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1.
Eur J Clin Invest ; 51(1): e13443, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33131070

RESUMO

BACKGROUND: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient. MATERIALS AND METHODS: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis. RESULTS: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3+ CD4- T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1ß and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation. CONCLUSIONS: Our results provide novel insight that the significant neutrophil/ CD3+ CD4- T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis.


Assuntos
/patologia , Hemorragia/patologia , Transplante de Pulmão , Pulmão/patologia , Linfonodos/patologia , Fibrose Pulmonar/patologia , Linfócitos B/patologia , Linfócitos B/ultraestrutura , Linfócitos B/virologia , /metabolismo , Cromatografia Líquida , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Células Matadoras Naturais/patologia , Células Matadoras Naturais/ultraestrutura , Células Matadoras Naturais/virologia , Pulmão/metabolismo , Pulmão/ultraestrutura , Pulmão/virologia , Linfonodos/metabolismo , Linfonodos/ultraestrutura , Linfonodos/virologia , Macrófagos Alveolares/patologia , Macrófagos Alveolares/ultraestrutura , Macrófagos Alveolares/virologia , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Monócitos/ultraestrutura , Monócitos/virologia , Neutrófilos/patologia , Neutrófilos/ultraestrutura , Neutrófilos/virologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteômica , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/cirurgia , RNA-Seq , Índice de Gravidade de Doença , Linfócitos T/patologia , Linfócitos T/ultraestrutura , Linfócitos T/virologia , Espectrometria de Massas em Tandem
2.
Life Sci ; 264: 118664, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33127511

RESUMO

Etoposide-induced protein 2.4 (EI24) is an autophagy-associated protein and acts as a tumor suppressor. However, its role in tissue fibrosis remains unknown. Herein, a downregulation of EI24 levels in the lungs from mouse pulmonary fibrosis (PF) model and lung epithelial cells was observed in response to bleomycin (BLM) or transforming growth factor-ß1 (TGF-ß1). Then, the role of EI24 in PF was investigated through the upregulation of EI24 in vitro and in vivo. EI24 inhibited epithelial-mesenchymal transition (EMT) process and extracellular matrix (ECM) production in EI24-overexpressing cells after stimulation with BLM or TGF-ß1. The overexpression of EI24 at 14 days after the establishment of the PF model through tail vein injection delayed the progression of PF. Moreover, the administration of EI24-overexpressing plasmid promoted the autophagy level in the lungs of the PF mouse model. In addition, the inhibition of autophagy by 3-methyladenine limited the role of EI24 in these processes. Thus, the current data indicated that EI24 attenuates PF through inhibition of EMT process and ECM production by promoting autophagy.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas Nucleares/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Bleomicina , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Transição Epitelial-Mesenquimal , Camundongos , Proteínas Nucleares/genética , Plasmídeos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
3.
Sci Rep ; 10(1): 16181, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999350

RESUMO

In spite of many compounds identified as antifibrotic in preclinical studies, pulmonary fibrosis remains a life-threatening condition for which highly effective treatment is still lacking. Towards improving the success-rate of bench-to-bedside translation, we investigated in vivo µCT-derived biomarkers to repeatedly quantify experimental silica-induced pulmonary fibrosis and assessed clinically relevant readouts up to several months after silicosis induction. Mice were oropharyngeally instilled with crystalline silica or saline and longitudinally monitored with respiratory-gated-high-resolution µCT to evaluate disease onset and progress using scan-derived biomarkers. At weeks 1, 5, 9 and 15, we assessed lung function, inflammation and fibrosis in subsets of mice in a cross-sectional manner. Silica-instillation increased the non-aerated lung volume, corresponding to onset and progression of inflammatory and fibrotic processes not resolving with time. Moreover, total lung volume progressively increased with silicosis. The volume of healthy, aerated lung first dropped then increased, corresponding to an acute inflammatory response followed by recovery into lower elevated aerated lung volume. Imaging results were confirmed by a significantly decreased Tiffeneau index, increased neutrophilic inflammation, increased IL-13, MCP-1, MIP-2 and TNF-α concentration in bronchoalveolar lavage fluid, increased collagen content and fibrotic nodules. µCT-derived biomarkers enable longitudinal evaluation of early onset inflammation and non-resolving pulmonary fibrosis as well as lung volumes in a sensitive and non-invasive manner. This approach and model of non-resolving lung fibrosis provides quantitative assessment of disease progression and stabilization over weeks and months, essential towards evaluation of fibrotic disease burden and antifibrotic therapy evaluation in preclinical studies.


