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In development of an embryo, healing of a wound, or progression of a carcinoma, a requisite event is collective epithelial cellular migration. For example, cells at the advancing front of a wound edge tend to migrate collectively, elongate substantially, and exert tractions more forcefully compared with cells many ranks behind. With regards to energy metabolism, striking spatial gradients have recently been reported in the wounded epithelium, as well as in the tumor, but within the wounded cell layer little is known about the link between mechanical events and underlying energy metabolism. Using the advancing confluent monolayer of MDCKII cells as a model system, here we report at single cell resolution the evolving spatiotemporal fields of cell migration speeds, cell shapes, and traction forces measured simultaneously with fields of multiple indices of cellular energy metabolism. Compared with the epithelial layer that is unwounded, which is non-migratory, solid-like and jammed, the leading edge of the advancing cell layer is shown to become progressively more migratory, fluid-like, and unjammed. In doing so the cytoplasmic redox ratio becomes progressively smaller, the NADH lifetime becomes progressively shorter, and the mitochondrial membrane potential and glucose uptake become progressively larger. These observations indicate that a metabolic shift toward glycolysis accompanies collective cellular migration but show, further, that this shift occurs throughout the cell layer, even in regions where associated changes in cell shapes, traction forces, and migration velocities have yet to penetrate. In characterizing the wound healing process these morphological, mechanical, and metabolic observations, taken on a cell-by-cell basis, comprise the most comprehensive set of biophysical data yet reported. Together, these data suggest the novel hypothesis that the unjammed phase evolved to accommodate fluid-like migratory dynamics during episodes of tissue wound healing, development, and plasticity, but is more energetically expensive compared with the jammed phase, which evolved to maintain a solid-like non-migratory state that is more energetically economical.

Summary It is well established that the early malignant tumor invades surrounding extracellular matrix (ECM) in a manner that depends upon material properties of constituent cells, surrounding ECM, and their interactions. Recent studies have established the capacity of the invading tumor spheroids to evolve into coexistent solid-like, fluid-like, and gas-like phases. Using breast cancer cell lines invading into engineered ECM, here we show that the spheroid interior develops spatial and temporal heterogeneities in material phase which, depending upon cell type and matrix density, ultimately result in a variety of phase separation patterns at the invasive front. Using a computational approach, we further show that these patterns are captured by a novel jamming phase diagram. We suggest that non-equilibrium phase separation based upon jamming and unjamming transitions may provide a unifying physical picture to describe cellular migratory dynamics within, and invasion from, a tumor., Graphical abstract, Highlights • Tumor spheroids invading into a 3D matrix exhibit coexistent material phases • Invasion leads to spatiotemporal heterogeneities consistent with jamming dynamics • Phase separation patterns are captured by a model-based jamming phase diagram • Effective thermodynamic variables Teff and Pconf capture cell and matrix properties, Biophysics; Cancer; Mechanobiology

Control of the structure and function of three-dimensional multicellular tissues depends critically on the spatial and temporal coordination of cellular physical properties, yet the organizational principles that govern these events and their disruption in disease remain poorly understood. Using a multicellular mammary cancer organoid model, we map here the spatial and temporal evolution of positions, motions and physical characteristics of individual cells in three dimensions. Compared with cells in the organoid core, cells at the organoid periphery and the invasive front are found to be systematically softer, larger and more dynamic. These mechanical changes are shown to arise from supracellular fluid flow through gap junctions, the suppression of which delays the transition to an invasive phenotype. These findings highlight the role of spatiotemporal coordination of cellular physical properties in tissue organization and disease progression. A platform for probing the mechanics and migratory dynamics of a growing model breast cancer reveals that cells at the invasive edge are faster, softer and larger than those in the core. Eliminating the softer cells delays the transition to invasion.

