RESUMEN
Objective: To study the tissue-infiltrating immune cells of the emphysema phenotype of chronic obstructive pulmonary disease (COPD) and find the molecular mechanism related to the development of emphysema to offer potential targets for more precise treatment of patients with COPD. Methods: Combined analyses of COPD emphysema phenotype lung tissue-related datasets, GSE47460 and GSE1122, were performed. CIBERSORT was used to assess the distribution of tissue-infiltrating immune cells. Weighted gene co-expression network analysis (WGCNA) was used to select immune key genes closely related to clinical features. Rt-qPCR experiments were used for the validation of key genes. Emphysema risk prediction models were constructed by logistic regression analysis and a nomogram was developed. Results: In this study, three immune cells significantly associated with clinical features of emphysema (FEV1 post-bronchodilator % predicted, GOLD Stage, and DLCO) were found. The proportion of neutrophils (p=0.025) infiltrating in the emphysema phenotype was significantly increased compared with the non-emphysema phenotype, while the proportions of M2 macrophages (p=0.004) and resting mast cells (p=0.01) were significantly decreased. Five immune-related differentially expressed genes (DEGs) were found. WGCNA and clinical lung tissue validation of patients with emphysema phenotype were performed to further screen immune-related genes closely related to clinical features. A key gene (SERPINA3) was selected and included in the emphysema risk prediction model. Compared with the traditional clinical prediction model (AUC=0.923), the combined prediction model, including SERPINA3 and resting mast cells (AUC=0.941), had better discrimination power and higher net benefit. Conclusion: This study comprehensively analyzed the tissue-infiltrating immune cells significantly associated with emphysema phenotype, including M2 macrophages, neutrophils, and resting mast cells, and identified SERPINA3 as a key immune-related gene.
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Enfisema , Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Broncodilatadores , Enfisema/genética , Humanos , Modelos Estadísticos , Fenotipo , Pronóstico , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfisema Pulmonar/genéticaRESUMEN
BACKGROUND: Long-term exposure to PM2.5 (particulate matter with an aerodynamic diameter of ≤ 2.5 µm) is associated with pulmonary injury and emphysema in patients with chronic obstructive pulmonary disease (COPD). We investigated mechanisms through which the long noncoding RNA lnc-IL7R contributes to cellular damage by inducing oxidative stress in COPD patients exposed to PM2.5. METHODS: Associations of serum lnc-IL7R levels with lung function, emphysema, and previous PM2.5 exposure in COPD patients were analyzed. Reactive oxygen species and lnc-IL7R levels were measured in PM2.5-treated cells. The levels of lnc-IL7R and cellular senescence-associated genes, namely p16INK4a and p21CIP1/WAF1, were determined through lung tissue section staining. The effects of p16INK4a or p21CIP1/WAF1 regulation were examined by performing lnc-IL7R overexpression and knockdown assays. The functions of lnc-IL7R-mediated cell proliferation, cell cycle, senescence, colony formation, and apoptosis were examined in cells treated with PM2.5. Chromatin immunoprecipitation assays were conducted to investigate the epigenetic regulation of p21CIP1/WAF1. RESULTS: Lnc-IL7R levels decreased in COPD patients and were negatively correlated with emphysema or PM2.5 exposure. Lnc-IL7R levels were upregulated in normal lung epithelial cells but not in COPD cells exposed to PM2.5. Lower lnc-IL7R expression in PM2.5-treated cells induced p16INK4a and p21CIP1/WAF1 expression by increasing oxidative stress. Higher lnc-IL7R expression protected against cellular senescence and apoptosis, whereas lower lnc-IL7R expression augmented injury in PM2.5-treated cells. Lnc-IL7R and the enhancer of zeste homolog 2 (EZH2) synergistically suppressed p21CIP1/WAF1 expression through epigenetic modulation. CONCLUSION: Lnc-IL7R attenuates PM2.5-mediated p21CIP1/WAF1 expression through EZH2 recruitment, and its dysfunction may augment cellular injury in COPD.
