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1.
Am J Respir Cell Mol Biol ; 50(6): 1053-63, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24393021

RESUMO

Airway smooth muscle (ASM) cells are thought to contribute to the pathogenesis of allergic asthma by orchestrating and perpetuating airway inflammation and remodeling responses. In this study, we evaluated the IL-17RA signal transduction and gene expression profile in ASM cells from subjects with mild asthma and healthy individuals. Human primary ASM cells were treated with IL-17A and probed by the Affymetrix GeneChip array, and gene targets were validated by real-time quantitative RT-PCR. Genomic analysis underlined the proinflammatory nature of IL-17A, as multiple NF-κB regulatory factors and chemokines were induced in ASM cells. Transcriptional regulators consisting of primary response genes were overrepresented and displayed dynamic expression profiles. IL-17A poorly enhanced IL-1ß or IL-22 gene responses in ASM cells from both subjects with mild asthma and healthy donors. Interestingly, protein modifications to the NF-κB regulatory network were not observed after IL-17A stimulation, although oscillations in IκBε expression were detected. ASM cells from subjects with mild asthma up-regulated more genes with greater overall variability in response to IL-17A than from healthy donors. Finally, in response to IL-17A, ASM cells displayed rapid activation of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway and increased nuclear levels of phosphorylated extracellular signal-regulated kinase. Taken together, our results suggest that IL-17A mediated modest gene expression response, which, in cooperation with the NF-κB signaling network, may regulate the gene expression profile in ASM cells.


Assuntos
Asma/genética , Asma/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Miócitos de Músculo Liso/metabolismo , Adulto , Asma/patologia , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Miócitos de Músculo Liso/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais , Transcriptoma , Regulação para Cima , Interleucina 22
2.
Proc Natl Acad Sci U S A ; 106(26): 10775-80, 2009 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-19541629

RESUMO

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)). A rise in [Ca(2+)](i) is normalized by rapid reuptake of Ca(2+) into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca(2+) (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca(2+)](i), cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca(2+)](i) following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca(2+) re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca](i) after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.


Assuntos
Asma/metabolismo , Brônquios/metabolismo , Retículo Sarcoplasmático/enzimologia , Asma/patologia , Asma/fisiopatologia , Western Blotting , Brônquios/patologia , Brônquios/fisiopatologia , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocina CCL11/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Homeostase , Humanos , Imuno-Histoquímica , Interleucina-13/farmacologia , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático
3.
Am J Respir Cell Mol Biol ; 43(3): 286-95, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19843707

RESUMO

Matrix metalloproteinases (MMPs) recently appeared as key regulators of inflammation, allowing the recruitment and clearance of inflammatory cells and modifying the biological activity of many peptide mediators by cleavage. MMP-19 is newly described, and it preferentially cleaves matrix proteins such as collagens and tenascin-C. The role of MMP-19 in asthma has not been described to date. The present study sought to assess the expression of MMP-19 in a murine asthma model, and to address the biological effects of MMP-19 deficiency in mice. Allergen-exposed, wild-type mice displayed increased expression of MMP-19 mRNA and an increased number of MMP-19-positive cells in the lungs, as detected by immunohistochemistry. After an allergen challenge of MMP-19 knockout (MMP-19(-/-)) mice, exacerbated eosinophilic inflammation was detected in bronchoalveolar lavage fluid and bronchial tissue, along with increased airway responsiveness to methacholine. A shift toward increased T helper-2 lymphocyte (Th2)-driven inflammation in MMP-19(-/-) mice was demonstrated by (1) increased numbers of cells expressing the IL-33 receptor T(1)/ST(2) in lung parenchyma, (2) increased IgG(1) levels in serum, and (3) higher levels of IL-13 and eotaxin-1 in lung extracts. Tenascin-C was found to accumulate in peribronchial areas of MMP-19(-/-) after allergen challenges, as assessed by Western blot and immunohistochemistry analyses. We conclude that MMP-19 is a new mediator in asthma, preventing tenascin-C accumulation and directly or indirectly controlling Th2-driven airway eosinophilia and airway hyperreactivity. Our data suggest that MMP-19 may act on Th2 inflammation homeostasis by preventing the accumulation of tenascin protein.


