Detrimental Actions of Chlorinated Nucleosides on the Function and Viability of Insulin-Producing Cells.

Neutrophils are innate immune cells that play a key role in pathogen clearance. They contribute to inflammatory diseases, including diabetes, by releasing pro-inflammatory cytokines, reactive oxygen species, and extracellular traps (NETs). NETs contain a DNA backbone and catalytically active myeloperoxidase (MPO), which produces hypochlorous acid (HOCl). Chlorination of the DNA nucleoside 8-chloro-deoxyguanosine has been reported as an early marker of inflammation in diabetes. In this study, we examined the reactivity of different chlorinated nucleosides, including 5-chloro-(deoxy)cytidine (5ClC, 5CldC), 8-chloro-(deoxy)adenosine (8ClA, 8CldA) and 8-chloro-(deoxy)guanosine (8ClG, 8CldG), with the INS-1E ß-cell line. Exposure of INS-1E cells to 5CldC, 8CldA, 8ClA, and 8CldG decreased metabolic activity and intracellular ATP, and, together with 8ClG, induced apoptotic cell death. Exposure to 8ClA, but not the other nucleosides, resulted in sustained endoplasmic reticulum stress, activation of the unfolded protein response, and increased expression of thioredoxin-interacting protein (TXNIP) and heme oxygenase 1 (HO-1). Exposure of INS-1E cells to 5CldC also increased TXNIP and NAD(P)H dehydrogenase quinone 1 (NQO1) expression. In addition, a significant increase in the mRNA expression of NQO1 and GPx4 was seen in INS-1E cells exposed to 8ClG and 8CldA, respectively. However, a significant decrease in intracellular thiols was only observed in INS-1E cells exposed to 8ClG and 8CldG. Finally, a significant decrease in the insulin stimulation index was observed in experiments with all the chlorinated nucleosides, except for 8ClA and 8ClG. Together, these results suggest that increased formation of chlorinated nucleosides during inflammation in diabetes could influence ß-cell function and may contribute to disease progression.

Identification and quantification of protein nitration sites in human coronary artery smooth muscle cells in the absence and presence of peroxynitrous acid/peroxynitrite.

Peroxynitrous acid/peroxynitrite (ONOOH/ONOO-) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, and particularly proteins. Here, we show that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with LC-MS peptide mass mapping providing data on the sites and extents of changes on cellular and extracellular matrix (ECM) proteins. Evidence is presented for selective and specific nitrations at Tyr and Trp on 11 cellular proteins (out of 3668, including 205 ECM species) in the absence of added reagent ONOOH/ONOO-, with this being consistent with low-level endogenous nitration. A number of these have key roles in cell signaling/sensing and protein turnover. With added ONOOH/ONOO-, more proteins were modified (84 total; with 129 nitrated Tyr and 23 nitrated Trp, with multiple modifications on some proteins), with this occurring at the same and additional sites to endogenous modification. With low concentrations of ONOOH/ONOO- (50 µM) nitration occurs on specific proteins at particular sites, and is not driven by protein or Tyr/Trp abundance, with modifications detected on some low abundance proteins. However, with higher ONOOH/ONOO- concentrations (500 µM), modification is primarily driven by protein abundance. ECM species are major targets and over-represented in the pool of modified proteins, with fibronectin and thrombospondin-1 being particularly heavily modified (12 sites in each case). Both endogenous and exogenous nitration of cell- and ECM-derived species may have significant effects on cell and protein function, and potentially be involved in the development and exacerbation of diseases such as atherosclerosis.

Exposure to peroxynitrite impacts the ability of anastellin to modulate the structure of extracellular matrix.

