8-Chloroadenosine Alters the Metabolic Profile and Downregulates Antioxidant and DNA Damage Repair Pathways in Macrophages.

The exposure of RNA and DNA nucleobases to the oxidant hypochlorous acid (HOCl) results in the generation of different stable chlorinated products. These chlorinated nucleobases are formed in vivo, particularly in chronic inflammatory pathologies, which are characterized by the overproduction of HOCl by myeloperoxidase. As such, chlorinated nucleosides are used as biomarkers of inflammation. However, these compounds have also attracted attention as potential chemotherapeutic agents with 8-chloro-adenosine (8ClA), for example, currently in clinical trials for the treatment of hematological cancers, including chronic lymphocytic leukemia. 8ClA has mainly RNA-directed effects in malignant cells, with exposure resulting in ATP depletion and apoptotic cell death. Whether 8ClA has significant reactivity with nonmalignant cells has not been widely studied. Here we show that prolonged incubation of J774A.1 macrophage-like cells with 8ClA results in the perturbation of cellular metabolism and apoptotic cell death. These effects are associated with an accumulation of 8-chloroadenosine triphosphate (8Cl-ATP), an effect not seen in experiments utilizing other chlorinated nucleosides. Exposure of the macrophages to 8ClA did not significantly change basal mitochondrial respiration or glycolysis but resulted in an increase in maximal mitochondrial respiration as well as spare respiratory capacity within these cells. Additionally, 8ClA exposure also altered the mRNA expression of a range of antioxidant and DNA damage repair genes in the macrophages in a manner consistent with a reduction in the capacity of the cells to cope with oxidative stress and repair DNA damage. Taken together, these results provide new insight into pathways by which the production of HOCl during chronic inflammation could perturb immune cell function and may also have implications for the use of 8ClA as a chemotherapeutic drug.

Nitroxides Mitigate Neutrophil-Mediated Damage to the Myocardium after Experimental Myocardial Infarction in Rats.

Reperfusion therapy increases survival post-acute myocardial infarction (AMI) while also stimulating secondary oxidant production and immune cell infiltration. Neutrophils accumulate within infarcted myocardium within 24 h post-AMI and release myeloperoxidase (MPO) that catalyses hypochlorous acid (HOCl) production while increasing oxidative stress and inflammation, thereby enhancing ventricular remodelling. Nitroxides inhibit MPO-mediated HOCl production, potentially ameliorating neutrophil-mediated damage. Aim: Assess the cardioprotective ability of nitroxide 4-methoxyTEMPO (4MetT) within the setting of AMI. Methods: Male Wistar rats were separated into 3 groups: SHAM, AMI/R, and AMI/R + 4MetT (15 mg/kg at surgery via oral gavage) and subjected to left descending coronary artery ligation for 30 min to generate an AMI, followed by reperfusion. One cohort of rats were sacrificed at 24 h post-reperfusion and another 28 days post-surgery (with 4MetT (15 mg/kg) administration twice daily). Results: 3-chlorotyrosine, a HOCl-specific damage marker, decreased within the heart of animals in the AMI/R + 4-MetT group 24 h post-AMI, indicating the drug inhibited MPO activity; however, there was no evident difference in either infarct size or myocardial scar size between the groups. Concurrently, MPO, NfκB, TNFα, and the oxidation marker malondialdehyde increased within the hearts, with 4-MetT only demonstrating a trend in decreasing MPO and TNF levels. Notably, 4MetT provided a significant improvement in cardiac function 28 days post-AMI, as assessed by echocardiography, indicating potential for 4-MetT as a treatment option, although the precise mechanism of action of the compound remains unclear.

Characterization of the cellular effects of myeloperoxidase-derived oxidants on H9c2 cardiac myoblasts.

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury, which is in part due to the release of the enzyme myeloperoxidase (MPO) from activated infiltrating leukocytes, and the subsequent production of the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Although exposure of various cell types to either oxidant is known to cause cellular dysfunction within a variety of pathological settings, the precise role of HOCl and HOSCN in the initiation of tissue damage evident following cardiac I/R injury remains unclear. In this study, we have employed the use of the cardiac myoblast cell line H9c2 as a model for cardiac myocytes and demonstrate that exposure to either oxidant elicits a dose-dependent increase in cytosolic calcium accumulation, depletion of the cellular thiol pool, reduction of glutathione (GSH) levels and loss of mitochondrial inner trans-membrane potential, concomitant with increased necrotic cell death. H9c2 cell recovery from the initial oxidant exposure involves the initiation of cell survival signalling pathways centred around Nrf2-antioxidant response element (ARE) and activator protein 1 (AP-1) activation, with cell survival accompanied by restoration of mitochondrial function following exposure to HOSCN, but not HOCl. These data highlight the cellular responses elicited by HOCl and HOSCN in cardiac myocytes furthering our understanding of the pathogenesis of oxidant injury following cardiac I/R injury.

