RESUMO
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
Double-stranded RNA adenosine deaminase 1 (ADAR1) is significantly down-regulated in fibroblasts derived from Idiopathic Pulmonary Fibrosis (IPF) patients, and its overexpression restored levels of miRNA-21, PELI1, and SPRY2. There are two ADAR1 isoforms in humans, ADAR1-p110 and ADAR1-p150, generated by an alternative promoter. Let-7d is considered an essential microRNA in Pulmonary Fibrosis (PF). In silico analysis revealed COL3A1 and SMAD2, proteins involved in the development of IPF, as Let-7d targets. We analyzed the role of ADAR1-p110 and ADAR1-p150 isoforms in the regulation of Let-7d maturation and the effect of this regulation on the expression of COL3A1 and SMAD2 in IPF fibroblast. We demonstrated that differential expression and subcellular distribution of ADAR1 isoforms in fibroblasts contribute to the up-regulation of pri-miR-Let-7d and down-regulation of mature Let-7d. Induction of overexpression of ADAR1 reestablishes the expression of pri-miR-Let-7d and Let-7d in lung fibroblasts. The reduction of mature Let-7d upregulates the expression of COL3A1 and SMAD2. Thus, ADAR1 isoforms and Let-7d could have a synergistic role in IPF, which is a promising explanation in the mechanisms of fibrosis development, and the regulation of both molecules could be used as a therapeutic approach in IPF.
Assuntos
Adenosina Desaminase , Fibrose Pulmonar Idiopática , MicroRNAs , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNARESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease of unknown etiology. Myofibroblasts are organized in peculiar subepithelial fibroblasts foci (FF), where they abnormally persist and exclude lymphocytes by unclear mechanisms. FF are the source of an excessive extracellular matrix, which results in progressive stiffening and destruction of the lung architecture. We hypothesized that the absence of T cells inside the FF could be related, at least partially, to an inefficient function of lymphocytes induced by IPF fibroblasts. Here, we evaluated the effect of a supernatant from IPF fibroblasts on T-cell apoptosis and migration capacity. Data showed that IPF fibroblasts secrete pro-apoptotic molecules (both from extrinsic and intrinsic pathways), generating a microenvironment that induces apoptosis of T cells at 3 h of culture, despite a weak anti-apoptotic profile exhibited by these T cells. At 24 h of culture, the supernatants from both IPF and control fibroblasts provoked T-cell death. However, at this time of culture, IPF fibroblasts caused a marked decrease in T-cell migration; in contrast, control lung fibroblasts induced an increase of T-cell migration. The reduction of T-cell migratory capacity provoked by IPF fibroblasts was associated with a negative regulation of RHOA and ROCK, two essential GTPases for migration, and was independent of the expression of chemokine receptors. In conclusion, our findings demonstrate that IPF fibroblasts/myofibroblasts induce apoptosis and affect T-cell migration, revealing a mechanism involved in the virtual absence of T lymphocytes inside the FF.
Assuntos
Fibrose Pulmonar Idiopática , Apoptose , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Miofibroblastos/metabolismo , Linfócitos T/metabolismoRESUMO
Exposure to air pollutants in wildfire smoke and indoor pollution causes lung diseases. Short-term exposure to wood smoke (WS) is partially known to alter the expression of human matrix metalloproteinases (MMPs), inflammatory cytokines, and tissue inhibitors of metalloproteinases (TIMPs). Accordingly, we investigated the effect of exposing guinea pigs to WS for two and four three-hour periods on different days. The daily content of particles reported by indoor pollution was produced by 60 g of pinewood. We analyzed the cell profile and collagen content in bronchoalveolar lavages (BAL). The mRNA expression of pro-inflammatory cytokines, MMPs, and TIMPs was studied in lung tissue. Cytokines and gelatinolytic activity were analyzed in BAL and serum. The results showed that total cells, macrophages, neutrophils, and collagen increased in BAL, whereas neutrophils and lymphocytes decreased. TGF-ß1, TNF-α, IFN-γ, IL-1ß, IL-6, IL-8, MMP-2, MMP-9, TIMP-1, and TIMP-2 were upregulated in lungs, downregulating IL-12. TNF-α, IFN-γ, TGF-ß1, IL-1ß, IL-6, and IL-8 were increased in BAL and serum, decreasing IL-12. Gelatinase activity was increased in serum. Thus, guinea pigs exposed to short-term domestic doses of WS overexpressed pro-inflammatory cytokines, MMPs, and TIMPs. These results are similar to ECM remodeling and pulmonary and systemic inflammation reported in humans.
RESUMO
Wood smoke (WS) contains many harmful compounds, including polycyclic aromatic hydrocarbons (PAHs). WS induces inflammation in the airways and lungs and can lead to the development of various acute and chronic respiratory diseases. Pulmonary fibroblasts are the main cells involved in the remodeling of the extracellular matrix (ECM) during the WS-induced inflammatory response. Although fibroblasts remain in a low proliferation state under physiological conditions, they actively participate in ECM remodeling during the inflammatory response in pathophysiological states. Consequently, we used normal human lung fibroblasts (NHLFs) to assess the potential effects of the PAHs-containing wood smoke extract (WSE) on the growth rate, total collagen synthesis, and the expression levels of collagen I and III, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and the transforming growth factor (TGF)-ß1. We also assessed MMPs activity. The results showed that WSE induced a trimodal behavior in the growth rate curves in NHLFs; the growth rate increased with 0.5-1 % WSE and decreased with 2.5% WSE, without causing cell damage; 5-20% WSE inhibited the growth and induced cell damage. After 3 hours of exposure, 2.5% WSE induced an increase in total collagen synthesis and upregulation of TGF-ß1, collagen I and III, MMP-1, TIMP-1, and TIMP-2 expression. However, MMP-2 expression was downregulated and MMP-9 was not expressed. The gelatinase activity of MMP-2 was also increased. These results suggest that WSE affects the ECM remodeling in NHLFs and indicate the potential involvement of PAHs in this process.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Pneumopatias/induzido quimicamente , Extratos Vegetais/efeitos adversos , Fumaça/efeitos adversos , Proliferação de Células/efeitos dos fármacos , Humanos , Magnoliopsida/química , Madeira/químicaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.
