Airway remodeling in asthma amplifies heterogeneities in smooth muscle shortening causing hyperresponsiveness.

Although airway remodeling and inflammation in asthma can amplify the constriction response of a single airway, their influence on the structural changes in the whole airway network is unknown. We present a morphometric model of the human lung that incorporates cross-sectional wall areas corresponding to the adventitia, airway smooth muscle (ASM), and mucosa for healthy and mildly and severely asthmatic airways and the influence of parenchymal tethering. A heterogeneous ASM percent shortening stimulus is imposed, causing distinct constriction patterns for healthy and asthmatic airways. We calculate lung resistance and elastance from 0.1 to 5 Hz. We show that, for a given ASM stimulus, the distribution of wall area in asthmatic subjects will amplify not only the mean but the heterogeneity of constriction in the lung periphery. Moreover, heterogeneous ASM shortening that would produce only mild changes in the healthy lung can cause hyperresponsive changes in lung resistance and elastance at typical breathing rates in the asthmatic lung, even with relatively small increases in airway resistance. This condition arises when airway closures occur randomly in the lung periphery. We suggest that heterogeneity is a crucial determinant of hyperresponsiveness in asthma and that acute asthma is more a consequence of extensive airway wall inflammation and remodeling, predisposing the lung to produce an acute pattern of heterogeneous constriction.     

Tumstatin regulates the angiogenic and inflammatory potential of airway smooth muscle extracellular matrix

The extracellular matrix (ECM) creates the microenvironment of the tissue; an altered ECM in the asthmatic airway may be central in airway inflammation and remodelling. Tumstatin is a collagen IV‐derived matrikine reduced in the asthmatic airway wall that reverses airway inflammation and remodelling in small and large animal models of asthma. This study hypothesized that the mechanisms underlying the broad asthma‐resolving effects of tumstatin were due to autocrine remodelling of the ECM. Neutrophils and endothelial cells were seeded on decellularized ECM of non‐asthmatic (NA) or asthmatic (A) airway smooth muscle (ASM) cells previously exposed to tumstatin in the presence or absence of a broad matrix metalloproteinase inhibitor, Marimastat. Gene expression in NA and A ASM induced by tumstatin was assessed using RT‐PCR arrays. The presence of tumstatin during ECM deposition affected neutrophil and endothelial cell properties on both NA and A ASM‐derived matrices and this was only partly due to MMP activity. Gene expression patterns in response to tumstatin in NA and A ASM cells were different. Tumstatin may foster an anti‐inflammatory and anti‐angiogenic microenvironment by modifying ASM‐derived ECM. Further work is required to examine whether restoring tumstatin levels in the asthmatic airway represents a potential novel therapeutic approach.     

Differences in airway structure in immature and mature rabbits.

Our laboratory has previously demonstrated that maximal bronchoconstriction produces a greater degree of airway narrowing in immature than in mature rabbit lungs (33). To determine whether these maturational differences could be related to airway structure, we compared the fraction of the airway wall occupied by airway smooth muscle (ASM) and cartilage, the proportion of wall area internal to ASM, and the number of alveolar attachments to the airways, from mature and immature (6-mo- and 4-wk-old, respectively) rabbit lungs that were formalin fixed at total lung capacity. The results demonstrate that the airway walls of immature rabbits had a greater percentage of smooth muscle, a lower percentage of cartilage, and fewer alveolar attachments compared with mature rabbit airways; however, we did not find maturational differences in the airway wall thickness relative to airway size. We conclude that structural differences in the airway wall may contribute to the greater airway narrowing observed in immature rabbits during bronchoconstriction.     

Heterogeneous airway tone in asthmatic subjects.

