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Background A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma. Methods Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds. Results Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo. Conclusions This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

The principal constituent of the living cell is water. The role of the hydration shell and bulk H(2)O solvent is well recognized in the dynamics of isolated proteins, but the role of water in the dynamics of the integrated living cytoskeleton (CSK) remains obscure. Here we report a direct connection of dynamics of water to dynamics of the integrated CSK. The latter are known to be scale-free and to hinge upon a frequency f(0) that is roughly invariant across cell types. Although f(0) is comparable in magnitude to the rotational relaxation frequency of water (gigahertz range), the physical basis of f(0) remains unknown. Using the human airway smooth muscle cell as a model system, we show here that replacing water acutely with deuterium oxide impacts CSK dynamics in major ways, slowing CSK remodeling dynamics appreciably, and lowering f(0) by up to four orders of magnitude. Although these observations do not distinguish contributions of bulk solvent versus hydration shell, they suggest a unifying hypothesis, namely, that dynamics of integrated CSK networks are slaved in a direct fashion to fluctuations arising in intracellular water.

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

Total lung resistance (RL), airway resistance (Raw), and tissue resistance (Rti) were measured in unconstricted and methacholine (MCh)-constricted guinea pigs while tidal volume, lung volume, and breathing frequency were varied. Measurements were made in tracheostomized ventilated guinea pigs with use of alveolar capsules. Relationships between Raw and Rti at different breathing frequencies, lung volumes, tidal volumes, and levels of constriction were compared with previously reported values in other species. Our results demonstrate that, at fixed tidal volume, Rti was inversely related to breathing frequency (Rti approximately f-0.64, where f is breathing frequency in Hz) and increased with increasing lung volume. Rti was a significantly greater percentage of RL after MCh administration (40–50%) than at baseline (15–35%), indicating a greater tissue than airway constrictor response. Rti was also 0.5 log dose more responsive to intravenous MCh than Raw on the basis of the dose required to produce 100% increase in resistance from baseline (PD100). These data show that, in the guinea pig, Rti changes with lung volume, breathing frequency, and constrictor tone in a manner similar to other species previously reported and that Rti can be an important determinant of lung dysfunction during constriction, even in species for which it is small in relation to Raw at baseline.

This report deals with noninvasive imaging of airway geometry based upon information contained in acoustic reflections measured at the mouth. Here we describe a new theoretical approach that enables development of a new miniaturized apparatus. Unlike the single-transducer systems used currently, this new strategy is based upon a two-transducer system that is a variant of that suggested originally by Shroeder (1967). We have developed, implemented, and tested computational algorithms necessary to reconstruct airway dimensions from acoustic reflection data using this two-transducer strategy.

When challenged with a contractile agonist in increasing graded concentrations, lung parenchymal tissue assumes a sequence of mechanical states. That sequence is mapped here. Isolated lung parenchymal strips from male Hartley guinea pigs were mounted in a bath containing Krebs solution at 37 degrees C, aerated with 95% O2–5% CO2. One end was attached to a force transducer and the other to a servo-controlled lever arm. After stress adaptation, sinusoidal length oscillations (1% strain at 0.31 Hz) yielded force-length loops from which we computed induced changes in active tension (F), tissue stiffness (E), and hysteresivity (eta) (J. J. Fredberg and D. Stamenovic. J. Appl. Physiol. 67:2408–2419, 1989). Changes of tissue resistance (R) were, by definition, governed by those of eta and E. Histamine (10(-6) -10(-3) M), prostaglandin D2 (10(-5) -10(-4) M), and prostaglandin F2 alpha (10(-5) -10(-4) M) caused dose-related increases of F, eta, and E. Plotting induced changes of E vs. those of F revealed a unique relationship that was identical for these as well as a wider panel of contractile agonists; changes of E and F were closely associated. However, plotting induced changes of E vs. those of eta revealed relationships that differed distinctly between agonists; changes of eta were dissociated from those of F and E. This latter observation demonstrated the existence of distinct mechanical states that differed according to the specific agonist by which the tissue was stimulated. In producing agonist-induced changes in R, changes of E were of equal or greater importance compared with those of eta. We conclude that guinea pig lung parenchyma, viewed as an integrated physiological tissue system, exhibits different kinds as well as varying intensities of mechanical response according to the specific agonist present in the cellular microenvironment. These differences in contractile state reveal themselves principally in the hysteretic nature of the tissue.

The most abundant proteins in our cells are there to generate mechanical forces, and measurement of these forces has just become possible.

