RESUMO
A characteristic feature of asthma is exaggerated airway narrowing, termed airway hyper-responsiveness (AHR) due to contraction of airway smooth muscle (ASM). Although smooth muscle (SM)-specific asthma susceptibility genes have been identified, it is not known whether asthmatic ASM is phenotypically different from nonasthmatic ASM in terms of subcellular structure or mechanical function. The present study is the first to systematically quantify, using electron microscopy, the ultrastructure of tracheal SM from subjects with asthma and nonasthmatic subjects. Methodological details concerning tissue sample preparation, ultrastructural quantification, and normalization of isometric force by appropriate morphometric parameters are described. We reasoned that genetic and/or acquired differences in the ultrastructure of asthmatic ASM could be associated with functional changes. We recently reported that asthmatic ASM is better able to maintain and recover active force generation after length oscillations simulating deep inspirations. The present study was designed to seek structural evidence to account for this observation. Contrary to our hypotheses, no significant qualitative or quantitative differences were found in the subcellular structure of asthmatic versus nonasthmatic tracheal SM. Specifically, there were no differences in average SM cell cross-sectional area; fraction of the cell area occupied by nonfilamentous area; amounts of mitochondria, dense bodies, and dense plaques; myosin and actin filament densities; basal lamina thickness; and the number of microtubules. These results indicate that functional differences in ASM do not necessarily translate into observable structural changes.
Assuntos
Asma/fisiopatologia , Músculo Liso/ultraestrutura , Traqueia/ultraestrutura , Actinas/metabolismo , Actinas/ultraestrutura , Adolescente , Adulto , Asma/metabolismo , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Miosinas/metabolismo , Miosinas/ultraestrutura , Traqueia/metabolismo , Adulto JovemRESUMO
Increased airway responsiveness is linked to lung function decline and mortality in subjects with chronic obstructive pulmonary disease (COPD); however, the genetic contribution to airway responsiveness remains largely unknown. A genome-wide association study (GWAS) was performed using the Illumina (San Diego, CA) Human660W-Quad BeadChip on European Americans with COPD from the Lung Health Study. Linear regression models with correlated meta-analyses, including data from baseline (n = 2,814) and Year 5 (n = 2,657), were used to test for common genetic variants associated with airway responsiveness. Genotypic imputation was performed using reference 1000 Genomes Project data. Expression quantitative trait loci (eQTL) analyses in lung tissues were assessed for the top 10 markers identified, and immunohistochemistry assays assessed protein staining for SGCD and MYH15. Four genes were identified within the top 10 associations with airway responsiveness. Markers on chromosome 9p21.2 flanked by LINGO2 met a predetermined threshold of genome-wide significance (P < 9.57 × 10(-8)). Markers on chromosomes 3q13.1 (flanked by MYH15), 5q33 (SGCD), and 6q21 (PDSS2) yielded suggestive evidence of association (9.57 × 10(-8) < P ≤ 4.6 × 10(-6)). Gene expression studies in lung tissue showed single nucleotide polymorphisms on chromosomes 5 and 3 to act as eQTL for SGCD (P = 2.57 × 10(-9)) and MYH15 (P = 1.62 × 10(-6)), respectively. Immunohistochemistry confirmed localization of SGCD protein to airway smooth muscle and vessels and MYH15 to airway epithelium, vascular endothelium, and inflammatory cells. We identified novel loci associated with airway responsiveness in a GWAS among smokers with COPD. Risk alleles on chromosomes 5 and 3 acted as eQTLs for SGCD and MYH15 messenger RNA, and these proteins were expressed in lung cells relevant to the development of airway responsiveness.
