RESUMO
Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Doenças Pulmonares Intersticiais , Humanos , Camundongos , Animais , Bleomicina/farmacologia , Plasmócitos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/metabolismo , Doenças Pulmonares Intersticiais/induzido quimicamenteRESUMO
Malignant mesothelioma is an aggressive fibrous tumor, predominantly of the pleura, with a very poor prognosis. Cell-matrix interactions are recognized important determinants of tumor growth and invasiveness but the role of the extracellular matrix in mesothelioma is unknown. Mesothelioma cells synthesize collagen as well as transforming growth factor-beta (TGF-ß), a key regulator of collagen production. This study examined the effect of inhibiting collagen production on mesothelioma cell proliferation in vitro and tumor growth in vivo. Collagen production by mesothelioma cells was inhibited by incubating cells in vitro with the proline analogue thiaproline (thiazolidine-4-carboxylic acid) or by oral administration of thiaproline in a murine tumor model. Cell cytotoxicity was measured using neutral red uptake and lactate dehydrogenase assays. Proliferation was measured by tritiated thymidine incorporation, and inflammatory cell influx, proliferation, apoptosis and angiogenesis in tumors examined by immunohistochemical labelling. Tumor size was determined by tumor weight and collagen production was measured by HPLC. Thiaproline at non-toxic doses significantly reduced basal and TGF-ß-induced collagen production by over 50% and cell proliferation by over 65%. In vivo thiaproline administration inhibited tumor growth at 10 days, decreasing the median tumor weight by 80%. The mean concentration of collagen was 50% lower in the thiaproline-treated tumors compared with the controls. There were no significant differences in vasculature or inflammatory cell infiltration but apoptosis was increased in thiaproline treated tumors at day 10. In conclusion, these observations strongly support a role for collagen in mesothelioma growth and establish the potential for inhibitors of collagen synthesis in mesothelioma treatment.
Assuntos
Colágeno/biossíntese , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Colágeno/antagonistas & inibidores , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Humanos , Inflamação , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , Camundongos Endogâmicos CBA , Neoplasias Pleurais/patologia , Tiazolidinas/farmacologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Numerous compounds have shown efficacy in limiting development of pulmonary fibrosis using animal models, yet few of these compounds have replicated these beneficial effects in clinical trials. Given the challenges associated with performing clinical trials in patients with idiopathic pulmonary fibrosis (IPF), it is imperative that preclinical data packages be robust in their analyses and interpretations to have the best chance of selecting promising drug candidates to advance to clinical trials. The American Thoracic Society has convened a group of experts in lung fibrosis to discuss and formalize recommendations for preclinical assessment of antifibrotic compounds. The panel considered three major themes (choice of animal, practical considerations of fibrosis modeling, and fibrotic endpoints for evaluation). Recognizing the need for practical considerations, we have taken a pragmatic approach. The consensus view is that use of the murine intratracheal bleomycin model in animals of both genders, using hydroxyproline measurements for collagen accumulation along with histologic assessments, is the best-characterized animal model available for preclinical testing. Testing of antifibrotic compounds in this model is recommended to occur after the acute inflammatory phase has subsided (generally after Day 7). Robust analyses may also include confirmatory studies in human IPF specimens and validation of results in a second system using in vivo or in vitro approaches. The panel also strongly encourages the publication of negative results to inform the lung fibrosis community. These recommendations are for preclinical therapeutic evaluation only and are not intended to dissuade development of emerging technologies to better understand IPF pathogenesis.
Assuntos
Congressos como Assunto , Modelos Animais de Doenças , Fibrose Pulmonar/terapia , Sociedades Médicas , Animais , Determinação de Ponto Final , Feminino , Humanos , Masculino , Organismos Geneticamente Modificados , Reprodutibilidade dos TestesRESUMO
Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-ß (TGF-ß) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-ß activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-ß activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-ß activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-ß activity following bleomycin, above their already elevated levels, although global TGF-ß activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-ß activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not markedly modify the development of lung fibrosis following bleomycin-induced lung injury.