Structure and function of the adherent cell depend in a crucial way on its microenvironment, including the stiffness of its substrate. It is often asserted that substrate thickness (as opposed to stiffness) plays a negligible role and therefore may be considered semi-infinite. This assertion has been recently challenged, but the characteristic length scale to consider in this regard is poorly understood. We show here that this characteristic length scale is the lateral cell size. As substrate thickness approaches the lateral dimension of the cell, the apparent stiffness of the substrate is amplified to levels much greater than the intrinsic stiffness of the substrate. This change in apparent stiffness is readily sensed by the cell, leading to changes in cell spreading area, stiffness, and contractile forces. In contrast to these responses that occur over the length of the cell, mechanosensing around an isolated point force is influenced greatly by intrinsic substrate stiffness but to a negligible extent by substrate thickness. We conclude that mechanosensing of substrate thickness is dominated in large part by traction forces spread over the lateral dimension of the cell.

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40–100 nm and less than 44 μm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 μm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 μM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Cell mechanical properties on a whole cell basis have been widely studied, whereas local intracellular variations have been less well characterized and are poorly understood. To fill this gap, here we provide detailed intracellular maps of regional cytoskeleton (CSK) stiffness, loss tangent, and rate of structural rearrangements, as well as their relationships to the underlying regional F-actin density and the local cytoskeletal prestress. In the human airway smooth muscle cell, we used micropatterning to minimize geometric variation. We measured the local cell stiffness and loss tangent with optical magnetic twisting cytometry and the local rate of CSK remodeling with spontaneous displacements of a CSK-bound bead. We also measured traction distributions with traction microscopy and cell geometry with atomic force microscopy. On the basis of these experimental observations, we used finite element methods to map for the first time the regional distribution of intracellular prestress. Compared with the cell center or edges, cell corners were systematically stiffer and more fluidlike and supported higher traction forces, and at the same time had slower remodeling dynamics. Local remodeling dynamics had a close inverse relationship with local cell stiffness. The principal finding, however, is that systematic regional variations of CSK stiffness correlated only poorly with regional F-actin density but strongly and linearly with the regional prestress. Taken together, these findings in the intact cell comprise the most comprehensive characterization to date of regional variations of cytoskeletal mechanical properties and their determinants.

Tidal breathing, and especially deep breathing, is known to antagonise bronchoconstriction caused by airway smooth muscle (ASM) contraction; however, this bronchoprotective effect of breathing is impaired in asthma. Force fluctuations applied to contracted ASM in vitro cause it to relengthen, force-fluctuation-induced relengthening (FFIR). Given that breathing generates similar force fluctuations in ASM, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. Thus it is of considerable interest to understand what modulates FFIR, and how ASM might be manipulated to exploit this phenomenon. It was demonstrated previously that p38 mitogen-activated protein kinase (MAPK) signalling regulates FFIR in ASM strips. Here, it was hypothesised that the MAPK kinase (MEK) signalling pathway also modulates FFIR. In order to test this hypothesis, changes in FFIR were measured in ASM treated with the MEK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Increasing concentrations of U0126 caused greater FFIR. U0126 reduced extracellular signal-regulated kinase 1/2 phosphorylation without affecting isotonic shortening or 20-kDa myosin light chain and p38 MAPK phosphorylation. However, increasing concentrations of U0126 progressively blunted phosphorylation of high-molecular-weight caldesmon (h-caldesmon), a downstream target of MEK. Thus changes in FFIR exhibited significant negative correlation with h-caldesmon phosphorylation. The present data demonstrate that FFIR is regulated through MEK signalling, and suggest that the role of MEK is mediated, in part, through caldesmon.

The ability of cells to respond to external mechanical stimulation is a complex and robust process involving a diversity of molecular interactions. Although mechanotransduction has been heavily studied, many questions remain regarding the link between physical stimulation and biochemical response. Of significant interest has been the contribution of the transmembrane proteins involved, and integrins in particular, because of their connectivity to both the extracellular matrix and the cytoskeleton. Here, we demonstrate the existence of a mechanically based initiation molecule, syndecan-4. We first demonstrate the ability of syndecan-4 molecules to support cell attachment and spreading without the direct extracellular binding of integrins. We also examine the distribution of focal adhesion-associated proteins through controlling surface interactions of beads with molecular specificity in binding to living cells. Furthermore, after adhering cells to elastomeric membranes via syndecan-4-specific attachments we mechanically strained the cells via our mechanical stimulation and polymer surface chemical modification approach. We found ERK phosphorylation similar to that shown for mechanotransductive response for integrin-based cell attachments through our elastomeric membrane-based approach and optical magnetic twisting cytometry for syndecan-4. Finally, through the use of cytoskeletal disruption agents, this mechanical signaling was shown to be actin cytoskeleton dependent. We believe that these results will be of interest to a wide range of fields, including mechanotransduction, syndecan biology, and cell–material interactions.

