RESUMEN
Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.
Asunto(s)
Envejecimiento/inmunología , Envejecimiento/fisiología , Sistema Inmunológico/inmunología , Sistema Inmunológico/fisiología , Inmunosenescencia/inmunología , Inmunosenescencia/fisiología , Especificidad de Órganos/inmunología , Especificidad de Órganos/fisiología , Envejecimiento/efectos de los fármacos , Envejecimiento/patología , Animales , Daño del ADN/inmunología , Daño del ADN/fisiología , Reparación del ADN/inmunología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Femenino , Envejecimiento Saludable/inmunología , Envejecimiento Saludable/fisiología , Homeostasis/inmunología , Homeostasis/fisiología , Sistema Inmunológico/efectos de los fármacos , Inmunosenescencia/efectos de los fármacos , Masculino , Ratones , Especificidad de Órganos/efectos de los fármacos , Rejuvenecimiento , Sirolimus/farmacología , Bazo/citología , Bazo/trasplanteRESUMEN
BACKGROUND: Microbial dysbiosis is a potential mediator of air pollution-induced adverse outcomes. However, a systemic comparison of the lung and gut microbiome alterations and lung-gut axis following air pollution exposure is scant. In this study, we exposed male C57BL/6J mice to inhaled air, CB (10 mg/m3), O3 (2 ppm) or CB + O3 mixture for 3 h/day for either one day or four consecutive days and were euthanized 24 h post last exposure. The lung and gut microbiome were quantified by 16 s sequencing. RESULTS: Multiple CB + O3 exposures induced an increase in the lung inflammatory cells (neutrophils, eosinophils and B lymphocytes), reduced absolute bacterial load in the lungs and increased load in the gut. CB + O3 exposure was more potent as it decreased lung microbiome alpha diversity just after a single exposure. CB + O3 co-exposure uniquely increased Clostridiaceae and Prevotellaceae in the lungs. Serum short chain fatty acids (SCFA) (acetate and propionate) were increased significantly only after CB + O3 co-exposure. A significant increase in SCFA producing bacterial families (Ruminococcaceae, Lachnospiraceae, and Eubacterium) were also observed in the gut after multiple exposures. Co-exposure induced significant alterations in the gut derived metabolite receptors/mediator (Gcg, Glp-1r, Cck) mRNA expression. Oxidative stress related mRNA expression in lungs, and oxidant levels in the BALF, serum and gut significantly increased after CB + O3 exposures. CONCLUSION: Our study confirms distinct gut and lung microbiome alterations after CB + O3 inhalation co-exposure and indicate a potential homeostatic shift in the gut microbiome to counter deleterious impacts of environmental exposures on metabolic system.
Asunto(s)
Microbiota , Ozono , Ratones , Animales , Masculino , Ozono/toxicidad , Hollín/toxicidad , Ratones Endogámicos C57BL , Pulmón/metabolismo , ARN Mensajero/metabolismoRESUMEN
Recent studies have identified skeletal muscle as a tissue compartment where nitrate and nitrite can be stored and utilized to potentially maintain nitric oxide (NO) homeostasis. Given its capacity to reduce nitrate and nitrite, the molybdopterin-containing enzyme, xanthine oxidoreductase (XOR) has been suggested as a key enzyme within skeletal muscle which catalytically reduces these N-oxides; however, there remains limited insight into the role of XOR in this process as well as how different conditions (e.g. health vs disease and rest vs exercise) may determine when and where, within skeletal muscle, XOR could serve as a significant source of NO. A key factor that determines the extent by which XOR may or may not contribute to NO generation in a biologically relevant manner is the biochemical landscape (e.g. oxygen tension, pH, isoform of XOR (XDH vs. XO) and substrate levels of the microenvironment in normal versus stressed skeletal muscle. As such, a critical focus of this review is the evaluation of the biochemical and physiologic data supporting the role of XOR within skeletal muscle for supplying nitrite and/or NO from endogenous and exogenous sources during pathophysiologic conditions and/or exercise stress.