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Enfisema , Enfermedad Pulmonar Obstructiva Crónica , ARN Largo no Codificante , Humanos , Apoptosis/genética , Senescencia Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Enfisema/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Material Particulado/toxicidad , Enfermedad Pulmonar Obstructiva Crónica/genética , ARN Largo no Codificante/genéticaRESUMEN
The simplification of alveoli leads to various lung pathologies such as bronchopulmonary dysplasia and emphysema. Deep insight into the process of emergence of the secondary septa during development and regeneration after pneumonectomy, and into the contribution of the drivers of alveologenesis and neo-alveolarization is required in an efficient search for therapeutic approaches. In this review, we describe the formation of the gas exchange units of the lung as a multifactorial process, which includes changes in the actomyosin cytoskeleton of alveocytes and myofibroblasts, elastogenesis, retinoic acid signaling, and the contribution of alveolar mesenchymal cells in secondary septation. Knowledge of the mechanistic context of alveologenesis remains incomplete. The characterization of the mechanisms that govern the emergence and depletion of αSMA will allow for an understanding of how the niche of fibroblasts is changing. Taking into account the intense studies that have been performed on the pool of lung mesenchymal cells, we present data on the typing of interstitial fibroblasts and their role in the formation and maintenance of alveoli. On the whole, when identifying cell subpopulations in lung mesenchyme, one has to consider the developmental context, the changing cellular functions, and the lability of gene signatures.
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Actomiosina/genética , Pulmón/crecimiento & desarrollo , Organogénesis/genética , Alveolos Pulmonares/crecimiento & desarrollo , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patología , Linaje de la Célula/genética , Citoesqueleto/genética , Enfisema/genética , Enfisema/patología , Gases/metabolismo , Humanos , Pulmón/patología , Mesodermo/citología , Mesodermo/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Tretinoina/metabolismoRESUMEN
BACKGROUND: Emphysema is a crucial pathological characteristic of chronic obstructive pulmonary disease (COPD). Oxidative stress, apoptosis and epigenetic mechanisms contribute to the pathogenesis of emphysema. However, an attempt to accurately identify whether these mechanisms interact with each other and how they are triggered has never been conducted. METHOD: The total reactive oxygen species (ROS) level, pulmonary apoptosis and B-cell lymphoma/leukemia-2 (Bcl-2) expression, an apoptosis regulator, were detected in samples from COPD patients. Bisulfite sequencing PCR (BSP) was conducted to observe the alterations in the methylation of the Bcl-2 promoter in specimens. The dysregulation of DNA methyltransferase enzyme 1 (DNMT1), a vital DNA methyltransferase enzyme, in the lungs of patients was confirmed through western blotting. To find out interactions between oxidative stress and DNA methylation in emphysema, mouse models were built with antioxidant treatment and DNMT1 silencing, and were examined with the pulmonary apoptosis, Bcl-2 and DNMT1 levels, and epigenetic alterations of Bcl-2. RESULTS: Higher ROS levels and pulmonary apoptosis were observed in COPD patients than in healthy controls. Downregulated Bcl-2 expression with increased promoter methylation and DNMT1 protein expression was found in COPD patients. Antioxidant treatment reduced the level of ROS, DNMT1 protein and emphysematous progression in the smoking models. Following DNMT1 blockade, smoking models showed improved lung function, pulmonary apoptosis, emphysematous progression, and increased Bcl-2 protein level with less promoter methylation than emphysema mice. CONCLUSION: Cigarette-induced oxidative stress mediates pulmonary apoptosis and hypermethylation of the Bcl-2 promoter in emphysema models through DNMT1.
