RESUMEN
The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor S1PR3 are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African- and ED samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Because of limited sample size, conclusive findings could not be drawn in African-descent ARDS subjects; however, significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two ED samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter; reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge; and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.
Asunto(s)
Regiones Promotoras Genéticas , Receptores de Lisoesfingolípidos/genética , Síndrome de Dificultad Respiratoria/genética , Sepsis/complicaciones , Secuencia de Bases , Biomarcadores/sangre , Estudios de Casos y Controles , Ensayo de Cambio de Movilidad Electroforética , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Lisofosfolípidos/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Receptores de Lisoesfingolípidos/sangre , Síndrome de Dificultad Respiratoria/sangre , Síndrome de Dificultad Respiratoria/etiología , Análisis de Secuencia de ADN , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Rayos gamma/efectos adversos , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/prevención & control , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Envejecimiento/efectos de la radiación , Animales , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Peróxido de Hidrógeno/efectos adversos , Peróxido de Hidrógeno/farmacología , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/farmacología , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/genética , Oxidantes/efectos adversos , Oxidantes/farmacología , Traumatismos Experimentales por Radiación/genética , Traumatismos Experimentales por Radiación/patología , Traumatismos Experimentales por Radiación/prevención & controlRESUMEN
We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.
Asunto(s)
Cortactina/metabolismo , Células Endoteliales/enzimología , Hiperoxia/enzimología , Pulmón/enzimología , Quinasa de Cadena Ligera de Miosina/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Cortactina/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Activación Enzimática , Humanos , Hiperoxia/genética , Hiperoxia/metabolismo , Pulmón/citología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasa de Cadena Ligera de Miosina/genética , NADPH Oxidasas/genética , Unión ProteicaRESUMEN
Reactive oxygen species (ROS) generated by vascular endothelial and smooth muscle cells contribute to the development and progression of vascular diseases. We have recently shown that hyperoxia enhances NADPH oxidase 4 (Nox4) expression, which regulates lung endothelial cell migration and angiogenesis. Regulation of Nox4 in the vasculature is poorly understood. The objective of this study was to identify the transcriptional factor(s) involved in regulation of endothelial Nox4. We found that hyperoxia-induced Nox4 expression was markedly reduced in Nrf2(-/-) mice, compared to Nrf2(+/+) mice. Exposure of human lung microvascular endothelial cells (HLMVECs) to hyperoxia stimulated Nrf2 translocation from the cytoplasm to the nucleus and increased Nox4 expression. Knockdown of Nrf2 expression using an siRNA approach attenuated basal Nox4 expression; however, it enhanced superoxide/ROS generation under both normoxia and hyperoxia. In silico analysis revealed the presence of at least three consensus sequences for the antioxidant response element (ARE) in the promoter region of Nox4. In transient transfections, hyperoxia stimulated Nox4 promoter activity in HLMVECs, and deletion of the -438 to -458 and -619 to -636 sequences markedly reduced hyperoxia-stimulated Nox4 promoter activation. ChIP analysis revealed an enhanced recruitment of Nrf2 to the endogenous Nox4 promoter spanning these two AREs after hyperoxic insult. Collectively, these results demonstrate, for the first time, a novel role for Nrf2 in regulating hyperoxia-induced Nox4 transcription via AREs in lung endothelium.
