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.

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.

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.

Fetal Growth Restriction and Asthma: Is the Damage Done?

Trajectories of airway remodeling and functional impairment in asthma are consistent with the notion that airway pathology precedes or coincides with the onset of asthma symptoms and may be present at birth. An association between intrauterine growth restriction (IUGR) and asthma development has also been established, and there is value in understanding the underlying mechanism. This review considers airway pathophysiology as a consequence of IUGR that increases susceptibility to asthma.

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.

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.

Fatty airways: implications for obstructive disease.

Epidemiological studies report that overweight or obese asthmatic subjects have more severe disease than those of a healthy weight. We postulated that accumulation of adipose tissue within the airway wall may occur in overweight patients and contribute to airway pathology. Our aim was to determine the relationship between adipose tissue within the airway wall and body mass index (BMI) in individuals with and without asthma.Transverse airway sections were sampled in a stratified manner from post mortem lungs of control subjects (n=15) and cases of nonfatal (n=21) and fatal (n=16) asthma. The relationship between airway adipose tissue, remodelling and inflammation was assessed. The areas of the airway wall and adipose tissue were estimated by point count and expressed as area per mm of basement membrane perimeter (Pbm). The number of eosinophils and neutrophils were expressed as area densities.BMI ranged from 15 to 45 kg·m-2 and was greater in nonfatal asthma cases (p<0.05). Adipose tissue was identified in the outer wall of large airways (Pbm >6 mm), but was rarely seen in small airways (Pbm <6 mm). Adipose tissue area correlated positively with eosinophils and neutrophils in fatal asthma (Pbm >12 mm, p<0.01), and with neutrophils in control subjects (Pbm >6 mm, p=0.04).These data show that adipose tissue is present within the airway wall and is related to BMI, wall thickness and the number of inflammatory cells. Therefore, the accumulation of airway adipose tissue in overweight individuals may contribute to airway pathophysiology.

The role of miRNA regulation in fetal cardiomyocytes, cardiac maturation and the risk of heart disease in adults.

Myocardial infarction is a primary contributor towards the global burden of cardiovascular disease. Rather than repairing the existing damage of myocardial infarction, current treatments only address the symptoms of the disease and reducing the risk of a secondary infarction. Cardiac regenerative capacity is dependent on cardiomyocyte proliferation, which concludes soon after birth in humans and precocial species such as sheep. Human fetal cardiac tissue has some ability to repair following tissue damage, whereas a fully matured human heart has minimal capacity for cellular regeneration. This is in contrast to neonatal mice and adult zebrafish hearts, which retain the ability to undergo cardiomyocyte proliferation and can regenerate cardiac tissue after birth. In mice and zebrafish models, microRNAs (miRNAs) have been implicated in the regulation of genes involved in cardiac cell cycle progression and regeneration. However, the significance of miRNA regulation in cardiomyocyte proliferation for humans and other large mammals, where the timing of heart development in relation to birth is similar, remains unclear. miRNAs may be valuable targets for therapies that promote cardiac repair after injury. Therefore, elucidating the role of specific miRNAs in large animals, where heart development closely resembles that of humans, remains vitally important for identifying therapeutic targets that may be translated into clinical practice focused on tissue repair.

Foetal growth restriction in mice modifies postnatal airway responsiveness in an age and sex-dependent manner.

Epidemiological studies demonstrate an association between intrauterine growth restriction (IUGR) and asthma; however the underlying mechanism is unknown. We investigated the impact of maternal hypoxia-induced IUGR on airway responsiveness in male and female mice during juvenility and adulthood. Pregnant BALB/c mice were housed under hypoxic conditions for gestational days 11-17.5 and then returned to normoxic conditions for the remainder of pregnancy. A control group was housed under normoxic conditions throughout pregnancy. Offspring were studied at 2 weeks (juveniles) and 8 weeks (adults), where lung volume was assessed by plethysmography, airway responsiveness to methacholine determined by the forced oscillation technique and lungs fixed for morphometry. IUGR offspring were lighter at birth, exhibited "catch-up growth" by 2 weeks, but were again lighter in adulthood. IUGR males were "hyper-responsive" at 2 weeks and "hypo-responsive" as adults, in contrast with IUGR females who were hyper-responsive in adulthood. IUGR males had increased inner and total wall thickness at 2 weeks which resolved by adulthood, while airways in IUGR females were structurally normal throughout life. There were no differences in lung volume between Control and IUGR offspring at any age. Our data demonstrate changes in airway responsiveness as a result of IUGR that could influence susceptibility to asthma development and contribute to sexual dimorphism in asthma prevalence which switches from a male dominated disease in early life to a female dominated disease in adulthood.