Assuntos
Doença Pulmonar Obstrutiva Crônica/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Polimorfismo de Nucleotídeo Único , Doença Pulmonar Obstrutiva Crônica/metabolismo , Locos de Características Quantitativas , Risco , Sarcoglicanas/genética , Sarcoglicanas/metabolismoRESUMO
RATIONALE: Airway narrowing is maintained for a prolonged period after acute bronchoconstriction in humans in the absence of deep inspirations (DIs). OBJECTIVES: To determine whether maintenance of airway smooth muscle (ASM) shortening is responsible for the persistence of airway narrowing in healthy subjects following transient methacholine (MCh)-induced bronchoconstriction. METHODS: On two separate visits, five healthy subjects underwent MCh challenges until respiratory system resistance (Rrs) had increased by approximately 1.5 cm H2O/L/s. Subjects took a DI either immediately after or 30 minutes after the last dose. The extent of renarrowing following the bronchodilator effect of DI was used to assess the continued action of MCh (calculated as percent change in Rrs from the pre-DI Rrs). We then used human bronchial rings to determine whether ASM can maintain shortening during a progressive decrease of carbachol concentration. MEASUREMENTS AND MAIN RESULTS: The increased Rrs induced by MCh was maintained for 30 minutes despite waning of MCh concentration over that period, measured as attenuated renarrowing when the DI was taken 30 minutes after compared with immediately after the last dose (7 min post-DI, -36.2 ± 11.8 vs. 14.4 ± 13.2%; 12 min post-DI, -39.5 ± 9.8 vs. 15.2 ± 17.8%). Ex vivo, ASM shortening was largely maintained during a progressive decrease of carbachol concentration, even down to concentrations that would not be expected to induce shortening. CONCLUSIONS: The maintenance of airway narrowing despite MCh clearance in humans is attributed to an intrinsic ability of ASM to maintain shortening during a progressive decrease of contractile stimulation.
Assuntos
Resistência das Vias Respiratórias/efeitos dos fármacos , Testes de Provocação Brônquica , Broncoconstrição/efeitos dos fármacos , Broncoconstritores/farmacologia , Inalação/fisiologia , Cloreto de Metacolina/farmacologia , Músculo Liso/efeitos dos fármacos , Adulto , Resistência das Vias Respiratórias/fisiologia , Brônquios/efeitos dos fármacos , Brônquios/fisiologia , Broncoconstrição/fisiologia , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/fisiologiaRESUMO
Asthma is an obstructive airway disease, with a heterogeneous and multifactorial pathogenesis. Although generally considered to be a disease principally driven by chronic inflammation, it is becoming increasingly recognised that the immune component of the pathology poorly correlates with the clinical symptoms of asthma, thus highlighting a potentially central role for non-immune cells. In this context airway smooth muscle (ASM) may be a key player, as it comprises a significant proportion of the airway wall and is the ultimate effector of acute airway narrowing. Historically, the contribution of ASM to asthma pathogenesis has been contentious, yet emerging evidence suggests that ASM contractile activation imparts chronic effects that extend well beyond the temporary effects of bronchoconstriction. In this review article we describe the effects that ASM contraction, in combination with cellular mechanotransduction and novel contraction-inflammation synergies, contribute to asthma pathogenesis. Specific emphasis will be placed on the effects that ASM contraction exerts on the mechanical properties of the airway wall, as well as novel mechanisms by which ASM contraction may contribute to more established features of asthma such as airway wall remodelling.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Asma/fisiopatologia , Mecanotransdução Celular/fisiologia , Contração Muscular/fisiologia , Músculo Liso/fisiopatologia , Animais , Asma/etiologia , Broncoconstrição/fisiologia , Humanos , Fenômenos Fisiológicos RespiratóriosRESUMO
Inflammasomes have emerged as playing key roles in inflammation and innate immunity. A growing body of evidence has suggested that the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Inflammasome activation results, in part, in pro-IL-1ß processing and the secretion of the proinflammatory cytokine IL-1ß. Because asthma exacerbations are associated with elevated concentrations of secreted IL-1ß, we addressed whether the NLRP3 inflammasome is activated under in vitro conditions that mimic infectious exacerbations in asthma. Primary cultures of airway smooth muscle (ASM) cells were treated with infectious stimuli (mimicked using the Toll-like receptor-2 agonist Pam3CSK4, a synthetic bacterial lipopeptide). Whereas Pam3CSK4 robustly up-regulated ASM cytokine expression in response to TNF-α and significantly enhanced IL-1ß mRNA expression, we were unable to detect IL-1ß in the cell supernatants. Thus, IL-1ß was not secreted and therefore was unable to act in an autocrine manner to promote the amplification of ASM inflammatory responses. Moreover, Toll-like receptor-2 ligation did not enhance NLRP3 or caspase-1 expression in ASM cells, and NLRP3 and caspase-1 protein were not present in the ASM layer of tracheal sections from human donors. In conclusion, these data demonstrate that the enhanced synthetic function of ASM cells, induced by infectious exacerbations of airway inflammation, is NLRP3 inflammasome-independent and IL-1ß-independent. Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma.