Assuntos
Alérgenos/farmacologia , Asma/metabolismo , Eosinófilos/metabolismo , Pulmão/metabolismo , Metaloproteinases da Matriz Secretadas/deficiência , Tenascina/metabolismo , Adulto , Animais , Asma/patologia , Western Blotting , Medula Óssea/metabolismo , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Líquido da Lavagem Broncoalveolar , Células Cultivadas , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Interleucina-13/farmacologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/genética , Sistema Respiratório/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Tenascina/genética , Células Th2/metabolismo
4.
Pulm Pharmacol Ther ; 22(5): 446-54, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19022391

RESUMO

SCOPE OF THE REVIEW: Our knowledge of the multifunctional nature of airway smooth muscle (ASM) has expanded rapidly in the last decade, but the underlying molecular mechanisms and how current therapies for obstructive airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD), affect these are still being elucidated. Our current knowledge has built on the pharmacology of human ASM contraction and relaxation established prior to that and which is reviewed in detail elsewhere in this issue. The advent of methods to isolate and culture ASM cells, especially human ASM cells, has made it possible to study how they may contribute to airway remodelling through their synthetic, proliferative, and migratory capacities. Now the underlying molecular mechanisms of ASM growth factor secretion, extracellular matrix (ECM) production, proliferation and migration, as well as contraction and relaxation, are being determined. A complex network of signalling pathways leading to gene transcription in ASM cells permits this functional plasticity in healthy and diseased airways. This review is an overview of the effects of current therapies, and some of those in development, on key signalling pathways and transcription factors involved in these ASM functions.


Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Músculo Liso/efeitos dos fármacos , Sistema Respiratório/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Agonistas Adrenérgicos beta/uso terapêutico , Animais , Antiasmáticos/uso terapêutico , Asma/metabolismo , Antagonistas Colinérgicos/farmacologia , Antagonistas Colinérgicos/uso terapêutico , Cromolina Sódica/farmacologia , Cromolina Sódica/uso terapêutico , Quimioterapia Combinada , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Humanos , Antagonistas de Leucotrienos/farmacologia , Antagonistas de Leucotrienos/uso terapêutico , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Nedocromil/farmacologia , Nedocromil/uso terapêutico , Sistema Respiratório/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
5.
Pulm Pharmacol Ther ; 22(5): 455-65, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19393759

RESUMO

Asthma is a complex disease that involves chronic inflammation and subsequent decline in airway function. The widespread use of animal models has greatly contributed to our understanding of the cellular and molecular pathways underlying human allergic asthma. Animal models of allergic asthma include smaller animal models which offer 'ease of use' and availability of reagents, and larger animal models that may be used to address aspects of allergic airways disease not possible in humans or smaller animal models. This review examines the application and suitability of various animal models for studying mechanisms of airway inflammation and tissue remodelling in allergic asthma, with a specific focus on airway smooth muscle.


Assuntos
Asma/fisiopatologia , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Inflamação/fisiopatologia , Músculo Liso/fisiopatologia , Animais , Asma/tratamento farmacológico , Hormônios Esteroides Gonadais/farmacologia , Hormônios Esteroides Gonadais/uso terapêutico , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/fisiopatologia , Inflamação/tratamento farmacológico , Músculo Liso/imunologia , Músculo Liso/patologia , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Sistema Respiratório/fisiopatologia , Caracteres Sexuais
6.
Pulm Pharmacol Ther ; 22(5): 417-25, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19409504

RESUMO

Altered bronchial vascular reactivity and remodelling including angiogenesis are documented features of asthma and other chronic inflammatory airway diseases. Expansion of the bronchial vasculature under these conditions involves both functional (vasodilation, hyperperfusion, increased microvascular permeability, oedema formation, and inflammatory cell recruitment) and structural changes (tissue and vascular remodelling) in the airways. These changes in airway vascular reactivity and vascularisation have significant pathophysiological consequences, which are manifest in the clinical symptoms of airway disease. Airway vascular reactivity is regulated by a wide variety of neurotransmitters and inflammatory mediators. Similarly, multiple growth factors are implicated in airway angiogenesis, with vascular endothelial growth factor amongst the most important. Increasing attention is focused on the complex interplay between angiogenic growth factors, airway smooth muscle and the various collagen-derived fragments that exhibit anti-angiogenic properties. The balance of these dynamic influences in airway neovascularisation processes and their therapeutic implications is just beginning to be elucidated. In this review article, we provide an account of recent developments in the areas of vascular reactivity and airway angiogenesis in chronic airway diseases.


