RESUMEN
Cardiovascular disease has been associated with increased levels of reactive oxygen species (ROS). Recently, we have shown that a critical balance between cytosolic ROS and mitochondrial ROS is crucial in cardiovascular health and that modulation of mitochondrial ROS helps prevent detrimental effects of cytosolic ROS on endothelial cells (EC) in transgenic animals. Here, we report the development of a controlled delivery system for a mitochondria-targeted antioxidant, JP4-039, from an electrospun scaffold made of FDA-approved biocompatible polymeric nanofibers. We demonstrate that the active antioxidant moiety was preserved in released JP4-039 for over 72 h using electron paramagnetic resonance. We also show that both the initial burst release of the drug within the first 20 min and the ensuing slow and sustained release that occurred over the next 24 h improved tube formation in human coronary artery ECs (HCAEC) in vitro. Taken together, these findings suggest that electrospinning methods can be used to upload mitochondrial antioxidant (JP4-039) onto a biocompatible nanofibrous PLGA scaffold, and the uploaded drug (JP4-039) retains nitroxide antioxidant properties upon release from the scaffold, which in turn can reduce mitochondrial ROS and improve EC function in vitro.
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Antioxidantes/administración & dosificación , Portadores de Fármacos/química , Nanofibras/química , Óxidos de Nitrógeno/administración & dosificación , Antioxidantes/farmacocinética , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/patología , Línea Celular , Vasos Coronarios/citología , Vasos Coronarios/patología , Liberación de Fármacos , Células Endoteliales , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxidos de Nitrógeno/farmacocinética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
INTRODUCTION: Free radicals and reactive oxygen species are related to deteriorating pathological conditions after head trauma because of their secondary effects. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) scavenges free radicals; however, this molecule is also toxic. Here, we have evaluated the neuroprotective effect of antioxidant nanoparticles, which consisted of a novel core-shell type nanoparticle containing 4-amino-TEMPO, that is, redox-active nitroxide radical-containing nanoparticles (RNPs). METHODS: Institute of Cancer Research mice were subjected to a head-impact procedure, randomly divided into four groups and intravenously (3 mg/kg) administered phosphate-buffered saline, TEMPO, micelle (a self-assembling block copolymer micelle without a TEMPO moiety), or RNP through the tail vein immediately thereafter and intraperitoneally at days 1, 3, and 5 after traumatic brain injury (TBI). The RNP distribution was detected by rhodamine labeling. Cognitive behavior was assessed using the neurological severity score and a rotarod test at days 1, 3, and 7 following TBI, and contusion volume was measured at day 7 after TBI. Free radical-scavenging capacity was analyzed by electron paramagnetic resonance on day 1 after TBI, and immunostaining was used to observe mobilization of microglia (Iba-1) and rescued neuronal cells (NeuN). RESULTS: Redox-active nitroxide radical-containing nanoparticle was detected in the microvessels around the injured area in the brain. Cognitive behavior assessment was significantly better, and contusion volume was significantly smaller in the RNP group compared with the other groups. Superoxide anion scavenging capacity was significantly higher in the RNP group, and neuronal loss was significantly suppressed around the injured area at day 7 after TBI. Furthermore, in the RNP group, neurodegenerative microglia production was suppressed at days 3 and 7 after TBI, whereas neuroprotective microglia production was higher at day 7 after TBI. CONCLUSION: The RNP administration after TBI improved cognitive behavior and reduced contusion volume by improving reactive oxygen species scavenging capacity. Therefore, RNP may have a neuroprotective effect after TBI. LEVEL OF EVIDENCE: Therapeutic test.