Assuntos
Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Músculo Liso/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Humanos , Inflamassomos/genética , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Myosin molecules from smooth muscle and non-muscle cells are known to self-assemble into side-polar filaments in vitro. However, the in situ mechanism of filament assembly is not clear and the question of whether there is a unique length for myosin filaments in smooth muscle is still under debate. In this study we measured the lengths of 16,587 myosin filaments in three types of smooth muscle cells using serial electron microscopy (EM). Sheep airway and pulmonary arterial smooth muscle as well as rabbit carotid arterial smooth muscle were fixed for EM and serial ultra-thin (50-60 nm) sections were obtained. Myosin filaments were traced in consecutive sections to determine their lengths. The results indicate that there is not a single length for the myosin filaments; instead there is a wide variation in lengths. The plots of observation frequency versus myosin filament length follow an exponential decay pattern. Analysis suggests that in situ assembly of myosin filaments in smooth muscle is governed by random processes of linear polymerization and de-polymerization, and that the dynamic equilibrium of these processes determines the observed length distribution.
Assuntos
Miócitos de Músculo Liso/ultraestrutura , Miosinas/ultraestrutura , Animais , Artérias Carótidas/citologia , Diafragma/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Artéria Pulmonar/citologia , Coelhos , Ovinos , Traqueia/citologiaRESUMO
Respiratory symptoms are largely caused by obstruction of the airways. In asthma, airway narrowing mediated by airway smooth muscle (ASM) contraction contributes significantly to obstruction. The spasmogens produced following exposure to environmental triggers, such as viruses or allergens, are initially responsible for ASM activation. However, the extent of narrowing of the airway lumen due to ASM shortening can be influenced by many factors and it remains a real challenge to decipher the exact role of ASM in causing asthmatic symptoms. Innovative tools, such as the forced oscillation technique, continue to develop and have been proven useful to assess some features of ASM function in vivo. Despite these technologic advances, it is still not clear whether excessive narrowing in asthma is driven by ASM abnormalities, by other alterations in non-muscle factors or simply because of the overexpression of spasmogens. This is because a multitude of forces are acting on the airway wall, and because not only are these forces constantly changing but they are also intricately interconnected. To counteract these limitations, investigators have utilized in vitro and ex vivo systems to assess and compare asthmatic and non-asthmatic ASM contractility. This review describes: 1- some muscle and non-muscle factors that are altered in asthma that may lead to airway narrowing and asthma symptoms; 2- some technologies such as the forced oscillation technique that have the potential to unveil the role of ASM in airway narrowing in vivo; and 3- some data from ex vivo and in vitro methods that probe the possibility that airway hyperresponsiveness is due to the altered environment surrounding the ASM or, alternatively, to a hypercontractile ASM phenotype that can be either innate or acquired.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Asma/fisiopatologia , Músculo Liso/metabolismo , Obstrução das Vias Respiratórias/fisiopatologia , Animais , Hiper-Reatividade Brônquica/fisiopatologia , Humanos , Testes de Função RespiratóriaRESUMO
The biological responses of airway smooth muscle (ASM) are diverse, in part due to ASM phenotype plasticity. ASM phenotype plasticity refers to the ability of ASM cells to change the degree of a variety of functions, including contractility, proliferation, migration and secretion of inflammatory mediators. This plasticity occurs due to intrinsic or acquired abnormalities in ASM cells, and these abnormalities or predisposition of the ASM cell may alter the ASM response and in some cases recapitulate disease hallmarks of asthma. These phenotypic changes are ultimately determined by multiple stimuli and occur due to alterations in the intricate balance or reversible state that maintains ASM cells in either a contractile or synthetic