We examined the effect of volume history on the dynamic relationship between airways and lung parenchyma (relative hysteresis) in 20 asthmatic subjects. The acoustic reflection technique was employed to evaluate changes in airway cross-sectional areas during a slow continuous expiration from total lung capacity to residual volume and inspiration back to total lung capacity. Lung volume was measured continuously during this quasi-static maneuver. We studied three anatomic airway segments: extra- and intrathoracic tracheal and main bronchial segments. Plots of airway area vs. lung volume were obtained for each segment to assess the relative magnitude and direction of the airway and parenchymal hysteresis. We also performed maximal expiratory flow-volume and partial expiratory flow-volume curves and calculated the ratio of maximal to partial flow rates (M/P) at 30% of the vital capacity. We found that 10 subjects (group I) showed a significant predominance of airway over parenchymal hysteresis (P < 0.005) at the extra- and intrathoracic tracheal and main bronchial segments; these subjects had high M/P ratios [1.53 +/- 0.27 (SD)]. The other 10 subjects (group II) showed similar airway and parenchymal hysteresis for all three segments and significantly lower M/P ratios (1.16 +/- 0.20, P < 0.01). We conclude that the effect of volume history on the relative hysteresis of airway and lung parenchyma and M/P ratio at 30% of vital capacity in nonprovoked asthmatic subjects is variable. We suggest that our findings may result from heterogeneous airway tone in asthmatic subjects.     

Stiffness of peripheral airway folding membrane in rabbits.

We have observed that small, membranous bronchioles from rabbits, in which the smooth muscle is not activated, experience a critical elastic buckling involving the whole airway wall during deflation of the lung. This implies that, at some point during the deflation, the airway wall goes from being in a state of tension to a state of compression. At the transition, there is neither net tension nor net compression in the wall, and the transmural pressure difference must, therefore, be zero. Thus at this point, the pressure difference across the muscle that results from the passive stress in the muscle is just balanced by the pressure difference across the folded mucosal membrane. We estimated the muscle stress, and hence the pressure across the muscle, from published data on rabbit trachealis (Opazo-Saez A and Paré PD, J Appl Physiol 77: 1638-1643, 1994) and equated this to the pressure across the folded membrane. By using a theoretical prediction of this pressure (Lambert RK, Codd SL, Alley MR, and Pack RJ, J Appl Physiol 77: 1206-1216, 1994), together with the results of our morphometric measurements on these airways, we estimated that the flexural rigidity of the folding membrane in peripheral rabbit airways is of the order of 10(-12) Pa x m3. This value implies that, in these airways, membrane folding provides significant resistance to airway smooth muscle shortening.     

Cellular biomechanics in the lung

Mechanical forces affect both the function and phenotype of cells in the lung. In this symposium, recent studies were presented that examined several aspects of biomechanics in lung cells and their relationship to disease. Wound healing and recovery from injury in the airways involve epithelial cell spreading and migration on a substrate that undergoes cyclic mechanical deformation; enhanced green fluorescent protein-actin was used in a stable cell line to examine cytoskeletal changes in airway epithelial cells during wound healing. Eosinophils migrate into the airways during asthmatic attacks and can also be exposed to cyclic mechanical deformation; cyclic mechanical stretch caused a decrease in leukotriene C(4) synthesis that may be dependent on mechanotransduction mechanisms involving the production of reactive oxygen species. Recent studies have suggested that proinflammatory cytokines are increased in ventilator-induced lung injury and may be elevated by overdistention of the lung tissue; microarray analysis of human lung epithelial cells demonstrated that cyclic mechanical stretch alone profoundly affects gene expression. Finally, airway hyperresponsiveness is a basic feature of asthma, but the relationship between airway hyperresponsiveness and changes in airway smooth muscle (ASM) function remain unclear. New analysis of the behavior of the ASM cytoskeleton (CSK) suggests, however, that the CSK may behave as a glassy material and that glassy behavior may account for the extensive ASM plasticity and remodeling that contribute to airway hyperresponsiveness. Together, the presentations at this symposium demonstrated the remarkable and varied roles that mechanical forces may play in both normal lung physiology as well as pathophysiology.     

Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Airway basement membrane perimeter distensibility and airway smooth muscle area in asthma.

The perimeter of the basement membrane (Pbm) of an airway viewed in cross section is used as a marker of airway size because in normal lungs it is relatively constant, despite variations in airway smooth muscle (ASM) shortening and airway collapse. In vitro studies (McParland BE, Pare PD, Johnson PR, Armour CL, Black JL. J Appl Physiol 97: 556-563, 2004; Noble PB, Sharma A, McFawn PK, Mitchell HW. J Appl Physiol 99: 2061-2066, 2005) have suggested that differential stretch of the Pbm between asthmatic and nonasthmatic airways fixed in inflation may occur and lead to an overestimation of ASM thickness in asthma. The relationships between the Pbm and the area of ASM were compared in transverse sections of airways from cases of fatal asthma (F) and from nonasthmatic control (C) cases where the lung tissue had been fixed inflated (Fi; Ci) or uninflated (Fu; Cu). When all available airways were used, the regression slopes were increased in Fu and Cu, compared with Fi and Ci, and increased in Fu and Fi, compared with Cu and Ci, suggesting effects of both inflation and asthma group, respectively. When analyses were limited to airway sizes that were available for all groups (Pbm < 15 mm), the slopes of Fi and Fu were similar, but both were greater than Ci and Cu, which were also similar. It was calculated that the effect of asthma group accounted for 80% and inflation for 20% of the differences between Fi and Ci. We conclude that the effects of inflation on the relationship between Pbm and ASM are small and do not account for the differences observed in ASM between cases of asthma and nonasthmatic controls.     

Angiogenic regulatory influence of extracellular matrix deposited by resting state asthmatic and non-asthmatic airway smooth muscle cells is similar

The extracellular matrix (ECM) is the tissue microenvironment that regulates the characteristics of stromal and systemic cells to control processes such as inflammation and angiogenesis. Despite ongoing anti-inflammatory treatment, low levels of inflammation exist in the airways in asthma, which alters ECM deposition by airway smooth muscle (ASM) cells. The altered ECM causes aberrant behaviour of cells, such as endothelial cells, in the airway tissue. We therefore sought to characterize the composition and angiogenic potential of the ECM deposited by asthmatic and non-asthmatic ASM. After 72 hours under non-stimulated conditions, the ECM deposited by primary human asthmatic ASM cells was equal in total protein, collagen I, III and fibronectin content to that from non-asthmatic ASM cells. Further, the matrices of non-asthmatic and asthmatic ASM cells were equivalent in regulating the growth, activity, attachment and migration of primary human umbilical vein endothelial cells (HUVECs). Under basal conditions, asthmatic and non-asthmatic ASM cells intrinsically deposit an ECM of equivalent composition and angiogenic potential. Previous findings indicate that dysregulation of the airway ECM is driven even by low levels of inflammatory provocation. This study suggests the need for more effective anti-inflammatory therapies in asthma to maintain the airway ECM and regulate ECM-mediated aberrant angiogenesis.     

Lung parenchymal shear modulus, airway wall remodeling, and bronchial hyperresponsiveness

Lambert, Rodney K., and Peter D. Paré. Lungparenchymal shear modulus, airway wall remodeling, and bronchialhyperresponsiveness. J. Appl. Physiol.83(1): 140-147, 1997.When airways narrow, either through theaction of smooth muscle shortening or during forced expiration, thelung parenchyma is locally distorted and provides an increasedperibronchial stress that resists the narrowing. Although thisinterdependence has been well studied, the quantitative significance ofairway remodeling to interdependence has not been elucidated. We haveused an improved computational model of the bronchial response tosmooth muscle agonists to investigate the relationships between airwaynarrowing (as indicated by airway resistance), parenchymal shearmodulus, adventitial thickening, and inner wall thickening at lungrecoil pressures of 4, 5, and 8 cmH₂O. We have found that, at lowrecoil pressures, decreases in parenchymal shear modulus have asignificant effect that is comparable to that of moderate thickening ofthe airway wall. At higher lung recoil pressures, the effect isnegligible.

