Airway smooth muscle is presumed to be the major effector of the airway narrowing that occurs during bronchial provocation testing. The sensitivity of induced bronchoconstriction to lung volume in humans and animals has been interpreted as indicating that the limitation of airway narrowing in vivo is caused by an inability of airway smooth muscle to overcome the impedance imposed by the parenchymal tethering. A number of potentially interesting candidate growth-promoting substances have been identified by in vitro testing. Of particular interest are those associated with allergic inflammation since allergy is one of the best-characterised mechanisms by which asthma is induced. The increase in airway smooth muscle induced by allergen challenge is attenuated by nedocromil, an antiallergic drug which effectively reduces allergic airway responses in the rat. Hyperoxic exposure of neonatal rats causes a variety of changes in airway structure and function. The mechanism of hyperoxia-induced changes in rat airways is not known.

ABSTRACTBackground and objectiveThe pathology of asthma is characterized by airway inflammation (granulocytic (GA) or paucigranulocytic (PGA)) and remodelling of airway structures. However, the relationship between inflammatory phenotypes and remodelling is unclear. We hypothesized that some features of airway remodelling are dependent on granulocytic airway inflammation while others are not.MethodsPost‐mortem airway sections from control subjects (n = 48) and cases of asthma with (n = 51) or without (n = 29) granulocytic inflammation in the inner airway wall were studied. The thickness of the airway smooth muscle (ASM) layer, basement membrane and inner and outer airway walls, the size and number of ASM cells, the volume fraction of extracellular matrix within the ASM layer, ASM shortening and luminal mucus were estimated. Airway dimensions were compared between the three subject groups.ResultsIn cases of PGA, only the thickness of the ASM layer and basement membrane was increased compared with control subjects. In cases of GA, not only the ASM and basement membrane were increased in thickness, but there was also increased inner and outer airway wall thickness and increased narrowing of the airway lumen due to ASM shortening and mucus obstruction, compared with control subjects. Granulocytic inflammation was observed more often in cases of fatal asthma.ConclusionThese findings suggest that inner and outer wall thickening coexists with inflammation, whereas thickening of the ASM layer and basement membrane may be present even in the absence of inflammation. Remodelling of the ASM layer and basement membrane may therefore be less susceptible to anti‐inflammatory therapy.

In asthma, it is unclear if the airway smooth muscle cells proliferate more or are increased at the onset of asthma and remain stable. This study aimed to compare smooth muscle cell proliferation in individuals with and without asthma and correlate proliferation rates with cell size and number and with granulocytic airway inflammation. Postmortem airway sections were labeled with proliferating cell nuclear antigen (PCNA) and percent positive muscle cells calculated. On the same sections, smooth muscle cell size and number and the number of eosinophils and neutrophils were estimated and compared in cases of nonfatal ( n = 15) and fatal ( n = 15) asthma and control subjects ( n = 15). The %PCNA+ muscle cells was not significantly different in fatal (29.4 ± 7.7%, mean ± SD), nonfatal asthma (28.6 ± 8.3%), or control subjects (24.6 ± 6.7%) and not related to mean muscle cell size ( r = 0.09), number ( r = 0.36), thickness of the muscle layer ( r = 0.05), or eosinophil numbers ( r = 0.04) in the asthma cases. These data support the hypothesis that in asthma the increased thickness of the smooth muscle layer may be present before or at the onset of asthma and independent of concurrent granulocytic inflammation or exacerbation. NEW & NOTEWORTHY There is debate regarding the origins of the increased airway smooth muscle in asthma. It may be independent of inflammation or arise as a proliferative response to inflammation. The present study found no increase in the proportion of proliferating smooth muscle cells in asthma and no relation of proliferation to numbers of airway smooth muscle cells or inflammation. These results support a stable increase in smooth muscle in asthma that is independent of airway inflammation.

Background and objectivePathological phenotypes of asthma have been based predominantly on inflammation, rather than airway wall remodelling. Differences in the distribution of airway smooth muscle (ASM) remodelling between large and small airways may affect clinical outcomes in asthma. The aim of this study was to examine the distribution of ASM remodelling and its relation to airway inflammation.MethodsPost-mortem cases of asthma (n=68) were categorized by the distribution of increased thickness of the ASM layer (relative to nonasthmatic controls, n=37), into large only' (LO, n=15), small only' (SO, n=4) large/small' (LS, n=24) or no increase (NI, n=25). Subject characteristics, ASM and airway wall dimensions and inflammatory cell numbers were compared between groups.ResultsApart from reduced clinical severity of asthma in NI cases (P=0.002), subject characteristics did not distinguish asthma groups. Compared with control subjects, ASM cell number, reticular basement membrane thickness, airway wall thickness, percent muscle shortening and eosinophil number were increased (P< 0.05) in both large and small airways in LS cases and only the large airways in LO cases. Increased numbers of neutrophils were observed only in the small airways of LO cases.ConclusionsDistinct distributions of ASM remodelling are seen in asthma. Pathology limited to the small airways was uncommon. Increased thickness of the ASM layer was associated with airway remodelling and eosinophilia, but not neutrophilia. These data support the presence of distinct pathological phenotypes based on the site of increased ASM.