Assuntos
Artérias Brônquicas/fisiopatologia , Broncopatias/fisiopatologia , Hiper-Reatividade Brônquica/fisiopatologia , Músculo Liso/irrigação sanguínea , Músculo Liso/fisiopatologia , Neovascularização Patológica/metabolismo , Moduladores da Angiogênese/metabolismo , Artérias Brônquicas/metabolismo , Artérias Brônquicas/patologia , Hiper-Reatividade Brônquica/patologia , Doença Crônica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Músculo Liso/patologia , Doenças Respiratórias/metabolismo , Doenças Respiratórias/patologia , Doenças Respiratórias/fisiopatologia
7.
Am J Respir Crit Care Med ; 178(5): 460-8, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18556625

RESUMO

RATIONALE: Airway remodeling in asthma involves accumulation of airway smooth muscle (ASM) and increased vascularity due to angiogenesis. Bronchial blood vessels and ASM are found in close proximity, and ASM releases multiple proinflammatory mediators, including vascular endothelial growth factor (VEGF). OBJECTIVES: We examined whether release of proangiogenic mediators is increased in ASM from subjects with asthma and whether this is translated to induction of angiogenesis. METHODS: Biopsy-derived ASM cells were cultured from 12 subjects with mild asthma, 8 with moderate asthma, and 9 healthy control subjects. Angiogenesis induced by cell-conditioned medium (CM) from ASM was evaluated in a tubule formation assay. Anti-CD31-labeled tubules were quantified by image analysis. Angiogenic factors in CM were quantified by antibody arrays and by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Induction of angiogenesis by CM from unstimulated ASM was increased in subjects with mild asthma (twofold) and moderate asthma (threefold), compared with healthy CM (P < 0.001). Levels of angiogenic factors (VEGF, angiopoietin [Ang]-1, angiogenin) were similarly elevated in CM from subjects with asthma compared with that from healthy subjects (P < 0.05), whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. VEGF, Ang-1, and angiogenin in combination increased vascularity (twofold, P < 0.01) in cultured intact biopsies. Selective VEGF immunodepletion abolished enhanced tubule formation by CM from asthmatic ASM (P < 0.01), but CM depletion of Ang-1 or angiogenin had no effect. CONCLUSIONS: ASM cultured from subjects with mild or moderate asthma, but not from healthy control subjects, promotes angiogenesis in vitro. This proangiogenic capacity resides in elevated VEGF release and suggests that ASM regulates airway neovascularization in asthma.


Assuntos
Indutores da Angiogênese/metabolismo , Proteínas Angiogênicas/metabolismo , Asma/fisiopatologia , Músculo Liso/metabolismo , Neovascularização Patológica , Adulto , Angiopoietina-1 , Brônquios/irrigação sanguínea , Brônquios/patologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Masculino , Miócitos de Músculo Liso/metabolismo , Análise Serial de Proteínas , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Br J Pharmacol ; 146(3): 370-7, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16025136

RESUMO

Accumulation of airway smooth muscle (ASM) and its infiltration by mast cells are key pathological features of airway remodelling in asthma. Heparin, a major component of mast cell granules, inhibits ASM proliferation by an unknown mechanism. Here, unfractionated heparins and related glycosaminoglycans having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule that were required for its antiproliferative activity in cultured human ASM cells. Proliferation induced by 10% fetal bovine serum (FBS) was abrogated by two unfractionated commercial heparin preparations (Sigma and Multiparin) and this effect was reproduced with each of three low-molecular weight heparin preparations (3, 5 and 6 kDa, respectively), demonstrating that antiproliferative activity resided in at least a 3 kDa heparin fraction. N-desulphated 20% re-acetylated (N-de) heparin (anticoagulant) and O-desulphated heparin (O-de) (non-anticoagulant) fractions also inhibited FBS-dependent proliferation (rank potency: Sigma heparin > O-de > N-de) suggesting that the antiproliferative action of heparin involved N-sulphation but was independent of its anticoagulant activity. Other sulphated molecules with variable anionic charge (dextran sulphate, fucoidan, chondroitin sulphates A or B, heparan sulphate) inhibited proliferation to varying degrees, as did the non-sulphated molecules hyaluronic acid and poly-L-glutamic acid. However, nonsulphated dextran had no effect. In summary, attenuation of FBS-dependent proliferation of human ASM by heparin involves but does not depend upon sulphation, although loss of N-sulphation reduces antiproliferative activity. This antiproliferative effect is independent of anionic charge and the anticoagulant actions of heparin.