Asunto(s)
Nitritos , Xantina Oxidasa , Nitratos , Oxidorreductasas , Xantina Deshidrogenasa , Óxido Nítrico , Músculo EsqueléticoRESUMEN
OBJECTIVE: Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration-approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD. CONCLUSIONS: Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.
Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Eritrocitos/efectos de los fármacos , Febuxostat/farmacología , Hemodinámica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Xantina Oxidasa/antagonistas & inhibidores , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/enzimología , Anemia de Células Falciformes/fisiopatología , Animales , Modelos Animales de Enfermedad , Eritrocitos/enzimología , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Arteria Pulmonar/enzimología , Arteria Pulmonar/fisiopatología , Función Ventricular/efectos de los fármacos , Xantina Oxidasa/genética , Xantina Oxidasa/metabolismoRESUMEN
NEW FINDINGS: What is the central question of this study? Thoracic perivascular adipose tissue (tPVAT) is known to, in part, regulate aortic function: what are the effects of unpredictable chronic mild stress (UCMS) on the tPVAT regulation of aortic function and what is the role of exercise training in alleviating the potential negative actions of UCMS on tPVAT? What is the main finding and its importance? UCMS causes tPVAT to disrupt endothelium-dependent dilatation, increases inflammatory cytokine production and diminishes tPVAT-adiponectin. Exercise training proved efficacious in preventing tPVAT-mediated disruption of aortic function. The data support a tPVAT mechanism through which chronic stress negatively impacts vascular health, which adds to our knowledge of how psychological disorders might increase the risk of cardiovascular disease. ABSTRACT: Chronic stress is a major risk for cardiovascular disease. Perivascular adipose tissue (PVAT) has been shown to regulate vascular function; however, the impact of chronic stress and the comorbidity of metabolic syndrome (MetS) on thoracic (t)PVAT is unknown. Additionally, aerobic exercise training (AET) is known to combat the pathology of MetS and chronic stress, but the role of tPVAT in these actions is also unknown. Therefore, the purpose of this study was to examine the effects of unpredictable chronic mild stress (UCMS) on the tPVAT regulation of aortic function and the preventative effect of AET. Lean (LZR) and obese (OZR) Zucker rats (16-17 weeks old) were exposed to 8 weeks of UCMS with and without treadmill exercise (AET). In LZR, UCMS impaired aortic endothelium-dependent dilatation (EDD) (assessed ex vivo by wire myography) and aortic stiffness (assessed by elastic modulus) with no change in OZR subject to UCMS. However, both LZR and OZR UCMS tPVAT impaired EDD compared to respective controls. LZR and OZR subject to UCMS had higher oxidative stress production, diminished adiponectin and impaired aortic nitric oxide levels. Divergently, UCMS induced greater inflammatory cytokine production in LZR UCMS tPVAT, but not in OZR UCMS tPVAT. AET prevented the tPVAT impairment of aortic relaxation with UCMS in LZR and OZR. Additionally, AET reduced aortic stiffness in both LZR and OZR. These beneficial effects on tPVAT regulation of the aorta are likely due to AET preservation of adiponectin, reduced oxidative stress and inflammation, and enhanced nitric oxide. UCMS impaired tPVAT-regulated aortic function in LZR, and augmented MetS-induced EDD in OZR. Conversely, AET in combination with UCMS largely preserved aortic function and the tPVAT environment, in both groups.