A Role for Chlorinated Nucleosides in the Perturbation of Macrophage Function and Promotion of Inflammation.

During inflammation, myeloperoxidase released from activated phagocytes generates the highly reactive oxidant hypochlorous acid (HOCl). This oxidant plays an important role in the immune response but can also promote tissue damage and has been strongly linked with the development of numerous inflammatory diseases. HOCl reacts with cellular DNA forming chlorinated nucleobases, which induce strand breaks, mutations, and cross-links. Although it has been shown that chlorinated nucleosides are present within inflammatory pathologies and diseased tissue, whether or not these species are biomarkers formed as a byproduct of chronic inflammation or play a role in the disease progression has not been ascertained. In this study, we show that exposure of J774A.1 macrophage-like cells to chlorinated ribose and deoxyribose nucleosides results in the incorporation of 5-chloro-cytidine (5ClC), 8-chloro-adenosine (8ClA), and 8-chloro-guanosine (8ClG) into the cellular RNA and 5-chloro-deoxycytidine (5CldC) but not 8-chloro-deoxyguanosine (8CldG) or 8-chloro-deoxyadenosine (8CldA) into cellular DNA. Evidence was obtained for the clearance of 5ClC from the RNA, with a loss of 8ClA and 8ClG observed to a lesser extent, whereas an increase in the level of 5CldC in DNA was seen on further incubation of treated cells in the absence of chlorinated nucleosides. Importantly, exposure of the macrophages to chlorinated nucleosides, particularly 8ClG and 5ClC, resulted in the increased expression of interleukin-1ß, and other pro-inflammatory cytokines and chemokines. With 5ClC, this inflammatory response was associated with the increased nuclear translocation of the NF-κB subunit, p65, rather than inflammasome activation. This alteration in gene expression appeared to be unrelated to the extent of incorporation of the chlorinated nucleosides into RNA or DNA and was not associated with any significant changes in cell viability or proliferation. Taken together, these results highlight a potential biological role for chlorinated nucleosides to promote inflammatory disease, in addition to their utility as biomarkers.

A pivotal role for NF-κB in the macrophage inflammatory response to the myeloperoxidase oxidant hypothiocyanous acid.

Atherosclerosis is characterised by the infiltration of macrophages at sites of inflammation within the vessel wall and the release of myeloperoxidase (MPO), which forms hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). HOCl is a damaging oxidant implicated in the development of atherosclerosis. Preferential formation of HOSCN occurs under conditions where thiocyanate ions are elevated, as is the case in smokers. HOSCN reacts selectively with thiols, which can result in more enzyme inactivation and damage than HOCl at susceptible sites, which may contribute to atherosclerosis in smokers. In this study, we show that exposure of macrophages to HOSCN results in a time- and dose-dependent increase in the mRNA expression and release of pro-inflammatory cytokines and chemokines, including monocyte chemotactic protein 1, tumour necrosis factor alpha, and interleukins 6, 8 and 1ß. At high oxidant concentrations (>200 µM), a significant loss of cellular thiols and increased cell death is observed. HOSCN-induced cytokine/chemokine expression and cell death were decreased on pharmacological inhibition of nuclear factor kappa B. These data highlight a pathway by which HOSCN could promote inflammation and the development of atherosclerosis, in the presence of supra-physiological levels of the precursor thiocyanate, which are achievable by cigarette smoking.

Multiplex analysis of mass imaging data: Application to the pathology of experimental myocardial infarction.

