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Objectives: To characterize the utility of nitrotyrosine (NT) as a biomarker for arthritis and joint injury., Design: Synovial fluid, plasma, and urine from patients diagnosed with osteoarthritis (OA), rheumatoid arthritis (RA), anterior cruciate ligament (ACL) injury, meniscus injury and pseudogout, and knee-healthy volunteers were analyzed for concentrations of NT, nitrate and nitrite (NO(x)), matrix metalloproteinase (MMP)-3, MMP-1, MMP-9, more than 40 chemokines and cytokines., Results: In OA, plasma and synovial fluid NT were increased versus healthy volunteers. Synovial fluid to plasma NT ratios were elevated in OA patients. Synovial fluid from patients with ACL and meniscus injury and pseudogout had increased levels of NT (P < 0.001). In these samples, NT levels significantly correlated with ARGS-aggrecan neoepitope generated by aggrecanase cleavage of aggrecan (P ≤ 0.001), cross-linked C-telopeptides of type II collagen (P < 0.001), MMP-1 (P = 0.008), and MMP-3 (P ≤ 0.001). In RA, plasma NT decreased following 6 months of anti-tumor necrosis factor (TNF) treatment. For every 1.1% change in log(10) NT, there was a 1.0% change in the log(10) disease activity scores (DAS28-3 CRP). Both predicted and observed DAS28-3 CRP showed a robust linear relationship with NT. RA plasma NT positively correlated with CRP, MMP-3 and interferon γ-induced protein 10., Conclusions: NT may serve as a useful biomarker for arthritis and joint injury. In RA, NT is highly correlated with several biomarkers and clinical correlates of disease activity and responds to anti-TNF therapy., (Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

A major feature of septic shock is the development of a vascular crisis characterized by nonresponsiveness to sympathetic vasoconstrictor agents and the subsequent irreversible fall in blood pressure. In addition, sepsis, like other inflammatory conditions, results in a large increase in the production of free radicals, including superoxide anions (O(2)) within the body. Here we show that O(2) reacts with catecholamines deactivating them in vitro. Moreover, this deactivation would appear to account for the hyporeactivity to exogenous catecholamines observed in sepsis, because administration of a superoxide dismutase (SOD) mimetic to a rat model of septic shock to remove excess O(2) restored the vasopressor responses to norepinephrine. This treatment with the SOD mimetic also reversed the hypotension in these animals; suggesting that deactivation of endogenous norepinephrine by O(2) contributes significantly to this aspect of the vascular crisis. Indeed, the plasma concentrations of both norepinephrine and epinephrine in septic rats treated with the SOD mimetic were significantly higher than in untreated rats. Interestingly, the plasma concentrations for norepinephrine and epinephrine were inversely related to the plasma concentrations of adrenochromes, the product of the autoxidation of catecholamines initiated by O(2). We propose, therefore, that the use of a SOD mimetic represents a new paradigm for the treatment of septic shock. By removing O(2), exogenous and endogenous catecholamines are protected from autoxidation. As a result, both hyporeactivity and hypotension are reversed, generation of potentially toxic adrenochromes is reduced, and survival rate is improved.

The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malonaldehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin localized mainly in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats and stained with antibody to ICAM-1 showed a diffuse staining. Administration of FeTMPS significantly reduced ischemia/reperfusion injury in the bowel, and reduced lipid and the production of peroxynitrite during reperfusion. Treatment with PN catalyst also markedly reduced the intensity and degree of P-selectin and ICAM-1 staining in tissue sections from SAO-shocked rats and improved survival. Our results clearly demonstrate that peroxynitrite decomposition catalysts exert a protective effect in SAO and that this effect may be due to inhibition of the expression of adhesion molecules and the tissue damage associated with peroxynitrite-related pathways.

Many human diseases are associated with the overproduction of oxygen free radicals that inflict cell damage. A manganese(II) complex with a bis(cyclohexylpyridine)-substituted macrocyclic ligand (M40403) was designed to be a functional mimic of the superoxide dismutase (SOD) enzymes that normally remove these radicals. M40403 had high catalytic SOD activity and was chemically and biologically stable in vivo. Injection of M40403 into rat models of inflammation and ischemia-reperfusion injury protected the animals against tissue damage. Such mimics may result in better clinical therapies for diseases mediated by superoxide radicals.