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Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Depuradores de Radicales Libres/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Administración Intravenosa , Animales , Conducta Animal/efectos de los fármacos , Lesiones Traumáticas del Encéfalo/patología , Cognición/efectos de los fármacos , Óxidos N-Cíclicos/administración & dosificación , Óxidos N-Cíclicos/química , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/química , Humanos , Masculino , Ratones , Nanopartículas/administración & dosificación , Nanopartículas/química , Fármacos Neuroprotectores/química , Óxidos de Nitrógeno/administración & dosificación , Óxidos de Nitrógeno/químicaRESUMEN
In diabetes, abnormal angiogenesis due to hyperglycemia and endothelial dysfunction impairs wound healing and results in high risks of diabetic foot ulcers and mortality. Alternative therapeutic methods were attempted to prevent diabetic complications through the activation of endothelial nitric oxide synthase. In this study, direct application of nitric oxide using dinitrosyl iron complexes (DNICs) to promote angiogenesis and wound healing under physiological conditions and in diabetic mice is investigated. Based on in vitro and in vivo studies, DNIC [Fe2(µ-SCH2CH2OH)2(NO)4] (DNIC-1) with a sustainable NO-release reactivity (t1/2 = 27.4 ± 0.5 h at 25 °C and 16.8 ± 1.8 h at 37 °C) activates the NO-sGC-cGMP pathway and displays the best pro-angiogenesis activity overwhelming other NO donors and the vascular endothelial growth factor. Moreover, this pro-angiogenesis effect of DNIC-1 restores the impaired angiogenesis in the ischemic hind limb and accelerates the recovery rate of wound closure in diabetic mice. This study translates synthetic DNIC-1 into a novel therapeutic agent for the treatment of diabetes and highlights its sustainable â¢NO-release reactivity on the activation of angiogenesis and wound healing.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Hierro/administración & dosificación , Neovascularización Patológica/prevención & control , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/prevención & control , Animales , Supervivencia Celular , Células Cultivadas , Embrión de Pollo , Membrana Corioalantoides/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Miembro Posterior , Humanos , Isquemia/patología , Isquemia/prevención & control , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Óxido Nítrico/química , Factor A de Crecimiento Endotelial Vascular/metabolismo , Heridas y Lesiones/patología , Pez CebraRESUMEN
AIMS: Repeated use of nonsteroidal anti-inflammatory drugs can induce changes in the redox status, including production of reactive oxygen species (ROS), but the specific details of these changes remain unknown. Overhauser-enhanced magnetic resonance imaging (OMRI) has been used in vivo to monitor the redox status in several diseases and map tissue oxygen concentrations. We monitored the intra- and extracellular redox status in the stomach of rats with indomethacin-induced gastric ulcers using OMRI and investigated the relationship with gastric mucosal damage. RESULTS: One hour after oral administration of indomethacin (30 mg/kg), OMRI measurements in the stomach were made following nitroxyl probe administration. OMRI with the membrane-permeable nitroxyl probe, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPOL), demonstrated a redox change toward oxidation, which was reversed by a membrane-permeable antioxidant. Conversely, imaging with the impermeable probe, 4-trimethylammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (CAT-1), demonstrated little redox change. Redox imbalance imaging of a live rat stomach with indomethacin-induced gastric ulcers was produced by dual imaging of 15N-labeled TEMPOL and 14N-labeled CAT-1, in addition to imaging with another membrane-permeable 15N-labeled probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL), and 14N-labeled CAT-1. Pretreatment with MC-PROXYL suppressed gastric mucosal damage, whereas pretreatment with CAT-1 did not suppress ulcer formation. INNOVATION: OMRI combined with a dual probe is a less invasive imaging technique for evaluation of intracellular ROS production contributing to the formation of gastric ulcers in the stomach of indomethacin-treated rats, which cannot be done with other methods. CONCLUSION: This method may be a very powerful tool for characterizing the pathogenesis of various diseases and may have medical applications.
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Indometacina/efectos adversos , Espectroscopía de Resonancia Magnética/métodos , Especies Reactivas de Oxígeno/metabolismo , Úlcera Gástrica/diagnóstico por imagen , Animales , Óxidos N-Cíclicos/administración & dosificación , Masculino , Imagen Molecular/métodos , Óxidos de Nitrógeno/administración & dosificación , Oxidación-Reducción , Pirrolidinas/administración & dosificación , Ratas , Marcadores de Spin , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/metabolismoRESUMEN
BACKGROUND/AIM: The mitochondrial targeted GS-nitroxide, JP4-039, is an effective total body irradiation (TBI) mitigator when delivered intravenously (IV) up to 72 h after exposure. Effective systemic and localized administration to oral cavity/oropharynx and esophagus has been demonstrated. The objective of the study was to establish alternatives to IV administration suitable for JP4-039 delivery to mass casualties. MATERIALS AND METHODS: JP4-039 was administered to C57BL/6 mice by topically applied carboxy-methyl-cellulose microneedle arrays (MNAs) or by intramuscular (IM) injection. Three different formulations that have passed Food and Drug Administration review, namely Captisol, 2-hydroxypropyl-ß-cyclodextrin (cyclodextrin), and Miglyol-812-N, were used for drug delivery. Intraoral (IO) administration with each formulation was also evaluated. RESULTS: All tested formulations and MNAs successfully delivered JP4-039. However, IM delivery of the Miglyol-812-N displayed very efficient and highly reproducible radiation mitigation. CONCLUSION: Effective IM delivery of JP4-039 in animal models after TBI or partial-body irradiation suggested the use of the Miglyol-812-N formulation in both medical indications and radiation countermeasures.
