Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels.

In obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), the extracellular matrix (ECM) protein amount and composition of the airway smooth muscle (ASM) is often remodelled, likely altering tissue stiffness. The underlying mechanism of how human ASM cell (hASMC) mechanosenses the aberrant microenvironment is not well understood. Physiological stiffnesses of the ASM were measured by uniaxial compression tester using porcine ASM layers under 0, 5 and 10% longitudinal stretch above in situ length. Linear stiffness gradient hydrogels (230 kPa range) were fabricated and functionalized with ECM proteins, collagen I (ColI), fibronectin (Fn) and laminin (Ln), to recapitulate the above-measured range of stiffnesses. Overall, hASMC mechanosensation exhibited a clear correlation with the underlying hydrogel stiffness. Cell size, nuclear size and contractile marker alpha-smooth muscle actin (αSMA) expression showed a strong correlation to substrate stiffness. Mechanosensation, assessed by Lamin-A intensity and nuc/cyto YAP, exhibited stiffness-mediated behaviour only on ColI and Fn-coated hydrogels. Inhibition studies using blebbistatin or Y27632 attenuated most mechanotransduction-derived cell morphological responses, αSMA and Lamin-A expression and nuc/cyto YAP (blebbistatin only). This study highlights the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.

Bronchial Thermoplasty Improves Ventilation Heterogeneity Measured by Functional Respiratory Imaging in Severe Asthma.

Purpose: Bronchial thermoplasty (BT) is a bronchoscopic intervention for the treatment of severe asthma. Despite demonstrated symptomatic benefit, the underlying mechanisms by which this is achieved remain uncertain. We hypothesize that the effects of BT are driven by improvements in ventilation heterogeneity as assessed using functional respiratory imaging (FRI). Patient and Methods: Eighteen consecutive patients with severe asthma who underwent clinically indicated BT were recruited. Patients were assessed at baseline, 4-week after treatment of the left lung, and 12-month after treatment of the right lung. Data collected included short-acting beta-agonist (SABA) and oral prednisolone (OCS) use, asthma control questionnaire (ACQ-5) and exacerbation history. Patients also underwent lung function tests and chest computed tomography. Ventilation parameters including interquartile distance (IQD; measure of ventilation heterogeneity) were derived using FRI. Results: 12 months after BT, significant improvements were seen in SABA and OCS use, ACQ-5, and number of OCS-requiring exacerbations. Apart from pre-bronchodilator FEV1, no other significant changes were observed in lung function. Ventilation heterogeneity significantly improved after treatment of the left lung (0.18 ± 0.04 vs 0.20 ± 0.04, p=0.045), with treatment effect persisting up to 12 months later (0.18 ± 0.05 vs 0.20 ± 0.04, p=0.028). Ventilation heterogeneity also improved after treatment of the right lung, although this did not reach statistical significance (0.18 ± 0.05 vs 0.19 ± 0.04, p=0.06). Conclusion: Clinical benefits after BT are accompanied by improvements in ventilation heterogeneity, advancing our understanding of its mechanism of action. Beyond BT, FRI has the potential to be expanded into other clinical applications.

Adipose tissue in the small airways: How much is enough to drive functional changes?

Obesity is a contributing factor to asthma severity; while it has long been understood that obesity is related to greater asthma burden, the mechanisms though which this occurs have not been fully elucidated. One common explanation is that obesity mechanically reduces lung volume through accumulation of adipose tissue external to the thoracic cavity. However, it has been recently demonstrated that there is substantial adipose tissue within the airway wall itself, and that the presence of adipose tissue within the airway wall is related to body mass index. This suggests the possibility of an additional mechanism by which obesity may worsen asthma, namely by altering the behaviour of the airways themselves. To this end, we modify Anafi & Wilson's classic model of the bistable terminal airway to incorporate adipose tissue within the airway wall in order to answer the question of how much adipose tissue would be required in order to drive substantive functional changes. This analysis suggests that adipose tissue within the airway wall on the order of 1%-2% of total airway cross-sectional area could be sufficient to drive meaningful changes, and further that these changes may interact with volume effects to magnify the overall burden.

Quantification of smooth muscle in human airways by polarization-sensitive optical coherence tomography requires correction for perichondrium.

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.

Postnatal steroids as lung protective and anti-inflammatory in preterm lambs exposed to antenatal inflammation.