Assuntos
Fibrose Pulmonar Idiopática/etiologia , Lesão Pulmonar/etiologia , Inibidor Secretado de Peptidases Leucocitárias/deficiência , Animais , Bleomicina/toxicidade , Colágeno/genética , Colágeno/metabolismo , Deleção de Genes , Hidroxiprolina/metabolismo , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.
Assuntos
Ciclo-Oxigenase 2/genética , Metilação de DNA , Epigênese Genética , Fibroblastos/enzimologia , Pulmão/enzimologia , Proteínas de Neoplasias/genética , Fibrose Pulmonar/genética , Escleroderma Sistêmico/genética , Idoso , Sítios de Ligação , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/patologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Escleroderma Sistêmico/enzimologia , Escleroderma Sistêmico/patologia , Transcrição Gênica , TransfecçãoRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-ß signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1ß, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis. METHODS: We investigated BARD1 expression as a function of TGF-ß in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients. RESULTS: FL BARD1 and BARD1ß were upregulated in response to TGF-ß in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1ß in fibrotic tissues. CONCLUSION: Our data suggest that FL BARD1 and BARD1ß might be mediators of pleiotropic effects of TGF-ß. In particular BARD1ß might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.
Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Pulmão/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bleomicina , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Sarcoidosis is a multisystem granulomatous disease of unknown aetiology characterized by increased inflammation, and results from gene-environment interactions. Proteinase-activated receptor-1 mediates the interplay between coagulation and inflammation. The rs2227744G > A promoter single nucleotide polymorphism has been linked to inflammation, cardiovascular disease and chronic obstructive pulmonary disease exacerbations. Using a case-control study (184 cases with sarcoidosis and 368 controls), we show that the rs2227744A allele significantly associates with protection from sarcoidosis (P = 0.003, OR = 0.68 (0.52-0.88)).
Assuntos
Receptor PAR-1/genética , Sarcoidose/genética , Adulto , Alelos , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de ProteçãoRESUMO
Proteinase-activated receptor-1 (PAR-1) plays a key role in mediating the interplay between coagulation and inflammation in response to injury. The aim of this study was to investigate the role of the promoter single-nucleotide polymorphism (SNP) rs2227744G>A in modulating PAR-1/F2R gene expression in the context of chronic obstructive pulmonary disease (COPD) and COPD exacerbations. The function of the rs2227744G>A SNP was investigated by using reporter gene assays. The frequency of the polymorphism in the UK population was assessed by genotyping 8,579 healthy individuals from the Whitehall II and English Longitudinal Study of Ageing cohorts. The rs2227744G>A SNP was genotyped in a carefully phenotyped cohort of 203 COPD cases and matched controls. The results were further replicated in two different COPD cohorts. The minor allele of the rs2227744G>A polymorphism was found to increase F2R expression by 2.6-fold (P < 0.001). The rs2227744G>A SNP was not significantly associated with COPD, or with lung function, in all cohorts. The minor allele of the SNP was found to be associated with protection from frequent exacerbations (P = 0.04) in the cohort of COPD patients for which exacerbation frequency was available. Considering exacerbations as a continuous variable, the presence of the minor allele was associated with a significantly lower COPD exacerbation rate (3.03 vs. 1.98 exacerbations/year, Mann-Whitney U-test P = 0.04). Taken together, these data do not support a role for the rs2227744G>A F2R polymorphism in the development of COPD but suggest a protective role for this polymorphism from frequent exacerbations. Studies in separate cohorts to replicate these findings are warranted.
