The second experimental design involved pre-mixing the VEGF (25 ng/mL) and polyphenol (1 M EGCG or dp4) for only 5min prior to treating the HUVECs with the mixture for 5 min. Remarkably, both polyphenols caused complete inhibition of VEGF-induced VEGFR-2 activation (Fig. 1B)without affecting VEGFR-2 total protein (Fig. 1C). These data support the notion that both polyphenols interact directly with the VEGF protein, and that the result of this interaction is complete
inhibition of the VEGFR-2-activating capacity of VEGF. The data also demonstrate that at 1 Mconcentrations of the two polyphenols, direct interaction with VEGF is the only effective
mechanism resulting in inhibition of VEGF-induced VEGF activation by dp4 and is the dominant mechanism for EGCG.