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Ventilation heterogeneity is an important marker of small airway dysfunction in asthma. The frequency dependence of respiratory system resistance ([R.sub.rs]) from oscillometry is used as a measure of this heterogeneity. However, this has not been quantitatively assessed or compared with other outcomes from oscillometry, including respiratory system reactance ([X.sub.rs]) and the associated elastance ([E.sub.rs]). Here, we used a multibranch model of the human lung, including an upper airway shunt, to match previously reported respiratory mechanics in mild to severe asthma. We imposed heterogeneity by narrowing a proportion of the peripheral airways to account for patient [E.sub.rs] at 5 Hz, and then narrowed central airways to account for the remaining [R.sub.rs] at 18 Hz. The model required >75% of the small airways to be occluded to reproduce severe asthma. While the model produced frequency dependence in [R.sub.rs], it was upward-shifted below 5 Hz compared with in-vivo results, indicating that other factors, including more distributed airway narrowing or central airway wall compliance, are required. However, [E.sub.rs] quantitatively reflected the imposed heterogeneity better than the frequency dependence of [R.sub.rs], independent of the frequency range for the estimation, and thus was a more robust measure of small-airway function. Thus, [E.sub.rs] appears to have greater potential as a clinical measure of early small-airway disease in asthma. Key words: small airway heterogeneity, ventilation defects, elastance, frequency dependence of resistance, oscillometry. L'heterogeneite ventilatoire constitue un marqueur important de la dysfonction des petites voies respiratoires dans l'asthme. La dependance en fonction de la frequence de la resistance du systeme respiratoire ([R.sub.rs]) a partir de l'oscillometrie est utilisee pour mesurer cette heterogeneite. Cependant, elle n'a pas ete evaluee de maniere quantitative ou comparee a d'autres resultats de l'oscillometrie, incluant la reactance du systeme respiratoire ([X.sub.rs]) et l'elastance associee ([E.sub.rs]). Les auteurs ont utilize ici un modele ramifie de poumon humain incluant une obstruction des voies aeriennes superieures qui reproduit la mecanique respiratoire de l'asthme leger a severe rapportee precedemment. Les auteurs ont impose une heterogeneite en retrecissant une partie des voies respiratoires peripheriques pour representer l'[E.sub.rs] a 5 Hz d'un patient, et en retrecissant ensuite les voies respiratoires centrales pour representer la [R.sub.rs] residuelle a 18 Hz. Une occlusion de >75 % des petites voies respiratoires etait necessaire pour representer l'asthme severe. Alors que le modele produisait une dependance en fonction de la frequence de la [R.sub.rs], elle se deplacait sous les 5 Hz relativement a la situation in vivo, indiquant que d'autres facteurs sont requis, incluant une meilleure distribution du retrecissement des voies respiratoires ou une compliance des parois des voies respiratoires centrales. Cependant, la [E.sub.rs] refletait quantitativement l'heterogeneite imposee davantage que la dependance en fonction de la frequence de la [R.sub.rs], de maniere independante du spectre de frequence pour l'estimation, et constituait ainsi une mesure plus robuste de la fonction des petites voies respiratoires. La [E.sub.rs] semble alors avoir un meilleur potentiel comme mesure clinique de la maladie precoce des petites voies respiratoires dans l'asthme. [Traduit par la Redaction] Mots-cles: heterogeneite des petites voies respiratoires, defauts de ventilation, elastance, dependance en fonction de la frequence de la resistance, oscillometrie., Introduction Heterogeneous airway narrowing is a hallmark feature of asthma; a fact also supported by the occurrence of 'patchy ventilation,' characterized by ventilation defects in asthmatic subjects as seen in [...]

Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and 'relaxability' (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness. asthma; mechanical stress; cytoskeletal remodeling; myosin light-chain kinase; optical magnetic twisting cytometry

Abstract: Extracellular matrix proteins (ECMs) play a significant role in the transfer of mechanical strain to monocyte derived macrophages (MDMs) affecting morphological changes in a foreign body reaction. This study [...]