The extracellular matrix (ECM) of tissues consists of multiple proteins, proteoglycans and glycosaminoglycans that form a 3-dimensional meshwork structure. This ECM is exposed to oxidants including peroxynitrite (ONOO-/ONOOH) generated by activated leukocytes at sites of inflammation. Fibronectin, a major ECM protein targeted by peroxynitrite, self-assembles into fibrils in a cell-dependent process. Fibrillation of fibronectin can also be initiated in a cell-independent process in vitro by anastellin, a recombinant fragment of the first type-III module in fibronectin. Previous studies demonstrated that modification of anastellin by peroxynitrite impairs its fibronectin polymerization activity. We hypothesized that exposure of anastellin to peroxynitrite would also impact on the structure of ECM from cells co-incubated with anastellin, and influence interactions with cell surface receptors. Fibronectin fibrils in the ECM of primary human coronary artery smooth muscle cells exposed to native anastellin are diminished, an effect which is reversed to a significant extent by pre-incubation of anastellin with high (200-fold molar excess) concentrations of peroxynitrite. Treatment with low or moderate levels of peroxynitrite (2-20 fold molar excess) influences interactions between anastellin and heparin polysaccharides, as a model of cell-surface proteoglycan receptors, and modulates anastellin-mediated alterations in fibronectin cell adhesiveness. Based on these observations it is concluded that peroxynitrite has a dose-dependent influence on the ability of anastellin to modulate ECM structure via interactions with fibronectin and other cellular components. These observations may have pathological implications since alterations in fibronectin processing and deposition have been associated with several pathologies, including atherosclerosis.

New insight into the composition of extracellular traps released by macrophages exposed to different types of inducers.

Neutrophil extracellular trap (NET) release plays a key role in many chronic disease settings, including atherosclerosis. They are critical to innate immune defence, but also contribute to disease by promoting thrombosis and inflammation. Macrophages are known to release extracellular traps or "METs", but their composition and role in pathological processes are less well defined. In this study, we examined MET release from human THP-1 macrophages exposed to model inflammatory and pathogenic stimuli, including tumour necrosis factor α (TNFα), hypochlorous acid (HOCl) and nigericin. In each case, there was release of DNA from the macrophages, as visualized by fluorescence microscopy with the cell impermeable DNA binding dye SYTOX green, consistent with MET formation. Proteomic analysis on METs released from macrophages exposed to TNFα and nigericin reveals that they are composed of linker and core histones, together with a range of cytosolic and mitochondrial proteins. These include proteins involved in DNA binding, stress responses, cytoskeletal organisation, metabolism, inflammation, anti-microbial activity, and calcium binding. Quinone oxidoreductase in particular, was highly abundant in all METs but has not been reported previously in NETs. Moreover, there was an absence of proteases in METs in contrast to NETs. Some of the MET histones, contained post-translational modifications, including acetylation and methylation of Lys but not citrullination of Arg. These data provide new insight into the potential implications of MET formation in vivo and their contributions to immune defence and pathology.

Hypochlorous Acid and Chloramines Induce Specific Fragmentation and Cross-Linking of the G1-IGD-G2 Domains of Recombinant Human Aggrecan, and Inhibit ADAMTS1 Activity.

Atherosclerosis is a chronic inflammatory disease and a leading cause of mortality. It is characterized by arterial wall plaques that contain high levels of cholesterol and other lipids and activated leukocytes covered by a fibrous cap of extracellular matrix (ECM). The ECM undergoes remodelling during atherogenesis, with increased expression of aggrecan, a proteoglycan that binds low-density-lipoproteins (LDL). Aggrecan levels are regulated by proteases, including a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1). Activated leukocytes release myeloperoxidase (MPO) extracellularly, where it binds to proteins and proteoglycans. Aggrecan may therefore mediate colocalization of MPO and LDL. MPO generates hypochlorous acid (HOCl) and chloramines (RNHCl species, from reaction of HOCl with amines on amino acids and proteins) that damage LDL and proteins, but effects on aggrecan have not been examined. The present study demonstrates that HOCl cleaves truncated (G1-IGD-G2) recombinant human aggrecan at specific sites within the IGD domain, with these being different from those induced by ADAMTS1 which also cleaves within this region. Irreversible protein cross-links are also formed dose-dependently. These effects are limited by the HOCl scavenger methionine. Chloramines including those formed on amino acids, proteins, and ECM materials induce similar damage. HOCl and taurine chloramines inactivate ADAMTS1 consistent with a switch from proteolytic to oxidative aggrecan fragmentation. Evidence is also presented for colocalization of aggrecan and HOCl-generated epitopes in advanced human atherosclerotic plaques. Overall, these data show that HOCl and chloramines can induce specific modifications on aggrecan, and that these effects are distinct from those of ADAMTS1.

Antioxidant Strategies to Modulate NETosis and the Release of Neutrophil Extracellular Traps during Chronic Inflammation.

