RESUMEN
Carbon monoxide (CO) provides protection against oxidative stress via anti-inflammatory and cytoprotective actions. In this study, we tested the hypothesis that a low concentration of exogenous (inhaled) CO would protect transplanted lung grafts from cold ischemia-reperfusion injury via a mechanism involving the mitogen-activated protein kinase (MAPK) signaling pathway. Lewis rats underwent orthotopic syngeneic or allogeneic left lung transplantation with 6 h of cold static preservation. Exposure of donors and recipients (1 h before and then continuously post-transplant) to 250 ppm CO resulted in significant improvement in gas exchange, reduced leukocyte sequestration, preservation of parenchymal and endothelial cell ultrastructure and reduced inflammation compared to animals exposed to air. The beneficial effects of CO were associated with p38 MAPK phosphorylation and were significantly prevented by treatment with a p38 MAPK inhibitor, suggesting that CO's efficacy is at least partially mediated by activation of p38 MAPK. Furthermore, CO markedly suppressed inflammatory events in the contralateral naïve lung. This study demonstrates that perioperative exposure of donors and recipients to CO at a low concentration can impart potent anti-inflammatory and cytoprotective effects in a clinically relevant model of lung transplantation and support further evaluation for potential clinical use.
Asunto(s)
Monóxido de Carbono/uso terapéutico , Trasplante de Pulmón/fisiología , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Daño por Reperfusión/prevención & control , Animales , Antiinflamatorios/uso terapéutico , Ciclooxigenasa 2/genética , Interleucinas/genética , Pulmón/ultraestructura , Masculino , Neutrófilos/fisiología , Óxido Nítrico Sintasa de Tipo II/genética , ARN Mensajero/genética , Ratas , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Homólogo , Trasplante IsogénicoRESUMEN
Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1-5%) (CO-UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO-UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO-UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO-UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO-UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO-UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant-induced I/R injury.
Asunto(s)
Antimetabolitos/farmacología , Monóxido de Carbono/farmacología , Intestino Delgado/irrigación sanguínea , Intestino Delgado/trasplante , Soluciones Preservantes de Órganos/farmacología , Trasplante de Órganos/efectos adversos , Daño por Reperfusión/prevención & control , Adenosina/química , Adenosina/farmacocinética , Adenosina/farmacología , Alopurinol/química , Alopurinol/farmacocinética , Alopurinol/farmacología , Animales , Antimetabolitos/análisis , Antimetabolitos/farmacocinética , Monóxido de Carbono/análisis , Monóxido de Carbono/farmacocinética , Modelos Animales de Enfermedad , Glutatión/química , Glutatión/farmacocinética , Glutatión/farmacología , Supervivencia de Injerto/efectos de los fármacos , Insulina/química , Insulina/farmacocinética , Insulina/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructura , Intestino Delgado/metabolismo , Masculino , Microscopía Electrónica de Transmisión , Preservación de Órganos , Soluciones Preservantes de Órganos/química , Soluciones Preservantes de Órganos/farmacocinética , Rafinosa/química , Rafinosa/farmacocinética , Rafinosa/farmacología , Ratas , Ratas Endogámicas Lew , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Resultado del TratamientoRESUMEN
We utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human pulmonary artery endothelial cells (HPAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HPAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same-passage cells cultured under standard (5% CO2-95% air) conditions. Hierarchical clustering of significant hypoxia-responsive genes identified temporal changes in the expressions of a number of well-described gene families including those encoding proteins involved in thrombosis, stress response, apoptosis, angiogenesis, and cell proliferation. These experiments build on previously published data describing the transcriptomic response of human aortic endothelial cells (HAECs) obtained from the same donor and cultured under identical conditions, and we have thus taken advantage of the immortality of SAGE data to make direct comparisons between these two data sets. This approach revealed comprehensive information relating to the similarities and differences at the level of mRNA expression between HAECs and HPAECs. For example, we found differences in the cell type-specific response to hypoxia among genes encoding cytoskeletal factors, including paxillin, and proteins involved in metabolic energy production, the response to oxidative stress, and vasoreactivity (e.g., endothelin-1). These efforts contribute to the expanding collection of publicly available SAGE data and provide a foundation on which to base further efforts to understand the characteristics of the vascular response to hypoxia in the pulmonary circulation relative to systemic vasculature.
Asunto(s)
Aorta/citología , Células Endoteliales/citología , Perfilación de la Expresión Génica/métodos , Hipoxia , Pulmón/citología , Aorta/patología , Células Cultivadas , Análisis por Conglomerados , Etiquetas de Secuencia Expresada , Femenino , Humanos , Pulmón/patología , Persona de Mediana Edad , Factores de Tiempo , Transcripción GenéticaRESUMEN
The stress-inducible protein heme oxygenase-1 exerts potent antiinflammatory, antiapoptotic and cytoprotective effects in vitro and in vivo. Another important mediator of cytoprotection, the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway activates many proteins involved in the maintenance of cellular homeostasis. Since activation of heme oxygenase-1 and PI3K/Akt both protect the cellular environment, we postulated that PI3K/Akt can regulate the induction of heme oxygenase-1 by proinflammatory stress. The treatment of primary murine macrophage cells (RAW 264.7) with lipopolysaccharide induced heme oxygenase-1 protein and mRNA expression, and increased the phosphorylation of Akt and p38 mitogen activated protein kinase (p38 MAPK). These cellular effects of lipopolysaccharide were markedly diminished by pre-treatment with wortmannin, a specific inhibitor of PI3K. Furthermore, lipopolysaccharide-inducible heme oxygenase expression was blocked by SB203580, a specific inhibitor of p38 MAPK. Both wortmannin and SB203580 decreased lipopolysaccharide-inducible NF-E2-related factor (Nrf2) DNA binding activity. Transfection of macrophages with dominant negative mutants of PI3K, Akt and Nrf2, as well as wortmannin treatment, significantly reduced the transcriptional activity of a minimal heme oxygenase-1 promoter luciferase construct (D33HO-1luc). We demonstrate, to our knowledge for the first time, that upon proinflammatory stimulation heme oxygenase-1 gene expression in macrophages depends on PI3K/Akt and p38 MAPK acting upstream of Nrf2-dependent promoter activation.
