In asthma, augmented airway wall smooth muscle (ASM) bulk is a major remodeling feature, promoted by increased transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF). Runt-related transcription factor-2 (RUNX2) represses TGF-β1-induced CTGF through interactions with SMAD3. This study aimed to investigate the expression and role of RUNX2 in asthmatic and nonasthmatic ASM cells. mRNA and protein were detected by microarray, PCR, and western blot in nonasthmatic and asthmatic ASM cells. Immunohistochemistry identified RUNX2 in lung tissues from asthmatic patients and nonasthmatic subjects. Different RUNX2 isoforms were transfected into immortalized-asthmatic ASM cells, and markers of inflammation and airway remodeling were measured. RUNX2 alternatively spliced forms were examined in bronchial biopsies from asthmatic and healthy subjects. The abundance of RUNX2 was decreased in isolated ASM cells from asthmatic compared with nonasthmatic subjects. The ASM layer around airways in lung tissue sections from asthmatic and nonasthmatic patients had a heterogeneous pattern of RUNX2 protein detection. TGF-β1 stimulation increased RUNX2/RUNX2 variant 1 mRNA in nonasthmatic but not asthmatic ASM cells, facilitating SMAD3 activation and nuclear translocation in asthmatic ASM cells. RUNX2 isoform overexpression in immortalized asthmatic ASM cells failed to alter markers of inflammation (IL-6) but significantly reduced markers of remodeling (CTGF), ASM cell hypertrophy (GSK-3β and desmin), and proliferation (pSer795 Rb and α-tubulin). In bronchial biopsies, RUNX2 mRNA splicing was higher in asthmatic patients compared with healthy subjects. These data suggest RUNX2 plays a role in the homeostasis of healthy airways. Restoring RUNX2 may provide a new therapeutic approach for asthma.

Rationale Airway smooth muscle (ASM) remodelling is a central feature of asthma pathology but cannot currently be measured in vivo with adequate coverage, limiting the development of targeted therapies. The objective of the present study was to trial bronchoscopic polarisation-sensitive optical coherence tomography (PS-OCT) to assess ASM remodelling and its distribution in vivo throughout the lungs in individuals with and without asthma. Methods Participants with (n=7) and without (n=13) doctor-diagnosed asthma undergoing bronchoscopy were recruited to obtain PS-OCT imaging data during the procedure. ASM area in large and small airways was measured and normalised for airway size (lumen perimeter). ASM remodelling was defined as ASM mass (ASM area/perimeter 2 ) >1.96 sd above a histological reference control group. Measurements and main results PS-OCT quantified ASM mass was greater in participants with asthma (median 0.0072, Q1–Q3 0.0064–0.0089) compared with the control group (0.0039, 0.0035–0.0049), (p=0.012). ASM remodelling was also more prevalent in the asthma group (44% of airways examined, 24–53%) than in the control group (0%, 0–4%) (p=0.007). ASM mass was heterogeneously distributed within airways, lungs and the sample population. Phenotypes of ASM remodelling (based on location in small or large airways) were apparent in the asthma group, compared with the control group where participants were all classified as non-remodelled. Conclusions PS-OCT is a minimally invasive, accurate, efficient and effective modality to measure ASM mass. Visualising and quantifying ASM in patients with asthma provides an opportunity to make ASM remodelling a treatable trait and may facilitate the development of novel therapeutics for the treatment of asthma.

