RESUMO
Acute respiratory distress syndrome (ARDS) is a life-threatening ailment characterized by severe lung injury involving inflammatory cell recruitment to the lung, cytokine production, surfactant dysfunction, and up-regulation of nitric oxide synthase 2 (NOS2) resulting in nitric oxide (NO) production. We hypothesized that NO production from NOS2 expressed in lung parenchymal cells in a murine model of ARDS would correlate with abnormal surfactant function and reduced surfactant protein-B (SP-B) expression. Pulmonary responses to nebulized endotoxin (lipopolysaccharide, LPS) were evaluated in wild-type (WT) mice, NOS2 null (-/-) mice, and NOS2-chimeric animals derived from bone marrow transplantation. NOS2-/- animals exhibited significantly less physiologic lung dysfunction and loss of SP-B expression than did WT animals. However, lung neutrophil recruitment and bronchoalveolar lavage cytokine levels did not significantly differ between NOS2-/- and WT animals. Chimeric animals for NOS2 exhibited the phenotype of the recipient and therefore demonstrated that parenchymal production of NOS2 is critical for the development of LPS-induced lung injury. Furthermore, administration of NO donors, independent of cytokine stimulation, decreased SP-B promoter activity and mRNA expression in mouse lung epithelial cells. This study demonstrates that expression of NOS2 in lung epithelial cells is critical for the development of lung injury and mediates surfactant dysfunction independent of NOS2 inflammatory cell expression and cytokine production.
Assuntos
Lipopolissacarídeos/toxicidade , Pulmão/patologia , Óxido Nítrico Sintase/fisiologia , Proteína B Associada a Surfactante Pulmonar/biossíntese , Síndrome do Desconforto Respiratório/enzimologia , Aerossóis , Animais , Transplante de Medula Óssea , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Inflamação , Interleucina-6/análise , Contagem de Leucócitos , Lipopolissacarídeos/administração & dosagem , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Proteína A Associada a Surfactante Pulmonar/farmacologia , Proteína B Associada a Surfactante Pulmonar/genética , Proteína B Associada a Surfactante Pulmonar/fisiologia , Proteína C Associada a Surfactante Pulmonar/farmacologia , RNA Mensageiro/biossíntese , Quimera por Radiação , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/patologia , Organismos Livres de Patógenos Específicos , Tensão Superficial/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análiseRESUMO
The full extent to which hypoxia produces gene expression changes in human cells is unknown. We used late-generation oligonucleotide arrays to catalog hypoxia-induced changes in gene expression in HepG2 cells. Five paired sets of cultures were subjected to either control (room air-5% CO(2)) or hypoxic (1% O(2)-5% CO(2)) conditions for 24 h, and RNA was analyzed on an Affymetrix cDNA array containing approximately 12,600 sequences. A statistically significant change in expression was shown by 2,908 sequences (1,255 increased and 1,653 decreased). The observed changes were highly concordant with published literature on hypoxic stress but showed relatively little overlap (12-22%) with changes in gene expression that have been reported to occur after heat stress in other systems. Of note, of these 2,908 sequences, only 387 (213 increased and 174 decreased) both exhibited changes in expression of twofold or greater and were highly expressed in at least three of the five experiments. We conclude that the effect of hypoxia on gene expression by HepG2 cells is broad, has a significant component of downregulation, and includes a relatively small number of genes whose response is truly independent of cell and stress type.
Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica/genética , Carcinoma Hepatocelular/patologia , Hipóxia Celular/fisiologia , Chaperoninas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temperatura , Células Tumorais CultivadasRESUMO
We studied the effect of heat shock on gene expression by normal human cells. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adults. Paired samples from each subject were subjected to either 20 min of heat shock (43 degrees C) or control (37 degrees C) conditions and then returned to 37 degrees C. RNA was isolated 160 min later, and five representative samples were analyzed on Affymetrix gene chip arrays containing approximately 12,600 probes. A biologically meaningful effect was defined as a statistically significant, twofold or greater difference in expression of sequences that were detected in all five experiments under control (downregulated sequences) or heat shock (upregulated sequences) conditions. Changes occurred in 395 sequences (227 increased by heat shock, 168 decreased), representing 353 Unigene numbers, in every functional category previously implicated in the heat shock response. By RT-PCR, we confirmed the findings for one upregulated sequence (Rad, a G protein) and one downregulated sequence (osteopontin, a cytokine). We conclude that heat shock causes extensive gene expression changes in PBMCs, affecting all functional categories of the heat shock response.