Assuntos
Apoptose , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Citocromos c/metabolismo , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Ionomicina , Mitocôndrias/patologia , Mitomicina , Oxigênio/metabolismo , Cultura Primária de CélulasRESUMO
. In passages above ten and growing very actively, we observed that some human lung fibroblasts cultured under standard conditions were transformed into a lineage of epithelial-like cells (ELC). To systematically evaluate the possible mesenchymal-epithelial transition (MET) occurrence, fibroblasts were obtained from normal lungs and also from lungs affected by idiopathic interstitial diseases. When an unusual epithelial-like phenotypic change was observed, cultured cells were characterized by confocal immunofluorescence microscopy, immunoblotting, immunocytochemistry, cytofluorometry, gelatin zymography, RT-qPCR, and hybridization in a whole-transcript human microarray. Additionally, microvesicles fraction (MVs) from ELC and fibroblasts were used to induce MET, while the microRNAs (miRNAs) contained in the MVs were identified. Pattern-gene expression of the original fibroblasts and the derived ELC revealed profound changes, upregulating characteristic epithelial-cell genes and downregulating mesenchymal genes, with a marked increase of E-cadherin, cytokeratin, and ZO-1, and the loss of expression of α-SMA, collagen type I, and Thy-1 cell surface antigen (CD90). Fibroblasts, exposed to culture media or MVs from the ELC, acquired ELC phenotype. The miRNAs in MVs shown six expressed exclusively in fibroblasts, and three only in ELC; moreover, twelve miRNAs were differentially expressed between fibroblasts and ELC, all of them but one was overexpressed in fibroblasts. These findings suggest that the MET-like process can occur in human lung fibroblasts, either from normal or diseased lungs. However, the biological implication is unclear.
Assuntos
Transição Epitelial-Mesenquimal , Fibroblastos/patologia , Doenças Pulmonares Intersticiais/patologia , Pulmão/patologia , Actinas/metabolismo , Idoso , Biomarcadores/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Análise por Conglomerados , Colágeno Tipo I/metabolismo , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Humanos , Doenças Pulmonares Intersticiais/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fenótipo , Antígenos Thy-1/metabolismoRESUMO
BACKGROUND: Around 8-10% of individuals over 50â years of age present interstitial lung abnormalities (ILAs), but their risk factors are uncertain. METHODS: From 817 individuals recruited in our lung ageing programme at the Mexican National Institute of Respiratory Diseases, 80 (9.7%) showed ILAs and were compared with 564 individuals of the same cohort with normal high-resolution computed tomography to evaluate demographic and functional differences, and with 80 individuals randomly selected from the same cohort for biomarkers. We evaluated MUC5B variant rs35705950, telomere length, and serum levels of matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, interleukin (IL)-6, surfactant protein (SP)-D, α-Klotho and resistin. RESULTS: Individuals with ILAs were usually males (p<0.005), older than controls (p<0.0001), smokers (p=0.01), with a greater frequency of MUC5B rs35705950 (OR 3.5, 95% CI 1.3-9.4; p=0.01), and reduced diffusing capacity of the lung for carbon monoxide and oxygen saturation. Resistin, IL-6, SP-D, MMP-1, MMP-7 and MMP-13 were significantly increased in individuals with ILAs. Resistin (12±5 versus 9±4â ng·mL-1; p=0.0005) and MMP-13 (357±143 versus 298±116â pg·mL-1; p=0.004) were the most increased biomarkers. On follow-up (24±18â months), 18 individuals showed progression which was associated with gastro-oesophageal reflux disease (OR 4.1, 95% CI 1.2-12.9; p=0.02) and in females with diabetes mellitus (OR 5.3, 95% CI 1.0-27.4; p=0.01). CONCLUSIONS: Around 10% of respiratory asymptomatic individuals enrolled in our lung ageing programme show ILAs. Increased serum concentrations of pro-inflammatory molecules and MMPs are associated with ILAs.
Assuntos
Doenças Pulmonares Intersticiais , Feminino , Humanos , Pulmão/diagnóstico por imagem , Masculino , Metaloproteinase 7 da Matriz , Mucina-5B , Fatores de RiscoRESUMO
Idiopathic pulmonary fibrosis is a chronic and progressive disease of unknown cause. It is characterized by the aberrant activation of the bronchioalveolar epithelium, the formation of fibroblast foci and the excessive production extracellular matrix. The cellular and molecular mechanisms that contribute to the pathobiology of the disease are unclear. The CX3CL1-CX3CR1 axis regulates cellular responses that are known to be relevant in IPF, such as proliferation and collagen production. In this study, we characterize for the first time the expression of CX3CL1 and its receptor in lung tissue from patients with IPF; and its effect on collagen production in IPF fibroblasts. We found that CX3CL1-CX3CR1 axis has a modified expression in the lung tissue, importantly this axis is expressed on fibroblasts, and CX3CL1 decreased the collagen production in pulmonary fibroblasts derived from IPF patients.
Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Colágeno/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Receptor 1 de Quimiocina CX3C/análise , Linhagem Celular , Proliferação de Células , Quimiocina CX3CL1/análise , Colágeno/análise , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Pulmão/citologia , Cultura Primária de Células , Transdução de SinaisRESUMO
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.