Asunto(s)
Hemo-Oxigenasa 1/efectos de los fármacos , Lipopolisacáridos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Inflamación/metabolismo , Macrófagos , Ratones , Factor 2 Relacionado con NF-E2/genética , Fosfatidilinositol 3-Quinasas/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/genética , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We have utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human aortic endothelial cells (HAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same passage cells cultured under standard (5% CO2-95% air) conditions. A total of 121,446 tags representing 37,096 unique tags were sequenced and genes whose expression levels were modulated by hypoxia identified by novel statistical analyses. Hierarchical clustering of genes displaying statistically significant hypoxia-responsive alterations in expression revealed temporal modulation of a number of major functional gene families including those encoding heat shock factors, glycolytic enzymes, extracellular matrix factors, cytoskeletal factors, apoptotic factors, cell cycle regulators and angiogenic factors. Within these families we documented the coordinated modulation of both previously known hypoxia-responsive genes, numerous genes whose expressions have not been previously shown to be altered by hypoxia, tags matching uncharacterized UniGene entries and entirely novel tags with no UniGene match. These preliminary data, which indicate a reduction in cell cycle progression, elevated metabolic stress and increased cytoskeletal remodeling under acute hypoxic stress, provide a foundation for further analyses of the molecular mechanisms underlying the endothelial response to short-term chronic hypoxia.
Asunto(s)
Hipoxia de la Célula/genética , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Transcripción Genética , Aorta , Apoptosis/genética , Células Cultivadas/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , ADN Complementario/genética , Metabolismo Energético/genética , Etiquetas de Secuencia Expresada , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Femenino , Biblioteca de Genes , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnica de SustracciónAsunto(s)
Hemo Oxigenasa (Desciclizante)/análisis , Macrófagos Alveolares/enzimología , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Líquido del Lavado Bronquioalveolar/química , Estudios de Casos y Controles , Recuento de Células , Hemo-Oxigenasa 1 , Humanos , Pulmón/enzimología , Proteínas de la Membrana , Persona de Mediana Edad , Fumar/patologíaRESUMEN
The acute inflammatory response elicited by adenovirus vectors results in loss of gene expression and tissue injury in the target organ. This acute inflammation is now believed to be the major limiting factor for the use of adenovirus vectors in gene therapy. While exploring the level of acute inflammation caused by the adenovirus encoding the gene for the anti-inflammatory enzyme heme oxygenase-1, we discovered that this adenovirus not only did not elicit acute inflammation, but could prevent the inflammation caused by a second adenovirus. Here we describe a new approach to gene therapy, which uses the encoding of the potent anti-inflammatory enzyme heme oxygenase-1 to prevent early host inflammatory responses normally associated with adenovirus vectors.
Asunto(s)
Adenoviridae/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Hemo Oxigenasa (Desciclizante)/genética , Hepatitis/prevención & control , Transfección/métodos , Enfermedad Aguda , Adenoviridae/inmunología , Animales , Vectores Genéticos/inmunología , Hemo-Oxigenasa 1 , Hepatitis/inmunología , Hígado/inmunología , Hígado/virología , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Microscopía por Video , Microsomas Hepáticos/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Intestinal transplantation provokes an intense inflammatory response within the graft muscularis that causes intestinal ileus. We hypothesised that endogenously produced anti-inflammatory substances could be utilised as novel therapeutics. Therefore, we tested the protective effects of inhaled carbon monoxide (CO) and an endogenous haeme oxygenase 1 (HO-1) anti-inflammatory mediator on transplant induced inflammatory responses and intestinal ileus in the rat. METHODS: Gastrointestinal transit of non-absorbable FITC labelled dextran and in vitro jejunal circular muscle contractions were measured in controls and syngeneic orthotopic transplanted animals with and without CO inhalation (250 ppm for 25 hours). Inflammatory mRNAs for interleukin (IL)-6, IL-1beta, tumour necrosis factor alpha (TNF-alpha), intercellular adhesion molecule 1 (ICAM-1), inducible nitric oxide (iNOS), cyclooxygenase 2 (COX-2), and IL-10 were quantified by real time reverse transcriptase-polymerase chain reaction and HO-1 by northern blot. Histochemical stains characterised neutrophil infiltration and enterocyte apoptosis. RESULTS: Transplantation delayed transit and suppressed jejunal circular muscle contractility. Transplantation induced dysmotility was significantly improved by CO inhalation. Transplantation initiated a significant upregulation in IL-6, IL-1beta, TNF-alpha, ICAM-1, iNOS, COX-2, and HO-1 mRNAs with the graft muscularis. CO inhalation significantly decreased expression of IL-6, IL-1beta, iNOS, and COX-2 mRNAs. CO also significantly decreased serum nitrite levels (iNOS activity). CONCLUSIONS: CO inhalation significantly improved post-transplant motility and attenuated the inflammatory cytokine milieu in the syngeneic rat transplant model. Thus clinically providing CO, the end product of the anti-inflammatory HO-1 pathway, may prove to be an effective therapeutic adjunct for clinical small bowel transplantation.