Viscoelasticity of human alveolar epithelial cells subjected to stretch. Am J Physiol Lung Cell Mol Physiol 287: L1025-L1034, 2004. First published July 9, 2004; doi:10.1152/ajplung.00077.2004.--Alveolar epithelial cells undergo stretching during breathing and mechanical ventilation. Stretch can modify cell viscoelastic properties, which may compromise the balance of forces in the alveolar epithelium. We studied the viscoelasticity of alveolar epithelial cells (A549) subjected to equibiaxial distention with a novel experimental approach. Cells were cultured on flexible substrates and subjected to stepwise deformations of up to 17% with a device built on an inverted microscope. Simultaneously, cell storage (G') and loss (G') moduli were measured (0.1-100 Hz) with optical magnetic twisting cytometry. G' and G' increased with strain up to 64 and 30%, respectively, resulting in a decrease in G'/G' (15%). This stretch-induced response was inhibited by disruption of the actin cytoskeleton with latrunculin A. G' increased with frequency following a power law with exponent [alpha] = 0.197. G' increased proportionally to G' but exhibited a more marked frequency dependence at high frequencies. Stretching (14%) caused a fall in [alpha] (13%). At high stretching amplitudes, actual cell strain (14.4%) was lower than the applied substrate strain (17.3%), which could indicate a partial cell detachment. These data suggest that cytoskeletal prestress modulates the elastic and frictional properties of alveolar epithelial cells in a coupled manner, according to soft glassy rheology. Stretch-induced cell stiffening could compromise the balance of forces at the cell-cell and cell-matrix adhesions. cell mechanics; cell stretching; magnetic twisting cytometry; prestress; power law

Mechanical stress (MS) causes cytoskeletal (CSK) and phenotypic changes in ceils. Such changes in airway smooth muscle (ASM) cells might contribute to the pathophysiology of asthma. We have shown that periodic mechanical strain applied to cultured ASM cells alters the structure and expression of CSK proteins and increases cell stiffness and contractility (Smith PG, Moreno R, and Ikebe M. Am J Physiol Lung Cell Mol Physiol 272: L20-L27, 1997; and Smith PG, Deng L, Fredberg JJ, and Maksym GN. Am J Physiol Lung Cell Mol Physiol 285: L456-L463, 2003). However, the mechanically induced CSK changes, altered cell function, and their time courses are not well understood. Here we applied MS to the CSK by magnetically oscillating ferrimagnetic beads bound to the CSK. We quantified CSK remodeling by measuring actin accumulation at the sites of applied MS using fluorescence microscopy. We also measured CSK stiffness using optical magnetic twisting cytometry. We found that, during MS of up to 120 min, the percentage of beads associated with actin structures increased with time. At 60 min, 68.1 [+ or -] 1.6% of the beads were associated with actin structures compared with only 6.7 [+ or -] 2.8% before MS and 38.4 [+ or -] 5.5% in time-matched controls (P < 0.05). Similarly, CSK stiffness increased more than twofold in response to the MS compared with time-matched controls. These changes were more pronounced than observed with contractile stimulation by 80 mM KCl or [10.sup.-4] M acetylcholine. Together, these findings imply that MS is a potent stimulus to enhance stiffness and contractility of ASM cells through CSK remodeling, which may have important implications in airway narrowing and dilation in asthma. mechanical stress; actin cytoskeleton; stiffness; airway smooth muscle cell; optical magnetic twisting cytometry; airway constriction and dilation; asthma