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Fumar Cigarrillos/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Enfisema/metabolismo , Epigénesis Genética/fisiología , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Anciano , Animales , Apoptosis/fisiología , Fumar Cigarrillos/efectos adversos , Fumar Cigarrillos/genética , ADN (Citosina-5-)-Metiltransferasa 1/genética , Enfisema/etiología , Enfisema/genética , Femenino , Humanos , Exposición por Inhalación/efectos adversos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
The dominant pathogenesis underlying the combined pulmonary fibrosis and emphysema (CPFE) remains unresolved. The receptor for advanced glycation end-products (RAGE) is highly expressed in lung tissues and interacts with distinct multiple ligands, implicating it in certain lung diseases. To elucidate the pathogenesis of CPFE, we genotyped three single nucleotide polymorphisms (SNPs: rs2070600, rs1800625, and rs2853807) of the gene encoding RAGE (AGER) in 111 CPFE patients and 337 chronic obstructive pulmonary disease (COPD) patients of Japanese by using StepOne Real-Time PCR System for SNP genotyping assay. Serum levels of soluble RAGE (sRAGE) were measured by ELISA. We found that the allele frequency of rs2070600 was significantly different between the two groups [corrected P (Pc) = 0.015]. In addition, the minor allele was associated with CPFE patients relative to COPD patients in a dominant effect model (Odds Ratio = 1.93; Pc = 0.018). Moreover, the serum sRAGE level was significantly lower in the CPFE group than the COPD group (P = 0.014). The rs2070600 minor allele was significantly associated with reduced sRAGE level in CPFE patients and independently affected sRAGE level reduction in this group (P = 0.020). We concluded that the AGER rs2070600 minor allele (Gly82Ser mutation) is associated with the pathogenesis of pulmonary fibrosis in CPFE in Japanese patients.
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Enfisema/genética , Mutación , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfisema Pulmonar/genética , Fibrosis Pulmonar/genética , Receptor para Productos Finales de Glicación Avanzada/genética , Anciano , Alelos , Pueblo Asiatico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Receptor para Productos Finales de Glicación Avanzada/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.
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Proteínas Adaptadoras Transductoras de Señales/efectos de los fármacos , Enfisema/tratamiento farmacológico , Enfisema/genética , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/efectos de los fármacos , Enfisema/etiología , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Transducción de Señal/genética , Superóxidos/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Wnt/ß-catenin signaling regulates progenitor cell fate decisions during lung development and in various adult tissues. Ectopic activation of Wnt/ß-catenin signaling promotes tissue repair in emphysema, a devastating lung disease with progressive loss of parenchymal lung tissue. The identity of Wnt/ß-catenin responsive progenitor cells and the potential impact of Wnt/ß-catenin signaling on adult distal lung epithelial progenitor cell function in emphysema are poorly understood. Here, we used TCF/Lef:H2B/GFP reporter mice to investigate the role of Wnt/ß-catenin signaling in lung organoid formation. We identified an organoid-forming adult distal lung epithelial progenitor cell population characterized by a low Wnt/ß-catenin activity, which was enriched in club and alveolar epithelial type (AT)II cells. Endogenous Wnt/ß-catenin activity was required for the initiation of multiple subtypes of distal lung organoids derived from the Wntlow epithelial progenitors. Further ectopic Wnt/ß-catenin activation specifically led to an increase in alveolar organoid number; however, the subsequent proliferation of alveolar epithelial cells in the organoids did not require constitutive Wnt/ß-catenin signaling. Distal lung epithelial progenitor cells derived from the mouse model of elastase-induced emphysema exhibited reduced organoid forming capacity. This was rescued by Wnt/ß-catenin signal activation, which largely increased the number of alveolar organoids. Together, our study reveals a novel mechanism of lung epithelial progenitor cell activation in homeostasis and emphysema.
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Enfisema/genética , Homeostasis/fisiología , Células Madre/metabolismo , beta Catenina/metabolismo , Animales , Enfisema/patología , Humanos , Ratones , Vía de Señalización WntRESUMEN
OBJECTIVE AND DESIGN: Gallic acid (GA) a naturally occurring phenolic compound, known to possess antioxidant/anti-inflammatory activities. The aim of the present work was to investigate the beneficial effects of GA against COPD-linked lung inflammation/emphysema by utilizing elastase (ET) and cigarette smoke (CS)-induced mice model. MATERIALS: Male BALB/c mice were treated with ET (1U/mouse) or exposed to CS (9 cigarettes/day for 4 days). GA administration was started 7 days (daily) prior to ET/CS exposure. Broncho-alveolar lavage was analyzed for inflammatory cells and pro-inflammatory cytokines. Lung homogenate was assessed for MPO activity/GSH/MDA/protein carbonyls. Further, Lung tissue was subjected to semi-quantitative RT-PCR, immunoblotting, and histological analysis. RESULTS: GA suppressed the ET-induced neutrophil infiltration, elevated MPO activity and production of pro-inflammatory cytokines (IL-6/TNF-α/IL-1ß) at 24 h. Reduced inflammation was accompanied with normalization of redox balance as reflected by ROS/GSH/MDA/protein carbonyl levels. Further, GA suppressed phosphorylation of p65NF-κB and IκBα along with down-regulation of IL-1ß/TNF-α/KC/MIP-2/GCSF genes. Furthermore, GA offered protection against ET-induced airspace enlargement and ameliorated MMP-2/MMP-9. Finally, GA suppressed the CS-induced influx of neutrophils and macrophages and blunted gene expression of TNF-α/MIP-2/KC. CONCLUSION: Overall, our data show that GA effectively modulates pulmonary inflammation and emphysema associated with COPD pathogenesis in mice.