Extracellular traps are released by neutrophils and other immune cells as part of the innate immune response to combat pathogens. Neutrophil extracellular traps (NETs) consist of a mesh of DNA and histone proteins decorated with various anti-microbial granule proteins, such as elastase and myeloperoxidase (MPO). In addition to their role in innate immunity, NETs are also strongly linked with numerous pathological conditions, including atherosclerosis, sepsis and COVID-19. This has led to significant interest in developing strategies to inhibit NET release. In this study, we have examined the efficacy of different antioxidant approaches to selectively modulate the inflammatory release of NETs. PLB-985 neutrophil-like cells were shown to release NETs on exposure to phorbol myristate acetate (PMA), hypochlorous acid or nigericin, a bacterial peptide derived from Streptomyces hygroscopicus. Studies with the probe R19-S indicated that treatment of the PLB-985 cells with PMA, but not nigericin, resulted in the production of HOCl. Therefore, studies were extended to examine the efficacy of a range of antioxidant compounds that modulate HOCl production by MPO to prevent NETosis. It was shown that thiocyanate, selenocyanate and various nitroxides could prevent NETosis in PLB-985 neutrophils exposed to PMA and HOCl, but not nigericin. These results were confirmed in analogous experiments with freshly isolated primary human neutrophils. Taken together, these data provide new information regarding the utility of supplementation with MPO inhibitors and/or HOCl scavengers to prevent NET release, which could be important to more specifically target pathological NETosis in vivo.

Anastellin impacts on the processing of extracellular matrix fibronectin and stimulates release of cytokines from coronary artery smooth muscle cells.

Anastellin, a recombinant protein fragment from the first type III module of fibronectin, mimics a partially unfolded intermediate implicated in the assembly of fibronectin fibrils. Anastellin influences the structure of fibronectin and initiates in vitro fibrillation, yielding "superfibronectin", a polymer with enhanced cell-adhesive properties. This ability is absent in an anastellin double mutant, L37AY40A. Here we demonstrate that both wild-type and L37AY40A anastellin affect fibronectin processing within the extracellular matrix (ECM) of smooth muscle cells. Fibronectin fibrils are diminished in the ECM from cells treated with anastellin, but are partially rescued by supplementation with plasma fibronectin in cell media. Proteomic analyses reveal that anastellin also impacts on the processing of other ECM proteins, with increased collagen and decreased laminin detected in media from cells exposed to wild-type anastellin. Moreover, both anastellin forms stimulate release of inflammatory cytokines, including interleukin 6. At the molecular level, L37AY40A does not exhibit major perturbations of structural features relative to wild-type anastellin, though the mutant showed differences in heparin binding characteristics. These findings indicate that wild-type and L37AY40A anastellin share similar molecular features but elicit slightly different, but partially overlapping, responses in smooth muscle cells resulting in altered secretion of cytokines and proteins involved in ECM processing.

Mapping the modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl).

Histones are critical for the packaging of nuclear DNA and chromatin assembly, which is facilitated by the high abundance of Lys and Arg residues within these proteins. These residues are also the site of a range of post-translational modifications, which influence the regulatory function of histones. Histones are also present in the extracellular environment, following release by various pathways, particularly neutrophil extracellular traps (NETs). NETs contain myeloperoxidase, which retains its enzymatic activity and produces hypochlorous acid (HOCl). This suggests that histones could be targets for HOCl under conditions where aberrant NET release is prevalent, such as chronic inflammation. In this study, we examine the reactivity of HOCl with a mixture of linker (H1) and core (H2A, H2B, H3 and H4) histones. HOCl modified the histones in a dose- and time-dependent manner, resulting in structural changes to the proteins and the formation of a range of post-translational modification products. N-Chloramines are major products following exposure of the histones to HOCl and decompose over 24 h forming Lys nitriles and carbonyls (aminoadipic semialdehydes). Chlorination and dichlorination of Tyr, but not Trp residues, is also observed. Met sulfoxide and Met sulfones are formed, though these oxidation products are also detected albeit at a lower extent, in the non-treated histones. Evidence for histone fragmentation and aggregation was also obtained. These results could have implications for the development of chronic inflammatory diseases, given the key role of Lys residues in regulating histone function.

Activation and Inhibition of Human Matrix Metalloproteinase-9 (MMP9) by HOCl, Myeloperoxidase and Chloramines.