Background Pulmonary fibrosis is a progressive disease characterized by structural distortion of the lungs. Transforming growth factor-beta (TGF-beta) is a key cytokine implicated in the pathogenesis of pulmonary fibrosis. TGF-beta-induced myofibroblast differentiation characterized by expression of smooth muscle alpha-actin and extracellular matrix proteins is a key process in pathogenesis of fibrotic disease. Tannic acid is a natural polyphenol with diverse applications. In this study, we investigated the effect of tannic acid on myofibroblast differentiation and pulmonary fibrosis in cultured cells and in bleomycin model of the disease. Methods Primary cultured human lung fibroblasts (HLF) were used. The relative levels of proteins were determined by Western blotting. HLF contraction was measured by traction microscopy. Bleomycin-induced pulmonary fibrosis in mice was used as the disease model. Results Tannic acid inhibited TGF-beta-induced expression of collagen-1 and smooth muscle alpha-actin (SMA) as well as force generation by HLF. Tannic acid did not affect initial phosphorylation of Smad2 in response to TGF-beta, but significantly inhibited sustained Smad2 phosphorylation, which we recently described to be critical for TGF-beta-induced myofibroblast differentiation. Accordingly, tannic acid inhibited Smad-dependent gene transcription in response to TGF-beta, as assessed using luciferase reporter for the activity of Smad-binding elements. Finally, in mouse model of bleomycin-induced pulmonary fibrosis, therapeutic application of tannic acid resulted in a significant reduction of lung fibrosis, decrease in collagen-1 content and of Smad2 phosphorylation in the lungs. Conclusions This study demonstrates the anti-fibrotic effect of tannic acid in vitro and in vivo through a regulation of sustained Smad2 phosphorylation. Electronic supplementary material The online version of this article (10.1186/s12931-019-1141-8) contains supplementary material, which is available to authorized users.

Every organ surface and body cavity is lined by a confluent collective of epithelial cells. In homeostatic circumstances the epithelial collective remains effectively solid-like and sedentary. But during morphogenesis, remodeling or repair, as well as during malignant invasion or metastasis, the epithelial collective becomes fluid-like and migratory1–4. This conversion from sedentary to migratory behavior has traditionally been understood as a manifestation of the epithelial-to-mesenchymal transition (EMT) or the partial EMT (pEMT)5–8. However, in certain contexts this conversion has been attributed to the recently discovered unjamming transition (UJT), in which epithelial cells move collectively and cooperatively9–11. UJT and pEMT share certain aspects of collective cellular migration, but the extent to which these processes are distinct, overlapping or perhaps even identical has remained undefined. Using the confluent layer of well-differentiated primary human bronchial epithelial (HBE) cells, here we triggered UJT by exposing the sedentary layer to mechanical compression9–12. Cells thereafter migrated cooperatively, aligned into packs locally, and elongated systematically. Nevertheless, cell-cell junctions, apico-basal polarity, and barrier function remained intact in response, and mesenchymal markers remained unapparent. As such, pEMT was not evident. When we triggered pEMT and associated cellular migration by exposing the sedentary layer to TGF-β1, metrics of UJT versus pEMT diverged. To account for these striking physical observations a new mathematical model attributes the effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Together, these findings establish that UJT is sufficient to account for vigorous epithelial layer migration even in the absence of pEMT. Distinct gateways to cellular migration therefore become apparent – UJT as it might apply to migration of epithelial sheets, and EMT/pEMT as it might apply to migration of mesenchymal cells on a solitary or collective basis, activated during development, remodeling, repair or tumor invasion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, living tissue is therefore seen to comprise an active engineering material whose modules for plasticity, self-repair and regeneration, are far richer than had been previously appreciated.

Collective cellular migration plays central roles in development, regeneration, and metastasis. In these processes mechanical interactions between cells are fundamental, but measurement of these interactions is often hampered by technical limitations. To overcome some of these limitations here we describe a system that integrates microfluidics with traction force microscopy (TFM). Using this system we can measure simultaneously, and in real time, cellular migration speeds, traction forces, and intercellular tension throughout a multicellular confluent island of confluent Madin-Darby canine kidney (MDCK) cells. The cell island is exposed to hepatocyte growth factor (HGF) at controlled concentrations and controlled gradients; HGF is known to elicit epithelial-to-mesenchymal transition (EMT) and cell scattering. As expected, the rate of expansion of the cell island was dependent on the concentration of HGF. Higher concentrations of HGF reduced intercellular tensions, as expected during EMT. A novel finding, however, is that the effects of HGF concentration and its gradient were seen to be cooperative. This integrated experimental system thus provides an improved window to better understand cellular forces during collective cellular migration and EMT.

