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Microtissues in the shape of toroidal rings provide an ideal geometry to better represent the structure and function of the airway smooth muscle present in the small airways, and to better understand diseases such as asthma. Here, polydimethylsiloxane devices consisting of a series of circular channels surrounding central mandrels are used to form microtissues in the shape of toroidal rings by way of the self-aggregation and -assembly of airway smooth muscle cell (ASMC) suspensions. Over time, the ASMCs present in the rings become spindle-shaped and axially align along the ring circumference. Ring strength and elastic modulus increase over 14 d in culture, without significant changes in ring size. Gene expression analysis indicates stable expression of mRNA for extracellular matrix-associated proteins, including collagen I and laminins α 1 and α 4 over 21 d in culture. Cells within the rings respond to TGF- β 1 treatment, leading to dramatic decreases in ring circumference, with increases in mRNA and protein levels for extracellular matrix and contraction-associated markers. These data demonstrate the utility of ASMC rings as a platform for modeling diseases of the small airways such as asthma., (Creative Commons Attribution license.)

Recently, "Technical standards for respiratory oscillometry" was published, which reviewed the physiological basis of oscillometric measures and detailed the technical factors related to equipment and test performance, quality assurance and reporting of results. Here we present a review of the clinical significance and applications of oscillometry. We briefly review the physiological principles of oscillometry and the basics of oscillometry interpretation, and then describe what is currently known about oscillometry in its role as a sensitive measure of airway resistance, bronchodilator responsiveness and bronchial challenge testing, and response to medical therapy, particularly in asthma and COPD. The technique may have unique advantages in situations where spirometry and other lung function tests are not suitable, such as in infants, neuromuscular disease, sleep apnoea and critical care. Other potential applications include detection of bronchiolitis obliterans, vocal cord dysfunction and the effects of environmental exposures. However, despite great promise as a useful clinical tool, we identify a number of areas in which more evidence of clinical utility is needed before oscillometry becomes routinely used for diagnosing or monitoring respiratory disease., Competing Interests: Conflict of interest: D.A. Kaminsky reports personal payments made as faculty speaker for Cardiorespiratory Diagnostics Seminar from MGC Diagnostics, Inc. outside the submitted work. Past Chair of ATS Proficiency Standards for Pulmonary Function Laboratories Committee, unpaid. Conflict of interest: S.J. Simpson has nothing to disclose. Conflict of interest: K.I. Berger has nothing to disclose. Conflict of interest: P. Calverley reports receiving consulting fees paid by Phillips Respironics for advisory work on a novel COPD ventilator. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events received from Phillips Respironics, outside the submitted work. Conflict of interest: P.L. de Melo reports patent 28727 issued. Conflict of interest: R.J. Dandurand reports grants or contracts paid to the institution from AstraZeneca, Boehringer-Ingelheim, Covis Pharma, Grifols, MGC Diagnostics, Teva Pharma, Thorasys, and Vyaire, outside the submitted work. Speaking payment from Novartis for L'oscillométrie en clinique: qu'ajoute-t-elle aux évaluations pulmonaires?, 18 September 2019, Boehringer-Ingelheim for L'oscillométrie: vieille physiologie avec un avenir brilliant, 17 November 2020, and Latin American Respiratory Physiology Society for Oscillometry in Asthma and COPD: Interpretation Strategies, 14 November 2020, outside the submitted work. Chairman, Oscillometry Harmonisation Study Group, an international committee academic and industry experts working to standardise oscillometry devices, and Chairman, Respiratory Effectiveness Group Technologies Working Group, Cambridge, U.K. (https://www.regresearchnetwork.org). Conflict of interest: R.L. Dellacà reports royalties or licenses from Restech, Philips and Vyaire. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events received from Restech and Philips, outside the submitted work. Support for attending meetings from Philips and Vyaire. Patent issued, owned and licensed from Politecnico di Milano University. Member of the Board of Directors for Restech. Stocks owned for Restech. Free loan of equipment for studies received from Vyaire and Restech. Conflict of interest: C.S. Farah has nothing to disclose. Conflict of interest: R. Farré has nothing to disclose. Conflict of interest: G.L. Hall has nothing to disclose. Conflict of interest: I. Ioan has nothing to disclose. Conflict of interest: C.G. Irvin received consulting fees from Medical Graphics Corporation, outside the submitted work. Conflict of interest: D.W. Kaczka reports support for the present manuscript from University of Iowa. Grants or contracts from Dept of Defence and NIH, outside the submitted work. Consulting fees received from ZOLL Medical, Inc. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events received from CHEST, ASME, Medical Society of New Zealand, and Johns Hopkins University, outside the submitted work. US patent 10,675,423 B2 (patent on MFOV technique, inventor) and PCT patent pending, patent on MFOV technique pending. Stock or stock options held for OscillaVent, Inc. Loan of ventilator for other projects from ZOLL Medical Inc. Conflict of interest: G.G. King reports grants or contracts from Restech Italy, NHMRC, Boehringer Ingelheim, CycloPharm, GlaxoSmithKline, Menarini, MundiPharma, Philanthropic individuals and societies, outside the submitted work. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events received from AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, CycloPharm, GlaxoSmithKline, Menarini, MundiPharma and Novartis, outside the submitted work. Leadership or fiduciary role in other board, society, committee or advocacy group for ERS Technical Standards for Respiratory Oscillometry. Conflict of interest: H. Kurosawa reports receiving a grant from CHEST Co. Ltd. Royalties or licence for CHEST Co. Ltd. Payment or honoraria for lectures received from CHEST Co. Ltd. Nippon, Boehringer Ingelheim, Novartis, and Teijin Pharma, outside the submitted work. Conflict of interest: E. Lombardi reports grants or contracts from Restech and Sanofi, outside the submitted work. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events received from Angelini, Chiesi, GSK, Novartis and Sanofi, outside the submitted work. Participation on a Data Safety Monitoring Board or Advisory Board for GSK and Novartis. Conflict of interest: G.N. Maksym reports grants or contracts from National Research Council of Canada, Cyclomedica Inc. Australia, and Lung Association of Nova Scotia, outside the submitted work. Accommodation Expenses received from Thorasys, Thoracic Medical Systems Inc. for attending European Society Meeting 2019. Patents planned, issued or pending for Method and system to acquire oscillometry measurements, owned by Thorasys, Thoracic Medical Systems Inc. Stock or stock options held for Thorasys, Thoracic Medical Systems Inc. Conflict of interest: F. Marchal has nothing to disclose. Conflict of interest: E. Oostveen has nothing to disclose. Conflict of interest: B.W. Oppenheimer has nothing to disclose. Conflict of interest: P.D. Robinson has nothing to disclose. Conflict of interest: M. van den Berge reports grants or contracts from GlaxoSmithKline, Novartis, Astra Zeneca, Roche, and Genentech, outside the submitted work. Conflict of interest: C. Thamrin report grants or contracts from Restech SRL and THORASYS Thoracic. Equipment on loan for research studies from Restech SRL and THORASYS Thoracic, outside the submitted work., (Copyright ©The authors 2022.)