Assuntos
Doença Pulmonar Obstrutiva Crônica/genética , Receptor PAR-1/genética , Predisposição Genética para Doença , Humanos , Estudos Longitudinais , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Receptor PAR-1/fisiologiaRESUMO
Receptor-targeted nanocomplexes are nonviral vectors developed for gene delivery to the airway epithelium for the treatment of pulmonary disease associated with cystic fibrosis. The present study aimed to optimize the delivery of the nanocomplex by nebulization, and to monitor the in vivo deposition of radiolabeled vector in the airways of a large animal model by γ-camera scintigraphy. Large White weaner pigs were nebulized with nanocomplexes mixed with technetium-99m radiopharmaceuticals. The aerosol deposition scans suggested that the nebulized radiovectors were deposited mainly in the trachea-main bronchi and in the midregion of the lungs. The plasmid biodistribution, assessed by real-time PCR, correlated with the scintigraphy images. The highest plasmid copy numbers were found in the bronchial areas and in the tissues proximal to the main bronchi bifurcation. Immunohistochemistry detected transgene expression in the tracheal and bronchial ciliated epithelium. Histological analysis of lung tissue showed no evidence of inflammation, and no increase in inflammatory cytokines or inflammatory cells was detected in the bronchoalveolar lavage. The deposition of nebulized nanocomplexes coassociated with technetium-99m can be monitored by nuclear medicine techniques. The use of a noninvasive strategy to follow the delivery of the vector could improve the clinical management of patients undergoing cystic fibrosis gene therapy.
Assuntos
Técnicas de Transferência de Genes , Imagem Molecular/métodos , Compostos Radiofarmacêuticos/farmacocinética , Mucosa Respiratória/diagnóstico por imagem , Sistema Respiratório/diagnóstico por imagem , Tecnécio/farmacocinética , Animais , Fibrose Cística/diagnóstico por imagem , Feminino , Terapia Genética , Humanos , Injeções Intravenosas , Masculino , Nanoconjugados/administração & dosagem , Nanoconjugados/química , Nebulizadores e Vaporizadores , Plasmídeos , Cintilografia , Compostos Radiofarmacêuticos/administração & dosagem , Mucosa Respiratória/ultraestrutura , Sistema Respiratório/ultraestrutura , Suínos , Tecnécio/administração & dosagemRESUMO
Pulmonary fibrosis is the ultimate outcome of various interstitial lung diseases, many of which have a dismal prognosis. Pulmonary fibrosis, therefore, represents a critical unmet medical need. Progress in research over the past 30 years has been encouraging. This work, which has been funded by governments, charitable trusts, industries and patient groups, has resulted in clinical trials testing novel drugs, giving hope to patients. In late September 2012, representatives from academia, industry and funding agencies met at the 17th International Colloquium on Airway and Lung Fibrosis to discuss state-of-the-art knowledge of pulmonary fibrosis. This manuscript summarises the outcomes of the meeting, highlighting the most relevant results and discoveries. It also attempts to provide a roadmap for future studies. It is hoped that such a roadmap may help interested parties to generate new research, which will be vital to continued progress. We are encouraged by the commitment expressed by all participants at this meeting and the shared vision of promoting future progress through international collaboration, the pooling of valuable resources, and the involvement of a new generation of physicians and scientists.
Assuntos
Fibrose Pulmonar/diagnóstico , Fibrose Pulmonar/terapia , Pesquisa Biomédica/tendências , Humanos , Cooperação Internacional , Itália , Fibrose Pulmonar/genética , Pneumologia/métodos , Pneumologia/tendências , Sociedades MédicasRESUMO
STAT3 is a latent transcription factor that plays a role in regulating fibroblast function in fibrotic lung diseases. To further understand the role of STAT3 in the phenotypic divergence and function of human lung fibroblasts (LFs), we investigated the effect of basal and cytokine-induced STAT3 activity on indices of LF differentiation and activation, including expression of α-smooth muscle actin (α-SMA), collagen, and adhesion molecules Thy-1/CD90 and α(v) ß(3) and ß(5) integrins. We identified a population of fibroblasts from usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) lungs characterized by constitutively phosphorylated STAT3, lower proliferation rates, and diminished expression of α-SMA, Thy-1/CD90, and ß(3) integrins compared with control LFs. Staining of UIP lung biopsy specimens demonstrated that phosphorylated STAT3 was not present in α-SMA-positive fibroblastic foci but was observed in the nuclei of cells located in the areas of dense fibrosis. STAT3 activation in LFs did not significantly influence basal or transforming growth factor ß(1)-induced collagen I expression but inhibited expression of α-SMA, Thy-1/CD90, and αv ß(3) integrins. Suppression of STAT3 signaling diminished resistance of IPF LFs to staurosporine-induced apoptosis and responsiveness to transforming growth factor ß(1) but increased basal α-SMA and restored ß(3) integrin expression in LFs via an ALK-5-dependent, SMAD3/7-independent mechanism. These data suggest that STAT3 activation regulates several pathways in human LFs associated with normal wound healing, whereas aberrant STAT3 signaling plays a critical role in UIP/IPF pathogenesis.
