RESUMEN
Inhalation of high levels of sulfur mustard (SM), a potent vesicating and alkylating agent used in chemical warfare, results in acutely lethal pulmonary damage. Sodium 2-mercaptoethane sulfonate (mesna) is an organosulfur compound that is currently Food and Drug Administration (FDA)-approved for decreasing the toxicity of mustard-derived chemotherapeutic alkylating agents like ifosfamide and cyclophosphamide. The nucleophilic thiol of mesna is a suitable reactant for the neutralization of the electrophilic group of toxic mustard intermediates. In a rat model of SM inhalation, treatment with mesna (three doses: 300 mg/kg intraperitoneally 20 minutes, 4 hours, and 8 hours postexposure) afforded 74% survival at 48 hours, compared with 0% survival at less than 17 hours in the untreated and vehicle-treated control groups. Protection from cardiopulmonary failure by mesna was demonstrated by improved peripheral oxygen saturation and increased heart rate through 48 hours. Additionally, mesna normalized arterial pH and pACO2 Airway fibrin cast formation was decreased by more than 66% in the mesna-treated group at 9 hour after exposure compared with the vehicle group. Finally, analysis of mixtures of a mustard agent and mesna by a 5,5'-dithiobis(2-nitrobenzoic acid) assay and high performance liquid chromatography tandem mass spectrometry demonstrate a direct reaction between the compounds. This study provides evidence that mesna is an efficacious, inexpensive, FDA-approved candidate antidote for SM exposure. SIGNIFICANCE STATEMENT: Despite the use of sulfur mustard (SM) as a chemical weapon for over 100 years, an ideal drug candidate for treatment after real-world exposure situations has not yet been identified. Utilizing a uniformly lethal animal model, the results of the present study demonstrate that sodium 2-mercaptoethane sulfonate is a promising candidate for repurposing as an antidote, decreasing airway obstruction and improving pulmonary gas exchange, tissue oxygen delivery, and survival following high level SM inhalation exposure, and warrants further consideration.
Asunto(s)
Sustancias para la Guerra Química , Gas Mostaza , Ratas , Animales , Gas Mostaza/toxicidad , Mesna/farmacología , Mesna/uso terapéutico , Antídotos/farmacología , Antídotos/uso terapéutico , Pulmón , Sodio , Sustancias para la Guerra Química/toxicidadRESUMEN
Administration of high concentrations of oxygen (hyperoxia) is one of few available options to treat acute hypoxemia-related respiratory failure, as seen in the current coronavirus disease (COVID-19) pandemic. Although hyperoxia can cause acute lung injury through increased production of superoxide anion (O2â¢-), the choice of high-concentration oxygen administration has become a necessity in critical care. The objective of this study was to test the hypothesis that UCP2 (uncoupling protein 2) has a major function of reducing O2â¢- generation in the lung in ambient air or in hyperoxia. Lung epithelial cells and wild-type; UCP2-/-; or transgenic, hTrx overexpression-bearing mice (Trx-Tg) were exposed to hyperoxia and O2â¢- generation was measured by using electron paramagnetic resonance, and lung injury was measured by using histopathologic analysis. UCP2 expression was analyzed by using RT-PCR analysis, Western blotting analysis, and RNA interference. The signal transduction pathways leading to loss of UCP2 expression were analyzed by using IP, phosphoprotein analysis, and specific inhibitors. UCP2 mRNA and protein expression were acutely decreased in hyperoxia, and these decreases were associated with a significant increase in O2â¢- production in the lung. Treatment of cells with rhTrx (recombinant human thioredoxin) or exposure of Trx-Tg mice prevented the loss of UCP2 protein and decreased O2â¢- generation in the lung. Trx is also required to maintain UCP2 expression in normoxia. Loss of UCP2 in UCP2-/- mice accentuated lung injury in hyperoxia. Trx activates the MKK4-p38MAPK (p38 mitogen-activated protein kinase)-PGC1α (PPARγ [peroxisome proliferator-activated receptor γ] coactivator 1α) pathway, leading to rescue of UCP2 and decreased O2â¢- generation in hyperoxia. Loss of UCP2 in hyperoxia is a major mechanism of O2â¢- production in the lung in hyperoxia. rhTrx can protect against lung injury in hyperoxia due to rescue of the loss of UCP2.