In asthma, the airway epithelium has an impaired capacity to differentiate and plays a key role in the development of airway inflammation and remodeling through mediator release. The study objective was to investigate the release of (IL)-1 family members from primary airway epithelial-cells during differentiation, and how they affect primary airway fibroblast (PAF)-induced inflammation, extracellular matrix (ECM) production, and collagen I remodeling. The release of IL-1α/β and IL-33 during airway epithelial differentiation was assessed over 20-days using air-liquid interface cultures. The effect of IL-1 family cytokines on airway fibroblasts grown on collagen-coated well-plates and 3-dimensional collagen gels was assessed by measurement of inflammatory mediators and ECM proteins by ELISA and western blot, as well as collagen fiber formation using non-linear optical microscopy after 24-hours. The production of IL-1α is elevated in undifferentiated asthmatic-PAECs compared to controls. IL-1α/β induced fibroblast pro-inflammatory responses (CXCL8/IL-8, IL-6, TSLP, GM-CSF) and suppressed ECM-production (collagen, fibronectin, periostin) and the cell's ability to repair and remodel fibrillar collagen I via LOX, LOXL1 and LOXL2 activity, as confirmed by inhibition with β-aminopropionitrile. These data support a role for epithelial-derived-IL-1 in the dysregulated repair of the asthmatic-EMTU and provides new insights into the contribution of airway fibroblasts in inflammation and airway remodeling in asthma.