It is commonly believed that the time scale governing the rheology of airway smooth muscle (ASM) is set by the internal viscosity and elasticity of the muscle. We show here, to the contrary, that this time scale is set by the externally imposed rate of strain.For any fixed strain rate amplitude (SRA), the elastic modulus of the ASM showed a sigmoidal dependence upon frequency. Remarkably, as the SRA was increased over a range spanning almost four decades, sigmoidal response curves demonstrated little change of shape but shifted dramatically to higher frequencies. As such, the time scale of underlying molecular processes is set not by any internal viscosity, elasticity, or any spontaneous internal rate process, but instead is set by the imposed rate of strain. When the muscle is loaded at a small strain-rate, the molecular dynamics are slow; when loaded at a large strain-rate, the dynamics are fast.Using numerical computations, we then assessed the contribution of myosin bridge kinetics to this behavior. In the regime where frequency was the highest, a good agreement between data and computations was obtained; ASM dynamics could, therefore, be attributed to forced acto-myosin crossbridge dynamics. But at the lowest frequencies, the slopes differed dramatically and stiffness values differed by an order of magnitude, exposing a new domain of slow dynamics that cannot be accounted for by acto-myosin interactions.Interestingly, these results unify scale-free dynamics, fluidization, and length adaptation. While this unification is not explained by any traditional physical picture of cell rheology or polymer dynamics, it deepens substantially the analogy between living and inert soft matter, and in doing so, reveals a central role for microstructural fragility.

Background Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood. Principal Findings In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize. Conclusions Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin.

We tested the hypothesis that particle deposition in the pulmonary acinus is substantially influenced by alveolar duct structure. A realistic geometric model of the alveolated duct was generated and the low Reynolds number velocity field of carrier gas in that conduit was solved numerically. To compute particle trajectories, analytic expressions of particle dynamics were developed based on integration of the Langevin equation. Using Monte Carlo methods, conditional probabilities of the trajectories for a given initial condition were calculated and an eigenfunction expansion technique was developed to derive a generalized expression of deposition kinetics. Brownian motion dominated deposition for small particles (≤1 μm), while gravitational effects dominated larger particles. For intermediate particles, both gravity and diffusion played important roles in deposition. Overall, the deposition rate was lower in the alveolated duct than in a non-alveolated duct, the deposition pattern within alveolus was nonuniform with higher deposition density near the alveolar entrance ring.

Mechanical robustness of the cell under different modes of stress and deformation is essential to its survival and function. Under tension, mechanical rigidity is provided by the cytoskeletal network; with increasing stress, this network stiffens, providing increased resistance to deformation. However, a cell must also resist compression, which will inevitably occur whenever cell volume is decreased during such biologically important processes as anhydrobiosis and apoptosis. Under compression, individual filaments can buckle, thereby reducing the stiffness and weakening the cytoskeletal network. However, the intracellular space is crowded with macromolecules and organelles that can resist compression. A simple picture describing their behavior is that of colloidal particles; colloids exhibit a sharp increase in viscosity with increasing volume fraction, ultimately undergoing a glass transition and becoming a solid. We investigate the consequences of these 2 competing effects and show that as a cell is compressed by hyperosmotic stress it becomes progressively more rigid. Although this stiffening behavior depends somewhat on cell type, starting conditions, molecular motors, and cytoskeletal contributions, its dependence on solid volume fraction is exponential in every instance. This universal behavior suggests that compression-induced weakening of the network is overwhelmed by crowding-induced stiffening of the cytoplasm. We also show that compression dramatically slows intracellular relaxation processes. The increase in stiffness, combined with the slowing of relaxation processes, is reminiscent of a glass transition of colloidal suspensions, but only when comprised of deformable particles. Our work provides a means to probe the physical nature of the cytoplasm under compression, and leads to results that are universal across cell type.