Assuntos
Pulmão/diagnóstico por imagem , Fibrose Pulmonar/diagnóstico por imagem , Silicose/diagnóstico por imagem , Animais , Biomarcadores , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Dióxido de Silício , Silicose/metabolismo , Silicose/patologia , Microtomografia por Raio-X
4.
Am J Emerg Med ; 38(10): 2134-2138, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33071084

RESUMO

OBJECTIVE: To investigate chest computed tomography (CT) findings associated with severe COVID-19 pneumonia in the early recovery period. METHODS: We retrospectively analyzed the cases of patients diagnosed with severe COVID-19 pneumonia at a single center between January 12, 2020, and March 16, 2020. The twelve ICU patients studied had been diagnosed SARS-CoV-2 (COVID-19) nucleic acid positive. Patient clinical symptoms were relieved or disappeared, and basic clinical information and laboratory test results were collected. The study focused on the most recent CT imaging characteristics. RESULTS: The average age of the 12 patients was 58.8 ± 16.2 years. The most prevalent symptoms were fever (100%), dyspnea (100%), and cough (83.3%). All patients experienced acute respiratory distress syndrome (ARDS), of which 9 were moderate to severe. Six patients used noninvasive ventilators, and 4 patients used mechanical ventilation. One patient was treated with extracorporeal membrane oxygenation (ECMO). The lymphocyte count decreased to 0.67 ± 0.3 (× 10 9/L). The average day from illness onset to the last follow-up CT was 56.1 ± 7.7 d. The CT results showed a decrease in ground glass opacities (GGO), whereas fibrosis gradually increased. The common CT features included GGO (10/12, 83.3%), subpleural line (10/12, 83.3%), fibrous stripes (12/12, 100%), and traction bronchiectasis (10/12, 83.3%). Eight patients (66.7%) showed predominant reticulation and interlobular thickening. Four patients (33.3%) showed predominant GGO. Lung segments involved were 174/216 (80.6%). CONCLUSIONS: Fibrous stripes and GGO are common CT signs in critically ill patients with COVID-19 pneumonia in the early recovery period. Signs of pulmonary fibrosis in survivors should be carefully monitored.


Assuntos
/complicações , Fibrose Pulmonar/diagnóstico por imagem , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/patologia , /etiologia , Estudos Retrospectivos , Tomografia Computadorizada por Raios X/métodos
5.
Signal Transduct Target Ther ; 5(1): 235, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037188

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to respiratory illness and multi-organ failure in critically ill patients. Although the virus-induced lung damage and inflammatory cytokine storm are believed to be directly associated with coronavirus disease 2019 (COVID-19) clinical manifestations, the underlying mechanisms of virus-triggered inflammatory responses are currently unknown. Here we report that SARS-CoV-2 infection activates caspase-8 to trigger cell apoptosis and inflammatory cytokine processing in the lung epithelial cells. The processed inflammatory cytokines are released through the virus-induced necroptosis pathway. Virus-induced apoptosis, necroptosis, and inflammation activation were also observed in the lung sections of SARS-CoV-2-infected HFH4-hACE2 transgenic mouse model, a valid model for studying SARS-CoV-2 pathogenesis. Furthermore, analysis of the postmortem lung sections of fatal COVID-19 patients revealed not only apoptosis and necroptosis but also massive inflammatory cell infiltration, necrotic cell debris, and pulmonary interstitial fibrosis, typical of immune pathogenesis in the lung. The SARS-CoV-2 infection triggered a dual mode of cell death pathways and caspase-8-dependent inflammatory responses may lead to the lung damage in the COVID-19 patients. These discoveries might assist the development of therapeutic strategies to treat COVID-19.


