage to apolipoprotein A-I, the major HDL protein, and the ability of HDL to remove cellular cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway. Methods and Results: We quantified both site-specific oxidation of apolipoprotein A-I and HDL’s ABCA1 cholesterol efflux capacity in control subjects and subjects with stable coronary artery disease or acute coronary syndrome. Subjects with coronary artery disease and acute coronary syndrome had higher levels of chlorinated tyrosine 192 and oxidized methionine 148 compared with control subjects. In contrast, plasma levels of myeloperoxidase did not differ between the groups. HDL from the subjects with coronary artery disease and acute coronary syndrome was less able to accept cholesterol from cells expressing ABCA1 compared with HDL from control subjects. Levels of chlorinated tyrosine and oxidized methionine associated inversely with ABCA1 efflux capacity and positively with atherosclerotic disease status. These differences remained significant after adjusting for HDL-cholesterol levels. Conclusions: Our observations indicate that myeloperoxidase may contribute to the generation of dysfunctional HDL with impaired ABCA1 efflux capacity in humans with atherosclerosis. Quantification of chlorotyrosine and oxidized methionine in circulating HDL might be useful indicators of the risk of cardiovascular disease that are independent of HDL-cholesterol.