Breathing is known to functionally antagonize bronchoconstriction caused by airway muscle contraction. During breathing, tidal lung inflation generates force fluctuations that are transmitted to the contracted airway muscle. In vitro, experimental application of force fluctuations to contracted airway smooth muscle strips causes them to relengthen. Such force fluctuation-induced relengthening (FFIR) likely represents the mechanism by which breathing antagonizes bronchoconstriction. Thus, understanding the mechanisms that regulate FFIR of contracted airway muscle could suggest novel therapeutic interventions to increase FFIR, and so to enhance the beneficial effects of breathing in suppressing bronchoconstriction. Here we propose that the connectivity between actin filaments in contracting airway myocytes is a key determinant of FFIR, and suggest that disrupting actin-myosin-actin connectivity by interfering with actin polymerization or with myosin polymerization merits further evaluation as a potential novel approach for preventing prolonged bronchoconstriction in asthma.

Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment. © 2009 Krishnan et al.

Tracheal instillation of pentamidine in a surfactant vehicle may be an effective direct method of antibiotic delivery to the lungs. In 10 healthy hamsters, we compared the pulmonary distribution of 99mTc sulfur colloid (TcSC) mixed with pentamidine, using as a vehicle either surfactant (n = 5) or saline (n = 5). Each animal was instilled with 0.25 ml/kg of suspension containing 0.0018 mCi TcSC and pentamidine mixed with either surfactant or saline. After 4 h of spontaneous respiration, the lungs were excised, inflated to TLC, dried, and sliced into 3-mm cross sections from apex to base. Autoradiographs were examined to evaluate 99mTc distribution. The surfactant group had detectable radioactivity in 93% of all slices compared with 72% in the saline group (p = 0.02). Six slices per animal (43% of total) and their corresponding autoradiographs were analyzed for distribution of radioactivity. Lung slice area was determined by planimetry, and autoradiograph area was determined by video densitometry. We calculated the fraction of each lung slice with detectable radioactivity. The surfactant group had 41% of the lung slice areas exposed compared with 21% in the saline group (p = 0.02). The coefficient of variation of radioactive intensities within each slice was used as an index of spatial uniformity. There was a trend towards more uniform distribution in the surfactant group, with a narrower range of variation of intensities (1.51 to 2.56) than the saline group (1.95 to 6.47). We conclude that a surfactant vehicle significantly increases airspace deposition of TcSC and pentamidine instilled intratracheally in normal hamster lungs, and may improve uniformity of spread.

Inspiratory airflow in the avian lung completely bypasses the most cranial secondary bronchi (the ventrobronchi), and instead enters bronchi arising more caudally (the dorsobronchi). Dotterweich (1936) proposed that ‘aerodynamic valves’ prevented entry into the ventrobronchi. We have recently provided evidence that inspiratory aerodynamic valving in avian lungs depends on convective inertia in the primary bronchus (Banzett et al., 1987). Theoretical and physical models (Butler et al., 1988; Wang et al., 1988) showed that convective inertia could effect valving, but the effectiveness of valving at resting flows was less than that observed in the bird. This leads us to hypothesize that a segment of the primary bronchus is constricted, accelerating the gas and enhancing the convective inertia. To test this hypothesis in the present work we measured pressures throughout the airways and air sacs in anesthetized, pump-ventilated geese at different flow rates and gas densities. Our data show: (1) there is a large pressure drop in the primary bronchus close to the ventrobronchial junction, indicating the presence of a constriction; (2) this pressure drop increases with gas density and flow; (3) the convective inertia at this site is more than 10 times downstream opposing pressures. We conclude that the primary bronchus just cranial to the first ventrobronchus forms a constriction which accelerates inspired air. Furthermore, we conclude that the convective inertia of gas leaving this segment is sufficient to achieve inspiratory valving.

Although volumetric displacements of the chest wall are often analyzed in terms of two independent parallel pathways (rib cage and abdomen), Loring and Mead have argued that these pathways are not mechanically independent (J. Appl. Physiol. 53: 756-760, 1982). Because of its apposition with the diaphragm, the rib cage is exposed to two distinct pressure differences, one of which depends on abdominal pressure. Using the analysis of Loring and Mead as a point of departure, we developed a complementary analysis in which mechanical coupling of the rib cage, abdomen, and diaphragm is modeled by a linear translational transformer. This model has the advantage that it possesses a precise electrical analogue. Pressure differences and compartmental displacements are related by the transformation ratio (n), which is the mechanical advantage of abdominal over pleural pressure changes in displacing the rib cage. In the limiting case of very high lung volume, n----0 and the pathways uncouple. In the limit of very small lung volume, n----infinity and the pathways remain coupled; both rib cage and abdomen are driven by abdominal pressure alone, in accord with the Goldman-Mead hypothesis. A good fit was obtained between the model and the previously reported data for the human chest wall from 0.5 to 4 Hz (J. Appl. Physiol. 66:350-359, 1989). The model was then used to estimate rib cage, diaphragm, and abdominal elastance, resistance, and inertance. The abdomen was a high-elastance high-inertance highly damped compartment, and the rib cage a low-elastance low-inertance more lightly damped compartment. Our estimate that n = 1.9 is consistent with the findings of Loring and Mead and suggests substantial pathway coupling.

