RESUMO
AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis and a therapeutic target for metabolic diseases. Co/post-translational N-myristoylation of glycine-2 (Gly2) of the AMPK ß subunit has been suggested to regulate the distribution of the kinase between the cytosol and membranes through a "myristoyl switch" mechanism. However, the relevance of AMPK myristoylation for metabolic signaling in cells and in vivo is unclear. Here, we generated knockin mice with a Gly2-to-alanine point mutation of AMPKß1 (ß1-G2A). We demonstrate that non-myristoylated AMPKß1 has reduced stability but is associated with increased kinase activity and phosphorylation of the Thr172 activation site in the AMPK α subunit. Using proximity ligation assays, we show that loss of ß1 myristoylation impedes colocalization of the phosphatase PPM1A/B with AMPK in cells. Mice carrying the ß1-G2A mutation have improved metabolic health with reduced adiposity, hepatic lipid accumulation, and insulin resistance under conditions of high-fat diet-induced obesity.
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Proteínas Quinases Ativadas por AMP , Fígado Gorduroso , Animais , Camundongos , Fosforilação , Proteínas Quinases Ativadas por AMP/metabolismo , Dieta Hiperlipídica , Processamento de Proteína Pós-Traducional , Obesidade , Ácido Mirístico/metabolismo , Camundongos Endogâmicos C57BL , Proteína Fosfatase 2C/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD) is characterized by long-term airflow obstruction with cigarette smoke as a key risk factor. Extracellular matrix (ECM) alterations in COPD may lead to small airway wall fibrosis. Altered collagen cross-linking, potentially mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4), orchestrates disturbed ECM homeostasis. In this study, we investigated the effects of smoking status and presence and severity of COPD on LOs gene and protein expression in the airways and the impact of LOs inhibition on airway contraction in an ex vivo mouse model. We used gene expression data from bronchial brushings, airway smooth muscle (ASM) cells in vitro and immunohistochemistry in lung tissue to assess smoke- and COPD-associated differences in LOs gene and protein expression in the small airways. We found higher LOX expression in current- compared to ex-smokers and higher LOXL1 expression in COPD compared to non-COPD patients. LOX and LOXL2 expression were upregulated in COPD ASM cells treated with cigarette smoke extract. LOXL1 and LOXL2 protein levels were higher in small airways from current- compared to non-smokers. In COPD patients, higher LOXL1 and lower LOX protein levels were observed, but no differences for LOXL2, LOXL3, and LOXL4 protein were detected in small airways. Inhibiting LOs activity increased airway contraction in murine lung slices. COPD-associated changes in LOs, in particular LOX and LOXL1, may be related to smoking and contribute to impaired airway function, providing potential novel targets for preventing or treating small airways changes in COPD.
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Proteína-Lisina 6-Oxidase , Doença Pulmonar Obstrutiva Crônica , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Humanos , Pulmão/metabolismo , Camundongos , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Fumar/efeitos adversosRESUMO
The extracellular matrix (ECM) is the tissue microenvironment that regulates the characteristics of stromal and systemic cells to control processes such as inflammation and angiogenesis. Despite ongoing anti-inflammatory treatment, low levels of inflammation exist in the airways in asthma, which alters ECM deposition by airway smooth muscle (ASM) cells. The altered ECM causes aberrant behaviour of cells, such as endothelial cells, in the airway tissue. We therefore sought to characterize the composition and angiogenic potential of the ECM deposited by asthmatic and non-asthmatic ASM. After 72 hours under non-stimulated conditions, the ECM deposited by primary human asthmatic ASM cells was equal in total protein, collagen I, III and fibronectin content to that from non-asthmatic ASM cells. Further, the matrices of non-asthmatic and asthmatic ASM cells were equivalent in regulating the growth, activity, attachment and migration of primary human umbilical vein endothelial cells (HUVECs). Under basal conditions, asthmatic and non-asthmatic ASM cells intrinsically deposit an ECM of equivalent composition and angiogenic potential. Previous findings indicate that dysregulation of the airway ECM is driven even by low levels of inflammatory provocation. This study suggests the need for more effective anti-inflammatory therapies in asthma to maintain the airway ECM and regulate ECM-mediated aberrant angiogenesis.
