The role of peroxidases in the pathogenesis of atherosclerosis

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*Corresponding author. Tel: 82-2-3277-4128; Fax: 82-2-3277-3760;

http://dx.doi.org/10.5483/BMBRep.2011.44.8.497 Received 2 August 2011

Keywords: Atherosclerosis, Catalase, Glutathione peroxidase, Oxida- tive stress, Peroxidase, Peroxiredoxin, Reactive oxygen species

The role of peroxidases in the pathogenesis of atherosclerosis

Jong-Gil Park^1,2 & Goo Taeg Oh^1,*

1Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, ²College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea

Reactive oxygen species (ROS), which include superoxide anions and peroxides, induce oxidative stress, contributing to the initiation and progression of cardiovascular diseases in- volving atherosclerosis. The endogenous and exogenous fac- tors hypercholesterolemia, hyperglycemia, hypertension, and shear stress induce various enzyme systems such as nic- otinamide adenine dinucleotide (phosphate) oxidase, xanthine oxidase, and lipoxygenase in vascular and immune cells, which generate ROS. Besides inducing oxidative stress, ROS mediate signaling pathways involved in monocyte adhesion and infiltration, platelet activation, and smooth muscle cell migration. A number of antioxidant enzymes (e.g., superoxide dismutases, catalase, glutathione peroxidases, and peroxir- edoxins) regulate ROS in vascular and immune cells. Atheros- clerosis results from a local imbalance between ROS pro- duction and these antioxidant enzymes. In this review, we will discuss 1) oxidative stress and atherosclerosis, 2) ROS-depend- ent atherogenic signaling in endothelial cells, macrophages, and vascular smooth muscle cells, 3) roles of peroxidases in atherosclerosis, and 4) antioxidant drugs and therapeutic pers- pectives. [BMB reports 2011; 44(8): 497-505]

INTRODUCTION

Cardiovascular disease, the current leading cause of death and illness in developed countries, will soon become the pre-emi- nent health problem worldwide (1). The primary cause of cardiovascular diseases (e.g., coronary artery diseases, stroke, and peripheral vascular disease) is atherosclerosis and inflammation (2-4). Atherosclerosis is initiated by lipids deposited in the subendothelial layer of the artery wall. These lipids are modified to oxidized low-density lipoprotein (oxLDL) and minimally oxidized LDL (mmLDL) (5, 6). These lipids and modified LDL damage endothelial cells, inducing the expression of adhesion molecules such as P- and E-selectin, vascular cell adhesion

molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) (7-10), and chemotactic molecules including monocyte chemoattractant protein-1 (MCP-1/CCL2) and macrophage colony-stimulating factor (11, 12). After activating endothelial cells in this way, leukocytes such as monocytes/ macrophage enter the intima by diapedesis (13) and undergo differentiation (14). Lipid-laden macrophages, known as foam cells, promote the progression of the atherosclerotic plaque (15). Specifically, pro-inflammatory cytokines and growth factors secreted by foam cells induce local inflammatory responses, increase reactive oxygen species (ROS) in the lesion, and accelerate the migration of vascular smooth muscle cells (VSMCs) from the media to the intima (16). Intimal VSMCs also take up modified lipoproteins, contributing to foam cell formation, and synthe- size extracellular matrix proteins (collagen, elastin, and proteoglycans) that lead to the development of a fibrous cap (2, 17).

Vulnerable plaques are characterized by lipid accumulation that expands the intima, degradation of the extracellular matrix, a decrease in connective tissue proteins, and a necrotic core.

Plaque rupture and thrombosis induce acute cardiovascular events that result in myocardial infarction and stroke (18).

