Insulin-like growth factor binding protein-2 (IGFBP-2) has displayed pro-angiogenic properties in metabolic diseased states. Renowned for modulating IGF, IGFBP-2 also exerts IGF-independent effects. Angiogenic signalling mechanisms activated by IGFBP-2’s interactions with integrins (RGD), heparin (HBD) and nuclear entry, as well as IGF, remain unestablished. Therapeutic angiogenesis is a potential acute treatment to aid restoration of tissue perfusion in ischaemic diseases. With clinical trials of vascular endothelial growth factor (VEGF) failing, we aim to exploit the pro-angiogenic effects of IGFBP-2 therapeutically via one or a combination of its structural domains.
Real-time (RT) PCR and Laser Doppler imaging was carried out on the limb, 24 hours after hind limb ischemia (HLI) surgery from wild-type (WT) and globally over-expressing hIGFBP-2 (TG) mice. Mutant variants of WT IGFBP-2 were generated containing a non-functional IGFΔ, RGDΔ or HBDΔ binding site. Functional angiogenic assays were explored in vitro using human umbilical vein endothelial cells (HUVECs) and the mutants or recombinant IGFBP-2 (REBP2).
We identified elevated mIGFBP-2 expression following hind limb ischemia in WT mice. This increase does not correlate with VEGF-A expression in the ischaemic muscle. Global over-expression of hIGFBP-2 significantly enhanced perfusion at day 7 (p<0.05) compared to WT littermates. Elevated hIGFBP-2 levels are localised in the Soleus muscle compared to the Gastrocnemius and Tibialis.
RGDΔIGFBP-2 failed to replicate the significant enhancement in tube formation following stimulation with WTIGFBP-2, IGFΔIGFBP-2 and HBDΔIGFBP-2 (p<0.05). HUVEC adhesion to fibronectin was significantly increased following stimulation with WTIGFBP-2 and mutants (approximately 2 fold), except RGDΔIGFBP-2. HUVEC wound closure was significantly disrupted in the presence of RGDΔIGFBP-2 compared to WTIGFBP-2. WTIGFBP-2 does not initiate a hyperpermeability response to the HUVEC cell barrier as exhibited by VEGF-A.
Stimulation of endothelial cells with WTIGFBP-2 (500 ng/ml; 15 min) induced phosphorylation of Akt (1.4 fold) and MAPK. RGDΔIGFBP-2 failed to induce Akt and MAPK phosphorylation in HUVECs, whereas the other mutants replicated the effect of the complete functional protein. Neither WTIGFBP-2, nor the mutants activated other angiogenic factors, eNOS or FAK.
Elevated IGFBP-2 levels in response to ischemia confirm its role in angiogenic recovery, also supported by in vitro and in vivo findings. Mechanistic studies confirm the RGD domain is critical in the angiogenic activity exerted by IGFBP-2 via Akt and MAPK activation. IGFBP-2’s RGD domain may provide a potential acute therapeutic treatment for ischemia without the negative effects exhibited by VEGF.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.