RESUMEN
Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2, 0.15%) or allopurinol (30 µg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fed mice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.
Asunto(s)
Cicatrización de Heridas/fisiología , Xantina Deshidrogenasa/metabolismo , Aldehído Oxidasa/metabolismo , Animales , Arginasa/genética , Arginasa/metabolismo , Proliferación Celular , Suplementos Dietéticos , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Expresión Génica , Tejido de Granulación/metabolismo , Peróxido de Hidrógeno/metabolismo , Queratinocitos/metabolismo , Masculino , Ratones , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tungsteno/metabolismo , Tungsteno/farmacología , Xantina Deshidrogenasa/antagonistas & inhibidores , Xantina Deshidrogenasa/genéticaRESUMEN
Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) that contribute to vascular inflammation. Binding of XO to vascular endothelial cell glycosaminoglycans (GAGs) results in significant resistance to inhibition by traditional pyrazolopyrimidine-based inhibitors such as allopurinol. Therefore, we compared the extent of XO inhibition (free and GAG-bound) by allopurinol to that by febuxostat, a newly approved nonpurine XO-specific inhibitor. In solution, febuxostat was 1000-fold more potent than allopurinol at inhibiting XO-dependent uric acid formation (IC50= 1.8 nM vs 2.9 µM). Association of XO with heparin-Sepharose 6B (HS6B-XO) had minimal effect on the inhibition of uric acid formation by febuxostat (IC50= 4.4 nM) while further limiting the effect of allopurinol (IC50= 64 µM). Kinetic analysis of febuxostat inhibition revealed K(i) values of 0.96 (free) and 0.92 nM (HS6B-XO), confirming equivalent inhibition for both free and GAG-immobilized enzyme. When XO was bound to endothelial cell GAGs, complete enzyme inhibition was observed with 25 nM febuxostat, whereas no more than 80% inhibition was seen with either allopurinol or oxypurinol, even at concentrations above those tolerated clinically. The superior potency for inhibition of endothelium-associated XO is predictive of a significant role for febuxostat in investigating pathological states in which XO-derived ROS are contributive and traditional XO inhibitors are only slightly effective.
Asunto(s)
Células Endoteliales/metabolismo , Inhibidores Enzimáticos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Tiazoles/farmacología , Xantina Oxidasa/antagonistas & inhibidores , Alopurinol/farmacología , Animales , Bovinos , Células Cultivadas , Febuxostat , Glicosaminoglicanos/metabolismo , Cinética , Oxipurinol/farmacología , Xantina Oxidasa/metabolismoRESUMEN
Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.
Asunto(s)
Arginina/fisiología , Óxido Nítrico Sintasa de Tipo III/fisiología , Óxido Nítrico/fisiología , Nitrito de Sodio/administración & dosificación , Túnica Íntima/fisiología , Animales , Arginasa/metabolismo , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Hiperplasia/patología , Hiperplasia/fisiopatología , Hiperplasia/prevención & control , Masculino , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Óxido Nítrico/biosíntesis , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Túnica Íntima/efectos de los fármacos , Túnica Íntima/lesiones , Túnica Íntima/patología , Xantina Deshidrogenasa/metabolismoRESUMEN
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.
Asunto(s)
Peróxido de Hidrógeno/farmacología , Oxidantes/química , Xantina Oxidasa/metabolismo , Animales , Tampones (Química) , Bovinos , Supervivencia Celular , Células Cultivadas , Células Endoteliales/enzimología , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Modelos Biológicos , Oxígeno/química , Consumo de Oxígeno , Transducción de Señal , Xantina Oxidasa/químicaRESUMEN
Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 microM, limiting both purine oxidation and formation of superoxide (O2.). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2. production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácidos Oléicos/metabolismo , Xantina Deshidrogenasa/metabolismo , Animales , Aorta/citología , Bovinos , Ditiotreitol/metabolismo , Células Endoteliales/citología , Ácidos Grasos/química , Glutatión/metabolismo , Concentración 50 Inhibidora , Mercaptoetanol/metabolismo , Oxígeno/química , Transducción de Señal , Superóxidos/metabolismoRESUMEN
Xanthine oxidoreductase (XOR) activity has been previously noted to be responsive to changes in O2 tension. While prior studies have focused on the extremes (0-3% and 95-100%) of O2 tensions, we report the influence of 10% O2 on endothelial cell XOR, a concentration resembling modest arterial hypoxia commonly found in patients with chronic cardiopulmonary diseases. Exposure of bovine aortic endothelial cells to 10% O2 increased XOR mRNA and protein abundance by 50%. Concomitantly, there was a 3-fold increase in XOR activity, XOR-dependent reactive oxygen species production, and cellular export of active enzyme. Although increases in mRNA and immunoreactive protein levels were observed, inhibition of transcription, translation, or protein degradation did not significantly alter cellular XOR specific activity, suggesting only modest contributions to 10% O2-induced effects. Exposure to 10% O2 did not increase cellular HIF-1alpha protein levels and hypoxia mimics did not alter XOR activity. Treatment of control cells with adenosine resulted in increased XOR activity similar to hypoxia. Exposure to the adenosine receptor agonist NECA increased enzymatic activity 4-fold while 8SPT, an adenosine receptor antagonist, reduced hypoxic induction of XOR activity approximately 50%. Combined, these data reveal that moderate hypoxia significantly enhances endothelial XOR specific activity, release, and XOR-derived reactive oxygen species generation. These effects appear to be mediated in part via adenosine-dependent processes.