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Antiinflamatorios/uso terapéutico , Enfisema/tratamiento farmacológico , Ácido Gálico/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Animales , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/inmunología , Enfisema/genética , Enfisema/inmunología , Pulmón/efectos de los fármacos , Pulmón/inmunología , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones Endogámicos BALB C , FN-kappa B/inmunología , Elastasa Pancreática , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Humo/efectos adversos , Inhibidor Tisular de Metaloproteinasa-1/genética , NicotianaRESUMEN
Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with multiple molecular mechanisms. To investigate and contrast the molecular processes differing between bronchiolitis and emphysema phenotypes of COPD, we downloaded the GSE69818 microarray data set from the Gene Expression Omnibus (GEO), which based on lung tissues from 38 patients with emphysema and 32 patients with bronchiolitis. Then, weighted gene coexpression network analysis (WGCNA) and differential coexpression (DiffCoEx) analysis were performed, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG) analysis. Modules and hub genes for bronchiolitis and emphysema were identified, and we found that genes in modules linked to neutrophil degranulation, Rho protein signal transduction and B cell receptor signalling were coexpressed in emphysema. DiffCoEx analysis showed that four hub genes (IFT88, CCDC103, MMP10 and Bik) were consistently expressed in emphysema patients; these hub genes were enriched, respectively, for functions of cilium assembly and movement, proteolysis and apoptotic mitochondrial changes. In our re-analysis of GSE69818, gene expression networks in relation to emphysema deepen insights into the molecular mechanism of COPD and also identify some promising therapeutic targets.
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Bronquiolitis/genética , Enfisema/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Enfermedad Pulmonar Obstructiva Crónica/genética , Análisis por Conglomerados , Ontología de Genes , Humanos , Anotación de Secuencia Molecular , Fenotipo , Transducción de Señal/genéticaRESUMEN
Murine studies have linked TGF-ß signaling to emphysema, and human genome-wide association studies (GWAS) studies of lung function and COPD have identified associated regions near genes in the TGF-ß superfamily. However, the functional regulatory mechanisms at these loci have not been identified. We performed the largest GWAS of emphysema patterns to date, identifying 10 GWAS loci including an association peak spanning a 200 kb region downstream from TGFB2. Integrative analysis of publicly available eQTL, DNaseI, and chromatin conformation data identified a putative functional variant, rs1690789, that may regulate TGFB2 expression in human fibroblasts. Using chromatin conformation capture, we confirmed that the region containing rs1690789 contacts the TGFB2 promoter in fibroblasts, and CRISPR/Cas-9 targeted deletion of a ~ 100 bp region containing rs1690789 resulted in decreased TGFB2 expression in primary human lung fibroblasts. These data provide novel mechanistic evidence linking genetic variation affecting the TGF-ß pathway to emphysema in humans.