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B lymphocytes are crucial for the body's humoral immune response, secreting antibodies generated against foreign antigens to fight infection. Adult murine B lymphopoiesis is initiated in the bone marrow and additional maturation occurs in the spleen. In both these organs, B lymphopoiesis involves interactions with numerous different non-hematopoietic cells, also known as stromal or microenvironment cells, which provide migratory, maturation, and survival signals. A variety of conditional knockout and transgenic mouse models have been used to identify the roles of distinct microenvironment cell types in the regulation of B lymphopoiesis. These studies have revealed that mesenchymal lineage cells and endothelial cells comprise the non-hematopoietic microenvironment cell types that support B lymphopoiesis in the bone marrow. In the spleen, various types of stromal cells and endothelial cells contribute to B lymphocyte maturation. More recently, comprehensive single cell RNA-seq studies have also been used to identify clusters of stromal cell types in the bone marrow and spleen, which will aid in further identifying key regulators of B lymphopoiesis. Here, we review the different types of microenvironment cells and key extrinsic regulators that are known to be involved in the regulation of murine B lymphopoiesis in the bone marrow and spleen.
Assuntos
Células Endoteliais , Linfopoese , Animais , Linfócitos B , Medula Óssea , Células da Medula Óssea , Camundongos , Células EstromaisRESUMO
Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited because of a lack of methods to isolate these cells. We found that murine Lin-CD31-Sca-1-CD51+ cells can be divided into 4 subpopulations by using flow cytometry based on their expression of the platelet-derived growth factor receptors ⺠and ß (PDGFR⺠and PDGFRß). The use of different skeletal lineage reporters confirmed the skeletal origin of the 4 populations. Multiplex immunohistochemistry studies revealed that all 4 populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit-fibroblast capacity, and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the 4 populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that 1 of these 4 different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B-lymphocyte precursors were also observed in these distinct skeletal sites. This cell population also supported pre-B lymphopoiesis in culture. Our method of isolating 4 distinct maturing skeletal populations will help elucidate the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.
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Linfócitos B/imunologia , Diferenciação Celular/imunologia , Linfopoese/imunologia , Músculo Esquelético/imunologia , Animais , Diferenciação Celular/genética , Linfopoese/genética , Camundongos , Camundongos TransgênicosRESUMO
The generation of adult hematopoietic stem and progenitor cells (HSPCs) from embryonic and induced pluripotent stem cells (iPSCs) will provide therapeutic benefits but is currently elusive. In this issue of Developmental Cell, Frame et al. reveal that the inflammasome has a key role in HSPCs specification during endothelial-to-hematopoietic transition (EHT).