Asunto(s)
Endotelio Vascular/metabolismo , Hiperoxia/genética , Pulmón/metabolismo , NADPH Oxidasas/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Elementos de Respuesta/genética , Animales , Movimiento Celular , Células Cultivadas , Células Endoteliales/metabolismo , Radicales Libres/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Pulmón/citología , Ratones , Ratones Noqueados , NADPH Oxidasa 4 , NADPH Oxidasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
Adipose-specific inactivation of both AP-1 and CCAAT-enhancer-binding protein (C/EBP) families of B-ZIP transcription factors in transgenic mice causes severe lipoatrophy. To evaluate whether inactivation of only C/EBP members was critical for lipoatrophy, A-C/EBP, a dominant-negative protein that specifically inhibits the DNA binding of the C/EBP members, was expressed in adipose tissue. For the first 2 weeks after birth, aP2-A-C/EBP mice had no white adipose tissue (WAT), drastically reduced brown adipose tissue (BAT), and exhibited marked hepatic steatosis, hyperinsulinemia, and hyperlipidemia. However, WAT appeared during the third week, coinciding with significantly improved metabolic functioning. In adults, BAT remained reduced, causing cold intolerance. At 30 weeks, the aP2-A-C/EBP mice had only 35% reduced WAT, with clear morphological signs of lipodystrophy in subcutaneous fat. Circulating leptin and adiponectin levels were less than the wild-type levels, and these mice exhibited impaired triglyceride clearance. Insulin resistance, glucose intolerance, and reduced free fatty acid release in response to ß3-adrenergic agonist suggest improper functioning of the residual WAT. Gene expression analysis of inguinal WAT identified reduced mRNA levels of several enzymes involved in fatty acid synthesis and glucose metabolism that are known C/EBPα transcriptional targets. There were increased levels for genes involved in inflammation and muscle differentiation. However, when dermal fibroblasts from aP2-A-C/EBP mice were differentiated into adipocytes in tissue culture, muscle markers were elevated more than the inflammatory markers. These results demonstrate that the C/EBP family is essential for adipose tissue development during the early postnatal period, the regulation of glucose and lipid homeostasis in adults, and the suppression of the muscle lineage.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Lipodistrofia/etiología , Lipodistrofia/metabolismo , Proteínas/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Western Blotting , Composición Corporal/genética , Composición Corporal/fisiología , Proteínas Potenciadoras de Unión a CCAAT/genética , Calorimetría Indirecta , Células Cultivadas , Ingestión de Alimentos/genética , Ingestión de Alimentos/fisiología , Proteínas de Unión a Ácidos Grasos/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Lipodistrofia/genética , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Proteínas/genética , Triglicéridos/metabolismoRESUMEN
Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non-muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (~50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody-conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (â¼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (â¼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6-signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Terapia Genética/métodos , Pulmón/efectos de los fármacos , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Lesión Pulmonar Inducida por Ventilación Mecánica/prevención & control , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/enzimología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/inmunología , Animales , Anticuerpos , Líquido del Lavado Bronquioalveolar/inmunología , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Inyecciones Intravenosas , Lipopolisacáridos , Liposomas , Pulmón/irrigación sanguínea , Pulmón/enzimología , Pulmón/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/deficiencia , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Peptidil-Dipeptidasa A/inmunología , Fosforilación , Inhibidores de Proteínas Quinasas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Transducción de Señal/genética , Factores de Tiempo , Lesión Pulmonar Inducida por Ventilación Mecánica/enzimología , Lesión Pulmonar Inducida por Ventilación Mecánica/genética , Lesión Pulmonar Inducida por Ventilación Mecánica/inmunologíaRESUMEN
BACKGROUND: The genetic mechanisms underlying asthma remain unclear. Increased permeability of the microvasculature is a feature of asthma, and the sphingosine-1-phosphate receptor (S1PR1) is an essential participant regulating lung vascular integrity and responses to lung inflammation. OBJECTIVE: We explored the contribution of polymorphisms in the S1PR1 gene to asthma susceptibility. METHODS: A combination of gene resequencing for single nucleotide polymorphism (SNP) discovery, case-control association, functional evaluation of associated SNPs, and protein immunochemistry studies was used. RESULTS: Immunohistochemistry studies demonstrated significantly decreased S1PR1 protein expression in pulmonary vessels in lungs of asthmatic patients compared with those of nonasthmatic subjects (P < .05). Direct DNA sequencing of 27 multiethnic samples identified 39 S1PR1 variants (18 novel SNPs). Association studies were performed based on genotyping results from cosmopolitan tagging SNPs in 3 case-control cohorts from Chicago and New York totaling 1,061 subjects (502 cases and 559 control subjects). The promoter SNP rs2038366 (-1557G/T) was found to be associated with asthma (P = .03) in European Americans. In African Americans an association was found for both asthma and severe asthma for intronic SNP rs3753194 (c.-164+170A/G; P = .006 and P = .040, respectively) and for promoter SNP rs59317557 (-532C/G) with severe asthma (P = .028). Consistent with predicted in silico functionality, alleles of the promoter SNPs rs2038366 (-1557G/T) and rs59317557 (-532C/G) influenced the activity of a luciferase S1PR1 reporter vector in transfected endothelial cells exposed to growth factors (epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor) known to be increased in asthmatic airways. CONCLUSION: These data provide strong support for a role for S1PR1 gene variants in asthma susceptibility and severity.