Asunto(s)
Síndrome Metabólico , Tejido Adiposo/metabolismo , Animales , Aorta/metabolismo , Obesidad/metabolismo , Ratas , Ratas ZuckerRESUMEN
PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC50 of 5.95 µM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ataque Isquémico Transitorio/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Animales , Moléculas de Adhesión Celular Neuronal/uso terapéutico , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Humanos , Inyecciones Intraperitoneales , Proteínas de Unión a Hierro/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
RATIONALE: Soluble guanylate cyclase (sGC) heme iron, in its oxidized state (Fe3+), is desensitized to NO and limits cGMP production needed for downstream activation of protein kinase G-dependent signaling and blood vessel dilation. OBJECTIVE: Although reactive oxygen species are known to oxidize the sGC heme iron, the basic mechanism(s) governing sGC heme iron recycling to its NO-sensitive, reduced state remain poorly understood. METHODS AND RESULTS: Oxidant challenge studies show that vascular smooth muscle cells have an intrinsic ability to reduce oxidized sGC heme iron and form protein-protein complexes between cytochrome b5 reductase 3, also known as methemoglobin reductase, and oxidized sGC. Genetic knockdown and pharmacological inhibition in vascular smooth muscle cells reveal that cytochrome b5 reductase 3 expression and activity is critical for NO-stimulated cGMP production and vasodilation. Mechanistically, we show that cytochrome b5 reductase 3 directly reduces oxidized sGC required for NO sensitization as assessed by biochemical, cellular, and ex vivo assays. CONCLUSIONS: Together, these findings identify new insights into NO-sGC-cGMP signaling and reveal cytochrome b5 reductase 3 as the first identified physiological sGC heme iron reductase in vascular smooth muscle cells, serving as a critical regulator of cGMP production and protein kinase G-dependent signaling.
Asunto(s)
GMP Cíclico/metabolismo , Citocromo-B(5) Reductasa/fisiología , Transducción de Señal/fisiología , Guanilil Ciclasa Soluble/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Benzoatos/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Oxidación-Reducción/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
Inhibition of xanthine oxidoreductase (XOR) has proven beneficial in a plethora of inflammatory disease processes due to a net reduction in pro-inflammatory oxidants and secondary nitrating species. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2) are also noted to display a broad spectrum of anti-inflammatory effects via interaction with critical signaling pathways. An alternative process in which nitrated fatty acids may extend anti-inflammatory actions is via inactivation of XOR, a process that is more effective than allo/oxypurinol-mediated inhibition. Herein, we describe the molecular aspects of nitrated fatty acid-associated inactivation of XOR, identify specificity via structure function relationships and discuss XOR as a crucial component of the anti-inflammatory portfolio of nitrated fatty acids.
Asunto(s)
Ácidos Grasos/farmacología , Inflamación , Nitratos/química , Oxidantes/química , Xantina Deshidrogenasa/antagonistas & inhibidores , HumanosRESUMEN
Accumulation of myeloid cells in the liver, notably dendritic cells (DCs) and monocytes/macrophages (MCs), is a major component of the metainflammation of obesity. However, the mechanism(s) stimulating hepatic DC/MC infiltration remain ill defined. Herein, we addressed the hypothesis that adipose tissue (AT) free fatty acids (FFAs) play a central role in the initiation of hepatic DC/MC accumulation, using a number of mouse models of altered FFA supply to the liver. In two models of acute FFA elevation (lipid infusion and fasting) hepatic DC/MC and triglycerides (TGs) but not AT DC/MC were increased without altering plasma cytokines (PCs; TNFα and monocyte chemoattractant protein 1) and with variable effects on oxidative stress (OxS) markers. However, fasting in mice with profoundly reduced AT lipolysis (AT-specific deletion of adipose TG lipase; AAKO) failed to elevate liver DC/MC, TG, or PC, but liver OxS increased. Livers of obese AAKO mice that are known to be resistant to steatosis were similarly protected from inflammation. In high-fat feeding studies of 1, 3, 6, or 20-wk duration, liver DC/MC accumulation dissociated from PC and OxS but tracked with liver TGs. Furthermore, decreasing OxS by ~80% in obese mice failed to decrease liver DC/MC. Therefore, FFA and more specifically AT-derived FFA stimulate hepatic DC/MC accumulation, thus recapitulating the pathology of the obese liver. In a number of cases the effects of FFA can be dissociated from OxS and PC but match well with liver TG, a marker of FFA oversupply.
Asunto(s)
Tejido Adiposo/metabolismo , Ayuno/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Hígado/metabolismo , Células Mieloides/metabolismo , Animales , Citocinas/sangre , Dieta Alta en Grasa , Ácidos Grasos no Esterificados/farmacología , Lipasa/genética , Lipasa/metabolismo , Lipólisis/fisiología , Hígado/efectos de los fármacos , Ratones , Ratones Noqueados , Obesidad/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Triglicéridos/metabolismoRESUMEN
Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.