BACKGROUND: Lung inflammation and impaired alveolarization precede bronchopulmonary dysplasia (BPD). Glucocorticoids are anti-inflammatory and reduce ventilator requirements in preterm infants. However, high-dose glucocorticoids inhibit alveolarization. The effect of glucocorticoids on lung function and structure in preterm newborns exposed to antenatal inflammation is unknown. We hypothesise that postnatal low-dose dexamethasone reduces ventilator requirements, prevents inflammation and BPD-like lung pathology, following antenatal inflammation. METHODS: Pregnant ewes received intra-amniotic LPS (E.coli, 4 mg/mL) or saline at 126 days gestation; preterm lambs were delivered 48 h later. Lambs were randomised to receive either tapered intravenous dexamethasone (LPS/Dex, n = 9) or saline (LPS/Sal, n = 10; Sal/Sal, n = 9) commencing <3 h after birth. Respiratory support was gradually de-escalated, using a standardised protocol aimed at weaning from ventilation towards unassisted respiration. Tissues were collected at day 7. RESULTS: Lung morphology and mRNA levels for inflammatory mediators were measured. Respiratory support requirements were not different between groups. Histological analyses revealed higher tissue content and unchanged alveolarization in LPS/Sal compared to other groups. LPS/Dex lambs exhibited decreased markers of pulmonary inflammation compared to LPS/Sal. CONCLUSION: Tapered low-dose dexamethasone reduces the impact of antenatal LPS on ventilation requirements throughout the first week of life and reduces inflammation and pathological thickening of the preterm lung IMPACT: We are the first to investigate the combination of antenatal inflammation and postnatal dexamethasone therapy in a pragmatic study design, akin to contemporary neonatal care. We show that antenatal inflammation with postnatal dexamethasone therapy does not reduce ventilator requirements, but has beneficial maturational impacts on the lungs of preterm lambs at 7 days of life. Appropriate tapered postnatal dexamethasone dosing should be explored for extuabtion of oxygen-dependant neonates.

Airway-associated adipose tissue accumulation is increased in a kisspeptin receptor knockout mouse model.

Airway-associated adipose tissue increases with body mass index and is a local source of pro-inflammatory adipokines that may contribute to airway pathology in asthma co-existing with obesity. Genetic susceptibility to airway adiposity was considered in the present study through kisspeptin/kisspeptin receptor signalling, known to modulate systemic adiposity and potentially drive airway remodelling. Therefore, the aim of the study was to determine the effects of kisspeptin/kisspeptin receptor signalling in the lung, focusing on airway-associated adipose tissue deposition and impact on airway structure-function. Wild-type, heterozygous and kisspeptin receptor knockout mice were studied at 6 or 8 weeks of age. Lung mechanics were assessed before and after methacholine challenge and were subsequently fixed for airway morphometry. A separate group of mice underwent glucose tolerance testing and bronchoalveolar lavage. At 6 weeks of age, kisspeptin/kisspeptin receptor signalling did not affect body adiposity, airway inflammation, wall structure or function. Despite no differences in body adiposity, there was a greater accumulation of airway-associated adipose tissue in knockout mice. By 8 weeks of age, female knockout mice displayed a non-diabetic phenotype with increased body adiposity but not males. Airway-associated adipose tissue area was also increased in both knockout females and males at 8 weeks of age, but again no other respiratory abnormality was apparent. In summary, airway-associated adipose tissue is decoupled from body adiposity in prepubescent mice which supports a genetic susceptibility to fatty deposits localised to the airway wall. There was no evidence that airway-associated adipose tissue drives pathology or respiratory impairment in the absence of other environmental exposures.

Dose-related cardiac outcomes in response to postnatal dexamethasone treatment in premature lambs.

BACKGROUND: Postnatal corticosteroids are used in the critical care of preterm infants for the prevention and treatment of bronchopulmonary dysplasia. We aimed to investigate the effects of early postnatal dexamethasone therapy and dose on cardiac maturation and morphology in preterm lambs. METHODS: Lambs were delivered prematurely at ~128 days of gestational age and managed postnatally according to best clinical practice. Preterm lambs were administered dexamethasone daily at either a low-dose (n = 9) or a high-dose (n = 7), or were naïve to steroid treatment and administered saline (n = 9), over a 7-day time-course. Hearts were studied at postnatal Day 7 for gene expression and assessment of myocardial structure. RESULTS: High-dose dexamethasone treatment in the early postnatal period led to marked differences in cardiac gene expression, altered cardiomyocyte maturation and reduced cardiomyocyte endowment in the right ventricle, as well as increased inflammatory infiltrates into the left ventricle. Low-dose exposure had minimal effects on the preterm heart. CONCLUSION: Neonatal dexamethasone treatment led to adverse effects in the preterm heart in a dose-dependent manner within the first week of life. The observed cardiac changes associated with high-dose postnatal dexamethasone treatment may influence postnatal growth and remodeling of the preterm heart and subsequent long-term cardiac function.

From Beneath the Skin to the Airway Wall: Understanding the Pathological Role of Adipose Tissue in Comorbid Asthma-Obesity.

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.

Distribution, composition, and activity of airway-associated adipose tissue in the porcine lung.

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-1ß, 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.

Heterogeneity of Airway Smooth Muscle Remodeling in Asthma.

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.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs.

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Impact of fetal treatments for congenital diaphragmatic hernia on lung development.