Assuntos
Apoptose/imunologia , Betacoronavirus/patogenicidade , Caspase 8/imunologia , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/imunologia , Necroptose/imunologia , Pneumonia Viral/imunologia , Fibrose Pulmonar/imunologia , Animais , Caspase 8/genética , Linhagem Celular Tumoral , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/patologia , Síndrome da Liberação de Citocina/virologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-7/genética , Interleucina-7/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Transgênicos , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Fibrose Pulmonar/virologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
6.
Respir Res ; 21(1): 286, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33126869

RESUMO

It has been recently hypothesized that infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may lead to fibrotic sequelae in patients recovering from coronavirus disease 2019 (COVID-19). In this observational study, hospitalized patients with COVID-19 had a HRCT of the chest performed to detect the extension of fibrotic abnormalities via Hounsfield Units (HU). At follow-up, the lung density significantly improved in both lungs and in each lobe of all patients, being in the normal range (- 950 to - 700 HU). This study provides preliminary evidence that hospitalized patients with mild-to-moderate forms of COVID-19 are not at risk of developing pulmonary fibrosis.


Assuntos
Infecções por Coronavirus/complicações , Progressão da Doença , Pneumonia Viral/complicações , Fibrose Pulmonar/diagnóstico por imagem , Síndrome Respiratória Aguda Grave/complicações , Síndrome Respiratória Aguda Grave/terapia , Idoso , Estudos de Coortes , Terapia Combinada , Intervalos de Confiança , Infecções por Coronavirus/diagnóstico , Feminino , Seguimentos , Hospitalização/estatística & dados numéricos , Hospitais Universitários , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , Estudos Prospectivos , Fibrose Pulmonar/epidemiologia , Fibrose Pulmonar/patologia , Radiografia Torácica/métodos , Medição de Risco , Síndrome Respiratória Aguda Grave/diagnóstico
7.
Am J Physiol Cell Physiol ; 319(6): C1059-C1069, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33026833

RESUMO

CXC chemokine receptor 3 (CXCR3) A and its IFN-inducible ligands CXCL9 and CXCL10 regulate vascular remodeling and fibroblast motility. IL-13 is a profibrotic cytokine implicated in the pathogenesis of inflammatory and fibroproliferative conditions. Previous work from our laboratory has shown that CXCR3A is negatively regulated by IL-13 and is necessary for the basal regulation of the IL-13 receptor subunit IL-13Rα2. This study investigates the regulation of fibroblast phenotype, function, and downstream IL-13 signaling by CXCR3A in vitro. CXCR3A was overexpressed via transient transfection. CXCR3A-/- lung fibroblasts were isolated for functional analysis. Additionally, the contribution of CXCR3A to tissue remodeling following acute lung injury was assessed in vivo with wild-type (WT) and CXCR3-/- mice challenged with IL-13. CXCR3 and IL-13Rα2 displayed a reciprocal relationship after stimulation with either IL-13 or CXCR3 ligands. CXCR3A reduced expression of fibroblast activation makers, soluble collagen production, and proliferation. CXCR3A enhanced the basal expression of pERK1/2 while inducing IL-13-mediated downregulation of NF-κB-p65. CXCR3A-/- pulmonary fibroblasts were increasingly proliferative and displayed reduced contractility and α-smooth muscle actin expression. IL-13 challenge regulated expression of the CXCR3 ligands and soluble IL-13Rα2 levels in lungs and bronchoalveolar lavage fluid (BALF) of WT mice; this response was absent in CXCR3-/- mice. Alveolar macrophage accumulation and expression of genes involved in lung remodeling was increased in CXCR3-/- mice. We conclude that CXCR3A is a central antifibrotic factor in pulmonary fibroblasts, limiting fibroblast activation and reducing extracellular matrix (ECM) production. Therefore, targeting of CXCR3A may be a novel approach to regulating fibroblast activity in lung fibrosis and remodeling.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Fibrose Pulmonar/patologia , Receptores CXCR3/metabolismo , Células 3T3 , Animais , Linhagem Celular , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Interleucina-13/genética , Interleucina-13/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/genética , Pulmão/citologia , Pulmão/metabolismo , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Transcrição RelA/metabolismo
8.
Am J Pathol ; 190(12): 2355-2375, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33039355