Asunto(s)
Citoprotección/fisiología , Sulfuro de Hidrógeno/metabolismo , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Transducción de Señal/fisiología , Alanina Transaminasa/sangre , Análisis de Varianza , Animales , Aspartato Aminotransferasas/sangre , Western Blotting , Cromatografía Líquida de Alta Presión , Cistationina gamma-Liasa/genética , Citoprotección/efectos de los fármacos , Sulfuro de Hidrógeno/farmacología , Inmunohistoquímica , Ratones , Ratones Noqueados , Mitocondrias/fisiología , Daño por Reperfusión Miocárdica/metabolismo , Estrés Oxidativo/fisiología , Consumo de Oxígeno/fisiología , Troponina I/metabolismoRESUMEN
Nitrite acts as an ischemic reservoir of nitric oxide (NO) and a potent S-nitrosating agent which reduced histologic brain injury after rat asphyxial cardiac arrest (ACA). The mechanism(s) of nitrite-mediated neuroprotection remain to be defined. We hypothesized that nitrite-mediated brain mitochondrial S-nitrosation accounts for neuroprotection by reducing reperfusion reactive oxygen species (ROS) generation. Nitrite (4 µmol) or placebo was infused IV after normothermic (37°C) ACA in randomized, blinded fashion with evaluation of neurologic function, survival, brain mitochondrial function, and ROS. Blood and CSF nitrite were quantified using reductive chemiluminescence and S-nitrosation by biotin switch. Direct neuroprotection was verified in vitro after 1 and 4 h neuronal oxygen glucose deprivation measuring neuronal death with inhibition studies to examine mechanism. Mitochondrial ROS generation was quantified by live neuronal imaging using mitoSOX. Nitrite significantly reduced neurologic disability after ACA. ROS generation was reduced in brain mitochondria from nitrite- versus placebo-treated rats after ACA with congruent preservation of brain ascorbate and reduction of ROS in brain sections using immuno-spin trapping. ATP generation was maintained with nitrite up to 24 h after ACA. Nitrite rapidly entered CSF and increased brain mitochondrial S-nitrosation. Nitrite reduced in vitro mitochondrial superoxide generation and improved survival of neurons after oxygen glucose deprivation. Protection was maintained with inhibition of soluble guanylate cyclase but lost with NO scavenging and ultraviolet irradiation. Nitrite therapy results in direct neuroprotection from ACA mediated by reductions in brain mitochondrial ROS in association with protein S-nitrosation. Neuroprotection is dependent on NO and S-nitrosothiol generation, not soluble guanylate cyclase.
Asunto(s)
Paro Cardíaco/fisiopatología , Neuroprotección/efectos de los fármacos , Nitritos/farmacología , Animales , Ácido Ascórbico/metabolismo , Asfixia/fisiopatología , Química Encefálica , Supervivencia Celular , Depuradores de Radicales Libres/farmacología , Glucosa/deficiencia , Guanilato Ciclasa/metabolismo , Paro Cardíaco/tratamiento farmacológico , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/metabolismo , Nitritos/administración & dosificación , Nitritos/farmacocinética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Análisis de SupervivenciaRESUMEN
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.
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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
Superoxide (O2(â¢-)) promotes neointimal hyperplasia following arterial injury. Conversely, nitric oxide ((â¢)NO) inhibits neointimal hyperplasia through various cell-specific mechanisms, including redox regulation. What remains unclear is whether (â¢)NO exerts cell-specific regulation of the vascular redox environment following arterial injury to inhibit neointimal hyperplasia. Therefore, the aim of the present study was to assess whether (â¢)NO exerts cell-specific, differential modulation of O2(â¢-) levels throughout the arterial wall, establish the mechanism of such modulation, and determine if it regulates (â¢)NO-dependent inhibition of neointimal hyperplasia. In vivo, (â¢)NO increased superoxide dismutase-1 (SOD-1) levels following carotid artery balloon injury in a rat model. In vitro, (â¢)NO increased SOD-1 levels in vascular smooth muscle cells (VSMC), but had no effect on SOD-1 in endothelial cells or adventitial fibroblasts. This SOD-1 increase was associated with an increase in sod1 gene expression, increase in SOD-1 activity, and decrease in O2(â¢-) levels. Lastly, to determine the role of SOD-1 in (â¢)NO-mediated inhibition of neointimal hyperplasia, we performed the femoral artery wire injury model in wild type and SOD-1 knockout (KO) mice, with and without (â¢)NO. Interestingly, (â¢)NO inhibited neointimal hyperplasia only in wild type mice, with no effect in SOD-1 KO mice. In conclusion, these data show the cell-specific modulation of O2(â¢-) by (â¢)NO through regulation of SOD-1 in the vasculature, highlighting its importance on the inhibition of neointimal hyperplasia. These results also shed light into the mechanism of (â¢)NO-dependent redox balance, and suggest a novel VSMC redox target to prevent neointimal hyperplasia.
