Abstract
Purpose :
Vascular endothelial growth factor (VEGF) plays an important role in retinal vasculogenesis and vascular diseases. However, it is technically difficult to quantify VEGF-binding activity of cells because of possible ligand-receptor dissociation. The purpose of this study is to develop a rapid and convenient method to quantify cell binding activity to VEGF.
Methods :
Three hVEGFA isoforms, hVEGF-111, hVEGF-165, and hVEGF-206 were expressed on the surface of T7 bacteriophage and characterized in vitro by binding to immobilized aflibercept, a decoy VEGF receptor. Binding of three hVEGF isoforms to immobilized heparan sulfate (HS) was compared by phage binding assay. Binding of hVEGF isoforms to human umbilical vein endothelial cells (HUVECs), HEK293 and SK-N-AS neuron cell lines was rapidly washed and quantified by an in vitro phage-cell binding assay.
Results :
All three hVEGF-Phage clones showed increased binding to immobilized aflibercept over hIgG. In contrast, the control phage did not show significant binding to either aflibercept or hIgG. Binding to immobilized HS showed that all three hVEGF-Phages bound significantly to HS compared to the control phage, but hVEGF-206 showed the highest binding. In vitro cell binding showed that hVEGF-206-Phage has the highest binding to all three cell types, whereas hVEGF-111 and 165 phages showed relatively low and intermediate binding, respectively. hVEGF-206 has the highest specific binding to HUVECs, followed by HEK293, and SK-N-AS. The trend is similar for hVEGF-165. On the other hand, hVEGF-111 phage showed similar specific binding to both HUVEC and HEK293 followed by SK-N-AS. Whereas hVEGF-111-Phage showed the lowest specific binding to all three cells, hVEGF-206-Phage has the highest specific binding to cells but with high background binding to neurons, probably due to cell surface heparan.
Conclusions :
All three hVEGF-Phage isoforms bind to immobilized aflibercept, suggesting the display of their native conformation on the phage surface. hVEGF-165-Phage is the most appropriate isoform for VEGF binding activity quantification because of its biological activity, relatively high specific binding and moderate non-specific binding. This method can be applied to any isolated or cultured cells to quantify their VEGF binding activity to facilitate VEGF research. This approach can also be applied to other cell-binding ligands.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.