Asunto(s)
Alelos , Asma/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Receptores de Lisoesfingolípidos/genética , Negro o Afroamericano/genética , Asma/epidemiología , Asma/metabolismo , Asma/patología , Estudios de Casos y Controles , Línea Celular , Chicago , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/etnología , Genotipo , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/farmacología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ciudad de Nueva York , Receptores de Lisoesfingolípidos/biosíntesis , Índice de Severidad de la Enfermedad , Receptores de Esfingosina-1-Fosfato , Transfección , Población Blanca/genéticaRESUMEN
Vascular barrier regulation is intimately linked to alterations in the distribution and configuration of the endothelial cell (EC) cytoskeleton in response to angiogenic and edemagenic agonists. Critical actin cytoskeletal rearrangement includes spatially directed increases in myosin light chain (MLC) phosphorylation, catalyzed by Ca(2+)/calmodulin-dependent non-muscle myosin light chain kinase variants (nmMLCK1- and -2), as well as association of nmMLCK with the actin-binding protein, cortactin. As these associations have proven difficult to quantify in a spatially specific manner, we now describe the utility of intensity correlation image analysis and the intensity correlation quotient (ICQ) to quantify colocalization in fixed and live cell imaging assays in human pulmonary artery EC. From baseline ICQ values averaging 0.216 reflecting colocalization of cortactin-DsRed with EGFP-nmMLCK fusion proteins in resting EC, thrombin-induced EC contraction significantly reduced cortactin-DsRed-EGFP-nmMLCK colocalization (nmMLCK1: ICQ=0.118; nmMLCK2: ICQ=0.091) whereas the potent EC barrier-protective agonist, sphingosine 1-phosphate (S1P), significantly increased nmMLCK-cortactin colocalization within lamellipodia (nmMLCK1: ICQ=0.275; nmMLCK2: ICQ=0.334). Over-expression of a cortactin-DsRed mutant fusion protein lacking the SH3 domain, known to be essential for cortactin-nmMLCK association, reduced baseline and S1P-mediated live cell colocalization with each nmMLCK variant (nmMLCK1: ICQ=0.160; nmMLCK2: ICQ=0.157). Similarly, expression of a truncated EGFP-nmMLCK2 mutant lacking cortactin- and actin-binding domains, markedly reduced basal localization in lamellipodia and abolished colocalization with cortactin-DsRed in lamellipodia after S1P (ICQ=-0.148). These data provide insights into the molecular basis for vascular barrier-regulatory cytoskeletal responses and support the utility of sophisticated imaging analyses and methodological assessment to quantify the critical nmMLCK and cortactin interaction during vascular barrier regulation.