     

Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms.

The development of severe childhood asthma may be influenced by several factors including environmental and infectious stimuli. The causal relationship between infectious viral responses, such as respiratory syncytial virus (RSV), and severe asthma during early childhood is unclear. In these studies, the ability for an initial RSV infection to exacerbate and promote a more severe asthmatic-type response was investigated by combining established murine models of disease. We examined the ability of RSV to induce exacerbation of allergic disease over a relatively long period, leading to development of severe airway responses including airway inflammation and hyperreactivity. The preferential production of IL-13 during a primary RSV infection appears to play a critical role for the exacerbation of cockroach allergen-induced disease. The depletion of IL-13 during RSV infections inhibited the exacerbation and acceleration of severe allergen-induced airway hyperreactivity. This was indicated by decreases in airway hyperreactivity and changes in lung chemokine production. These data suggest that the airway responses during asthma can be greatly affected by a previous RSV infection, even when infection occurs before allergen sensitization. Overall, infection of the airways with RSV can induce an IL-13-dependent change in airway function and promotes an environment that contributes to the development of severe allergic asthmatic responses.     

Effects of decorin and biglycan on human airway smooth muscle cell proliferation and apoptosis

Proteoglycans (PG) are altered in the asthmatic airway wall. Because PGs are known to affect cell proliferation and apoptosis, we hypothesized that alterations in PG might influence the airway smooth muscle (ASM) hyperplasia observed in the asthmatic airway. Human ASM cells were seeded on plastic or plates coated with decorin (Dcn), biglycan (Bgn), or collagen type I (Col I) (1, 3, and 10 microg/ml). Cells were stimulated with platelet-derived growth factor (PDGF), and cell number was assessed at 0, 48, and 96 h. Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation and apoptosis by annexin V and propidium iodide staining at 48 h post-PDGF stimulation. A significant decrease in cell number was observed with cells seeded on Dcn (10 microg/ml) at 0, 48, and 96 h (P < 0.01). Dcn induced both decreases in BrdU incorporation and increases in annexin V staining (P < 0.05). Bgn decreased cell number at time 0 only (P < 0.05) and affected neither proliferation nor apoptosis. Col I (10 mug/ml) caused a significant increase in cell number at 48 and 96 h (P < 0.01). Adding exogenous Dcn (1-30 microg/ml) to the medium had no effect on cell number. Exposing Dcn-coated matrices to chondroitinase ABC, an enzyme that degrades glycosaminoglycan side chains, reversed the Dcn-induced decrease in cell number. These studies demonstrate that different PGs have variable effects on ASM cell proliferation and apoptosis. Recently described decreases in Dcn in the asthmatic airway wall could potentially permit more exuberant ASM growth.     

Dual ERK and phosphatidylinositol 3-kinase pathways control airway smooth muscle proliferation: differences in asthma

Hyperplasia of airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely due to increased muscle proliferation. We have shown that ASM cells obtained from asthmatic patients proliferate faster than those obtained from non-asthmatic patients. In ASM from non-asthmatics, mitogens act via dual signaling pathways (both ERK- and PI 3-kinase-dependent) to control growth. In this study we are the first to examine whether dual pathways control the enhanced proliferation of ASM from asthmatics. When cells were incubated with 0.1% or 1% FBS, ERK activation was significantly greater in cells from asthmatic subjects (P < 0.05). In contrast, when cells were stimulated with 10% FBS, ERK activity was significantly greater in the non-asthmatic cells. However, cell proliferation in asthmatic cells was still significantly higher in cells stimulated by both 1% and 10% FBS. Pharmacological inhibition revealed that although dual proliferative pathways control ASM growth in cells from non-asthmatics stimulated with 10% FBS to an equal extent ([(3)H]-thymidine incorporation reduced to 57.2 +/- 6.9% by the PI 3-kinase inhibitor LY294002 and 57.8 +/- 1.1% by the ERK-pathway inhibitor U0126); in asthmatics, the presence of a strong proliferative stimulus (10% FBS) reduces ERK activation resulting in a shift to the PI 3-kinase pathway. The underlying mechanism appears to be upregulation of an endogenous MAPK inhibitor--MKP-1--that constrains ERK signaling in asthmatic cells under strong mitogenic stimulation. This study suggests that the PI 3-kinase pathway may be an attractive target for reversing hyperplasia in asthma.     