Assuntos
Transplante de Células-Tronco Hematopoéticas , Inflamassomos , Células Sanguíneas , Diferenciação Celular , Hematopoese , AçúcaresRESUMO
Tissue remodeling/fibrosis is a major feature of all fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). It is underpinned by accumulating extracellular matrix (ECM) proteins. Fibulin-1c (Fbln1c) is a matricellular ECM protein associated with lung fibrosis in both humans and mice, and stabilizes collagen formation. Here we discovered that Fbln1c was increased in the lung tissues of IPF patients and experimental bleomycin-induced pulmonary fibrosis. Fbln1c-deficient (-/-) mice had reduced pulmonary remodeling/fibrosis and improved lung function after bleomycin challenge. Fbln1c interacted with fibronectin, periostin and tenascin-c in collagen deposits following bleomycin challenge. In a novel mechanism of fibrosis Fbln1c bound to latent transforming growth factor (TGF)-ß binding protein-1 (LTBP1) to induce TGF-ß activation, and mediated downstream Smad3 phosphorylation/signaling. This process increased myofibroblast numbers and collagen deposition. Fbln1 and LTBP1 co-localized in lung tissues from IPF patients. Thus, Fbln1c may be a novel driver of TGF-ß-induced fibrosis involving LTBP1 and may be an upstream therapeutic target.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Fibrose Pulmonar Idiopática/patologia , Proteínas de Ligação a TGF-beta Latente/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Animais , Bleomicina/toxicidade , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/cirurgia , Pulmão/citologia , Pulmão/patologia , Transplante de Pulmão , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Isoformas de Proteínas/metabolismo , Adulto JovemRESUMO
Bone marrow contains numerous different cell types arising from hematopoietic stem cells (HSCs) and non-hematopoietic mesenchymal/skeletal stem cells, in addition to other cell types such as endothelial cells- these non-hematopoietic cells are commonly referred to as stromal cells or microenvironment cells. HSC function is intimately linked to complex signals integrated by their niches, formed by combinations of hematopoietic and stromal cells. Studies of hematopoietic cells have been significantly advanced by flow cytometry methods, enabling the quantitation of each cell type in normal and perturbed situations, in addition to the isolation of these cells for molecular and functional studies. Less is known, however, about the specific niches for distinct developing hematopoietic lineages, or the changes occurring in the niche size and function in these distinct anatomical sites in the bone marrow under stress situations and ageing. Significant advances in imaging technology during the last decade have permitted studies of HSC niches in mice. Additional imaging technologies are emerging that will facilitate the study of human HSC niches in trephine BM biopsies. Here we provide an overview of imaging technologies used to study HSC niches, in addition to highlighting emerging technology that will help us to more precisely identify and characterize HSC niches in normal and diseased states.
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Células-Tronco Hematopoéticas/citologia , Imagem Molecular/métodos , Nicho de Células-Tronco , Animais , Medula Óssea/fisiologia , Humanos , Imageamento Tridimensional , Camundongos , Análise Serial de TecidosRESUMO
Retinoic acid receptor (RAR) signaling regulates bone structure and hematopoiesis through intrinsic and extrinsic mechanisms. This study aimed to establish how early in the osteoblast lineage loss of RARγ (Rarg) disrupts the bone marrow microenvironment. Bone structure was analyzed by micro-computed tomography (µCT) in Rarg-/- mice and mice with Rarg conditional deletion in Osterix-Cre-targeted osteoblast progenitors or Prrx1-Cre-targeted mesenchymal stem cells. Rarg-/- tibias exhibited less trabecular and cortical bone and impaired longitudinal and radial growth. The trabecular bone and longitudinal, but not radial, growth defects were recapitulated in Prrx1:RargΔ/Δ mice but not Osx1:RargΔ/Δ mice. Although both male and female Prrx1:RargΔ/Δ mice had low trabecular bone mass, males exhibited increased numbers of trabecular osteoclasts and Prrx1:RargΔ/Δ females had impaired mineral deposition. Both male and female Prrx1:RargΔ/Δ growth plates were narrower than controls and their epiphyses contained hypertrophic chondrocyte islands. Flow cytometry revealed that male Prrx1:RargΔ/Δ bone marrow exhibited elevated pro-B and pre-B lymphocyte numbers, accompanied by increased Cxcl12 expression in bone marrow cells. Prrx1:RargΔ/Δ bone marrow also had elevated megakaryocyte-derived Vegfa expression accompanied by smaller sinusoidal vessels. Thus, RARγ expression by Prrx1-Cre-targeted cells directly regulates endochondral bone formation and indirectly regulates tibial vascularization. Furthermore, RARγ expression by Prrx1-Cre-targeted cells extrinsically regulates osteoclastogenesis and B lymphopoiesis in male mice. © 2018 American Society for Bone and Mineral Research.