Fredberg and Stamenovic (J. Appl. Physiol. 67: 2408-2419, 1989) demonstrated a relatively robust phenomenological relationship between resistance (R) and elastance (E) of lung tissue during external forcing. The relationship can be expressed as omega R = eta E, where omega = 2 pi times forcing frequency and eta is hysteresivity; they found eta to be remarkably invariant under a wide range of circumstances. From data gathered in previous experiments, we have tested the adequacy and utility of this phenomenological description for the chest wall (eta w) and its major compartments, the rib cage (eta rc), diaphragm-abdomen (eta d-a), and belly wall (eta bw+). For forcing frequencies and tidal volumes within the normal range of breathing, we found that eta w remained in a relatively narrow range (0.27-0.37) and that neither eta w nor the compartmental eta's changed much with frequency or tidal volume. Compared with eta w, eta rc tended to be slightly low, whereas eta d-a tended to be slightly higher than eta w. However, at higher frequencies (greater than 1 Hz) all eta's increased appreciably with frequency. During various static nonrespiratory maneuvers involving use of respiratory muscles, eta w increased up to twofold. We conclude that in the normal ranges of breathing frequency and tidal volume 1) elastic and dissipative processes within the chest wall appear to be coupled, 2) eta's of the various component parts of the chest wall are well matched, 3) respiratory muscle contraction increases the ratio of cyclic dissipative losses to energy storage, and 4) R of the relaxed chest wall can be estimated from E.(ABSTRACT TRUNCATED AT 250 WORDS)

Axial dispersion in alveolated channels was studied experimentally. Our motivation was to improve understanding of the physics of gas mixing in the pulmonary acinus. The apparatus consisted of a quasi-two-dimensional central convective channel surrounded top and bottom by dead-end cells (alveoli). Quasi-steady oscillatory bulk flow and a small steady-flow of tracer gas (He, SF 6 ) were introduced upstream of the apparatus. The steady-state axial concentration distribution was measured by mass spectrometry, and its gradient was used to calculate an axial dispersion coefficient ( D *) from a generalized Fick's law. We found that for small Peclet ( Pe ) numbers D * was appreciably smaller than molecular diffusivity of the tracer gas, while for large Pe, D * was substantially greater than the Taylor—Aris result for flow enhanced dispersion in non-alveolated parallel plates. D * was sensitive to the ratio of alveolar volume to central channel volume, which was varied from 0.75 to 4.64. These results are consistent with theoretical predictions. We conclude that the structure of the alveolated channel alters the interaction between lateral diffusion and axial convection, and as a result, conditions axial dispersion phenomena.

Excessive force generation by airway smooth muscle is the main culprit in excessive airway narrowing during an asthma attack. The maximum force the airway smooth muscle can generate is exquisitely sensitive to muscle length fluctuations during breathing, and is governed by complex mechanotransduction events that can best be studied by a hybrid approach in which the airway wall is modeled in silico so as to set a dynamic muscle load comparable to that experienced in vivo.

In a remarkably broad range of eukaryotic cell types, the cytoskeleton exhibits physical properties and remodelling dynamics strongly reminiscent of soft inert condensed systems, although with important differences as well. This unexpected intersection of condensed matter physics and cytoskeletal biology suggests that trapping, intermittency, and approach to kinetic arrest represent central mesoscale features linking underlying molecular events to integrative cellular functions such as crawling, contraction and remodelling.

Parasitic infection with Plasmodium falciparum is responsible for the most severe form of human malaria in which patients suffer from periodic fever. It is well established that during intra-erythrocytic maturation of the parasite in the red blood cell (RBC), the RBC becomes significantly more cytoadhesive and less deformable; these and other biochemical factors together with human host factors such as compromised immune status are important contributors to the disease pathology. There is currently substantial interest in understanding the loss of RBC deformability due to P. falciparum infection, but few results are available concerning effects of febrile conditions or parasitization on RBC membrane rheology. Here, for the first time, we report rheology of the single, isolated RBC with and without P. falciparum merozoite invasion, spanning a range from room temperature to febrile conditions (41°C), over all the stages of parasite maturation. As expected, stiffness increased with parasite maturation. Surprisingly, however, stiffness increased acutely with temperature on a scale of minutes, particularly in late trophozoite and schizont stages. This acute stiffening in late falciparum stages may contribute to fever-dependent pathological consequences in the microcirculation.