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Vías de Administración de Medicamentos , Composición de Medicamentos , Óxidos de Nitrógeno/administración & dosificación , Óxidos de Nitrógeno/química , Protectores contra Radiación/administración & dosificación , Protectores contra Radiación/química , Administración Intravenosa , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Estabilidad de Medicamentos , Femenino , Inyecciones Intramusculares , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Estructura Molecular , Óxidos de Nitrógeno/farmacocinética , Traumatismos Experimentales por Radiación , Radiación Ionizante , Protectores contra Radiación/farmacocinética , Reproducibilidad de los Resultados , Tasa de Supervivencia , Irradiación Corporal TotalRESUMEN
Tissue damage and the impairment of regenerative processes by excessive reactive oxygen species (ROS) contributes to the pathogenesis of various diseases in soft tissues including diabetes, atherosclerosis, Parkinson's disease and myocardial ischemic/reperfusion injury. In this study, a thermally responsive injectable hydrogel poly(NIPAAm-co-VP-co-MAPLA-co-MATEMPO) (pNVMT, NIPAAm: N-isopropylacrylamide, VP: vinylpyrrolidone, MAPLA: methacrylate-polylactide, MATEMPO: methacrylate-TEMPO, TEMPO: 4-amino-TEMPO or 4-Amino-2,2,6,6-tetramethylpiperidine-1-oxyl) incorporating recyclable ROS scavenging nitroxide radicals on the polymer backbone was developed to locally control adverse tissue effects from free radical generation. In an in vitro oxidative environment, TEMPO Gel significantly preserved cell viability. In a rat myocardial infarction/reperfusion model, TEMPO Gel diffused through the infarcted myocardium, integrated with the tissue upon gelation, and remained for over one week as visualized by MRI. The TEMPO Gel reduced infarction/reperfusion injury and preserved left ventricle geometry. This thermally responsive hydrogel was demonstrated to have properties desirable for local application to soft tissue beds where oxidative damage by ROS is of concern in pathological mechanisms.
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Acrilamidas/química , Antioxidantes/administración & dosificación , Óxidos N-Cíclicos/química , Hidrogeles/química , Metacrilatos/química , Óxidos de Nitrógeno/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/uso terapéutico , Materiales Biocompatibles/química , Preparaciones de Acción Retardada/química , Femenino , Inyecciones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Óxidos de Nitrógeno/uso terapéutico , Pirrolidinonas/química , Ratas Endogámicas Lew , TemperaturaRESUMEN
BACKGROUND: During exercise as pulmonary blood flow rises, pulmonary capillary blood volume increases and gas exchange surface area expands through distention and recruitment. We have previously demonstrated that pulmonary capillary recruitment is limited in COPD patients with poorer exercise tolerance. Hypoxia and endothelial dysfunction lead to pulmonary vascular dysregulation possibly in part related to nitric oxide related pathways. PURPOSE: To determine if increasing dietary nitrate might influence lung surface area for gas exchange and subsequently impact exercise performance. METHODS: Subjects had stable, medically treated COPD (nâ¯=â¯25), gave informed consent, filled out the St George Respiratory Questionnaire (SGRQ), had a baseline blood draw for Hgb, performed spirometry, and had exhaled nitric oxide (exNO) measured. Then they performed the intra-breath (IB) technique for lung diffusing capacity for carbon monoxide (DLCO) as well as pulmonary blood flow (Qc). Subsequently they completed a progressive semi-recumbent cycle ergometry test to exhaustion with measures of oxygen saturation (SpO2) and expired gases along with DLCO and Qc measured during the 1st work load only. Subjects were randomized to nitrate supplement group (beetroot juice) or placebo group (black currant juice) for 8 days and returned for repeat of the above protocol. RESULTS: Exhaled nitric oxide levels rose >200% in the nitrate group (pâ¯<â¯0.05) with minimal change in placebo group. The SGRQ suggested a small fall in perceived symptom limitation in the nitrate group, but no measure of resting pulmonary function differed post nitrate supplementation. With exercise, there was no influence of nitrate supplementation on peak VO2 or other measures of respiratory gas exchange. There was a tendency for the exercise DLCO to increase slightly in the nitrate group with a trend towards a rise in the DLCO/Qc relationship (pâ¯=â¯0.08) but not in the placebo group. The only other significant finding was a fall in the exercise blood pressure in the nitrate group, but not placebo group (pâ¯<â¯0.05). CONCLUSION: Despite evidence of a rise in exhaled nitric oxide levels with nitrate supplementation, there was minimal evidence for improvement in exercise performance or pulmonary gas exchange surface area in a stable medically treated COPD population.