Rationale: Histologic stains have been used as the gold standard to visualize extracellular matrix (ECM) changes associated with airway remodeling in asthma, yet they provide no information on the biochemical and structural characteristics of the ECM, which are vital to understanding alterations in tissue function. Objectives: To demonstrate the use of nonlinear optical microscopy (NLOM) and texture analysis algorithms to image fibrillar collagen (second harmonic generation) and elastin (two-photon excited autofluorescence), to obtain biochemical and structural information on the remodeled ECM environment in asthma. Methods: Nontransplantable donor lungs from donors with asthma ( n  = 13) and control ( n  = 12) donors were used for the assessment of airway collagen and elastin fibers by NLOM, and extraction of lung fibroblasts for in vitro experiments. Measurements and Main Results: Fibrillar collagen is not only increased but also highly disorganized and fragmented within large and small asthmatic airways compared with control subjects, using NLOM imaging. Furthermore, such structural alterations are present in pediatric and adult donors with asthma, irrespective of fatal disease. In vitro studies demonstrated that asthmatic airway fibroblasts are deficient in their packaging of fibrillar collagen-I and express less decorin, important for collagen fibril packaging. Packaging of collagen fibrils was found to be more disorganized in asthmatic airways compared with control subjects, using transmission electron microscopy. Conclusions: NLOM imaging enabled the structural assessment of the ECM, and the data suggest that airway remodeling in asthma involves the progressive accumulation of disorganized fibrillar collagen by airway fibroblasts. This study highlights the future potential clinical application of NLOM to assess airway remodeling in vivo .

Developed over six decades ago, pulmonary oscillometry has re-emerged as a noninvasive and effort-independent method for evaluating respiratory-system impedance in patients with obstructive lung disease. Here, we evaluated the relationships between hyperpolarized 3 He ventilation-defect-percent (VDP) and respiratory-system resistance, reactance and reactance area (A X ) measurements in 175 participants including 42 never-smokers without respiratory disease, 56 ex-smokers with chronic-obstructive-pulmonary-disease (COPD), 28 ex-smokers without COPD and 49 asthmatic never-smokers. COPD participants were dichotomized based on x-ray computed-tomography (CT) evidence of emphysema (relative-area CT-density-histogram ≤ 950HU (RA 950 ) ≥ 6.8%). In asthma and COPD subgroups, MRI VDP was significantly related to the frequency-dependence of resistance (R 5-19 ; asthma: ρ = 0.48, P = 0.0005; COPD: ρ = 0.45, P = 0.0004), reactance at 5 Hz (X 5 : asthma, ρ = -0.41, P = 0.004; COPD: ρ = -0.38, P = 0.004) and A X (asthma: ρ = 0.47, P = 0.0007; COPD: ρ = 0.43, P = 0.0009). MRI VDP was also significantly related to R 5-19 in COPD participants without emphysema (ρ = 0.54, P = 0.008), and to X 5 in COPD participants with emphysema (ρ = -0.36, P = 0.04). A X was weakly related to VDP in asthma (ρ = 0.47, P = 0.0007) and COPD participants with (ρ = 0.39, P = 0.02) and without (ρ = 0.43, P = 0.04) emphysema. A X is sensitive to obstruction but not specific to the type of obstruction, whereas the different relationships for MRI VDP with R 5-19 and X 5 may reflect the different airway and parenchymal disease-specific biomechanical abnormalities that lead to ventilation defects., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