Matrix metalloproteinase-9 (MMP9, gelatinase B) plays a key role in the degradation of extracellular-matrix (ECM) proteins in both normal physiology and multiple pathologies, including those linked with inflammation. MMP9 is excreted as an inactive proform (proMMP9) by multiple cells, and particularly neutrophils. The proenzyme undergoes subsequent processing to active forms, either enzymatically (e.g., via plasmin and stromelysin-1/MMP3), or via the oxidation of a cysteine residue in the prodomain (the "cysteine-switch"). Activated leukocytes, including neutrophils, generate O2- and H2O2 and release myeloperoxidase (MPO), which catalyzes hypochlorous acid (HOCl) formation. Here, we examine the reactivity of HOCl and a range of low-molecular-mass and protein chloramines with the pro- and activated forms of MMP9. HOCl and an enzymatic MPO/H2O2/Cl- system were able to generate active MMP9, as determined by fluorescence-activity assays and gel zymography. The inactivation of active MMP9 also occurred at high HOCl concentrations. Low (nM-low µM) concentrations of chloramines formed by the reaction of HOCl with amino acids (taurine, lysine, histidine), serum albumin, ECM proteins (laminin and fibronectin) and basement membrane extracts (but not HEPES chloramines) also activate proMMP9. This activation is diminished by the competitive HOCl-reactive species, methionine. These data indicate that HOCl-mediated oxidation and MMP-mediated ECM degradation are synergistic and interdependent. As previous studies have shown that modified ECM proteins can also stimulate the cellular expression of MMP proteins, these processes may contribute to a vicious cycle of increasing ECM degradation during disease development.

Influence of plasma halide, pseudohalide and nitrite ions on myeloperoxidase-mediated protein and extracellular matrix damage.

Myeloperoxidase (MPO) mediates pathogen destruction by generating the bactericidal oxidant hypochlorous acid (HOCl). Formation of this oxidant is however associated with host tissue damage and disease. MPO also utilizes H2O2 to oxidize other substrates, and we hypothesized that mixtures of other plasma anions, including bromide (Br-), iodide (I-), thiocyanate (SCN-) and nitrite (NO2-), at normal or supplemented concentrations, might modulate MPO-mediated HOCl damage. For the (pseudo)halide anions, only SCN- significantly modulated HOCl formation (IC50 ∼33 µM), which is within the normal physiological range, as judged by damage to human plasma fibronectin or extracellular matrix preparations detected by ELISA and LC-MS. NO2- modulated HOCl-mediated damage, in a dose-dependent manner, at physiologically-attainable anion concentrations. However, this was accompanied by increased tyrosine and tryptophan nitration (detected by ELISA and LC-MS), and the overall extent of damage remained approximately constant. Increasing NO2- concentrations (0.5-20 µM) diminished HOCl-mediated modification of tyrosine and methionine, whereas tryptophan loss was enhanced. At higher NO2- concentrations, enhanced tyrosine and methionine loss was detected. These analytical data were confirmed in studies of cell adhesion and metabolic activity. Together, these data indicate that endogenous plasma levels of SCN- (but not Br- or I-) can modulate protein modification induced by MPO, including the extent of chlorination. In contrast, NO2- alters the type of modification, but does not markedly decrease its extent, with chlorination replaced by nitration. These data also indicate that MPO could be a major source of nitration in vivo, and particularly at inflammatory sites where NO2- levels are often elevated.

Role of macrophage extracellular traps in innate immunity and inflammatory disease.

Macrophages play an integral role in initiating innate immune defences and regulating inflammation. They are also involved in maintaining homeostasis and the resolution of inflammation, by promoting tissue repair and wound healing. There is evidence that like neutrophils, macrophages can release extracellular traps following exposure to a range of pathogenic and pro-inflammatory stimuli. Extracellular traps are released by a specialised cell death pathway termed 'ETosis', and consist of a backbone of DNA and histones decorated with a range of other proteins. The composition of extracellular trap proteins can be influenced by both the cell type and the local environment in which the traps are released. In many cases, these proteins have an antimicrobial role and assist with pathogen killing. Therefore, the release of extracellular traps serves as a means to both immobilise and destroy invading pathogens. In addition to their protective role, extracellular traps are also implicated in disease pathology. The release of neutrophil extracellular traps (NETs) is causally linked to the development of wide range of human diseases. However, whether macrophage extracellular traps (METs) play a similar role in disease pathology is less well established. Moreover, macrophages are also involved in the clearance of extracellular traps, which could assist in the resolution of tissue damage associated with the presence of extracellular traps. In this review, we will provide an overview of the pathways responsible for macrophage extracellular trap release, and discuss the role of these structures in innate immunity and disease pathology and possible therapeutic strategies.