RESUMO

Despite many reports about pulmonary blood vessels in lung fibrosis, the contribution of lymphatics to fibrosis is unknown. We examined the mechanism and consequences of lymphatic remodeling in mice with lung fibrosis after bleomycin injury or telomere dysfunction. Widespread lymphangiogenesis was observed after bleomycin treatment and in fibrotic lungs of prospero homeobox 1-enhanced green fluorescent protein (Prox1-EGFP) transgenic mice with telomere dysfunction. In loss-of-function studies, blocking antibodies revealed that lymphangiogenesis 14 days after bleomycin treatment was dependent on vascular endothelial growth factor (Vegf) receptor 3 signaling, but not on Vegf receptor 2. Vegfc gene and protein expression increased specifically. Extensive extravasated plasma, platelets, and macrophages at sites of lymphatic growth were potential sources of Vegfc. Lymphangiogenesis peaked at 14 to 28 days after bleomycin challenge, was accompanied by doubling of chemokine (C-C motif) ligand 21 in lung lymphatics and tertiary lymphoid organ formation, and then decreased as lung injury resolved by 56 days. In gain-of-function studies, expansion of the lung lymphatic network by transgenic overexpression of Vegfc in club cell secretory protein (CCSP)/VEGF-C mice reduced macrophage accumulation and fibrosis and accelerated recovery after bleomycin treatment. These findings suggest that lymphatics have an overall protective effect in lung injury and fibrosis and fit with a mechanism whereby lung lymphatic network expansion reduces lymph stasis and increases clearance of fluid and cells, including profibrotic macrophages.


Assuntos
Proliferação de Células/fisiologia , Fibrose/patologia , Lesão Pulmonar/patologia , Linfangiogênese/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Fibrose/metabolismo , Vasos Linfáticos/patologia , Macrófagos/metabolismo , Camundongos Transgênicos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
PLoS One ; 15(10): e0241310, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33119648

RESUMO

Silicosis is a systemic disease characterized by chronic persistent inflammation and incurable pulmonary fibrosis with the underlying molecular mechanisms to be fully elucidated. In this study, we employed tandem mass tag (TMT) based on quantitative proteomics technology to detect differentially expressed proteins (DEPs) in lung tissues of silica-exposed rats. A total of 285 DEPs (145 upregulated and 140 downregulated) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the biological pathway and functional classification of the proteins. Results showed that these DEPs were mainly enriched in the phagosome, lysosome function, complement and the coagulation cascade, glutathione metabolism, focal adhesion and ECM-receptor interactions. To validate the proteomics data, we selected and analyzed the expression trends of six proteins including CD14, PSAP, GM2A, COL1A1, ITGA8 and CLDN5 using parallel reaction monitoring (PRM). The consistent result between PRM and TMT indicated the reliability of our proteomic data. These findings will help to reveal the pathogenesis of silicosis and provide potential therapeutic targets. Data are available via ProteomeXchange with identifier PXD020625.


Assuntos
Bases de Dados de Proteínas , Regulação da Expressão Gênica/efeitos dos fármacos , Proteômica , Fibrose Pulmonar/metabolismo , Dióxido de Silício/toxicidade , Animais , Masculino , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Ratos , Ratos Wistar
10.
Nanotoxicology ; 14(9): 1175-1197, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32924694

