Abstract
The proinflammatory and chemoattractant chemokine interleukin-8 (IL-8) inhibits cell proliferation induced by basic fibroblast growth factor (bFGF) in mouse endothelial cells isolated from subcutaneous sponge implant (sponge-induced mouse endothelial cells) and in bovine aortic endothelial GM 7373 cells. The mechanism of action of IL-8 was investigated in GM 7373 cells. IL-8 did not prevent the binding of bFGF to its tyrosine kinase FGF receptors (FGFRs) nor to cell surface heparan sulfate proteoglycans (HSPGs). A transient interaction of IL-8 with the cell before the addition of the growth factor was sufficient to prevent bFGF activity. The inhibitory activity of IL-8 was abolished by protein kinase C (PKC) inhibitors and was mimicked by the PKC activator 12-O-tetradecanoylphorbol-13-acetate. Accordingly, both IL-8 and 12-O-tetradecanoylphorbol-13-acetate caused a approximately 60% decrease of the binding capacity of GM 7373 cells due to the down-regulation of FGFRs. Several C-X-C and C-C chemokines exerted an inhibitory action on bFGF activity similar to IL-8. Soluble heparin, 6-O-desulfated heparin, N-desulfated heparin, and heparan sulfate but not 2-O-desulfated heparin, chondroitin-4-sulfate, hyaluronic acid, and K5 polysaccharide abrogated IL-8 inhibitory activity consistently with the presence of low affinity, high capacity HSPG-like chemokine-binding sites on GM 7373 cells. Finally, neovascularization induced by bFGF in murine subcutaneous sponge implants was reduced significantly by IL-8. In conclusion, IL-8 inhibits the mitogenic activity exerted by bFGF on cultured endothelial cells by a PKC-dependent, noncompetitive mechanism of action that causes FGFR down-regulation. This activity is shared by several chemokines and requires endothelial cell surface HSPGs. The endothelial cell line utilized in the present study may help to elucidate the complex interplay among chemokines, HSPGs, growth factors, and receptors in endothelial cells.
Highlights
Proliferation of endothelial cells is low compared with many other cell types
Several molecules including basic fibroblast growth factor1 have been demonstrated to be positive mediators of angiogenesis in vitro and in vivo [1]. bFGF belongs to the FGF family that includes at least nine different gene products that share a high affinity for heparin and a partial amino acid sequence homology [2]. bFGF exerts angiogenic activity in vivo and induces cell proliferation, chemotaxis, and protease production in cultured endothelial cells [3] by interacting with high affinity tyrosine kinase FGF receptors (FGFRs) and low affinity proteoglycans (HSPGs) containing heparan sulfate (HS) polysaccharides [4, 5]
Heat inactivation abolished the capacity of IL-8 to inhibit the mitogenic activity of bFGF, indicating that an appropriate threedimensional conformation is required for the chemokine to elicit its inhibitory activity
Summary
Human recombinant bFGF was expressed and purified from transformed Escherichia coli cells by heparin-Sepharose affinity chromatography [24]. Epidermal growth factor, dibutyryl cAMP, and 5Ј-methyl-thioadenosine were from Sigma. Bisindolylmaleimide GF 109203X was a gift of Dr M. Pizzi (Institute of Pharmacology, University of Brescia, Brescia, Italy). Heparin was obtained from a commercial batch preparation of unfractionated sodium heparin from beef mucosa (1131/900 from Laboratori Derivati Organici S.p.A., Milan, Italy). Type I heparan sulfate (HS) was from Opocrin (Corlo, Italy). K5 polysaccharide was a gift of Dr G. Chondroitin4-sulfate and hyaluronic acid were a gift of Dr M. Del Rosso (University of Florence, Florence, Italy). 2-O-desulfated, 6-O-desulfated, totally Odesulfated, and N-desulfated heparins were provided by Dr B. The characteristics of the GAGs utilized in the present study have been described elsewhere [25]
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