Abstract
Purpose::
VEGF-A in mice is produced as three alternatively spliced isoforms (VEGF-A120, VEGF-A164 and VEGF-A188) that differ in their binding affinity for heparan sulfate proteoglycan and thus in their extracellular localization following secretion. Transgenic mice that express single VEGF-A isoforms display distinct phenotypes. The goal of this work was to compare the biological activities of the three murine isoforms.
Methods::
Conditioned media (CM) containing the VEGF-A120, VEGF-A164 and VEGF-A188 produced by transient transfection of 293 cells with vectors expressing murine VEGF-A isoforms or media conditioned by 293 cells transfected with empty vectors were assayed for their effects on human umbilical vein endothelial cell (HUVEC) proliferation and tube formation. Levels of VEGFR-2 phosphorylation in response to the three isoforms were assessed using Phospho-VEGFR-2 (Tyr1175) and Total-VEGFR2 sandwich ELISA assays. Ability to bind neuropilin-1 (NP-1) was assessed in an in vitro binding assay. Effects on permeability were assessed in vivo using the Miles assay.
Results::
All three isoforms stimulated the proliferation of HUVEC to an equivalent extent, 2.7-fold increase over the control CM. Each isoform induced a dose-dependent increase in phosphorylation; however, the level of phosphorylation varied significantly: VEGF-A120> VEGF-A188> VEGF-A164. The induction of cord formation by HUVECs in response to the VEGF isoforms varied; VEGF-A188 was the most potent followed by VEGF-A164 and VEGF-A120. Binding of VEGF-A164, a known NP-1 ligand, to NP-1 was effectively competed by VEGF-A188 but not by VEGF-A120. Subcutaneous injection of each CM led to a dose dependent increase in permeability as detected by Evans blue extravasation, with no apparent difference among the isoforms.
Conclusions::
VEGF-A120, VEGF-A164 and VEGF-A188 have equal potency in stimulating endothelial proliferation and vascular permeability. However, the ability to induce VEGFR2 phosphorylation and formation of capillary-like structures in vitro differs and may be related to their abilities to be locally sequestered.
Keywords: vascular cells • growth factors/growth factor receptors • signal transduction