Role of myeloperoxidase and oxidant formation in the extracellular environment in inflammation-induced tissue damage.

The heme peroxidase family generates a battery of oxidants both for synthetic purposes, and in the innate immune defence against pathogens. Myeloperoxidase (MPO) is the most promiscuous family member, generating powerful oxidizing species including hypochlorous acid (HOCl). Whilst HOCl formation is important in pathogen removal, this species is also implicated in host tissue damage and multiple inflammatory diseases. Significant oxidant formation and damage occurs extracellularly as a result of MPO release via phagolysosomal leakage, cell lysis, extracellular trap formation, and inappropriate trafficking. MPO binds strongly to extracellular biomolecules including polyanionic glycosaminoglycans, proteoglycans, proteins, and DNA. This localizes MPO and subsequent damage, at least partly, to specific sites and species, including extracellular matrix (ECM) components and plasma proteins/lipoproteins. Biopolymer-bound MPO retains, or has enhanced, catalytic activity, though evidence is also available for non-catalytic effects. These interactions, particularly at cell surfaces and with the ECM/glycocalyx induce cellular dysfunction and altered gene expression. MPO binds with higher affinity to some damaged ECM components, rationalizing its accumulation at sites of inflammation. MPO-damaged biomolecules and fragments act as chemo-attractants and cell activators, and can modulate gene and protein expression in naïve cells, consistent with an increasing cycle of MPO adhesion, activity, damage, and altered cell function at sites of leukocyte infiltration and activation, with subsequent tissue damage and dysfunction. MPO levels are used clinically both diagnostically and prognostically, and there is increasing interest in strategies to prevent MPO-mediated damage; therapeutic aspects are not discussed as these have been reviewed elsewhere.

Oral pre-treatment with thiocyanate (SCN-) protects against myocardial ischaemia-reperfusion injury in rats.

Despite improvements in revascularization after a myocardial infarction, coronary disease remains a major contributor to global mortality. Neutrophil infiltration and activation contributes to tissue damage, via the release of myeloperoxidase (MPO) and formation of the damaging oxidant hypochlorous acid. We hypothesized that elevation of thiocyanate ions (SCN-), a competitive MPO substrate, would modulate tissue damage. Oral dosing of rats with SCN-, before acute ischemia-reperfusion injury (30 min occlusion, 24 h or 4 week recovery), significantly reduced the infarct size as a percentage of the total reperfused area (54% versus 74%), and increased the salvageable area (46% versus 26%) as determined by MRI imaging. No difference was observed in fractional shortening, but supplementation resulted in both left-ventricle end diastolic and left-ventricle end systolic areas returning to control levels, as determined by echocardiography. Supplementation also decreased antibody recognition of HOCl-damaged myocardial proteins. SCN- supplementation did not modulate serum markers of damage/inflammation (ANP, BNP, galectin-3, CRP), but returned metabolomic abnormalities (reductions in histidine, creatine and leucine by 0.83-, 0.84- and 0.89-fold, respectively), determined by NMR, to control levels. These data indicate that elevated levels of the MPO substrate SCN-, which can be readily modulated by dietary means, can protect against acute ischemia-reperfusion injury.

Role of myeloperoxidase-derived oxidants in the induction of vascular smooth muscle cell damage.

Myeloperoxidase (MPO) is released by activated immune cells and forms the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) from the competing substrates chloride and thiocyanate. MPO and the overproduction of HOCl are strongly linked with vascular cell dysfunction and inflammation in atherosclerosis. HOCl is highly reactive and causes marked cell dysfunction and death, whereas data with HOSCN are conflicting, and highly dependent on the nature of the cell type. In this study we have examined the reactivity of HOCl and HOSCN with human coronary artery smooth muscle cells (HCASMC), given the key role of this cell type in maintaining vascular function. HOCl reacts rapidly with the cells, resulting in extensive cell death by both necrosis and apoptosis, and increased levels of intracellular calcium. In contrast, HOSCN reacts more slowly, with cell death occurring only after prolonged incubation, and in the absence of the accumulation of intracellular calcium. Exposure of HCASMC to HOCl also influences mitochondrial respiration, decreases glycolysis, lactate release, the production of ATP, cellular thiols and glutathione levels. These changes occurred to varying extents on exposure of the cells to HOSCN, where evidence was also obtained for the reversible modification of cellular thiols. HOCl also induced alterations in the mRNA expression of multiple inflammatory and phenotypic genes. Interestingly, the extent and nature of these changes was highly dependent on the specific cell donor used, with more marked effects observed in cells isolated from diseased compared to healthy vessels. Overall, these data provide new insight into pathways promoting vascular dysfunction during chronic inflammation, support the use of thiocyanate as a means to modulate MPO-induced cellular damage in atherosclerosis.