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Suplementos Dietéticos , Ejercicio Físico , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Óxidos de Nitrógeno/farmacología , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Intercambio Gaseoso Pulmonar/efectos de los fármacos , Anciano , Femenino , Humanos , Pulmón/metabolismo , Masculino , Óxidos de Nitrógeno/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
Squamous cell carcinomas of the head and neck are appearing with increased frequency in both marrow transplanted and non-transplanted Fanconi anemia (FA) patients. FA patients commonly display radiosensitivity of epithelial tissues, complicating effective radiotherapy. Fancd2-/- mice (C57BL/6J and 129/Sv background) demonstrate epithelial tissue sensitivity to single-fraction or fractionated irradiation to the head and neck and distant marrow suppression (abscopal effect), both ameliorated by intraoral administration of the mitochondrial-targeted antioxidant, GS-nitroxide, JP4-039. We now report that mice of two other FA genotypes, Fancg-/- (B6) and the most prevalent human genotype Fanca-/- (129/Sv), also demonstrate: 1. reduced longevity of hematopoiesis in long-term bone marrow cultures; 2. radiosensitivity of bone marrow stromal cell lines; and 3. head and neck radiation-induced severe mucositis and abscopal suppression of distant marrow hematopoiesis. Intraoral administration of JP4-039/F15, but not non-mitochondrial-targeted 4-amino-Tempo/F15 or F15 alone, prior to each radiation treatment ameliorated both local and abscopal radiation effects. Head and neck irradiated TGF-ß-resistant SMAD3-/- (129/Sv) mice and double-knockout SMAD3-/- Fancd2-/- (129/Sv) mice treated daily with TGF-ß receptor antagonist, LY364947, still displayed abscopal bone marrow suppression, implicating a non-TGF-ß mechanism. Thus, amelioration of both local normal tissue radiosensitivity and distant marrow suppression by intraoral administration of JP4-039 in Fancg-/- and Fanca-/- mice supports a clinical trial of this locally administered normal tissue radioprotector and mitigator during head and neck irradiation in FA patients.
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Médula Ósea/efectos de los fármacos , Neoplasias de Cabeza y Cuello/radioterapia , Mucositis/tratamiento farmacológico , Óxidos de Nitrógeno/administración & dosificación , Óxidos de Nitrógeno/farmacología , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Protectores contra Radiación/farmacología , Administración Oral , Animales , Médula Ósea/patología , Médula Ósea/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Proteína del Grupo de Complementación A de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación G de la Anemia de Fanconi/deficiencia , Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Interleucina-3/metabolismo , Ratones , Mitomicina/farmacología , Mucositis/metabolismo , Mucositis/patología , Óxidos de Nitrógeno/uso terapéutico , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Protectores contra Radiación/administración & dosificación , Protectores contra Radiación/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50µL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.
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Cromatografía de Fase Inversa , Monitoreo de Drogas/métodos , Óxidos de Nitrógeno/sangre , Protectores contra Radiación/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Administración Intravenosa , Animales , Calibración , Cromatografía de Fase Inversa/normas , Frío , Monitoreo de Drogas/normas , Estabilidad de Medicamentos , Masculino , Ratones Endogámicos C57BL , Óxidos de Nitrógeno/administración & dosificación , Óxidos de Nitrógeno/farmacocinética , Protectores contra Radiación/administración & dosificación , Protectores contra Radiación/farmacocinética , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/normasRESUMEN
The use of nitroglycerin in the treatment of angina pectoris began not long after its original synthesis in 1847. Since then, the discovery of nitric oxide as a biological effector and better understanding of its roles in vasodilation, cell permeability, platelet function, inflammation, and other vascular processes have advanced our knowledge of the hemodynamic (mostly mediated through vasodilation of capacitance and conductance arteries) and nonhemodynamic effects of organic nitrate therapy, via both nitric oxide-dependent and -independent mechanisms. Nitrates are rapidly absorbed from mucous membranes, the gastrointestinal tract, and the skin; thus, nitroglycerin is available in a number of preparations for delivery via several routes: oral tablets, sublingual tablets, buccal tablets, sublingual spray, transdermal ointment, and transdermal patch, as well as intravenous formulations. Organic nitrates are commonly used in the treatment of cardiovascular disease, but clinical data limit their use mostly to the treatment of angina. They are also used in the treatment of subsets of patients with heart failure and pulmonary hypertension. One major limitation of the use of nitrates is the development of tolerance. Although several agents have been studied for use in the prevention of nitrate tolerance, none are currently recommended owing to a paucity of supportive clinical data. Only 1 method of preventing nitrate tolerance remains widely accepted: the use of a dosing strategy that provides an interval of no or low nitrate exposure during each 24-h period. Nitric oxide's important role in several cardiovascular disease mechanisms continues to drive research toward finding novel ways to affect both endogenous and exogenous sources of this key molecular mediator.