It is self-evident that acute narrowing of the asthmatic airways and shortening of the airway smooth muscle are inextricably linked. Nonetheless, it was many years ago that research on the asthmatic airways and research on the biophysics of airway smooth muscle had a parting of the ways (Seow and Fredberg, 2001). The study of smooth muscle biophysics took on a life of its own and pursued a deeply reductionist agenda, one that became focused to a large extent on myosin II and regulation of the actomyosin cycling rate. The study of airway biology pursued a reductionist agenda as well, but one that became focused less and less on contractile functions of muscle and instead emphasized immune responses, inflammatory cells and mediators, and, to the extent that smooth muscle remained

We review here four recent findings that have altered in a fundamental way our understanding of airways smooth muscle (ASM), its dynamic responses to physiological loading, and their dominant mechanical role in bronchospasm. These findings highlight ASM remodeling processes that are innately out-of-equilibrium and dynamic, and bring to the forefront a striking intersection between topics in condensed matter physics and ASM cytoskeletal biology. By doing so, they place in a new light the role of enhanced ASM mass in airway hyper-responsiveness as well as in the failure of a deep inspiration to relax the asthmatic airway. These findings have established that (i) ASM length is equilibrated dynamically, not statically; (ii) ASM dynamics closely resemble physical features exhibited by so-called soft glassy materials; (iii) static force-length relationships fail to describe dynamically contracted ASM states; (iv) stretch fluidizes the ASM cytoskeleton. Taken together, these observations suggest that at the origin of the bronchodilatory effect of a deep inspiration, and its failure in asthma, may lie glassy dynamics of the ASM cell.

Obesity is a risk factor for asthma. The purpose of this study was to determine whether metformin, an agent used in the treatment of an obesity-related condition (type II diabetes), might have therapeutic potential for modifying the effects of obesity on airway smooth muscle (ASM) function. Metformin acts via activation of AMP-activated protein kinase (AMPK), a cellular sensor of energy status. In cultured murine ASM cells, metformin (0.2-2 mM) caused a dose-dependent inhibition of cell proliferation induced by PDGF (10(-8) M) and serotonin (10(-4) M). Another AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-Driboruranoside (AICAR), also inhibited PDGF-induced proliferation. Furthermore, cells treated with metformin or AICAR, also exhibited an attenuation in the rate of cytoskeletal remodeling, as quantified by spontaneous nanoscale motions of microbeads tightly anchored to the cytoskeleton (CSK) of the ASM cell. ASM cells treated with metformin or AICAR, however, exhibited no appreciable differences in stiffness as measured by optical magnetic twisting cytometry (OMTC) or their abilities to stiffen in response to contractile agonist serotonin. Taken together, these findings suggest that metformin, probably through activation of AMPK, reduces the rate of ongoing reorganization of the CSK and inhibits ASM cell proliferation.

Low-frequency mechanical behavior of various respiratory tissues shows certain similarities. In this study we test the hypothesis that rate-independent plastic processes along with rate-dependent viscoelastic processes are responsible. We considered oscillatory responses of several respiratory tissues measured over prescribed ranges of frequency (up to 6 Hz) and amplitude of forcing. These included the excised cat lung, the human chest wall in vivo, and two components of the chest wall: the excised dog rib cage and the excised rabbit abdominal viscera; some data were previously reported and some are new. We analyzed these data using the viscoplastic model of Hildebrandt (J. Appl. Physiol. 28: 365-372, 1970). It consists of three compartments: a plastoelastic compartment mechanically in parallel with a viscoelastic compartment, both in series with a lumped inertia. We fitted oscillatory data of the above respiratory tissues to the model by a least-squares technique. The fit was qualitatively consistent with the observations and exhibited moderately good to very good quantitative correspondence. As an independent verification of this approach, we obtained the stress relaxation after a step-volume change. Based on the oscillatory response of cat lungs, the calculated stress relaxation function was found to be generally consistent with corresponding observations. This study indicates that both plasticity and viscoelasticity appear to be important determinants of mechanical behavior of respiratory tissues at low frequencies and that inertial effects are negligible.