AIM: Imaging mass cytometry (IMC) affords simultaneous immune-labelling/imaging of multiple antigens in the same tissue. Methods utilizing multiplex data beyond co-registration are lacking. This study developed and applied an innovative spatial analysis workflow for multiplex imaging data to IMC data determined from cardiac tissues and revealed the mechanism(s) of neutrophil-mediated post-myocardial-infarction damage. METHODS: IMC produced multiplex images with various redox/inflammatory markers. The cardiac peri-infarct zone (PIZ) was determined to be up to 240 µm from the infarct border based on the presence of neutrophils. The tissue region beyond the infarct was defined as the remote area (RA). ImageJ was used to quantify the immunoreactivity. Functional assessments included infarct size, cell necro/apoptosis, total thiol assay and echocardiogram. RESULTS: Expression of damage markers decreased in order from the infarct area to PIZ and then RA, reflecting the neutrophil density in the regions. Concentrically spaced "shoreline contour analysis" around the cardiac infarct extending into the PIZ showed that immunoreactivity for damage markers decreased linearly with increasing distance from the infarct, concomitant with a decreasing neutrophil-myeloperoxidase (MPO) gradient from the infarct to the PIZ. Stratifying by concentric bands around individual MPO+ -signal identified that the immunoreactivity of haem-oxygenase-1 (HO-1) and phosphorylated-p38 mitogen-activated protein kinase (pP38) peaked near neutrophils. Furthermore, spatial dependence between neutrophils and markers of cardiac cellular damage was confirmed by nearest-neighbour distance analysis. Post-infarction tissue exhibited declined functional parameters that were associated with neutrophil migration from the infarct to PIZ. CONCLUSION: This image-based quantitative protocol revealed the spatial association and provided potential molecular pathways responsible for neutrophil-mediated damage post-infarction.

Evaluation of the diagnostic accuracy of current biomarkers in heart failure with preserved ejection fraction: A systematic review and meta-analysis.

BACKGROUND: A number of circulating biomarkers are currently utilized for the diagnosis of chronic heart failure with preserved ejection fraction (HFpEF). However, due to HFpEF heterogeneity, the accuracy of these biomarkers remains unclear. AIMS: This study aimed to systematically determine the diagnostic accuracy of currently available biomarkers for chronic HFpEF. METHODS: PubMed, Web of Science, MEDLINE and SCOPUS databases were searched systematically to identify studies assessing the diagnostic accuracy of biomarkers of chronic HFpEF with left ventricular ejection fraction (LVEF) ≥50%. All included studies were independently assessed for quality and relevant information was extracted. Random-effects models were used to estimate the pooled diagnostic accuracy of HFpEF biomarkers. RESULTS: The search identified 6145 studies, of which 19 were included. Four biomarkers were available for meta-analysis. The pooled sensitivity of B-type natriuretic peptide (BNP) (0.787, 95% confidence interval [CI] 0.719-0.842) was higher than that of N-terminal pro-BNP (NT-proBNP) (0.696, 95% CI 0.599-0.779) in chronic HFpEF diagnosis. However, NT-proBNP showed improved specificity (0.882, 95% CI 0.778-0.941) compared to BNP (\0.796, 95% CI 0.672-0.882). Galectin-3 (Gal-3) exhibited a reliable diagnostic adequacy for HFpEF (sensitivity 0.760, 95% CI 0.631-0.855; specificity 0.803, 95% CI 0.667-0.893). However, suppression of tumorigenesis-2 (ST2) displayed limited diagnostic performance for chronic HFpEF diagnosis (sensitivity 0.636, 95% CI 0.465-0.779; specificity 0.595, 95% CI 0.427-0.743). CONCLUSION: NT-proBNP and BNP appear to be the most reliable biomarkers in chronic HFpEF with NT-proBNP showing higher specificity and BNP showing higher sensitivity. Although Gal-3 appears more reliable than ST2 in HFpEF diagnosis, the conclusions are limited as only three studies were included in this meta-analysis.

Therapeutic Targets in Diffuse Midline Gliomas-An Emerging Landscape.

Diffuse midline gliomas (DMGs) are invariably fatal pediatric brain tumours that are inherently resistant to conventional therapy. In recent years our understanding of the underlying molecular mechanisms of DMG tumorigenicity has resulted in the identification of novel targets and the development of a range of potential therapies, with multiple agents now being progressed to clinical translation to test their therapeutic efficacy. Here, we provide an overview of the current therapies aimed at epigenetic and mutational drivers, cellular pathway aberrations and tumor microenvironment mechanisms in DMGs in order to aid therapy development and facilitate a holistic approach to patient treatment.

