Nicholas K.H. Khoo

Hydrogen peroxide is the major oxidant product of xanthine oxidase.

Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) in inflammatory disease. Focus, however, has centered almost exclusively on XO-derived superoxide (O(2)(*-)), whereas direct H(2)O(2) production from XO has been less well investigated. Therefore, we examined the relative quantities of O(2)(*-) and H(2)O(2) produced by XO under a range (1-21%) of O(2) tensions. At O(2) concentrations between 10 and 21%, H(2)O(2) accounted for approximately 75% of ROS production. As O(2) concentrations were lowered, there was a concentration-dependent increase in H(2)O(2) formation, accounting for 90% of ROS production at 1% O(2). Alterations in pH between 5.5 and 7.4 did not affect the relative proportions of H(2)O(2) and O(2)(*-) formation. Immobilization of XO, by binding to heparin-Sepharose, further enhanced relative H(2)O(2) production by approximately 30%, under both normoxic and hypoxic conditions. Furthermore, XO bound to glycosaminoglycans on the apical surface of bovine aortic endothelial cells demonstrated a similar ROS production profile. These data establish H(2)O(2) as the dominant (70-95%) reactive product produced by XO under clinically relevant conditions and emphasize the importance of H(2)O(2) as a critical factor when examining the contributory roles of XO-catalyzed ROS in inflammatory processes as well as cellular signaling.

Oxidases and Peroxidases in Cardiovascular and Lung Disease: New Concepts in Reactive Oxygen Species Signaling

Reactive oxygen species (ROS) are involved in numerous physiological and pathophysiological responses. Increasing evidence implicates ROS as signaling molecules involved in the propagation of cellular pathways. The NADPH oxidase (Nox) family of enzymes is a major source of ROS in the cell and has been related to the progression of many diseases and even environmental toxicity. The complexity of this familys effects on cellular processes stems from the fact that there are seven members, each with unique tissue distribution, cellular localization, and expression. Nox proteins also differ in activation mechanisms and the major ROS detected as their product. To add to this complexity, mounting evidence suggests that other cellular oxidases or their products may be involved in Nox regulation. The overall redox and metabolic status of the cell, specifically the mitochondria, also has implications on ROS signaling. Signaling of such molecules as electrophilic fatty acids has an impact on many redox-sensitive pathologies and thus, as anti-inflammatory molecules, contributes to the complexity of ROS regulation. This review is based on the proceedings of a recent international Oxidase Signaling Symposium at the University of Pittsburghs Vascular Medicine Institute and Department of Pharmacology and Chemical Biology and encompasses further interaction and discussion among the presenters.

Covalent Peroxisome Proliferator-activated Receptor γ Adduction by Nitro-fatty Acids SELECTIVE LIGAND ACTIVITY AND ANTI-DIABETIC SIGNALING ACTIONS

The peroxisome proliferator-activated receptor-γ (PPARγ) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARγ include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARγ ligands are intermediates of lipid metabolism and oxidation that bind PPARγ with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO2-FA) are endogenous products of nitric oxide (•NO) and nitrite (NO2−)-mediated redox reactions that activate PPARγ at nanomolar concentrations. We report that NO2-FA act as partial agonists of PPARγ and covalently bind PPARγ at Cys-285 via Michael addition. NO2-FA show selective PPARγ modulator characteristics by inducing coregulator protein interactions, PPARγ-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO2-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.

Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via β-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (•NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and β-oxidation reactions that terminate at the site of acyl-chain nitration.

Conjugated Linoleic Acid is a Preferential Substrate for Fatty Acid Nitration

Background: Nitroalkene fatty acids are electrophilic cell metabolites that mediate anti-inflammatory signaling actions. Results: Conjugated linoleic acid is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative inflammatory conditions and digestion. Conclusion: Nitro-fatty acid formation in vivo occurs during metabolic and inflammatory reactions and modulates cell signaling. Significance: Nitro-conjugated linoleic acid transduces signaling actions of nitric oxide, nitrite, and conjugated linoleic acid. The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 105 greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO2-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.