We measured the energy dissipation associated with large-amplitude periodic flow through airway bifurcation models. Each model consisted of a single asymmetric bifurcation with a different branching angle and area ratio, with each branch terminated into an identical elastic load. Sinusoidal volumetric oscillations were applied at the parent duct so that the upstream Reynolds number (Re) varied from 30 to 77,000 and the Womersley parameter (alpha) from 4 to 30. Pressures were measured continuously at the parent duct and at both terminals, and instantaneous branch flow rates were calculated. Time-averaged energy dissipation in the bifurcation was computed from an energy budget over a control volume integrated over a cycle and was expressed as a friction factor, F. We found that when tidal volume was small [ratio of tidal volume to resident (dead space) volume, VT/VD less than 1], F was independent of branching angle and fell with increasing alpha and VT/VD. When tidal volume was large (VT/VD greater than 1), F increased with increasing branching angle and varied less strongly with alpha and VT/VD. No simple benchmark flow represented the data well over the entire experimental range. This study demonstrates that only two nondimensional parameters, alpha and VT/VD, are necessary and are sufficient to describe time-averaged energy dissipation in a given bifurcation geometry during sinusoidal flow.

In studies of large-amplitude periodic flows at an airway bifurcation, we found an appreciable steady-state pressure difference between the terminal units. To elucidate the fluid dynamic origins of such steady-state pressure differences, we studied single asymmetric bifurcation models with various area ratios and branching angles. The daughter ducts were identical in size and were terminated into identical elastic loads. Sinusoidal flow oscillations were applied at the parent duct so that the upstream Reynolds number ranged from 30 to 77,000 and the Womersley parameter from 2 to 30. The steady-state component (time averaged) of the pressure measured at the terminal with the smaller branching angle was found to be consistently higher than that at the other terminal. This steady-state pressure difference scaled approximately as a fixed fraction of the parent duct dynamic head. Guided by the results of flow-visualization studies, we modeled such behavior based on the temporal and spatial differences of head loss between the two branches of the bifurcation. Our results suggest that interlobar heterogeneity of mean alveolar-pressure observed in excised canine lungs during high frequency oscillation (Allen et al., J. Appl. Physiol. 62: 223-228, 1987) arises solely from fluid dynamic origins: differential head loss due to asymmetry of central airway branching structure.

The interrupter method for measuring respiratory system resistance involves interrupting flow at the airway opening and measuring the resultant changes in pressure. We have recently shown (J. Appl. Physiol. 65: 408-414, 1988) that in open-chest mongrel dogs, under control conditions, the initial rapid pressure change (delta Pinit) reflects conducting airway resistance and the subsequent gradual pressure change (delta Pdif) reflects stress recovery of the tissues. We questioned whether the same interpretation would apply after induced constriction. Accordingly, we performed interruption experiments on anesthetized, paralyzed, tracheostomized, open-chest mongrel dogs during passive expiration, measuring pressure at the trachea and in three different alveolar regions with alveolar capsules. We recorded measurements before and after the administration of increasing concentrations of histamine aerosol (0.1-30.0 mg/ml). We found a significant increase in the heterogeneity of alveolar pressures during the relaxed expiration with increasing concentrations of histamine. Despite the introduction of significant mechanical heterogeneities, delta Pinit still reflected the pressure drop as the result of the resistance of the conducting airways. delta Pdif, however, reflected a combination of the stress recovery of the tissues and pendelluft.

To understand how bical mechanical chest wall (CW) properties are related to those of the CW as a whole, we measured esophageal and gastric pressures, CW volume changes (measured with a head-out body plethysmograph), and anteroposterior and transverse CW diameter changes (measured with magnetometers attached to the surface) during sinusoidal forcing at the mouth (2.5% vital capacity, 0.5-10 Hz) in four healthy subjects. Total CW resistance decreased sharply as frequency rose to 3-4 Hz and remained relatively constant at higher frequencies. Total CW reactance became less negative with increasing frequency but showed no tendency to change sign. Above 2 Hz, diameters measured at different locations changed asynchronously between and within the rib cage and abdomen. “Local pathway impedances” (ratios of esophageal or gastric pressure to a rate of diameter change) showed frequency dependence similar to that of the total CW less than 3 Hz. Local pathway impedances increased during contraction of respiratory muscles acting on the pathway. We conclude that 1) total CW behavior is mainly a reflection of its individual local properties at less than or equal to 3 Hz, 2) local impedances within the rib cage or within the abdomen can change independently in some situations, and 3) asynchronies that develop within the CW during forcing greater than 3 Hz suggest that two compartments may be insufficient to describe CW properties from impedance measurements.