Asunto(s)
Pulmón/metabolismo , Oxígeno/metabolismo , Tiorredoxinas/metabolismo , Proteína Desacopladora 2/metabolismo , Animales , COVID-19/metabolismo , COVID-19/terapia , Línea Celular , Humanos , Hiperoxia/metabolismo , Pulmón/citología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oxígeno/toxicidad , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fosforilación , Transducción de Señal , Superóxidos/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/farmacología , Proteína Desacopladora 2/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
E-cigarette vaping is a major aspect of nicotine consumption, especially for children and young adults. Although it is branded as a safer alternative to cigarette smoking, murine and rat models of subacute and chronic e-cigarette vaping exposure have shown many proinflammatory changes in the respiratory tract. An acute vaping exposure paradigm has not been demonstrated in the golden Syrian hamster, and the hamster is a readily available small animal model that has the unique benefit of becoming infected with and transmitting respiratory viruses, including SARS-CoV-2, without genetic alteration of the animal or virus. Using a 2-day, whole body vaping exposure protocol in male golden Syrian hamsters, we evaluated serum cotinine, bronchoalveolar lavage cells, lung, and nasal histopathology, and gene expression in the nasopharynx and lung through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Depending on the presence of nonnormality or outliers, statistical analysis was performed by ANOVA or Kruskal-Wallis tests. For tests that were statistically significant (P < 0.05), post hoc Tukey-Kramer and Dunn's tests, respectively, were performed to make pairwise comparisons between groups. In nasal tissue, RT-qPCR analysis revealed nicotine-dependent increases in gene expression associated with type 1 inflammation (CCL-5 and CXCL-10), fibrosis [transforming growth factor-ß (TGF-ß)], nicotine-independent increase oxidative stress response (SOD-2), and a nicotine-independent decrease in vasculogenesis/angiogenesis (VEGF-A). In the lung, nicotine-dependent increases in the expression of genes involved in the renin-angiotensin pathway [angiotensin-converting enzyme (ACE), ACE2], coagulation (tissue factor, Serpine-1), extracellular matrix remodeling (MMP-2, MMP-9), type 1 inflammation (IL-1ß, TNF-α, and CXCL-10), fibrosis (TGF-ß and Serpine-1), oxidative stress response (SOD-2), neutrophil extracellular traps release (ELANE), and vasculogenesis and angiogenesis (VEGF-A) were identified. To our knowledge, this is the first demonstration that the Syrian hamster is a viable model of e-cigarette vaping. In addition, this is the first report that e-cigarette vaping with nicotine can increase tissue factor gene expression in the lung. Our results show that even an acute exposure to e-cigarette vaping causes significant upregulation of mRNAs in the respiratory tract from pathways involving the renin-angiotensin system, coagulation, extracellular matrix remodeling, type 1 inflammation, fibrosis, oxidative stress response, neutrophil extracellular trap release (NETosis), vasculogenesis, and angiogenesis.
Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Transcriptoma , Vapeo , Animales , Cricetinae , Masculino , Enzima Convertidora de Angiotensina 2 , Angiotensinas , Cotinina , Fibrosis , Inflamación/patología , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Mesocricetus , Nicotina/farmacología , Renina , Superóxido Dismutasa , Tromboplastina , Factor de Crecimiento Transformador beta , Factor de Necrosis Tumoral alfa , Vapeo/efectos adversos , Factor A de Crecimiento Endotelial VascularRESUMEN
The importance of cell phenotype in determining the molecular mechanisms underlying ß2 -adrenoceptor (ß2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged ß2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged ß2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface ß2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface ß2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting ß2AR agonists.