Loss of function of the tumor suppressor p53 is known to increase the rate of migration of cells transiting the narrow pores of the traditional Boyden chamber assay. Here by contrast we investigate how p53 impacts the rate of cellular migration within a 2D confluent cell layer and a 3D collagen-embedded multicellular spheroid. We use two human carcinoma cell lines, the bladder carcinoma EJ and the colorectal carcinoma HCT116. In the confluent 2-D cell layer, for both EJ and HCT cells the migratory speeds and effective diffusion coefficients for the p53 null cells were significantly smaller than in p53-expressing cells. Compared to p53 expressers, p53-null cells exhibited more organized cortical actin rings together with reduced front-rear cell polarity. Furthermore, loss of p53 caused cells to exert smaller traction forces upon their substrates, and reduced formation of cryptic lamellipodia. In the 3D multicellular spheroid, loss of p53 consistently reduced collective cellular migration into surrounding collagen matrix. As regards the role of p53 in cellular migration, extrapolation from the Boyden chamber assay to other cellular microenvironments is seen to be fraught even in terms of the sign of the effect. Together, these paradoxical results show that the effects of p53 on cellular migration are context-dependent.

BackgroundAmong the most basic measures of respiratory function is the total lung capacity (TLC). TLC is the pulmonary gas volume at maximal lung inflation, which is the sum of the volume of gas that can be exhaled –the vital capacity (VC)– and the volume of gas that cannot –the residual volume (RV). Determination of VC requires only spirometry whereas determination of RV or TLC requires body plethysmography, gas dilution or washout, or thoracic imaging, each of which is more complex than spirometry, and none of which is suited to routine office practice, population screening, or community medicine. To fill this gap, we describe here a new approach to determine TLC without plethysmography.MethodsIn a heterogeneous population of 434 volunteers (265 male, 169 female; 201 healthy, 170 with airflow obstruction, and 63 with ventilatory restriction), we determined TLC in the standard fashion using conventional body plethysmography (TLCpleth). In the same individuals, we also determined TLC in a novel fashion using the MiniBox ™ (TLCMB). To obtain TLCMB, population-based data from traditional spirometry together with flow-interruption transients were subjected to data mining and machine-learning to create for each individual subject an unbiased statistical determination of TLC.ResultsFor the combined heterogeneous population, we found TLCpleth = 1.02TLCMB −0.091 L, adjusted r2=0.824. For the heterogeneous population as a whole, and for each subpopulation, TLCMB closely tracked TLCpleth. For 26 healthy subjects measured on different days, the coefficient of variation for repeated measurements in was 3.3% for TLCpleth versus 1.6% for TLCMB.ConclusionsThese results establish the validity and potential utility of a new method for rapid, accurate, and repeatable determination of TLC in a heterogeneous patient population, but without the need of a plethysmograph.

As a wound heals, or a body plan forms, or a tumour invades, observed cellular motions within the advancing cell swarm are thought to stem from yet to be observed physical stresses that act in some direct and causal mechanical fashion. Here we show that such a relationship between motion and stress is far from direct. Using monolayer stress microscopy, we probed migration velocities, cellular tractions and intercellular stresses in an epithelial cell sheet advancing towards an island on which cells cannot adhere. We found that cells located near the island exert tractions that pull systematically towards this island regardless of whether the cells approach the island, migrate tangentially along its edge, or paradoxically, recede from it. This unanticipated cell-patterning motif, which we call kenotaxis, represents the robust and systematic mechanical drive of the cellular collective to fill unfilled space.

[This corrects the article on p. 309 in vol. 7, PMID: 27559314.].

Collective cellular migration within the epithelial layer impacts upon development, wound healing and cancer invasion, but remains poorly understood. Prevailing conceptual frameworks tend to focus on the isolated role of each particular underlying factor – taken one at a time or at most a few at a time – and thus might not be tailored to describe a cellular collective that embodies a wide palette of physical and molecular interactions that are both strong and complex. To bridge this gap, we shift the spotlight to the emerging concept of cell jamming, which points to only a small set of parameters that govern when a cellular collective might jam and rigidify like a solid, or instead unjam and flow like a fluid. As gateways to cellular migration, the unjamming transition (UJT) and the epithelial-to-mesenchymal transition (EMT) share certain superficial similarities, but their congruence – or lack thereof – remains unclear. In this Commentary, we discuss aspects of cell jamming, its established role in human epithelial cell layers derived from the airways of non-asthmatic and asthmatic donors, and its speculative but emerging roles in development and cancer cell invasion.

Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell-cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. This stress landscape becomes increasingly rugged, sluggish, and cooperative with increasing system density. Within that landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behavior, as do breast cancer cell lines before but not after the epithelial-mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighboring cells join forces to transmit appreciable normal stress across the cell-cell junction, but migrate along orientations of minimal intercellular shear stress.