Assuntos
Proliferação de Células/efeitos dos fármacos , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Adulto , Idoso , Brônquios , Células Cultivadas , Feminino , Heparina/química , Heparina de Baixo Peso Molecular , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/citologia , Sulfatos/análise
9.
J Appl Physiol (1985) ; 97(6): 2029-34, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15531570

RESUMO

The observation that the length-force relationship in airway smooth muscle can be shifted along the length axis by accommodating the muscle at different lengths has stimulated great interest. In light of the recent understanding of the dynamic nature of length-force relationship, many of our concepts regarding smooth muscle mechanical properties, including the notion that the muscle possesses a unique optimal length that correlates to maximal force generation, are likely to be incorrect. To facilitate accurate and efficient communication among scientists interested in the function of airway smooth muscle, a revised and collectively accepted nomenclature describing the adaptive and dynamic nature of the length-force relationship will be invaluable. Setting aside the issue of underlying mechanism, the purpose of this article is to define terminology that will aid investigators in describing observed phenomena. In particular, we recommend that the term "optimal length" (or any other term implying a unique length that correlates with maximal force generation) for airway smooth muscle be avoided. Instead, the in situ length or an arbitrary but clearly defined reference length should be used. We propose the usage of "length adaptation" to describe the phenomenon whereby the length-force curve of a muscle shifts along the length axis due to accommodation of the muscle at different lengths. We also discuss frequently used terms that do not have commonly accepted definitions that should be used cautiously.


Assuntos
Contração Muscular/fisiologia , Músculo Liso/fisiologia , Terminologia como Assunto , Traqueia/fisiologia , Animais , Humanos
11.
Respir Physiol Neurobiol ; 137(2-3): 309-26, 2003 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-14516734

RESUMO

General agreement exists that in asthma, airway smooth muscle contracts, narrowing the airway lumen and thereby causing airflow obstruction and dyspnoea. New evidence is emerging that airway smooth muscle may also fulfil an immunomodulatory role by providing a rich source of pro-inflammatory cytokines and chemokines, polypeptide growth factors, extracellular matrix (ECM) proteins, cell adhesion receptors and co-stimulatory molecules. Together, the available data support a role for airway smooth muscle in actively perpetuating airway mucosal inflammatory processes including mast cell and leukocyte (T cell, neutrophil, eosinophil) activation and recruitment. Production of anti-inflammatory mediators by airway smooth muscle such as prostaglandin E(2) suggests that it is also capable of exerting a 'braking' effect on local inflammation. Recognition of this newly described property of airway smooth muscle makes it important to consider therapeutic targets for suppressing the synthesis and secretion of immunomodulatory mediators from this cell. However, it remains imperative to establish to what extent the secretory potential of airway smooth muscle is quantitatively important in vivo and in asthmatic subjects.


Assuntos
Adjuvantes Imunológicos/metabolismo , Asma/imunologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Miócitos de Músculo Liso/metabolismo , Traqueia/imunologia , Traqueia/metabolismo , Animais , Antiasmáticos/farmacologia , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Brônquios/citologia , Brônquios/imunologia , Brônquios/metabolismo , Quimiocinas/imunologia , Citocinas/imunologia , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Traqueia/citologia
12.
Proc Am Thorac Soc ; 6(8): 673-7, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20008874

RESUMO

Expansion of the airway wall vascular compartment has recently been established as a feature of asthma involving both enlargement of existing vascular structures and the formation of new vessels (angiogenesis). Both processes are likely to occur together and are fundamental for supporting the many aspects of tissue inflammation and remodeling manifest in the clinical symptoms of airway disease. Multiple growth factors are implicated in airway angiogenesis, with vascular endothelial growth factor among the most important. Other asthma-associated stimuli, including ADAM33, environmental tobacco smoke, and rhinovirus infection, are emerging as proangiogenic regulators. Increasing attention is also focused on the complex interplay of airway wall inflammatory and structural cells, including airway smooth muscle in driving expansion of the bronchial submucosal vascular plexus in asthma. Here, we provide a brief update on recent developments in this emerging area and highlight the potential role played by airway smooth muscle.