Asunto(s)
Cortactina/metabolismo , Células Endoteliales/metabolismo , Isoenzimas/metabolismo , Pulmón/citología , Quinasa de Cadena Ligera de Miosina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Células Cultivadas , Cortactina/genética , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Humanos , Isoenzimas/genética , Proteínas Luminiscentes/genética , Lisofosfolípidos/farmacología , Quinasa de Cadena Ligera de Miosina/genética , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Seudópodos/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia/genética , Esfingosina/análogos & derivados , Esfingosina/farmacología , Trombina/farmacología , Transfección , Dominios Homologos src/genéticaRESUMEN
The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1-phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR(1), we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR(1)) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar-capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR(1), intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar-capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR(1) regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR(1) inverse agonist, SB-649146, or S1PR(1)(+/-) mice exhibited reduced S1P/SEW-2871-mediated barrier protection after challenge with LPS. In contrast, S1PR(2)(-/-) knockout mice as well as mice with reduced S1PR(3) expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR(1) receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR(1) agonist concentration, and S1PR(1) expression in target tissues.
Asunto(s)
Barrera Alveolocapilar/fisiopatología , Pulmón/irrigación sanguínea , Pulmón/fisiopatología , Receptores de Lisoesfingolípidos/metabolismo , Lesión Pulmonar Aguda/fisiopatología , Animales , Barrera Alveolocapilar/efectos de los fármacos , Líquidos Corporales , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Agonismo Inverso de Drogas , Eliminación de Gen , Silenciador del Gen/efectos de los fármacos , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxadiazoles/agonistas , Oxadiazoles/farmacología , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Tiofenos/agonistas , Tiofenos/farmacologíaRESUMEN
Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.
Asunto(s)
Caveolina 1/metabolismo , Dinamina II/metabolismo , Células Endoteliales , Microdominios de Membrana , NADPH Oxidasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Líquido del Lavado Bronquioalveolar/citología , Caveolina 1/genética , Células Cultivadas , Dinamina II/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Activación Enzimática , Humanos , Hiperoxia/metabolismo , Pulmón/citología , Pulmón/metabolismo , Masculino , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasas/genética , Proteínas Proto-Oncogénicas c-abl/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
We used a double transgenic tetracycline system to conditionally express A-CREB, a dominant negative protein that prevents the DNA binding and function of cAMP-responsive element binding protein (CREB) family members, in mouse basal epidermis using the keratin 5 promoter. There was no phenotype in the adult. However, following a 7,12-dimethylbenz(a)anthracene (DMBA)/phorbol-12-myristate-13-acetate two-stage skin carcinogenesis experiment, A-CREB-expressing epidermis develop 5-fold fewer papillomas than wild-type controls. However, A-CREB expression one month after DMBA treatment does not prevent papilloma formation, suggesting that CREB functions at an early stage of papilloma formation. Oncogenic H-Ras genes with A-->T mutations in codon 61 were found in wild-type skin but not in A-CREB-expressing skin 2 days after DMBA treatment, suggesting that A-CREB either prevents DMBA mutagenesis or kills oncogenic H-Ras cells. In primary keratinocyte cultures, A-CREB expression induced apoptosis of v-Ras(Ha)-infected cells and suppressed the expression of cell cycle proteins cyclin B1 and cyclin D1. These results suggest that inhibiting CREB function is a valuable cancer prevention strategy.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Epidermis/metabolismo , Papiloma/metabolismo , Neoplasias Cutáneas/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Western Blotting , Carcinógenos/toxicidad , Ciclo Celular/fisiología , Proliferación Celular , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Epidermis/efectos de los fármacos , Epidermis/patología , Femenino , Genes ras/genética , Etiquetado Corte-Fin in Situ , Queratinocitos/citología , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Mutación , Papiloma/inducido químicamente , Papiloma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