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Enfisema/genética , Fibroblastos/fisiología , Regulación de la Expresión Génica , Sitios Genéticos , Predisposición Genética a la Enfermedad , Pulmón/patología , Factor de Crecimiento Transformador beta2/biosíntesis , Anciano , Anciano de 80 o más Años , Estudio de Asociación del Genoma Completo , Humanos , Persona de Mediana Edad , Factor de Crecimiento Transformador beta2/genética , Estados UnidosRESUMEN
Dysregulated matrix metalloproteinase (MMP) gene expression is a major cause of the degradation of lung tissue that is integral to emphysema pathogenesis. Cigarette smoking (CS) increases MMP gene expression, a major contributor to emphysema development. We previously reported that Zbtb7c is a transcriptional repressor of several Mmp genes (Mmps-8, -10, -13, and -16). Here, we show that Zbtb7c knockout mice have mild emphysema-like phenotypes, including alveolar wall destruction, enlarged alveoli, and upregulated Mmp genes. Alveolar size and Mmp gene expression in Zbtb7c-/- mouse lungs are increased more severely upon exposure to CS, compared to those of Zbtb7c+/+ mouse lungs. These observations suggest that Zbtb7c degradation or absence may contribute to the pathogenesis of emphysema.
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Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Enfisema/genética , Pulmón/patología , Metaloproteinasas de la Matriz/genética , Fenotipo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética/genética , Animales , Fumar Cigarrillos/efectos adversos , Técnicas de Inactivación de Genes , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , RatonesRESUMEN
BACKGROUND: The study was conducted to evaluate the clinical and computed tomography (CT) findings of non-small cell lung cancer (NSCLC) patients to distinguish between ALK gene rearrangement, EGFR mutation, and non-ALK/EGFR (no genetic abnormalities). METHODS: We enrolled 201 patients with primary NSCLC who had undergone molecular testing for both ALK gene rearrangement and EGFR mutation. The clinical features and CT findings of the main lesion and associated pulmonary abnormalities were investigated. RESULTS: Female gender (P = 0.0043 vs. non-ALK/EGFR), young age (P = 0.0156 vs. EGFR), and a light or never smoking history (P = 0.0039 vs. non-ALK/EGFR) were significant clinical characteristics of NSCLC with ALK gene rearrangement. The significant CT characteristics compared to NSCLC with EGFR mutation were a large mass (P = 0.0155), solid mass (P = 0.0048), and no air bronchogram (P = 0.0148). A central location (P = 0.0322) and lymphadenopathy (P = 0.0353) were also more frequently observed. Coexisting emphysema was significantly less frequent in NSCLC patients with ALK gene rearrangement (P = 0.0135) than non-ALK/EGFR. CONCLUSIONS: NSCLC with ALK gene rearrangement was more likely to develop in younger women with a light or never smoking history. The characteristic CT findings of NSCLC with ALK gene rearrangement were a large solid mass, less air bronchogram, a central location, and lymphadenopathy.
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Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Reordenamiento Génico , Neoplasias Pulmonares/diagnóstico por imagen , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/genética , Enfisema/epidemiología , Enfisema/genética , Receptores ErbB/genética , Femenino , Humanos , Linfadenopatía/epidemiología , Linfadenopatía/genética , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tomografía Computarizada por Rayos XRESUMEN
In ATP6V0A2-related cutis laxa, the skin phenotype varies from a wrinkly skin to prominent cutis laxa and typically associates with skeletal and neurological manifestations. The phenotype remains incompletely characterized, especially in adult patients. Glycosylation defects and reduced acidification of secretory vesicles contribute to the pathogenesis, but the consequences at the clinical level remain to be determined. Moreover, the morphology of the elastic fibres has not been studied in ATP6V0A2-related cutis laxa, nor its relation with potential clinical risks. We report on the extreme variability in ATP6V0A2-related cutis laxa in 10 novel patients, expand the phenotype with emphysema and von Willebrand disease and hypothesize on the pathogenesis that might link both with deficiency of glycosylation and with elastic fibre anomalies. Our data will affect clinical management of patients with ATP6V0A2-related cutis laxa.