Assuntos
Linfócitos B/metabolismo , Osso e Ossos/metabolismo , Linfopoese , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Receptores do Ácido Retinoico/metabolismo , Animais , Desenvolvimento Ósseo , Medula Óssea/irrigação sanguínea , Medula Óssea/patologia , Osso Esponjoso/metabolismo , Osso Cortical/metabolismo , Feminino , Masculino , Camundongos Transgênicos , Tamanho do Órgão , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Tíbia/patologia , Receptor gama de Ácido RetinoicoRESUMO
Chronic mucus hypersecretion (CMH) contributes to the morbidity and mortality of asthma, and remains uncontrolled by current therapies in the subset of patients with severe, steroid-resistant disease. Altered cross-talk between airway epithelium and airway smooth muscle cells (ASMCs), driven by pro-inflammatory cytokines such as interleukin (IL)-1ß, provides a potential mechanism that influences CMH. This study investigated mechanisms underlying CMH by comparing IL-1ß-induced gene expression profiles between asthma and control-derived ASMCs and the subsequent paracrine influence on airway epithelial mucus production in vitroIL-1ß-treated ASMCs from asthmatic patients and healthy donors were profiled using microarray analysis and ELISA. Air-liquid interface (ALI)-cultured CALU-3 and primary airway epithelial cells were treated with identified candidates and mucus production assessed.The IL-1ß-induced CCL20 expression and protein release was increased in ASMCs from moderate compared with mild asthmatic patients and healthy controls. IL-1ß induced lower MIR146A expression in asthma-derived ASMCs compared with controls. Decreased MIR146A expression was validated in vivo in bronchial biopsies from 16 asthmatic patients versus 39 healthy donors. miR-146a-5p overexpression abrogated CCL20 release in ASMCs. CCL20 treatment of ALI-cultured CALU-3 and primary airway epithelial cells induced mucus production, while CCL20 levels in sputum were associated with increased levels of CMH in asthmatic patients.Elevated CCL20 production by ASMCs, possibly resulting from dysregulated expression of the anti-inflammatory miR-146a-5p, may contribute to enhanced mucus production in asthma.
Assuntos
Asma/metabolismo , Quimiocina CCL20/metabolismo , Interleucina-1beta/farmacologia , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Adolescente , Adulto , Idoso , Asma/tratamento farmacológico , Estudos de Casos e Controles , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Muco/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Escarro/metabolismo , Adulto JovemRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with few effective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linking mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4)] might contribute to disease pathogenesis and represent a therapeutic target. This study aimed to further our understanding of the mechanisms by which LO inhibitors might improve lung fibrosis. Lung tissues from IPF and non-IPF subjects were examined for collagen structure (second harmonic generation imaging) and LO gene (microarray analysis) and protein (immunohistochemistry and western blotting) levels. Functional effects (collagen structure and tissue stiffness using atomic force microscopy) of LO inhibitors on collagen remodelling were examined in two models, collagen hydrogels and decellularized human lung matrices. LOXL1/LOXL2 gene expression and protein levels were increased in IPF versus non-IPF. Increased collagen fibril thickness in IPF versus non-IPF lung tissues correlated with increased LOXL1/LOXL2, and decreased LOX, protein expression. ß-Aminoproprionitrile (ß-APN; pan-LO inhibitor) but not Compound A (LOXL2-specific inhibitor) interfered with transforming growth factor-ß-induced collagen remodelling in both models. The ß-APN treatment group was tested further, and ß-APN was found to interfere with stiffening in the decellularized matrix model. LOXL1 activity might drive collagen remodelling in IPF lungs. The interrelationship between collagen structural remodelling and LOs is disrupted in IPF lungs. Inhibition of LO activity alleviates fibrosis by limiting fibrillar collagen cross-linking, thereby potentially impeding the formation of a pathological microenvironment in IPF.
