3-D structure of bFGF

We propose to investigate the mechanism of action of the angiogenic heparin-binding growth factor bFGF, the ultimate objective of this project being the definition of new bFGF antagonists to be utilized as inhibitors of tumor neovascularization and of the growth of Kaposi's sarcoma and hemangioma. Goals of the present project are:

1) The structural characterization of the interaction of bFGF with glycosaminoglycans (GAGs) and glycosphingolipids of the cell surface and with high affinity tyrosine-kinase (TK) receptors (FGFRs) [we will utilize chemically modified GAGs, gangliosides, soluble FGFRs on cultures of NIH 3T3 cells and of wild-type or sulfated GAG-deficient CHO cells transfected with each of the four FGFRs].

2) The characterization of the FGFR signal transduction pathway [we will use CHO cells transfected with FGFR-1 cDNA mutagenized by deletion of the TK domain or by substitution of its Tyr residues; we will also investigate the effect of Src, Ras, Raf, Mapk or Jun dominant negative mutants on the biological activity exerted by bFGF in these cells].

3) The study of the modulation of the expression and biological activity of bFGF in endothelial cells by chemokines [we will investigate the capacity of different heparin-binding chemokines to affect bFGF and FGFR expression in endothelial cells and to interact with bFGF for the binding to low affinity heparan-sulfate binding sites and to high affinity TK receptors].

4) The study of the angiogenic and tumorogenic activity induced in vitro and/or in vivo by overexepression of human bFGF cDNA in tumor cells (paracrine model of angiogenesis) or in immortalized or polyoma middle T-transformed endothelial cells (autocrine model of angiogenesis and hemangioma).

5) The study of the sinergy between bFGF and HIV-1 tat protein in endothelial cells [different bFGF isoforms will be overexpressed in endothelial cells transfected with HIV-1 tat cDNA and their angiogenic and tumorogenic potential will be evaluated in vitro and/or in vivo].

This project will allow the characterization of the mechanism(s) responsible for the angiogenic activity of bFGF and will represent the rationale for the development of new bFGF antagonists (e.g. polysulfonated compounds, gangliosides, synthetic peptides, soluble receptors). Moreover, this project will allow the definition of new experimental models for the in vitro investigation of the paracrine and/or autocrine mode of action of bFGF, thus reducing the requirement for in vivo experimentation on animal models.