We tested the hypothesis that cytoskeletal reorganization induced by cyclic strain increases cytoskeletal stiffness (G'). G' was measured by optical magnetic twisting cytometry in control cells and cells that had received mechanical strain for 10-12 days. G' was measured before and after both contractile and relaxant agonists, and in the strained cells both parallel (Para) and perpendicular (Perp) to the aligned cytoskeleton. Before activation, G' Para was 24 [+ or -] 5% ([+ or -] SE) greater compared with Perp (P < 0.05), and 35% [+ or -] 6 greater compared with control (Cont, P < 0.01). The difference between strained and control cells was enhanced by KCl, increasing G' 171 [+ or -] 7% Para compared with 125 [+ or -] 6% Perp and 129 [+ or -] 8% Cont (P < [10.sup.-5] both cases). The decrease in G' from baseline due to relaxant agonists isoproterenol and dibutyryl cAMP was similar in all groups. Long-term oscillatory loading of airway smooth muscle (ASM) cells caused stiffness to increase and become anisotropic. These findings are consistent with the hypothesis that cytoskeletal reorganization can enhance ASM stiffness and contractility. They imply, furthermore, that oscillatory loading of ASM may contribute to airway narrowing and failure of airway dilation in asthma. smooth muscle contraction; plasticity; optical magnetic twisting cytometry; anisotropy; airway smooth muscle cell

Laporte, Johanne D., Paul E. Moore, Joseph H. Abraham, Geoffrey N. Maksym, Ben Fabry, Reynold A. Panettieri, Jr., and Stephanie A. Shore. Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1[Beta]. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L943-L951, 1999.--We have previously reported that interleukin (IL)-1[Beta] causes [Beta]-adrenergic hyporesponsiveness in cultured human airway smooth muscle cells by increasing cyclooxygenase-2 (COX-2) expression and prostanoid formation. The purpose of this study was to determine whether extracellular signal-regulated kinases (ERKs) are involved in these events. Levels of phosphorylated ERK (p42 and p44) increased 8.3- and 13-fold, respectively, 15 min after treatment with IL-1[Beta] (20 ng/ml) alone. Pretreating cells with the mitogen-activated protein kinase kinase inhibitor PD-98059 or U-126 (2 h before IL-1[Beta] treatment) decreased ERK phosphorylation. IL-1[Beta] (20 ng/ml for 22 h) alone caused a marked induction of COX-2 and increased basal [PGE.sub.2] release 28-fold (P [is less than] 0.001). PD-98059 (100 [micro]M) and U-126 (10 [micro]M) each decreased COX-2 expression when administered before IL-1[Beta] treatment. In control cells, PD-98059 and U-126 had no effect on basal or arachidonic acid (AA; 10 [micro]M)-stimulated [PGE.sub.2] release, but both inhibitors caused a significant decrease in bradykinin (BK; 1 [micro]M)-stimulated [PGE.sub.2] release, consistent with a role for ERK in the activation of phospholipase [A.sub.2] by BK. In IL-1[Beta]-treated cells, prior administration of PD-98059 caused 81, 92 and 40% decreases in basal and BK- and AA-stimulated [PGE.sub.2] release, respectively (P [is less than] 0.01), whereas administration of PD-98059 20 h after IL-1[Beta] resulted in only 38 and 43% decreases in basal and BK-stimulated [PGE.sub.2] release, respectively (P [is less than] 0.02) and had no effect on AA-stimulated [PGE.sub.2] release. IL-1[Beta] attenuated isoproterenol-induced decreases in human airway smooth muscle stiffness as measured by magnetic twisting cytometry, and PD-98059 or U-126 abolished this effect in a concentration-dependent manner. These results are consistent with the hypothesis that ERKs are involved early in the signal transduction pathway through which IL-1[Beta] induces [PGE.sub.2] synthesis and [Beta]-adrenergic hyporesponsiveness and that ERKs act by inducing COX-2 and activating phospholipase [A.sub.2]. extracellular signal-regulated kinase; mitogen-activated protein; interleukin-1[Beta]; prostaglandin [E.sub.2]; [Beta]-adrenergic responses; PD-98059; U-126; magnetic twisting cytometry; cyclooxygenase