The phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) by engineered surfaces with electrostatically or covalently immobilized VEGF.
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 stems 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). 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. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm2 and 100 pg/cm2, respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature.
Anderson
Sean M
SM
University of California, Los Angeles, Chemical and Biomolecular Engineering Department, Los Angeles, CA 90095, USA.
Chen
Tom T
TT
Iruela-Arispe
M Luisa
ML
Segura
Tatiana
T
D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006046:Gold / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010084:Oxidation-Reduction / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D013055:Spectrophotometry, Infrared; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D013499:Surface Properties / Q000187:drug effects; D034421:Sus scrofa; D023822:Tissue Engineering / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D055767:Immobilized Proteins; D042461:Vascular Endothelial Growth Factor A; D006046:Gold; D006493:Heparin; D040301:Vascular Endothelial Growth Factor Receptor-2