Abstract
Vascular endothelial growth factor-A165 (VEGF-A165) is the key mediator of angiogenesis which has been explored as a therapeutic agent to control angiogenesis and revascularisation of ischaemic tissues. Unfortunately, the use of VEGF-A165 has produced poor outcomes in clinical trials, despite showing promising results in vitro and in vivo. We hypothesise that these failures occurred due to therapies using VEGF-A165 lacking appropriate glycosylation. The aim of this project was to test the angiogenic capacity of differentially glycosylated isoforms (glycoforms) of VEGF-A165.
Chemically-synthesised (unglycosylated), Escherichia coli-produced (unglycosylated) and human embryonic kidney (HEK)293 cell-produced (glycosylated) forms of recombinant human VEGF-A165 were evaluated in in vitro angiogenic assays. A Ba/F3 bioassay was employed to quantify cell proliferation upon VEGF receptor (VEGFR)-2 binding. A Matrigel® assay was utilised to assess endothelial cell tube formation upon stimulation from VEGF-A165 glycoforms. An endothelial cell barrier assay was used to measure vascular permeability. A hydrogel release assay was used to measure bioactivity of each VEGF-A165 following incorporation and release from a light-mediated PVA-tyramine gelatine hydrogel. Three replicates were conducted for each assay except for hydrogel release.
VEGFR-2 receptor binding of the VEGF-A165 glycoforms induced similar upregulation of cell proliferation, increasing cell number with the Emax ranging from 2.37 to 3.54-fold compared to control. All VEGF-A165 glycoforms induced a change in endothelial tube formation but not in a dose-response fashion. All VEGF-A165 glycoforms at 25 ng/mL induced a 2.79 to 3.24-fold increase in vascular permeability compared to assay control on the endothelial cell monolayer. Bioactivity was retained for VEGF-A165 glycoforms upon incorporation and release from the hydrogel.
These findings show that the chemically-synthesised VEGF-A165 is biologically active and suggests that VEGF-A165 glycosylation may impact its angiogenic capacity but has resulted in slightly different outcomes compared to the other glycoforms, needing further studies to determine changes in other characteristics such as pharmacokinetics. These may have utility for the treatment of vascular diseases such as diabetic ulcers, ischaemia, and osteonecrosis of the femoral head.