Asunto(s)
Células Madre Embrionarias/fisiología , Fibroblastos/fisiología , Miocitos Cardíacos/fisiología , Receptores Adrenérgicos beta 2/metabolismo , Espectrometría de Fluorescencia/métodos , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Diferenciación Celular , Colorantes Fluorescentes/química , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Proteínas de la Membrana , Propranolol/farmacología , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genéticaRESUMEN
Rationale: Antenatal inflammation with placental dysfunction is strongly associated with high bronchopulmonary dysplasia (BPD) risk in preterm infants. Whether antenatal or postnatal HIF (hypoxia-inducible factor) augmentation can preserve lung structure and function and prevent pulmonary hypertension after intrauterine inflammation is controversial.Objectives: To determine whether antenatal or postnatal prolyl-hydroxylase inhibitor (PHi) therapy increases lung HIF expression, preserves lung growth and function, and prevents pulmonary hypertension in a rat model of chorioamnionitis-induced BPD caused by antenatal inflammation.Methods: Endotoxin (ETX) was administered to pregnant rats by intraamniotic injection at Embryonic Day 20, and pups were delivered by cesarean section at Embryonic Day 22. Selective PHi drugs, dimethyloxalylglycine or GSK360A, were administered into the amniotic space at Embryonic Day 20 or after birth by intraperitoneal injection for 2 weeks. Placentas and lung tissue were collected at birth for morphometric and Western blot measurements of HIF-1a, HIF-2a, VEGF (vascular endothelial growth factor), and eNOS (endothelial nitric oxide synthase) protein contents. At Day 14, lung function was assessed, and tissues were harvested to determine alveolarization by radial alveolar counts, pulmonary vessel density, and right ventricle hypertrophy (RVH).Measurements and Main Results: Antenatal PHi therapy preserves lung alveolar and vascular growth and lung function and prevents RVH after intrauterine ETX exposure. Antenatal administration of PHi markedly upregulates lung HIF-1a, HIF-2a, VEGF, and eNOS expression after ETX exposure.Conclusions: HIF augmentation improves lung structure and function, prevents RVH, and improves placental structure following antenatal ETX exposure. We speculate that antenatal or postnatal PHi therapy may provide novel strategies to prevent BPD due to antenatal inflammation.
Asunto(s)
Displasia Broncopulmonar/tratamiento farmacológico , Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/efectos de los fármacos , Péptido PHI/farmacología , Preñez , Aminoácidos Dicarboxílicos/farmacología , Animales , Animales Recién Nacidos , Western Blotting , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patología , Modelos Animales de Enfermedad , Endotoxinas/efectos adversos , Endotoxinas/farmacología , Femenino , Factor 1 Inducible por Hipoxia/efectos de los fármacos , Inmunohistoquímica , Técnicas In Vitro , Inyecciones Intralesiones , Pulmón/embriología , Embarazo , Atención Prenatal , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/crecimiento & desarrollo , Circulación Pulmonar/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Pruebas de Función Respiratoria , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Methyl mercaptan occurs naturally in the environment and is found in a variety of occupational settings, including the oil, paper, plastics, and pesticides industries. It is a toxic gas and deaths from methyl mercaptan exposure have occurred. The Department of Homeland Security considers it a high threat chemical agent that could be used by terrorists. Unfortunately, no specific treatment exists for methyl mercaptan poisoning. METHODS: We conducted a randomized trial in 12 swine comparing no treatment to intramuscular injection of the vitamin B12 analog cobinamide (2.0 mL, 12.5 mg/kg) following acute inhalation of methyl mercaptan gas. Physiological and laboratory parameters were similar in the control and cobinamide-treated groups at baseline and at the time of treatment. RESULTS: All six cobinamide-treated animals survived, whereas only one of six control animals lived (17% survival) (p = 0.0043). The cobinamide-treated animals returned to a normal breathing pattern by 3.8 ± 1.1 min after treatment (mean ± SD), while all but one animal in the control group had intermittent gasping, never regaining a normal breathing pattern. Blood pressure and arterial oxygen saturation returned to baseline values within 15 minutes of cobinamide-treatment. Plasma lactate concentration increased progressively until death (10.93 ± 6.02 mmol [mean ± SD]) in control animals, and decreased toward baseline (3.79 ± 2.93 mmol [mean ± SD]) by the end of the experiment in cobinamide-treated animals. CONCLUSION: We conclude that intramuscular administration of cobinamide improves survival and clinical outcomes in a large animal model of acute, high dose methyl mercaptan poisoning.
