(670a) A Bind-and-Lock VEGF Immobilization Strategy to Study VEGFR-2 Signaling

Growth factors are a class of signaling proteins that direct cell fate through interaction with cell surface receptors. Although a myriad of possible cell fates stem from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor - soluble or immobilized (matrix bound). For example, studies have shown that soluble VEGF, which can be internalized, enables endothelial cells to bypass confluent monolayer growth inhibition and continue to proliferate resulting in large, leaky, tumor-like blood vessels. In contrast, immobilized VEGF with a slower internalization rate causes endothelial cells to adhere to confluent monolayer growth inhibition, and results in the formation of small, structured capillaries. Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Amine (-NH₂) and alcohol (-OH) terminated self-assembled monolayers of thiols on gold (SAMs) were used for immobilization. Heparin was oxidized to generate aldehyde groups to be used for Schiff base chemistry. Infrared (IR) spectroscopy confirmed aldehyde groups with the generation of a characteristic peak at 1730 cm^-1. Surface plasmon resonance (SPR) confirmed heparin and VEGF binding with surface densities of 44 pmol/cm² and 3 fmol/cm², respectively. Negligible amounts of heparin and VEGF were observed with surfaces containing no amines and negligible amounts of VEGF were observed with surfaces containing no heparin. VEGF immobilization was further confirmed with direct ELISA readings. To prepare the surfaces for cellular experiments, the surfaces were incubated with fibronectin. Cell studies showed that cells are able to bind to the heparin only surfaces, but showed enhanced binding to fibronectin-coated surfaces (p < 0.05). Further, cell spreading was most pronounced on surfaces that contained both heparin and fibronectin with a two-fold increase in cell area when compared to heparin alone. Last, endothelial cells plated on the VEGF-modified surfaces showed enhanced VEGFR-2 phosphorylation when compared to non-covalently bound VEGF and soluble VEGF (p < 0.05). This data suggest that immobilized VEGF signaling is different from soluble VEGF signaling. We believe that growth factor signaling is, at least, in part modulated by the physical nature of the growth factor - immobilized or soluble. The use of engineered surfaces such as the one described here are ideal for studying signaling and the resulting cell fates of immobilized growth factors, which could offer new insight into how nature regulates tissue formation and how to design scaffolds to guide tissue formation.