We explored the mechanistic involvement of the growth arrest and DNA damage-inducible gene GADD45a in lipopolysaccharide (LPS)- and ventilator-induced inflammatory lung injury (VILI). Multiple biochemical and genomic parameters of inflammatory lung injury indicated that GADD45a(-/-) mice are modestly susceptible to intratracheal LPS-induced lung injury and profoundly susceptible to high tidal volume VILI, with increases in microvascular permeability and bronchoalveolar lavage levels of inflammatory cytokines. Expression profiling of lung tissues from VILI-challenged GADD45a(-/-) mice revealed strong dysregulation in the B-cell receptor signaling pathway compared with wild-type mice and suggested the involvement of PI3 kinase/Akt signaling components. Western blot analyses of lung homogenates confirmed approximately 50% reduction in Akt protein levels in GADD45a(-/-) mice accompanied by marked increases in Akt ubiquitination. Electrical resistance measurements across human lung endothelial cell monolayers with either reduced GADD45a or Akt expression (siRNAs) revealed significant potentiation of LPS-induced human lung endothelial barrier dysfunction, which was attenuated by overexpression of a constitutively active Akt1 transgene. These studies validate GADD45a as a novel candidate gene in inflammatory lung injury and a significant participant in vascular barrier regulation via effects on Akt-mediated endothelial signaling.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/fisiología , Transducción de Señal/fisiología , Lesión Pulmonar Inducida por Ventilación Mecánica/fisiopatologíaRESUMEN
RATIONALE: We previously demonstrated pre-B-cell colony enhancing factor (PBEF) as a biomarker in sepsis and sepsis-induced acute lung injury (ALI) with genetic variants conferring ALI susceptibility. OBJECTIVES: To explore mechanistic participation of PBEF in ALI and ventilator-induced lung injury (VILI). METHODS: Two models of VILI were utilized to explore the role of PBEF using either recombinant PBEF or PBEF(+/-) mice. MEASUREMENTS AND MAIN RESULTS: Initial in vitro studies demonstrated recombinant human PBEF (rhPBEF) as a direct rat neutrophil chemotactic factor with in vivo studies demonstrating marked increases in bronchoalveolar lavage (BAL) leukocytes (PMNs) after intratracheal injection in C57BL/6J mice. These changes were accompanied by increased BAL levels of PMN chemoattractants (KC and MIP-2) and modest increases in lung vascular and alveolar permeability. We next explored the potential synergism between rhPBEF challenge (intratracheal) and a model of limited VILI (4 h, 30 ml/kg tidal volume) and observed dramatic increases in BAL PMNs, BAL protein, and cytokine levels (IL-6, TNF-alpha, KC) compared with either challenge alone. Gene expression profiling identified induction of ALI- and VILI-associated gene modules (nuclear factor-kappaB, leukocyte extravasation, apoptosis, Toll receptor pathways). Heterozygous PBEF(+/-) mice were significantly protected (reduced BAL protein, BAL IL-6 levels, peak inspiratory pressures) when exposed to a model of severe VILI (4 h, 40 ml/kg tidal volume) and exhibited significantly reduced expression of VILI-associated gene expression modules. Finally, strategies to reduce PBEF availability (neutralizing antibody) resulted in significant protection from VILI. CONCLUSIONS: These studies implicate PBEF as a key inflammatory mediator intimately involved in both the development and severity of ventilator-induced ALI.
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Citocinas/fisiología , Nicotinamida Fosforribosiltransferasa/fisiología , Síndrome de Dificultad Respiratoria/fisiopatología , Ventiladores Mecánicos , Animales , Líquido del Lavado Bronquioalveolar , Quimiocina CXCL1/metabolismo , Quimiocina CXCL2/metabolismo , Quimiotaxis de Leucocito/fisiología , Perfilación de la Expresión Génica , Interleucina-6/análisis , Macrófagos Alveolares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Síndrome de Dificultad Respiratoria/etiología , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Ventiladores Mecánicos/efectos adversosRESUMEN
We have generated genetically engineered mice that are uniquely susceptible to lipopolysaccharide (LPS)-induced and mechanical ventilation-induced lung injury in a sex-specific and age-specific manner. These mice express a nonmuscle isoform of the myosin light chain kinase gene (nmMLCK2) targeted to the endothelium. Homozygous mice have significantly reduced fecundity and litter survival until weaning, and they are initially growth delayed but eventually exceed the size of wild-type littermates. Mice at all ages show increased protein transport across the lung barrier; however, the phenotype is most discernible in 8-12-week-old male mice. When subjected to a clinically relevant LPS-induced lung injury model, 8-12-week-old young females and 30-36-week-old males seem to be the most significantly injured group. In contrast, 30-36-week-old males remain the most significantly injured group when mechanically ventilated at high tidal volumes, which is a clinically relevant model of mechanical stress lung injury. These data reveal that nmMLCK2 overexpression in the endothelium exacerbates lung injury in vivo in a sexually dimorphic and age-dependent manner.