Background: Excessive airway narrowing, the cause of asthma symptoms, may arise from different mechanisms in cases of asthma with eosinophils (EA) or without eosinophils (NEA). Aim: To compare the ratio of the airway lumen occupied by mucus (MOR), percent airway smooth muscle shortening (PMS) and airway wall dimensions in cases of EA (n=38), NEA (n=43) and control subjects (n=48). Methods: On sections of airway taken from post-mortem lungs, the area densities of eosinophils and neutrophils within the inner airway wall were calculated (H&E, 5μm). Asthmatics with a mean eosinophil density >5cells/mm 2 were classified as EA. On the same section, MOR, PMS and airway wall dimensions were determined. Results: There were no significant differences in duration, age of onset of asthma or smoking history between case groups, however EA had more severe asthma (more cases of fatal versus nonfatal asthma) than NEA. The thickness of the airway smooth muscle layer was significantly increased in EA cases compared with control subjects in all airway size groups (p<0.05). The thickness of the inner airway wall and reticular basement membrane, MOR, PMS and neutrophil area density were increased in medium and large airways in the EA cases compared with control subjects (p<0.05). These differences persisted when fatal and nonfatal cases were analysed separately. Conclusion : Eosinophilic asthma is characterized by airway wall and airway smooth muscle remodeling with increased mucus within the airway lumen and increased percent muscle shortening. Support: NHMRC of Australia Project Grant #618700.

Epidemiological associations of worse respiratory outcomes from combined exposure to ambient particulate matter (PM) and respiratory viral infection suggest possible interactions between PM and viruses. To characterize outcomes of such exposures, we developed an in vitro mimic of the in vivo event of exposure to PM contaminated with respiratory syncytial virus (RSV). Concentration of infectious RSV stocks and a particle levitation apparatus were the foundations of the methodology developed to generate specific numbers of PM mimics (PM(Mimics)) of known composition for dry, direct deposition onto airway epithelial cell cultures. Three types of PM(Mimics) were generated for this study: (i) carbon alone (P(C)), (ii) carbon and infectious RSV (P(C+RSV)), and (iii) aerosols consisting of RSV (A(RSV)). P(C+RSV) were stable in solution and harbored infectious RSV for up to 6 months. Unlike A(RSV) infection, P(C+RSV) infection was found to be dynamin dependent and to cause lysosomal rupture. Cells dosed with PM(Mimics) comprised of RSV (A(RSV)), carbon (P(C)), or RSV and carbon (P(C+RSV)) responded differentially as exemplified by the secretion patterns of IL-6 and IL-8. Upon infection, and prior to lung cell death due to viral infection, regression analysis of these two mediators in response to incubation with A(RSV), P(C), or P(C+RSV) yielded higher concentrations upon infection with the latter and at earlier time points than the other PM(Mimics). In conclusion, this experimental platform provides an approach to study the combined effects of PM-viral interactions and airway epithelial exposures in the pathogenesis of respiratory diseases involving inhalation of environmental agents.

RATIONALE:Increased thickness of the airway smooth muscle (ASM) layer in asthma may result from hyperplasia or hypertrophy of muscle cells or increased extracellular matrix (ECM).OBJECTIVES:To relate ASM hypertrophy, ASM hyperplasia, and deposition of ECM to the severity and duration of asthma.METHODS:Airways from control subjects (n = 51) and from cases of nonfatal (n = 49) and fatal (n = 55) asthma were examined postmortem. Mean ASM cell volume (V(C)), the number of ASM cells per length of airway (N(L)), and the volume fraction of extracellular matrix (f(ECM)) within the ASM layer were estimated. Comparisons between subject groups were made on the basis of general linear regression models.MEASUREMENTS AND MAIN RESULTS:Mean V(C) was increased in the large airways of cases of nonfatal asthma (P = 0.015) and fatal asthma (P < 0.001) compared with control subjects. N(L) was similar in nonfatal cases and control subjects but increased in large (P < 0.001), medium (P < 0.001), and small (P = 0.034) airways of cases of fatal asthma compared with control subjects and with nonfatal cases (large and medium airways, P ≤ 0.003). The f(ECM) was similar in cases of asthma and control subjects. Duration of asthma was associated with a small increase in N(L).CONCLUSIONS:Hypertrophy of ASM cells occurs in the large airways in both nonfatal and fatal cases of asthma, but hyperplasia of ASM cells is present in the large and small airways in fatal asthma cases only. Both are associated with an absolute increase in ECM. Duration of asthma has little or no effect on ASM hypertrophy or hyperplasia or f(ECM).

