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.

Increased thickness of the airway smooth muscle (ASM) layer is a characteristic feature of airway remodelling in both asthma and chronic obstructive pulmonary disease (COPD) [1, 2]. The composition of the ASM layer has been well studied in asthma where the increased thickness of the ASM layer is due to hypertrophy and/or hyperplasia [3–5] of ASM cells. These changes are associated with a concomitant increase in absolute volumes of both ASM and extracellular matrix (ECM), without changes in their relative volume fractions [5]. Although ASM cell hypertrophy has been reported in the large airways of patients with COPD [6], these findings did not exclude the effect of an increase in the volume fraction of ECM on the estimated average volume of the ASM cells [7]. Remodelling of the layer of airway smooth muscle in COPD is characterised by an increase in the extracellular matrix http://ow.ly/YZqhz

Background: Protective effects of deep inspiration (DI) fail in chronic obstructive pulmonary disease (COPD). It is unknown if this reflects abnormal properties of the airway wall. Aim: To determine if the response of the airway wall to stretch is abnormal in COPD. Methods: Subjects undergoing lung surgery (65±3y) had lung function assessed before surgery and a bronchial segment was dissected from resected lung tissue for in vitro study. Subjects with COPD had a post-bronchodilator FEV1/FVC ratio and FEV1 below lower limit of normal. Response to DI was assessed from maximal and partial (M/P) flow ratio. In bronchial segments, bronchodilation to simulated DI was measured from the change in airway volume after a transient stretch. Wall dimensions were measured on fixed airways. Results: The M/P ratio positively correlated with FEV1/FVC (%predicted, p=0.002, r=0.72) and was reduced from 0.87±0.14 (mean±SE) in the control group (n=9) to 0.50±0.08 in the COPD group (n=7, P=0.047). An M/P ratio of <1 indicated that the dominant effect of DI was bronchoconstriction, and the lower ratio in the COPD group reflected greater constriction. Bronchodilation (%volume) to DI in vitro positively correlated with M/P ratio in vivo (p=0.03, r=0.53). However the magnitude of bronchodilation to DI was not different between groups (p=0.42). Airway wall thickness was not related to M/P ratio (p=0.61) or bronchodilation (p=0.96) and was not different between groups (p=0.81). Conclusion: A DI produces bronchoconstriction in subjects not undergoing bronchial challenge, which is greater in subjects with COPD. While the intrinsic response of the airway wall to stretch contributes to the respiratory effect of DI, it is not different in subjects with COPD.

COPD is defined as airflow limitation that is not reversed by treatment. In asthma, airflow limitation is not only reversible, but also inducible. This is called 'airway hyperresponsiveness' (AHR) and is associated with thickening of the airway wall, predominantly the layer of airway smooth muscle, due to more cells, bigger cells and more extracellular matrix (ECM) in proportion to the increase in smooth muscle. AHR is also observed in COPD if the changes in airflow are expressed as a percent of the baseline lung function. However, the absolute change in baseline lung function that can be induced in COPD is actually less than that seen in normal subjects, suggesting that the airways in COPD are resistant not only to opening, but also to closing. This observation agrees with physiological measures showing increased airway wall stiffness in COPD. Like asthma, airway wall thickness is increased in COPD, including the layer of smooth muscle. Unlike asthma, however, fixed airflow obstruction appears to be characterized by a disproportionate increase in the ECM within the smooth muscle layer. In this review, we summarize the studies of airway matrix deposition in COPD and put forward the proposal that the airway remodelling in COPD is different from that in asthma and call for a systematic analysis of airway matrix deposition in COPD.

