Mara S. Ludwig

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Differences in proteoglycan deposition in the airways of moderate and severe asthmatics.

Excess deposition of proteoglycans (PGs) has been described in the subepithelial layer of the asthmatic airway wall. However, less is known about deposition in the airway smooth muscle (ASM) layer, and whether the pattern of deposition is altered depending upon disease severity. Endobronchial biopsies were performed in patients with severe or moderate asthma (defined using American Thoracic Society criteria) and in control subjects. Biopsies were immunostained for the PGs biglycan, lumican, versican and decorin. PG deposition was measured in the subepithelial and ASM layers, the former by calculating the area of positive staining, and the latter by determining the percentage area stained using point counting. Immunostaining for PGs was prominent in biopsies from both moderate and severe asthmatics, compared with control subjects. While there was no difference in the amount of PG in the subepithelial layer between the two asthmatic groups, the percentage area of biglycan and lumican staining in the ASM layer was significantly greater in moderate versus severe asthmatics. Differences in the deposition of proteoglycans within the airway smooth muscle layer of moderate versus severe asthmatics potentially impact on the functional behaviour of the airway smooth muscle in these two groups of patients.

Airway remodeling: lessons from animal models.

Airway remodeling, an array of persistent tissue structural changes that occurs through a process of injury and dysregulated repair linked to airway chronic inflammation, is presently believed to largely account for the disease mechanisms of asthma. Increases in airway smooth muscle mass are probably the main mechanism causing airway hyperresponsiveness, and changes in the extracellular matrix may stimulate smooth muscle growth and contribute to the mechanics of airway obstruction. The various components of airway remodeling described in human asthma have been successfully reproduced in animal models of several species. Most of the data have been contributed by rat models of allergic sensitization and repeated challenge, transgenic mouse models of cytokine overexpression localized to the lung and, more recently, allergen-driven mouse models using wild-type inbred strains. Overall, animal models have provided significant insights into the mechanisms of airway remodeling and recent technological developments allow us to exploit these models in new directions. However, the challenge of finding new therapeutic strategies that prevent or control airway remodeling, thus providing etiopathogenically oriented treatments for asthma, still stands. Experimental airway remodeling in animals should be an essential tool for treatment discovery in the near future.

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.

Mechanical strain enhances proteoglycan message in fibroblasts from asthmatic subjects.

Background Remodelling of the asthmatic airway includes increased deposition of proteoglycan (PG) molecules. One of the stimuli driving airway remodelling may be excessive mechanical stimulation.

Psychologic Distress and Maladaptive Coping Styles in Patients With Severe vs Moderate Asthma

BACKGROUND Though several biologic factors have been suggested to play a role in the development and persistence of severe asthma, those associated with psychologic factors remain poorly understood. This study assessed levels of psychologic distress and a range of disease-relevant emotional and behavioral coping styles in patients with severe vs moderate asthma. METHODS Eighty-four patients (50% women, mean [M] age 46 years) with severe (n = 42) and moderate (n = 42) asthma were recruited. Severe asthma was defined according to American Thoracic Society criteria. Patients underwent demographic and medical history interviews and pulmonary function and allergy testing. Patients also completed questionnaires measuring asthma symptoms and the Millon Behavioral Medicine Diagnostic Inventory, which assesses psychologic distress and emotional/behavioral coping factors that influence disease progression and treatment. RESULTS After adjustment for covariates and applying a correction factor that reduced the significant P level to < .01, patients with severe vs moderate asthma reported experiencing more psychologic distress, including worse cognitive dysfunction (F = 6.72, P < .01) and marginally worse anxiety-tension (F = 4.02, P < .05). They also reported worse emotional coping (higher illness apprehension [F = 9.57, P < .01], pain sensitivity [F = 10.65, P < .01], future pessimism [F= 8.53, P < .01], and interventional fragility [F = 7.18, P < .01]), and marginally worse behavioral coping (more functional deficits [F = 5.48, P < .05] and problematic compliance [F = 4.32, P < .05]). CONCLUSIONS Patients with severe asthma have more psychologic distress and difficulty coping with their disease, both emotionally and behaviorally, relative to patients with moderate asthma. Future treatment studies should focus on helping patients with severe asthma manage distress and cope more effectively with their illness, which may improve outcomes in these high-risk patients.

