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Airway smooth muscle (ASM) plays a central role in the excessive narrowing of the airway that characterizes the primary functional impairment in asthma. This phenomenon is known as airway hyper-responsiveness (AHR). Emerging evidence suggests that the development and maintenance of ASM force involves dynamic reorganization of the subcellular filament network in both the cytoskeleton and the contractile apparatus. In this review, evidence is presented to support the view that regulation of ASM contraction extends beyond the classical actomyosin interaction and involves processes within the cytoskeleton and at the interfaces between the cytoskeleton, the contractile apparatus, and the extracellular matrix. These processes are initiated when the muscle is activated, and collectively they cause the cytoskeleton and the contractile apparatus to undergo structural transformation, resulting in a more connected and solid state that allows force generated by the contractile apparatus to be transmitted to the extracellular domain. Solidification of the cytoskeleton also serves to stiffen the muscle and hence the airway. Oscillatory strain from tidal breathing and deep inspiration is believed to be the counter balance that prevents hypercontraction and stiffening of ASM in vivo. Dysregulation of this balance could lead to AHR seen in asthma. Key words: asthma, Rho-kinase, cytoskeleton, cross-bridge, length adaptation, force adaptation. Le muscle lisse respiratoire (MLR) joue un role central dans le retrecissement excessif des voies respiratoires qui caracterise la principale deterioration fonctionnelle associee a l'asthme. Ce phenomene est connu sous le nom d'hyperreactivite bronchique (HRB). Une donnee nouvelle suggere que le developpement et le maintien de la force du MLR implique la reorganization dynamique du reseau de filaments subcellulaires du cytosquelette et de l'appareil contractile. Dans cet article de revue, les auteurs presentent une preuve qui appuie la vision que la contraction du MLR va au-dela de l'interaction classique de l'actomyosine et implique des processus a l'interieur du cytosquelette et a l'interface entre le cytosquelette, l'appareil contractile et la matrice extracellulaire. Ces processus sont inities lorsque le muscle est active, et ils provoquent collectivement une transformation structurale du cytosquelette et de l'appareil contractile resultant en un etat mieux connecte et solide qui permet a la force generee par l'appareil contractile de se transmettre au domaine extracellulaire. La solidification du cytosquelette sert aussi a durcir le muscle et, consequemment, les voies respiratoires. On croit que la pression oscillatoire de la respiration courante et de l'inspiration profonde constitue la contrebalance qui previent l'hyper-contraction et le durcissement du MLR in vivo. La deregulation de cette balance pourrait conduire a l'HRB observee dans l'asthme. [Traduit par la Redaction] Mots-cles: asthme, Rho-kinase, cytosquelette, ponts transversaux, adaptation de la longueur, adaptation de la force., Introduction The regulation of the mechanical properties of smooth muscle, such as the ability to generate force and maintain tensile stiffness, is fundamental to the proper functioning of the muscle. [...]