OXIDATIVE STRESS AND ATHEROSCLEROSIS

ROS include hydrogen peroxide (H2O2), peroxynitrite (ON- OO⁻), and hypochlorous acid (HOCl) and free radicals such as superoxide anion (O2ㆍ−), hydroxyl radical (HO^ㆍ), and nitric oxide (NO^ㆍ). Increased ROS production has been implicated in hypertension, diabetes mellitus, hypercholesterolemia, reste- nosis, heart failure, and atherosclerosis (19-23). Specifically, overproduction of ROS leads to oxidative stress, which contrib- utes to the pathogenesis of atherosclerosis (Fig. 1). A number of in vitro and in vivo studies have demonstrated a critical role for ROS or enzyme systems involved in ROS production, including endothelial NO synthases, xanthine oxidase, enzymes of the respiratory chain, cytochrome P450 monooxygenases, and NAD(P)H oxidase in the vasculature (Fig. 1) (16, 24). Upregu- lation of the NAD(P)H oxidase subunits gp91phox and Nox4 increases intracellular oxidative stress in macrophages and non- phagocytic vascular cells of human coronary atherosclerosis, respectively (25). In addition, smooth muscle cells in mice deficient in the NAD(P)H oxidase subunit p47phox exhibit decreased superoxide production and proliferative responses to growth factors compared with wild-type cells. Accordingly, de- Invited Mini Review

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Fig. 1. Generation and function of ROS in the vasculature. Endoge- nous and exogenous factors (e.g., hypercholesterolemia, hyperglycemia, hypertension, and shear stress) induce enzyme systems including NAD(P)H, xanthine oxidase, and lipoxygenase in the vasculature to generate reactive oxygen species, which promote atherosclerosis via intracellular signaling and oxidative stress. eNOS, endothelial nitric oxide synthase; NAD(P)H, nicotinamide adenine dinucleotide (phosphate) oxidase; O2ㆍ−, superoxide anion; ROS, reactive oxygen species; SOD, superoxide dismutase.

Fig. 2. Intracellular signaling cascades involved in ROS in atheros- clerosis. Atherogenic stimuli (e.g., oxLDL, mmLDL, PDGF, shear stress, and TNF-α) activate their cognate receptors, which triggers intracellular signaling pathways and ROS generation. Finally, redox-dependent transcriptional factors such as Elk1, NF-κB, and AP-1 regulate the expression of proinflammatory genes. COX, cyclooxygenase; ERK, extracellular signal-related kinase; FAK, focal adhesion kinase; JNK, c-Jun N-terminal kinase; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; mmLDL, minimally oxidized LDL; NF-κB, nuclear factor kappa-B; NOS, nitric oxide synthase; oxLDL, oxidized low density lipoprotein; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; PLCγ1, phospholipase Cγ1; PTP, protein tyrosine phosphatase; ROS, reactive oxygen species; SR-A, scavenger receptor-A; TLR, toll-like receptor; TNF-α, tumor necrosis factor α; TRAF, TNF receptor-associated factor; VE- GF, vascular endothelial growth factor.

letion of p47phox in apolipoprotein E-deficient (ApoE^−/−) mice ameliorated atherosclerotic plaques (26).

　A Western diet was found to increase the mRNA and protein levels of xanthine dehydrogenase in the liver and convert xanthine dehydrogenase to xanthine oxidase; however, tungsten (a xanthine oxidase inhibitor) decreased plasma xanthine oxidase activity and vascular superoxide anion formation, preventing endothelial dysfunction and atherosclerosis in the aorta of ApoE^−/− mice fed a Western diet (27). Recently, we reported that the benzylidenethiazole analog BHB-TZD is a dual inhibitor for cyclooxygenase and 5-lipoxygenase. BHB-TZD showed anti-atherogenic effects in hyperlipidemic mice by reducing macrophage infiltration into atherosclerotic lesions, thereby increasing plaque stability (28).

Another benzylidenethiazole analog, HMB-TZD, inhibits 5-lipoxygenase, reduces cell migration and adhesion by decreasing proinflammatory molecule production, and ameliorates atherosclerotic lesion formation in LDL receptor-deficient mice (29).

　Although increased ROS production directly causes oxidative stress in the vasculature, ROS, especially H2O2, has another important role in the pathophysiological signaling pathway of atherosclerosis.