Asunto(s)
Antídotos/farmacología , Cobamidas/farmacología , Compuestos de Sulfhidrilo/toxicidad , Animales , Antídotos/administración & dosificación , Cobamidas/administración & dosificación , Femenino , Exposición por Inhalación , Inyecciones Intramusculares , Masculino , Distribución Aleatoria , PorcinosAsunto(s)
Mastocitos , Proteínas del Tejido Nervioso , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido , Humanos , Mastocitos/inmunología , Mastocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/inmunología , Receptores de Neuropéptido/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/inmunología , Degranulación de la Célula/inmunologíaRESUMEN
Methyl isocyanate (MIC) is a highly toxic industrial chemical causing acute lethality after inhalation. The objective of this study was to determine whether alterations in hemostasis also occur in the immediate hours after exposure. Male rats were exposed to MIC (125-500 ppm) by nose-only vapor inhalation for 30 min. Arterial O2 saturation was monitored prior to exposure, and hourly thereafter. Rats were euthanized at 1, 2, 4, and 8 hr and plasma analyzed for recalcification clotting time, tissue factor (TF) activity, and protein levels. Hypoxemia, as assessed by pulse oximetry, was an early feature of MIC inhalation. In contrast to sham or low (125 ppm) concentrations, 250 and 500 ppm MIC caused significant declines in blood oxygen saturation (% SpO2) at 1 hr, which remained at deficit during the postexposure period. Commensurate with hypoxemia, plasma clotting time was significantly accelerated 1 hr after MIC inhalation (sham treatment: 955 ± 62.8 s; 125 ppm MIC: 790 ± 62 s; 250 ppm: 676 ± 28.0 s; 500 ppm: 581 ± 175 s). This procoagulant effect was transient, with no difference observed between sham and all MIC groups by 8 hr. Similarly, elevated TF activity and protein were detected in plasma 1 hr after MIC inhalation, each of which showed a progressive decline back to control levels at later timepoints. This study demonstrates that MIC inhalation resulted in hypoxemia and transient hypercoagulability of blood. Accelerated clotting occurred rapidly and was likely due to intravascular TF, which initiates the extrinsic coagulation pathway.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Exposición por Inhalación/efectos adversos , Isocianatos/toxicidad , Tromboplastina/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Hipoxia/sangre , Hipoxia/inducido químicamente , Masculino , Oxígeno/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
Chlorine is a highly reactive gas that can cause significant injury when inhaled. Unfortunately, its use as a chemical weapon has increased in recent years. Massive chlorine inhalation can cause death within 4 hours of exposure. Survivors usually require hospitalization after massive exposure. No countermeasures are available for massive chlorine exposure and supportive-care measures lack controlled trials. In this work, adult rats were exposed to chlorine gas (LD58-67) in a whole-body exposure chamber, and given oxygen (0.8 FiO2) or air (0.21 FiO2) for 6 hours after baseline measurements were obtained. Oxygen saturation, vital signs, respiratory distress and neuromuscular scores, arterial blood gases, and hemodynamic measurements were obtained hourly. Massive chlorine inhalation caused severe acute respiratory failure, hypoxemia, decreased cardiac output, neuromuscular abnormalities (ataxia and hypotonia), and seizures resulting in early death. Oxygen improved survival to 6 hours (87% versus 42%) and prevented observed seizure-related deaths. However, oxygen administration worsened the severity of acute respiratory failure in chlorine-exposed rats compared with controls, with increased respiratory acidosis (pH 6.91 ± 0.04 versus 7.06 ± 0.01 at 2 h) and increased hypercapnia (180.0 ± 19.8 versus 103.2 ± 3.9 mm Hg at 2 h). In addition, oxygen did not improve neuromuscular abnormalities, cardiac output, or respiratory distress associated with chlorine exposure. Massive chlorine inhalation causes severe acute respiratory failure and multiorgan damage. Oxygen administration can improve short-term survival but appears to worsen respiratory failure, with no improvement in cardiac output or neuromuscular dysfunction. Oxygen should be used with caution after massive chlorine inhalation, and the need for early assisted ventilation should be assessed in victims.