RESUMO

We and other groups have demonstrated that exposure to nickel nanoparticles (Nano-Ni) results in severe and persistent lung inflammation and fibrosis, but the underlying mechanisms remain unclear. Here, we propose that miR-21 may play an important role in Nano-Ni-induced lung inflammation, injury, and fibrosis. Our dose- and time-response studies demonstrated that exposure of C57BL/6J (WT) mice to Nano-Ni resulted in upregulation of miR-21, proinflammatory cytokines, and profibrotic mediators. Histologically, exposure to Nano-Ni caused severe pulmonary inflammation and fibrosis. Based on the dose- and time-response studies, we chose a dose of 50 µg of Nano-Ni per mouse to compare the effects of Nano-Ni on WT with those on miR-21 KO mouse lungs. At day 3 post-exposure, Nano-Ni caused severe acute lung inflammation and injury that were reflected by increased neutrophil count, CXCL1/KC level, LDH activity, total protein concentration, MMP-2/9 protein levels and activities, and proinflammatory cytokines in the BALF or lung tissues from WT mice, which were confirmed histologically. Although Nano-Ni had similar effects on miR-21 KO mice, the above-mentioned levels were significantly lower than those in WT mice. Histologically, lungs from WT mice exposed to Nano-Ni had infiltration of a large number of polymorphonuclear (PMN) cells and macrophages in the alveolar space and interstitial tissues. However, exposure of miR-21 KO mice to Nano-Ni only caused mild acute lung inflammation and injury. At day 42 post-exposure, Nano-Ni caused extensive pulmonary fibrosis and chronic inflammation in the WT mouse lungs. However, exposure of miR-21 KO mice to Nano-Ni only caused mild lung fibrosis and chronic lung inflammation. Our results also showed that exposure to Nano-Ni caused upregulation of TGF-ß1, phospho-Smad2, COL1A1, and COL3A1 in both WT and miR-21 KO mouse lungs. However, levels were significantly lower in miR-21 KO mice than in WT mice, except TGF-ß1, which was similar in both kinds of mice. Decreased expression of Smad7 was observed in WT mouse lungs, but not in miR-21 KO mice. Our results demonstrated that knocking out miR-21 ameliorated Nano-Ni-induced pulmonary inflammation, injury, and fibrosis, suggesting the important role of miR-21 in Nano-Ni-induced pulmonary toxicity.


Assuntos
Lesão Pulmonar/induzido quimicamente , MicroRNAs/metabolismo , Nanopartículas/toxicidade , Níquel/toxicidade , Pneumonia/induzido quimicamente , Fibrose Pulmonar/induzido quimicamente , Animais , Citocinas/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Nanopartículas/química , Níquel/química , Pneumonia/metabolismo , Pneumonia/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Regulação para Cima
11.
Am J Respir Cell Mol Biol ; 63(6): 806-818, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32915635

RESUMO

Excessive release of neutrophil extracellular traps (NETs) has been implicated in several organ fibrosis, including pulmonary fibrosis. NETs constitute a phenomenon in which decorated nuclear chromatin with cytosolic proteins is released into the extracellular space. PAD4 (peptidylarginine deiminase 4) plays an important role in the formation of NETs. However, the role of NETs in the pathogenesis of pulmonary fibrosis remains undefined. Here, we identified NETs in the alveolar and interstitial lung space of mice undergoing bleomycin (BLM)-induced lung fibrosis, which was suppressed by a pan-PAD inhibitor, Cl-amidine. In vitro, BLM directly induced NETs in blood neutrophils, which was also inhibited by Cl-amidine. Furthermore, Padi4 gene knockout (PAD4-KO) in mice led to the alleviation of BLM-induced NETs and pulmonary fibrosis and to the expression of inflammatory and fibrotic genes. PAD4 deficiency prevented decreases in alveolar epithelial and pulmonary vascular endothelial cell numbers and increases in ACTA2-positive mesenchymal cells and S100A4-positive fibroblasts in the lung. Hematopoietic cell grafts from PAD4-KO mice, not wild-type mice, resolved BLM-induced lung fibrosis and fibrotic gene expression in wild-type and PAD4-KO mice, suggesting that expression of PAD4 in hematopoietic cells may be involved in the development of lung fibrosis. These data suggest that PAD4 deficiency could ameliorate BLM-induced formation of NETs and lung fibrosis, suggesting that this pathway could serve as a therapeutic target for pulmonary fibrosis treatment.


Assuntos
Armadilhas Extracelulares/genética , Pulmão/patologia , Neutrófilos/metabolismo , Proteína-Arginina Desiminase do Tipo 4/deficiência , Fibrose Pulmonar/patologia , Animais , Bleomicina/farmacologia , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/patologia , Fibrose Pulmonar/metabolismo
13.
Am J Pathol ; 190(12): 2427-2435, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32919981

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease with the histology of usual interstitial pneumonia (UIP). Although the pathologist's visual inspection is central in histologic assessments, three-dimensional microcomputed tomography (microCT) assessment may complement the pathologist's scoring. We examined associations between the histopathologic features of UIP and IPF in explanted lungs and quantitative microCT measurements, including alveolar surface density, total lung volume taken up by tissue (%), and terminal bronchiolar number. Sixty frozen samples from 10 air-inflated explanted lungs with severe IPF and 36 samples from 6 donor control lungs were scanned with microCT and processed for histologic analysis. An experienced pathologist scored three major UIP criteria (patchy fibrosis, honeycomb, and fibroblastic foci), five additional pathologic changes, and immunohistochemical staining for CD68-, CD4-, CD8-, and CD79a-positive cells, graded on a 0 to 3+ scale. The alveolar surface density and terminal bronchiolar number decreased and the tissue percentage increased in lungs with IPF compared with controls. In lungs with IPF, lower alveolar surface density and higher tissue percentage were correlated with greater scores of patchy fibrosis, fibroblastic foci, honeycomb, CD79a-positive cells, and lymphoid follicles. A decreased number of terminal bronchioles was correlated with honeycomb score but not with the other scores. The three-dimensional microCT measurements reflect the pathological UIP and IPF criteria and suggest that the reduction in the terminal bronchioles may be associated with honeycomb cyst formation.


Assuntos
Bronquíolos/patologia , Fibrose Pulmonar Idiopática/patologia , Imuno-Histoquímica , Pulmão/patologia , Fibrose Pulmonar/patologia , Idoso , Bronquíolos/metabolismo , Feminino , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Imuno-Histoquímica/métodos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Microtomografia por Raio-X
14.
Life Sci ; 260: 118399, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32918977

RESUMO

Pulmonary fibrosis is the end stage of many interstitial lung diseases, characterized by the deposition of excess extracellular matrix (ECM), destruction of normal alveolar structure, and resulting in the obstruction of gas exchange and respiratory failure. The idiopathic pulmonary fibrosis (IPF) is the most common form of pulmonary fibrosis with little effective therapies. 5-Methoxytryptophan (5-MTP) is a newly found tryptophan metabolite. Previous studies suggested that 5-MTP has the effects of anti-inflammatory, anti-tumorigenesis, vascular protection and anti-fibrosis in renal disease. Whether 5-MTP has therapeutic effect on pulmonary fibrosis is not clear. In our study, we used TGF-ß1 to stimulate human lung fibroblasts (HLFs) and bleomycin (BLM) induced pulmonary fibrosis model to investigate the effect of 5-MTP on pulmonary fibrosis. Our study demonstrated that 5-MTP could improve the lung function and attenuate the destruction of alveolar structure in BLM-induced pulmonary fibrosis mice. Furthermore, 5-MTP significantly decreased accumulation of myofibroblasts and the deposition of ECM by inhibiting the differentiation of fibroblasts to myofibroblasts and suppressing the protein expression of the ECM both in vivo and in vitro. Our results also revealed 5-MTP could inhibit the proliferation and migration of the fibroblasts in vitro, which played an important role in the progressive pulmonary fibrosis. To further investigate the mechanism of the anti-fibrosis of 5-MTP, several canonical and noncanonical signaling pathways were examined. Our results revealed that 5-MTP could inhibit the pulmonary fibrosis through downregulating the phosphorylation of TGF-ß/SMAD3, PI3K/AKT signaling pathways. Together, our study indicated that 5-MTP promises to be therapeutic agent of pulmonary fibrosis.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Triptofano/análogos & derivados , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Diferenciação Celular , Matriz Extracelular , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Triptofano/farmacologia
15.
Sci Rep ; 10(1): 15073, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934322

RESUMO

Somatic stem cells have been isolated from multiple human tissues for their potential usefulness in cell therapy. Currently, mesenchymal stromal cells (MSCs) are prepared after several passages requiring a few months of cell culture. In this study, we used a prospective isolation method of somatic stem cells from gestational or fat tissues, which were identified using CD73 antibody. CD73-positive population from various tissues existed individually in flowcytometric pattern, especially subcutaneous fat- and amniotic-derived cells showed the highest enrichment of CD73-positive cells. Moreover, the cell populations isolated with the prospective method showed higher proliferative capacity and stem cell marker expression, compared to the cell populations which isolated through several passages of culturing whole living cells: which we named "conventional method" in this paper. Furthermore, the therapeutic potential of CD73-positive cells was evaluated in vivo using a mouse model of pulmonary fibrosis. After intranasal administration, murine CD73-positive cells reduced macrophage infiltration and inhibited fibrosis development. These results suggest that further testing using CD73-positive cells may be beneficial to help establish the place in regenerative medicine use.


Assuntos
5'-Nucleotidase/metabolismo , Citometria de Fluxo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Fibrose Pulmonar , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Especificidade de Órgãos , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/terapia
16.
Ecotoxicol Environ Saf ; 205: 111283, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32977282

RESUMO

Fine particulate matter (PM2.5) airborne pollution increases the risk of chronic respiratory diseases, such as idiopathic pulmonary fibrosis (IPF), which is characterized by non-specific inflammation of the interstitial lung and extensive deposition of collagen fibers. Type 2 alveolar epithelial cells (AEC2s) are alveolar stem cells in the adult lung that contribute to the lung repair process through complex signaling. Our previous studies demonstrated that OGG1, a kind of DNA repair enzyme, have a critical role in protecting cells from oxidative damage and apoptosis induced by PM2.5, but the contribution of OGG1 in proliferation and self-renewal of AEC2s is not known. Here, we constructed OGG1-/-mice to test the effect and mechanism of OGG1 on PM2.5-induced pulmonary fibrosis and injury in vivo. We detected proliferation and self-renewal of OGG1 overexpression or OGG1 knockout AEC2s after PM2.5 injury by flow cytometry and clone formation. We observed that knockout of OGG1 aggravated pulmonary fibrosis, oxidative stress, and AEC2 cell death in PM2.5-injured mice. In addition, OGG1 is required for the proliferation and renewal of AEC2s after PM2.5 injury. Overexpression of OGG1 promotes the proliferation and self-renewal of AEC2s by inhibiting PM2.5-mediated oxidative stress and NF-κB signaling hyperactivation in vitro. Furthermore, NF-κB inhibitors promoted proliferation and self-renewal of OGG1-deficient AEC2s cells after PM2.5 injury, and attenuated PM2.5-induced pulmonary fibrosis and injury in mice. These data establish OGG1 as a regulator of NF-κB signal that serves to regulate AEC2 cell proliferation and self-renewal, and suggest a mechanism that inhibition of the NF-κB signaling pathway may represent a potential therapeutic strategy for IPF patients with low-expression of OGG1.


Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais Alveolares/efeitos dos fármacos , Autorrenovação Celular/genética , DNA Glicosilases/metabolismo , Material Particulado/toxicidade , Fibrose Pulmonar/induzido quimicamente , Células-Tronco/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , DNA Glicosilases/genética , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia
17.
Phytomedicine ; 78: 153298, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32781391

RESUMO

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-ß mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease. PURPOSE: The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-ß mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects. METHODS: The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-ß to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA. RESULTS: In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-ß modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-ß1/BLM-mediated increase of TGF-ß/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations. CONCLUSION: Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-ß/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.