ROS-DEPENDENT SIGNALING

In addition to killing invading pathogens, H2O2 is a second

messenger in the activation of various cell surface receptors (30, 31). The mechanism of receptor-mediated H2O2 generation has been studied in phagocytic cells (32), endothelial cells, and vascular smooth muscle cells (33). Superoxide dismutase generates H2O2 from O2ㆍ−, and xanthine oxidase and glucose oxidase directly produce H2O2 by donating two elec- trons to oxygen (34). These H2O2 molecules induce pro- grammed cell death or necrosis, regulate the expression of many genes, and activate cell signaling molecules such as nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) (35, 36). In the presence of the transition metal Fe^2＋, H2O2 spontaneously converts to HO^ㆍ by the Fenton reaction (37) and can also be detoxified via catalase, glutathione peroxidase (Gpx), and peroxiredoxin (Prdx) to H2O and O2 (24).

　The H2O2 structure is too simple to be recognized specifically by a protein (30); however, H2O2 can oxidize the thiol group of cysteine residues to a sulfenic acid, sulfinic acid, or disulfide, all of which are readily reduced by various cellular reductants to restore cysteine (31). Oxidation of cysteine residues by H2O2 changes the conformation and catalytic activity of protein tyrosine phosphatases, thereby increasing the level

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of tyrosine phosphorylation (30). Activating receptor protein tyrosine kinase by binding ligands (e.g., platelet-derived growth factor, epidermal growth factor, and insulin) requires H2O2

production and oxidation of protein tyrosine phosphatases (38-41). In atherosclerosis, ROS, especially H2O2, also mediate signaling pathways that upregulate monocyte adhesion and infiltration, platelet activation, and smooth muscle cell migration (Fig. 2).

ROS signaling pathway for atherogenic responses in endothelial cell

In endothelial cells, H2O2 regulates numerous signaling pathways including those regulating growth, proliferation, vaso-relaxation, inflammatory responses, barrier function, and vascular remodeling (34). Endothelial cell proliferation is increased by glucose oxidase-mediated H2O2 generation and abolished by removing H2O2 with catalase or Gpx overexpression (42-44). This H2O2-mediated endothelial cell proliferation was associated with vascular endothelial growth factor receptor 2/extracellular signal-regulated kinase (ERK), Ras/

Raf/ERK and Fyn/Ras/p90 ribosomal S6 kinase signaling (45-47). Previous reports showed that H2O2 induces endothelium-dependent vasorelaxation via NO, Ca^2＋, and cyclic gua- nosine monophosphate (48, 49).

　The endothelial cell responses to various proinflammatory mediators such as oxLDL and mmLDL are critical for initiating atherosclerosis (17). OxLDL activates the lectin-like oxidized low-density lipoprotein receptor-1 on endothelial cells, and upregulates the expression of MCP-1 and adhesion molecules (e.g., P-selectin, ICAM-1, and VCAM-1) via ROS and NF-κB activation (7-11, 50, 51). Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) activate NF-κB and activator protein-1 (AP-1), which regulate genes involved in the expression of adhesion molecules on activated endothelial cells (52-54).

TNF-α also induces expression of adhesion molecules associated with the TNF receptor-associated factor 2 (TRAF2)/apoptosis signal-regulated kinase 1/stress-activated protein kinase ERK kinase 1/c-Jun N-terminal kinase (JNK) signaling pathway (55). We confirmed that H2O2 and NF-κB signaling are important for the TNF-α-mediated expression of VCAM-1 and ICAM-1 using antioxidants and inhibitors of NF-κB and MAPK in mouse aortic endothelial cells (56). However, the activation of MAPKs (e.g., p38 MAPK, JNKs, and ERKs) is involved in the expression of adhesion molecules under increasing endogenous H2O2 conditions (56). The junction integrity of endothelial cells in blood vessels regulates leukocyte transmigration. In this process, ICAM-1 and VCAM-1 clustering induces actin remodeling and junction disruption via ROS production (13).