Nitric oxide stimulates myoglobin gene and protein expression in vascular smooth muscle.

Mb (myoglobin) is a haemoprotein present in cardiac, skeletal and smooth muscle and is primarily responsible for the storage and 'facilitated transfer' of molecular oxygen from the cell membrane to mitochondria. Also, Mb plays a role in regulating *NO (nitric oxide) homoeostasis through (i) binding *NO (Mb-NO complex); (ii) oxidation of *NO to nitrate; and (iii) formation of vasoactive S-nitroso-Mb [Rayner, B.S., Wu, B.-J., Raftery, M., Stocker, R. and Witting, P.K. (2005) J. Biol. Chem. 280, 9985-9993]. Pathological *NO concentrations affect mitochondrial function and decrease cell viability through inducing apoptosis. Treatment of cultured rat VSMCs (vascular smooth muscle cells) with cumulative doses (0.1, 1 or 10 microM) of *NO from the donors diethylamineNONOate or spermineNONOate (N-[2-aminoethyl]-N-[2-hydroxy-3-nitrosohydrazine]-1,2-ethelenediamine) yielded a time-dependent increase in Mb gene expression. Concomitant transcriptional activation increased the concentration of Mb within cultured rat or primary human VSMCs as judged by Western blot analysis and indirect immunofluorescence microscopy. Cell viability did not decrease in these cells at the *NO doses tested. Importantly, sub-culturing isolated rat aortic segments for 7 days in the presence of L-arginine at 37 degrees C stimulated *NO production with a parallel increase in Mb in the underlying VSMCs. Overall, exposure of VSMCs (either in cell culture or intact vessels) to pathological *NO promotes an up-regulation of the Mb gene and protein, suggesting a feedback relationship between *NO and Mb that regulates the concentration of the potent cell signalling molecule in the vessel wall, similar to the role haemoglobin plays in the vessel lumen.

Selenomethionine supplementation reduces lesion burden, improves vessel function and modulates the inflammatory response within the setting of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the vasculature characterised by the infiltration of activated neutrophils and macrophages at sites of damage within the vessel wall, which contributes to lesion formation and plaque progression. Selenomethionine (SeMet) is an organic form of selenium (Se), an essential trace element that functions in the regulation of the immune response by both bolstering the endogenous thioredoxin and glutathione antioxidant defence systems and by directly scavenging damaging oxidant species. This study evaluated the effect of dietary SeMet supplementation within a high fat diet fed apolipoprotein E deficient (ApoE-/-) mouse model of atherosclerosis. Dietary supplementation with SeMet (2 mg/kg) increased the tissue concentration of Se, and the expression and activity of glutathione peroxidase, compared to non-supplemented controls. Supplementation with SeMet significantly reduced atherosclerotic plaque formation in mouse aortae, resulted in a more stable lesion phenotype and improved vessel function. Concurrent with these results, SeMet supplementation decreased lesion accumulation of M1 inflammatory type macrophages, and decreased the extent of extracellular trap release from phorbol myristate acetate (PMA)-stimulated mouse bone marrow-derived cells. Importantly, these latter results were replicated within ex-vivo experiments on cultured neutrophils isolated from acute coronary syndrome patients, indicating the ability of SeMet to alter the acute inflammatory response within a clinically-relevant setting. Together, these data highlight the potential beneficial effect of SeMet supplementation as a therapeutic strategy for atherosclerosis.

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

8-Chloroadenosine induces apoptosis in human coronary artery endothelial cells through the activation of the unfolded protein response.

Infiltration of leukocytes within the vessel at sites of inflammation and the subsequent generation of myeloperoxidase-derived oxidants, including hypochlorous acid, are key characteristics of atherosclerosis. Hypochlorous acid is a potent oxidant that reacts readily with most biological molecules, including DNA and RNA. This results in nucleic acid modification and the formation of different chlorinated products. These products have been used as biomarkers of inflammation, owing to their presence in elevated amounts in different inflammatory fluids and diseased tissue, including atherosclerotic lesions. However, it is not clear whether these materials are simply biomarkers, or could also play a role in the development of chronic inflammatory pathologies. In this study, we examined the reactivity of different chlorinated nucleosides with human coronary artery endothelial cells (HCAEC). Evidence was obtained for the incorporation of each chlorinated nucleoside into the cellular RNA or DNA. However, only 8-chloro-adenosine (8ClA) had a significant effect on the cell viability and metabolic activity. Exposure of HCAEC to 8ClA decreased glycolysis, and resulted in a reduction in ATP, with a corresponding increase in the chlorinated analogue, 8Cl-ATP in the nucleotide pool. 8ClA also induced sustained endoplasmic reticulum stress within the HCAEC, which resulted in activation of the unfolded protein response, the altered expression of antioxidant genes and culminated in the release of calcium into the cytosol and cell death by apoptosis. Taken together, these data provide new insight into pathways by which myeloperoxidase activity and resultant hypochlorous acid generation could promote endothelial cell damage during chronic inflammation, which could be relevant to the progression of atherosclerosis.

