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Background and objective Decorin ( Dcn), an extracellular matrix proteoglycan, has several important biological functions, and its deposition is altered in the airway wall of humans with asthma and animal models of asthma. Due to its high affinity for transforming growth factor beta ( TGF)-β, Dcn can function as part of a negative feedback mechanism, resulting in the regulation of this factor's bioavailability. Dcn deficient (Dcn−/−) mice develop reduced airway inflammation, hyperresponsiveness and remodeling in response to repeated allergen challenge; we investigated whether regulatory T cells play a role in the diminished airway response of Dcn−/− mice. Methods Dcn−/− and Dcn+/+ mice ( C57 Bl/6) were sensitized with ovalbumin ( OVA) and challenged intra-nasally 3 days/week × 3 weeks. After allergen challenge, bronchoalveolar lavage was collected to quantify total and differential cell counts and cytokine levels. Inflammatory cell number and cytokine messenger ribonucleic acid ( mRNA) production were assessed in lung tissues. Cells from lung and spleen were extracted to evaluate regulatory T cells. Results Tissue inflammation and interleukin ( IL)-13 mRNA expression were significantly increased in OVA-challenged Dcn+/+ mice, only. The increased expression of Foxp3 in CD4+ CD25+ T cells found in lung of OVA-challenged Dcn−/− mice was accompanied by an increase in IL-10 mRNA. Conclusions Our data demonstrated that a diminished lung inflammation in OVA challenged Dcn−/− mice was accompanied by a higher expression of regulatory T cells and IL-10 mRNA levels. These results reinforce the importance of Dcn in biological processes, particularly in an allergic model of asthma. [ABSTRACT FROM AUTHOR]

Background: Epithelial-to-mesenchymal transition (EMT), which involves changes in cellular morphology of highly polarized epithelial cells and the gain of mesenchymal cell phenotype with migratory and invasive capacities, is implicated in smoking-related chronic obstructive pulmonary disease (COPD). However, the interactions of fibroblasts and epithelial cells and the participation of fibroblasts in the EMT processes in COPD are poorly understood. Here, we investigated the hypothesis that EMT is active in human bronchial epithelial (HBE) cells of COPD patients, and that mediators secreted by lung fibroblasts from COPD patients induce EMT. Methods: Primary HBE cells from normal subjects and COPD patients were purchased from LONZA. HLFs were derived from resected lung obtained from normal (N) and COPD (D) subjects and their conditioned medium (CM) was collected after 2-day culture in serum-free medium. The expression of epithelial and mesenchymal markers as well as EMT-related transcription factors in lung biopsies, and in HBE cells following stimulation with CM from both normal human lung fibroblasts (NHLF) and COPD human lung fibroblasts (DHLF) was evaluated by immunohistochemistry, qRT-PCR and western blot. Results: Basal mRNA expression of mesenchymal markers and EMT-related transcription factors were increased in DHBE cells compared to normal human bronchial epithelial cells (NHBE) cells as well as in COPD lungs. CM from NHLF significantly induced vimentin expression in both NHBE and COPD human bronchial epithelial cells (DHBE) cells, but only increased N-cadherin expression in DHBE cells. CM from NHLF significantly induced Twist1 and Twist2 expression in NHBE cells and increased Snai2 (Slug) expression in DHBE cells. While CM from NHLF had no effect on such EMT markers, CM from DHLF significantly increased the protein expression of E-cadherin and vimentin in NHBE cells compared to control. N-cadherin expression was upregulated to a greater degree in NHBE cells than DHBE cells. Only CM from DHLF significantly increased E-/N-cadherin ratio in DHBE cells. Conclusions: Our results suggest that DHBE cells have partially undergone EMT under baseline conditions. DHLF-CM promoted EMT in NHBE, suggesting that interactions between fibroblast and epithelial cells may play an important role in the EMT process in COPD. [ABSTRACT FROM AUTHOR]