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Cutis Laxo/genética , ATPasas de Translocación de Protón/genética , Piel/patología , Adulto , Anciano , Agenesia del Cuerpo Calloso/genética , Catarata/genética , Niño , Preescolar , Codón sin Sentido , Consanguinidad , Cutis Laxo/patología , Tejido Elástico/patología , Enfisema/genética , Cara/anomalías , Femenino , Glicosilación , Trastornos Hemorrágicos/genética , Humanos , Masculino , Fenotipo , Procesamiento Proteico-Postraduccional , Sitios de Empalme de ARN/genética , Adulto JovenRESUMEN
In this study, we aimed to investigate precise mechanism(s) of sphingolipid-imbalance and resulting ceramide-accumulation in COPD-emphysema. Where, human and murine emphysema lung tissues or human bronchial epithelial cells (Beas2b) were used for experimental analysis. We found that lungs of smokers and COPD-subjects with increasing emphysema severity demonstrate sphingolipid-imbalance, resulting in significant ceramide-accumulation and increased ceramide/sphingosine ratio, as compared to non-emphysema/non-smoker controls. Next, we found a substantial increase in emphysema chronicity-related ceramide-accumulation in murine (C57BL/6) lungs, while sphingosine levels only slightly increased. In accordance, the expression of the acid ceramidase decreased after CS-exposure. Moreover, CS-induced (sub-chronic) ceramide-accumulation was significantly (pâ¯<â¯0.05) reduced by treatment with TFEB/autophagy-inducing drug, gemfibrozil (GEM), suggesting that autophagy regulates CS-induced ceramide-accumulation. Next, we validated experimentally that autophagy/lipophagy-induction using an anti-oxidant, cysteamine, significantly (pâ¯<â¯0.05) reduces CS-extract (CSE)-mediated intracellular-ceramide-accumulation in p62â¯+â¯aggresome-bodies. In addition to intracellular-accumulation, we found that CSE also induces membrane-ceramide-accumulation by ROS-dependent acid-sphingomyelinase (ASM) activation and plasma-membrane translocation, which was significantly controlled (pâ¯<â¯0.05) by cysteamine (an anti-oxidant) and amitriptyline (AMT, an inhibitor of ASM). Cysteamine-mediated and CSE-induced membrane-ceramide regulation was nullified by CFTR-inhibitor-172, demonstrating that CFTR controls redox impaired-autophagy dependent membrane-ceramide accumulation. In summary, our data shows that CS-mediated autophagy/lipophagy-dysfunction results in intracellular-ceramide-accumulation, while acquired CFTR-dysfunction-induced ASM causes membrane ceramide-accumulation. Thus, CS-exposure alters the sphingolipid-rheostat leading to the increased membrane- and intracellular- ceramide-accumulation inducing COPD-emphysema pathogenesis that is alleviated by treatment with cysteamine, a potent anti-oxidant with CFTR/autophagy-augmenting properties.
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Autofagia/efectos de los fármacos , Ceramidas/metabolismo , Mezclas Complejas/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Enfisema/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Fumar/efectos adversos , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Animales , Antioxidantes/farmacología , Autofagia/genética , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Bronquios/patología , Estudios de Casos y Controles , Línea Celular , Mezclas Complejas/aislamiento & purificación , Cisteamina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Enfisema/tratamiento farmacológico , Enfisema/metabolismo , Enfisema/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Gemfibrozilo/farmacología , Expresión Génica , Humanos , Hipolipemiantes/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Nicotiana/efectos adversos , Nicotiana/químicaRESUMEN
Our current knowledge about the cellular mechanisms underlying serpin-related disorders, the serpinopathies, is predominantly based on studies in cell culture models of disease, particularly for alpha-1 antitrypsin (AAT, SERPINA1) deficiency causing emphysema and the familial encephalopathy with neuroserpin (NS, SERPINI1) inclusion bodies (FENIB). FENIB, a neurodegenerative dementia, is caused by polymerization of NS (Miranda and Lomas, Cell Mol Life Sci 63:709-722, 2006; Roussel BD et al., Epileptic Disor 18:103-110, 2016), while AAT deficiency presents as a result of several divergent mutations in the AAT gene that cause lack of protein synthesis or complete intracellular degradation (null variants) or polymer formation (polymerogenic variants) (Lomas et al., J Hepatol 65:413-424, 2016; Greene et al., Nat Rev Dis Primers 2:16051, 2016; Ferrarotti et al. Orphanet J Rare D 9:172, 2014). Both diseases have been extensively modeled in cell culture systems by expressing mutant variants in a variety of ways. Here we describe the methodologies we follow in our cell model systems used to examine serpin disorders.