It has long been known that airway smooth muscle (ASM) contraction contributes significantly to the reversible airflow obstruction that defines asthma. It has also been postulated that phenotypic changes in ASM contribute to the airway hyperresponsiveness (AHR) that is a characteristic feature of asthma. Although there is agreement that the mass of ASM surrounding the airways is significantly increased in asthmatic compared with non-asthmatic airways, it is still uncertain whether there are quantitative or qualitative changes in the level of expression of the genes and proteins involved in the canonical contractile pathway in ASM that could account for AHR. This review will summarize past attempts at quantifying gene expression changes in the ASM of asthmatic lungs as well as non-asthmatic ASM cells stimulated with various inflammatory cytokines. The lack of consistent findings in asthmatic samples coupled with the relative concordance of results from stimulated ASM cells suggests that changes to the contractility of ASM tissues in asthma may be dependent on the presence of an inflammatory environment surrounding the ASM layer. Removal of the ASM from this environment could explain why hypercontractility is rarely seen ex vivo. Key words: asthma, airway smooth muscle, gene expression, protein expression, cytokines. On sait depuis longtemps que la contraction du muscle lisse respiratoire (MLR) contribue de maniere significative a l'obstruction reversible du debit d'air qui definit l'asthme. Il a aussi ete postule que des changements phenotypiques dans le MLR contribue a l'hyperreactivite bronchique (HRB) qui caracterise l'asthme. Meme si l'on s'accorde sur le fait que la masse de MLR qui entoure les voies respiratoires est significativement plus importante en situation d'asthme comparativement a l'absence d'asthme, on ne sait pas encore avec certitude si des changements quantitatifs ou qualitatifs du niveau d'expression de genes et de proteines impliques dans les voies standards de regulation de la contraction chez le MLR pourraient expliquer l'HRB. Cet article de revue resumera les tentatives passees de quantification de changements d'expression genique dans des cellules de MLR de poumons asthmatiques et non asthmatiques, stimulees a l'aide de differentes cytokines inflammatoires. L'absence de donnees coherentes issues d'echantillons asthmatiques, couplee a la concordance relative des resultats obtenus a partir de cellules de MLR stimulees suggere que les changements de contractilite du MLR dans l'asthme peuvent etre dependants de la presence d'un environnement inflammatoire autour de la couche de MLR. Le fait de retirer le MLR de son environnement pourrait expliquer pourquoi l'hyper-contractilite est rarement observee ex vivo. [Traduit par la Redaction] Mots-cles: asthme, muscle lisse respiratoire, expression genique, expression proteique, cytokines., Introduction Asthma is a disease that affects nearly 300 million people worldwide (Masoli et al. 2004) and is characterized by periods of breathlessness and wheezing caused by narrowing of the [...]

The standard method for measuring the phosphorylation of the regulatory myosin light chain (MLC20) in smooth muscle is extraction of the light chain using a urea extraction buffer, urea-glycerol gel electrophoresis of the soluble portion of the extract (supernatant) and Western blot analysis. The undissolved portion of the tissue during extraction (the pellet) is usually discarded. Because the pellet contains a finite amount of MLC20, omission of the pellet could result in inaccurate measurement of MLC20 phosphorylation. In this study we compared the level of tracheal smooth muscle MLC20 phosphorylation in the supernatant alone, with that in the complete tissue homogenate (supernatant and pellet) using the standard method. The supernatant fraction showed the well-known double bands representing phosphorylated and un-phosphorylated MLC20. The dissolved pellet fraction showed varying amounts of un-phosphorylated and phosphorylated MLC20. There was a small but statistically significant overestimation of the percent MLC20 phosphorylation if the pellet was not taken into consideration. The overestimation was 7% ± 2% (mean ± SEM) (p < 0.05) in unstimulated muscle and 2% ± 1% (p < 0.05) in acetylcholine (10-6 mol/L) stimulated muscle. This finding suggests that for accurate estimation of the stoichiometry of MLC20 phosphorylation it is necessary to consider the contribution from the pellet portion of the muscle tissue homogenate. Key words: urea-glycerol gel electrophoresis, MLC20 phosphorylation, supernatant and pellet, Western blots. La methode standard pour mesurer la phosphorylation de la chaine legere de la myosine regulatrice (MLC20) dans le muscle lisse consiste en une extraction de la chaine legere dans un tampon d'extraction comprenant de l'uree, une electrophorese sur gel d'uree-glycerol de la portion soluble de l'extrait (surnageant) et d'un buvardage Western. La portion tissulaire non dissoute lors de l'extraction (culot) est habituellement jetee. Parce que le culot contient une faible quantite de MLC20, l'omission du culot pourrait resulter en une mesure imprecise de la phosphorylation de la MLC20. Dans cette etude, les auteurs ont compare le niveau de phosphorylation de MLC20 du muscle lisse tracheal dans le surnageant seul a celui de l'homogenat tissulaire complet (surnageant et culot) a l'aide de la methode standard. La fraction du surnageant montrait les doubles bandes bien connues representant la MLC20 phosphorylee et non phosphorylee. La fraction dissoute du culot contenait des quantites variables de MLC20 phosphorylee et non phosphorylee. Il y avait une legere mais significative surestimation du pourcentage de MLC20 phosphorylee si le culot n'etait pas considere. La surestimation etait de7%±2% (moyenne ± ESM) (p < 0,05) dans le muscle non stimule et de 2%[+ or -]1%(p < 0,05) dans le muscle stimule a l'acetylcholine (10-6 mol/L). Ces donnees suggerent que l'estimation precise de la strechiometrie de la phosphorylation de la MLC20 necessite de considerer la contribution de la portion du culot issue de l'homogenat de tissu musculaire. [Traduit par la Redaction] Mots-cles: electrophorese sur gel uree-glycerol, phosphorylation de la MLC20, surnageant et culot, buvardages Western., Introduction The crucial step for the initiation of the ATP-dependent actomyosin interaction in smooth muscle is the phosphorylation of the myosin light chain 20 (MLC20) (Frearson et al. 1976; Sobieszek [...]