Quantifying airway smooth muscle (ASM) in patients with asthma raises the possibility of improved and personalized disease management. Endobronchial polarization-sensitive optical coherence tomography (PS-OCT) is a promising quantitative imaging approach that is in the early stages of clinical translation. To date, only animal tissues have been used to assess the accuracy of PS-OCT to quantify absolute (rather than relative) ASM in cross sections with directly matched histological cross sections as validation. We report the use of whole fresh human and pig airways to perform a detailed side-by-side qualitative and quantitative validation of PS-OCT against gold-standard histology. We matched and quantified 120 sections from five human and seven pig (small and large) airways and linked PS-OCT signatures of ASM to the tissue structural appearance in histology. Notably, we found that human cartilage perichondrium can share with ASM the properties of birefringence and circumferential alignment of fibers, making it a significant confounder for ASM detection. Measurements not corrected for perichondrium overestimated ASM content several-fold (P < 0.001, paired t test). After careful exclusion of perichondrium, we found a strong positive correlation (r = 0.96, P < 0.00001) of ASM area measured by PS-OCT and histology, supporting the method's application in human subjects. Matching human histology further indicated that PS-OCT allows conclusions on the intralayer composition and in turn potential contractile capacity of ASM bands. Together these results form a reliable basis for future clinical studies.NEW & NOTEWORTHY Polarization-sensitive optical coherence tomography (PS-OCT) may facilitate in vivo measurement of airway smooth muscle (ASM). We present a quantitative validation correlating absolute ASM area from PS-OCT to directly matched histological cross sections using human tissue. A major confounder for ASM quantification was observed and resolved: fibrous perichondrium surrounding hyaline cartilage in human airways presents a PS-OCT signature similar to ASM for birefringence and optic axis orientation. Findings impact the development of automated methods for ASM segmentation.

Patients with comorbid asthma-obesity experience greater disease severity and are less responsive to therapy. We have previously reported adipose tissue within the airway wall that positively correlated with body mass index. Accumulation of biologically active adipose tissue may result in the local release of adipokines and disrupt large and small airway function depending on its anatomical distribution. This study therefore characterized airway-associated adipose tissue distribution, lipid composition, and adipokine activity in a porcine model. Airway segments were systematically dissected from different locations of the bronchial tree in inflation-fixed lungs. Cryosections were stained with hematoxylin and eosin (H&E) for airway morphology, oil red O to distinguish adipose tissue, and Nile blue A for lipid subtype delineation. Excised airway-associated adipose tissue was cultured for 72 h to quantify adipokine release using immunoassays. Results showed that airway-associated adipose tissue extended throughout the bronchial tree and occupied an area proportionally similar to airway smooth muscle within the wall area. Lipid composition consisted of pure neutral lipids (61.7 ± 3.5%), a mixture of neutral and acidic lipids (36.3 ± 3.4%), or pure acidic lipids (2.0 ± 0.8%). Following tissue culture, there was rapid release of IFN-γ, IL-1β, and TNF-α at 12 h. Maximum IL-4 and IL-10 release was at 24 and 48 h, and peak leptin release occurred between 48 and 72 h. These data extend previous findings and demonstrate that airway-associated adipose tissue is prevalent and biologically active within the bronchial tree, providing a local source of adipokines that may be a contributing factor in airway disease.

Rationale: Ventilatory defects in asthma are heterogeneous and may represent the distribution of airway smooth muscle (ASM) remodeling. Objectives: To determine the distribution of ASM remodeling in mild-severe asthma. Methods: The ASM area was measured in nine airway levels in three bronchial pathways in cases of nonfatal (n = 30) and fatal asthma (n = 20) and compared with control cases without asthma (n = 30). Correlations of ASM area within and between bronchial pathways were calculated. Asthma cases with 12 large and 12 small airways available (n = 42) were classified on the basis of the presence or absence of ASM remodeling (more than two SD of mean ASM area of control cases, n = 86) in the large or small airway or both. Measurements and Main Results: ASM remodeling varied widely within and between cases of nonfatal asthma and was more widespread and confluent and more marked in fatal cases. There were weak correlations of ASM between levels within the same or separate bronchial pathways; however, predictable patterns of remodeling were not observed. Using mean data, 44% of all asthma cases were classified as having no ASM remodeling in either the large or small airway despite a three- to 10-fold increase in the number of airways with ASM remodeling and 81% of asthma cases having ASM remodeling in at least one large and small airway. Conclusions: ASM remodeling is related to asthma severity but is heterogeneous within and between individuals and may contribute to the heterogeneous functional defects observed in asthma. These findings support the need for patient-specific targeting of ASM remodeling.