Assessing the Efficacy of Dietary Selenomethionine Supplementation in the Setting of Cardiac Ischemia/Reperfusion Injury.

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing myeloperoxidase (MPO) and its production of hypochlorous acid (HOCl). The dietary supplement selenomethionine (SeMet) has been shown to bolster endogenous antioxidant processes as well as readily react with MPO-derived oxidants. The aim of this study was to assess whether supplementation with SeMet could modulate the extent of cellular damage observed in an in vitro cardiac myocyte model exposed to (patho)-physiological levels of HOCl and an in vivo rat model of cardiac I/R injury. Exposure of the H9c2 cardiac myoblast cell line to HOCl resulted in a dose-dependent increase in necrotic cell death, which could be prevented by SeMet supplementation and was attributed to SeMet preventing the HOCl-induced loss of mitochondrial inner trans-membrane potential, and the associated cytosolic calcium accumulation. This protection was credited primarily to the direct oxidant scavenging ability of SeMet, with a minor contribution arising from the ability of SeMet to bolster cardiac myoblast glutathione peroxidase (GPx) activity. In vivo, a significant increase in selenium levels in the plasma and heart tissue were seen in male Wistar rats fed a diet supplemented with 2 mg kg-1 SeMet compared to controls. However, SeMet-supplementation demonstrated only limited improvement in heart function and did not result in better heart remodelling following I/R injury. These data indicate that SeMet supplementation is of potential benefit within pathological settings where excessive HOCl is known to be generated but has limited efficacy as a therapeutic agent for the treatment of heart attack.

Role of hypochlorous acid (HOCl) and other inflammatory mediators in the induction of macrophage extracellular trap formation.

The infiltration of activated leukocytes, including macrophages, at sites of inflammation and the formation and presence of hypochlorous acid (HOCl) are interlinked hallmarks of many debilitating disease processes, including atherosclerosis, arthritis, neurological and renal disease, diabetes and obesity. The production of extracellular traps by activated leukocytes in response to a range of inflammatory stimuli is increasingly recognised as an important process within a range of disease settings. We show that exposure of human monocyte-derived macrophages to pathophysiological levels of HOCl results in the dose-dependent extrusion of DNA and histones into the cellular supernatant, consistent with extracellular trap formation. Concurrent with, but independent of these findings, macrophage exposure to HOCl also resulted in an immediate and sustained cytosolic accumulation of Ca2+, culminating in the increased production of cytokines and chemokines. Polarisation of the macrophages prior to HOCl exposure revealed a greater propensity for inflammatory M1 macrophages to produce extracellular traps, whereas alternatively-activated M2 macrophages were less susceptible to HOCl insult. M1 macrophages also produced extracellular traps on exposure to phorbol myristate acetate (PMA), interleukin-8 (IL-8) and tumour necrosis factor α (TNFα). Taken together, these data indicate a potential role for macrophages in mediating extracellular trap formation, which may be relevant in pathological conditions characterised by chronic inflammation or excessive HOCl formation.

TRAIL protects against endothelial dysfunction in vivo and inhibits angiotensin-II-induced oxidative stress in vascular endothelial cells in vitro.