Background An imbalance between proteolytic enzymes and their inhibitors is thought to be involved in the pathogenesis of chronic obstructive pulmonary disease. Matrix metalloproteinase-1, also known as interstitial collagenase, has been implicated as a potentially important proteinase in the genesis of chronic obstructive pulmonary disease and, more specifically, emphysema. Methods We performed quantitative immunohistochemical assessment of matrix metalloproteinase-1 expression in the resected lung of 20 smokers/ex-smokers who had varying severity of airflow obstruction and emphysema and compared this with the lungs of 5 nonsmokers. Emphysema was measured using a morphometric measure of the lungs' surface area/volume ratio and with qualitative and quantitative computed tomography (CT) measures of emphysema. Results There were significantly more matrix metalloproteinase-1-expressing alveolar macrophages and type II pneumocytes as well as a greater percentage of small airways that stained positively for matrix metalloproteinase-1 in the lungs of smokers than in those of nonsmokers (p \ 0.0001, p \ 0.0001, and p = 0.0003, respectively). The extent of staining of type II pneumocytes and airways for matrix metalloproteinase-1 was significantly related to the extent of smoking (p = 0.012 and p = 0.013, respectively). In addition, the extent of matrix metalloproteinase-1 staining of alveolar macrophages was related to the lung surface area/volume ratio and to qualitative estimates of emphysema on CT. Conclusion These findings suggest that cigarette smoking increases expression of matrix metalloproteinase-1 in alveolar macrophages as well as in alveolar and small airway epithelial cells. Smokers who develop emphysema have increased alveolar macrophage expression of matrix metalloproteinase-1. Keywords Computed tomography * Emphysema * Expression * Immunohistochemistry * Metalloproteinase * Lung, Introduction Cigarette smoke is the primary risk factor for chronic obstructive pulmonary disease (COPD), a disease state characterized by airflow limitation that is not fully reversible [1]. Not everyone who [...]

The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells. muscle mechanics; stiffness; ultrastructure; three-dimensional electron microscopy doi: 10.1152/ajplung.00087.2010.