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Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Neumonía/metabolismo , Factores de Edad , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Susceptibilidad a Enfermedades/metabolismo , Endotelio Vascular/patología , Femenino , Expresión Génica , Homocigoto , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Quinasa de Cadena Ligera de Miosina/genética , Neumonía/inducido químicamente , Neumonía/genética , Respiración Artificial/efectos adversos , Caracteres Sexuales , Estrés MecánicoRESUMEN
Quantifying the amount of albumin conjugated to Evans Blue dye (EBA) fluxing across organ-specific vascular barriers is a popular technique to measure endothelial monolayer integrity in rodent and murine models of human diseases. We have re-evaluated this technique with a specific focus of assessing the commonly used turbidity correction factors. These factors, originally developed and required in a spectrophotometric assay to quantify Evans Blue (EB) in human infant or dog serum, produced negative numbers when applied to murine models of acute lung injury. We next sought to determine tissue-specific correction factors for murine tissues and experimentally derived such factors, which allow estimation of the amount of EB in formamide extracts of murine tissues as positive numbers. Utilization of a best fit correction factor in a lipopolysaccharide (LPS)-induced murine model of acute lung injury resulted in significantly increased sensitivity and repeatability of the EB dye tissue extravasation assay. This factor may be of significant utility in animal models of inflammatory injury.
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Permeabilidad Capilar , Colorantes/análisis , Azul de Evans/análisis , Pulmón/patología , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/patología , Albúmina Sérica/farmacocinética , Animales , Biomarcadores/análisis , Centrifugación/instrumentación , Centrifugación/métodos , Modelos Animales de Enfermedad , Extravasación de Materiales Terapéuticos y Diagnósticos/metabolismo , Hipergravedad , Lipopolisacáridos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Síndrome de Dificultad Respiratoria/inducido químicamente , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodos , Distribución TisularRESUMEN
The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.
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Permeabilidad Capilar , Cortactina/metabolismo , Dinamina II/metabolismo , Células Endoteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Receptores de Hialuranos/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Síndrome de Dificultad Respiratoria/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Anticuerpos/farmacología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Permeabilidad Capilar/genética , Cortactina/genética , Modelos Animales de Enfermedad , Dinamina II/genética , Células Endoteliales/patología , Factores de Intercambio de Guanina Nucleótido/genética , Factor de Crecimiento de Hepatocito/genética , Homeostasis/genética , Humanos , Receptores de Hialuranos/genética , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Noqueados , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neuropéptidos/genética , Neuropéptidos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/patología , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, the MIF gene and protein expression were examined in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared with healthy controls (African- and European-descent). The association of 8 MIF gene polymorphisms (single-nucleotide polymorphisms (SNPs)) (within a 9.7-kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European-descent and African-descent populations was studied next. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3' end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI.
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Biomarcadores/sangre , Oxidorreductasas Intramoleculares/sangre , Oxidorreductasas Intramoleculares/genética , Factores Inhibidores de la Migración de Macrófagos/sangre , Factores Inhibidores de la Migración de Macrófagos/genética , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/metabolismo , Adulto , Negro o Afroamericano/genética , Anciano , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Estudios de Casos y Controles , Femenino , Haplotipos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Síndrome de Dificultad Respiratoria/etnología , Factores de Riesgo , Población Blanca/genéticaRESUMEN
Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALI), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and Dahl salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 ml/kg, 85 breaths/min, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as "transcription" and "chemotaxis/cell motility." We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI.