1. The relative contributions of superoxide anion (O2-) and peroxynitrite (PN) were evaluated in the pathogenesis of intestinal microvascular damage caused by the intravenous injection of E. coli lipopolysaccharide (LPS) in rats. The superoxide dismutase mimetic (SODm) SC-55858 and the active peroxynitrite decomposition catalysts 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulphonatophenyl)-por phyrinato iron (III) and 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)-porphyrinato iron (III) (FeTMPS, FeTMPyP respectively) were used to assess the roles of O2- and PN respectively. 2. The intravenous injection of LPS elicited an inflammatory response that was characterized by a time-dependent infiltration of neutrophils, lipid peroxidation, microvascular leakage (indicative of microvascular damage), and epithelial cell injury in both the duodenum and jejunum. 3. Administration of the SODm SC-55858, FeTMPS or FeTMPyP at 3 h post LPS reduced the subsequent increase in microvascular leakage, lipid peroxidation and epithelial cell injury. Inactive peroxynitrite decomposition catalysts exhibited no protective effects. Only, SC-55858 inhibited neutrophil infiltration. 4. Our results suggest that O2 and peroxynitrite play a significant role in the pathogenesis of duodenal and intestinal injury during endotoxaemia and that their remoyal by SODm and peroxynitrite decomposition catalysts offers a novel approach to the treatment of septic shock or clinical conditions of gastrointestinal inflammation. Furthermore, the remarkable protection of the intestinal epithelium by these agents suggests their use during chemo- and radiation therapy, cancer treatments characterized by gastrointestinal damage. Potential mechanisms through which these radicals evoke damage are discussed.

Interest in peroxynitrite (PN) chemistry soared after recognition in 1987 of the biological role of nitric oxide, and PN has recently emerged as a possible key mediator of in vivo oxidative stress and disease. The role of PN in disease processes can be dissected both pharmacologically and biochemically, and several laboratories have examined the cellular production of peroxynitrite. In vitro demonstrations of PN-mediated cellular injury have served to strengthen the case for peroxynitrite's proposed role in the pathogenesis of human neurodegenerative disorders as examined in animal models and in diseased human tissue. Among the nervous system disorders in which PN is strongly implicated in pathology are stroke, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's and Parkinson's diseases. Pathologies driven by the formation of PN are amenable to pharmacological intervention at either the reactant (nitric oxide, superoxide anions) or the product (peroxynitrite). Strategies for blocking the deleterious biochemistry of peroxynitrite must aim to decrease either the flux or the intrinsic lifetime of the peroxynitrite; three particular tactics would accomplish such purposes. A novel class of antiinflammatory agents has recently been identified: PN decomposition catalysts. Identification of these catalysts offers the scientific community the opportunity to elucidate and further our understanding of the roles of peroxynitrite in animal models of diseases, which may lead to a major breakthrough in understanding the physiopathological importance of this molecule., ((c) 1998 Prous Science. All rights reserved.)

Inflamed tissue is often characterized by the production of NO and superoxide. These radicals react at diffusion-limited rates to form the powerful oxidant peroxynitrite (PN). When protonated, PN decomposes into either nitrate or reactive intermediates capable of mediating tissue damage by oxidation of protein, lipid, and nucleic acid. We recently have identified porphyrin derivatives capable of catalyzing an increase in nitrate formation with a concomitant decrease in the HO.-like and NO2.-like reactivity of PN. Here, we present evidence for the efficacy of these PN decomposition catalysts both in vitro and in vivo. Cells in culture were protected from exogenously added PN by the catalyst 5,10,15,20-tetrakis(2,4, 6-trimethyl-3,5-disulfonatophenyl)porphyrinato iron (III), whereas free iron and the structurally related compound without iron 5,10,15, 20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)porphyrin did not protect. Cytoprotection correlated well with a reduction in the nitrotyrosine content of released cytosolic proteins, a biochemical marker for PN formation. Carrageenan-induced paw edema is a model of acute inflammation in which PN may play a major role. When tested in this system, both 5,10,15,20-tetrakis(2,4,6-trimethyl-3, 5-disulfonatophenyl)porphyrinato iron (III) and 5,10,15, 20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron (III) caused a dose-dependent reduction in swelling and lactate dehydrogenase release as well as a detectable shift to nitrate formation in paw tissue. In addition, the catalysts did not elevate mean arterial pressure, suggesting a lack of interaction with NO. Taken together, our data provide compelling evidence supporting the therapeutic value of manipulating PN pharmacologically. Thus, PN decomposition catalysts may represent a unique class of anti-inflammatory agents.

Tumor necrosis factor (TNF-alpha) and nitric oxide (NO) are important vasoactive mediators of septic shock. This study used a well-characterized quantitative permeation method to examine the effect of TNF-alpha and NO on systemic vascular barrier function in vivo, without confounding endotoxemia, hypotension, or organ damage. Our results showed 1) TNF-alpha reversibly increased albumin permeation in the systemic vasculature (e.g., lung, liver, brain, etc.); 2) TNF-alpha did not affect hemodynamics or blood flow or cause significant tissue injury; 3) pulmonary vascular barrier dysfunction was associated with increased lung water content and impaired oxygenation; 4) TNF-alpha caused inducible nitric oxide synthase (iNOS) mRNA expression in the lung and increased in vivo NO production; 5) selective inhibition of iNOS with aminoguanidine prevented TNF-alpha-induced lung and liver vascular barrier dysfunction; 6) aminoguanidine prevented increased tissue water content in TNF-alpha-treated lungs and improved oxygenation; and 7) nonselective inhibition of NOS with NG-monomethly-L-arginine increased vascular permeation in control lungs and caused severe lung injury in TNF-alpha-treated animals. We conclude that 1) TNF-alpha reversibly impairs vascular barrier integrity through NO-dependent and -independent mechanisms; 2) nonselective NOS inhibition increased vascular barrier dysfunction and caused severe lung injury, whereas selective inhibition of iNOS prevented impaired endothelial barrier integrity and pulmonary dysfunction; and 3) selective inhibition of iNOS may be beneficial in treating increased vascular permeability that complicates endotoxemia and cytokine immunotherapy.