PAR-2 activation,PGE2, and COX-2 in human asthmatic and nonasthmatic airway smooth muscle cells

The protease-activated receptor-2 (PAR-2) is present on human airway smooth muscle (ASM) cells and can be activated by mast cell tryptase, trypsin, or an activating peptide (AP). Trypsin induced significant increases in PGE2 release from human ASM cells after 6 and 24 h and also induced cyclooxygenase (COX)-2 mRNA expression and COX-2 protein. Tryptase and the PAR-2 AP did not alter PGE2 release or COX-2 protein levels, suggesting a lack of PAR-2 involvement. When we compared results in asthmatic and nonasthmatic muscle cells, both trypsin and bradykinin induced less PGE2 from asthmatic ASM cells, and bradykinin induced significantly less COX-2 mRNA in asthmatic cells. Significantly less PGE2 was released from proliferating ASM cells from asthmatic patients. In conclusion, trypsin induces PGE2 release and COX-2 in human ASM cells, which is unlikely to be via PAR-2 activation. In addition, ASM cells from asthmatic patients produce significantly less PGE2 and COX-2 compared with nonasthmatic cells. These findings may contribute to the increase in muscle mass evident in asthmatic airways.     

Parenchymal interdependence and airway response to methacholine in excised dog lobes

The objective of this investigation was to determine the minimum transpulmonary pressure (PL) at which the forces of interdependence between the airways and the lung parenchyma can prevent airway closure in response to maximal stimulation of the airways in excised canine lobes. We first present an analysis of the relationship between PL and the transmural pressure (Ptm) that airway smooth muscle must generate to close the airways. This analysis predicts that airway closure can occur at PL less than or equal to 10 cmH2O with maximal airway stimulation. We tested this prediction in eight excised canine lobes by nebulizing 50% methacholine into the airways while the lobe was held at constant PL values ranging from 25 to 5 cmH2O. Airway closure was assessed by comparing changes in alveolar pressure (measured by an alveolar capsule technique) and pressure at the airway opening during low-amplitude oscillations in lobar volume. Airway closure occurred in two of the eight lobes at PL = 10 cmH2O; in an additional five it occurred at PL = 7.5 cmH2O. We conclude that the forces of parenchymal interdependence per se are not sufficient to prevent airway closure at PL less than or equal to 7.5 cmH2O in excised canine lobes.     

Small airway pressure-diameter relationships during nonhomogeneous lung lobe inflation

The pressure-diameter behavior of airways within a collaterally ventilating segment of lung was evaluated radiographically in 12 excised dog lung lobes. The results were compared with the pressure-diameter behavior of airways in a lung region adjacent to the collaterally ventilating segment. Airways in each lung region were dusted with powdered tantalum, and airway diameters were measured during homogeneous and nonhomogeneous lobe inflation. Intrasegmental and extrasegmental airways behaved similarly during homogeneous lobe inflation; airway diameter increased as alveolar pressure increased. The lobe was inflated nonhomogeneously by raising pressure in the collaterally ventilating segment (Ps) while maintaining pressure at the lobar bronchus (Pao) constant at 5, 10, or 15 cmH2O. Increasing Ps at constant Pao reciprocally affected intrasegmental and extrasegmental airways. When Pao was low, intrasegmental airways were expanded, and extrasegmental airways were compressed when Ps was raised. When Pao was high, airway diameter was unaffected by increasing Ps presumably because the airways were already maximally expanded. A comparison of diameters during homogenous and nonhomogenous lobe inflation suggests a very small interdependence effect from the parenchyma surrounding the collaterally ventilating segment. These results demonstrate the combined effects of parenchymal properties and airway pressure-diameter relationships in determining the effect of local lung distortion on airway function.     