Asunto(s)
Gasto Cardíaco/efectos de los fármacos , Sustancias para la Guerra Química/toxicidad , Cloro/toxicidad , Oxígeno/farmacología , Insuficiencia Respiratoria , Enfermedad Aguda , Animales , Hipercapnia/inducido químicamente , Hipercapnia/tratamiento farmacológico , Hipercapnia/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , Insuficiencia Respiratoria/inducido químicamente , Insuficiencia Respiratoria/tratamiento farmacológico , Insuficiencia Respiratoria/fisiopatologíaRESUMEN
Inhalation of powerful chemical agents, such as sulfur mustard (SM), can have debilitating pulmonary consequences, such as bronchiolitis obliterans (BO) and parenchymal fibrosis (PF). The underlying pathogenesis of disorders after SM inhalation is not clearly understood, resulting in a paucity of effective therapies. In this study, we evaluated the role of profibrotic pathways involving transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) in the development of BO and PF after SM inhalation injury using a rat model. Adult Sprague-Dawley rats were intubated and exposed to SM (1.0 mg/kg), then monitored daily for respiratory distress, oxygen saturation changes, and weight loss. Rats were killed at 7, 14, 21, or 28 days, and markers of injury were determined by histopathology; pulmonary function testing; and assessment of TGF-ß, PDGF, and PAI-1 concentrations. Respiratory distress developed over time after SM inhalation, with progressive hypoxemia, respiratory distress, and weight loss. Histopathology confirmed the presence of both BO and PF, and both gradually worsened with time. Pulmonary function testing demonstrated a time-dependent increase in lung resistance, as well as a decrease in lung compliance. Concentrations of TGF-ß, PDGF, and PAI-1 were elevated at 28 days in lung, BAL fluid, and/or plasma. Time-dependent development of BO and PF occurs in lungs of rats exposed to SM inhalation, and the elevated concentrations of TGF-ß, PDGF, and PAI-1 suggest involvement of these profibrotic pathways in the aberrant remodeling after injury.
Asunto(s)
Bronquiolitis Obliterante/inducido químicamente , Gas Mostaza/administración & dosificación , Gas Mostaza/toxicidad , Fibrosis Pulmonar/inducido químicamente , Administración por Inhalación , Animales , Bronquiolitis Obliterante/metabolismo , Bronquiolitis Obliterante/mortalidad , Bronquiolitis Obliterante/patología , Líquido del Lavado Bronquioalveolar , Sustancias para la Guerra Química/toxicidad , Relación Dosis-Respuesta a Droga , Inhibidor 1 de Activador Plasminogénico/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/mortalidad , Ratas Sprague-Dawley , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pruebas de Función Respiratoria , Factor de Crecimiento Transformador beta1/metabolismo , Pérdida de Peso/efectos de los fármacosRESUMEN
Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.
Asunto(s)
Células Epiteliales/citología , Pulmón/citología , Trasplante de Células Madre , Células Madre/citología , Animales , Diferenciación Celular , Linaje de la Célula , Epitelio/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Naftalenos , RatasRESUMEN
Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive.
Asunto(s)
Anticoncepción , Infertilidad Masculina/genética , Receptores Adrenérgicos alfa 1 , Receptores Purinérgicos P2X1 , Espermatozoides/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Barorreflejo , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Ratones Noqueados , Antagonistas del Receptor Purinérgico P2X/farmacología , Motilidad Espermática/fisiología , Espermatozoides/citologíaRESUMEN
Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.
