Insulin-like growth factor binding protein-2 and its role in angiogenesis

Therapeutic angiogenesis is currently under investigation to restore tissue perfusion in peripheral arterial disease (PAD). However, clinical trials have proved to be disappointing in stimulating the development of functional blood vessels and reducing adverse events. Insulin-like growth factor binding protein-2 (IGFBP-2) has demonstrated potential in proangiogenic activity but the molecular mechanisms remain unestablished. Structurally, IGFBP-2 possesses domains which can interact with insulin-like growth factor (IGF), receptor protein tyrosine phosphatase-β, glycosaminoglycans, integrins and can potentially translocate into the nucleus to activate cellular and pathological processes. In this project, we used two IGFBP-2 over-expressing mouse models, namely a global and an endothelial specific model, to determine whether increasing IGFBP-2 can increase perfusion in an experimental model of ischemia in vivo. The angiogenic potential of IGFBP-2 was investigated in an array of in vitro angiogenic signalling and functionality studies in vascular endothelial cells. Recombinant IGFBP-2 was generated, in which site-directed mutagenesis was employed to disrupt the integrin binding site (RGD), IGF binding site, or the heparin binding domain-1/nuclear localisation signal. These mutants were used to determine the primary mechanism IGFBP-2 may use to exert in vitro angiogenic effects. Upregulation of IGFBP-2 in ischemic muscles was confirmed in wild-type mice following hind limb ischemia surgery. IGFBP-2 over-expression significantly enhanced perfusion at early stages of recovery. In vitro angiogenic signalling and functional assays demonstrated IGFBP-2 increased phosphorylation of Akt and ERK/MAPK, as well as enhancing endothelial cell adhesion, wound closure and tube formation. Site-directed mutagenesis identified the RGD domain to be critical in IGFBP-2-stimulated in vitro angiogenic activity. In contrast to VEGF, exposure of IGFBP-2 to endothelial cells did not affect endothelial monolayer permeability. In conclusion, IGFBP-2, via its RGD domain displays promising potential as a new therapeutic angiogenic treatment.