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Fármacos Cardiovasculares/administración & dosificación , Enfermedades Cardiovasculares/tratamiento farmacológico , Óxidos de Nitrógeno/administración & dosificación , Nitroglicerina/administración & dosificación , Animales , Fármacos Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Vías de Administración de Medicamentos , Humanos , Óxidos de Nitrógeno/metabolismo , Nitroglicerina/metabolismoRESUMEN
AIMS: Nitroxyl anion (HNO) has recently become an emerging candidate in vascular regulation. NO- is a potent vasodilator of both conduit and small resistance vessels and mediates relaxation in a soluble guanylate cyclase-dependent manner. Interestingly, HNO activates voltage-dependent K+ (K+ V) channels, whereas Nitric Oxide (NO) activates calcium-activated K+ Ca channels. To date, there are few studies investigating the role of HNO in hypertension, and the possible mechanisms, which may be altered during this condition. We hypothesized that mesenteric arteries from angiotensin II-induced (AngII) hypertensive mice would exhibit an increased dependence upon NO- for relaxation, which may be mediated through K+ V channels. Methods and Key Results: C57/Bl6 mice, aged 12-14 weeks were implanted with mini-pumps containing angiotensin II (AngII, 3600ng/kg/min) for 14 days. For this study, we proposed to investigate the role of HNO in the resistance vasculature, and so first order mesenteric arteries were isolated and used in functional studies, or were frozen for Western blot analysis. We observed that mesenteric arteries from AngII mice (AngII) exhibited a decrease in HNO-mediated relaxation, which was endotheliumindependent. With HNO scavenging by L-cysteine [3mM], the maximal acetylcholine (ACh)-mediated relaxation response was decreased in sham, whereas mesenteric arteries from AngII exhibited a decrease in sensitivity. Incubation with the K+ V channel inhibitor, 4-aminopyridine [1mM], decreased AChmediated relaxation responses in sham, but almost completely abolished relaxation in AngII. CONCLUSION: We reveal that exogenous HNO-mediated relaxation, via Angeli's Salt, is impaired in mesenteric arteries from AngII-treated mice, yet endogenous HNO-mediated relaxation may be more important during hypertension.
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Hipertensión/fisiopatología , Arterias Mesentéricas/metabolismo , Óxidos de Nitrógeno/administración & dosificación , Vasodilatación/fisiología , 4-Aminopiridina/farmacología , Acetilcolina/farmacología , Angiotensina II/administración & dosificación , Animales , Modelos Animales de Enfermedad , Masculino , Arterias Mesentéricas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Nitritos/farmacología , Óxidos de Nitrógeno/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Oxidative stress and inflammation play important roles in the pathogenesis of ischemia/reperfusion (I/R)-injury. The administration of antioxidants and anti-inflammatory agents has been applied to prevent I/R-injury for several decades. Of the numerous compounds administrated therapeutically in anti-oxidative stress, nitronyl nitroxide has gained increasing attention due to its continuous ability to scavenge active oxygen radicals. However, its effect is not ideal in clinical therapy. In previous study, we linked the anti-inflammatory amino acid glycine to nitronyl nitroxide and developed a novel glycine-nitronyl nitroxide (GNN) conjugate, which showed a synergetic protection against renal ischemia/reperfusion injury. However, the underlying mechanism remains unclear. In this study, a hypoxia/reoxygenation (H/R) injury model was established in human umbilical vein endothelial cells (HUVECs) and we found that the GNN conjugate significantly elevated the cell viability via reducing the apoptosis rate in H/R-treated HUVECs. Meanwhile, GNN conjugate attenuated H/R induced mitochondrial fragmentation, mitochondrial membrane potential reduction, Cytochrome c release and autophagy. To determine the extensive applicability of GNN conjugate in different I/R models and its effect in remote organs, an in vivo hind limb I/R model was established. As expected, GNN conjugate ameliorated damages of muscle and remote organs. These results demonstrate that GNN conjugate may be an effective agent against ischemia/reperfusion injury in clinical therapy.
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Antioxidantes/farmacología , Glicina/farmacología , Miembro Posterior/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Óxidos de Nitrógeno/farmacología , Sustancias Protectoras/farmacología , Daño por Reperfusión/tratamiento farmacológico , Animales , Antioxidantes/administración & dosificación , Apoptosis/efectos de los fármacos , Células Cultivadas , Glicina/administración & dosificación , Humanos , Hipoxia , Inyecciones Intraperitoneales , Masculino , Estructura Molecular , Óxidos de Nitrógeno/administración & dosificación , Sustancias Protectoras/administración & dosificación , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patologíaRESUMEN
Hepatic fibrosis is a chronic disorder caused by viral infection and/or metabolic, genetic and cholestatic disorders. A noninvasive procedure that enables the detection of liver fibrosis based on redox status would be useful for disease identification and monitoring, and the development of treatments. However, an appropriate technique has not been reported. This study describes a novel method for assessing the redox status of the liver using in vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) with the nitroxyl radical carbamoyl-PROXYL as a molecular imaging probe, which was tested in dimethylnitrosamine-treated mice as a model of liver fibrosis. Based on the pharmacokinetics of carbamoyl-PROXYL in control livers, reduction rate mapping was performed in fibrotic livers. Reduction rate maps demonstrated a clear difference between the redox status of control and fibrotic livers according to the expression of antioxidants. These findings indicate that in vivo DNP-MRI with a nitroxyl radical probe enables noninvasive detection of changes in liver redox status.
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Cirrosis Hepática/diagnóstico , Cirrosis Hepática/metabolismo , Imagen por Resonancia Magnética , Animales , Óxidos N-Cíclicos/sangre , Dimetilnitrosamina , Inyecciones Intravenosas , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/inducido químicamente , Masculino , Ratones Endogámicos BALB C , Óxidos de Nitrógeno/administración & dosificación , Oxidación-ReducciónRESUMEN
Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli's salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7-14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1ß (IL-1ß), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1ß, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1ß and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.
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Analgésicos/uso terapéutico , Neuralgia/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Óxidos de Nitrógeno/uso terapéutico , Nervio Ciático/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Analgésicos/administración & dosificación , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Interleucina-1beta/genética , Interleucina-33/genética , Masculino , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Neuralgia/genética , Neuralgia/metabolismo , Neuralgia/fisiopatología , Fármacos Neuroprotectores/administración & dosificación , Óxidos de Nitrógeno/administración & dosificación , Nervio Ciático/fisiopatología , Transducción de Señal/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Nitroxyl anion (HNO) donors are currently being assessed for their therapeutic utility in several cardiovascular disorders including heart failure. Here, we examine their effect on factors that precede atherosclerosis including endothelial cell and monocyte activation, leucocyte adhesion to the endothelium and macrophage polarization. Similar to the NO donor glyceryl trinitrate (GTN), the HNO donors Angeli's salt (AS) and isopropylamine NONOate (IPA/NO) decreased leucocyte adhesion to activated human umbilical vein endothelial cells (HUVECs) and mouse isolated aorta. This reduction in adhesion was accompanied by a reduction in intercellular adhesion molecule-1 (ICAM-1) and the cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) which was inhibitor of nuclear factor κB (NFκB) α (IκBα)- and subsequently NFκB-dependent. Intriguingly, the effects of AS on leucocyte adhesion, like those on vasodilation, were found to not be susceptible to pharmacological tolerance, unlike those observed with GTN. As well, HNO reduces monocyte activation and promotes polarization of M2 macrophages. Taken together, our data demonstrate that HNO donors can reduce factors that are associated with and which precede atherosclerosis and may thus be useful therapeutically. Furthermore, since the effects of the HNO donors were not subject to tolerance, this confers an additional advantage over NO donors.