Independent and combined effects of airway remodelling and allergy on airway responsiveness.

Airway remodelling and allergic inflammation are key features of airway hyperresponsiveness (AHR) in asthma; however, their interrelationships are unclear. The present study investigated the separate and combined effects of increased airway smooth muscle (ASM) layer thickness and allergy on AHR. We integrated a protocol of ovalbumin (OVA)-induced allergy into a non-inflammatory mouse model of ASM remodelling induced by conditional and airway-specific expression of transforming growth factor-α (TGF-α) in early growth response-1 (Egr-1)-deficient transgenic mice, which produced thickening of the ASM layer following ingestion of doxycycline. Mice were sensitised to OVA and assigned to one of four treatment groups: Allergy - normal chow diet and OVA challenge; Remodelling - doxycycline in chow and saline challenge; Allergy and Remodelling - doxycycline in chow and OVA challenge; and Control - normal chow diet and saline challenge. Airway responsiveness to methacholine (MCh) and histology were assessed. Compared with the Control group, airway responsiveness to MCh was increased in the Allergy group, independent of changes in wall structure, whereas airway responsiveness in the Remodelling group was increased independent of exposure to aeroallergen. The combined effects of allergy and remodelling on airway responsiveness were greater than either of them alone. There was a positive relationship between the thickness of the ASM layer with airway responsiveness, which was shifted upward in the presence of allergy. These findings support allergy and airway remodelling as independent causes of variable and excessive airway narrowing.

Increased heterogeneity of airway calibre in adult rats after hypoxia-induced intrauterine growth restriction.

BACKGROUND AND OBJECTIVE: Intrauterine growth restriction (IUGR) is associated with asthma development. We hypothesized that IUGR disrupts airway development leading to postnatal structural abnormalities of the airway that predispose to disease. This study therefore examined structural changes to the airway and lung in a rat model of maternal hypoxia-induced IUGR. METHODS: Pregnant rats were housed under hypoxic conditions (11.5% O2 ) from gestational days (GDs) 13 to 20 (pseudoglandular-canalicular stages, i.e. period of airway development) and then returned to normoxic conditions (21% O2 ). A control group of pregnant rats was housed under normoxic conditions throughout pregnancy. Weights of male offspring were recorded at birth and 7 weeks of age (adulthood), at which point lungs were fixed for morphometry and stereology (n = 6/group), or bronchoalveolar lavage fluid (BALF) was collected for cell counts (n = 6/group). RESULTS: IUGR offspring were lighter at birth compared with control, but not at 7 weeks. While there was no difference in mean airway dimensions or lung volume, there was greater anatomical variation in airway lumen area in the IUGR group. A mathematical model of the human lung was used to show that greater heterogeneity in lumen area in IUGR-affected individuals increases bronchoconstriction during simulated bronchial challenge. More macrophages were identified in the BALF of IUGR offspring. CONCLUSION: The rat model demonstrates that IUGR leads to a more heterogeneous distribution of airway lumen calibre in adulthood with potential implications for bronchoconstriction in human subjects. Together with increased lung macrophages, these findings support a phenotypic shift after IUGR that may impact disease susceptibility.

IGF-2R-Gαq signaling and cardiac hypertrophy in the low-birth-weight lamb.

The cardiac insulin-like growth factor 2 receptor (IGF-2R) can induce cardiomyocyte hypertrophy in a heterotrimeric G protein receptor-coupled manner involving αq (Gαq) or αs (Gαs). We have previously shown increased left ventricular weight and cardiac IGF-2 and IGF-2R gene expression in low-birth-weight (LBW) compared with average-birth-weight (ABW) lambs. Here, we have investigated the cardiac expression of IGF-2 gene variants, the degree of histone acetylation, and the abundance of proteins in the IGF-2R downstream signaling pathway in ABW and LBW lambs. Samples from the left ventricle of ABW and LBW lambs were collected at 21 days of age. There was increased phospho-CaMKII protein with decreased HDAC 4 abundance in the LBW compared with ABW lambs. There was increased GATA 4 and decreased phospho-troponin I abundance in LBW compared with ABW lambs, which are markers of pathological cardiac hypertrophy and impaired or reduced contractility, respectively. There was increased histone acetylation of H3K9 at IGF-2R promoter and IGF-2R intron 2 differentially methylated region in the LBW lamb. In conclusion, histone acetylation of IGF-2R may lead to increased IGF-2R mRNA expression and subsequently mediate Gαq signaling early in life via CaMKII, resulting in an increased risk of left ventricular hypertrophy and cardiovascular disease in adult life.

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.