ROS signaling pathway for atherogenic responses in macrophage

Macrophages infiltrate into atherosclerotic plaques, where they take up oxLDL through scavenger receptors (3,17). An extracellular domain of the scavenger receptor CD36 binds to

oxidized phosphatidyl cholines of oxLDL, which initiates the interaction of the CD36 intracytoplasmic domain with src family kinases. The resulting activation of focal adhesion kinase, MAPK cascades, ROS production, and phosphatase SHP2 inactivation leads to ligand internalization and foam cell formation. Foam cells are then trapped in the atherosclerotic plaque by actin polymerization and loss of cell polarity (57). A recent study showed that oxLDL-induced inflammatory signaling is generated through a complex consisting of CD36, a Toll-like receptor 4 (TLR4)/TLR6 heterodimer, and ROS. Accordingly, a mutation of the cytoplasmic C terminus or Tyr463 of CD36 blocked ligand-induced NF-κB activation (58). In addition, the CD14/TLR4/MD-2 complex interacts with mmLDL, inducing cytoskeletal rearrangements and secretion of macrophage inflammatory protein-2, MCP-1, TNF-α, and interleukin-6 (59, 60) via ROS generation from spleen tyrosine kinase/Nox2 signaling (61).

　NF-κB and AP-1, the most well-known redox-dependent transcriptional factors, regulate a number of genes involved in inflammatory responses in macrophages (62-65). AP-1 is a heterodimer of Jun and Fos and is regulated by ROS through conserved cysteine residues in the DNA-binding domain of each protein (66). The activation of NF-κB, homodimeric or hetero- meric complexes of the Rel family, is mediated by ROS in response to specific stimuli (15, 67, 68).

ROS signaling pathway for atherogenic responses in vascular smooth muscle cells

In VSMCs, ROS mediates various functions including growth, migration, matrix regulation, inflammation, and contraction (69), which are critical factors in the progression and compli- cation of atherosclerosis. Platelet-derived growth factor (PDGF) increases the intracellular concentration of H2O2 in VSMCs, leading to tyrosine phosphorylation, MAPK stimulation, DNA synthesis, and chemotaxis (38). By increasing H2O2, the PDGF receptor triggers a cascade of signaling events, including activation of phosphatidylinositol 3-kinase, the Ras GTPase-activating protein, the protein-tyrosine phosphatase SHP-2, phospholipase C-γ1 (PLC-γ1) (70, 71). Weber et al. reported that PDGF-dependent VSMC migration is associated with ROS and the Src/p21-activated protein kinase 1/phosphoinositide-dependent kinase-1 signaling pathway (72). VSMCs regulate plaque stability through modulation of inflammation and apoptosis, and the production or degradation of matrix proteins (3,17).

Under atherogenic conditions, VSMCs in the media of the artery undergo phenotypic changes from the normal contractile state to the active synthetic state, which produces collagen, elastin, proteoglycans. Activated VSMCs migrate to the intima and proliferate (73). In addition, ROS produced via NAD(P)H oxidase induce VSMCs to produce and secrete matrix metalloproteinases involved in the degradation and reorganization of the extracellular matrix (74, 75). In VSMCs, ROS also mediate inflammation (e.g., MCP-1 expression via TNF-α) (76) and apoptosis via p53 and Bax/Bad (77).

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Fig. 3. The role of peroxidases in the pathogenesis of atheroscler- osis. Glutathione peroxidase, catalase, and peroxiredoxin regulate ROS as an inducer of oxidative stress and second messenger in atherogenesis. COX, cyclooxygenase; JNK, c-Jun N-terminal kinase;

LDL, low-density lipoprotein; 5-LO, 5-lipoxygenase; MAPK, mitogen-activated protein kinase; NAD(P)H, nicotinamide adenine dinucleotide (phosphate) oxidase; NK-κB, nuclear factor kappa-B; PKC, protein kinase C; PP2B, protein phosphatase 2B; PPAR-γ, perox- isome proliferator-activated receptor; PTP, protein tyrosine phosphatase; ROS, reactive oxygen species; SR-A, scavenger receptor-A;

TRAF, TNF receptor-associated factor.

