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Background: Hypersensitivity pneumonitis (HP) is an inflammatory disorder affecting lung parenchyma and often evolves into fibrosis (fHP). The altered regulation of genes involved in the pathogenesis of the disease is not well comprehended, while the role of microRNAs in lung fibroblasts remains unexplored. Methods: We used integrated bulk RNA-Seq and enrichment pathway bioinformatic analyses to identify differentially expressed (DE)-miRNAs and genes (DEGs) associated with HP lungs. In vitro, we evaluated the expression and potential role of miR-155-5p in the phenotype of fHP lung fibroblasts. Loss and gain assays were used to demonstrate the impact of miR-155-5p on fibroblast functions. In addition, mir-155-5p and its target TP53INP1 were analyzed after treatment with TGF-ß, IL-4, and IL-17A. Results: We found around 50 DEGs shared by several databases that differentiate HP from control and IPF lungs, constituting a unique HP lung transcriptional signature. Additionally, we reveal 18 DE-miRNAs that may regulate these DEGs. Among the candidates likely associated with HP pathogenesis was miR-155-5p. Our findings indicate that increased miR-155-5p in fHP fibroblasts coincides with reduced TP53INP1 expression, high proliferative capacity, and a lack of senescence markers compared to IPF fibroblasts. Induced overexpression of miR-155-5p in normal fibroblasts remarkably increases the proliferation rate and decreases TP53INP1 expression. Conversely, miR-155-5p inhibition reduces proliferation and increases senescence markers. TGF-ß, IL-4, and IL-17A stimulated miR-155-5p overexpression in HP lung fibroblasts. Conclusion: Our findings suggest a distinctive signature of 53 DEGs in HP, including CLDN18, EEF2, CXCL9, PLA2G2D, and ZNF683, as potential targets for future studies. Likewise, 18 miRNAs, including miR-155-5p, could be helpful to establish differences between these two pathologies. The overexpression of miR-155-5p and downregulation of TP53INP1 in fHP lung fibroblasts may be involved in his proliferative and profibrotic phenotype. These findings may help differentiate and characterize their pathogenic features and understand their role in the disease.
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Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFß-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated ß Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.
Assuntos
Proteínas de Ligação a DNA , Fibrose Pulmonar Idiopática , Fatores de Transcrição , Animais , Bleomicina/toxicidade , Senescência Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibrose , Histonas , Humanos , Hidroxiprolina , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53 , beta-Galactosidase/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a lethal age-related lung disease whose pathogenesis involves an aberrant response of alveolar epithelial cells (AEC). Activated epithelial cells secrete mediators that participate in the activation of fibroblasts and the excessive deposition of extracellular matrix proteins. Previous studies indicate that matrix metalloproteinase 14 (MMP14) is increased in the lung epithelium in patients with IPF, however, the role of this membrane-type matrix metalloproteinase has not been elucidated. In this study, the role of Mmp14 was explored in experimental lung fibrosis induced with bleomycin in a conditional mouse model of lung epithelial MMP14-specific genetic deletion. Our results show that epithelial Mmp14 deficiency in mice increases the severity and extension of fibrotic injury and affects the resolution of the lesions. Gain-and loss-of-function experiments with human epithelial cell line A549 demonstrated that cells with a deficiency of MMP14 exhibited increased senescence-associated markers. Moreover, conditioned medium from these cells increased fibroblast expression of fibrotic molecules. These findings suggest a new anti-fibrotic mechanism of MMP14 associated with anti-senescent activity, and consequently, its absence results in impaired lung repair. Increased MMP14 in IPF may represent an anti-fibrotic mechanism that is overwhelmed by the strong profibrotic microenvironment that characterizes this disease.
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Células Epiteliais/patologia , Fibrose Pulmonar Idiopática/genética , Metaloproteinase 14 da Matriz/genética , Alvéolos Pulmonares/metabolismo , Células A549 , Actinas/genética , Actinas/metabolismo , Animais , Bleomicina/administração & dosagem , Senescência Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Metaloproteinase 14 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Several lung structural and functional abnormalities may occur associated with aging, including emphysema. In this study, we evaluated the frequency and risk factors associated with emphysema in respiratory asymptomatic individuals enrolled in our Lung Aging Program. From a cohort of 687 subjects, we found by high-resolution computed tomography (HRCT) 29 individuals (4%) with emphysematous changes that were compared with 87 controls (3:1) randomly selected from the same cohort. METHODS: This was a transversal, observational, case-control study where we examined demographics and functional characteristics, as well as telomere length and serum Klotho concentration, two conditions that have been associated with aging and some aging-associated diseases including emphysema. RESULTS: Individuals with subclinical pulmonary emphysema were older (72 ± 9 versus 67 ± 6 years), and primarily smoker males with low body mass index. Despite that they were asymptomatic, two of them exhibited a decrease of forced expiratory volume in 1 s (FEV1), with a lower FEV1/FVC suggesting airway obstruction. Cigarette smoking (OR = 5.43, CI95% 1.8-16.7), family history of lung disease (OR = 4.32, CI95% 1.0-19.0) and lower body mass index (OR 7.22, CI95% 1.2-3.5) were risk factors for the development of lung emphysematous changes. No association was found with telomere length and Klotho serum concentration. CONCLUSION: Our findings reveal that a small but important percentage of older people without respiratory symptoms, present pulmonary emphysema and indicate that smoking exposure and genetic background may contribute to etiological factors.