Asunto(s)
Enfisema , Epilepsias Mioclónicas , Trastornos Heredodegenerativos del Sistema Nervioso , Modelos Biológicos , Mutación , Neuropéptidos , Serpinas , alfa 1-Antitripsina , Animales , Células COS , Chlorocebus aethiops , Enfisema/genética , Enfisema/metabolismo , Enfisema/patología , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/patología , Células HEK293 , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/patología , Humanos , Ratones , Neuropéptidos/genética , Neuropéptidos/metabolismo , Células PC12 , Ratas , Serpinas/genética , Serpinas/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , NeuroserpinaRESUMEN
Airway inflammation has been implicated in evoking progressive pulmonary disorders including chronic obstructive pulmonary disease (COPD) and asthma as a result of exposure to inhaled irritants, characterized by airway fibrosis, mucus hypersecretion, and loss of alveolar integrity. The current study examined whether oleuropein, a phenylethanoid found in olive leaves, inhibited pulmonary inflammation in experimental models of interleukin (IL)-4-exposed bronchial BEAS-2B epithelial cells and ovalbumin (OVA)- or cigarette smoke (CS)-exposed BALB/c mice. Nontoxic oleuropein at 1-20 µM diminished eotaxin-1-mediated induction of α-smooth muscle actin and mucin 5AC in epithelial cells stimulated by IL-4 at the transcriptional levels. Oral supplementation of 10-20 mg/kg oleuropein reduced the airway influx of eosinophils and lymphocytes as well as IL-4 secretion in lung promoted by OVA inhalation or CS. In addition, oleuropein suppressed infiltration of macrophages and neutrophils through blocking OVA inhalation- and CS-promoted induction of ICAM-1, F4/80, CD68, and CD11b in airways. OVA-exposed pulmonary fibrosis was detected, while alveolar emphysema was evident in CS-exposed mouse lungs. In alveolar epithelial A549 cells exposed to CS extracts, oleuropein attenuated apoptotic cell loss. Collectively, oleuropein inhibited pulmonary inflammation leading to asthmatic fibrosis and alveolar emphysema driven by influx of inflammatory cells in airways exposed OVA or CS. Therefore, oleuropein may be a promising anti-inflammatory agent for treating asthma and COPD.
Asunto(s)
Asma/tratamiento farmacológico , Enfisema/tratamiento farmacológico , Iridoides/administración & dosificación , Neumonía/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Asma/etiología , Asma/genética , Asma/inmunología , Fumar Cigarrillos/efectos adversos , Enfisema/etiología , Enfisema/genética , Enfisema/inmunología , Humanos , Inmunoglobulina E/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Glucósidos Iridoides , Masculino , Ratones , Ratones Endogámicos BALB C , Neumonía/etiología , Neumonía/genética , Neumonía/inmunologíaRESUMEN
The most common genotype associated with severe α-1-antitrypsin deficiency (AATD) is the Z homozygote. The Z variant (Glu342Lys) of α-1-antitrypsin (AAT) undergoes a conformational change and is retained within the endoplasmic reticulum (ER) of hepatocytes leading to the formation of ordered polymeric chains and inclusion bodies. Accumulation of mutated protein predisposes to cirrhosis whilst plasma AAT deficiency leads to emphysema. Increased risk of liver and lung disease has also been reported in heterozygous subjects who carry Z in association with the milder S allele (Glu264Val) or even with wild-type M. However, it is unknown whether Z AAT can co-polymerize with other AAT variants in vivo. We co-expressed two AAT variants, each modified by a different tag, in cell models that replicate AAT deficiency. We used pull-down assays to investigate interactions between co-expressed variants and showed that Z AAT forms heteropolymers with S and with the rare Mmalton (Phe52del) and Mwurzburg (Pro369Ser) mutants, and to a lesser extent with the wild-type protein. Heteropolymers were recognized by the 2C1 mAb that binds to Z polymers in vivo. There was increased intracellular accumulation of AAT variants when co-expressed with Z AAT, suggesting a dominant negative effect of the Z allele. The molecular interactions between S and Z AAT were confirmed by confocal microscopy showing their colocalization within dilated ER cisternae and by positivity in Proximity Ligation Assays. These results provide the first evidence of intracellular co-polymerization of AAT mutants and contribute to understanding the risk of liver disease in SZ and MZ heterozygotes.