This article provides a contemporary report on the role of adipose tissue in respiratory dysfunction. Adipose tissue is distributed throughout the body, accumulating beneath the skin (subcutaneous), around organs (visceral), and importantly in the context of respiratory disease, has recently been shown to accumulate within the airway wall: "airway-associated adipose tissue." Excessive adipose tissue deposition compromises respiratory function and increases the severity of diseases such as asthma. The mechanisms of respiratory impairment are inflammatory, structural, and mechanical in nature, vary depending on the anatomical site of deposition and adipose tissue subtype, and likely contribute to different phenotypes of comorbid asthma-obesity. An understanding of adipose tissue-driven pathophysiology provides an opportunity for diagnostic advancement and patient-specific treatment. As an exemplar, the potential impact of airway-associated adipose tissue is highlighted, and how this may change the management of a patient with asthma who is also obese. © 2023 American Physiological Society. Compr Physiol 13:4321-4353, 2023.

Tweetable abstract Low birth weight infants born at term have structurally abnormal airway smooth muscle which may contribute to an increased risk of obstructive disease https://bit.ly/3F0GmOd

The perimeter of the basement membrane (Pbm), as seen on whole cross-sections of airways, has become a standard index of airway size since it is independent of the effects of lung inflation, bronchoconstriction and the presence of asthma.1, 2 This has allowed the comparison of airway wall dimensions, especially airway smooth muscle (ASM), between airways of different size within individuals (human or animal) and airways of the same size between individuals with and without disease. However, the relationship between ASM (or gross airway wall area) and Pbm is not necessarily a simple one and the question arises - how should we normalize measures of airway wall components with respect to airway size (as measured by Pbm)? The above question has far-reaching implications for both basic science and respiratory medicine. ‘Over-normalizing’ might reduce the apparent differences in the large airways between experimental groups or clinical cases, relative to the small airways. Conversely, ‘under-normalizing’ may introduce the opposite effect. These errors in turn affect conclusions drawn from morphological data such as, in an animal model, does a particular allergen exposure produce ASM remodelling in both small and large airways? Does a patient with asthma, who may be short or tall, with an airway diameter that will scale accordingly, exhibit clinically significant ASM remodelling? Since sampled or diagnostically examined airways will always vary in size (between and within an organism), the correct form of normalization is therefore necessary. This issue of normalization is separate from any concerns that the length of an apparently indistensible membrane may not be constant with respect to fixation procedures or other factors.1, 3 As clearly stated by Chin et al.,4 regarding measurements of ASM, ‘(the results) could have been confounded if we were comparing airways of different size since the ratio of airway wall area/Pbm increases as airways get smaller’. The uncertain relationship between areas of wall components and Pbm has long been acknowledged and many authors prefer to assume that ASM varies as Pbm2, thus normalizing as ASM/Pbm2 (or equivalently ASM / P bm ).4, 5 This has the added advantage that the normalized quantity is dimensionless. Both this approach, and a simple ASM/Pbm ratio, can be thought of as special cases of assuming that ASM is related to Pbma by a so-called power law, where ASM ∝ P bm a for some constant ‘a’. The simple ratio ASM/Pbm is equivalent to taking a = 1, while the use of ASM/Pbm2 (or ASM / P bm ) is to assume that a = 2. Both ASM and gross wall area do follow a power law to a large degree6, 7 (Figure 1). Recall that the usual approach to visualizing a power law is to use logarithmic axes (Figure 1B), in which case the power law relationship becomes a straight line with slope ‘a’ (the power law exponent). However, the power law exponent appears not to be constant, either with respect to airway development8 or disease. Take ASM for example, which has a power law exponent slightly above 1.0 just before birth, rising throughout early childhood to a value of approximately 1.8 in a non-asthma adult population; the exponent in fatal asthma is higher still, approaching 2 (Figure 1C). A similar trend occurs for gross wall area, though the exponent values are not identical. Healthy adult power-law exponents are relatively close to 2 (though not exactly), providing good support for the use of ASM/Pbm2 as the conventional normalization approach. Given the choice between ASM/Pbm and ASM/Pbm2, the latter is the better option in almost all situations (pre-natal and very early childhood being the exceptions in this dataset). However, it is worth noting that the normalization is imperfect: in most cases, ASM/Pbm2 will overcorrect slightly, meaning that a sample skewed towards larger airways would be biased lower. With the problem now apparent, the solution is less definitive. Assuming an exponent of 1.8 might be closer in some situations, but still biased in others. In some cases, a more rigorous approach to fit the power law parameters directly8 may be justified, particularly since factors specific to a certain experimental design or clinical scenario will alter the power law exponent. These include, but are not limited to, age, disease and choice of animal model. At a minimum, when using ASM/Pbm2 (or ASM / P bm ) one must be aware that there will be some over-correction and that the composition of airway sizes in the sample influences the results. The question of how best to normalize airway measurements, and accompanying analysis, is far from a scientific niche. The need for direct and accurate measurement of ASM dimensions has been proposed to expand treatment of asthma,9, 10 optimize current approaches11, 12 and to add an additional dimension to patient phenotyping.13 Specifically, newer in vivo approaches such as polarization-sensitive optical coherence tomography are being developed for identifying and mapping ASM remodelling,14 and will require an effective normalization method for airway size. Open access publishing facilitated by The University of Western Australia, as part of the Wiley - The University of Melbourne agreement via the Council of Australian University Librarians. None declared.