In patients with asthma, magnetic resonance imaging (MRI) provides direct measurements of regional ventilation heterogeneity, the etiology of which is not well-understood, nor is the relationship of ventilation abnormalities with lung mechanics. In addition, respiratory resistance and reactance are often abnormal in asthmatics and the frequency dependence of respiratory resistance is thought to reflect ventilation heterogeneity. We acquiredMRIventilation defect maps, forced expiratory volume in one-second (FEV1), and airways resistance (Raw) measurements, and used a computational airway model to explore the relationship of ventilation defect percent (VDP) with simulated measurements of respiratory system resistance (Rrs) and reactance (Xrs).MRIventilation defect maps were experimentally acquired in 25 asthmatics before, during, and after methacholine challenge and these were nonrigidly coregistered to the airway tree model. Using the model coregistered to ventilation defect maps, we narrowed proximal (9th) and distal (14th) generation airways that were spatially related to theMRIventilation defects. The relationships forVDPwith Raw measured using plethysmography (r = 0.79), and model predictions of Rrs>14(r = 0.91,P < 0.0001) and Rrs>9(r = 0.88,P < 0.0001) were significantly stronger (P = 0.005;P = 0.03, respectively) than withFEV1(r = -0.68,P = 0.0001). The slopes for the relationship ofVDPwith simulated lung mechanics measurements were different (P < 0.0001); among these, the slope for theVDP-Xrs0.2relationship was largest, suggesting thatVDPwas dominated by peripheral airway heterogeneity in these patients. In conclusion, as a first step toward understanding potential links between lung mechanics and ventilation defects, impedance predictions were made using a computational airway tree model with simulated constriction of airways related to ventilation defects measured in mild-moderate asthmatics., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Ventilation heterogeneity is an important marker of small airway dysfunction in asthma. The frequency dependence of respiratory system resistance (Rrs) from oscillometry is used as a measure of this heterogeneity. However, this has not been quantitatively assessed or compared with other outcomes from oscillometry, including respiratory system reactance (Xrs) and the associated elastance (Ers). Here, we used a multibranch model of the human lung, including an upper airway shunt, to match previously reported respiratory mechanics in mild to severe asthma. We imposed heterogeneity by narrowing a proportion of the peripheral airways to account for patient Ers at 5 Hz, and then narrowed central airways to account for the remaining Rrs at 18 Hz. The model required >75% of the small airways to be occluded to reproduce severe asthma. While the model produced frequency dependence in Rrs, it was upward-shifted below 5 Hz compared with in-vivo results, indicating that other factors, including more distributed airway narrowing or central airway wall compliance, are required. However, Ers quantitatively reflected the imposed heterogeneity better than the frequency dependence of Rrs, independent of the frequency range for the estimation, and thus was a more robust measure of small-airway function. Thus, Ers appears to have greater potential as a clinical measure of early small-airway disease in asthma.

Spirometry is one of the most widely used tests in the assessment and monitoring of asthma. However, spirometry cannot be performed in very young children and some adult patients, and is poorly sensitive to small airways, which are primarily involved in the pathophysiology of asthma. The forced oscillation technique (FOT) has emerged as a powerful alternative technique that instead characterizes respiratory mechanics during normal breathing with no forced maneuver. In this review we highlight the current state of the art of the FOT and its utility in the assessment of lung function in asthma. First we briefly discuss the clinical features and characteristics of asthma. This is followed by a discussion of the assessment of airway obstruction and airway hyperresponsiveness using spirometry. We then review the basics of FOT and its application in respiratory diseases. FOT data are particularly amenable to modeling as an aide to physiological interpretation, and we review several common approaches. This is followed by an in-depth discussion of the assessment of airway variability and heterogeneity using FOT in asthma. Finally, we speculate on the potential clinical utility of FOT in asthma.