Asunto(s)
Cloro/toxicidad , Cardiopatías/enzimología , Exposición por Inhalación , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis , Señalización del Calcio , Cardiotónicos/farmacología , Células Cultivadas , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacología , Cardiopatías/inducido químicamente , Masculino , Mitocondrias Cardíacas , Miocardio/enzimología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ranolazina/farmacología , Ratas Sprague-Dawley , Tiocianatos/farmacologíaRESUMEN
Currently, there is no effective antidote to prevent skin injuries by sulfur mustard (SM) and nitrogen mustard (NM), which are vesicating agents with potential relevance to chemical warfare, terrorist attacks, or industrial/laboratory accidents. Our earlier report has demonstrated the therapeutic efficacy of silibinin, a natural flavanone, in reversing monofunctional alkylating SM analog 2-chloroethyl ethyl sulfide-induced toxic effects in mouse skin. To translate this effect to a bifunctional alkylating vesicant, herein, efficacy studies were carried out with NM. Topical application of silibinin (1 or 2mg) 30 min after NM exposure on the dorsal skin of male SKH-1 hairless mice significantly decreased NM-induced toxic lesions at 24, 72 or 120 h post-exposure. Specifically, silibinin treatment resulted in dose-dependent reduction of NM-induced increase in epidermal thickness, dead and denuded epidermis, parakeratosis and microvesication. Higher silibinin dose also caused a 79% and 51%reversal in NM-induced increases in myeloperoxidase activity and COX-2 levels, respectively. Furthermore, silibinin completely prevented NM-induced H2A.X phosphorylation, indicating reversal of DNA damage which could be an oxidative DNA damage as evidenced by high levels of 8-oxodG in NM-exposed mouse skin that was significantly reversed by silibinin. Together, these findings suggest that attenuation of NM-induced skin injury by silibinin is due to its effects on the pathways associated with DNA damage, inflammation, vesication and oxidative stress. In conclusion, results presented here support the optimization of silibinin as an effective treatment of skin injury by vesicants.
Asunto(s)
Antídotos/farmacología , Irritantes/toxicidad , Mecloretamina/toxicidad , Silimarina/farmacología , Piel/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Administración Cutánea , Animales , Antídotos/administración & dosificación , Apoptosis/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Citoprotección , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relación Dosis-Respuesta a Droga , Histonas/metabolismo , Masculino , Ratones Pelados , Necrosis , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Silibina , Silimarina/administración & dosificación , Piel/metabolismo , Piel/patología , Factores de TiempoRESUMEN
BACKGROUND: Ozone pollution has adverse effects on respiratory health in children and adults. This study was carried out in the mouse model to investigate the influence of age and to define the role of toll-like receptor four (TLR4) in the lung response to ozone exposure during postnatal development. METHODS: Female mice (1 to 6 weeks of age) were exposed for 3 h to ozone (1 part per million) or filtered air. Analyses were carried out at six and 24 h after completion of exposure, to assess the effects on lung permeability, airway neutrophilia, expression of antioxidants and chemokines, and mucus production. The role of TLR4 was defined by examining TLR4 expression in the lung during development, and by investigating the response to ozone in tlr4-deficient mice. RESULTS: Metallothionein-1, calcitonin gene-related product, and chemokine C-X-C ligand (CXCL) five were consistent markers induced by ozone throughout development. Compared with adults, neonates expressed lower levels of pulmonary TLR4 and responded with increased mucus production, and developed an attenuated response to ozone characterized by reduced albumin leakage and neutrophil influx into the airways, and lower expression of CXCL1 and CXCL2 chemokines. Examination of the responses in tlr4-deficient mice indicated that ozone-mediated airway neutrophilia, but not albumin leakage or mucus production were dependent on TLR4. CONCLUSIONS: Collectively, the data demonstrate that the response to ozone is determined by age and is partially dependent on TLR4 signaling. The reduced responsiveness of the neonatal lung to ozone may be due at least in part to insufficient pulmonary TLR4 expression.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Pulmón/efectos de los fármacos , Ozono/toxicidad , Receptor Toll-Like 4/efectos de los fármacos , Factores de Edad , Animales , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Quimiocinas/metabolismo , Femenino , Exposición por Inhalación , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Ratones Endogámicos BALB C , Ratones Noqueados , Moco/metabolismo , Neuropéptidos/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Albúmina Sérica/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
AIMS: An age-related increase in prostatic smooth muscle tone is partly responsible for the lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH). Changes in the effectors of prostatic smooth muscle contraction with age may play a role in the development of these symptoms. Using a mouse model of prostate contractility, this study investigated the effect of age on the different components of contractility in the prostate gland. METHODS: The isometric force developed in response to electrical field stimulation or exogenously applied agonists by mouse prostates mounted in organ baths, was evaluated to determine the effect of age on contractile mechanisms. Changes with age in the rate of ATP breakdown and levels of the P2rx1 gene and P2X1-purinoceptor expression in mouse prostate were measured by a modified luciferin-luciferase assay, RT-PCR and western blot, respectively. RESULTS: Nerve mediated contractile responses containing a component elicited by P2X1-purinoceptors were observed in prostates taken from aged mice, but not in prostates taken from young adult mice. Furthermore, the potency of the endogenous purinoceptor agonist ATP was 50-fold greater in aged mice, whereas the potency of its stable analogue α,ß-metATP was unchanged. An age-related decrease in ATP metabolism was also observed. CONCLUSIONS: With age, a purinergic contractile response to nerve stimulation develops in the mouse prostate gland due to a decrease in the rate of ATP breakdown. This may contribute to the increase in muscular tone observed in BPH and suggests that P2X1-purinoceptors are an additional target for the treatment of BPH.