Nitro–Fatty Acids Reduce Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Inflammatory processes and foam cell formation are key determinants in the initiation and progression of atherosclerosis. Electrophilic nitro–fatty acids, byproducts of nitric oxide- and nitrite-dependent redox reactions of unsaturated fatty acids, exhibit antiinflammatory signaling actions in inflammatory and vascular cell model systems. The in vivo action of nitro–fatty acids in chronic inflammatory processes such as atherosclerosis remains to be elucidated. Methods and Results—Herein, we demonstrate that subcutaneously administered 9- and 10-nitro-octadecenoic acid (nitro-oleic acid) potently reduced atherosclerotic lesion formation in apolipoprotein E–deficient mice. Nitro–fatty acids did not modulate serum lipoprotein profiles. Immunostaining and gene expression analyses revealed that nitro-oleic acid attenuated lesion formation by suppressing tissue oxidant generation, inhibiting adhesion molecule expression, and decreasing vessel wall infiltration of inflammatory cells. In addition, nitro-oleic acid reduced foam cell formation by attenuating oxidized low-density lipoprotein–induced phosphorylation of signal transducer and activator of transcription-1, a transcription factor linked to foam cell formation in atherosclerotic plaques. Atherosclerotic lesions of nitro-oleic acid-treated animals also showed an increased content of collagen and α-smooth muscle actin, suggesting conferral of higher plaque stability. Conclusion—These results reveal the antiatherogenic actions of electrophilic nitro–fatty acids in a murine model of atherosclerosis.

Dietary flavonoid quercetin stimulates vasorelaxation in aortic vessels

Considerable epidemiological evidence indicates that dietary consumption of moderate levels of polyphenols decreases both the incidence of cardiovascular disease and the mortality associated with myocardial infarction. Molecular mechanisms of this cardiovascular protection remain uncertain but can involve changes in rates of nitric oxide (NO) generation by endothelial nitric oxide synthase (eNOS). We examined the vascular responses to quercetin using a combination of biochemical and vessel function criteria. Quercetin treatment for 30min enhanced relaxation of rat aortic ring segments. Moreover, the addition of L-NAME (100muM) or charybdotoxin (ChTx) blocked quercetin-mediated vasorelaxation thus demonstrating the effect was partially dependent on NOS and endothelium-derived hyperpolarizing factor (EDHF). Additionally, bovine aortic endothelial cells (BAEC) treated with quercetin showed a rapid increase of intracellular Ca(2+) concentrations as well as a dose- and time-dependent stimulation of eNOS phosphorylation with a concomitant increase in NO production. These results demonstrate that quercetin-mediated stimulation of eNOS phosphorylation increases NO bioavailability in endothelial cells and can thus play a role in the vascular protective effects associated with improved endothelial cell function.

Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids.

Reactive oxygen species mediate a decrease in nitric oxide (NO) bioavailability and endothelial dysfunction, with secondary oxidized and nitrated by-products of these reactions contributing to the pathogenesis of numerous vascular diseases. While oxidized lipids and lipoproteins exacerbate inflammatory reactions in the vasculature, in stark contrast the nitration of polyunsaturated fatty acids and complex lipids yields electrophilic products that exhibit pluripotent anti-inflammatory signaling capabilities acting via both cGMP-dependent and -independent mechanisms. Herein we report that nitro-oleic acid (OA-NO(2)) treatment increases expression of endothelial nitric oxide synthase (eNOS) and heme oxygenase 1 (HO-1) in the vasculature, thus transducing vascular protective effects associated with enhanced NO production. Administration of OA-NO(2) via osmotic pump results in a significant increase in eNOS and HO-1 mRNA in mouse aortas. Moreover, HPLC-MS/MS analysis showed that NO(2)-FAs are rapidly metabolized in cultured endothelial cells (ECs) and treatment with NO(2)-FAs stimulated the phosphorylation of eNOS at Ser(1179). These posttranslational modifications of eNOS, in concert with elevated eNOS gene expression, contributed to an increase in endothelial NO production. In aggregate, OA-NO(2)-induced eNOS and HO-1 expression by vascular cells can induce beneficial effects on endothelial function and provide a new strategy for treating various vascular inflammatory and hypertensive disorders.

Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion

AIMS Nitrite (NO2(-)), a dietary constituent and nitric oxide (NO) oxidation product, mediates cardioprotection after ischaemia/reperfusion (I/R) in a number of animal models when administered during ischaemia or as a pre-conditioning agent hours to days prior to the ischaemic episode. When present during ischaemia, the reduction of nitrite to bioactive NO by deoxygenated haem proteins accounts for its protective effects. However, the mechanism of nitrite-induced pre-conditioning, a normoxic response which does not appear to require reduction of nitrite to NO, remains unexplored. METHODS AND RESULTS Using a model of hypoxia/reoxygenation (H/R) in cultured rat H9c2 cardiomyocytes, we demonstrate that a transient (30 min) normoxic nitrite treatment significantly attenuates cell death after a hypoxic episode initiated 1 h later. Mechanistically, this protection depends on the activation of protein kinase A, which phosphorylates and inhibits dynamin-related protein 1, the predominant regulator of mitochondrial fission. This results morphologically, in the promotion of mitochondrial fusion and functionally in the augmentation of mitochondrial membrane potential and superoxide production. We identify AMP kinase (AMPK) as a downstream target of the mitochondrial reactive oxygen species (ROS) generated and show that its oxidation and subsequent phosphorylation are essential for cytoprotection, as scavenging of ROS prevents AMPK activation and inhibits nitrite-mediated protection after H/R. The protein kinase A-dependent protection mediated by nitrite is reproduced in an intact isolated rat heart model of I/R. CONCLUSIONS These data are the first to demonstrate nitrite-dependent normoxic modulation of both mitochondrial morphology and function and reveal a novel signalling pathway responsible for nitrite-mediated cardioprotection.

Nitro-Fatty Acid Inhibition of Neointima Formation After Endoluminal Vessel Injury

Rationale: Fatty acid nitroalkenes are endogenously generated electrophilic byproducts of nitric oxide and nitrite-dependent oxidative inflammatory reactions. Existing evidence indicates nitroalkenes support posttranslational protein modifications and transcriptional activation that promote the resolution of inflammation. Objective: The aim of this study was to assess whether in vivo administration of a synthetic nitroalkene could elicit antiinflammatory actions in vivo using a murine model of vascular injury. Methods and Results: The in vivo administration (21 days) of nitro-oleic acid (OA-NO2) inhibited neointimal hyperplasia after wire injury of the femoral artery in a murine model (OA-NO2 treatment resulted in reduced intimal area and intima to media ratio versus vehicle- or oleic acid (OA)-treated animals, P<0.0001). Increased heme oxygenase (HO)-1 expression accounted for much of the vascular protection induced by OA-NO2 in both cultured aortic smooth muscle cells and in vivo. Inhibition of HO by Sn(IV)-protoporphyrin or HO-1 small interfering RNA reversed OA-NO2–induced inhibition of platelet-derived growth factor-stimulated rat aortic smooth muscle cell migration. The upregulation of HO-1 expression also accounted for the antistenotic actions of OA-NO2 in vivo, because inhibition of neointimal hyperplasia following femoral artery injury was abolished in HO-1−/− mice (OA-NO2–treated wild-type versus HO-1−/− mice, P=0.016). Conclusions: In summary, electrophilic nitro-fatty acids induce salutary gene expression and cell functional responses that are manifested by a clinically significant outcome, inhibition of neointimal hyperplasia induced by arterial injury.