Purpose: To compare hyperpolarized helium-3 ((3) He) and xenon-129 ((129) Xe) MRI in asthmatics before and after salbutamol inhalation., Materials and Methods: Seven asthmatics provided written informed consent and underwent spirometry, plethysmography, and MRI before and after salbutamol inhalation. (3) He and (129) Xe ventilation defect percent (VDP) and ventilation coefficient of variation (COV) were measured. To characterize the airways spatially related to ventilation defects, wall area percent (WA%) and lumen area (LA) were evaluated for two subjects who had thoracic x-ray computed tomography (CT) acquired 1 year before MRI., Results: Before salbutamol inhalation, (129) Xe VDP (8 ± 5%) was significantly greater than (3) He VDP (6 ± 5%, P = 0.003). Post-salbutamol, there was a significant improvement in both (129) Xe (5 ± 4%, P < 0.0001) and (3) He (4 ± 3%, P = 0.001) VDP, and the improvement in (129) Xe VDP was significantly greater (P = 0.008). (129) Xe MRI COV (Pre: 0.309 ± 0.028, Post: 0.296 ± 0.036) was significantly greater than (3) He MRI COV (Pre: 0.282 ± 0.018, Post: 0.269 ± 0.024), pre- (P < 0.0001) and post-salbutamol (P < 0.0001) and the decrease in COV post-salbutamol was significant ((129) Xe, P = 0.002; (3) He, P < 0.0001). For a single asthmatic, a sub-segmental (129) Xe MRI ventilation defect that was visible only before salbutamol inhalation but not visible using (3) He MRI was spatially related to a remodeled fourth generation sub-segmental airway (WA% = 78%, LA = 2.9 mm(2) )., Conclusion: In asthma, hyperpolarized (129) Xe MRI may help reveal ventilation abnormalities before bronchodilation that are not observed using hyperpolarized (3) He MRI., (Copyright © 2013 Wiley Periodicals, Inc.)

Respiratory symptoms are largely caused by obstruction of the airways. In asthma, airway narrowing mediated by airway smooth muscle (ASM) contraction contributes significantly to obstruction. The spasmogens produced following exposure to environmental triggers, such as viruses or allergens, are initially responsible for ASM activation. However, the extent of narrowing of the airway lumen due to ASM shortening can be influenced by many factors and it remains a real challenge to decipher the exact role of ASM in causing asthmatic symptoms. Innovative tools, such as the forced oscillation technique, continue to develop and have been proven useful to assess some features of ASM function in vivo. Despite these technologic advances, it is still not clear whether excessive narrowing in asthma is driven by ASM abnormalities, by other alterations in non-muscle factors or simply because of the overexpression of spasmogens. This is because a multitude of forces are acting on the airway wall, and because not only are these forces constantly changing but they are also intricately interconnected. To counteract these limitations, investigators have utilized in vitro and ex vivo systems to assess and compare asthmatic and non-asthmatic ASM contractility. This review describes: 1- some muscle and non-muscle factors that are altered in asthma that may lead to airway narrowing and asthma symptoms; 2- some technologies such as the forced oscillation technique that have the potential to unveil the role of ASM in airway narrowing in vivo; and 3- some data from ex vivo and in vitro methods that probe the possibility that airway hyperresponsiveness is due to the altered environment surrounding the ASM or, alternatively, to a hypercontractile ASM phenotype that can be either innate or acquired., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Objective: Spirometry is insensitive to small airway abnormalities in asthma. Our objective was to evaluate regional lung structure and function using hyperpolarized (3)He magnetic resonance imaging (MRI) before, during and after a methacholine challenge (MCh)., Methods: Twenty-five asthmatics (mean age = 34 ± 11 years) and eight healthy volunteers (HV) (mean age = 33 ± 11 years) underwent spirometry, plethysmography and hyperpolarized (3)He MRI prior to a MCh. MRI was repeated following the MCh and again 25 min after salbutamol administration. (3)He MRI gas distribution was quantified using semiautomated segmentation of the ventilation defect percent (VDP). Tissue microstructure was measured using the (3)He apparent diffusion coefficient (ADC). Analysis of variance with repeated measures was used to evaluate changes at each time point as well as to determine interactions between regions of interest (ROI) and subject group. Pearson's correlations were performed to evaluate associations between (3)He MRI measurements and established clinical measures., Results: In asthmatics, but not HV, whole-lung ADC was increased post-MCh (P < 0.01). In asthmatics only, ADC was increased post-MCh in posterior ROI (P < 0.01) and all ROI in the superior-inferior direction (P < 0.01). VDP was increased in posterior and inferior ROI (P < 0.001). There was a correlation between VDP and specific airway resistance (r = 0.74, P < 0.0001), dyspnoea score (r = 0.66, P < 0.01) and fractional exhaled nitric oxide (r = 0.45, P < 0.05)., Conclusions: We evaluated the regional pulmonary response to methacholine and salbutamol using (3)He MRI and showed heterogeneous VDP and ADC consistent with bronchoconstriction and gas trapping, respectively, post-MCh. These regional alterations resolved post-salbutamol., (© 2012 The Authors. Respirology © 2012 Asian Pacific Society of Respirology.)