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Modelos Animales de Enfermedad , Genómica/métodos , Respiración Artificial/efectos adversos , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/fisiopatología , Animales , Biología Computacional , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Dahl , Ratas Sprague-Dawley , Síndrome de Dificultad Respiratoria/etiología , Especificidad de la EspecieRESUMEN
To examine the consequences of inhibiting activator protein-1 (AP-1) transcription factors in skin, transgenic mice were generated, which use the tetracycline system to conditionally express A-FOS, a dominant negative that inhibits AP-1 DNA binding. Older mice develop mild alopecia and hyperplasia of sebaceous glands, particularly around the eyes. When A-FOS was expressed during chemical-induced skin carcinogenesis, mice do not develop characteristic benign and malignant squamous lesions but instead develop benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity after tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, reactivating AP-1 in sebaceous tumors results in a reciprocal transdifferentiation into squamous tumors. In both cases of transdifferentiation, individual cells express molecular markers for both cell types, indicating individual tumor cells have the capacity to express multiple lineages. Molecular characterization of cultured keratinocytes and tumor material indicates that AP-1 regulates the balance between the wnt/beta-catenin and hedgehog signaling pathways that determine squamous and sebaceous lineages, respectively. Chromatin immunoprecipitation analysis indicates that c-Jun binds several wnt promoters, which are misregulated by A-FOS expression, suggesting that members of the wnt pathway can be a primary targets of AP-1 transcriptional regulation. Thus, AP-1 activity regulates tumor cell lineage and is essential to maintain the squamous tumor cell identity.
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Adenocarcinoma Sebáceo/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Cutáneas/metabolismo , Factor de Transcripción AP-1/metabolismo , Adenocarcinoma Sebáceo/patología , Animales , Carcinoma de Células Escamosas/patología , ADN de Neoplasias/metabolismo , Hiperplasia , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Glándulas Sebáceas/patología , Neoplasias Cutáneas/patología , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/genética , Activación Transcripcional , Proteínas Wnt/genéticaRESUMEN
Epidemiologic studies show a positive association between obesity and cancer risk. In addition to increased body adiposity and secretion of fat-derived hormones, obesity is also linked to insulin resistance, type 2 diabetes, and chronic inflammation. We used the fatless A-ZIP/F-1 transgenic mouse to dissociate the relative role of each of these underlying factors in the development of cancer. These mice are unique in that they do not have white fat but do develop type 2 diabetes. In two cancer models, the classic two-stage skin carcinogenesis protocol and the C3(1)/T-Ag transgenic mouse mammary tumor model, A-ZIP/F-1 mice displayed higher tumor incidence, tumor multiplicity, and decreased tumor latency than wild-type mice. We examined circulating levels of adipokines, growth factors, and cytokines. As expected, adipokines (i.e., leptin, adiponectin, and resistin) were undetectable or found at very low levels in the blood of fatless mice. However, insulin, insulin-like growth factor-I, growth hormone, vascular endothelial growth factor, and proinflammatory Th2 cytokines, such as interleukin (IL)-1beta, IL-4, and IL-6, were elevated in A-ZIP/F-1 mice. Additionally, we examined multiple phosphorylated proteins (i.e., protein kinase B/Akt and ErbB2/HER-2 kinase) associated with cancer development. Results show that many of these phosphorylated proteins were activated specifically in the A-ZIP/F-1 skin but not in the wild-type skin. These findings suggest that adipokines are not required for the promotion of tumor development and thus contradict the epidemiologic data linking obesity to carcinogenesis. We postulate that insulin resistance and inflammation are responsible for the positive correlation with cancer observed in A-ZIP/F-1 mice.