Assuntos
Fibroblastos/patologia , Fibrose Pulmonar Idiopática/patologia , Fator de Transcrição STAT3/fisiologia , Actinas/metabolismo , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Fibrose Pulmonar Idiopática/fisiopatologia , Integrina alfaVbeta3/metabolismo , Interleucina-6/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Oncostatina M/farmacologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Antígenos Thy-1/metabolismo , Transdução Genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Although empyema affects more than 65,000 people each year in the United States and in the United Kingdom, there are limited data on the pathogenesis of pleural infection. We investigated the pathogenesis of empyema using animal and cell culture models of Streptococcus pneumoniae infection. The pathological processes during the development of empyema associated with murine pneumonia due to S. pneumoniae (strain D39) were investigated. Lungs were examined using histology, and pleural fluid and blood bacterial colony-forming units, cytokine levels, and cellular infiltrate were determined over time. Bacterial migration across mesothelial monolayers was investigated using cell culture techniques, flow cytometry, and confocal microscopy. After intranasal inoculation with 10(7) S. pneumoniae D39 strain, mice developed pneumonia associated with rapid bacterial invasion of the pleural space; raised intrapleural IL-8, VEGF, MCP-1, and TNF-α levels; and caused significant intrapleural neutrophilia followed by the development of fibrinous pleural adhesions. Bacterial clearance from the pleural space was poor, and in vitro assays demonstrated that S. pneumoniae crossed mesothelial layers by translocation through cells rather than by a paracellular route. This study describes key events during the development of S. pneumoniae empyema using a novel murine model of pneumonia-associated empyema that closely mimics human disease. The model allows for future assessment of molecular mechanisms involved in the development of empyema and evaluation of potential new therapies. The data suggest that transmigration of bacteria through mesothelial cells could be important in empyema development. Furthermore, upon entry the pleural cavity offers a protected compartment for the bacteria.
Assuntos
Modelos Animais de Doenças , Empiema/fisiopatologia , Pneumopatias/microbiologia , Pleura/microbiologia , Doenças Pleurais/microbiologia , Streptococcus pneumoniae/patogenicidade , Animais , Empiema/microbiologia , CamundongosRESUMO
BRCA1 mRNA overexpression is correlated with poor survival in NSCLC. However, BRCA1 functions depend on the interaction with BARD1 for its stability, nuclear localization and ubiquitin ligase activity. Expression of alternatively spliced BARD1 isoforms that lack the BRCA1-interaction domain was found upregulated and correlated with poor prognosis in breast and ovarian cancer. These BARD1 isoforms are essential for proliferation of cancer cells in vitro. We investigated whether BARD1 isoforms are expressed in NSCLC. While in lung tissues from healthy controls BARD1 expression was undetectable on the mRNA level and protein level, we found two novel isoforms in addition to previously identified mRNAs expressed in all NSCLC samples tested. Furthermore, the pattern of BARD1 isoform expression was similar in tumor and morphologically normal peri-tumor tissues, and only one novel isoform π was specifically upregulated in tumors. Immunohistochemistry revealed that all 100 NSCLC cases tested expressed isoform-specific BARD1 epitopes, while BARD1 expression was undetectable in biopsies from healthy controls. Statistical analysis showed that the expression of epitopes PVC and WFS, present on isoform π, or epitope WFS alone, expressed on isoforms π, κ and ß, were significantly correlated with decreased patient survival. These findings were corroborated in a mouse model of chemically induced lung cancer. Immunostaining of mouse tumors showed that BARD1 epitopes PVC and WFS were specifically upregulated in invasive, but not in confined lung tumors. Thus, BARD1 isoforms might be involved in tumor initiation and invasive progression and might represent a novel prognostic marker for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Processamento Alternativo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis. OBJECTIVES: To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance. METHODS: Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed. MEASUREMENTS AND MAIN RESULTS: IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses. CONCLUSIONS: The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.