state, through processes termed maturation or modulation, respectively. To elucidate the role of ASM phenotype in disease states, numerous in vitro studies have suggested a phenotypic switch in ASM primary cell cultures as an explanation for the plethora of responses mediated by ASM cells. Moreover, there is overwhelming evidence suggesting that the immunomodulatory response of ASM is due to the acquisition of a synthetic phenotype; however, whether this degree of plasticity is present in vivo as opposed to cell culture-based models remains speculative. Nonetheless, this review will give an overall scope of ASM phenotypic markers, triggers of ASM phenotype modulation and novel therapeutic approaches to control ASM phenotype plasticity.
Assuntos
Asma/fisiopatologia , Músculo Liso/patologia , Miócitos de Músculo Liso/patologia , Animais , Movimento Celular , Proliferação de Células , Humanos , Mediadores da Inflamação/metabolismo , Contração Muscular/fisiologia , Músculo Liso/citologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , FenótipoRESUMO
In asthma, the airway smooth muscle (ASM) cell plays a central role in disease pathogenesis through cellular changes which may impact on its microenvironment and alter ASM response and function. The answer to the long debated question of what makes a 'healthy' ASM cell become 'asthmatic' still remains speculative. What is known of an 'asthmatic' ASM cell, is its ability to contribute to the hallmarks of asthma such as bronchoconstriction (contractile phenotype), inflammation (synthetic phenotype) and ASM hyperplasia (proliferative phenotype). The phenotype of healthy or diseased ASM cells or tissue for the most part is determined by expression of key phenotypic markers. ASM is commonly accepted to have different phenotypes: the contractile (differentiated) state versus the synthetic (dedifferentiated) state (with the capacity to synthesize mediators, proliferate and migrate). There is now accumulating evidence that the synthetic functions of ASM in culture derived from asthmatic and non-asthmatic donors differ. Some of these differences include an altered profile and increased production of extracellular matrix proteins, pro-inflammatory mediators and adhesion receptors, collectively suggesting that ASM cells from asthmatic subjects have the capacity to alter their environment, actively participate in repair processes and functionally respond to changes in their microenvironment.
Assuntos
Asma/fisiopatologia , Inflamação/patologia , Miócitos de Músculo Liso/patologia , Animais , Broncoconstrição , Microambiente Celular , Humanos , Hiperplasia/patologia , Contração Muscular , Miócitos de Músculo Liso/metabolismo , FenótipoRESUMO
Airway wall remodeling and lung hyperinflation are two typical features of asthma that may alter the contractility of airway smooth muscle (ASM) by affecting its operating length. The aims of this study were as follows: 1) to describe in detail the "length dependency of ASM force" in response to different spasmogens; and 2) to predict, based on morphological data and a computational model, the consequence of this length dependency of ASM force on airway responsiveness in asthmatic subjects who have both remodeled airway walls and hyperinflated lungs. Ovine tracheal ASM strips and human bronchial rings were isolated and stimulated to contract in response to increasing concentrations of spasmogens at three different lengths. Ovine tracheal strips were more sensitive and generated greater force at longer lengths in response to acetylcholine (ACh) and K(+). Equipotent concentrations of ACh were approximately a log less for ASM stretched by 30% and approximately a log more for ASM shortened by 30%. Similar results were observed in human bronchi in response to methacholine. Morphometric and computational analyses predicted that the ASM of asthmatic subjects may be elongated by 6.6-10.4% (depending on airway generation) due to remodeling and/or hyperinflation, which could increase ACh-induced force by 1.8-117.8% (depending on ASM length and ACh concentration) and enhance the increased resistance to airflow by 0.4-4,432.8%. In conclusion, elongation of ASM imposed by airway wall remodeling and/or hyperinflation may allow ASM to operate at a longer length and to consequently generate more force and respond to lower concentration of spasmogens. This phenomenon could contribute to airway hyperresponsiveness.
Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Músculo Liso/fisiologia , Músculo Liso/fisiopatologia , Acetilcolina/farmacologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Remodelação das Vias Aéreas/fisiologia , Asma/fisiopatologia , Brônquios/efeitos dos fármacos , Brônquios/fisiologia , Brônquios/fisiopatologia , Humanos , Cloreto de Metacolina/farmacologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Potássio/farmacologia , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Traqueia/fisiopatologiaRESUMO
The lung is a dynamic organ and the oscillating stress applied to the airway wall during breathing maneuvers can decrease airway smooth muscle (ASM) contractility. However, it is unclear whether it is the stress or the attendant strain that is responsible for the decline of ASM force associated with breathing maneuvers, and whether tone can prevent the decline of force by attenuating the strain. To investigate these questions, ovine tracheal strips were subjected to oscillating stress that simulates breathing maneuvers, and the resulting strain and decline of force were measured in the absence or presence of different levels of tone elicited by acetylcholine. In relaxed ASM, high stress simulating 20 cm H(2)O-transpulmonary pressure excursions strained ASM strips by 20.7% and decreased force by 17.1%. When stress oscillations were initiated during measurement of ACh concentration-response curves, tone almost abrogated strain at an ACh concentration of 10(-6) M (1.1%) but the decline of force was not affected (18.9%). When stress oscillations were initiated after ACh-induced contraction had reached its maximal force, strain was almost abrogated at an ACh concentration of 10(-6) M (0.9%) and the decline of force was attenuated (10.1%). However, even at the highest ACh concentration (10(-4) M), substantial decline of force (6.1%) was still observed despite very small strain (0.7%). As expected, the results indicate that tone attenuated the strain experienced by ASM during breathing maneuver simulations. More surprisingly, the reduction of strain induced by tone was not proportional to its effect on the decline of force induced by simulated breathing maneuvers.
Assuntos
Contração Muscular/fisiologia , Músculo Liso/fisiologia , Traqueia/fisiologia , Acetilcolina/farmacologia , Animais , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Respiração/efeitos dos fármacos , Ovinos , Traqueia/efeitos dos fármacosRESUMO
The structurally dynamic cytoskeleton is important in many cell functions. Large gaps still exist in our knowledge regarding what regulates cytoskeletal dynamics and what underlies the structural plasticity. Because Rho-kinase is an upstream regulator of signaling events leading to phosphorylation of many cytoskeletal proteins in many cell types, we have chosen this kinase as the focus of the present study. In detergent skinned tracheal smooth muscle preparations, we quantified the proteins eluted from the muscle cells over time and monitored the muscle's ability to respond to acetylcholine (ACh) stimulation to produce force and stiffness. In a partially skinned preparation not able to generate active force but could still stiffen upon ACh stimulation, we found that the ACh-induced stiffness was independent of calcium and myosin light chain phosphorylation. This indicates that the myosin light chain-dependent actively cycling crossbridges are not likely the source of the stiffness. The results also indicate that Rho-kinase is central to the ACh-induced stiffness, because inhibition of the kinase by H1152 (1 µM) abolished the stiffening. Furthermore, the rate of relaxation of calcium-induced stiffness in the skinned preparation was faster than that of ACh-induced stiffness, with or without calcium, suggesting that different signaling pathways lead to different means of maintenance of stiffness in the skinned preparation.