The vascular endothelium is critical for maintenance of cardiovascular homeostasis. Endothelial dysfunction is a key event of atherosclerosis, with oxidative stress mediated by reactive oxygen species (ROS) playing a major role. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is increasingly recognised to play a protective role in atherosclerosis, however the molecular mechanisms by which it exerts its beneficial effects are unclear. Here we examined if TRAIL could attenuate vascular oxidative stress and improve endothelial cell (EC) function. In coronary artery disease patients, plasma TRAIL levels were significantly reduced compared to healthy individuals, and negatively correlated with the levels of circulating 8-iso Prostaglandin F2α, a marker of in vivo oxidative stress. In vivo, high-fat fed, atherosclerotic Trail-/-Apoe-/- mice exhibited a significant impairment in endothelial-dependent vasorelaxation, which correlated with increased vascular ROS and 4-hydroxynonenal compared to Apoe-/- mice. Endothelial permeability measured by Evan's blue dye extravasation was increased in several organs of Trail-/- mice compared to wild-type mice, which correlated with a decrease in VE-cadherin expression. In vitro in ECs, angiotensin II (AngII)-induced ROS generation involving the mitochondria, NADPH oxidase-4 (NOX-4) and eNOS, was inhibited by pre-treatment with TRAIL. Furthermore, AngII-augmented VCAM-1 expression and monocyte adhesion to ECs was inhibited by TRAIL. Finally, AngII reduced VE-cadherin expression and redistributed this protein, all of which was brought back to baseline by TRAIL pre-treatment. These findings demonstrate for the first time that TRAIL protects against several forms of endothelial dysfunction involving its ability to control EC ROS generation. Understanding the role TRAIL plays in normal physiology and disease, may lead to potential new therapies to improve endothelial function and atherosclerosis.

Low-density lipoprotein modified by myeloperoxidase oxidants induces endothelial dysfunction.

Low-density lipoprotein (LDL) modified by hypochlorous acid (HOCl) produced by myeloperoxidase (MPO) is present in atherosclerotic lesions, where it is implicated in the propagation of inflammation and acceleration of lesion development by multiple pathways, including the induction of endothelial dysfunction. Thiocyanate (SCN-) ions are utilised by MPO to produce the oxidant hypothiocyanous acid (HOSCN), which reacts with LDL in a different manner to HOCl. Whilst the reactivity of HOCl-modified LDL has been previously studied, the role of HOSCN in the modification of LDL in vivo is poorly defined, although emerging evidence suggests that these particles have distinct biological properties. This is important because elevated plasma SCN- is linked with both the propagation and prevention of atherosclerosis. In this study, we demonstrate that both HOSCN- and HOCl-modified LDL inhibit endothelium-mediated vasorelaxation ex vivo in rat aortic ring segments. In vitro experiments with human coronary artery endothelial cells show that HOSCN-modified LDL decreases in the production of nitric oxide (NO•) and induces the loss of endothelial nitric oxide synthase (eNOS) activity. This occurs to a similar extent to that seen with HOCl-modified LDL. In each case, these effects are related to eNOS uncoupling, rather than altered expression, phosphorylation or cellular localisation. Together, these data provide new insights into role of MPO and LDL modification in the induction of endothelial dysfunction, which has implications for both the therapeutic use of SCN- within the setting of atherosclerosis and for smokers, who have elevated plasma levels of SCN-, and are more at risk of developing cardiovascular disease.

Regio- and stereo-chemical oxidation of linoleic acid by human myoglobin and hydrogen peroxide: Tyr(103) affects rate and product distribution.

Mb (myoglobin) plus H2O2 catalyses the oxidation of various substrates via a peroxidase-like activity. A Y103F (Tyr103-->Phe) variant of human Mb has been constructed to assess the effect of exchanging an electron-rich oxidizable amino acid on the peroxidase activity of human Mb. Steady-state analyses of reaction mixtures containing Y103F Mb, purified linoleic acid and H2O2 revealed a lower total yield of lipid oxidation products than mixtures containing the wild-type protein, consistent with the reported decrease in the rate constant for reaction of Y103F Mb with H2O2 [Witting, Mauk and Lay (2002) Biochemistry 41, 11495-11503]. Irrespective of the Mb employed, lipid oxidation yielded 9(R/S)-HODE [9(R,S)-hydroxy-10E,12Z-octadecadienoic acid] in preference to 13(R/S)-HODE [13(R,S)-hydroxy-9Z,11E-octadecadienoic acid], while 9- and 13-keto-octadecadienoic acid were formed in trace amounts. However, lipid oxidation by the Y103F variant of Mb proceeded with a lower V(max) value and an increased K(m) value relative to the wild-type control. Consistent with the increased K(m), the product distribution from reactions with Y103F Mb showed decreased selectivity compared with the wild-type protein, as judged by the decreased yield of 9(S)-relative to 9(R)-HODE. Together, these data verify that Tyr103 plays a significant role in substrate binding and orientation in the haem pocket of human Mb. Also, the midpoint potential for the Fe(III)/(II) one-electron reduction was shifted slightly, but significantly, to a higher potential, confirming the importance of Tyr103 to the hydrogen-bonding network involving residues that line the haem crevice of human Mb.