A substantial proportion of healthcare cost associated with asthma is attributable to exacerbations of the disease. Within the airway, the epithelium forms the mucosal immune barrier, the first structural cell defense against common environmental insults such as respiratory syncytial virus (RSV) and particulate matter. We sought to characterize the phenotype of differentiated asthmatic-derived airway epithelial cultures and their intrinsic inflammatory responses to environmental challenges. Air-liquid interface (ALI) cultures were generated from asthmatic (n = 6) and nonasthmatic (n = 6) airway epithelial cells. Airway tissue and ALI cultures were analyzed by immunohistochemistry for cytokeratin-5, E-cadherin, Ki67, Muc5AC, NF-κB, the activation of p38, and apoptosis. ALI cultures were exposed to RSV (4 × 10(6) plaque forming unit/ml), particulate matter collected by Environmental Health Canada (EHC-93, 100 μg/ml), or mechanically wounded for 24, 48, and 96 hours and basolateral supernatants analyzed for inflammatory cytokines, using Luminex and ELISA. The airway epithelium in airway sections of patients with asthma as well as in vitro ALI cultures demonstrated a less differentiated epithelium, characterized by elevated numbers of basal cells marked by the expression of cytokeratin-5, increased phosphorylation of p38 mitogen-activated protein kinase, and less adherens junction protein E-cadherin. Transepithelial resistance was not different between asthmatic and nonasthmatic cultures. In response to infection with RSV, exposure to EHC-93, or mechanical wounding, asthmatic ALI cultures released greater concentrations of IL-6, IL-8, and granulocyte macrophage colony-stimulating factor, compared with nonasthmatic cultures (P < 0.05). This parallel ex vivo and in vitro study of the asthmatic epithelium demonstrates an intrinsically altered phenotype and aberrant inflammatory response to common environmental challenges, compared with nonasthmatic epithelium.

Purpose of review This review focuses on recent findings in relation to potential functional consequences of structural changes in the asthmatic airway. Recent findings Increases in smooth muscle mass have been shown to be an early finding in childhood asthma, related to clinical severity and predictive of greater airflow obstruction. Both hyperplasia and hypertrophy contribute to the increase in smooth muscle mass. A phenotypic shift in the epithelium of asthmatic airways related to stress and injury is suggested by recent data, with likely direct transformation of epithelial cells into mesenchymal cells. Fibrocyte in-migration from the vasculature may be an additional source of increased smooth muscle mass. The increased smooth muscle may contribute to neovascularization via vascular endothelial growth factor. Computed tomography studies continue to show some correlations between wall thickness and airway physiology. Exacerbations are predictive of greater lung function decline and hence remodeling. Summary On balance, recent evidence continues to show that structural changes contribute to asthma persistence, airflow obstruction, lung function decline, and clinical severity, though there is increased recognition of the heterogeneity of asthma and in some phenotypes inflammatory cell influx or vascular effects may be more important than structural effects.

PURPOSE: The utility of evaluation of the lipid content of induced macrophages (lipid macrophage index, LMI) in guiding treatment in patients with asthma or chronic cough (CC) has not been extensively evaluated. It has been suggested to indicate occult aspiration. We evaluated patient characteristics predictive of an elevated LMI in an extended cohort of patients with CC or asthma, hypothesizing if due to microaspiration LMI should be related to BMI and/or rhinosinusitis and a diagnosis of CC. If related to airway cell flux (macrophage ingestion of necrotic/ apoptotic cells) then LMI should be related to total sputum cells.

RATIONALE:Airway remodeling in asthma is associated with the accumulation of fibroblasts, the primary cell responsible for synthesis and secretion of extracellular matrix proteins. The process by which the number of fibroblasts increases in asthma is poorly understood, but epithelial-mesenchymal transition (EMT) may play a significant role.OBJECTIVES:To evaluate whether EMT occurs in primary airway epithelial cells (AECs), the mechanisms involved, and if this process is altered in asthmatic AECs.METHODS:AECs were obtained from subjects with asthma (n = 8) and normal subjects without asthma (n = 10). Monolayer and air-liquid interface-AEC (ALI-AEC) cultures were treated with transforming growth factor (TGF)-beta1 (10 ng/ml) for 72 hours and assayed for mesenchymal and epithelial markers using quantitative polymerase chain reaction, confocal microscopy, and immunoblot. The involvement of BMP-7, Smad3, and MAPK-mediated signaling were also evaluated.MEASUREMENTS AND MAIN RESULTS:TGF-beta1-induced EMT in AEC monolayers derived from subjects with asthma and normal donors. EMT was characterized by changes in cell morphology, increased expression of mesenchymal markers EDA-fibronectin, vimentin, alpha-smooth muscle actin, and collagen-1, and loss of epithelial markers E-cadherin and zonular occludin-1. Inhibition of TGF-beta1-induced signaling with Smad3-inhibiting siRNA or TGF-beta1-neutralizing antibodies prevented and reversed EMT, respectively, whereas BMP-7 had no effect. In ALI-AEC cultures derived from normal subjects, EMT was confined to basally situated cells, whereas in asthmatic ALI-AEC cultures EMT was widespread throughout the epithelium.CONCLUSIONS:TGF-beta1 induces EMT in a Smad3-dependent manner in primary AECs. However, in asthmatic-derived ALI-AEC cultures, the number of cells undergoing EMT is greater. These findings support the hypothesis that epithelial repair in asthmatic airways is dysregulated.