Assuntos
Remodelação das Vias Aéreas/fisiologia , Asma/patologia , Neovascularização Patológica/patologia , Asma/fisiopatologia , Humanos , Músculo Liso/patologia , Neovascularização Patológica/fisiopatologia
13.
Am J Respir Cell Mol Biol ; 36(6): 721-7, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17272821

RESUMO

Airway hyperresponsiveness (AHR) is associated with airway wall structural remodeling and alterations in airway smooth muscle (ASM) function. Previously, in bronchioles from Brown Norway rats challenged by repeated ovalbumin (OVA) inhalation, we have reported increased force generation and depletion of smooth muscle contractile proteins. Here, we investigated if cytoskeletal changes in smooth muscle could account for this paradox. Sensitized rats (n = 5/group) were repeatedly challenged with OVA or saline, and the lungs were removed 24 h after the last challenge. Levels of globular (G) and filamentous (F) actin in bronchioles were determined by DNase I inhibition and contraction assessed in intact small bronchioles using a myograph. DNase I inhibition assays showed that G-actin monomers were more abundant ( approximately 1F:2G) in extracts from resting small bronchioles from OVA- or saline-challenged animals. However, while contractile protein levels in bronchioles were reduced by OVA (P < 0.05), the proportion of F:G actin was 1.8-fold greater compared with saline challenge (P < 0.05). Consistent with induction of F-actin after OVA challenge, increases in maximum tension development to carbachol or KCl in small bronchioles from OVA-challenged animals were abrogated (P < 0.01) by actin cytoskeleton disruption with 0.5 microM latrunculin A. Cytoskeletal stabilization of F-actin with 0.1 microM jasplakinolide potentiated maximum contractions to carbachol or KCl (P < 0.05) in bronchioles from OVA- but not saline-treated rats. We conclude that alterations in the composition and/or arrangement of the contractile apparatus after OVA exposure confer enhanced contractile responses, possibly as a result of increased F-actin content. Such a mechanism may have relevance for AHR found in allergic asthma.


Assuntos
Alérgenos/imunologia , Brônquios/citologia , Brônquios/imunologia , Citoesqueleto/metabolismo , Músculo Liso/metabolismo , Actinas/metabolismo , Animais , Antineoplásicos/farmacologia , Asma/imunologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Brônquios/efeitos dos fármacos , Carbacol/farmacologia , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Depsipeptídeos/farmacologia , Exposição por Inalação , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Ratos , Tiazolidinas/farmacologia
14.
Am J Respir Crit Care Med ; 176(2): 146-53, 2007 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-17463417

RESUMO

RATIONALE: Asthmatic airways have an increased number and size of vascular structures, which contribute to airflow obstruction and hyperresponsiveness. OBJECTIVES: We examined whether proangiogenic mediators are elevated in bronchoalveolar lavage fluid (BALF) from subjects with asthma and if this translated to induction of angiogenesis. METHODS: Angiogenic activity in BALF from 12 healthy, nonatopic subjects and 10 atopic subjects with mild asthma was evaluated by examining tubule formation at 11 days in cocultures of human endothelial cells with dermal fibroblasts. Vascular structures were visualized by anti-CD31 labeling and quantified by image analysis. Angiogenic growth factors in BALF from healthy subjects and subjects with asthma were identified using antibody arrays and by ELISA. MEASUREMENTS AND MAIN RESULTS: Angiogenic activity induced by BALF from healthy subjects was not different from basal tubule formation (p>0.05). However, induction of tubular structures by asthmatic BALF was 2.5-fold greater (p<0.001) compared with healthy samples. Similarly, levels of proangiogenic growth factors (angiogenin, vascular endothelial growth factor [VEGF], monocyte chemotactic protein-1) were increased approximately 2.5-fold (p<0.05) in BALF from subjects with asthma, whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. A blocking anti-VEGF antibody abolished tubule formation induced by BALF from either healthy subjects or subjects with asthma (p<0.01). Immunodepletion of VEGF had no effect on basal tubule formation induced by healthy BALF but abrogated enhanced tubule formation by asthmatic BALF (p<0.01). CONCLUSIONS: BALF collected from subjects with asthma but not healthy subjects is functionally active in promoting angiogenesis in vitro. The proangiogenic capacity of BALF from subjects with asthma resides in elevated VEGF derived from asthmatic airways. This observation supports VEGF as a key factor in vascular remodeling in asthma.