Background: We recently demonstrated that inhibition of inducible nitric oxide synthase (iNOS) ameliorated severe acute lung allograft rejection. This study used a rat lung transplant model to determine (1) the time course and cellular localization of iNOS expression during the histological progression of unmodified acute rejection and (2) whether inhibition of iNOS prevented impaired gas exchange function of the allograft lung and/or ameliorated the histological changes of acute rejection., Methods and Results: iNOS mRNA and enzyme activity were expressed in allograft lungs during mild, moderate, and severe acute rejection, but not in normal, isograft, or allograft lungs before histological changes of mild acute rejection. iNOS expression in allografts resulted in elevated serum nitrite/nitrate levels, indicative of increased in vivo nitric oxide (NO) production. In situ hybridization demonstrated iNOS mRNA expression in infiltrating inflammatory cells, but not in allograft parenchymal cells. Allografts had significantly impaired gas exchange, which was prevented with the selective iNOS inhibitor aminoguanidine (PaO2 of 566+/-19, 76+/-22, and 504+/-105 mmHg for isograft, allograft, and aminoguanidine-treated allograft, respectively; P<0.0002). Aminoguanidine also significantly improved the histological rejection scores., Conclusions: (1) iNOS expression and increased NO production occurred during the early stages of acute rejection, persisted throughout the unmodified rejection process, and localized to infiltrating inflammatory cells, but not allograft parenchymal cells; (2) aminoguanidine ameliorated the histological and functional changes of acute rejection; and (3) increased NO production, detected by the presence of iNOS mRNA, protein, or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.

Background: We recently demonstrated that inhibition of nitric oxide (NO) production ameliorated acute pulmonary allograft rejection. This study examined whether inducible NO synthase (iNOS) was expressed in the transplanted lung during acute rejection., Methods: With a rat left lung transplant model, tissue from syngeneic (Fischer 344 to Fischer 344) and allogeneic (Brown Norway to Fischer 344) transplants were harvested on postoperative day 4 and analyzed for iNOS mRNA expression (ribonuclease protection assay), iNOS enzyme activity (conversion of L-[3H]-arginine to NO and L-[3H]-citrulline), and serum nitrite/nitrate levels., Results: The iNOS mRNA was expressed in allograft lungs but was not detected in isografts or controls. The iNOS protein was present in allograft lungs, as demonstrated by high levels of L-[3H]-citrulline production compared with minimal iNOS enzyme activity in isograft and control lungs (10.1 +/- 2.4 vs 0.6 +/- 0.2 and 0.7 +/- 0.2 pmol L-[3H]-citrulline.mg-1.min-1, respectively; n = 6, p < 0.001). Allografts had significantly elevated systemic serum nitrite/nitrate levels compared with isografts and controls (38 +/- 6 vs 18 +/- 2 and 16 +/- 1 mumol/L, respectively; n = 6; p < 0.005)., Conclusions: These results, together with our previous demonstration that iNOS inhibition ameliorated lung allograft rejection, suggest that (1) iNOS expression and increased NO production contributed to acute rejection of the transplanted lung, (2) iNOS inhibition may offer an alternative in management of acute lung allograft rejection, and (3) increased NO production, detected by the presence of iNOS mRNA or protein or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.

Background: We previously demonstrated that continuous treatment with aminoguanidine, a selective inhibitor of nitric oxide production by inducible nitric oxide synthase, attenuated acute cardiac allograft rejection., Methods: A rat transplant model was used to determine (1) when inducible nitric oxide synthase was expressed in the allograft heart during unmodified acute rejection and (2) whether pulse therapy with aminoguanidine attenuated the histologic changes of established acute rejection, in comparison with the effects of pulse therapy with corticosteroids., Results: Inducible nitric oxide synthase messenger RNA and protein were expressed during early and late acute rejection. Pulse therapy with aminoguanidine inhibited nitric oxide production and attenuated the histologic changes of acute rejection, but not as effectively as corticosteroid therapy (rejection scores of 4.1 +/- 0.4, 2.5 +/- 0.9, and 1.4 +/- 0.6 on postoperative day 8, for untreated, aminoguanidine-, and dexamethasone-treated allografts, respectively (scale, 0 to 5; p < 0.05)., Conclusions: (1) Inducible nitric oxide synthase expression first occurs during early acute allograft rejection and persists throughout rejection and (2) nitric oxide is an important effector molecule in acute rejection. Inducible nitric oxide synthase inhibition may offer a therapeutic adjunct in the management of acute rejection.

NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature.