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We previously reported pulmonary arterial remodelling and active endothelial to mesenchymal transition (EndMT) in smokers and patients with early COPD. In this study, we aimed to evaluate the role of different drivers of EndMT. Immunohistochemical staining for EndMT drivers, TGF-ß1, pSMAD-2/3, SMAD-7, and ß-catenin, was performed on lung resections from 46 subjects. Twelve were non-smoker-controls (NC), six normal lung function smokers (NLFS), nine patients with small-airway diseases (SAD), nine mild-moderate COPD-current smokers (COPD-CS) and ten COPD-ex-smokers (COPD-ES). Histopathological measurements were done using Image ProPlus softwarev7.0. We observed lower levels of total TGF-ß1 (p<0.05) in all smoking groups than in the non-smoking control (NC). Across arterial sizes, smoking groups exhibited significantly higher (p <0.05) total and individual layer pSMAD-2/3 and SMAD-7 than in the NC group. The ratio of SAMD-7 to pSMAD-2/3 was higher in COPD patients compared to NC. Total ß-catenin expression was significantly higher in smoking groups across arterial sizes (p <0.05), except for COPD-ES and NLFS groups in small and medium arteries, respectively. Increased total ß-catenin was positively correlated with total S100A4 in small and medium arteries (r= 0.35, 0.50; p=0.02, 0.01, respectively), with vimentin in medium arteries (r=0.42, p=0.07), and with arterial thickness of medium and large arteries (r= 0.34, 0.41, p=0.02, 0.01, respectively). This is the first study uncovering active endothelial SMAD pathway independent of TGF-ß1 in smokers, SAD, and COPD patients. Increased expression of ß-catenin indicates its potential interaction with SMAD pathway, warranting further research to identify the deviation of this classical pathway.
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Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification.
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Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Animais , Camundongos , Neutrófilos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Pulmão , InflamaçãoRESUMO
BACKGROUND: COPD is the third leading cause of death worldwide. Cigarette smoke (CS)-induced chronic inflammation inducing airway remodelling, emphysema and impaired lung function is the primary cause. Effective therapies are urgently needed. Human chymase (hCMA)1 and its orthologue mCMA1/mouse mast cell protease (mMCP)5 are exocytosed from activated mast cells and have adverse roles in numerous disorders, but their role in COPD is unknown. METHODS: We evaluated hCMA1 levels in lung tissues of COPD patients. We used mmcp5-deficient (-/-) mice to evaluate this protease's role and potential for therapeutic targeting in CS-induced experimental COPD. In addition, we used ex vivo/in vitro studies to define mechanisms. RESULTS: The levels of hCMA1 mRNA and CMA1+ mast cells were increased in lung tissues from severe compared to early/mild COPD patients, non-COPD smokers and healthy controls. Degranulated mast cell numbers and mMCP5 protein were increased in lung tissues of wild-type mice with experimental COPD. mmcp5 -/- mice were protected against CS-induced inflammation and macrophage accumulation, airway remodelling, emphysema and impaired lung function in experimental COPD. CS extract challenge of co-cultures of mast cells from wild-type, but not mmcp5 -/- mice with wild-type lung macrophages increased in tumour necrosis factor (TNF)-α release. It also caused the release of CMA1 from human mast cells, and recombinant hCMA-1 induced TNF-α release from human macrophages. Treatment with CMA1 inhibitor potently suppressed these hallmark features of experimental COPD. CONCLUSION: CMA1/mMCP5 promotes the pathogenesis of COPD, in part, by inducing TNF-α expression and release from lung macrophages. Inhibiting hCMA1 may be a novel treatment for COPD.
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Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Animais , Camundongos , Quimases/metabolismo , Mastócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Remodelação das Vias Aéreas , Enfisema Pulmonar/etiologia , Pulmão , Enfisema/complicações , Inflamação/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Structural changes involving tissue remodelling and fibrosis are major features of many pulmonary diseases, including asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Abnormal deposition of extracellular matrix (ECM) proteins is a key factor in the development of tissue remodelling that results in symptoms and impaired lung function in these diseases. Tissue remodelling in the lungs is complex and differs between compartments. Some pathways are common but tissue remodelling around the airways and in the parenchyma have different morphologies. Hence it is critical to evaluate both common fibrotic pathways and those that are specific to different compartments; thereby expanding the understanding of the pathogenesis of fibrosis and remodelling in the airways and parenchyma in asthma, COPD and IPF with a view to developing therapeutic strategies for each. Here we review the current understanding of remodelling features and underlying mechanisms in these major respiratory diseases. The differences and similarities of remodelling are used to highlight potential common therapeutic targets and strategies. One central pathway in remodelling processes involves transforming growth factor (TGF)-ß induced fibroblast activation and myofibroblast differentiation that increases ECM production. The current treatments and clinical trials targeting remodelling are described, as well as potential future directions. These endeavours are indicative of the renewed effort and optimism for drug discovery targeting tissue remodelling and fibrosis.
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Pneumopatias/tratamento farmacológico , Pneumopatias/fisiopatologia , Remodelação das Vias Aéreas/fisiologia , Asma/tratamento farmacológico , Asma/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos , Fibrose/fisiopatologia , Glicoproteínas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Metaloproteinases da Matriz/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fator de Crescimento Transformador betaRESUMO
BACKGROUND AND OBJECTIVE: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter AEC. We determined what factors are associated with ACE2 expression particularly in patients with asthma and COPD. METHODS: We obtained lower AEC from 145 people from two independent cohorts, aged 2-89 years, Newcastle (n = 115) and Perth (n = 30), Australia. The Newcastle cohort was enriched with people with asthma (n = 37) and COPD (n = 38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry was assessed by qPCR, and protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AEC. RESULTS: Increased gene expression of ACE2 was associated with older age (P = 0.03) and male sex (P = 0.03), but not with pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma patients (P = 0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in patients with asthma (P = 0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface, was increased (P = 0.02). ACE2 protein expression was also reduced in endobronchial biopsies from asthma patients. CONCLUSION: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.
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Asma/genética , COVID-19/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Peptidil Dipeptidase A/genética , SARS-CoV-2 , Asma/epidemiologia , Asma/metabolismo , Austrália/epidemiologia , COVID-19/epidemiologia , COVID-19/metabolismo , Comorbidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/biossínteseAssuntos
Infecções por Coronavirus , Pneumonia Viral , Betacoronavirus , COVID-19 , Humanos , Pandemias , Sistema Renina-Angiotensina , SARS-CoV-2RESUMO
Increased airway smooth muscle (ASM) mass is observed in chronic obstructive pulmonary disease (COPD), which is correlated with disease severity and negatively affects lung function in these patients. Thus, there is clear unmet clinical need for finding new therapies which can target airway remodeling and disease progression in COPD. Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) activated by various stress stimuli, including reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) and is known to regulate cell proliferation. ASM cells from COPD patients are hyperproliferative to mitogens in vitro However, the role of ASK1 in ASM growth is not established. Here, we aim to determine the effects of ASK1 inhibition on ASM growth and pro-mitogenic signaling using ASM cells from COPD patients. We found greater expression of ASK1 in ASM bundles of COPD lung when compared with non-COPD. Pre-treatment of ASM cells with highly selective ASK1 inhibitor, TC ASK 10 resulted in a dose-dependent reduction in mitogen (FBS, PDGF, and EGF; 72 h)-induced ASM growth as measured by CyQUANT assay. Further, molecular targetting of ASK1 using siRNA in ASM cells prevented mitogen-induced cell growth. In addition, to anti-mitogenic potential, ASK1 inhibitor also prevented TGFß1-induced migration of ASM cells in vitro Immunoblotting revealed that anti-mitogenic effects are mediated by C-Jun N-terminal kinase (JNK) and p38MAP kinase-signaling pathways as evident by reduced phosphorylation of downstream effectors JNK1/2 and p38MAP kinases, respectively, with no effect on extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2). Collectively, these findings establish the anti-mitogenic effect of ASK1 inhibition and identify a novel pathway that can be targetted to reduce or prevent excessive ASM mass in COPD.
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Movimento Celular/genética , Proliferação de Células/genética , Pulmão/metabolismo , MAP Quinase Quinase Quinase 5/genética , Miócitos de Músculo Liso/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Pulmão/citologia , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Mitógenos/farmacologia , Músculo Liso/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Interferência de RNA , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: The objective of this study was to enumerate total cells and the number of inflammatory cell differentials in large airways (LAs) versus small airways (SAs) of mild-moderate COPD, and against appropriate controls. METHODS: For LA, we used endobronchial biopsies and for SA resected lung tissues. Immunostaining was enumerated (cells per mm2 ) for macrophages, neutrophils, CD4 and CD8 T cells in the lamina propria (LP) up to 150 µM deep for LA and full wall thickness for SA. RESULTS: We confirmed hypocellularity in the LA and in the SA wall in smokers and COPD (P < 0.001). LA cellularity was least in current smokers with COPD (COPD-CS) (P < 0.01), while SA cellularity was similar across smoker/COPD groups. LA neutrophils were decreased in COPD-CS (P < 0.01), while SA neutrophil counts were unchanged. Compared with controls, LA macrophage numbers in COPD were significantly lower (P < 0.05), with SA macrophage numbers unchanged. A significant increase was observed in SA CD8+ cells in both normal smokers (P < 0.01) and COPD-CS (P < 0.001) but not in LA. CONCLUSION: These unique data indicate that the current model for airway wall inflammation in COPD is oversimplified, and contrast with innate inflammatory activation in the lumen, at least in mild-moderate disease. Any abnormalities in airway wall cell differentials are small, although exaggerated in percentage terms.
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Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Inflamação/patologia , Macrófagos , Neutrófilos , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Brônquios/patologia , Contagem de Linfócito CD4 , Feminino , Humanos , Inflamação/imunologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/imunologia , Fumar Tabaco/patologia , Adulto JovemRESUMO
Phosphodiesterases (PDE) are enzymes that catalyze the hydrolysis of cAMP/cGMP to 5'-AMP/GMP. In vitro assays have routinely assayed cAMP/cGMP levels as a direct indicator of PDE activity. Earlier PDE assays depended on radiometric detection of radiolabeled cAMP. Of late, nonradiometric cAMP detection systems have been developed that are cheaper and more amenable to high-throughput screening. Two such assays, namely the enzyme fragment complementation technology and homogeneous time-resolved fluorescence assays, are currently used for monitoring cAMP as a correlate for G-protein-coupled-receptor-induced cellular signaling events. Here, we have compared and validated both of these assays for the measurement of PDE4 enzyme activity in cell-free systems.
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AMP Cíclico/metabolismo , Ensaios Enzimáticos/métodos , Diester Fosfórico Hidrolases/metabolismo , Sistema Livre de Células , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Técnica de Imunoensaio Enzimático de Multiplicação , Fluorimunoensaio , Cinética , Limite de Detecção , Substâncias Luminescentes , Concentração Osmolar , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de TempoRESUMO
Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis-Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.
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Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/biossíntese , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/isolamento & purificação , Dictyostelium/metabolismo , Animais , Células Cultivadas , Clonagem Molecular , AMP Cíclico/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/química , Dictyostelium/química , Dictyostelium/citologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Targeting phosphodiesterase IV (PDE-IV) with small-molecule inhibitors as a therapeutic for chronic inflammatory disorders has been an active area of research interest for many years. The major drawback, however, has been to develop pharmacophores that would differentiate between targeting isoforms of PDE-IV associated with inflammation, as opposed to those that cause emesis, a major side effect associated with PDE-IV inhibition. Several different approaches have been employed, including designing subtype selective PDE-IV inhibitors. A recent approach has been to develop chemotypes that target PDE-VII, a cAMP-specific PDE, expressed widely in immune and pro-inflammatory cells. It is hypothesized that dual inhibitors, which function to inhibit both PDE-IV and VII, may achieve a higher therapeutic index and thereby exhibit a lower propensity to cause adverse side effects that are characteristic when targeting PDE-IV alone. This review focuses on the major classes of compounds that are presently being studied for their potential to inhibit PDE-VII and discusses the available data in the development of dual PDE-IV and -VII inhibitors, their biologic activity and their scope as a therapeutic choice in chronic inflammatory diseases.