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Traumatismos de las Arterias Carótidas/metabolismo , Hiperplasia/metabolismo , Neointima/metabolismo , Óxido Nítrico/farmacología , Superóxido Dismutasa/genética , Animales , Proliferación Celular , Células Cultivadas , Arteria Femoral/lesiones , Arteria Femoral/metabolismo , Arteria Femoral/patología , Hiperplasia/patología , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/citología , Neointima/patología , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/análisis , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Xanthine oxidoreductase (XOR), the molybdoflavin enzyme responsible for the terminal steps of purine degradation in humans, is also recognized as a significant source of reactive species contributory to inflammatory disease. In animal models and clinical studies, inhibition of XOR has resulted in diminution of symptoms and enhancement of function in a number of pathologies including heart failure, diabetes, sickle cell anemia, hypertension and ischemia-reperfusion injury. For decades, XOR involvement in pathologic processes has been established by salutary outcomes attained from treatment with the XOR inhibitor allopurinol. This has served to frame a working dogma that elevation of XOR-specific activity is associated with enhanced rates of reactive species generation that mediate negative outcomes. While adherence to this narrowly focused practice of designating elevated XOR activity to be "bad" has produced some benefit, it has also led to significant underdevelopment of the processes mediating XOR regulation, identification of alternative reactants and products as well as micro-environmental factors that alter enzymatic activity. This is exemplified by recent reports: (1) identifying XOR as a nitrite reductase and thus a source of beneficial nitric oxide ((â¢)NO) under in vivo conditions similar to those where XOR inhibition has been assumed an optimal treatment choice, (2) describing XOR-derived uric acid (UA) as a critical pro-inflammatory mediator in vascular and metabolic disease and (3) ascribing an antioxidant/protective role for XOR-derived UA. When taken together, these proposed and countervailing functions of XOR affirm the need for a more comprehensive evaluation of product formation as well as the factors that govern product identity. As such, this review will critically evaluate XOR-catalyzed oxidant, (â¢)NO and UA formation as well as identify factors that mediate their production, inhibition and the resultant impact on inflammatory disease.
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Inflamación/enzimología , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Úrico/metabolismo , Xantina Deshidrogenasa/antagonistas & inhibidores , Alopurinol/farmacología , Animales , Humanos , Inflamación/metabolismo , Xantina Deshidrogenasa/genética , Xantina Deshidrogenasa/metabolismoRESUMEN
Excessive vascular and colon epithelial reactive oxygen species production by NADPH oxidase isoform 1 (Nox1) has been implicated in a number of disease states, including hypertension, atherosclerosis, and neoplasia. A peptide that mimics a putative activation domain of the Nox1 activator subunit NOXA1 (NOXA1 docking sequence, also known as NoxA1ds) potently inhibited Nox1-derived superoxide anion (O2·-) production in a reconstituted Nox1 cell-free system, with no effect on Nox2-, Nox4-, Nox5-, or xanthine oxidase-derived reactive oxygen species production as measured by cytochrome c reduction, Amplex Red fluorescence, and electron paramagnetic resonance. The ability of NoxA1ds to cross the plasma membrane was tested by confocal microscopy in a human colon cancer cell line exclusively expressing Nox1 (HT-29) using FITC-labeled NoxA1ds. NoxA1ds significantly inhibited whole HT-29 carcinoma cell-derived O2·- generation. ELISA and fluorescence recovery after photobleaching experiments indicate that