Assuntos
Apoptose/fisiologia , Ciclo-Oxigenase 2/fisiologia , Dinoprostona/fisiologia , Fibroblastos/fisiologia , Fibrose Pulmonar Idiopática/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Células Cultivadas , Células Epiteliais/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/fisiologia , Cicatrização/fisiologiaRESUMO
The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5'-aza-2'-deoxycytidine' treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.
RESUMO
The morphological features of chronic obstructive pulmonary disease in man include emphysema and chronic bronchitis associated with mucus hypersecretion. These alterations can be induced in mice by a single intratracheal instillation of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), a chemoattractant and degranulating agent for neutrophils. The mechanisms underlying excessive mucus production and, in particular, goblet cell hyperplasia/metaplasia in chronic obstructive pulmonary disease remain poorly understood. The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties during inflammation. In this study, we examined whether PAR-1 contributes to inflammation and lung damage induced by fMLP by comparing the response of PAR-1-deficient (PAR-1(-/-)) mice with that of wild-type (WT) mice. Mice were killed at various time points after fMLP instillation (200 microg/50 microl). WT mice developed emphysema and goblet cell metaplasia. The onset of pulmonary lesions was preceded by an increase in thrombin immunoreactivity in bronchial airways and alveolar tissue. This was followed by a decrease in PAR-1 immunoreactivity, and by an increase in IL-13 immunostaining on the luminal surface of airway epithelial cells. In PAR-1(-/-) mice, fMLP administration induced similar responses in terms of inflammation and emphysema, but these mice were protected from the development of goblet cell metaplasia. The involvement of PAR-1 in airway epithelial cell transdifferentiation was confirmed by demonstrating that intratracheal instillation of the selective PAR-1 agonist (TFLLR) induced goblet cell metaplasia in the airways of WT mice only. These data suggest that emphysema and goblet cell metaplasia occur independently, and that PAR-1 signaling through IL-13 stimulation may play an important role in inducing goblet cell metaplasia.
Assuntos
Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , N-Formilmetionina Leucil-Fenilalanina/toxicidade , Receptor PAR-1/deficiência , Animais , Diferenciação Celular/efeitos dos fármacos , Enfisema/induzido quimicamente , Enfisema/metabolismo , Enfisema/patologia , Receptores ErbB/metabolismo , Células Caliciformes/patologia , Humanos , Interleucina-13/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligopeptídeos/farmacologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Receptor PAR-1/agonistas , Receptor PAR-1/genética , Transdução de SinaisRESUMO
Activation of latent TGF-beta by the alpha(v)beta6 integrin is a critical step in the development of acute lung injury. However, the mechanism by which alpha(v)beta6-mediated TGF-beta activation is regulated has not been identified. We show that thrombin, and other agonists of protease-activated receptor 1 (PAR1), activate TGF-beta in an alpha(v)beta6 integrin-specific manner. This effect is PAR1 specific and is mediated by RhoA and Rho kinase. Intratracheal instillation of the PAR1-specific peptide TFLLRN increases lung edema during high-tidal-volume ventilation, and this effect is completely inhibited by a blocking antibody against the alpha(v)beta6 integrin. Instillation of TFLLRN during high-tidal-volume ventilation is associated with increased pulmonary TGF-beta activation; however, this is not observed in Itgb6-/- mice. Furthermore, Itgb6-/- mice are also protected from ventilator-induced lung edema. We also demonstrate that pulmonary edema and TGF-beta activity are similarly reduced in Par1-/- mice following bleomycin-induced lung injury. These results suggest that PAR1-mediated enhancement of alpha(v)beta6-dependent TGF-beta activation could be one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury, and they identify PAR1 and the alpha(v)beta6 integrin as potential therapeutic targets in this condition.