Airway closure on imaging relates to airway hyperresponsiveness and peripheral airway disease in asthma

The regional pattern and extent of airway closure measured by three-dimensional ventilation imaging may relate to airway hyperresponsiveness (AHR) and peripheral airways disease in asthmatic subjects. We hypothesized that asthmatic airways are predisposed to closure during bronchoconstriction in the presence of ventilation heterogeneity and AHR. Fourteen asthmatic subjects (6 women) underwent combined ventilation single photon emission computed tomography/computed tomography scans before and after methacholine challenge. Regional airway closure was determined by complete loss of ventilation following methacholine challenge. Peripheral airway disease was measured by multiple-breath nitrogen washout from which S(cond) (index of peripheral conductive airway abnormality) was derived. Relationships between airway closure and lung function were examined by multiple-linear regression. Forced expiratory volume in 1 s was 87.5 ± 15.8% predicted, and seven subjects had AHR. Methacholine challenge decreased forced expiratory volume in 1 s by 23 ± 5% and increased nonventilated volume from 16 ± 4 to 29 ± 13% of computed tomography lung volume. The increase in airway closure measured by nonventilated volume correlated independently with both S(cond) (partial R(2) = 0.22) and with AHR (partial R(2) = 0.38). The extent of airway closure induced by methacholine inhalation in asthmatic subjects is greater with increasing peripheral airways disease, as measured by ventilation heterogeneity, and with worse AHR.     

Understanding airway pathophysiology with computed tomograpy.

Conventional pulmonary function tests are limited in the mechanistic insight that they can provide by the fact that they can only provide average measures of lung function. For example, a measurement of decreased expiratory flow assessed with conventional spirometry could result from narrowed large airways, narrowed small airways, closed airways, altered elasticity, or regional heterogeneities in parenchyma or airways. To examine specific mechanisms and pathology in the airways, a method is required that can actually look at specific individual airways. Over the past decade, several more direct methods of assessing specific mechanisms and structural alterations in normal airways and airway pathology in asthma have become available for such purposes. One such method is high-resolution computed tomography (HRCT), a method that allows the study of multiple individual airways during either contraction to closure or relaxation in real time, as well as changes in airway size with changes in lung volume. Although other imaging modalities have the potential to image airways in vivo, none presently has the convenience and the accessibility coupled with the resolution required to visualize the parenchymal airways in vivo. Although HRCT may never be widely utilized for routine measurements or screening, because of radiation exposure, cost issues, and a limited ability to follow changes over extended time periods, the method has distinct and unique advantages in quantifying the behavior of airways in vivo. In this mini-review, we focus on these capabilities of HRCT by briefly reviewing highlights of experimental results from several canine and human studies.     

Signaling and regulation of G protein-coupled receptors in airway smooth muscle

Signaling through G protein-coupled receptors (GPCRs) mediates numerous airway smooth muscle (ASM) functions including contraction, growth, and "synthetic" functions that orchestrate airway inflammation and promote remodeling of airway architecture. In this review we provide a comprehensive overview of the GPCRs that have been identified in ASM cells, and discuss the extent to which signaling via these GPCRs has been characterized and linked to distinct ASM functions. In addition, we examine the role of GPCR signaling and its regulation in asthma and asthma treatment, and suggest an integrative model whereby an imbalance of GPCR-derived signals in ASM cells contributes to the asthmatic state.