NoxA1ds, but not its scrambled control, binds Nox1. FRET experiments conducted using Nox1-YFP and NOXA1-CFP illustrate that NoxA1ds disrupts the binding interaction between Nox1 and NOXA1, whereas a control peptide did not. Moreover, hypoxia-induced human pulmonary artery endothelial cell O2·- production was completely inhibited by NoxA1ds. Human pulmonary artery endothelial cell migration under hypoxic conditions was also reduced by pretreatment with NoxA1ds. Our data indicate that a peptide recapitulating a putative activation subdomain of NOXA1 (NoxA1ds) is a highly efficacious and selective inhibitor of Nox1 activity and establishes a critical interaction site for Nox1-NOXA1 binding required for enzyme activation.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Dominio Catalítico , Movimiento Celular , Células Endoteliales/metabolismo , NADPH Oxidasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Secuencia Conservada , Células Endoteliales/enzimología , Células Endoteliales/fisiología , Activación Enzimática , Células HT29 , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Mutación , NADPH Oxidasa 1 , Unión Proteica , Arteria Pulmonar/citología , Superóxidos/metabolismoRESUMEN
Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.
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Lesión Pulmonar Aguda/metabolismo , Proteínas Portadoras/metabolismo , Células Endoteliales/enzimología , Glutatión/metabolismo , Lipopolisacáridos/toxicidad , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Superóxidos/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Células Endoteliales/patología , Glutatión/genética , Humanos , Pulmón/irrigación sanguínea , Pulmón/enzimología , Pulmón/patología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Procesamiento Proteico-Postraduccional/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ubiquitina-Proteína LigasasRESUMEN
The placenta plays a critical role in nutrient-waste exchange between the maternal and fetal circulation, and thus impacts fetal growth and development. We have previously shown that nano-titanium dioxide (nano-TiO2) inhalation exposure during gestation decreased fetal female pup and placenta mass [1], which persists in the following generation [2]. In utero exposed females, once mated, their offspring's placentas had increased capacity for H2O2 production. Generation of oxidants such as hydrogen peroxide (H2O2), have been shown to impact cyclooxygenase activity, specifically metabolites such as prostacyclin (PGI2) or thromboxane (TXA2). Therefore, we hypothesized that maternal nano-TiO2 inhalation exposure during gestation results in alterations in placental production of prostacyclin and thromboxane mediated by enhanced H2O2 production in a sexually dimorphic manner. Pregnant Sprague-Dawley rats were exposed to nano-TiO2 aerosols or filtered air (sham--control) from gestational day (GD) 10-19. Dams were euthanized on GD 20, and fetal serum and placental tissue were collected based on fetal sex. Fetal placental zones (junctional zone (JZ) and labyrinth zone (LZ)) were assessed for xanthine oxidoreductase (XOR) activity, H2O2, and catalase activity, as well as 6-keto-PGF1α and TXB2 levels. Nano-TiO2 exposed fetal female LZ demonstrated significantly greater XOR activity compared to exposed males. Exposed fetal female LZ also demonstrated significantly diminished catalase activity compared to sham-control females. Exposed fetal female LZ had significantly increased abundance of 6-keto-PGF1α compared to sham-control females and increased TXB2 compared to exposed males. In the aggregate these data indicate that maternal nano-TiO2 inhalation exposure has a greater impact on redox homeostasis and PGI2/TXA2 balance in the fetal female LZ. Future studies need to address if treatment with an XO inhibitor during gestation can prevent diminished fetal female growth during maternal nano-TiO2 inhalation exposure.