Assuntos
Proteínas Angiogênicas/biossíntese , Asma/metabolismo , Brônquios/metabolismo , Líquido da Lavagem Broncoalveolar , Neovascularização Fisiológica/fisiologia , Alvéolos Pulmonares/metabolismo , Adulto , Asma/patologia , Brônquios/irrigação sanguínea , Estudos de Casos e Controles , Técnicas de Cocultura , Células Endoteliais/fisiologia , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Alvéolos Pulmonares/irrigação sanguínea
15.
Am J Respir Crit Care Med ; 174(4): 379-85, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16709936

RESUMO

RATIONALE: Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with fibronectin and collagen are key features of asthma. Previously, we have reported these ECM proteins enhance ASM synthetic function. OBJECTIVE: We compared ASM cultured from endobronchial biopsies from subjects with and without asthma to assess if asthmatic cells were hypersecretory and determined whether the underlying mechanism involved autocrine ECM production. METHODS AND MEASUREMENTS: Cells from subjects with and without asthma were cultured on plastic or in plates precoated with ECM proteins. Cytokine production was evaluated by enzyme-linked immunosorbent assay and by reverse transcriptase-polymerase chain reaction. Function-blocking integrin antibodies were used to identify integrin involvement. RESULTS: Baseline eotaxin and its production after stimulation with interleukin (IL)-13, IL-1beta, or tumor necrosis factor-alpha was increased (2.5- to 6.0-fold) in ASM cells cultured from subjects with asthma compared with healthy subjects. When seeded on ECM from asthmatic ASM, IL-13-dependent eotaxin release from healthy or asthmatic ASM was enhanced compared with culture on healthy ECM. The ECM substrates fibronectin and type I collagen each enhanced IL-13-dependent eotaxin release, and Western immunoblot indicated that fibronectin expression was higher in asthmatic ASM cells. Integrin-blocking antibodies revealed that alpha5beta1 was required for more than 50% of the enhanced IL-13-dependent eotaxin release by ASM cells from subjects with asthma, whereas alpha2beta1 or alphavbeta3 neutralization lacked effect. CONCLUSION: The data indicate that ASM cells cultured from subjects with asthma are hypersecretory compared with cells from healthy donors and that autocrine fibronectin secretion acting via alpha5beta1 in part underlies this effect.


Assuntos
Asma/metabolismo , Quimiocinas CC/metabolismo , Matriz Extracelular/fisiologia , Miócitos de Músculo Liso/metabolismo , Adulto , Idoso , Western Blotting , Células Cultivadas , Quimiocina CCL11 , Colágeno Tipo I/metabolismo , Feminino , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Interleucina-13/metabolismo , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/fisiologia
16.
J Immunol ; 174(4): 2258-64, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15699160

RESUMO

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with interstitial collagen and fibronectin are major pathological features of airway remodeling in asthma. We have previously shown that these ECM components confer enhanced ASM proliferation in vitro, but their action on its newly characterized secretory function is unknown. Here, we examined the effects of fibronectin and collagen types I, III, and V on IL-1beta-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1beta-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that alpha2beta1, alpha5beta1, alphavbeta1, and alphavbeta3 integrins were required for up-regulation of IL-1beta-dependent ASM secretory responses by fibronectin, while alpha2beta1 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1beta-dependent ASM secretory responses through a beta1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma.


Assuntos
Adjuvantes Imunológicos/fisiologia , Colágeno Tipo I/fisiologia , Citocinas/metabolismo , Fibronectinas/fisiologia , Integrina beta1/fisiologia , Pulmão/metabolismo , Músculo Liso/metabolismo , Adulto , Idoso , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Quimiocina CCL11 , Quimiocinas CC/metabolismo , Citocinas/genética , Proteínas da Matriz Extracelular/fisiologia , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Integrina beta1/imunologia , Interleucina-1/fisiologia , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Pulmão/imunologia , Masculino , Pessoa de Meia-Idade , Músculo Liso/citologia , Músculo Liso/imunologia
17.
Am J Respir Crit Care Med ; 171(3): 217-23, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15502110