"Does “Skippy” Wheeze? Evidence of Airway Remodeling in the Australian Kangaroo." American Journal of Respiratory Cell and Molecular Biology, 67(1), pp. 125–127

Background and objective The airway smooth muscle (ASM) layer thickens during development. Identifying the mechanism(s) for normal structural maturation of the ASM reveals pathways susceptible to disease processes. This study characterized thickening of the ASM layer from foetal life to childhood and elucidated the underlying mechanism in terms of hypertrophy, hyperplasia and extracellular matrix (ECM) deposition. Methods Airways from post-mortem cases were examined from seven different age groups: 22-24 weeks gestation, 25-31 weeks gestation, term (37-41 weeks gestation), <0.5 year, 0.5-1 year, 2-5 years and 6-10 years. The ASM layer area (thickness), the number and size of ASM cells and the volume fraction of ECM were assessed by planimetry and stereology. Results From late gestation to the first year of life, normalized ASM thickness more than doubled as a result of ASM hypertrophy. Thereafter, until childhood, the ASM layer grew in proportion to airway size, which was mediated by ASM hyperplasia. Hypertrophy and hyperplasia of ASM were accompanied by a proportional change in ECM such that the broad composition of the ASM layer was constant across age groups. Conclusion These data suggest that the mechanisms of ASM growth from late gestation to childhood are temporally decoupled, with early hypertrophy and subsequent proliferation. We speculate that the developing airway is highly susceptible to ASM thickening in the first year of life and that the timing of an adverse event will determine structural phenotype.

Clinical visualization and quantification of the amount and distribution of airway smooth muscle (ASM) in the lungs of individuals with asthma has major implications for our understanding of airway wall remodeling as well as treatments targeted at the ASM. This paper theoretically investigates the feasibility of quantifying airway wall thickness (focusing on the ASM) throughout the lung in vivo by means of bronchoscopic polarization-sensitive optical coherence tomography (PS-OCT). Using extensive human biobank data from subjects with and without asthma in conjunction with a mathematical model of airway compliance, we define constraints that airways of various sizes pose to any endoscopic imaging technique and how this is impacted by physiologically relevant processes such as constriction, inflation and deflation. We identify critical PS-OCT system parameters and pinpoint parts of the airway tree that are conducive to successful quantification of ASM. We further quantify the impact of breathing and ASM contraction on the measurement error and recommend strategies for standardization and normalization