Asunto(s)
Hipoxia/enzimología , Xantina Oxidasa/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Adenosina/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Aorta/citología , Bovinos , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Inducción Enzimática , Hipoxia/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/farmacología , Oxígeno/farmacología , Antagonistas de Receptores Purinérgicos P1 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Although the binding of xanthine oxidase (XO) to glycosaminoglycans (GAGs) results in significant alterations in its catalytic properties, the consequence of XO/GAG immobilization on interactions with clinically relevant inhibitors is unknown. Thus, the inhibition kinetics of oxypurinol for XO was determined using saturating concentrations of xanthine. When XO was bound to a prototypical GAG, heparin-Sepharose 6B (HS6B-XO), the rate of inactivation for uric acid formation from xanthine was less than that for XO in solution (k(inact) = 0.24 versus 0.39 min(-1)). Additionally, the overall inhibition constant (K(i)) of oxypurinol for HS6B-XO was 2-5-fold greater than for free XO (451 versus 85 nm). Univalent electron flux (O(2)(.) formation) was diminished by the binding of XO to heparin from 28.5% for free XO to 18.7% for GAG-immobilized XO. Similar to the results obtained with HS6B-XO, the binding of XO to bovine aortic endothelial cells rendered the enzyme resistant to inhibition by oxypurinol, achieving approximately 50% inhibition. These results reveal that GAG immobilization of XO in both HS6B and cell models substantially limits oxypurinol inhibition of XO, an event that has important relevance for the use of pyrazolo inhibitors of XO in clinical situations where XO and its products may play a pathogenic role.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glicosaminoglicanos/metabolismo , Oxipurinol/farmacología , Xantina Oxidasa/metabolismo , Cinética , Unión Proteica , Superóxidos/metabolismo , Ácido Úrico/metabolismo , Xantina Oxidasa/antagonistas & inhibidoresRESUMEN
Xanthine oxidase (XO)-derived superoxide contributes to endothelial dysfunction in humans and animal models of hypercholesterolemia (HC). Since L-arginine supplementation prevents defects in NO signaling, we tested the hypothesis that L-arginine blunts the inhibitory effect of XO on vascular function. Acetylcholine-mediated relaxation was significantly impaired in ring segments of HC rabbits, a response that was associated with an increase in plasma XO activity. L-Arginine treatment of HC rabbits reduced plasma XO and improved endothelial function. L-Arginine also modestly prolonged the lag time for oxidation in isolated lipoprotein samples. These results reveal that the principal action of L-arginine is to protect against the XO-dependent inactivation of NO in arteries of HC rabbits.
Asunto(s)
Arginina/farmacología , Endotelio Vascular/efectos de los fármacos , Hipercolesterolemia/fisiopatología , Xantina Oxidasa/efectos de los fármacos , Acetilcolina/farmacología , Animales , Aorta/fisiología , Arginina/administración & dosificación , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiopatología , Hipercolesterolemia/tratamiento farmacológico , Óxido Nítrico/metabolismo , Conejos , Vasodilatación/efectos de los fármacos , Xantina Oxidasa/sangreRESUMEN
Facile detection of reactive oxygen and nitrogen species in biologic systems is often problematic. This is a result of the numerous cellular mechanisms, both enzymatic and nonenzymatic involved in their catabolism/decomposition, the complex and overlapping nature of their reactivities, as well as the often limited intracellular access of detector systems. This review describes approaches to the direct and indirect measurement of different reactive metabolites of oxygen and nitrogen. Particular attention to a method's applicability for in vivo determinations will be addressed.
