Cell Release of Bioactive Fibroblast Growth Factor 2 by Exon 6-encoded Sequence of Vascular Endothelial Growth Factor

Abstract

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor which is synthesized and secreted by many differentiated cells in response to various stimuli including hypoxia and growth factor exposure. Alternative splicing of vascular endothelial growth factor mRNA results in three distinct molecular forms: V189 and V165 or V121 which lack the exons 6 or 6 and 7, respectively. To clarify the functions of the 24-amino acid insertion, the biological activity of V165 was compared with that exerted by purified recombinant V189 and a synthetic peptide designed on the sequence encoded by exon 6 (Ex6P). V189 and Ex6P, but not V165, induced cell proliferation on corneal endothelial cells cultured in vitro. These effects were due to the release of fibroblast growth factor 2 (FGF2) stored in the extracellular matrix but not to direct interactions with FGF receptors since V189 was inefficient on heparan sulfatedeficient cells expressing constitutively FGF-R1. Moreover corneas incubated ex vivo with Ex6P solubilized 10-fold more FGF2 than a isocationic peptide containing a scrambled sequence. Ex6P elicited an angiogenic response in a corneal pocket assay which was totally inhibited by addition of anti-FGF2 IgG. Moreover the angiogenic response to V189, but not to V165, was inhibited by FGF2 immunoneutralization. These findings demonstrate that the presence of the exon 6-encoded sequence confers VEGF with the ability to exert its biological effects through FGF2 signaling pathways.

Multiple growth factors regulate coronary embryonic vasculogenesis

“…VEGF-induced tube formation by cultured bovine endothelial cells has been shown to be dependent upon bFGF (Jonca et al, 1997;Mandriota and Pepper, 1997). That study also revealed that the VEGF-dependence on bFGF is not via a decrease in VEGFR-2 (flk-1).…”

Netrin-4 inhibits angiogenesis via binding to neogenin and recruitment of Unc5B

“…This discrepancy might be explained by the fact that netrins have a strong affinity for heparan sulfates and thus may displace endogenous FGF-2 from its storage sites in the corneal stroma. We have previously noticed such a phenomenon for small peptides designed on the sequence of the exon 6 of VEGF 189 (26), which could induce a strong corneal angiogenesis despite binding to VEGF receptors in limbal endothelial cells. Recent evidence demonstrates that corneal vascularization relies on soluble VEGFR-1 expression (27), and it would be interesting to determine whether N1 or N4 could down-regulate VEGFR1 in the cornea.…”

“…HUVEC and HUAEC from the same donors were cultured in EBM (Clonetics) supplemented with 10% FCS and 2 ng/ml VEGF every other day. Migration assay was performed as previously described (13,26) using recombinant VEGF, N1, and N4 (R & D Systems) as modulators. A total of 30,000 HUAEC were applied on the top of the Matrigel (BD Biosciences) on a 24-well plate and incubated at 37°C; 1 h later the modulators were added.…”

Etiology and pathogenesis of uterine leiomyomas: a review.

“…VEGF could also have an indirect effect by inducing the proliferation of endothelial cells, which themselves produce a number of growth factors. In addition, VEGF acts synergistically with fibroblast growth factor (FGF) (Hyder et al 2000), and it can release the angiogenic factor bFGF from its storage on heparan sulfates of the extracellular matrix (Jonca et al 1997), with the resulting combination of the two angiogenic mitogens having a synergistic effect on angiogenesis (Asahara et al 1995;Goto et al 1993). Further, the resulting availability of bFGF permits the expression of its mitogenic effect upon the smooth muscle cells.…”