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P. A. D'Amore, A. M. Goodwin, S. K. Smith, E. Geretti, J. A. Nagy, E. B. Finkelstein, H. F. Dvorak, M. Saint-Geniez; Comparison of the Biological Activities of VEGF Isoforms. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1776.
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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.
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.
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.
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.
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