Angiogenesis in adults plays a critical role on the pathogenesis of the two main causes of death: cardiovascular disease and cancer. Angiogenesis drives tumour progression and development, as well as the development and destabilization of atherosclerotic plaques. Vascular endothelial growth factor (VEGF) is the most active endogenous pro-angiogenic factor in humans. Therefore, inhibition of VEGF activity is a plausible mechanism to decrease cardiovascular and cancer risk. The aim of the present study is to evaluate the anti-VEGF activity of different bioactive compounds, some of them present in food and/or human metabolites (melatonin, serotonin, 3-indoleacetic acid, 5-hydroxytryptophol and hydroxytyrosol) on the endothelial cell migration and the molecular mechanisms involved. Wound-healing assay was performed on HUVEC cells to evaluate the inhibitory effect of the bioactive compounds on migration process. The wounds (at 0 and 24 hours) were photographed using phase contrast microscopy on an inverted microscope. Western blot analysis was performed using antibodies directed against phospho-AKT (Ser473), AKT, phospho-PLCƔ1 (Tyr783), PLCƔ1, phospho-eNOS (Ser1177) and eNOS antibodies to evaluate their impact on the intracellular signaling cascade. A pathscan phospho-VEGFR-2 (Tyr1175) sandwich ELISA kit were used to evaluated the capability of the combined bioactives to inhibit VEGF-induced VEGFR-2 activation, as well as to determine hydroxytyrosol molecular target. This is the first time that serotonin and 5-hydroxytryptophol (1 mM) show to significantly inhibit HUVEC migration (98 and 50% respectively), following the same mechanism previously reported for melatonin and 3-indoleacetic acid (interaction between indolic compounds and surface components of the endothelial cell to prevent VEGF from activating its receptor). HUVEC migration inhibition have been previously reported for melatonin and 3-indoleacetic acid (87 and 99%, respectively). Our results reveal that the anti-angiogenic effect of melatonin, serotonin, 3-indoleacetic acid and 5-hydroxytryptophol is not mediated via PLCγ1, in contrast to other bioactive compounds. Therefore, other routes such as Shb-Sck-PI9K-Rac-IQGAPI, p38/MAPK-HSP27 y TSAd/Src might be involved. Simultaneously, only melatonin and serotonin significantly activated Akt and maintained eNOS phosphorylation. Thus, it would be expected that melatonin and serotonin maintained NO bioavailability, preventing for the hypertensive effect associated to the used of anti-VEGF drugs. Moreover, combination of melatonin and serotonin improved their anti-VEGF potential. Our data suggest for the first time, that, similarly to indolic compounds, the interaction between hydroxytyrosol and surface components of the endothelial cell membrane (VEGFR-2, etc.) in a way that prevents VEGF from activating its receptor is the most plausible molecular mechanism for its anti-VEGF activity.

P4-13-01 - Melatonin, serotonin and other bioactive compounds: anti-angiogenic mechanism against VEGF activity