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Abstract Background Mechanical strain alters protein expression. It results in phosphorylation of MAP kinases and up-regulation of extracellular matrix proteins. We investigated whether phosphorylation of MAP kinase family members was increased in response to mechanical strain in fibroblasts from asthmatic patients (AF) and normal controls (NF), and whether phosphorylation of these signaling molecules would be different in the two cell populations. Methods Fibroblasts were obtained from mild, atopic asthmatics and non-atopic volunteers using endobronchial biopsy. Cells were grown on flexible, collagen I-coated membranes, and subjected to mechanical strain (Flexercell). MAP kinase phosphorylation was measured at baseline, and during one hour of strain. We also examined the effect of strain on proteoglycan production. Results At baseline, there was increased phosphorylation of ERK1/2 and p38, and decreased phosphorylation of JNK in AF vs NF. During strain in NF, p38 phosphorylation was increased. Conversely in AF, strain resulted in an increase in JNK phosphorylation, had no effect on phosphorylation of p38, and resulted in a decrease in ERK1/2 phosphorylation. There was a significant increase in versican protein production after 24 h strain in both AF and NF. JNK inhibition reversed the strain-induced increase in versican in NF, but had no effect in AF. Conclusion These results show that there are phenotypic differences in MAP kinase phosphorylation in AF vs NF, and that different signaling pathways are involved in transducing mechanical stimuli in these two populations of cells.

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]

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) 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.

Decorin (Dcn), a small leucine-rich proteoglycan, is present in the extracellular matrix of the airways and lung tissues, contributes to lung mechanical properties, and its deposition is altered in asthma. The effect of Dcn deficiency on airway parenchymal interdependence was examined during induced bronchoconstriction. Studies were performed in C57Bl/6 mice in which the Dcn gene was disrupted by targeted deletion (Dcn(-/-)) and in wild-type controls (Dcn(+/+)). Mice were mechanically ventilated, and respiratory system impedance was measured during in vivo ventilation at positive end-expiratory pressure (PEEP) = 2 and 10 cmH(2)0, before and after aerosol delivery of methacholine (MCh). Length vs. tension curves in isolated tracheal rings were measured in vitro. Dcn distribution in +/+ mice airways was characterized by immunofluorescence; differences in collagen structure in Dcn(+/+) and Dcn(-/-) mouse lungs was examined by electron microscopy. MCh caused similar increases in airway resistance (Raw) and tissue elastance (H) in Dcn(+/+) and Dcn(-/-) mice. During MCh-induced constriction, increasing PEEP caused a decrease in Raw that was greater in Dcn(-/-) mice and a decrease in H in Dcn(-/-) mice only. Tracheal ring compliance was greater in Dcn (-/-) mice. Imaging studies showed that Dcn was deposited primarily in the airway adventitial layer in Dcn(+/+) mice; in Dcn(-/-) mice, collagen had an irregular appearance, especially in the lung periphery. These results show that lack of Dcn alters the normal interaction between airways and lung parenchyma; in asthma, changes in Dcn could potentially contribute to abnormal airway physiology.

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.

Decorin, a small leucine-rich proteoglycan with a widespread tissue distribution, is required for the normal fibrillogenesis of collagen in most tissues. Because collagen is important in determining the elastic behavior of the lung, we hypothesized that lung tissue mechanics would be altered in a mutant mouse in which the single decorin gene was abrogated by targeted deletion (Dcn-/-). Complex impedance of the respiratory system was measured in C57Bl/6 mice (Dcn-/- and Dcn+/+) using a small animal ventilator that delivers a volume signal with multiple frequencies to the trachea. A constant-phase model was fit to calculate airway resistance (R(aw)), tissue damping, and tissue elastance. Compliance of the respiratory system (C(rs)) was measured from a pressure volume curve during stepwise deflations. Lungs were excised, and parenchymal tissue strips were mounted in an organ bath for in vitro measurement of tissue impedance and quasistatic length-stress curves. In addition, pulmonary tissue was examined by immunohistochemistry and immunoblotting. In vivo, in the Dcn-/- mice, R(aw) was decreased and C(rs) was increased. Similarly, in vitro, length-stress curves showed increased compliance of the strips in the Dcn-/- mice. These alterations in lung tissue mechanical behavior in Dcn-/- mice support a critical role for decorin in the formation of the lung collagen network.

Bleomycin (BM)-induced pulmonary fibrosis involves excess production of proteoglycans (PGs). Because transforming growth factor-beta(1) (TGF-beta(1)) promotes fibrosis, and interferon-gamma (IFN-gamma) inhibits it, we hypothesized that TGF-beta(1) treatment would upregulate PG production in fibrotic lung fibroblasts, and IFN-gamma would abrogate this effect. Primary lung fibroblast cultures were established from rats 14 days after intratracheal instillation of saline (control) or BM (1.5 units). PGs were extracted and subjected to Western blot analysis. Bleomycin-exposed lung fibroblasts (BLF) exhibited increased production of versican (VS), heparan sulfate proteoglycan (HSPG), and biglycan (BG) compared with normal lung fibroblasts (NLF). Compared with NLF, BLF released significantly increased amounts of TGF-beta(1). TGF-beta(1) (5 ng/ml for 48 h) upregulated PG expression in both BLF and NLF. Incubation of BLF with anti-TGF-beta antibody (1, 5, and 10 microg/ml) inhibited PG expression in a dose-dependent manner. Treatment of BLF with IFN-gamma (500 U. ml(-1) x 48 h) reduced VS, HSPG, and BG expression. Furthermore, IFN-gamma inhibited TGF-beta(1)-induced increases in PG expression by these fibroblasts. Activation of fibroblasts by TGF-beta(1) promotes abnormal deposition of PGs in fibrotic lungs; downregulation of TGF-beta(1) by IFN-gamma may have potential therapeutic benefits in this disease.