The sorting of distinctly different cell types into specific tissue compartments has long been thought to be a problem in minimization of total free energy in immiscible fluids, wherein cell–cell adhesion, cell stiffness, and cell contraction combine to define an effective macroscopic tissue surface tension. Pawlizak et al (2015 New J. Phys. 17 083049) now show not only that adhesion forces at interfaces unexpectedly fail to correlate with the density of adhesion molecules, but also that certain cancer cell lines unexpectedly fail to behave as a fluid, with cells becoming kinetically trapped in what might be a jammed, solid-like non-equilibrium state.

Asthma is characterized by chronic inflammation, airway hyperresponsiveness, and progressive airway remodeling. The airway epithelium is known to play a critical role in the initiation and perpetuation of these processes. Here, we review how excessive epithelial stress generated by bronchoconstriction is sufficient to induce airway remodeling, even in the absence of inflammatory cells.

In vivo animal models can offer valuable information on several aspects of asthma pathogenesis and treatment. The mouse is increasingly used in these models, mainly because this species allows for the application in vivo of a broad range of immunological tools, including gene deletion technology. Mice, therefore, seem particularly useful to further elucidate factors influencing the response to inhaled allergens. Examples include: the role of immunoregulatory mechanisms that protect against T-helper cell type 2 cell development; the trafficking of T-cells; and the contribution of the innate immunity. However, as for other animal species, murine models also have limitations. Mice do not spontaneously develop asthma and no model mimics the entire asthma phenotype. Instead, mice should be used to model specific traits of the human disease. The present task force report draws attention to specific aspects of lung structure and function that need to be borne in mind when developing such models and interpreting the results. In particular, efforts should be made to develop models that mimic the lung function changes characteristic of asthma as closely as possible. A large section of this report is therefore devoted to an overview of airway function and its measurement in mice.

In wound healing, tissue growth, and certain cancers, the epithelial or the endothelial monolayer sheet expands. Within the expanding monolayer sheet, migration of the individual cell is strongly guided by physical forces imposed by adjacent cells. This process is called plithotaxis and was discovered using Monolayer Stress Microscopy (MSM). MSM rests upon certain simplifying assumptions, however, concerning boundary conditions, cell material properties and system dimensionality. To assess the validity of these assumptions and to quantify associated errors, here we report new analytical, numerical, and experimental investigations. For several commonly used experimental monolayer systems, the simplifying assumptions used previously lead to errors that are shown to be quite small. Out-of-plane components of displacement and traction fields can be safely neglected, and characteristic features of intercellular stresses that underlie plithotaxis remain largely unaffected. Taken together, these findings validate Monolayer Stress Microscopy within broad but well-defined limits of applicability.

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g′) and internal friction (g″) were measured over a wide frequency range. The dependencies of g′ and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x = 1) to fluid-like (x = 2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the micro-circulation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.

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.

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

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.

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.

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.

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.

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.

Obesity increases the risk of both asthma and obstructive sleep apnoea (OSA). By compromising upper airway anatomy, obesity contributes to compromised pharyngeal mechanics and the development of OSA. The mechanisms for how obesity contributes to the development of asthma are less well understood. The association between obesity and asthma, however, is undeniable and to date over 30 articles have shown a relationship between the two 1, 2. Obesity has effects on both inflammation and airway mechanics, which may be important in asthma. For example, obesity has been associated with increased inflammatory cytokines and adipokines 3. The role of leptin, a hormone that decreases appetite and increases metabolism, has been of particular interest in obesity and asthma. Obese subjects have increased levels of leptin, suggesting leptin resistance, at least at the level of hypothalamic appetite regulation. Leptin has been found to be pro-inflammatory and in animal models it may increase airway hyperresponsiveness 4, 5. However, the pro-inflammatory effects of obesity are not the only way in which obesity contributes to the development of asthma. The effect of obesity on lung mechanics may be just as or even more important. With obesity, lung volumes (especially functional residual capacity; FRC) decrease as a result of increased abdominal and chest wall pressure. Since airway calibre is a function of lung volume 6, 7, obesity contributes to smaller airway calibre. Smaller airway calibre is likely to unload the airway smooth muscle, increase actin–myosin cross-bridging and allow for increased shortening when activated 8, 9. Deep breathing has been found to be a potent bronchodilator by periodically stretching the airway smooth muscle and disturbing actin–myosin attachments, making contractions less forceful …

Adherent cells exert tractions on their surroundings. These tractions can be measured by observing the displacements of beads embedded on a flexible gel substrate on which the cells are cultured. This paper presents an exact solution to the problem of computing the traction field from the observed displacement field. The solution rests on recasting the relationship between displacements and tractions into Fourier space, where the recovery of the traction field is especially simple. We present two subcases of the solution, depending on whether or not tractions outside the observed cell boundaries are set to be zero. The implementation is computationally efficient. We also give the solution for the traction field in a representative human airway smooth muscle cell contracted by treatment with histamine. Finally, we give explicit formulas for reducing the traction and displacement fields to contraction moments, the orientation of the principal axes of traction, and the strain energy imparted by the cell to the substrate.

