Different effects of mucosal, bovine lung and chemically modified heparin on selected biological properties of basic fibroblast growth factor

Abstract

Heparins from bovine mucosa and lung, and chemically modified heparins were assayed for their capacity to: (i) protect human recombinant basic fibroblast growth factor (bFGF) from tryptic cleavage; (ii) prevent 125I-bFGF binding to heparan sulphate proteoglycans present in the extracellular matrix and on the cell surface of fetal bovine aortic endothelial GM 7373 cell cultures; (iii) affect 125I-bFGF binding to high-affinity tyrosine kinase FGF receptors present on the cell membrane of GM 7373 cells; (iv) inhibit the mitogenic activity exerted by bFGF in the same cells. The results demonstrate that the potency shown by mucosal heparins in the different assays is a direct function of size, very-low-molecular-mass heparin (2.0 kDa) being significantly less effective on a molar basis than unfractionated heparin (13.6 kDa). Increased flexibility of the backbone structure, as observed in reduced/oxidized heparins of different size, does not affect the capacity of the polysaccharide to interact with bFGF. In contrast, selective 2-O-desulphation, but not 6-O-desulphation, drastically reduced the capacity of heparin to protect bFGF from proteolytic cleavage, to affect its interaction with low- and high-affinity sites, and to inhibit its mitogenic activity. Two preparations of bovine lung heparin, differing in molecular mass, were as effective as mucosal heparin in the bFGF-tryptic-digestion assay and the endothelial-cell proteoglycan-binding assay, but they were highly inefficient at inhibiting the capacity of bFGF to interact with its tyrosine kinase receptors. Bovine lung heparins were also less effective than mucosal heparin as bFGF antagonists in GM 7373-cell-proliferation assays. N-Desulphated/N-acetylated bovine lung heparin retained only a significant capacity to protect bFGF from tryptic cleavage. The results demonstrate that different chemical features of the heparin molecule, including decrease in molecular mass, selective desulphation, disaccharide composition and clustering, affect differently the capacity of the glycosaminoglycan to interact with bFGF and to influence its biological behaviour in different assays in vitro and in endothelial cell cultures. Our findings should aid the design of synthetic oligosaccharides aimed at improving the bioavailability of bFGF when administered in vivo as a therapeutic agent.

Role of fibroblast growth factor-binding protein in the pathogenesis of HIV-associated hemolytic uremic syndrome

“…Primary glomerular endothelial cells were maintained in Cell System-Certified endothelial culture medium (ACBRI), containing the following endothelial cells growth supplement (ECGS): recombinant human epidermal growth factor (10 ng/ml), bovine brain extract, heparin (10 g/ml), amphotericine B (50 ng/ml), gentamycin (50 mg/ml), and 5% (vol/vol) fetal bovine serum. Transformed fetal bovine aortic endothelial GM7373 cells, corresponding to the BFA-1c 1BPT clone (4,19), were obtained from the National Institute of General Medical Sciences, Human Genetic Mutual Cell Repository, Coriell Institute for Medical Research (Camden, NJ) and were maintained in modified improved MEM (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum.…”

Paracrine and autocrine effects of fibroblast growth factor-4 in endothelial cells

“…Then, cells were washed with 2 M NaC1 in 20 mM HEPES bu er (pH 7.5) to remove [ 125 I]FGF-2 bound to low a nity HSPGs and with 2 M NaC1 in 20 mM sodium acetate (pH 4.0) to remove [ 125 I]FGF2 bound to high a nity FGFRs (Moscatelli, 1987). When indicated, 100 nM unmodi®ed or 2-O-desulfated heparin (Coltrini et al, 1994) were added to the binding medium.…”

Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin

“…Both the sulfation degree and disposition of sulfate groups along the GAG chain are important in determining the interactive capacity (18). In heparinbinding proteins, unique or multiple basic motifs can be found composed of linear stretches of basic amino acids as in HIV-1 Tat or of conformational cluster of scattered basic amino acids that crowd together in the properly folded protein as in fibroblast growth factor 2 (FGF2) (19).…”