Regulation of vascular tone by S-nitroso-myoglobin.

Myoglobin (Mb) is a haem protein present in skeletal, cardiac and smooth muscle where it facilitates the transfer of O(2) from the extracellular matrix to the cell cytosol in a cycle termed 'facilitated O(2)-diffusion'. In addition, we showed recently that recombinant human Mb binds endothelium-derived relaxant factor - nitric oxide ((.-)NO) - via formation of both nitrosyl-haem iron and S-nitroso-myoglobin (S-NO-Mb). S-NO-Mb represents a novel form of endothelium-derived relaxant factor (EDRF) that may be important in maintaining optimal (.-)NO concentrations in the human vasculature. In this study we aim to show that: (i) S-nitrosation of oxygenated ferrous myoglobin (oxyMb) can compete with the rapid oxidation of (.-)NO by oxyMb; and (ii) S-NO-Mb retains characteristics of physiological EDRF.

Comparative reactivity of myeloperoxidase-derived oxidants with mammalian cells.

Myeloperoxidase is an important heme enzyme released by activated leukocytes that catalyzes the reaction of hydrogen peroxide with halide and pseudo-halide ions to form various hypohalous acids. Hypohalous acids are chemical oxidants that have potent antibacterial, antiviral, and antifungal properties and, as such, play key roles in the human immune system. However, increasing evidence supports an alternative role for myeloperoxidase-derived oxidants in the development of disease. Excessive production of hypohalous acids, particularly during chronic inflammation, leads to the initiation and accumulation of cellular damage that has been implicated in many human pathologies including atherosclerosis, neurodegenerative disease, lung disease, arthritis, inflammatory cancers, and kidney disease. This has sparked a significant interest in developing a greater understanding of the mechanisms involved in myeloperoxidase-derived oxidant-induced mammalian cell damage. This article reviews recent developments in our understanding of the cellular reactivity of hypochlorous acid, hypobromous acid, and hypothiocyanous acid, the major oxidants produced by myeloperoxidase under physiological conditions.

Comparative reactivity of the myeloperoxidase-derived oxidants hypochlorous acid and hypothiocyanous acid with human coronary artery endothelial cells.

In the immune response, hypohalous acids are generated by activated leukocytes via the release of myeloperoxidase and the formation of H2O2. Although these oxidants have important bactericidal properties, they have also been implicated in causing tissue damage in inflammatory diseases, including atherosclerosis. Hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) are the major oxidants formed by myeloperoxidase under physiological conditions, with the ratio of these oxidants dependent on diet and smoking status. HOCl is highly reactive and causes marked cellular damage, but few data are available on the effects of HOSCN on mammalian cells. In this study, we have compared the actions of HOCl and HOSCN on human coronary artery endothelial cells (HCAEC). HOCl reacts rapidly with the cells, resulting in extensive cell death by both apoptosis and necrosis, with necrosis dominating at higher oxidant doses. In contrast, HOSCN is consumed more slowly, with cell death occurring only by apoptosis. Exposure of HCAEC to HOCl and HOSCN induces changes in mitochondrial membrane permeability, which, in the case of HOSCN, is associated with mitochondrial release of proapoptotic factors, including cytochrome c, apoptosis-inducing factor, and endonuclease G. With each oxidant, apoptosis appears to be caspase-independent, with the inactivation of caspases 3/7 observed, and pretreatment of the cells with the caspase inhibitor Z-VAD-fmk having no effect on the extent of cell death. Loss of cellular thiols, depletion of glutathione, and the inactivation of thiol-dependent enzymes, including glyceraldehyde-3-phosphate dehydrogenase, were seen with both oxidants, though to a much greater extent with HOCl. The ability of myeloperoxidase-derived oxidants to induce endothelial cell apoptosis may contribute to the formation of unstable lesions in atherosclerosis. The results with HOSCN may be particularly significant for smokers, who have elevated plasma levels of SCN(-), the precursor of this oxidant.