FIBROBLAST GROWTH FACTOR-2 ANTAGONIST ACTIVITY AND ANGIOSTATIC CAPACITY 
OF SULFATED ESCHERICHIA COLI K5 POLYSACCHARIDE DERIVATIVES

 
 
Daria Leali, Mirella Belleri, Chiara Urbinati, Daniela Coltrini, Pasqua Oreste*, Giorgio Zoppetti*, Domenico Ribatti§, Marco Rusnati, and Marco Presta 

 
Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, 25123 Brescia; *Glycores 2000 S.r.l., Milano, Italy; §Institute of Human Anatomy, Histology and Embryology, University of Bari, 70124 Bari, Italy.

 

 
 
 
 
 
 
 
 

Chemical structure
of K5 derivatives
 
 


 
 

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    ABSTRACT
 
Basic Fibroblast Growth Factor (FGF2) is a potent angiogenic growth factor. FGF2 interacts with tyrosine-kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Both interactions are required for FGF2-mediated biological responses.

Here, we report about the FGF2 antagonist and anti-angiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N-position, in O-position after N-deacetylation, or in both. 

O-sulfated and N,O-sulfated K5 derivatives with low and high degree of sulfation compete with immobilized heparin for the binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG/FGF2/FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient CHO cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to HSPGs and FGFRs in FGFR1-overexpressing, HSPG-bearing CHO cells and in adult bovine aortic endothelial cells. Also, K5 derivatives inhibited with different potency FGF2-mediated cell proliferation in fetal bovine aortic endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective.

Among all the derivatives tested, only highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected murine aortic endothelial FGF2-T-MAE cells in 3D fibrin gel and “spontaneous angiogenesis in vitro” on Matrigel of FGF2-T-MAE and HUVE cells. 

Finally, the highly N,O-sulfated K5 derivative exerted a potent anti-angiogenic activity in vivo when tested on the chick embryo chorioallantoic membrane. 

These data demonstrate the possibility to generate FGF2 antagonists endowed with anti-angiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative is effective in vitro and in vivo on endothelial cells of murine, bovine, avian, and human origin. This compound may provide the basis for the design of novel angiostatic compounds.
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J. Biol. Chem. (2001) 41, 37900-37908.
AIRC: Special Project Angiogenesis