Asunto(s)
Especies de Nitrógeno Reactivo/análisis , Especies Reactivas de Oxígeno/análisis , Tirosina/análogos & derivados , Algoritmos , Animales , Humanos , Peróxido de Hidrógeno/análisis , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Oxidantes/análisis , Estrés Oxidativo/fisiología , Ácido Peroxinitroso/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , S-Nitrosotioles/metabolismo , Superóxidos/análisis , Tirosina/análisis , Tirosina/metabolismoRESUMEN
Transforming growth factor-beta (TGF-beta) is a potent fibrogenic cytokine. The molecular mechanism underlying TGF-beta fibrogenesis, however, has not been completely elucidated. In this study, we showed that TGF beta decreased the intracellular GSH content in murine embryo fibroblasts (NIH 3T3), which was followed by an increase in collagen I mRNA content and collagen protein production. Prevention of GSH depletion with N-acetylcysteine (NAC), GSH, or GSH ester abrogated TGF-beta-stimulated collagen production, whereas a decrease in intracellular GSH content with L-buthionine-S,R-sulfoximine, an inhibitor of de novo GSH synthesis, enhanced TGF-beta-stimulated collagen production. These results suggest that GSH depletion induced by TGF-beta may mediate TGF-beta-stimulated collagen production. In addition, we showed that TGF-beta stimulated superoxide production and increased release of H2O2 from the cells, whereas GSH ester decreased basal and TGF-beta + glucose oxidase-stimulated H2O2 release. H2O2, exogenously added or continuously generated by glucose oxidase, enhanced TGF-beta-stimulated collagen production, whereas suppression of superoxide production by diphenyliodonium, an NAD(P)H oxidase inhibitor, blocked TGF-beta-stimulated collagen production. These data further suggest that reactive oxygen species are involved in TGF-beta-stimulated collagen production and that the effect of GSH depletion on TGF-beta-stimulated collagen production may be mediated by facilitating reactive oxygen species signaling.
Asunto(s)
Colágeno Tipo III/genética , Colágeno Tipo I/genética , Fibroblastos/fisiología , Glutatión/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Ratones , Células 3T3 NIH , ARN Mensajero/análisis , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , TritioRESUMEN
BACKGROUND: Human serum albumin is used clinically to maintain colloid osmotic pressure and is viewed to serve an antioxidant role in the vascular compartment via binding of redox-active metal complexes, transport of nitric oxide, and the oxidant-scavenging reactions of the single thiol of human serum albumin, cys34. Because of these potentially desirable adjunctive actions, we evaluated the purity and thiol redox state and compared the relative effects of clinically available 25% human serum albumin preparations with a starch-derived colloid, 6% hydroxyethyl starch, in in vitro models of inflammatory vascular injury. METHODS: Bovine aortic endothelial cell responses to chemical, enzymatic, and cell-derived reactive inflammatory mediators in the presence of human serum albumin or hydroxyethyl starch were assessed. RESULTS: The cys34 thiol of fresh human serum albumin preparations was 70-85% oxidized and contained a population of human serum albumin (approximately 25% of total) having the cys34 resistant to reduction by 2-mercaptoethanol and NaBH4. Treatment of bovine aortic endothelial cells with human serum albumin dose-dependently protected from HOCl-mediated 14C-adenine release, with this protective effect of human serum albumin not dependent on protein thiol status. Addition of human serum albumin to cell media provided no protection from the cytotoxic actions of peroxynitrite and xanthine oxidase-derived reactive species. Binding of activated polymorphonuclear leukocytes to bovine aortic endothelial cells was significantly amplified by hydroxyethyl starch and inhibited by human serum albumin administration. The binding of neutrophil-derived myeloperoxidase to bovine aortic endothelial cells, a mediator of multiple oxidative and nitric oxide-consuming reactions, was also inhibited by human serum albumin and enhanced by hydroxyethyl starch. CONCLUSIONS: Clinical human serum albumin preparations show modest intrinsic non-thiol-dependent antiinflammatory properties in vitro, a phenomenon that was not observed with hydroxyethyl starch.