ABSTRACT COPD is defined as airflow limitation that is not reversed by treatment. In asthma, airflow limitation is not only reversible, but also inducible. This is called ‘airway hyperresponsiveness’ ( AHR ) and is associated with thickening of the airway wall, predominantly the layer of airway smooth muscle, due to more cells, bigger cells and more extracellular matrix ( ECM ) in proportion to the increase in smooth muscle. AHR is also observed in COPD if the changes in airflow are expressed as a percent of the baseline lung function. However, the absolute change in baseline lung function that can be induced in COPD is actually less than that seen in normal subjects, suggesting that the airways in COPD are resistant not only to opening, but also to closing. This observation agrees with physiological measures showing increased airway wall stiffness in COPD . Like asthma, airway wall thickness is increased in COPD , including the layer of smooth muscle. Unlike asthma, however, fixed airflow obstruction appears to be characterized by a disproportionate increase in the ECM within the smooth muscle layer. In this review, we summarize the studies of airway matrix deposition in COPD and put forward the proposal that the airway remodelling in COPD is different from that in asthma and call for a systematic analysis of airway matrix deposition in COPD .

ABSTRACT COPD is defined as airflow limitation that is not reversed by treatment. In asthma, airflow limitation is not only reversible, but also inducible. This is called ‘airway hyperresponsiveness’ (AHR) and is associated with thickening of the airway wall, predominantly the layer of airway smooth muscle, due to more cells, bigger cells and more extracellular matrix (ECM) in proportion to the increase in smooth muscle. AHR is also observed in COPD if the changes in airflow are expressed as a percent of the baseline lung function. However, the absolute change in baseline lung function that can be induced in COPD is actually less than that seen in normal subjects, suggesting that the airways in COPD are resistant not only to opening, but also to closing. This observation agrees with physiological measures showing increased airway wall stiffness in COPD. Like asthma, airway wall thickness is increased in COPD, including the layer of smooth muscle. Unlike asthma, however, fixed airflow obstruction appears to be characterized by a disproportionate increase in the ECM within the smooth muscle layer. In this review, we summarize the studies of airway matrix deposition in COPD and put forward the proposal that the airway remodelling in COPD is different from that in asthma and call for a systematic analysis of airway matrix deposition in COPD.

Introduction/Aim: In patients with asthma, the airway smooth muscle (ASM) layer is thicker and is stable with age. Within the ASM layer, extracellular matrix (ECM) represents less than 20% of its total volume fraction (V V ). Departure from normal development of the ASM layer may alter V V ECM and result in airway hyperresponsiveness and asthma or contribute to fixed airflow obstruction later in life. The aim of this study was to compare the volume fractions of ECM, ASM and “Other” (space between ASM cells) within the ASM layer from infancy to adulthood in subjects with (n=84, age range 4-59yrs) and without (n=59, 0.2-57yrs) asthma. Methods: On transverse airways taken from formalin-fixed, post-mortem lungs, sections were cut from paraffin-embedded tissue blocks at 0.5microns and stained using the Masson9s trichrome technique. Volume fractions of ECM, ASM and Other within the ASM layer were measured (x1000) by point counting and then related to age in cases with and without asthma. Results: Cases of asthma had decreased V V ECM (17±5%, mean ± SD) compared with control subjects (19±4%, p=0.02) and no difference in V V ASM or V V Other. V V ECM and V V ASM positively correlated with age in control subjects (r 2 =0.13, p=0.005 and r 2 =0.11, p=0.01), but not in cases of asthma (r 2 =0.04, p=0.07 and r 2 =0.03, p=0.1). V V ASM and V V Other did not correlate with age. Discussion: These data suggest that the ASM layer normally undergoes structural change from infancy to adulthood and this involves changes in V V ECM. However in asthma, the ASM layer may not follow the same developmental trajectory as control subjects, possibly due to the confounding effect of early structural changes in the layer of ASM.

When comparing the pathology of airways in individuals with and without asthma, the perimeter of the basement membrane (Pbm) is used as a marker of airway size, as it is independent of airway smooth muscle shortening or airway collapse. The extent to which the Pbm is itself altered in asthma has not been quantified. The aim of this study was to compare the Pbm from the same anatomical sites in postmortem lungs from subjects with ( n = 55) and without ( n = 30) asthma (nonfatal or fatal). Large and small airways were systematically sampled at equidistant “levels” from the apical segment of the left upper lobes and anterior and basal segments of the left lower lobes of lungs fixed in inflation. The length of the Pbm was estimated from cross sections of airway at each relative level. Linear mixed models were used to investigate the relationships between Pbm and sex, age, height, smoking status, airway level, and asthma group. The final model showed significant interactions between Pbm and airway level in small (<3 mm) airways, in subjects having asthma ( P < 0.0001), and by sex ( P < 0.0001). No significant interactions for Pbm between asthma groups were observed for larger airways (equivalent to a diameter of ∼3 mm and greater) or smoking status. Asthma is not associated with remodeling of the Pbm in large airways. In medium and small airways, the decrease in Pbm in asthma (≤20%) would not account for the published differences in wall area or area of smooth muscle observed in cases of severe asthma.

Reduced FEV1 in smokers may be due to the combined effects of pre-existing airway remodeling and/or progressive loss of lung function (due to emphysema or airway remodeling). We have previously observed an inverse relationship between FEV1 and the volume fraction of extracellular matrix (VVECM) in the layer of airway smooth muscle (ASM). Given the increased decline in FEV1 in smokers, we hypothesized that this relationship reflects progressive remodeling of the ASM in smokers. Aim: To examine the relationship between smoke exposure and VVECM within the ASM layer in smokers and non-smokers undergoing lung resection. Methods: Prior to surgery, FEV1 was measured and smoking history was recorded. Post-operative tissue was obtained from current smokers (n=10), ex-smokers (n=20) and never smokers (n=9). Transverse sections (0.5μm thick) of large and small airway were stained with the Masson9s trichrome technique. VVECM within the ASM layer was estimated and related to age, duration of smoking and number cigarettes smoked per week. Results: Mean pack-years of smoking was 49±39 (range 4-160). VVECM was increased in current (p=0.001) and ex-smokers (p=0.016) compared with non-smokers in the large airways. VVECM was not related to pack years (r2=0.036, p=0.38), duration of smoking (r2=0.035, p=0.41), number of cigarettes smoked (r2=0.008, p=0.69) or age (r2=0.007, p=0.68) in large or small airways. Conclusion: Although increase VVECM in the ASM layer is related to FEV1 and current smoking, we could not detect a dose effect of smoking. This suggests a non-progressive effect of smoking and the presence of pre-existing remodeling of the ASM layer or a threshold effect of cigarette smoking.

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.

The present study presents preliminary findings on how structural/functional abnormalities of the airway wall relate to excessive airway narrowing and reduced bronchodilatory response to deep inspiration (DI) in subjects with a history of asthma. Bronchial segments were acquired from subjects undergoing surgery, mostly to remove pulmonary neoplasms. Subjects reported prior doctor-diagnosed asthma (n = 5) or had no history of asthma (n = 8). In vitro airway narrowing in response to acetylcholine was assessed to determine maximal bronchoconstriction and sensitivity, under static conditions and during simulated tidal and DI maneuvers. Fixed airway segments were sectioned for measurement of airway wall dimensions, particularly the airway smooth muscle (ASM) layer. Airways from subjects with a history of asthma had increased ASM (P = 0.014), greater maximal airway narrowing under static conditions (P = 0.003), but no change in sensitivity. Maximal airway narrowing was positively correlated with the area of the ASM layer (r = 0.58, P = 0.039). In tidally oscillating airways, DI produced bronchodilation in airways from the control group (P = 0.0001) and the group with a history of asthma (P = 0.001). While bronchodilation to DI was reduced with increased airway narrowing (P = 0.02; r = -0.64)), when the level of airway narrowing was matched, there was no difference in magnitude of bronchodilation to DI between groups. Results suggest that greater ASM mass in asthma contributes to exaggerated airway narrowing in vivo. In comparison, the airway wall in asthma may have a normal response to mechanical stretch during DI. We propose that increased maximal airway narrowing and the reduced bronchodilatory response to DI in asthma are independent.