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Envelhecimento , Pulmão/fisiopatologia , Enfisema Pulmonar/diagnóstico , Enfisema Pulmonar/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Fumar Cigarros/efeitos adversos , Feminino , Glucuronidase/sangue , Humanos , Proteínas Klotho , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Enfisema Pulmonar/sangue , Testes de Função Respiratória , Fatores de Risco , Telômero/fisiologia , Tomografia Computadorizada por Raios XRESUMO
Chronic Obstructive Pulmonary Disease (COPD) and Idiopathic Pulmonary Fibrosis (IPF) have contrasting clinical and pathological characteristics and interesting whole-genome transcriptomic profiles. However, data from public repositories are difficult to reprocess and reanalyze. Here, we present PulmonDB, a web-based database (http://pulmondb.liigh.unam.mx/) and R library that facilitates exploration of gene expression profiles for these diseases by integrating transcriptomic data and curated annotation from different sources. We demonstrated the value of this resource by presenting the expression of already well-known genes of COPD and IPF across multiple experiments and the results of two differential expression analyses in which we successfully identified differences and similarities. With this first version of PulmonDB, we create a new hypothesis and compare the two diseases from a transcriptomics perspective.
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Bases de Dados Genéticas , Redes Reguladoras de Genes , Fibrose Pulmonar Idiopática/genética , Doença Pulmonar Obstrutiva Crônica/genética , Curadoria de Dados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Internet , Sequenciamento do ExomaRESUMO
Interstitial lung abnormalities (ILA) represent aging-associated bilateral interstitial abnormalities in nondependent areas of the lung. However, the aging mechanisms associated with ILA remain uncertain. α-Klotho is an anti-aging molecule that decreases progressively with age, and abnormally low circulating levels of this protein have been revealed in several chronic-degenerative diseases. In this study, we evaluated α-Klotho serum concentrations in individuals with ILA, and examined whether its levels were associated with pulmonary function decline. α-Klotho was measured by ELISA in 50 respiratory asymptomatic adults with ILA and 150 healthy individuals over 60 years. Compared with controls, ILA subjects were predominantly older males, and showed lower lung diffusing capacity (DLCO), higher desaturation after exercise, and higher concentrations of serum matrix metalloprotease-7 (6.24 ± 4.1 versus 4.3 ± 1.7 ng/ml; p = 0.002). No differences were found in serum concentrations of α-Klotho. However, lower levels of this protein in ILA significantly correlated with lower values of forced vital capacity (Rho = 0.39; p = 0.005), forced expiratory volume in one second (Rho = 0.39; p = 0.005), and DLCO (Rho = 0.29, p = 0.04). These findings suggest that decreased concentrations of α-Klotho may be a predictive biomarker of accelerated decline of lung function in individuals with ILA.
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Glucuronidase/sangue , Doenças Pulmonares Intersticiais/sangue , Idoso , Biomarcadores/sangue , Feminino , Humanos , Proteínas Klotho , Doenças Pulmonares Intersticiais/fisiopatologia , Masculino , Metaloproteinase 7 da Matriz/sangue , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: microRNAs (miRNAs) are small non-coding 1RNAs that post-transcriptionally regulate gene expression. Recent evidence shows that adenosine deaminases that act on RNA (ADAR) can edit miRNAs. miRNAs are involved in the development of different diseases, such as idiopathic pulmonary fibrosis (IPF). In IPF, about 40% of the miRNAs are differentially expressed with respect to controls. Among these miRNAs, miRNA-21 has been found over-expressed in IPF and its targets are anti-fibrosing molecules such as PELI1 and SPRY2. The objective of this study is to determine the role of ADAR1 and 2 on the expression of miRNA-21 in human lung fibroblasts trough quantification of gene expression, protein levels, and overexpression of ADAR1 and 2. METHODS: Six control and six fibrotic primary fibroblast cell cultures were used for RNA extraction, ADAR1, ADAR2, PELI1, SPRY2, miRNA-21, and pri-miRNA-21 expression was measured. Subsequently, two fibrotic fibroblast cultures were used for overexpression of ADAR1 and ADAR2, and they were stimulated with TGFß1. Real-time PCR and Western blot were performed. RESULTS: ADAR1 is significantly downregulated in IPF fibroblasts; the overexpression of ADAR1 and ADAR2 reestablishes the expression levels of miRNA-21, PELI1, and SPRY2 in fibroblasts of patients with IPF. CONCLUSION: These changes in the processing of miRNAs have great value in pathology diagnosis, including lung diseases, and play an important role in the understanding of molecular mechanisms involved in the development of different pathologies, as well as representing new therapeutic targets.
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Adenosina Desaminase/metabolismo , Fibroblastos/enzimologia , Fibrose Pulmonar Idiopática/enzimologia , Pulmão/enzimologia , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação Enzimológica da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Fator de Crescimento Transformador beta1/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and devastating lung disorder of unknown origin, with very poor prognosis and no effective treatment. The disease is characterized by abnormal activation of alveolar epithelial cells, which secrete numerous mediators involved in the expansion of the fibroblast population, its differentiation to myofibroblasts, and in the exaggerated accumulation of extracellular matrix provoking the loss of lung architecture. Among the excessively produced mediators are several matrix metalloproteases (MMPs) which may contribute to modify the lung microenvironment by various mechanisms. Thus, these enzymes can not only degrade all the components of the extracellular matrix, but they are also able to release, cleave and activate a wide range of growth factors, cytokines, chemokines and cell surface receptors affecting numerous cell functions including adhesion, proliferation, differentiation, recruiting and transmigration, and apoptosis. Therefore, dysregulated expression of MMPs may have profound impact on the biopathological mechanisms implicated in the development of IPF. This review focuses on the current and emerging evidence regarding the role of MMPs on the fibrotic processes in IPF as well as in mouse models of lung fibrosis.