RESUMO

Airway smooth muscle (ASM) accumulation and enrichment of the extracellular matrix (ECM) with type I collagen and fibronectin are major pathologic features of airway remodeling in asthma. These ECM components confer enhanced ASM proliferation in vitro, but a requirement for specific integrin ECM receptors has not been examined. Here, we examined the mitogen platelet-derived growth factor (PDGF)-BB on beta1-integrin expression on human ASM cells cultured on these ECM substrates and defined the involvement of specific integrins in cell attachment and proliferation using integrin-neutralizing antibodies. PDGF-BB-dependent proliferation was enhanced two- to threefold by monomeric type I collagen or fibronectin and to a lesser extent by vitronectin; other interstitial ECM components (fibrillar type I and III collagen and tenascin-C) had no effect. Except for increased alpha3 expression induced by PDGF-BB and monomeric type I collagen or fibronectin, alpha1, alpha2, alpha4, alpha5, alphav, and alphavbeta3 integrins were unchanged compared with unstimulated cells on plastic. Blocking antibodies revealed alpha2beta1- and alphavbeta3-mediated attachment to monomeric type I collagen, whereas attachment to fibronectin required alpha5beta1. In contrast, enhancement of PDGF-BB-dependent proliferation by either monomeric type I collagen or fibronectin required alpha2beta1, alpha4beta1, and alpha5beta1 integrins. These data suggest multiple beta1-integrins regulate enhanced ASM proliferative responses.


Assuntos
Brônquios/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Fibronectinas/farmacologia , Integrina beta1/farmacologia , Músculo Liso/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Becaplermina , Brônquios/citologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo III/farmacologia , Feminino , Humanos , Integrina alfa2beta1/fisiologia , Integrina alfa4beta1/fisiologia , Integrina alfa5beta1/fisiologia , Integrina alfaVbeta3/fisiologia , Masculino , Pessoa de Meia-Idade , Mitógenos/farmacologia , Músculo Liso/citologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Tenascina/farmacologia , Vitronectina/farmacologia
18.
Am J Respir Crit Care Med ; 169(5): 596-603, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-14670803

RESUMO

Interleukin (IL)-13 receptor activation on airway smooth muscle cells induces eotaxin release and activates multiple signaling pathways including mitogen-activated protein kinases, and signal transducer and activator of transcription 6 (STAT6). To examine a requirement for STAT6 in mediating IL-13-stimulated eotaxin release we used antisense oligodeoxynucleotides (ODNs) to downregulate endogenous STAT6 protein. STAT6 antisense ODNs were taken up by about 85% of cells. Selective downregulation of STAT6 protein occurred with antisense ODNs, but not with sense or scrambled ODNs. Eotaxin release induced by IL-13 or IL-4 (10 ng/ml) was reduced by 81 +/- 4 and 75 +/- 7%, respectively, in cells transfected with antisense ODNs (p < 0.001), but not with a sense ODN or a scrambled ODN. Eotaxin release induced by IL-1beta was unaffected by STAT6 antisense ODN (p > 0.05). Finally, IL-13- or IL-4-dependent eotaxin release was abolished when inhibitors of both p42/p44 ERK (U0126, 10 microM) and p38 (SB202190, 10 microM) mitogen-activated protein kinase pathways were combined in STAT6 antisense ODN-transfected cells. In contrast, about 25% of the response remained when each inhibitor was examined alone in STAT6 antisense ODN-treated cells. These data support roles for both STAT6- and mitogen-activated protein kinase-dependent pathways in mediating eotaxin release from airway smooth muscle by IL-13 or IL-4.


Assuntos
Asma/imunologia , Quimiocinas/imunologia , Interleucina-13/imunologia , Músculo Liso , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia , Adulto , Idoso , Asma/prevenção & controle , Hiper-Reatividade Brônquica/imunologia , Butadienos/imunologia , Butadienos/farmacologia , Células Cultivadas , Quimiocina CCL11 , Quimiocinas CC/imunologia , Regulação para Baixo , Feminino , Humanos , Imidazóis/imunologia , Imidazóis/farmacologia , Inflamação , Interleucina-4/imunologia , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/imunologia , Músculo Liso/citologia , Músculo Liso/imunologia , Músculo Liso/metabolismo , Nitrilas/imunologia , Nitrilas/farmacologia , Oligodesoxirribonucleotídeos Antissenso/imunologia , Piridinas/imunologia , Piridinas/farmacologia , Mucosa Respiratória/metabolismo , Fator de Transcrição STAT6 , Transativadores/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno
19.
Exp Lung Res ; 29(6): 339-59, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12888448