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Aterosclerosis/tratamiento farmacológico , Polaridad Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Óxidos de Nitrógeno/administración & dosificación , Animales , Aorta/efectos de los fármacos , Aorta/inmunología , Aorta/fisiopatología , Aterosclerosis/inmunología , Aterosclerosis/fisiopatología , Quimiocina CCL2/inmunología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/inmunología , Interleucina-6/inmunología , Macrófagos/citología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunologíaRESUMEN
We evaluated normal tissue specific radioprotection of the oral cavity in radiosensitive Fanconi Anemia (FA) Fancd2(-/-) mice with orally established tumors using mitochondrial-targeted GS-nitroxide (JP4-039). Adult (10-12 weeks old) Fancd2(+/+), Fancd2(+/-) and Fancd2(-/-) mice (C57BL/6 background) and subgroups with orally established TC-1 epithelial cell tumors received a single fraction of 28 Gy or four daily fractions of 8 Gy to the head and neck. Subgroups received JP4-039 in F15 emulsion (F15/JP4-039; 0.4 mg/mouse), 4-amino-Tempo in F15 emulsion (F15/4-amino-Tempo; 0.2 mg/mouse) or F15 emulsion alone prior to each irradiation. Oral mucosa of Fancd2(-/-) mice showed baseline elevated RNA transcripts for Sod2, p53, p21 and Rad51 (all P < 0.0012) and suppressed levels of Nfkb and Tgfb, (all P < 0.0020) compared with Fancd2(+/+) mice. The oral mucosa in tumor-bearing mice of all genotypes showed decreased levels of p53 and elevated Tgfb and Gadd45a (P ≤ 0.0001 for all three genotypes). Intraoral F15/JP4-039, but not F15/4-amino-Tempo, modulated radiation-induced normal tissue transcript elevation, ameliorated mucosal ulceration and reduced the depletion of antioxidant stores in oral cavity tissue of all genotypes, but did not radioprotect tumors. Mitochondrial targeting makes F15/JP4-039 an effective normal tissue radioprotector for Fancd2(-/-) mice, as well as wild-type mice.
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Mitocondrias/efectos de los fármacos , Mitocondrias/efectos de la radiación , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/efectos de la radiación , Neoplasias de la Boca/radioterapia , Óxidos de Nitrógeno/administración & dosificación , Administración Oral , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta en la Radiación , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Ratones , Ratones Endogámicos C57BL , Mucosa Bucal/patología , Neoplasias de la Boca/patología , Órganos en Riesgo , Tolerancia a Radiación/efectos de los fármacos , Protectores contra Radiación/administración & dosificación , Resultado del TratamientoRESUMEN
Free radicals are involved in the development of reperfusion injuries. Using a spin trap, the intensity of such lesions can be reduced. Nitrones (effective in vivo spin traps) were tried in this work as in vivo nitric oxide donors. Nitrite and nitrate concentration values (rabbit blood) were used as biomarkers of nitric oxide production. Most nitrones did not increase plasma concentrations of nitrite and nitrate; on the contrary, reduced plasma concentrations of these indicators were noted. However, glyoxal isopropyldinitrone, in a dose of 50 mg kg-1, was highly effective in increasing nitric oxide production. At the same time, nitrones do not react with hepatic homogenates, proving that the release of nitric oxide takes place in the tissues and is not related to hepatic metabolism. Before using nitrones in vivo, they were tested in vitro for the ability to release nitric oxide following a reaction with the hydroxyl radical.
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Donantes de Óxido Nítrico/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrógeno/metabolismo , Vasodilatadores/metabolismo , Animales , Biomarcadores/sangre , Radical Hidroxilo/química , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Hígado/metabolismo , Estructura Molecular , Nitratos/sangre , Óxido Nítrico/sangre , Óxido Nítrico/química , Donantes de Óxido Nítrico/administración & dosificación , Donantes de Óxido Nítrico/sangre , Donantes de Óxido Nítrico/química , Nitritos/sangre , Óxidos de Nitrógeno/administración & dosificación , Óxidos de Nitrógeno/sangre , Óxidos de Nitrógeno/química , Conejos , Detección de Spin , Factores de Tiempo , Vasodilatadores/administración & dosificación , Vasodilatadores/sangre , Vasodilatadores/químicaRESUMEN
Earlier it has been found that the hypotensive drug Oxacom containing binuclear dinitrosyl iron complexes (B-DNIC) with glutathione can effectively decrease, as a nitric monooxide (NO) donor, the mean arterial pressure (ÐÐÐ ) in rats upon intravenous bolus injection in the form of an aqueous solution (Chazov et al., 2012). The aim of this study was to investigate the hypotensive effects of Oxacom administered to experimental rats by intravenous, intramuscular, subcutaneous, intraperitoneal, intragastric, rectal routes.MAP and heart rate (HR) were measured with the help of arterial catheters equipped with tensometric sensors. Oxacom was administered to rats at the dose of 2.0 µmole of B-DNIC/kg. The concentration of paramagnetic mononuclear protein-bound DNIC (Ð-DNIC) formed in the blood and tissues of various internal organs of the rat was determined by the EPR method. Upon subcutaneous, intramuscular or intraperitoneal administration of Oxacom, the maximum amplitude of the ÐÐÐ decrease varies from 30% to 70%, respectively, in comparison with the corresponding parameter for the intravenously injected Oxacom. Another difference is the lack of the fast phase in the initial stage of the ÐÐÐ decrease and the longer persistence of protein-bound M-DNIC formed in the circulating blood after intramuscular, subcutaneous or intraperitoneal administration of Oxacom. Thus, the NO donor Oxacom exerts pronounced hypotensive effects on rats not only upon intravenous, but also upon intramuscular, subcutaneous or intraperitoneal administration.