Assuntos
Colágenos Fibrilares/metabolismo , Fibrose Pulmonar Idiopática/enzimologia , Proteína-Lisina 6-Oxidase/metabolismo , Estudos de Casos e Controles , Colágeno Tipo I/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Hidrogéis/farmacologia , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/patologia , Proteína-Lisina 6-Oxidase/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The lung is composed of airways and lung parenchyma, and the extracellular matrix (ECM) contains the main building blocks of both components. The ECM provides physical support and stability to the lung, and as such it has in the past been regarded as an inert structure. More recent research has provided novel insights revealing that the ECM is also a bioactive environment that orchestrates the cellular responses in its environs. Changes in the ECM in the airway or parenchymal tissues are now recognized in the pathological profiles of many respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Only recently have we begun to investigate whether these ECM changes result from the disease process, or whether they constitute a driving factor that orchestrates the pathological outcomes. This review summarizes our current knowledge of the alterations in the ECM in asthma, COPD, and IPF, and the contributions of these alterations to the pathologies. Emerging data suggest that alterations in the composition, folding or rigidity of ECM proteins may alter the functional responses of cells within their environs, and in so doing change the pathological outcomes. These characteristics highlight potential avenues for targeting lung pathologies in the future. This may ultimately contribute to a better understanding of chronic lung diseases, and novel approaches for finding therapeutic solutions. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Matriz Extracelular/patologia , Pneumopatias/patologia , Asma/metabolismo , Asma/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pneumopatias/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologiaRESUMO
Airway and/or lung remodeling, involving exaggerated extracellular matrix (ECM) protein deposition, is a critical feature common to pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). Fibulin-1 (Fbln1), an important ECM protein involved in matrix organization, may be involved in the pathogenesis of these diseases. We found that Fbln1 was increased in COPD patients and in cigarette smoke-induced (CS-induced) experimental COPD in mice. Genetic or therapeutic inhibition of Fbln1c protected against CS-induced airway fibrosis and emphysema-like alveolar enlargement. In experimental COPD, this occurred through disrupted collagen organization and interactions with fibronectin, periostin, and tenascin-c. Genetic inhibition of Fbln1c also reduced levels of pulmonary inflammatory cells and proinflammatory cytokines/chemokines (TNF-α, IL-33, and CXCL1) in experimental COPD. Fbln1c-/- mice also had reduced airway remodeling in experimental chronic asthma and pulmonary fibrosis. Our data show that Fbln1c may be a therapeutic target in chronic respiratory diseases.
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Altered ECM protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or nonasthmatic HBECs and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)-ß1-regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol 3 kinase, and activin-like kinase 5 (ALK5). Asthmatic HBECs deposited higher levels of Fn in the ECM than nonasthmatic cells under basal conditions, whereas cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-ß1 increased mRNA levels and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in nonasthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells after TGF-ß1 stimulation persisted compared with nonasthmatic cells. Inhibition of ALK5 completely prevented TGF-ß1-induced Fn deposition. Importantly, ECM Fn increased HBEC proliferation and IL-6 release, decreased PGE2 secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM, is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represents a novel, therapeutic target.
Assuntos
Asma/metabolismo , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Asma/patologia , Brônquios/patologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Fibronectinas/genética , Expressão Gênica , Humanos , Mucosa Respiratória/patologia , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. The aim of this work was to identify altered proteins in LAM serum which may be potential biomarkers of disease. Serum from LAM patient volunteers and healthy control volunteers were pooled and analysis carried out using quantitative 4-plex iTRAQ technology. Differentially expressed proteins were validated using ELISAs and pathway analysis was carried out using Ingenuity Pathway Analysis. Fourteen proteins were differentially expressed in LAM serum compared to control serum (p<0.05). Further screening validated the observed differences in extracellular matrix remodelling proteins including fibronectin (30% decrease in LAM, pâ=â0.03), von Willebrand Factor (40% reduction in LAM, pâ=â0.03) and Kallikrein III (25% increase in LAM, pâ=â0.03). Pathway networks elucidated the relationships between the ECM and cell trafficking in LAM. This study was the first to highlight an imbalance in networks important for remodelling in LAM, providing a set of novel potential biomarkers. These understandings may lead to a new effective treatment for LAM in the future.