Modulation of hypochlorous acid (HOCl) induced damage to vascular smooth muscle cells by thiocyanate and selenium analogues.

The production of hypochlorous acid (HOCl) by myeloperoxidase (MPO) plays a key role in immune defense, but also induces host tissue damage, particularly in chronic inflammatory pathologies, including atherosclerosis. This has sparked interest in the development of therapeutic approaches that decrease HOCl formation during chronic inflammation, including the use of alternative MPO substrates. Thiocyanate (SCN-) supplementation decreases HOCl production by favouring formation of hypothiocyanous acid (HOSCN), which is more selectively toxic to bacterial cells. Selenium-containing compounds are also attractive therapeutic agents as they react rapidly with HOCl and can be catalytically recycled. In this study, we examined the ability of SCN-, selenocyanate (SeCN-) and selenomethionine (SeMet) to modulate HOCl-induced damage to human coronary artery smooth muscle cells (HCASMC), which are critical to both normal vessel function and lesion formation in atherosclerosis. Addition of SCN- prevented HOCl-induced cell death, altered the pattern and extent of intracellular thiol oxidation, and decreased perturbations to calcium homeostasis and pro-inflammatory signaling. Protection was also observed with SeCN- and SeMet, though SeMet was less effective than SeCN- and SCN-. Amelioration of damage was detected with sub-stoichiometric ratios of the added compound to HOCl. The effects of SCN- are consistent with conversion of HOCl to HOSCN. Whilst SeCN- prevented HOCl-induced damage to a similar extent to SCN-, the resulting product hyposelenocyanous acid (HOSeCN), was more toxic to HCASMC than HOSCN. These results provide support for the use of SCN- and/or selenium analogues as scavengers, to decrease HOCl-induced cellular damage and HOCl production at inflammatory sites in atherosclerosis and other pathologies.

Myeloperoxidase Modulates Hydrogen Peroxide Mediated Cellular Damage in Murine Macrophages.

Myeloperoxidase (MPO) is involved in the development of many chronic inflammatory diseases, in addition to its key role in innate immune defenses. This is attributed to the excessive production of hypochlorous acid (HOCl) by MPO at inflammatory sites, which causes tissue damage. This has sparked wide interest in the development of therapeutic approaches to prevent HOCl-induced cellular damage including supplementation with thiocyanate (SCN-) as an alternative substrate for MPO. In this study, we used an enzymatic system composed of glucose oxidase (GO), glucose, and MPO in the absence and presence of SCN-, to investigate the effects of generating a continuous flux of oxidants on macrophage cell function. Our studies show the generation of hydrogen peroxide (H2O2) by glucose and GO results in a dose- and time-dependent decrease in metabolic activity and cell viability, and the activation of stress-related signaling pathways. Interestingly, these damaging effects were attenuated by the addition of MPO to form HOCl. Supplementation with SCN-, which favors the formation of hypothiocyanous acid, could reverse this effect. Addition of MPO also resulted in upregulation of the antioxidant gene, NAD(P)H:quinone acceptor oxidoreductase 1. This study provides new insights into the role of MPO in the modulation of macrophage function, which may be relevant to inflammatory pathologies.

Myeloperoxidase-derived damage to human plasma fibronectin: Modulation by protein binding and thiocyanate ions (SCN-).

