A peptide derived from TIMP-3 inhibits multiple angiogenic growth factor receptors and tumour growth and inflammatory arthritis in mice

The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the surface of vascular endothelial cells stimulates many steps in the angiogenic pathway. Inhibition of this interaction is proving of value in moderating the neovascularization accompanying age-related macular degeneration and in the treatment of cancer. Tissue inhibitor of metalloproteinases-3 (TIMP-3) has been shown to be a natural VEGFR-2 specific antagonist—an activity that is independent of its ability to inhibit metalloproteinases. In this investigation we localize this activity to the C-terminal domain of the TIMP-3 molecule and characterize a short peptide, corresponding to part of this domain, that not only inhibits all three VEGF-family receptors, but also fibroblast growth factor and platelet-derived growth factor receptors. This multiple-receptor inhibition may explain why the peptide was also seen to be a powerful inhibitor of tumour growth and also a partial inhibitor of arthritic joint inflammation in vivo

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies

Contribution of vascular endothelial growth factor receptor-2 sialylation to the process of angiogenesis

Vascular endothelial growth factor receptor-2 (VEGFR2) is the main pro-angiogenic receptor expressed by endothelial cells (ECs). Using surface plasmon resonance, immunoprecipitation, enzymatic digestion, immunofluorescence and cross-linking experiments with specific sugar-binding lectins, we demonstrated that VEGFR2 bears both α,1-fucose and α(2,6)-linked sialic acid (NeuAc). However, only the latter is required for VEGF binding to VEGFR2 and consequent VEGF-dependent VEGFR2 activation and motogenic response in ECs. Notably, downregulation of β-galactoside α(2,6)-sialyltransferase expression by short hairpin RNA transduction inhibits VEGFR2 α(2,6) sialylation that is paralleled by an increase of β-galactoside α(2,3)-sialyltransferase expression. This results in an ex-novo α(2,3)-NeuAc sialylation of the receptor that functionally replaces the lacking α(2,6)-NeuAc, thus allowing VEGF/VEGFR2 interaction. In keeping with the role of VEGFR2 sialylation in angiogenesis, the α(2,6)-NeuAc-binding lectin Sambucus nigra (SNA) prevents VEGF-dependent VEGFR2 autophosphorylation and EC motility, proliferation and motogenesis. In addition, SNA exerts a VEGF-antagonist activity in tridimensional angiogenesis models in vitro and in the chick-embryo chorioallantoic membrane neovascularization assay and mouse matrigel plug assay in vivo. In conclusion, VEGFR2-associated NeuAc plays an important role in modulating VEGF/VEGFR2 interaction, EC pro-angiogenic activation and neovessel formation. VEGFR2 sialylation may represent a target for the treatment of angiogenesis-dependent diseases.Oncogene advance online publication, 7 August 2017; doi:10.1038/onc.2017.243