RESUMO

Culture of dispersed airway smooth muscle (ASM) cells with fetal bovine serum (FBS) induces rapid growth and modulation from a contractile to a synthetic phenotype, but this may be an artificial situation due to loss of tissue architecture. In this study, structural, functional and biochemical changes of ASM were examined in human intact bronchiole ring segments (200 to 600 micro m internal diameter) after organ culture for up to 6 days in 10% FBS or in D-STIM, an FBS-free medium formulated to maintain a contractile phenotype. ASM content was unchanged after culture for 3 or 6 days with D-STIM or FBS compared with fresh tissues. However, by 6 days culture with FBS reduced the maximum developed contraction to several agonists (carbachol, histamine, and KCl). Smooth muscle (sm)-alpha-actin and sm-myosin heavy chain (MHC) isoform 1 expression was similar for all culture conditions, though FBS reduced calponin and metavinculin content. Nonmuscle (nm) proteins, including total vinculin, beta-actin, and nm-MHC, were unchanged. Thus, ASM in human intact bronchioles maintains its functional, biochemical, and morphometric properties after culture in FBS-free (D-STIM) medium for at least 6 days, and for 3 days when cultured with FBS. These findings in organ culture may reflect more closely the in vivo situation in which tissue architecture is better preserved over cell culture, and may provide a basis for examining long-term effects of trophic or contractile stimuli on intact ASM in vitro that contribute to airway hyperresponsiveness and remodeling.


Assuntos
Brônquios/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Soro , Adulto , Idoso , Anticorpos Monoclonais , Western Blotting , Brônquios/efeitos dos fármacos , Carbacol/farmacologia , Proteínas Contráteis/metabolismo , Meios de Cultura Livres de Soro , Proteínas do Citoesqueleto/metabolismo , Feminino , Histamina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fenótipo
20.
Am J Respir Crit Care Med ; 165(8): 1161-71, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11956062

RESUMO

The biologic activities of interleukin (IL)-13 and IL-4 often overlap, and evidence supports their importance in atopic disease and airways hyperresponsiveness. Here, their capacity to release eosinophil-activating cytokines was examined in cultured human airway smooth muscle. IL-13 and IL-4 induced selective release of eotaxin with no effect on granulocyte-macrophage colony-stimulating factor, regulated upon activation, normal T-cell expressed and secreted (RANTES), or IL-8. A profound synergistic increase in eotaxin release occurred when IL-13 or IL-4 was combined with IL-1beta that was abrogated by a neutralizing antibody to the IL-4 receptor alpha (IL-4Ralpha)-chain but not to the IL-2 receptor gamma (IL-2Rgamma)-chain. Expression of cell surface IL-4 receptors and IL-4Ralpha in lysates was constitutive and unchanged by treatment with IL-13 or IL-4 alone or in combination with IL-1beta. Activation of IL-4Ralpha by IL-13 or IL-4 induced signal transducer and activation of transcription-6 (STAT6), p42/ p44 ERK, p38, and to a lesser extent, SAPK/JNK mitogen-activated protein kinase phosphorylation. STAT6 and MAP kinase activation by IL-13 or IL-4 was not further potentiated after combined stimulation with IL-1beta. However, eotaxin release induced by IL-13 or IL-4 alone, and in combination with IL-1beta, was prevented by the MEK inhibitor U 0126 and by the p38 inhibitor SB 202190. Collectively, the data suggest that selective eotaxin release induced either by IL-13 and IL-4 or when combined with IL-1beta is mediated by a constitutive cell surface IL-4Ralpha and the activation of multiple intracellular pathways.


Assuntos
Brônquios/metabolismo , Quimiocinas CC/metabolismo , Fatores Quimiotáticos de Eosinófilos/metabolismo , Interleucinas/fisiologia , Músculo Liso/metabolismo , Adulto , Idoso , Anticorpos Monoclonais/farmacologia , Células Cultivadas , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Ativação Enzimática , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-1/fisiologia , Interleucina-13/fisiologia , Interleucina-4/fisiologia , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Interleucina-4/imunologia , Receptores de Interleucina-4/fisiologia , Fator de Transcrição STAT6 , Transdução de Sinais , Transativadores/metabolismo
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