Endothelial cell dysfunction is an early event in cardiovascular disease and atherosclerosis. The origin of this dysfunction is unresolved, but accumulating evidence implicates damaging oxidants, including hypochlorous acid (HOCl), a major oxidant produced by myeloperoxidase (MPO), during chronic inflammation. MPO is released extracellularly by activated leukocytes and binds to extracellular molecules including fibronectin, a major matrix glycoprotein involved in endothelial cell binding. We hypothesized that MPO binding might influence the modifications induced on fibronectin, when compared to reagent HOCl, with this including alterations to the extent of damage to protein side-chains, modified structural integrity, changes to functional domains, and impact on naïve human coronary artery endothelial cell (HCAEC) adhesion and metabolic activity. The effect of increasing concentrations of the alternative MPO substrate thiocyanate (SCN-), which might decrease HOCl formation were also examined. Exposure of fibronectin to MPO/H2O2/Cl- is shown to result in damage to the functionally important cell-binding and heparin-binding fragments, gross structural changes to the protein, and altered HCAEC adhesion and activity. Differences were observed between stoichiometric, and above-stoichiometric MPO concentrations consistent with an effect of MPO binding to fibronectin. In contrast, MPO/H2O2/SCN- induced much less marked changes and limited protein damage. Addition of increasing SCN- concentrations to the MPO/H2O2/Cl- system provided protection, with 20 µM of this anion rescuing damage to functionally-important domains, decreasing chemical modification, and maintaining normal HCAEC behavior. Modulating MPO binding to fibronectin, or enhancing SCN- levels at sites of inflammation may therefore limit MPO-mediated damage, and be of therapeutic value.

Role of thiocyanate in the modulation of myeloperoxidase-derived oxidant induced damage to macrophages.

Myeloperoxidase (MPO) is a vital component of the innate immune system, which produces the potent oxidant hypochlorous acid (HOCl) to kill invading pathogens. However, an overproduction of HOCl during chronic inflammatory conditions causes damage to host cells, which promotes disease, including atherosclerosis. As such, there is increasing interest in the use of thiocyanate (SCN-) therapeutically to decrease inflammatory disease, as SCN- is the favoured substrate for MPO, and a potent competitive inhibitor of HOCl formation. Use of SCN- by MPO forms hypothiocyanous acid (HOSCN), which can be less damaging to mammalian cells. In this study, we examined the ability of SCN- to modulate damage to macrophages induced by HOCl, which is relevant to lesion formation in atherosclerosis. Addition of SCN- prevented HOCl-mediated cell death, altered the extent and nature of thiol oxidation and the phosphorylation of mitogen activated protein kinases. These changes were dependent on the concentration of SCN- and were observed in some cases, at a sub-stoichiometric ratio of SCN-: HOCl. Co-treatment with SCN- also modulated HOCl-induced perturbations in the expression of various antioxidant and inflammatory genes. In general, the data reflect the conversion of HOCl to HOSCN, which can induce reversible modifications that are repairable by cells. However, our data also highlight the ability of HOSCN to increase pro-inflammatory gene expression and cytokine/chemokine release, which may be relevant to the use of SCN- therapeutically in atherosclerosis. Overall, this study provides further insight into the cellular pathways by which SCN- could exert protective effects on supplementation to decrease the development of chronic inflammatory diseases, such as atherosclerosis.

The role of the myeloperoxidase-derived oxidant hypothiocyanous acid (HOSCN) in the induction of mitochondrial dysfunction in macrophages.

A host of chronic inflammatory diseases are accelerated by the formation of the powerful oxidant hypochlorous acid (HOCl) by myeloperoxidase (MPO). In the presence of thiocyanate (SCN-), the production of HOCl by MPO is decreased in favour of the formation of a milder oxidant, hypothiocyanous acid (HOSCN). The role of HOSCN in disease has not been fully elucidated, though there is increasing interest in using SCN- therapeutically in different disease settings. Unlike HOCl, HOSCN can be detoxified by thioredoxin reductase, and reacts selectively with thiols to result in reversible modifications, which could potentially reduce the extent of MPO-induced damage during chronic inflammation. In this study, we show that exposure of macrophages, a key inflammatory cell type, to HOSCN results in the reversible modification of multiple mitochondrial proteins, leading to increased mitochondrial membrane permeability, decreased oxidative phosphorylation and reduced formation of ATP. The increased permeability and reduction in ATP could be reversed by pre-treatment of the macrophages with cyclosporine A, implicating a role for the mitochondrial permeability transition pore. HOSCN also drives cells to utilise fatty acids as an energetic substrate after the inhibition of oxidative phosphorylation. Raman imaging studies highlighted the ability of HOSCN to perturb the electron transport chain of mitochondria and redistribute these organelles within the cell. Taken together, these data provide new insight into the pathways by which HOSCN can induce cytotoxicity and cellular damage, which may have relevance for the development of inflammatory disease, and therapeutic strategies to reduce HOCl-induced damage by supplementation with SCN-.

Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization.

The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-l-lysine, o,o'-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2-1000 pmol for parent amino acids, 80 fmol-20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o'-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.