Am. J. Respir. Crit. Care Med., Volume 161, Number 6, June 2000, 2066-2073

Alterations in Large and Small Proteoglycans in Bleomycin-Induced Pulmonary Fibrosis in Rats

NARAYANAN VENKATESAN, TAKAE EBIHARA, PETER J. ROUGHLEY, and MARA S. LUDWIG

Meakins-Christie Laboratories, Royal Victoria Hospital, and Genetics Unit, Shriners Hospital for Crippled Children, McGill University, Montreal, Quebec, Canada

In bleomycin (BM)-induced lung fibrosis, alterations have been shown in the expression and deposition of small proteoglycans (PGs). Less, however, is known about changes in large PGs. We investigated changes in large aggregating (versican [VS]), basement membrane (heparan sulfate proteoglycan [HSPG]), and small (biglycan and fibromodulin) PGs during the development of BM-induced pulmonary fibrosis. BM (1.5 U) was instilled intratracheally into male Sprague-Dawley rats. Control rats received saline. At 7, 14, and 28 d after administration of BM, lungs were excised; one lung was fixed in formalin and 5-µm sections were cut and stained with hematoxylin-eosin. The other lung was used for PG extraction. PGs were extracted with guanidine and were separated through composite gel polyacrylamide gel electrophoresis (PAGE) (large PGs) and sodium dodecylsulfate-PAGE (small PGs). Gels were either stained or electrophoretically transferred and probed with antibodies to VS, HSPG, biglycan, and fibromodulin. Histologic samples showed prominent inflammation, with abundant proteinaceous material, most evident in the samples obtained at 7 and 14 d after administration of BM. By 28 d after BM, much of the inflammatory response had resolved, and heterogeneous distribution of fibrosis was observed. Immunoblots showed a relative abundance of VS at 7 and 14 d. Control lungs stained minimally for VS. Levels of HSPG, biglycan, and fibromodulin were increased maximally at 14 d after administration of BM. Immunocytochemistry showed intense immunostaining of biglycan and fibromodulin in the areas of injured lung tissue from rats 14 and 28 d after BM administration. Control lungs revealed minimal staining for small PGs. Our findings indicate that changes in all subclasses of PGs occur during the development of BM-induced pulmonary fibrosis.

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