Asthmatic individuals typically experience exaggerated decrements in their ability to breathe after receiving standardized doses of smooth muscle agonist, a phenomenon known as airways hyperresponsiveness (AHR). Breathing difficulties are caused by excessive narrowing of the pulmonary airways, which is instigated by shortening of the airway smooth muscle (ASM). Exactly why many asthmatic individuals are hyperresponsive, however, remains controversial because of the many varied mechanisms that could possibly be involved. Nevertheless, much of the understanding of AHR comes down to a matter of considering the spatial configuration of the components that make up the airway, and the static and dynamic physical forces these components experience. In this review, we consider these mechanical factors, which are conveniently subdivided into three groups involving (i) the active forces construing to narrow the airways, (ii) the mechanical loads against which these forces must work, and (iii) the geometric transformation of a given degree of ASM shortening into airway narrowing. Each of these groups of factors has potent potential to influence AHR. It is likely, however, that they operate together to produce the AHR characteristic of severe asthma.

Airway smooth muscle (ASM) cells are constantly under mechanical strain as the lung cyclically expands and deflates, and this stretch is now known to modulate the contractile function of ASM. However, depending on the experimental conditions, stretch is either beneficial or harmful limiting or enhancing contractile force generation, respectively. Stretch caused by a deep inspiration is known to be beneficial in limiting or reversing airway constriction in healthy individuals, and oscillatory stretch lowers contractile force and stiffness or lengthens muscle in excised airway tissue strips. Stretch in ASM culture has generally been reported to cause increased contractile function through increases in proliferation, contractile protein content, and organization of the cell cytoskeleton. Recent evidence indicates the type of stretch is critically important. Growing cells on flexible membranes where stretch is non-uniform and anisotropic leads to pro-contractile changes, whereas uniform biaxial stretch causes the opposite effects. Furthermore, the role of contractile tone might be important in modulating the response to mechanical stretch in cultured cells. This report will review the contrasting evidence for modulation of contractile function of ASM, both in vivo and in vitro, and summarize the recent evidence that mechanical stress applied either acutely within 2 h or chronically over 11 d is a potent stimulus for cytoskeletal remodelling and stiffening. We will also point to new data suggesting that perhaps some of the difference in response to stretch might lie with one of the fundamental differences in the ASM environment in asthma and in culture--the presence of elevated contractile tone.

In patients with asthma, magnetic resonance imaging ( MRI) provides direct measurements of regional ventilation heterogeneity, the etiology of which is not well-understood, nor is the relationship of ventilation abnormalities with lung mechanics. In addition, respiratory resistance and reactance are often abnormal in asthmatics and the frequency dependence of respiratory resistance is thought to reflect ventilation heterogeneity. We acquired MRI ventilation defect maps, forced expiratory volume in one-second ( FEV1), and airways resistance (Raw) measurements, and used a computational airway model to explore the relationship of ventilation defect percent ( VDP) with simulated measurements of respiratory system resistance (Rrs) and reactance (Xrs). MRI ventilation defect maps were experimentally acquired in 25 asthmatics before, during, and after methacholine challenge and these were nonrigidly coregistered to the airway tree model. Using the model coregistered to ventilation defect maps, we narrowed proximal (9th) and distal (14th) generation airways that were spatially related to the MRI ventilation defects. The relationships for VDP with Raw measured using plethysmography ( r = 0.79), and model predictions of Rrs>14 ( r = 0.91, P < 0.0001) and Rrs>9 ( r = 0.88, P < 0.0001) were significantly stronger ( P = 0.005; P = 0.03, respectively) than with FEV1 ( r = −0.68, P = 0.0001). The slopes for the relationship of VDP with simulated lung mechanics measurements were different ( P < 0.0001); among these, the slope for the VDP-Xrs0.2 relationship was largest, suggesting that VDP was dominated by peripheral airway heterogeneity in these patients. In conclusion, as a first step toward understanding potential links between lung mechanics and ventilation defects, impedance predictions were made using a computational airway tree model with simulated constriction of airways related to ventilation defects measured in mild-moderate asthmatics. [ABSTRACT FROM AUTHOR]

