Agonist Activity Of VEGF165b In HEK 293 Cells Expressing The Human VEGF Receptor 2

AJ Wheal, JJ Carter, SJ Hill, J Woolard. University of Nottingham, Nottingham, UK

Vascular endothelial growth factor (VEGF) is an agonist at VEGF receptor 2 (VEGFR2) associated with angiogenesis (1). VEGF165b is a splice variant of this protein that has often been described as an inhibitory isoform or weak partial agonist with anti-angiogenic activity (1,2). Here we have compared the agonist activity of VEGF165a and VEGF165b in HEK 293 cells expressing human VEGFR2.

HEK 293 cells expressing VEGFR2 (wild type or containing an N-terminal NanoLuc® tag; Promega) and an NFAT (nuclear factor of activated T-cells) luciferase reporter gene, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% foetal calf serum. VEGFR2-NanoLuc® cells were grown on poly-d-lysine-coated 96-well white, clear-bottomed Greiner plates at a density of 25000 cells per well for 24h, then serum starved overnight before experimentation. VEGFR2 cells were grown to confluence in flasks before being seeded in a DMEM+0.1% BSA suspension of 40000 cells per poly-d-lysine-coated well. All cells were then pre-incubated with DMEM+0.1% BSA ± cediranib (a tyrosine kinase inhibitor, final assay concentrations: 1x10-11M to 1x10-7M) before a further 5h incubation with VEGF165a or VEGF165b (final assay concentrations: 1x10-12M to 3x10-8M). For VEGFR2 cells, luciferase activity was measured using the One-GloTM Luciferase Assay System (Promega) according to manufactures instructions. In the case of VEGFR2-NanoLuc® cells, medium was aspirated from the cells prior to addition of 50μl DMEM plus 50μl ONE-GloTM reagent. Luminescence was measured on a TopCount NXT plate reader.

In both cell lines, VEGF165a and VEGF165b produced a concentration-dependent increase in luminescence, with VEGF165b producing a lower maximal response compared to that obtained with VEGF165a (Table 1). Cediranib inhibited the response to 3nM VEGF165b in a concentration-dependent manor (Table 1). The pIC50 values obtained for cediranib were similar to those obtained with 1nM VEGF165a in VEGFR2-NanoLuc® (9.33 ± 0.19, n=6) and VEGFR2 (3) cells (9.13 ± 0.01, n=5).

Table 1. Concentration-response parameters for VEGF165a and VEGF165b.

These data show that VEGF165b is a partial agonist at VEGFR2 in HEK 293 cells expressing an NFAT reporter gene. These data, taken together with previously published work (1,2), raise the possibility that the effect of VEGF165b on signalling may vary between cell types and with expression level of VEGFR2.

1. Woolard J et al. (2004). Cancer Res. 64: 7822-7835.

2. Suarez SC et al. (2006). Cell Mol Life Sci. 63: 2067-77.

3. Carter JJ et al. (2014). This meeting.