A role for decorin in a murine model of allergen-induced asthma

Decorin (Dcn) is an extracellular matrix proteoglycan, which affects airway mechanics, airway-parenchymal interdependence, airway smooth muscle proliferation and apoptosis, and transforming growth factor-β bioavailability. As Dcn deposition is differentially altered in asthma, we questioned whether Dcn deficiency would impact the development of allergen-induced asthma in a mouse model. Dcn(-/-) and Dcn(+/+) mice (C57Bl/6) were sensitized with ovalbumin (OA) and challenged intranasally 3 days/wk × 3 wk. After OA challenge, mice were anesthetized, and respiratory mechanics measured under baseline conditions and after delivery of increasing concentrations of methacholine aerosol. Complex impedance was partitioned into airway resistance and tissue elastance and damping. Bronchoalveolar lavage was performed. Lungs were excised, and tissue sections evaluated for inflammatory cell influx, α-smooth muscle actin, collagen, biglycan, and Dcn deposition. Changes in TH-2 cytokine mRNA and protein were also measured. Airway resistance was increased in OA-challenged Dcn(+/+) mice only (P < 0.05), whereas tissue elastance and damping were increased in both OA-challenged Dcn(+/+) and Dcn(-/-), but more so in Dcn(+/+) mice (P < 0.001). Inflammation and collagen staining within the airway wall were increased with OA in Dcn(+/+) only (P < 0.001 and P < 0.01, respectively, vs. saline). IL-5 and IL-13 mRNA were increased in lung tissue of OA-challenged Dcn(+/+) mice. Dcn deficiency resulted in more modest OA-induced hyperresponsiveness, evident at the level of the central airways and distal lung. Differences in physiology were accompanied by differences in inflammation and remodeling. These findings may be, in part, due to the well-described ability of Dcn to bind transforming growth factor-β and render it less bioavailable.

Mechanical properties of mouse distal lung: in vivo versus in vitro comparison.

While measurements of lung tissue mechanics have been made in several species, relatively little has been reported in the mouse. Moreover, whether in vivo measurements truly reflect tissue properties is somewhat controversial. We measured complex impedance of the mouse respiratory system in vivo using a ventilator, which applies a multiple frequency volume signal to the airway opening. A constant phase model was fit to the impedance data, yielding parameters for tissue damping (G) and elastance (H). Hysteresivity (eta) was calculated as G/H. Quasistatic pressure-volume (P-V) curves were obtained during deflation. In vitro measurements of complex impedance and stress-strain curves were made in lung tissue strips. Values of eta were significantly higher in vivo than in vitro (0.111 +/- 0.004 versus 0.042 +/- 0.003). The higher values of eta in vivo may represent the effects of airway heterogeneities, surfactant, or changes in alveolar geometry. Measurement of mechanics in the tissue strip offers a better assessment of pure tissue properties.

Allergen-induced asthma in C57Bl/6 mice: hyper-responsiveness, inflammation and remodelling.

The relationship among airway responsiveness, inflammation and remodelling in asthma is incompletely understood. To investigate potential mechanistic associations, allergen-induced asthma was studied in C57Bl/6 mice. Mice were sensitized and challenged with ovalbumin (OVA) using sub-acute (SA) or chronic (C) protocols. Responsiveness was assessed by measuring respiratory impedence which was partitioned into airway resistance (Raw) and distal lung components (Gti, Hti) during methacholine-induced constriction. Inflammation, airway mucus, airway smooth muscle, collagen, biglycan and decorin were quantified. The airways were sub-divided into central or peripheral. In SA and C OVA, Raw, Gti and Hti responsiveness were significantly increased; the peripheral response was significantly greater in SA vs C OVA. Airway inflammation and mucus were increased in both groups, but more significantly in peripheral airways in SA OVA. In the SA OVA model, inflammation and mucus appear to drive the mechanical response, especially in the lung periphery; airway remodelling seems to contribute to hyper-responsiveness to an equivalent degree, after both challenge protocols.

Increased deposition of chondroitin/dermatan sulfate glycosaminoglycan and upregulation of β1,3-glucuronosyltransferase I in pulmonary fibrosis

Pulmonary fibrosis (PF) is characterized by increased deposition of proteoglycans (PGs), in particular core proteins. Glycosaminoglycans (GAGs) are key players in tissue repair and fibrosis, and we investigated whether PF is associated with changes in the expression and structure of GAGs as well as in the expression of β1,3-glucuronosyltransferase I (GlcAT-I), a rate-limiting enzyme in GAG synthesis. Lung biopsies from idiopathic pulmonary fibrosis (IPF) patients and lung tissue from a rat model of bleomycin (BLM)-induced PF were immunostained for chondroitin sulfated-GAGs and GlcAT-I expression. Alterations in disaccharide composition and sulfation of chondroitin/dermatan sulfate (CS/DS) were evaluated by fluorophore-assisted carbohydrate electrophoresis (FACE) in BLM rats. Lung fibroblasts isolated from control (saline-instilled) or BLM rat lungs were assessed for GAG structure and GlcAT-I expression. Disaccharide analysis showed that 4- and 6-sulfated disaccharides were increased in the lungs and lung fibroblasts obtained from fibrotic rats compared with controls. Fibrotic lung fibroblasts and transforming growth factor-β(1) (TGF-β(1))-treated normal lung fibroblasts expressed increased amounts of hyaluronan and 4- and 6-sulfated chondroitin, and neutralizing anti-TGF-β(1) antibody diminished the same. TGF-β(1) upregulated GlcAT-I and versican expression in lung fibroblasts, and signaling through TGF-β type I receptor/p38 MAPK was required for TGF-β(1)-mediated GlcAT-I and CS-GAG expression in fibroblasts. Our data show for the first time increased expression of CS-GAGs and GlcAT-I in IPF, fibrotic rat lungs, and fibrotic lung fibroblasts. These data suggest that alterations of sulfation isomers of CS/DS and upregulation of GlcAT-I contribute to the pathological PG-GAG accumulation in PF.