Asunto(s)
Adenosina Trifosfato/metabolismo , Contracción Muscular , Próstata/fisiología , Receptores Purinérgicos P2X1/fisiología , Factores de Edad , Animales , Masculino , Ratones , Próstata/metabolismo , Factores de TiempoRESUMEN
Acute lung injury in response to mustard gas (sulfur mustard [SM]) inhalation results in formation of fibrin casts, which obstruct the airway. The objective of this study was to identify fibrinolytic pathways that could be contributing to the persistence of airway casts after SM exposure. Rats were exposed to the SM analog, 2-chloroethyl ethyl sulfide, via nose-only aerosol inhalation. At 4 and 18 hours after exposure, animals were killed and airway-capillary leak estimated by measuring bronchoalveolar lavage fluid (BALF) protein and IgM content. The fibrin clot-degrading and plasminogen-activating capabilities of BALF were also assessed by activity assays, whereas Western blotting was used to determine the presence and activities of plasminogen activator inhibitor-1, thrombin activatable fibrinolytic inhibitor and α2-antiplasmin. Measurement of tissue-specific steady-state mRNA levels was also conducted for each fibrinolytic inhibitor to assess whether its synthesis occurs in lung or at extrapulmonary sites. The results of this study demonstrate that fibrin-degrading and plasminogen-activating capabilities of the airways become impaired during the onset of 2-chloroethyl ethyl sulfide-induced vascular leak. Findings of functionally active reservoirs of plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor, and α2-antiplasmin in BALF indicate that airway fibrinolysis is inhibited at multiple levels in response to SM.
Asunto(s)
Lesión Pulmonar Aguda/inducido químicamente , Antifibrinolíticos/toxicidad , Sustancias para la Guerra Química/toxicidad , Fibrinólisis/efectos de los fármacos , Exposición por Inhalación , Pulmón/efectos de los fármacos , Gas Mostaza/análogos & derivados , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Obstrucción de las Vías Aéreas/inducido químicamente , Obstrucción de las Vías Aéreas/metabolismo , Obstrucción de las Vías Aéreas/patología , Animales , Barrera Alveolocapilar/efectos de los fármacos , Barrera Alveolocapilar/metabolismo , Líquido del Lavado Bronquioalveolar/química , Permeabilidad Capilar/efectos de los fármacos , Carboxipeptidasa B2/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Gas Mostaza/toxicidad , Inhibidor 1 de Activador Plasminogénico/metabolismo , Ratas Sprague-Dawley , Factores de Tiempo , alfa 2-Antiplasmina/metabolismoRESUMEN
CXCL17 is a chemokine principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, and its expression is increased during viral infections of the lung. However, the exact role of CXCL17 in health and disease requires further investigation, and there is a need for confirmed molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET)-based assays, here we demonstrated that CXCL17 inhibited CXCR4-mediated signaling and ligand binding. Moreover, CXCL17 interacted with neuropillin-1, a VEGFR2 coreceptor. In addition, we found that CXCL17 only inhibited CXCR4 ligand binding in intact cells and demonstrated that this effect was mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. Disruption of putative GAG binding domains in CXCL17 prevented CXCR4 binding. This indicated that CXCL17 inhibited CXCR4 by a mechanism of action that potentially required the presence of a glycosaminoglycan-containing accessory protein. Together, our results revealed that CXCL17 is an endogenous inhibitor of CXCR4 and represents the next step in our understanding of the function of CXCL17 and regulation of CXCR4 signaling.
