RESUMEN
Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation, small airway remodeling, and emphysema. Airway remodeling in patients with COPD involves both the airway epithelium and the subepithelial extracellular matrix (ECM). However, it is currently unknown how epithelial remodeling in COPD airways depends on the relative influence from inherent defects in the epithelial cells and alterations in the ECM. To address this, we analyzed global gene expression in COPD human bronchial epithelial cells (HBEC) and normal HBEC after repopulation on decellularized bronchial scaffolds derived from patients with COPD or donors without COPD. COPD HBEC grown on bronchial scaffolds showed an impaired ability to initiate ciliated-cell differentiation, which was evident on all scaffolds regardless of their origin. In addition, although normal HBEC were less affected by the disease state of the bronchial scaffolds, COPD HBEC showed a gene expression pattern indicating increased proliferation and a retained basal-cell phenotype when grown on COPD bronchial scaffolds compared with normal bronchial scaffolds. By using mass spectrometry, we identified 13 matrisome proteins as being differentially abundant between COPD bronchial scaffolds and normal bronchial scaffolds. These observations are consistent with COPD pathology and suggest that both epithelial cells and the ECM contribute to epithelial-cell remodeling in COPD airways.
Asunto(s)
Bronquios/química , Diferenciación Celular , Células Epiteliales/metabolismo , Matriz Extracelular/química , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Andamios del Tejido/química , Células Epiteliales/patología , Femenino , Humanos , Masculino , Enfermedad Pulmonar Obstructiva Crónica/patologíaRESUMEN
The mitogen-activated protein kinase p38α pathway has been an attractive target for the treatment of inflammatory conditions such as rheumatoid arthritis. While a number of p38α inhibitors have been taken to the clinic, they have been limited by their efficacy and toxicological profile. A lead identification program was initiated to selectively target prevention of activation (PoA) of mitogen-activated protein kinase-activated protein kinaseâ 2 (MK2) rather than mitogen- and stress-activated protein kinaseâ 1 (MSK1), both immediate downstream substrates of p38α, to improve the efficacy/safety profile over direct p38α inhibition. Starting with a series of pyrazole amide PoA MK2 inhibitor leads, and guided by structural chemistry and rational design, a highly selective imidazole 9 (2-(3'-(2-amino-2-oxoethyl)-[1,1'-biphenyl]-3-yl)-N-(5-(N,N-dimethylsulfamoyl)-2-methylphenyl)-1-propyl-1H-imidazole-5-carboxamide) and the orally bioavailable imidazole 18 (3-methyl-N-(2-methyl-5-sulfamoylphenyl)-2-(o-tolyl)imidazole-4-carboxamide) were discovered. The PoA concept was further evaluated by protein immunoblotting, which showed that the optimized PoA MK2 compounds, despite their biochemical selectivity against MSK1 phosphorylation, behaved similarly to p38 inhibitors in cellular signaling. This study highlights the importance of selective tool compounds in untangling complex signaling pathways, and although 9 and 18 were not differentiated from p38α inhibitors in a cellular context, they are still useful tools for further research directed to understand the role of MK2 in the p38α signaling pathway.
Asunto(s)
Antiinflamatorios/síntesis química , Activación Enzimática/efectos de los fármacos , Imidazoles/síntesis química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/química , Antiinflamatorios/farmacología , Células Cultivadas , Humanos , Imidazoles/farmacología , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. SIGNIFICANCE: We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF.
Asunto(s)
Proteínas de la Matriz Extracelular/análisis , Matriz Extracelular/química , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/química , Proteómica/métodos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Fraccionamiento Químico/métodos , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/patologíaRESUMEN
Chronic obstructive pulmonary disease (COPD) is a serious global health problem characterized by chronic airway inflammation, progressive airflow limitation and destruction of lung parenchyma. Remodeling of the bronchial airways in COPD includes changes in both the bronchial epithelium and the subepithelial extracellular matrix (ECM). To explore the impact of an aberrant ECM on epithelial cell phenotype in COPD we developed a new ex vivo model, in which normal human bronchial epithelial (NHBE) cells repopulate and differentiate on decellularized human bronchial scaffolds derived from COPD patients and healthy individuals. By using transcriptomics, we show that bronchial ECM from COPD patients induces differential gene expression in primary NHBE cells when compared to normal bronchial ECM. The gene expression profile indicated altered activity of upstream mediators associated with COPD pathophysiology, including hepatocyte growth factor, transforming growth factor beta 1 and platelet-derived growth factor B, which suggests that COPD-related changes in the bronchial ECM contribute to the defective regenerative ability in the airways of COPD patients.
Asunto(s)
Matriz Extracelular/genética , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Transcriptoma/genética , Bronquios/metabolismo , Bronquios/patología , Factor de Crecimiento del Tejido Conjuntivo/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Matriz Extracelular/patología , Regulación de la Expresión Génica/genética , Factor de Crecimiento de Hepatocito/genética , Humanos , Pulmón/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-sis/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.