Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation by a peptide corresponding to the exon 7-encoded domain of VEGF(165)

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells (EC) in vitro and a major regulator of angiogenesis in vivo. VEGF(121) and VEGF(165) are the most abundant of the five known VEGF isoforms. The structural difference between these two is the presence in VEGF(165) of 44 amino acids encoded by exon 7 lacking in VEGF(121). It was previously shown that VEGF(165) and VEGF(121) both bind to KDR/Flk-1 and Flt-1 but that VEGF(165) binds in addition to a novel receptor (Soker, S., Bidder, Il., Neufeld, G., and Klagsbrun, M. (1996) J. Biol. Chem. 271, 5761-5767). The binding of VEGF(165) to this VEGF(165)-specific receptor (VEGF(165)R) is mediated by the exon 7-encoded domain. To investigate the biological role of this domain further, a glutathione S-transferase fusion protein corresponding to the VEQF(165) exon 7-encoded domain was prepared. The fusion protein inhibited binding of I-125-VEGF(165) to VEGF(165)R on human umbilical vein-derived EC (HUVEC) and MDA-MB-231 tumor cells. The fusion protein also inhibited significantly I-125-VEGF(165) binding to KDR/Flk-1 on HUVEC but not on porcine EC which express KDR/Flk-1 alone. VEGF(165) had a 2-fold higher mitogenic activity for HUVEC than did VEGF(121). The exon 7 fusion protein inhibited VEGF(165)-induced HUVEC proliferation by 60% to about the level stimulated by VEGF(121). Unexpectedly, the fusion protein also inhibited HUVEC proliferation in response to VEGF(121). Deletion analysis revealed that a core inhibitory domain exists within the C-terminal 23-amino acid portion of the exon 7-encoded domain and that a cysteine residue at position 22 in exon 7 is critical for inhibition. It was concluded that the exon 7-encoded domain of VEGF(165) enhances its mitogenic activity for HUVEC by interacting with VEGF(165)R and modulating KDR/Flk-1-mediated mitogenicity indirectly and that exon 7-derived peptides may be useful VEGF antagonists in ansogenesis-associated diseases.