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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 [micro]g/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 [micro]g/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 [micro]g/ml) caused a significant increase in cell number at 48 and 96 h (P < 0.01). Adding exogenous Dcn (1-30 [micro]g/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; biglycan; collagen; asthma; airway remodeling

Decorin (Dcn), a small leucinerich 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 C57B1/6 mice in which the Den gene was disrupted by targeted deletion ([Dcn.sup.-/-]) and in wild-type controls ([Dcn.sup.+/+]). Mice were mechanically ventilated, and respiratory system impedance was measured during in vivo ventilation at positive end-expiratory pressure (PEEP) = 2 and 10 cm[H.sub.2]O, 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.sup.+/+] and [Dcn.sup.-1-] mouse lungs was examined by electron microscopy. MCh caused similar increases in airway resistance (Raw) and tissue elastance (H) in [Dcn.sup.+/+] and [Dcn.sup.-/-] mice. During MCh-induced constriction, increasing PEEP caused a decrease in Raw that was greater in [Dcn.sup.-/-] mice and a decrease in H in [Dcn.sup.-/-] mice only. Tracheal ring compliance was greater in [Dcn.sup.-/-] mice. Imaging studies showed that Den was deposited primarily in the airway adventitial layer in [Dcn.sup.-/-] mice; in [Dcn.sup.-/-] 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. airway responsiveness; asthma; proteoglycan; collagen

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 ([less than or equal to] 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: biglycan; collagen

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.sub.aw]), tissue damping, and tissue elastance. Compliance of the respiratory system ([C.sub.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.sub.aw] was decreased and [C.sub.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. complex impedance; pressure-volume curves; length-stress curves

Bleomycin (BM)-induced pulmonary fibrosis involves excess production of proteoglycans (PGs). Because transforming growth factor-[[beta].sub.1] (TGF-[[beta].sub.1]) promotes fibrosis, and interferon-[gamma] (IFN-[gamma]) inhibits it, we hypothesized that TGF-[[beta].sub.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].sub.1]. TGF-[[beta].sub.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.sup.-1] x 48 h) reduced VS, HSPG, and BG expression. Furthermore, IFN-[gamma] inhibited TGF-[[beta].sub.1]-induced increases in PG expression by these fibroblasts. Activation of fibroblasts by TGF-[[beta].sub.1] promotes abnormal deposition of PGs in fibrotic lungs; downregulation of TGF-[[beta].sub.1] by IFN-[gamma] may have potential therapeutic benefits in this disease. lung fibrosis; versican; heparan sulfate proteoglycan; biglycan

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.

Effect of glycosaminoglycan degradation on lung tissue viscoelasticity. Am J Physiol Lung Cell Mol Physiol 280: L306-L315, 2001.-- We tested the hypothesis that matrix glycosaminoglycans contribute to lung tissue viscoelasticity. We exposed lung parenchymal strips to specific degradative enzymes (chondroitinase ABC, heparitinase I, and hyaluronidase) and determined whether the mechanical properties of the tissue were affected. Subpleural parenchymal strips were obtained from Sprague-Dawley rats and suspended in a Krebs-filled organ bath. One end of the strip was attached to a force transducer and the other to a servocontrolled lever arm that effected sinusoidal oscillations. Recordings of tension and length at different amplitudes and frequencies of oscillation were recorded before and after enzyme exposure. Resistance, dynamic elastance, and hysteresivity were estimated by fitting the equation of motion to changes in tension and length. Quasi-static stress-strain curves were also obtained. Exposure to chondroitinase and heparitinase I caused significant increases in hysteresivity, no decrement in resistance, and similar decreases in dynamic elastance relative to control strips exposed to Krebs solution only. Conversely, measures of static elastance were different in treated versus control strips. Hyaluronidase treatment did not alter any of the mechanical measures. These data demonstrate that digestion of chondroitin sulfate and heparan sulfate alters the mechanical behavior of lung parenchymal tissues. chondroitin sulfate; heparan sulfate; hyaluronidase; hysteresivity