The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness ( G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ( [Formula: see text]t) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18 [Formula: see text]t+ 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

We carried out a detailed mathematical analysis of the effects of length fluctuations on the dynamically evolving cross-bridge distributions, simulating those that occur in airway smooth muscle during breathing. We used the latch regulation scheme of Hai and Murphy (Am. J. Physiol. Cell Physiol. 255:C86–C94, 1988) integrated with Huxley’s sliding filament theory of muscle contraction. This analysis showed that imposed length fluctuations decrease the mean number of attached bridges, depress muscle force and stiffness, and increase force-length hysteresis. At frequencies >0.1Hz, the bond-length distribution of slowly cycling latch bridges changed little over the stretch cycle and contributed almost elastically to muscle force, but the rapidly cycling cross-bridge distribution changed substantially and dominated the hysteresis. By contrast, at frequencies

The newly discovered extensibility of actin and myosin filaments challenges the foundation of the theory of muscle mechanics. We have reformulated A. F. Huxley's sliding filament theory to explicitly take into account filament extensibility. During isometric force development, growing cross-bridge tractions transfer loads locally between filaments, causing them to extend and, therefore, to slide locally relative to one another. Even slight filament extensibility implies that 1) relative displacement between the two must be nonuniform along the region of filament overlap, 2) cross-bridge strain must vary systematically along the overlap region, and importantly, 3) the local shortening velocities, even at constant overall sarcomere length, reduce force below the level that would have developed if the filaments had been inextensible. The analysis shows that an extensible filament system with only two states (attached and detached) displays three important characteristics: 1) muscle stiffness leads force during force development; 2) cross-bridge stiffness is significantly higher than previously assessed by inextensible filament models; and 3) stiffness is prominently dissociated from the number of attached cross-bridges during force development. The analysis also implies that the local behavior of one myosin head must depend on the state of neighboring attachment sites. This coupling occurs exclusively through local sliding velocities, which can be significant, even during isometric force development. The resulting mechanical cooperativity is grounded in fiber mechanics and follows inevitably from filament extensibility.

We measured pressure excursions at the airway opening and at the alveoli (PA) as well as measured the regional distribution of PA during forced oscillations of six excised dog lungs while frequency (f[2-32 Hz]), tidal volume (VT [5-80 ml]), and mean transpulmonary pressure (PL [25, 10, and 6 cm H2O]) were varied. PA's were measured in four alveolar capsules glued to the pleura of different lobes. The apex-to-base ratio of PA's was used as an index of the distribution of dynamic lung distension. At low f, there was slight preferential distension of the lung base which was independent of VT, but at higher f, preferential distension of the lung apex was found when VT's were small, whereas preferential distension of the lung base was found when VT's approached or exceeded dead space. These VT-related changes in distribution at high frequencies seem to depend upon the branching geometry of the central airways and the relative importance of convective momentum flux vs. unsteady inertia of gas residing therein, which, in this study, we showed to be proportional to the ratio VT/VD*, where VD* is an index of dead space. Furthermore, they imply substantial alteration in the distribution of ventilation during high frequency ventilation as f, VT, and PL vary. The data also indicate that alveolar and airway opening pressure costs per unit flow delivered at the airway opening exhibit weakly nonlinear behavior and that resonant amplification of PA's, which has been described previously for the case of very small VT's, persists but is damped as VT's approach dead space values.

Airways hyperresponsiveness is a cardinal feature of asthma but remains unexplained. In asthma, the airway smooth muscle cell is the key end-effector of bronchospasm and acute airway narrowing, but in just the past five years our understanding of the relationship of responsiveness to muscle biophysics has dramatically changed. It has become well established, for example, that muscle length is equilibrated dynamically rather than statically, and that non-classical features of muscle biophysics come to the forefront, including unanticipated interactions between the muscle and its time-varying load, as well as the ability of the muscle cell to adapt rapidly to changes in its dynamic microenvironment. These newly discovered phenomena have been described empirically, but a mechanistic basis to explain them is only beginning to emerge.