Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-10-24
2004-06-15
Romeo, David S. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S070300, C435S355000, C435S366000, C424S282100, C424S531000, C536S055100, C530S350000
Reexamination Certificate
active
06750028
ABSTRACT:
BACKGROUND OF THE INVENTION
Transforming Growth Factor &bgr; (TGF-&bgr;) is a potent growth regulatory protein and a key molecule implicated in various fibrotic (scarring) disorders. Most of the cells secrete TGF-&bgr;1 in a predominantly inactive high molecular weight form, latent TGF-&bgr; (L-TGF-&bgr;). Latent TGF-&bgr; is composed of an amino-terminal latency-associated peptide (LAP) noncovalently associated with the carboxyl-terminal mature TGF-&bgr;. The latency-associated peptide, is disulfide-bonded to a second, structurally unrelated protein, latent TGF-&bgr; binding protein (LTBP), which plays a role in the processing and secretion of TGF-&bgr;1 (1).
A major mechanism of regulating TGF-&bgr; activity occurs through factors which control the processing of the latent to biologically active form of the molecule. Physiochemical activation can occur by extremes of pH, heat, chaotropic agents (sodium dodecyl sulfate, urea) and deglycosylation (2, 3, 4, 5). Activation in vivo is more complex and not well understood.
Cell-mediated activation was first achieved by co-cultures of either pericytes or smooth muscle cells with capillary endothelial cells (6, 7). This method requires interaction of two cell types from the same species. The activation is apparently mediated by plasmin since activation is blocked by plasmin/serine protease inhibitors (8). Cellular activation is thought to require binding of the latent TGF-&bgr; to the mannose-6-phosphate/insulin-like growth factor II receptor (9) via the mannose-6-phosphate molecules found on the LAP component of the latent form of TGF-&bgr; (10). Tissue type II transglutaminase has also been demonstrated as a requirement for activation in this cell-dependent model which may function to crosslink latent TGF-&bgr; to the matrix molecules (11). The final requirement for activation involves the LTBP. It has been proposed that LTBP is necessary for concentrating the latent TGF-&bgr; complex onto the cell surface where it is subsequently activated, presumably by tissue transglutaminase and/or plasmin (12). Three different LTBP's have been identified and cloned (13-15) which may indicate a potential mechanism by which cells may control interactions of the latent TGF-&bgr; complexes with specific tissue sites or cell types. In addition, there are reports of TGF-&bgr; activation occurring independently of these mechanisms by binding of the latent complex to thrombospondin, an extracellular matrix associated glycoprotein (16, 17). Although L-TGF-&bgr; is not activated under normal culture conditions unless co-cultures are prepared, cells in homotypic cultures can be induced to form active TGF-&bgr; by application of specific agents. Among these agents are retinoids, which induce the activation of latent-TGF-&bgr;1 in keratinocytes, endothelial cells and osteoclasts (18, 19, 20). Retinoid-induced activation of TGF-&bgr;1 is dependent upon plasmin (18, 19). Anti-estrogens (tamoxifen or toremifine) induced the production of active TGF-&bgr; in fetal fibroblasts and mammary carcinoma cells (21). Exposure of bovine arterial or capillary endothelial cells to bFGF (basic Fibroblast Growth Factor) in vitro also resulted in activation of TGF-&bgr; apparently by a plasmin-dependent mechanism (22). Growth zone costochondral chondrocyte matrix vesicles were able to activate latent TGF-&bgr; when incubated with 1,25-dihydroxy vitamin D3 through a direct action of the vitamin on the extracellular matrix vesicle membrane (23). In addition there are reports that small quantities of active TGF-&bgr;1 could be detected in conditioned media from human prostate epithelial cells (24), melanoma cell lines (25) and glioblastoma cells (26); however the mechanism of this activation is not understood.
To date, none of the systems generating active TGF-&bgr;1 has been utilized as a screening model for agents with the potential ability to sustain the latency of TGF-&bgr;1. The co-culture method has been studied extensively and the mechanisms responsible for activation of TGF-&bgr;1 in this system were elucidated. However, this co-culture method lacks reproducibility and has a strong requirement for screening large numbers of cell clones to produce an effective system.
The aim of these studies was to develop a new in vitro model for TGF-&bgr; activation for subsequent screening of molecules with potential abilities to interfere with its activation. Since TGF-&bgr; has been shown to be a key factor in scarring and fibrotic disorders, agents shown to be active in such an assay would be expected to function as anti-fibrotics and anti-scarring in vivo.
SUMMARY OF THE INVENTION
A novel method has been developed for screening and identifying modulators of scar formation, such as anti-scarring and anti-fibrotic agents. This method offers simplicity, it is reproducible and could be adopted to screen large number of modulator compounds to identify new potential anti-fibrotic agents.
This method has characteristics in common with the bovine arterial endothelial cells/bovine arterial smooth muscle cells (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 novel system, similar to the co-culture system, activation of L-TGF-&bgr;1 occurs by several independent mechanisms which involve binding of the latent complex to mannose-6 phosphate/insulin-like growth factor-II (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 were identified, such as insulin-like growth factor II defined as IGF-II, (used separately or in combination with insulin-like growth factor binding protein-2 (IGFBP-2) as a delivery vehicle), tissue type II transglutaminase inhibitors and anti-inflammatory agents (such as hydrocortisone).
A novel mechanism of action for M6P has been proposed herein which is based on downregulation of M6P/IGF-II receptor and TGF-&bgr;1 mRNAs.
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B. Cullen et al. “The Differential Regulation and Secretion of Proteinases from Fetal and Neonatal Fibroblasts by Growth Factor”,Int. J. Biochem. Cell Biol. (1997) vol. 29, pp. 241-250.
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Geesin Jeffrey C.
Gosiewska Anna
Ethicon, Inc
Romeo David S.
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