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Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma. [ABSTRACT FROM AUTHOR]

ABSTRACT Background and objective: Spirometry is insensitive to small airway abnormalities in asthma. Our objective was to evaluate regional lung structure and function using hyperpolarized 3He magnetic resonance imaging (MRI) before, during and after a methacholine challenge (MCh). Methods: Twenty-five asthmatics (mean age = 34 ± 11 years) and eight healthy volunteers (HV) (mean age = 33 ± 11 years) underwent spirometry, plethysmography and hyperpolarized 3He MRI prior to a MCh. MRI was repeated following the MCh and again 25 min after salbutamol administration. 3He MRI gas distribution was quantified using semiautomated segmentation of the ventilation defect percent (VDP). Tissue microstructure was measured using the 3He apparent diffusion coefficient (ADC). Analysis of variance with repeated measures was used to evaluate changes at each time point as well as to determine interactions between regions of interest (ROI) and subject group. Pearson's correlations were performed to evaluate associations between 3He MRI measurements and established clinical measures. Results: In asthmatics, but not HV, whole-lung ADC was increased post-MCh ( P < 0.01). In asthmatics only, ADC was increased post-MCh in posterior ROI ( P < 0.01) and all ROI in the superior-inferior direction ( P < 0.01). VDP was increased in posterior and inferior ROI ( P < 0.001). There was a correlation between VDP and specific airway resistance ( r = 0.74, P < 0.0001), dyspnoea score ( r = 0.66, P < 0.01) and fractional exhaled nitric oxide ( r = 0.45, P < 0.05). Conclusions: We evaluated the regional pulmonary response to methacholine and salbutamol using 3He MRI and showed heterogeneous VDP and ADC consistent with bronchoconstriction and gas trapping, respectively, post-MCh. These regional alterations resolved post-salbutamol. [ABSTRACT FROM AUTHOR]

A deep inspiration (DI) temporarily relaxes agonist-constricted airways in normal subjects, but in asthma airways are refractory and may rapidly renarrow, possibly due to changes in the structure and function of airway smooth muscle (ASM). Chronic largely uniaxial cyclic strain of ASM cells in culture causes several structural and functional changes in ASM similar to that in asthma, including increases in contractility, MLCK content, shortening velocity, and shortening capacity. However, changes in recovery from acute stretch similar to a DI have not been measured. We have therefore measured the response and recovery to large stretches of cells modified by chronic stretching and investigated the role of MLCK. Chronic, 10% uniaxial cyclic stretch, with or without a strain gradient, was administered for up to 11 days to cultured cells grown on Silastic membranes. Single cells were then removed from the membrane and subjected to 1 Hz oscillatory stretches up to 10% of the in situ cell length. These oscillations reduced stiffness by 66% in all groups (P < 0.05). Chronically strained cells recovered stiffness three times more rapidly than unstrained cells, while the strain gradient had no effect. The stiffness recovery in unstrained cells was completely inhibited by the MLCK inhibitor ML-7, but recovery in strained cells exhibiting increased MLCK was slightly inhibited. These data suggest that chronic strain leads to enhanced recovery from acute stretch, which may be attributable to the strain-induced increases in MLCK. This may also explain in part the more rapid renarrowing of activated airways following DI in asthma. [ABSTRACT FROM AUTHOR]