RESUMEN
Numerous inflammatory disorders are associated with elevated levels of xanthine oxidoreductase (XOR) and allied enhancement of reactive species formation contributory to systemic pathology. Despite a long standing association between increased XOR activity and negative clinical outcomes, recent reports describe a paradigm shift where XOR mediates beneficial actions by catalyzing the reduction of NO2(-) to NO. While provocative, these observations contradict reports of improved outcomes in similar models upon XOR inhibition as well as reports revealing strict anoxia as a requisite for XOR-mediated NO formation. To garner a more clear understanding of conditions necessary for in vivo XOR-catalyzed NO production, this review critically analyzes the impact of O2 tension, pH, substrate concentrations, glycoaminoglycan docking and inhibition strategies on the nitrite reductase activity of XOR and reveals a hypoxic milieu where this process may be operative. As such, information herein serves to link recent reports in which XOR activity has been identified as mediating the beneficial outcomes resulting from nitrite supplementation to a microenvironmental setting where XOR can serve as substantial source of NO.
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Óxido Nítrico/metabolismo , Nitritos/metabolismo , Xantina Deshidrogenasa/metabolismo , Animales , Humanos , Modelos Moleculares , Óxido Nítrico/química , Nitritos/química , Oxidación-Reducción , Xantina Deshidrogenasa/químicaRESUMEN
OBJECTIVE: Although the matricellular protein thrombospondin-1 (TSP1) is highly expressed in the vessel wall in response to injury, its pathophysiological role in the development of vascular disease is poorly understood. This study was designed to test the hypothesis that TSP1 stimulates reactive oxygen species production in vascular smooth muscle cells and induces vascular dysfunction by promoting oxidative stress. METHODS AND RESULTS: Nanomolar concentrations of TSP1 found in human vascular disease robustly stimulated superoxide (O(2)(â¢-)) levels in vascular smooth muscle cells at both cellular and tissue level as measured by cytochrome c and electron paramagnetic resonance. A peptide mimicking the C terminus of TSP1 known to specifically bind CD47 recapitulated this response. Transcriptional knockdown of CD47 and a monoclonal inhibitory CD47 antibody abrogated TSP1-triggered O(2)(â¢-) in vitro and ex vivo. TSP1 treatment of vascular smooth muscle cells activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase organizer subunit p47(phox) and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and NADPH oxidase 1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow after ischemia reperfusion. CONCLUSIONS: Our data suggest a highly regulated process of reactive oxygen species stimulation and blood flow regulation promoted through a direct TSP1/CD47-mediated activation of Nox1. This is the first report, to our knowledge, of a matricellular protein acting as a ligand for NADPH oxidase activation and through specific engagement of integrin-associated protein CD47.
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Antígeno CD47/fisiología , Músculo Liso Vascular/fisiología , NADH NADPH Oxidorreductasas/fisiología , Flujo Sanguíneo Regional/fisiología , Trombospondina 1/fisiología , Animales , Antígeno CD47/genética , Silenciador del Gen , Masculino , Ratones , Músculo Liso Vascular/efectos de los fármacos , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Proteína Quinasa C/fisiología , Ratas , Flujo Sanguíneo Regional/efectos de los fármacos , Transducción de Señal/fisiología , Superóxido Dismutasa/fisiología , Superóxido Dismutasa-1 , Trombospondina 1/farmacología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Xanthine oxidase (XO) mediates vascular function. Chronic stress impairs cerebrovascular function and increases the risk of stroke and cognitive decline. Our study determined the role of XO on stress-induced cerebrovascular dysfunction and cognitive decline. We measured middle cerebral artery (MCA) function, free radical formation, and working memory in 6-month-old C57BL/6 mice who underwent 8 weeks of control conditions or unpredictable chronic mild stress (UCMS) with or without febuxostat (50 mg/L), a XO inhibitor. UCMS mice had an impaired MCA dilation to acetylcholine vs. controls (p < 0.0001), and increased total free radical formation, XOR protein levels, and hydrogen peroxide production in the liver compared to controls. UCMS increased hydrogen peroxide production in the brain and cerebrovasculature compared to controls. Working memory, using the y-maze test, was impaired (p < 0.05) in UCMS mice compared to control mice. However, blocking XO using febuxostat prevented the UCMS-induced impaired MCA response, while free radical production and hydrogen peroxide levels were similar to controls in the liver and brain of UCMS mice treated with febuxostat. Further, UCMS + Feb mice did not have a significant reduction in working memory. These data suggest that the cerebrovascular dysfunction associated with chronic stress may be driven by XO, which leads to a reduction in working memory.