Assuntos
Colágeno/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Genisteína/farmacologia , Miofibroblastos/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina/toxicidade , Diferenciação Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Ratos Wistar , Reprodutibilidade dos Testes , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia
18.
Biochim Biophys Acta Mol Cell Res ; 1867(11): 118806, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32739525

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive and fibrosing interstitial pneumonia of unknown cause. The main feature of IPF is a heterogeneous appearance with areas of sub-pleural fibrosis. However, the mechanism of sub-pleural fibrosis was poorly understood. In this study, our in vivo study revealed that pleural mesothelial cells (PMCs) migrated into lung parenchyma and localized alongside lung fibroblasts in sub-pleural area in mouse pulmonary fibrosis. Our in vitro study displayed that cultured-PMCs-medium induced lung fibroblasts transforming into myofibroblast, cultured-fibroblasts-medium promoted mesothelial-mesenchymal transition of PMCs. Furthermore, these changes in lung fibroblasts and PMCs were prevented by blocking TGF-ß1/Smad2/3 signaling with SB431542. TGF-ß1 neutralized antibody attenuated bleomycin-induced pulmonary fibrosis. Similar to TGF-ß1/Smad2/3 signaling, wnt/ß-catenin signaling was also activated in the process of PMCs crosstalk with lung fibroblasts. Moreover, inhibition of CD147 attenuated cultured-PMCs-medium induced collagen-I synthesis in lung fibroblasts. Blocking CD147 signaling also prevented bleomycin-induced pulmonary fibrosis. Our data indicated that crosstalk between PMC and lung fibroblast contributed to sub-pleural pulmonary fibrosis. TGF-ß1, Wnt/ß-catenin and CD147 signaling was involved in the underling mechanism.


Assuntos
Epitélio/efeitos dos fármacos , Pulmão/metabolismo , Pleura/efeitos dos fármacos , Fibrose Pulmonar/genética , Animais , Benzamidas/farmacologia , Movimento Celular/genética , Dioxóis/farmacologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Epitélio/patologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Pleura/metabolismo , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Fator de Crescimento Transformador beta1/genética
19.
Am J Physiol Cell Physiol ; 319(5): C895-C905, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32755451

RESUMO

Interstitial lung disease (ILD) comprises of a group of diffuse parenchymal lung disorders that are strongly associated with substantial morbidity and mortality. Previous studies have highlighted the therapeutic significance of microRNAs (miRNAs) in the treatment of ILD. Thus this study aims to investigate the mechanism by which miR-140 affects ILD through the regulation of osteoglycin (OGN)-Wnt signaling pathway. Gene expression microarray analysis was performed to screen ILD-related differentially expressed genes and miRNAs that regulated OGN. The targeting relationship between miR-140 and OGN was verified. Ectopic expression and knockdown experiments were performed in lung fibroblasts to explore the potential mechanism of action of miR-140 in ILD. The expression of miR-140, OGN, as well as Wnt- and pulmonary fibrosis-related factors, was determined by RT-qPCR and Western blot analysis. In addition, cell viability and apoptosis were examined. OGN was found to be negatively regulated by miR-140. The ectopic expression of miR-140 and OGN silencing resulted in increased lung fibroblast apoptosis and Wnt3a expression, along with reduced proliferation and pulmonary fibrosis. Our results also revealed that miR-140 decreased OGN, thereby activating the Wnt signaling pathway, which was observed to further affect the expression of genes associated with the progression of pulmonary fibrosis in mouse fibroblasts. In conclusion, the key findings from our study suggest that overexpressed miR-140 suppresses ILD development via the Wnt signaling pathway by downregulating OGN, which could potentially be used as a therapeutic target for ILD.


Assuntos
Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Doenças Pulmonares Intersticiais/genética , Pulmão/metabolismo , MicroRNAs/genética , Fibrose Pulmonar/genética , Proteína Wnt3A/genética , Animais , Apoptose/genética , Pareamento de Bases , Sequência de Bases , Bleomicina/administração & dosagem , Estudos de Casos e Controles , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/patologia , Doenças Pulmonares Intersticiais/induzido quimicamente , Doenças Pulmonares Intersticiais/metabolismo , Doenças Pulmonares Intersticiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Cultura Primária de Células , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo
20.
Nat Commun ; 11(1): 4254, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848143

RESUMO

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFß1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFß1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF.


Assuntos
Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Tiorredoxinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Bleomicina/toxicidade , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Deleção de Genes , Humanos , Fibrose Pulmonar Idiopática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Estabilidade Proteica , Fibrose Pulmonar/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/química , Transdução de Sinais , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Regulação para Cima
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