Introduction: The remodelled asthmatic airway features an altered extracellular matrix (ECM) & increased vasculature. Previous studies found asthmatic (A) airway smooth muscle cells (ASMCs) to deposit an ECM with enhanced bioactivity. These studies however investigated ECM deposited in the presence of proliferative stimuli. Objective: This study examined the regulatory activity of the ECM generated by A & non-asthmatic (NA) ASMCs under quiescent conditions. Methods: ECM from primary human NA& AASMCs was isolated under quiescent conditions. NA-, AASMCs, or human umbilical vein endothelial cells (HUVECs) were seeded onto decellularised NA-or AASM-ECM. Metabolic activity was measured by MTT. ASMC proliferation (CyQUANT), HUVEC attachment & transwell migration (toluidine blue) were also quantified. Results: NAASMCs proliferated less on A-ECM compared to NA-ECM (N=3&5 P=0.04) but there were no differences in metabolic activity on NAor A-ECMs (N=4&3 P=0.21). AASMCs had no difference in proliferation or metabolic activity on A-ECM (N=3&5) versus the NA-ECM (N=4 for both P=0.44&0.28). HUVECs showed similar attachment to NA& A-ECMs (N=10&8 P=0.97). HUVEC metabolic activity on & migration through the quiescent ECM was not different between A-& NA-ECM (N=10&11 P=0.83, and N=3&5 P=0.30). Conclusion: A-ECM produced under quiescent conditions has similar bioactive properties to NA-derived ECM. This study contrasts reports of an altered ECM bioactivity generated by AASMCs under stimulated conditions. These data provide further evidence for maintaining AASMCs in a resting state to prevent deposition of an abnormal ECM which may contribute to the remodelling in the airways.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti–mouse CD49b-PE (phycoerythrin) antibody. Platelet–endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

Airway remodelling is a cardinal feature of asthma in which airways undergo structural changes - in particular, increased airway smooth muscle mass and total airway wall area. Remodelling has long been thought to have functional consequences in asthma due to geometric effects that can increase airway narrowing and luminal occlusion. Prior studies have examined the distribution of remodelling between and within patients, but none have yet considered the possibility for spatial correlations in airway remodelling. That is, is remodelling clustered locally, or interrelated along proximal and distal locations of the bronchial tree? In view of recent interest regarding airway remodelling produced by mechanical stimuli, we developed a mathematical model to examine whether spatial correlations in airway remodelling could arise due to cycles of bronchoconstriction and mechanotransduction. Further, we compared modelling predictions to the spatial distribution of airway remodelling in lungs from subjects with and without asthma. Results indicate that spatial correlations in airway remodelling do exist in vivo, and cycles of bronchoconstriction and mechanotransduction are one plausible mechanism for their origin. These findings offer insights into the evolution of airway remodelling in asthma, which may inform strategies for treatment and prevention.

The volume fraction of extracellular matrix (ECM) within the layer of airway smooth muscle (ASM) is increased in subjects with fixed airflow obstruction. We postulated that changes in ECM within the ASM layer will impact force transmission during induced contraction and/or in response to externally applied stresses like a deep inspiration (DI). Subjects were patients undergoing lung resection surgery who were categorized as unobstructed (n = 12) or "fixed" obstructed (n = 6) on the basis of preoperative spirometry. The response to a DI, assessed by the ratio of isovolumic flows from maximal and partial inspirations (M/P), was also measured preoperatively. M/P was reduced in the obstructed group (P = 0.02). Postoperatively, bronchial segments were obtained from resected tissue, and luminal narrowing to acetylcholine and bronchodilation to simulated DI were assessed in vitro. Airway wall dimensions and the volume fraction of ECM within the ASM were quantified. Maximal airway narrowing to acetylcholine (P = 0.01) and the volume fraction of ECM within the ASM layer (P = 0.02) were increased in the obstructed group, without a change in ASM thickness. Whereas bronchodilation to simulated DI in vitro was not different between obstructed and unobstructed groups, it was correlated with increased M/P (bronchodilation/less bronchoconstriction) in vivo (P = 0.03). The volume fraction of ECM was inversely related to forced expiratory volume in 1 s FEV1 %predicted (P = 0.04) and M/P (P = 0.01). Results show that in subjects with fixed airflow obstruction the mechanical behavior of the airway wall is altered and there is a contemporaneous shift in the structural composition of the ASM layer.NEW & NOTEWORTHY Cartilaginous airways from subjects with fixed airflow obstruction have an increase in the volume fraction of extracellular matrix within the airway smooth muscle layer. These airways are also intrinsically more reactive to a contractile stimulus, which is expected to contribute to airway hyperresponsiveness in this population, often attributed to geometric mechanisms. In view of these results, we speculate on how changes in extracellular matrix may impact airway mechanics.