Heparin affin regulatory peptide binds to vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis

Heparin affin regulatory peptide (HARP) is an heparin-binding molecule involved in the regulation of cell proliferation and differentiation. Here, we report that HARP inhibited the biological activity induced by the 165-amino-acid form of vascular endothelial growth factor (VEGF(165)) on human umbilical vein endothelial cells. Endothelial-cell proliferation induced by VEGF(165) showed about 50% inhibition in the presence of HARP in a concentration of 3 nM. In similar range of concentrations, HARP blocked tube formation induced by VEGF(165) in three-dimensional angiogenesis assay. In vivo studies showed that HARP inhibited the VEGF(165)-induced Matrigel(TM) infiltration of endothelial cells. We then investigated the mechanisms of this inhibition and shown that HARP inhibited the binding of I-125-VEGF(165) to the VEGF receptors of endothelial cells. Additional studies using VEGF soluble receptors indicated that binding of I-125-VEGF(165) to kinase insert domain-containing receptor and neuropilin receptor was inhibited by HARP, but conversely the binding of I-125-VEGF(165) to fms-like tyrosine kinase I receptor was unaffected. A competitive affinity-binding assay demonstrated that HARP interacted directly with VEGF(165) with a dissociation coefficient of 1.38 nM. Binding assay using deletion mutants of HARP revealed that the thrombospondin type-1 repeats domains were involved in this interaction. These data demonstrate for the first time that the angiogenic factor HARP can also negatively regulates the angiogenic activity of VEGF(165).