Changes in proteoglycans and lung tissue mechanics during excessive mechanical ventilation in rats. Am J Physiol Lung Cell Mol Physiol 281: L1078-L1087, 2001.--Excessive mechanical ventilation results in changes in lung tissue mechanics. We hypothesized that changes in tissue properties might be related to changes in the extracellular matrix component proteoglycans (PGs). The effect of different ventilation regimens on lung tissue mechanics and PGs was examined in an in vivo rat model. Animals were anesthetized, tracheostomized, and ventilated at a tidal volume of 8 (V[T.sub.8]), 20, or 30 (V[T.sub.30]) ml/kg, positive end-expiratory pressure of 0 (PEE[P.sub.0]) or 1.5 (PEE[P.sub.1.5]) cm[H.sub.2]O, and frequency of 1.5 Hz for 2 h. The constant-phase model was used to derive airway resistance, tissue elastance, and tissue damping. After physiological measurements, one lung was frozen for immunohistochemistry and the other was reserved for PG extraction and Western blotting. After 2 h of mechanical ventilation, tissue elastance and damping were significantly increased in rats ventilated at V[T.sub.30]PEE[P.sub.0] compared with control rats (ventilated at V[T.sub.8]PEE[P.sub.1.5]). Versican, basement membrane heparan sulfate PG, and biglycan were all increased in rat lungs ventilated at V[T.sub.30]PEE[P.sub.0] compared with control rats. At V[T.sub.30]PEE[P.sub.0], heparan sulfate PG and versican staining became prominent in the alveolar wall and airspace; biglycan was mostly localized in the airway wall. These data demonstrate that alterations in lung tissue mechanics with excessive mechanical ventilation are accompanied by changes in all classes of extracellular matrix PG. tissue resistance; viscoelasticity; extracellular matrix

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. [ABSTRACT FROM AUTHOR]

Examines the proteoglycan expression in bleomycin (BM) lung fibroblasts. Role of BM in transforming growth factor-Β[sub 1] and interferon-γ promoting fibrosis; Interactions of macromolecules with biological functions; Establishment of animal model to study human pulmonary fibrosis.

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.

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.

Proteoglycans (PG) have important effects on the mechanical properties of tissues and the phenotype of various structural cells. Little is known about changes in PG deposition in the airways in animal models of asthma. We studied changes in PG in the airway wall of Brown Norway rats sensitized to ovalbumin (OA) and exposed to repeated OA challenge. Control (Sal) animals were sensitized and challenged with saline. After the 3rd challenge, animals were killed and lungs fixed in formalin. Tissue sections were incubated with antibodies to the small, leucine-rich PG, decorin, and biglycan and collagen type I. Airways were classified according to basement membrane perimeter length (< or =0.99, 1-2.99, and > or =3 mm). Decorin, biglycan, and collagen type I were increased in the airways of OA vs. Sal rats. Remodeling was most prominent in central airways. The distribution of PG differed with respect to the subepithelial vs. airway smooth muscle (ASM) vs. adventitial layer. Whereas biglycan was readily detected within the ASM, decorin and collagen were detected outside the ASM and especially in the adventitial layer. Differences in the distribution of these molecules within the layers of the airway wall may reflect their specific functional roles.

We have observed striking differences in the mechanical properties of airway smooth muscle preparations among different species. In this study, we provide a novel analysis on the influence of tissue elastance on smooth muscle shortening using previously published data from our laboratory. We have found that isolated human airways exhibit substantial passive tension in contrast to airways from the dog and pig, which exhibit little passive tension (<5% of maximal active force versus approximately 60% for human bronchi). In the dog and pig, airway preparations shorten up to 70% from Lmax (the length at which maximal active force occurs), whereas human airways shorten by only approximately 12% from Lmax. Isolated airways from the rabbit exhibit relatively low passive tension (approximately 22% Fmax) and shorten by 60% from Lmax. Morphologic evaluation of airway cross sections revealed that 25-35% of the airway wall is muscle in canine, porcine, and rabbit airways in contrast to approximately 9% in human airway preparations. We postulate that the large passive tension needed to stretch the muscle to Lmax reflects the high connective tissue content surrounding the smooth muscle, which limits shortening during smooth muscle contraction by imposing an elastic load, as well as by causing radial constraint.