A novel physical perspective of molecular interactions within the cytoskeleton of the airway smooth muscle cell may help to explain why the most efficacious of all known bronchodilatory agencies—a simple deep inspiration—becomes abrogated during the spontaneous asthma attack and leads thereby to excessive airway narrowing. This perspective invites us to think of airway smooth muscle not only biochemically as a nidus of traditional cell signaling and immune modulation or mechanically as a motor for generation of active forces but also physically as a phase of soft condensed matter that can restrict airway stretch and dilation. This is perhaps a risky path and is surely an unconventional one, but it is where the trail of evidence leads. This line of investigation is unlikely by itself to provide an asthma cure but will lead to a new conceptual framework without which novel pathways, unsuspected phase transitions, and unanticipated mechanisms of action of target molecules would almost surely remain hidden. Glassy dynamics of the cytoskeleton are likely to be important in a wide range of biological functions and disease processes, but had it not been for their preeminent role in bronchospasm, they might never have been discovered.

We report here measurements of rheological properties of the human airway smooth muscle cell using forced nanoscale motions of Arg-Gly-Asp RGD-coated microbeads tightly bound to the cytoskeleton. With changes of forcing amplitude, the storage modulus showed small but systematic nonlinearities, especially after treatment with a contractile agonist. In a dose-dependent manner, a large oscillatory shear applied from a few seconds up to 400 s caused the cytoskeleton matrix to soften, a behavior comparable to physical rejuvenation observed in certain inert soft materials; the stiffness remained constant for as long as the large oscillatory shear was maintained, but suddenly fell with shear cessation. Stiffness then followed a slow scale-free recovery, a phenomenon comparable to physical aging. However, acetylated low-density lipoprotein acLDL-coated microbeads, which connect mainly to scavenger receptors, did not show similar out-of-equilibrium behaviors. Taken together, these data demonstrate in the cytoskeleton of the living cell behaviors with all the same signatures as that of soft inert condensed systems. This unexpected intersection of condensed matter physics and cytoskeletal biology suggests that trapping, intermittency, and approach to kinetic arrest represent central mesoscale features linking underlying molecular events to integrative cellular functions.

We report directional memory of spontaneous nanoscale displacements of an individual bead firmly anchored to the cytoskeleton of a living cell. A novel method of analysis shows that for shorter time intervals cytoskeletal displacements are antipersistent and thus provides direct evidence in a living cell of molecular trapping and caged dynamics. At longer time intervals displacements are persistent. The transition from antipersistence to persistence is indicative of a time-scale for cage rearrangements and is found to depend upon energy release due to ATP hydrolysis and proximity to a glass transition. Anomalous diffusion is known to imply memory, but we show here that memory is attributed to direction rather than step size. As such, these data are the first to provide a molecular-scale physical picture describing the cytoskeletal remodelling process and its rate of progression.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

We quantified the effects of airway wall remodeling upon airway smooth muscle (ASM) shortening. Isolated ASM from sheep was attached to a servo-controller that applied a physiologic load. This load could be altered to reflect specified changes of airway wall geometry, elasticity, parenchymal tethering, transpulmonary pressure (P(L)), and fluctuations in P(L) associated with breathing. Starting at a reference length (L(ref)), ASM was stimulated with acetlycholine and held at constant P(L) of 4 cm H(2)O for 2 h. When all compartments were thickened to simulate the asthmatic airway but P(L) was held fixed, ASM shortened much more than that in the normal airway (to 0.52 L(ref) versus 0.66 L(ref)). When breathing with deep inspirations (DIs) was initiated, within the first three DIs the ASM in the normal airway lengthened to 0.84 L(ref), whereas that in the asthmatic airway remained stuck at 0.53 L(ref). Thickening of the smooth muscle layer alone produced the greatest muscle shortening (to 0.47 L(ref)) when compared with thickening of only submucosal (to 0.67 L(ref)) or only adventitial (to 0.62 L(ref)) compartments. With increased ASM mass, the ASM failed to lengthen in response to DIs, whereas in the airway with thickened submucosal and adventitial layers ASM lengthened dramatically (to 0.83 L(ref)). These findings confirm the long-held conclusion that increased muscle mass is the functionally dominant derangement, but mechanisms accounting for this conclusion differ dramatically from those previously presumed. Furthermore, increased ASM mass explained both hyperresponsiveness and the failure of a DI to relax the asthmatic airway.