The amplitude of strain in airway smooth muscle (ASM) produced by oscillatory perturbations such as tidal breathing or deep inspiration (DI) influences the force loss in the muscle and is therefore a key determinant of the broncho-protective and bronchodilatory effects of these breathing maneuvers. The stiffness of unstimulated ASM (passive stiffness) directly influences the amplitude of strain. The nature of the passive stiffness is, however, not clear. In this study, we measured the passive stiffness of ovine ASM at different muscle lengths (relative to in situ length, which was used as a reference length, [L.sub.ref]) and states of adaptation to gain insights into the origin of this muscle property. The results showed that the passive stiffness was relatively independent of muscle length, possessing a constant plateau value over a length range from 0.62 to 1.25 [L.sub.ref]. Following a halving of ASM length, passive stiffness decreased substantially (by 71%) but redeveloped over time (~30 rain) at the shorter length to reach 65% of the stiffness value at [L.sub.ref], provided that the muscle was stimulated to contract at least once over a ~30-min period. The redevelopment and maintenance of passive stiffness were dependent on the presence of [Ca.sup.2+] but unaffected by latrunculin B, an inhibitor of actin filament polymerization. The maintenance of passive stiffness was also not affected by blocking myosin cross-bridge cycling using a myosin light chain kinase inhibitor or by blocking the Rho-Rho kinase (RhoK) pathway using a RhoK inhibitor. Our results suggest that the passive stiffness of ASM is labile and capable of redevelopment following length reduction. Redevelopment and maintenance of passive stiffness following muscle shortening could contribute to airway hyperresponsiveness by attenuating the airway wall strain induced by tidal breathing and DI. asthma; lung doi:10.1152/ajplung.00275.2009

Although cystic fibrosis (CF) is a monogenic disease, its clinical manifestations are influenced in a complex manner. Severity of lung disease, the main cause of mortality among CF patients, is likely modulated by several genes. The mannose-binding lectin 2 (MBL2) gene encodes an innate immune response protein and has been implicated as a pulmonary modifier in CF. However, reports have been conflicting, and interactions with other modifiers have not been investigated. We therefore evaluated the association of MBL2 with CF pulmonary phenotype in a cohort of 1,019 Canadian pediatric CF patients. MBL2 genotypes were combined into low-, intermediate-, and high-expression groups based on MBL2 levels in plasma. Analysis of age at first infection with Pseudomonas aeruginosa demonstrated that MBL2 deficiency was significantly associated with earlier onset of infection. This MBL2 effect was amplified in patients with high-producing genotypes of transforming growth factor beta 1 (TGFB1). Similarly, MBL2 deficiency was associated with more rapid decline of pulmonary function, most significantly in those carrying the high-producing TGFB1 genotype. These findings provide evidence of gene-gene interaction in the pathogenesis of CF lung disease, whereby high TGF-[beta]1 production enhances the modulatory effect of MBL2 on the age of first bacterial infection and the rate of decline of pulmonary function., Introduction Cystic fibrosis (CF) is an autosomal-recessive disease typically manifesting with progressive obstructive lung disease, insufficiency of the exocrine pancreas, and elevated electrolyte concentration in sweat glands (OMIM 219700). Although [...]

Abstract: The sliding filament theory of contraction that was developed for striated muscle is generally believed to be also applicable to smooth muscle. However, the well-organized myofilament lattice (i.e., the [...]

Abstract: Airway hyperresponsiveness, particularly the ability of airways to narrow excessively in response to stimuli that normally cause little airway narrowing in nonasthmatic subjects, is a characteristic feature of asthma [...]

Chronic obstructive pulmonary disease (COPD) is a major health problem, which is caused due to excessive exposure to toxic gases and particles. It is concluded that COPD is associated with a thickening of the airway wall, which results in the accumulation of inflammatory exudates in the lumen.

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.4% [+ or -] 5.7 (SE) and 76.0% [+ or -] 9.8, respectively, for a 100% increase in cell length. Muscle power output, ATPase rate, and myosin filament density also have the same dependence on muscle cell length: increased by 35.4% [+ or -] 6.7, 34.6% [+ or -] 3.4, and 35.6% [+ or -] 10.6, respectively, for a 50% increase in cell length. The data can be explained by a model in which additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length. muscle contraction; myosin filaments; ATPase activity; electron microscopy

Tissue macrophages and interstitial airways generate platelet-derived growth factor (PDGF), which activates human eosinophils to produce inflammation of the airways in asthma and chronic obstructive pulmonary diseases (COPD). PDGF multiplies smooth muscle cells and is mitogenic for cultured fibroblasts. Asthmatic patients reveal higher levels of mRNA for PDGF and PDGF-beta than COPD patients due to the glucocorticoid administration.