Asunto(s)
Quimiocinas CXC , Glicosaminoglicanos , Quimiocinas CXC/metabolismo , Glicosaminoglicanos/farmacología , Ligandos , Quimiocinas/metabolismo , Transducción de Señal , Receptores CXCR4/genética , Quimiocina CXCL12RESUMEN
Objective: Human and preclinical studies of sulfur mustard (SM)-induced acute and chronic lung injuries highlight the role of unremitting inflammation. We assessed the utility of targeting the novel DAMP and TLR4 ligand, eNAMPT (extracellular nicotinamide phosphoribosyltransferase), utilizing a humanized mAb (ALT-100) in rat models of SM exposure. Methods: Acute (SM 4.2 mg/kg, 24 hrs), subacute (SM 0.8 mg/kg, day 7), subacute (SM 2.1 mg/kg, day 14), and chronic (SM 1.2 mg/kg, day 29) SM models were utilized. Results: Each SM model exhibited significant increases in eNAMPT expression (lung homogenates) and increased levels of phosphorylated NFkB and NOX4. Lung fibrosis (Trichrome staining) was observed in both sub-acute and chronic SM models in conjunction with elevated smooth muscle actin (SMA), TGFß, and IL-1ß expression. SM-exposed rats receiving ALT-100 (1 or 4 mg/kg, weekly) exhibited increased survival, highly significant reductions in histologic/biochemical evidence of lung inflammation and fibrosis (Trichrome staining, decreased pNFkB, SMA, TGFß, NOX4), decreased airways strictures, and decreased plasma cytokine levels (eNAMPT, IL-6, IL-1ß. TNFα). Conclusion: The highly druggable, eNAMPT/TLR4 signaling pathway is a key contributor to SM-induced ROS production, inflammatory lung injury and fibrosis. The ALT-100 mAb is a potential medical countermeasure to address the unmet need to reduce SM-associated lung pathobiology/mortality.
RESUMEN
Hypoxia-inducible transcription factors HIF-1α and HIF-2α can contribute to pulmonary hypertension and vascular remodeling, but their mechanisms remain unknown. This study investigated the role of HIF-1α and HIF-2α in pulmonary artery endothelial and smooth muscle cells. The exposure of human pulmonary artery endothelial cells (HPAECs) to hypoxia (10% O2 or 5% O2) increased proliferation over 48 hours, compared with cells during normoxia (21% O2). The adenovirus-mediated overexpression of HIF-2α that is transcriptionally active during normoxia (mutHIF-2α) increased HPAEC proliferation, whereas the overexpression of HIF-1α, which is transcriptionally active during normoxia (mutHIF-1α), exerted no effect. The knockdown of HIF-2α decreased proliferation during both hypoxia and normoxia. Both HIFs increased migration toward fibrinogen, used as a chemoattractant. In an angiogenesis tube formation assay, mutHIF-2α-transduced cells demonstrated increased tube formation, compared with the mutHIF-1α-transduced cells. In addition, the tubes formed in HIF-2α-transduced cells were more enduring than those in the other groups. In human pulmonary artery smooth muscle cells (HPASMCs), chronic exposure to hypoxia increased proliferation, compared with cells during normoxia. For HPASMCs transduced with adenoviral HIFs, HIF-1α increased proliferation, whereas HIF-2α exerted no such effect. Thus, HIF-1α and HIF-2α exert differential effects in isolated cells of the human pulmonary vasculature. This study demonstrates that HIF-2α plays a predominant role in the endothelial growth pertinent to the remodeling process. In contrast, HIF-1α appears to play a major role in pulmonary smooth muscle growth. The selective targeting of each HIF in specific target cells may more effectively counteract hypoxic pulmonary hypertension and vascular remodeling.