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Antihipertensivos/administración & dosificación , Presión Arterial/efectos de los fármacos , Glutatión/administración & dosificación , Hierro/administración & dosificación , Donantes de Óxido Nítrico/administración & dosificación , Óxidos de Nitrógeno/administración & dosificación , Administración Cutánea , Animales , Presión Arterial/fisiología , Vías de Administración de Medicamentos , Glutatión/química , Inyecciones Intravenosas , Hierro/química , Masculino , Óxido Nítrico , Donantes de Óxido Nítrico/química , Óxidos de Nitrógeno/química , Ratas , Ratas WistarRESUMEN
Postsurgical tissue adhesion formation caused by inflammation and oxidative stress is one of the serious issues because it induces severe clinical disorders. In this study, we designed redox injectable gel (RIG) which covalently possesses nitroxide radicals as a reactive oxygen species (ROS) scavenger for high performance anti-adhesion agent. The redox flower micelles exhibiting gelation under physiological conditions were prepared by a polyion complex (PIC) between polyamine-PEG-polyamine triblock copolymer possessing nitroxide radicals as a side chain of polyamine segments and poly(acrylic acid). RIG showed prolonged local retention in the abdominal cavity of the mice, which was monitored by in vivo imaging system (IVIS). Compared with a commercial anti-adhesion agent (Seprafilm(®), Genzyme, Cambridge, MA), RIG dramatically inhibited the formation of tissue adhesions via a combination of physical separation and biological elimination of generated ROS in talc-induced adhesion model mice. Treatment with RIG suppressed inflammatory cytokines and neutrophil invasion, suppressing the increase in peritoneal membrane thickness. It is also emphasized that RIG suppressed the increase of white blood cells level, indicating that the present RIG treatment effectively prevents diffusion of local inflammation to entire body. These findings indicate that RIG has a great potential as a high performance anti-adhesion agent.
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Resinas Acrílicas/uso terapéutico , Antioxidantes/uso terapéutico , Geles/uso terapéutico , Óxidos de Nitrógeno/uso terapéutico , Poliaminas/uso terapéutico , Polietilenglicoles/uso terapéutico , Adherencias Tisulares/prevención & control , Resinas Acrílicas/administración & dosificación , Resinas Acrílicas/química , Animales , Antioxidantes/administración & dosificación , Antioxidantes/química , Geles/administración & dosificación , Geles/química , Inyecciones , Ratones Endogámicos BALB C , Micelas , Óxidos de Nitrógeno/administración & dosificación , Óxidos de Nitrógeno/química , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Poliaminas/administración & dosificación , Poliaminas/química , Polietilenglicoles/administración & dosificación , Polietilenglicoles/químicaRESUMEN
Prolonged hypotensive effect of oxacom (dinitrosyl iron complexes with ligand glutathione) has been well documented in animals and healthy volunteers, but effect on the heart is not defined. In this work, the blood pressure (BP), and the pressure in the left ventricle (LV) were recorded in rats. Intravenous bolus injection of oxacom (10 mg/kg) caused an immediate average BP decrease by 20-30 mm Hg followed by a slow recovery. The LV systolic and diastolic pressures did not change, but the maximal speed of pressure development, and contractility index at peak action of oxacom increased by 26-33%, while isovolumic relaxation constant fell by 30%. These changes gradually normalized within 10-15 minutes. Effect of oxacom on hearts with isoproterenol-induced diffuse ischemic myocardial lesions and diastolic heart failure was similar to that observed in the control group except the isovolumic relaxation constant rose 1.5-fold while elevated LV diastolic pressure fell. Results suggest that oxacom exerts the positive cardiotropic action especially at heart failure.