PEROXIDASES IN CARDIOVASCULAR DISEASES Catalase in cardiovascular diseases

Catalase, an important peroxidase located in the peroxisomes of all living aerobic organisms, catalyzes hydrogen peroxide to water and oxygen (78). Catalase appears to protect against oxidative stress in atherosclerosis (Fig. 3) (79). For example, ec- topic overexpression of catalase in human aortic smooth muscle cells decreases oxLDL-induced proliferation and decreases expression of cyclin D1, cyclin E, and cycline dependent kinase 2 and 4 (80). Catalase overexpression also suppresses oxLDL-induced phosphorylation of ERK and JNK and activation of AP-1 and NF-κB. Adenovirus-mediated overexpression of catalase in human aortic endothelial cells reduced oxLDL- induced ROS production and apoptosis, inhibiting the activa- tion of JNK, ERK, and AP-1 (43). In an in vivo model, catalase overexpression reduced lipid peroxidation and inhibited development of atherosclerosis (79). Interestingly, atherosclerosis in ApoE^−/− mice overexpressing Cu/Zn-superoxide dismutase was comparable to ApoE^−/− control mice, suggesting that endogenous H2O2 plays a more important role than endogenous superoxide anions in the pathogenesis of atherosclerosis.

However, individuals who have acatalasia, an autosomal re- cessive peroxisomal disorder caused by very low levels of catalase, exhibit few ill effects. Similarly, the rate of atherosclerosis in catalase-deficient ApoE^−/− mice did not differ from that of ApoE^−/− control mice (56).

Glutathione peroxidases in cardiovascular diseases

Glutathione peroxidases (Gpxs) catalyze H2O2 and lipid peroxides to water or their respective alcohols using reduced glutathione (81). The major isoform, Gpx1, is located in the cyto- sol and mitochondria. Wagner et al. reported that upregulation of GPX1 in human endothelial cells induced by stretch decreased expression of the proatherogenic genes GD40, MCP-1, and VCAM-1 (82). Among ApoE^−/− mice fed a Western-type diet for 24 weeks, those with Gpx1 deficiency showed more severe progression of atherosclerosis and reduced levels of bi- oactive nitric oxide than control mice (83). Similarly, Lewis et al. reported that lack of Gpx1 in diabetic ApoE-/- mice accel- erates atherosclerotic lesions and upregulates proinflammatory and profibrotic markers including VCAM-1, vascular endothelial growth factor, connective tissue growth factor, and receptors for advanced glycation end products (84). However, lack of Gpx1 in mice did not significantly influence the development of atherosclerosis under less severe atherogenic conditions (e.g., 6- or 12-week Western-type diet) or in non-diabetic mice. Nevertheless, many human studies have demonstrated the relationship between GPX1 activity and cardiovascular disease. For example, GPX1 activity is inversely corre- lated with coronary artery disease and acute myocardial infarction (85, 86), and decreased erythrocyte intracellular GPX1 activity is associated with an increase in the number of vascular beds with atherosclerotic manifestations (87). A recent study reported that Gpx4 overexpression in ApoE^−/− mice reduced plaque formation and signs of advanced lesions (i.e., fibrous caps and acellular areas) (88). In addition, overexpression of Gpx4 downregulated the expression of adhesion molecules and reduced necrosis in endothelial cells. Taken together, these studies suggest that Gpx may be a useful therapeutic target in atherosclerosis (Fig. 3).

Peroxiredoxins in cardiovascular diseases

Peroxiredoxins (Prdxs), a family of peroxidases, also play a role in regulating atherosclerotic pathophysiology (Fig. 3). Prd- xs are thiol-specific antioxidant proteins found in mammals, yeast, and bacteria that are classified largely on the basis of having either one or two conserved cysteine residues (89).

Wang et al. first reported that Prdx6-deficient mice with a B6;129 background showed significantly larger aortic root lesions than the wild-type control, but this protein does not play a major anti-atherogenic role in mice, or its potentially major anti-atherogenic function is redundant to other antioxidants (90). Although Prdx6 overexpression in an ApoE^−/−background does not affect atherosclerosis, a high-fat diet or Prdx6 deficiency in ApoE^−/− mice may increase plaque formation.

The effects of Prdx6 mutation may be masked by other Prdx isoforms; however, the anti-atherogenic actions of Prdx6 (e.g., inhibiting LDL oxidation and reducing plasma lipid peroxide levels) may still be important in reducing atherosclerosis.