Asunto(s)
Enfisema/genética , Cirrosis Hepática/genética , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Alelos , Enfisema/sangre , Enfisema/complicaciones , Enfisema/fisiopatología , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Regulación de la Expresión Génica/genética , Genotipo , Células HEK293 , Hepatocitos/metabolismo , Hepatocitos/patología , Heterocigoto , Humanos , Hígado , Cirrosis Hepática/complicaciones , Cirrosis Hepática/fisiopatología , Agregado de Proteínas/genética , Conformación Proteica , Multimerización de Proteína/genética , alfa 1-Antitripsina/sangre , alfa 1-Antitripsina/química , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/fisiopatologíaRESUMEN
Emphysema is characterized by irreversibly enlarged airspaces and destruction of alveolar walls. One of the factors contributing to this disease pathogenesis is an elevation in extracellular matrix (ECM) degradation in the lung. Alveolar type II (ATII) cells produce and secrete pulmonary surfactants and proliferate to restore the epithelium after damage. We isolated ATII cells from control non-smokers, smokers and patients with emphysema to determine the role of NFE2 (nuclear factor, erythroid-derived 2). NFE2 is a heterodimer composed of two subunits, a 45 kDa (p45 NFE2) and 18 kDa (p18 NFE2) polypeptides. Low expression of p45 NFE2 in patients with emphysema correlated with a high ECM degradation. Moreover, we found that NFE2 knockdown increased cell death induced by cigarette smoke extract. We also studied the cross talk between p45 NFE2 and DJ-1. DJ-1 protein is a redox-sensitive chaperone that protects cells from oxidative stress. We detected that cigarette smoke significantly increased p45 NFE2 levels in DJ-1 KO mice compared to wild-type mice. Our results indicate that p45 NFE2 expression is induced by exposure to cigarette smoke, has a cytoprotective activity against cell injury, and its downregulation in human primary ATII cells may contribute to emphysema pathogenesis.
Asunto(s)
Enfisema/genética , Pulmón/efectos de los fármacos , Subunidad p45 del Factor de Transcripción NF-E2/genética , Proteína Desglicasa DJ-1/genética , Animales , Proliferación Celular/genética , Fumar Cigarrillos/efectos adversos , Enfisema/fisiopatología , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pulmón/metabolismo , Pulmón/fisiopatología , Ratones Noqueados , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/fisiopatologíaRESUMEN
Alpha-1 antitrypsin (AAT) deficiency-related emphysema is the fourth leading indication for lung transplant. Chymotrypsin-like elastase 1 (Cela1) is a digestive protease that is expressed during lung development in association with regions of elastin remodeling, exhibits stretch-dependent expression during lung regeneration, and binds lung elastin in a stretch-dependent manner. AAT covalently neutralizes Cela1 in vitro. We sought to determine the role of Cela1 in postnatal lung physiology, whether it interacted with AAT in vivo, and to detect any effects it may have in the context of AAT deficiency. The lungs of Cela1-/- mice had aberrant lung elastin structure and higher elastance as assessed with the flexiVent system. On the basis of in situ zymography with ex vivo lung stretch, Cela1 was solely responsible for stretch-inducible lung elastase activity. By mass spectrometry, Cela1 degraded mature elastin similarly to pancreatic elastase. Cela1 promoter and protein sequences were phylogenetically distinct in the placental mammal lineage, suggesting an adaptive role for lung-expressed Cela1 in this clade. A 6-week antisense oligonucleotide mouse model of AAT deficiency resulted in emphysema with increased Cela1 mRNA and reduction of approximately 70 kD Cela1, consistent with covalent binding of Cela1 by AAT. Cela1-/- mice were completely protected against emphysema in this model. Cela1 was increased in human AAT-deficient emphysema. Cela1 is important in physiologic and pathologic stretch-dependent remodeling processes in the postnatal lung. AAT is an important regulator of this process. Our findings provide proof of concept for the development of anti-Cela1 therapies to prevent and/or treat AAT-deficient emphysema.