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

The application of mechanical stresses to the airway smooth muscle (ASM) cell causes time-dependent cytoskeletal stiffening and remodeling (Dang L, Fairbank NJ, Fabry B, Smith PG, and Maksym GN. Am J Physiol Cell Physiol 287: C440–C448, 2004). We investigated here the extent to winch these behaviors are modulated by the state of cell activation (tone). Localized mechanical stress was applied to the ASM cell in culture via oscillating beads (4.5 μm) that were tightly bound to the actin cytoskeleton (CSK). Tone was reduced from baseline level using a panel of relaxant agonists (10-3 M dibutyryl cAMP, 10-4 M forskolin, or 10-4 M formoterol). To assess functional changes, we measured cell stiffness (G′) using optical magnetic twisting cytometry, and to assess structural changes of the CSK we measured actin accumulation in the neighborhood of the bead. Applied mechanical stress caused a twofold increase in G′ at 120 rain. Afar cessation of applied stress, G′ diminished only 24 ± 6% (mean ± SE) at 1 h, leaving substantial residual effects that were largely irreversible. However, applied stress-induced stiffening could be prevented by ablation of tone. Ablation of tone also inhibited the amount of actin accumulation induced by applied mechanical stress (P < 0.05). Thus the greater the contractile tone, the greater was applied stress-induced CSK stiffening and remodeling. As regards pathobiology of asthma, this suggests a maladaptive positive feedback in winch tone potentiates ASM remodeling and stiffening that further increases stress and possibly leads to worsening airway function. [ABSTRACT FROM AUTHOR]

Mechanical stress (MS) causes cytoskeletal (CSK) and phenotypic changes in cells. Such changes in airway smooth muscle (ASM) cells might contribute to the pathophysiology of asthma. We have shown that periodic mechanical strain applied to cultured ASM cells alters the structure and expression of CSK proteins and increases cell stiffness and contractility (Smith PG, Moreno R, and Ikebe M. Am J Physiol Lung Cell Mol Physiol 272: L20-L27, 1997; and Smith PG, Deng L, Fredberg JJ, and Maksym GN. Am J Physiol Lung Cell Mol Physiol 285: L456-L463, 2003). However, the mechanically induced CSK changes, altered cell function, and their time courses are not well understood. Here we applied MS to the CSK by magnetically oscillating ferromagnetic beads bound to the CSK. We quantified CSK remodeling by measuring actin accumulation at the sites of applied MS using fluorescence microscopy. We also measured CSK stiffness using optical magnetic twisting cytometry. We found that, during MS of up to 120 min, the percentage of beads associated with actin structures increased with time. At 60 min, 68.1 ± 1.6% of the beads were associated with actin structures compared with only 6.7 ± 2.8% before MS and 38.4 ± 5.5% in time-matched controls (P < 0.05). Similarly, CSK stiffness increased more than twofold in response to the MS compared with time-matched controls. These changes were more pronounced than observed with contractile stimulation by 80 mM KC1 or 10-4 M acetylcholine. Together, these findings imply that MS is a potent stimulus to enhance stiffness and contractility of ASM cells through CSK remodeling, which may have important implications in airway narrowing and dilation in asthma. [ABSTRACT FROM AUTHOR]

Ventilation heterogeneity is an important marker of small airway dysfunction in asthma. The frequency dependence of respiratory system resistance ( Rrs) from oscillometry is used as a measure of this heterogeneity. However, this has not been quantitatively assessed or compared with other outcomes from oscillometry, including respiratory system reactance ( Xrs) and the associated elastance ( Ers). Here, we used a multibranch model of the human lung, including an upper airway shunt, to match previously reported respiratory mechanics in mild to severe asthma. We imposed heterogeneity by narrowing a proportion of the peripheral airways to account for patient Ers at 5 Hz, and then narrowed central airways to account for the remaining Rrs at 18 Hz. The model required >75% of the small airways to be occluded to reproduce severe asthma. While the model produced frequency dependence in Rrs, it was upward-shifted below 5 Hz compared with in-vivo results, indicating that other factors, including more distributed airway narrowing or central airway wall compliance, are required. However, Ers quantitatively reflected the imposed heterogeneity better than the frequency dependence of Rrs, independent of the frequency range for the estimation, and thus was a more robust measure of small-airway function. Thus, Ers appears to have greater potential as a clinical measure of early small-airway disease in asthma. [ABSTRACT FROM AUTHOR]

Abstract: Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK''s dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology. [Copyright &y& Elsevier]