RESUMO
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.
Assuntos
Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Broncodilatadores , Enfisema/genética , Humanos , Modelos Estatísticos , Fenótipo , Prognóstico , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Enfisema Pulmonar/genéticaRESUMO
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.
Assuntos
Enfisema , Doença Pulmonar Obstrutiva Crônica , RNA Longo não Codificante , Humanos , Apoptose/genética , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Enfisema/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Material Particulado/toxicidade , Doença Pulmonar Obstrutiva Crônica/genética , RNA Longo não Codificante/genéticaRESUMO
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.
Assuntos
Actomiosina/genética , Pulmão/crescimento & desenvolvimento , Organogênese/genética , Alvéolos Pulmonares/crescimento & desenvolvimento , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/patologia , Linhagem da Célula/genética , Citoesqueleto/genética , Enfisema/genética , Enfisema/patologia , Gases/metabolismo , Humanos , Pulmão/patologia , Mesoderma/citologia , Mesoderma/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Tretinoína/metabolismoRESUMO
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.
Assuntos
Fumar Cigarros/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Enfisema/metabolismo , Epigênese Genética/fisiologia , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Idoso , Animais , Apoptose/fisiologia , Fumar Cigarros/efeitos adversos , Fumar Cigarros/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Enfisema/etiologia , Enfisema/genética , Feminino , Humanos , Exposição por Inalação/efeitos adversos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
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.
Assuntos
Enfisema/genética , Mutação , Doença Pulmonar Obstrutiva Crônica/genética , Enfisema Pulmonar/genética , Fibrose Pulmonar/genética , Receptor para Produtos Finais de Glicação Avançada/genética , Idoso , Alelos , Povo Asiático , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Receptor para Produtos Finais de Glicação Avançada/metabolismoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Enfisema/tratamento farmacológico , Enfisema/genética , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Enfisema/etiologia , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Doença Pulmonar Obstrutiva Crônica/complicações , Transdução de Sinais/genética , Superóxidos/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
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.
Assuntos
Enfisema/genética , Homeostase/fisiologia , Células-Tronco/metabolismo , beta Catenina/metabolismo , Animais , Enfisema/patologia , Humanos , Camundongos , Via de Sinalização WntRESUMO
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.
Assuntos
Anti-Inflamatórios/uso terapêutico , Enfisema/tratamento farmacológico , Ácido Gálico/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Enfisema/genética , Enfisema/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , Elastase Pancreática , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Fumaça/efeitos adversos , Inibidor Tecidual de Metaloproteinase-1/genética , NicotianaRESUMO
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.
Assuntos
Bronquiolite/genética , Enfisema/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Doença Pulmonar Obstrutiva Crônica/genética , Análise por Conglomerados , Ontologia Genética , Humanos , Anotação de Sequência Molecular , Fenótipo , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Enfisema/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Loci Gênicos , Predisposição Genética para Doença , Pulmão/patologia , Fator de Crescimento Transformador beta2/biossíntese , Idoso , Idoso de 80 Anos ou mais , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta2/genética , Estados UnidosRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Enfisema/genética , Pulmão/patologia , Metaloproteinases da Matriz/genética , Fenótipo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica/genética , Animais , Fumar Cigarros/efeitos adversos , Técnicas de Inativação de Genes , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , CamundongosRESUMO
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.
Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Rearranjo Gênico , Neoplasias Pulmonares/diagnóstico por imagem , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Enfisema/epidemiologia , Enfisema/genética , Receptores ErbB/genética , Feminino , Humanos , Linfadenopatia/epidemiologia , Linfadenopatia/genética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
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.
Assuntos
Autofagia/efeitos dos fármacos , Ceramidas/metabolismo , Misturas Complexas/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Enfisema/genética , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversos , Ceramidase Ácida/genética , Ceramidase Ácida/metabolismo , Animais , Antioxidantes/farmacologia , Autofagia/genética , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/patologia , Estudos de Casos e Controles , Linhagem Celular , Misturas Complexas/isolamento & purificação , Cisteamina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Enfisema/tratamento farmacológico , Enfisema/metabolismo , Enfisema/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Genfibrozila/farmacologia , Expressão Gênica , Humanos , Hipolipemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Nicotiana/efeitos adversos , Nicotiana/químicaRESUMO
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.
Assuntos
Cútis Laxa/genética , ATPases Translocadoras de Prótons/genética , Pele/patologia , Adulto , Idoso , Agenesia do Corpo Caloso/genética , Catarata/genética , Criança , Pré-Escolar , Códon sem Sentido , Consanguinidade , Cútis Laxa/patologia , Tecido Elástico/patologia , Enfisema/genética , Face/anormalidades , Feminino , Glicosilação , Transtornos Hemorrágicos/genética , Humanos , Masculino , Fenótipo , Processamento de Proteína Pós-Traducional , Sítios de Splice de RNA/genética , Adulto JovemRESUMO
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.
Assuntos
Enfisema , Epilepsias Mioclônicas , Transtornos Heredodegenerativos do Sistema Nervoso , Modelos Biológicos , Mutação , Neuropeptídeos , Serpinas , alfa 1-Antitripsina , Animais , Células COS , Chlorocebus aethiops , Enfisema/genética , Enfisema/metabolismo , Enfisema/patologia , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/metabolismo , Epilepsias Mioclônicas/patologia , Células HEK293 , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Humanos , Camundongos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Células PC12 , Ratos , Serpinas/genética , Serpinas/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , NeuroserpinaRESUMO
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.
Assuntos
Asma/tratamento farmacológico , Enfisema/tratamento farmacológico , Iridoides/administração & dosagem , Pneumonia/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Asma/etiologia , Asma/genética , Asma/imunologia , Fumar Cigarros/efeitos adversos , Enfisema/etiologia , Enfisema/genética , Enfisema/imunologia , Humanos , Imunoglobulina E/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Glucosídeos Iridoides , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/etiologia , Pneumonia/genética , Pneumonia/imunologiaRESUMO
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.
Assuntos
Enfisema/genética , Cirrose Hepática/genética , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Alelos , Enfisema/sangue , Enfisema/complicações , Enfisema/fisiopatologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Regulação da Expressão Gênica/genética , Genótipo , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/patologia , Heterozigoto , Humanos , Fígado , Cirrose Hepática/complicações , Cirrose Hepática/fisiopatologia , Agregados Proteicos/genética , Conformação Proteica , Multimerização Proteica/genética , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/química , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/fisiopatologiaRESUMO
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.
Assuntos
Enfisema/genética , Pulmão/efeitos dos fármacos , Subunidade p45 do Fator de Transcrição NF-E2/genética , Proteína Desglicase DJ-1/genética , Animais , Proliferação de Células/genética , Fumar Cigarros/efeitos adversos , Enfisema/fisiopatologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/metabolismo , Pulmão/fisiopatologia , Camundongos Knockout , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/fisiopatologiaRESUMO
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.