Anti-fibrotic agent assay

Transforming Growth Factor (TGF- ) is a potent growth regulatory protein and a key molecule implicated in various fibrotic (scarring) disorders. Most of the cells secrete TGF- 1 in a predominantly inactive high molecular weight form, latent TGF- (L-TGF- ). Latent TGF- is composed of an amino-terminal latency-associated peptide (LAP) noncovalently associated with the carboxyl-terminal mature TGF- . The latency-associated peptide, is disulfide-bonded to a second, structurally unrelated protein, latent TGF- binding protein (LTBP), which plays a role in the processing and secretion of TGF- 1 (1). A novel method has been developed for screening anti-scarring and anti-fibrotic agents. This method offers simplicity, it is reproducible and could be adopted to screen large numbers of new potential ant-fibrotic agents. This method has characteristics in common with the BAEC/BASMC co-culture system, but is more sensitive and does not require screening large number of clonal lines for developing an effective method. In this system, similar to the co-culture system, activation of L-TGF- 1 occurs by several independent mechanisms which involve binding of the latent complex to M6P/IGF-II receptors, thrombospondin and/or tissue type II transglutaminase. But, in contrast to the co-culture system, this macrophage-dependent system does not appear to involve plasmin. Using this method, potential novel anti-fibrotic agents such as IGF-II, (used separately or in combination with IGFBP-2 as a delivery vehicle) tissue type II transglutaminase inhibitors and anti-inflammatory agents (such as hydrocortisone) were identified. A potential novel mechanism of action for Mannose 6-Phosphate has been proposed which is based on downregulation of M6P/IGF-II receptor and TGF- 1 mRNAs.