Perlecan, a multidomain heparan sulfate proteoglycan (PG), is an intrinsic component of basement membranes and extracellular matrices. We used a prokaryotic expression vector to generate fusion proteins encoding various domains of human perlecan protein core and these recombinant proteins were used as immunogens to produce mouse anti-human monoclonal antibodies (MAb). One MAb, designated 7B5, was characterized by Western blotting and ELISA and was shown to react specifically with the laminin-like region of perlecan (Domain III) but not with two other fusion proteins encoding Domain II or V. This perlecan epitope was detected by immunoenzymatic staining in the basement membranes of human tissues including pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. All vascular basement membranes tested contained this gene product. In addition, sinusoidal vessels of liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, using as probe the same human cDNA-encoding Domain III, localized perlecan mRNA to specific cell types within the tissues and demonstrated that in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer. The availability of MAb against precise regions of human perlecan will allow the investigation of this gene product in normal and diseased states.

The critical molecular mechanism in the development of the pulmonary fibrosis remains unknown, leaving diagnosed patients with a poor prognosis. To isolate the genes specifically up-regulated in pulmonary fibrosis, we established a rat silicosis model 360 d after treatment with crystalline silica suspension. Radiographs of chests showed that some scattered high-density shadows appeared in the lung field. Typical microscopic fibrosing silicotic nodules formed in the lung, alveolar epithelial cells and bronchial epithelial cells, particularly around the partial fibrosing silicotic nodules; some of them showed atypical hyperplasia that suggested a correlation between silicosis and lung cancer. Suppression subtractive hybridization analysis was performed to compare gene expression in lung tissue with silicosis and normal lung tissue. Reverse transcription-polymerase chain reaction showed that the expressions of seven novel cDNA sequences identified by suppression subtractive hybridization in lung tissue with silicosis differed from normal lung tissue. Bioinformatics analysis showed that 47 positive clones represented 35 genes containing two putative proteins and four predicted similar proteins. The analysis also showed that some screened genes in silicosis, such as prolyl 4-hydroxylases, actin-related protein-2/3 complex and acidic mammalian chitinase, have not been previously reported. These genes may provide new clues for investigating the molecular mechanisms in the development of pulmonary fibrosis.