Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

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Heparin, with or without the addition of an adrenocorticosteroid, can inhibit normal angiogenesis in the chick embryo chorioallantoic membrane. Low- or non-sulphated heparin fragments also have anti-angiogenic effect. Attempts to define a saccharide structure responsible for the anti-angiogenic effect implicated a -[GlcA beta 1,4-GlcNAc alpha 1,4]n-sequence. This structure represents the product of the initial polymerization reaction in heparin/heparan sulphate biosynthesis. It persists in the non-sulphated regions of heparan sulphate and also occurs in the Escherichia coli K5 capsular polysaccharide. The K5 polysaccharide, fragments thereof down to octasaccharide size and analogous N-acetylated fragments of heparan sulphate, all showed anti-angiogenic activity. Hyaluronan, however, with the isomeric -[GlcA beta 1,3-GlcNAc beta 1,4]n-structure was inactive. The anti-angiogenic activity of -[GlcA beta 1,4-GlcNAc alpha 1,4]n-containing saccharides was potentiated by the presence of L-iduronic acid and one or two O-sulphate groups in the non-reducing-terminal disaccharide unit. The anti-angiogenic effect of these non- or low-sulphated saccharides was unaffected by the addition of hydrocortisone. Endothelial cell surface-bound heparan sulphate proteoglycans may represent a pool of precursors of anti-angiogenic oligosaccharides which may be of primary importance in the regulation of angiogenesis.

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