title: VEGF internalization is not required for VEGFR-2 phosphorylation in bioengineered surfaces with covalently linked VEGF.
abstract: Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. Further, although growth factor internalization along with the receptor generally occurs in a physiological setting, whether this internalization is needed for receptor phosphorylation is not entirely clear. Here we show that VEGF covalently bound through a modified heparin molecule elicits an extended response of pVEGFR-2 in human umbilical vein endothelial cells (HUVECs) and that the covalent linkage reduces internalization of the growth factor during receptor endocytosis. Optical tweezer measurements show that the rupture force required to disrupt the heparin-VEGF-VEGFR-2 interaction increases from 3-8 pN to 6-12 pN when a covalent bond is introduced between VEGF and heparin. Importantly, by covalently binding VEGF to a heparin substrate, the stability (half-life) of VEGF is extended over three-fold. Here, mathematical models support the biological conclusions, further suggesting that VEGF internalization is significantly reduced when covalently bound, and indicating that VEGF is available for repeated phosphorylation events.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, 420 Westwood Plaza, 5531 Boelter Hall, Los Angeles, CA 90095, USA.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Anderson
authors.forename: Bhupinder
authors.initials: B
authors.lastname: Shergill
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Barry
authors.forename: Eleana
authors.initials: E
authors.lastname: Manousiouthakis
authors.forename: Tom T
authors.initials: TT
authors.lastname: Chen
authors.forename: Elliot
authors.initials: E
authors.lastname: Botvinick
authors.forename: Manu O
authors.initials: MO
authors.lastname: Platt
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Iruela-Arispe
authors.forename: Tatiana
authors.initials: T
authors.lastname: Segura
mesh_terms: D001672:Biocompatible Materials; D001698:Biomedical Engineering; D004705:Endocytosis; D005109:Extracellular Matrix / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D052898:Optical Tweezers; D010766:Phosphorylation; D055550:Protein Stability; D012995:Solubility; D013499:Surface Properties; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; D055767:Immobilized Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D006493:Heparin; D040301:Vascular Endothelial Growth Factor Receptor-2; D048051:p38 Mitogen-Activated Protein Kinases