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Proteínas Sanguíneas/metabolismo , Linfangioleiomiomatose/sangue , Linfangioleiomiomatose/metabolismo , Mapas de Interação de Proteínas , Adulto , Idoso , Proteínas Sanguíneas/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Transdução de SinaisRESUMO
We hypothesised that the response to cigarette smoke in airway smooth muscle (ASM) cells from smokers with chronic obstructive pulmonary disease (COPD) would be intrinsically different from smokers without COPD, producing greater pro-inflammatory mediators and factors relating to airway remodelling. ASM cells were obtained from smokers with or without COPD, and then stimulated with cigarette smoke extract (CSE) or transforming growth factor-ß1. The production of chemokines and matrix metalloproteinases (MMPs) were measured by ELISA, and the deposition of collagens by extracellular matrix ELISA. The effects of CSE on cell attachment and wound healing were measured by toluidine blue attachment and cell tracker green wound healing assays. CSE increased the release of CXCL8 and CXCL1 from human ASM cells, and cells from smokers with COPD produced more CSE-induced CXCL1. The production of MMP-1, -3 and -10, and the deposition of collagen VIII alpha 1 (COL8A1) were increased by CSE, especially in the COPD group which had higher production of MMP-1 and deposition of COL8A1. CSE decreased ASM cell attachment and wound healing in the COPD group only. ASM cells from smokers with COPD were more sensitive to CSE stimulation, which may explain, in part, why some smokers develop COPD.
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Brônquios/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/etiologia , Fumar/efeitos adversos , Remodelação das Vias Aéreas , Linhagem Celular , Movimento Celular , Sobrevivência Celular , Colágeno Tipo VIII/metabolismo , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Fluoresceínas/química , Humanos , Imuno-Histoquímica , Inflamação , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real , Sistema Respiratório/efeitos dos fármacos , Nicotiana/efeitos adversos , Cloreto de Tolônio/química , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , CicatrizaçãoRESUMO
Extracellular matrix (ECM) remodeling contributes to the pathogenic changes in chronic obstructive pulmonary disease (COPD) and is both complex and not well understood. Collagen I, a component of the ECM altered in COPD airways, has second harmonic generation (SHG) properties. The SHG signal is coherent, propagating both forward (F) (primarily organized/mature collagen fibrils) and backward (B) (primarily disorganized/immature collagen fibrils) parallel to the incident light. The F/B SHG ratio was used to determine the proportion of organized to disorganized collagen, with lower variation in F/B ratio between sampling regions within the same patient and between patients in the same disease group compared with analyzing F and B data alone. The F/B ratio was independent of laser power drift, regions analyzed within a tissue and tissue orientation during analysis. Using this method, we identified a significant difference in collagen organization in airway tissue between COPD and non diseased. We have developed a robust optimization and calibration methodology that will allow direct comparison of data obtained at different times and from multiple microscopes, which is directly adaptable for use with other tissue types. We report a powerful new tool for advancing our understanding of pathological ECM remodeling that may uncover new therapeutic targets in the future.
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Colágeno Tipo I/análise , Processamento de Imagem Assistida por Computador/métodos , Pulmão/química , Processamento de Sinais Assistido por Computador , Adulto , Remodelação das Vias Aéreas , Matriz Extracelular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Óptica , Doença Pulmonar Obstrutiva Crônica/patologiaRESUMO
BACKGROUND: Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors. OBJECTIVE: To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors. METHODS: Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay. RESULTS: Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p < 0.001). Levels of ENA-78, GRO-α and IL-8 were significantly elevated in CM of BSMC from asthma patients (p < 0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC. CONCLUSIONS: BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC. IMPLICATIONS: CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.