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.

Hypertrophy and hyperplasia of airway smooth muscle (ASM) cells are features of asthma that can be assessed in airway transverse sections using stereologically derived parameters. However, little is known about the variability of these parameters within and between airways. The aim of the present study was to assess sources of variation in the measurement of the area of the ASM layer (A(ASM)), and the volume fraction of ASM cells (V-VASM) and numerical density of ASM cells within that layer. A(ASM) increased by up to 12% in 4-mu m sections, and 28% in 30-mu m sections, compared with 0.5-mu m sections. A(ASM) was greater (P < 0.05) in large than in small airways, and varied by up to 28% along segments of large airways. Numerical density of ASM cell estimates around the airway circumference varied by less than 10% if 40 random high-power fields were sampled. V-VASM was most accurately estimated on 0.5-mu m, rather than 4- or 30-mu m sections, and was less (P < 0.05) in large than in small airways. The coefficients of variation for V-VASM were less than 10% along airway segments. We found that variation of parameters used to estimate ASM cell number or size could be minimized with adequate sampling frequency around or along airway segments. Section thickness was positively related to the measured area of ASM on transverse airway sections. Thin (0.5-mu m) sections should be used to estimate tissue volume fractions, which vary little within and between airways of similar size. Airway size contributes most to the variation in estimating parameters of the ASM layer.

Background Cigarette smoke exposure is likely to account for differences in pathophysiology in chronic obstructive pulmonary disease (COPD) and asthma. Aim To examine the relationship between smoking and the mechanical properties and structure of the airway smooth muscle (ASM) layer in human airways. Methods Bronchial segments were obtained from subjects (n=12, 66±6yr) undergoing lobectomy for pulmonary neoplasms. Bronchoconstriction (% volume change) to acetylcholine, airway compliance and pressure to collapse were measured in vitro. The area and volume fractions (V V ) of muscle and extracellular matrix (ECM) in the ASM layer were subsequently assessed in the fixed segments. Results Pack-years of smoking were inversely related to airway compliance (p V ECM within the ASM layer (p V muscle or the size or number of ASM cells. Conclusions The fraction of ECM within the ASM layer, rather than changes in ASM cells, is altered by smoking and this restricts radial movement. This may contribute to the functional differences between COPD and asthma. Supported by: NHMRC Project Grant #513842 Conflict of interest: No.

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).

In healthy individuals, deep inspiration produces bronchodilation and reduced airway responsiveness, which may be a response of the airway wall to mechanical stretch. The aim of this study was to examine the in vitro response of isolated human airways to the dynamic mechanical stretch associated with normal breathing. Human bronchial segments (n = 6) were acquired from patients without airflow obstruction undergoing lung resection for pulmonary neoplasms. The side branches were ligated and the airways were mounted in an organ bath chamber. Airway narrowing to cumulative concentrations of acetylcholine (3 × 10(-6) M to 3 × 10(-3) M) was measured under static conditions and in the presence of "tidal" oscillations with intermittent "deep inspiration." Respiratory maneuvers were simulated by varying transmural pressure using a motor-controlled syringe pump (tidal 5 to 10 cmH(2)O at 0.25 Hz, deep inspiration 5 to 30 cmH(2)O). Airway narrowing was determined from decreases in lumen volume. Tidal oscillation had no effect on airway responses to acetylcholine which was similar to those under static conditions. Deep inspiration in tidally oscillating, acetylcholine-contracted airways produced potent, transient (<1 min) bronchodilation, ranging from full reversal in airway narrowing at low acetylcholine concentrations to ∼50% reversal at the highest concentration. This resulted in a temporary reduction in maximal airway response (P < 0.001), without a change in sensitivity to acetylcholine. Our findings are that the mechanical stretch of human airways produced by physiological transmural pressures generated during deep inspiration produces bronchodilation and a transient reduction in airway responsiveness, which can explain the beneficial effects of deep inspiration in bronchial provocation testing in vivo.