The extent of lung hypoplasia impacts the survival and severity of morbidities associated with congenital diaphragmatic hernia (CDH). The alveoli of CDH infants and in experimental models of CDH have thickened septa with fewer type II pneumocytes and capillaries. Fetal treatments of CDH-risk preterm birth. Therefore, treatments must aim to balance the need for increased gas exchange surface area with the restoration of pulmonary epithelial type II cells and the long-term respiratory and neurodevelopmental consequences of prematurity. Achievement of sufficient lung development in utero for successful postnatal transition requires adequate intra-thoracic space for lung growth, maintenance of sufficient volume and appropriate composition of fetal lung fluid, regular fetal breathing movements, appropriate gas exchange area, and ample surfactant production. The review aims to examine the rationale for current and future therapeutic strategies to improve postnatal outcomes of infants with CDH.

Airway smooth muscle thickness and contraction are enhanced by intra-amniotic lipopolysaccharide in an ovine model of premature birth.

Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. We hypothesize that antenatal inflammation causes physiological abnormalities of the ASM that predisposes asthma. This study determined the short-term effects of antenatal inflammation on the developing ASM. Fourteen pregnant ewes were randomly assigned to one of three groups. Fetal lambs were exposed to intra-amniotic injections of lipopolysaccharide (LPS, n = 4) or saline (controls; n = 5) at 127 days' gestational age (GA). Preterm lambs were surgically delivered at 129 days' GA and received intensive care for 7 days before euthanasia. Naïve fetal controls (n = 5) were delivered and euthanized at 136 days' GA. ASM force to acetylcholine was measured in bronchial rings and normalized to ring length (tension) and ASM cross-sectional area (stress). Airway narrowing (% volume) to acetylcholine was assessed in bronchial segments. Fetal controls were structurally and functionally similar to saline-exposed lambs. Compared with saline, LPS-exposed lambs had increased macrophages in lung tissue (P = 0.0002) and interleukin-8 in alveolar wash (P = 0.003). LPS exposure increased ASM thickness (P = 0.005), airway narrowing (P = 0.003), ASM tension (P = 0.0002), and contractile stress (P < 0.0001). Notably, LPS-exposed lambs were more dependent on mechanical ventilation, and both LPS (P < 0.001) and ventilation (P = 0.012) were independent factors in increasing ASM stress. Only LPS independently increased ASM thickness (P = 0.045). Results indicate that antenatal exposure to LPS and subsequent mechanical ventilation promotes intrinsic changes to the ASM that enhances bronchoconstriction. If persistent into postnatal life, these developmental abnormalities may contribute to the known association between chorioamnionitis and asthma.NEW & NOTEWORTHY Abnormalities of the airway smooth muscle (ASM) layer in asthma may develop before birth. Using an ovine model of antenatal inflammation, we demonstrate thickening and increased contraction of the premature ASM layer. If such physiological abnormalities persist throughout postnatal life, this represents a predisposition to an asthma diagnosis.

Growth of the airway smooth muscle layer from late gestation to childhood is mediated initially by hypertrophy and subsequently hyperplasia.

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.

Azithromycin inhibits mucin secretion, mucous metaplasia, airway inflammation, and airways hyperresponsiveness in mice exposed to house dust mite extract.

Excessive production, secretion, and retention of abnormal mucus is a pathological feature of many obstructive airways diseases including asthma. Azithromycin is an antibiotic that also possesses immunomodulatory and mucoregulatory activities, which may contribute to the clinical effectiveness of azithromycin in asthma. The current study investigated these nonantibiotic activities of azithromycin in mice exposed daily to intranasal house dust mite (HDM) extract for 10 days. HDM-exposed mice exhibited airways hyperresponsiveness to aerosolized methacholine, a pronounced mixed eosinophilic and neutrophilic inflammatory response, increased airway smooth muscle (ASM) thickness, and elevated levels of epithelial mucin staining. Azithromycin (50 mg/kg sc, 2 h before each HDM exposure) attenuated HDM-induced airways hyperresponsiveness to methacholine, airways inflammation (bronchoalveolar lavage eosinophil and neutrophils numbers, and IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and RANTES levels), and epithelial mucin staining (mucous metaplasia) by at least 50% (compared with HDM-exposed mice, P < 0.05). Isolated tracheal segments of HDM-exposed mice secreted Muc5ac and Muc5b (above baseline levels) in response to exogenous ATP. Moreover, ATP-induced secretion of mucins was attenuated in segments obtained from azithromycin-treated, HDM-exposed mice (P < 0.05). In additional ex vivo studies, ATP-induced secretion of Muc5ac (but not muc5b) from HDM-exposed tracheal segments was inhibited by in vitro exposure to azithromycin. In vitro azithromycin also inhibited ATP-induced secretion of Muc5ac and Muc5b in tracheal segments from IL-13-exposed mice. In summary, azithromycin inhibited ATP-induced mucin secretion and airways inflammation in HDM-exposed mice, both of which are likely to contribute to suppression of airways hyperresponsiveness.