　Among the six Prdx isoforms, Prdx1 and Prdx2 are the most abundant, constituting 0.2% to 1% of total soluble protein in

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cultured mammalian cells (89). These isoforms regulate intracellular levels of H2O2 (91). Prdx1 is induced in macrophages by oxLDL exposure (92) and in endothelial cells by laminar shear stress (93). Prdx1 deficiency results in the excessive re- lease of P-selectin and von Willebrand factor, proinflammatory components of Weibel-Palade bodies (94). Prdx2 removes the H2O2 transiently produced by activation of various cell surface receptors (95). Prdx2 regulates PDGF signaling through enhanced activation of PDGF receptor and phospholipase Cγ1, and increases vascular remodeling (e.g., neointimal thickening of VSMCs) (70). In addition, Prdx2 inhibits general immune cell responsiveness by scavenging ROS (96), modulates LPS-induced proinflammatory responses, and protects against endo- toxin-induced lethal shock (97). Recently, we reported that Prdx2 ameliorates atherosclerosis in ApoE^−/− mice (56). Stro- ng expression of Prdx2 in endothelial and immune cells in the atherosclerotic lesion blocks the upregulation of endogenous H2O2 induced by proinflammatory cytokines. Conversely, Prdx2 deficiency in ApoE^−/− mice enhanced activation of p65, c-Jun, JNKs, and p38 MAPK, thereby accelerating plaque formation. These effects of Prdx2 deficiency were partially res- cued by the antioxidant ebselen and mediated by bone mar- row-derived inflammatory cells and vascular cells. Other effects of Prdx2 deficiency are increased expression of VCAM-1, ICAM-1, and MCP-1, immune cell infiltration into the plaque, and TNF-α generation by inflammatory cells.

　The function of human PRDXs in atherogenesis is unclear;

however, PRDX1 was suggested as a biomarker for abdominal aortic aneurysm (98), and PRDX2 expression was increased in ruptured abdominal aortic aneurysms compared with non- ruptured abdominal aortic aneurysms (99). Although the exact mechanisms underlying atherogenesis and aneurysm differ, the diseases share common factors including vascular disease, inflammation, and oxidative stress. Taken together, these findings suggest that investigations of PRDX1 and PRDX2 poly- morphisms and their roles in patients with atherosclerosis may help to identify a biomarker for cardiovascular events. In addition, the ability of PRDX to inhibit atherogenic responses in the vasculature and inflammatory cells suggests its potential usefulness as an effective anti-atherogenic therapy.

ANTIOXIDANT EFFECTS AND PERSPECTIVES FOR ATHEROSCLEROSIS

Ebselen, a seleno-organic compound Gpx1-mimetic, inhibits TNF-α-induced JNK activation following TRAF2/apoptosis signal-regulated kinase 1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1, and down- regulates TNF-α-induced expression of VCAM-1 and ICAM-1 via inactivation of AP-1 and NF-κB (55). Results from an in vivo model showed that ebselen produces a site-specific an- ti-atherogenic effect by reducing levels of nitrotyrosine, Nox2, and vascular endothelial growth factor (100). Administration of ebselen by osmotic pump reduced plaque formation more

than 40% in ApoE^−/−mice on an 8-week atherogenic diet (56). The novel compound KR-31378 is another potent antioxidant, ameliorating atherosclerosis in LDL receptor-deficient mice by blocking monocyte recruitment and downregulating VCAM-1 and interleukin-8 and (101). We also reported that ti- lianin, a major component of Agastache rugosa (Labiatae), suppresses the TNF-α-induced expression of VCAM-1 in human umbilical vein endothelial cells by inhibiting NO production via inducible nitric oxide synthase (102). However, despite numerous in vitro and animal studies, results of clin- ical trials evaluating the use of antioxidants (e.g., vitamin C and E, carotenoids, N-acetyl-cysteine) as chronic preventative therapy for cardiovascular diseases have been primarily neg- ative (103). Possible reasons for these findings include short trial periods, low bioavailability of the tested compounds, and limited antioxidant effects. However, the development of peroxidase mimetics may be a promising strategy as a therapeutic drug for atherosclerosis.

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