Assuntos
Integrinas/metabolismo , Receptor PAR-1/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Amidas/metabolismo , Animais , Antibióticos Antineoplásicos/metabolismo , Bleomicina/metabolismo , Células Cultivadas , Inibidores Enzimáticos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Integrinas/genética , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/citologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Edema Pulmonar/etiologia , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Piridinas/metabolismo , Receptor PAR-1/agonistas , Receptor PAR-1/genética , Síndrome do Desconforto Respiratório/patologia , Trombina/metabolismo , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Hepatic progenitor cells (HPCs) play an important regenerative role in acute and chronic liver pathologies. Liver disease research often necessitates the grading of disease severity, and pathologists' reports are the current gold-standard for assessment. However, it is often impractical to recruit pathologists in large cohort studies. In this study we utilise PerkinElmer's "InForm" software package to semi-automate the scoring of patient liver biopsies, and compare outputs to a pathologist's assessment. We examined a cohort of eleven acute hepatitis samples and three non-alcoholic fatty liver disease (NAFLD) samples, stained with HPC markers (GCTM-5 and Pan Cytokeratin), an inflammatory marker (CD45), Sirius Red to detect collagen and haematoxylin/eosin for general histology. InForm was configured to identify presumptive HPCs, CD45+ve inflammatory cells, areas of necrosis, fat and collagen deposition (p < 0.0001). Hepatitis samples were then evaluated both by a pathologist using the Ishak-Knodell scoring system, and by InForm through customised algorithms. Necroinflammation as evaluated by a pathologist, correlated with InForm outputs (r2 = 0.8192, p < 0.05). This study demonstrates that the InForm software package provides a useful tool for liver disease research, allowing rapid, and objective quantification of the presumptive HPCs and identifies histological features that assist with assessing liver disease severity, and potentially can facilitate diagnosis.
Assuntos
Hepatite/diagnóstico , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Software , Células-Tronco/patologia , Biomarcadores/análise , Estudos de Coortes , Colágeno/análise , Hepatite/patologia , Humanos , Queratinas/análise , Antígenos Comuns de Leucócito/análise , Fígado/citologia , Hepatopatia Gordurosa não Alcoólica/patologia , Células-Tronco/citologiaRESUMO
PURPOSE: Currently the screening for lung cancer for risk groups is based on Computed Tomography (CT) or low dose CT (LDCT); however, the lung cancer death rate has not decreased significantly with people undergoing LDCT. We aimed to develop a simple reliable blood test for early detection of all types of lung cancer based on the immunogenicity of aberrant forms of BARD1 that are specifically upregulated in lung cancer. METHODS: ELISA assays were performed with a panel of BARD1 epitopes to detect serum levels of antibodies against BARD1 epitopes. We tested 194 blood samples from healthy donors and lung cancer patients with a panel of 40 BARD1 antigens. Using fitted Lasso logistic regression we determined the optimal combination of BARD1 antigens to be used in ELISA for discriminating lung cancer from healthy controls. Random selection of samples for training sets or validations sets was applied to validate the accuracy of our test. RESULTS: Fitted Lasso logistic regression models predict high accuracy of the BARD1 autoimmune antibody test with an AUC = 0.96. Validation in independent samples provided and AUC = 0.86 and identical AUCs were obtained for combined stages 1-3 and late stage 4 lung cancers. The BARD1 antibody test is highly specific for lung cancer and not breast or ovarian cancer. CONCLUSION: The BARD1 lung cancer test shows higher sensitivity and specificity than previously published blood tests for lung cancer detection and/or diagnosis or CT scans, and it could detect all types and all stages of lung cancer. This BARD1 lung cancer test could therefore be further developed as i) screening test for early detection of lung cancers in high-risk groups, and ii) diagnostic aid in complementing CT scan.