Final Report Summary - VEGFENOL (Inhibition of VEGF signalling by dietary polyphenols as a plausible mechanism for their health benefits) The aim of this project is to advance our understanding of the inhibition of VEGF signalling by polyphenols and its relevance to dietary exposure, by investigating the chemical, structural and physical characteristics of the specific interaction that leads to the inhibition of VEGF signalling and the effect of human metabolism on the potency of inhibition. The final objective of the research project is to develop an advanced in vitro model that can be used for the routine assessment of the attenuation of VEGF signalling following normal human metabolism of polyphenols that would occur post-ingestion.The specific aims of this project are to: 1. determine the chemical, structural and physical features of polyphenols associated with potent inhibition of VEGF signalling;2. characterise the nature of the interactions that underlie the potent inhibition of VEGF signalling by specific polyphenols;3. examine how human metabolism of ingested polyphenols influences their potency;4. develop an in vitro model for assessing the effects of dietary polyphenols on VEGF signalling and other aspects of endothelial function.In order to better understand the physioco-chemical properties of polyphenols required for potent inhibition of VEGF signalling, an extensive list of polyphenols (> 50 compounds), including different classes (phenolic acids, stilbenes, flavanols, flavonols, isoflavones, flavanones, etc.), plant glycosides and human phase-2 conjugates was drawn up. These were tested at various concentrations (0.6 - 100 µM) in order to ascertain whether or not they are effective inhibitors of VEGF signalling. The IC50 of those polyphenols that potently inhibited VEGF signalling at sub-micromolar concentration were also estimated for several compounds (> 20 compounds). The experiments were carried out in HUVEC cells using a model developed by the host team, the details of which are being included in a patent to be filed shortly (July 2013). VEGFR2 phosphorylation was measured using an ELISA assay (PathScan Phospho-VEGFR2 (Tyr 1175) sandwich ELISA kit). These experiments have shown that certain polyphenols, but not the majority, including some present in strawberry, grapes, wine, apple and green tea are potent inhibitors of VEGF signalling at sub-micromolar concentrations. This is important because the concentrations required for inhibition are shown to be achievable through diet or are likely to be achievable in the future with appropriate functional foods. It has been demonstrated that the inhibitory effect on VEGF signalling is highly dependent on polyphenol structure, and the structural and chemical properties required for potent inhibition of VEGF signalling have been elucidated. These data are being written up for publication in a peer-reviewed journal.A series of key experiments were conducted in HUVEC cells to further explore the mechanism of inhibition of VEGFR2 activation. The combination of results obtained from these experimental designs demonstrated the molecular target involved in the VEGFR2-inhibitory activities of the polyphenols.We explored the nature of the polyphenol interaction with the molecular target further by means of different techniques including use of dialysis, binding kinetics assays, western blotting (for assessing effects on downstream signalling pathways), flow cytometry (assessment of binding to cells), mass spectrometry (adduct formation) and in silico molecular docking simulations. In addition, the ligand was purified from insect cell culture supernatants using FPLC and used to assess the nature of the physical interactions by atomic force microscopy and surface plasmon resonance (BiaCore).The results from the experiments have demonstrated how polyphenols interact with the ligand and the characteristics of the interaction. It is not possible to provide further details here because the novel findings are an important component of a patent filing. The ability of certain polyphenol human metabolites (glucuronides, sulfates, methylated derivates) to inhibit VEGF signalling was assessed using the unique collection of pure authentic human metabolites prepared by the host team. Those polyphenols that showed to be potent inhibitors of VEGF signalling were selected to use in studies to test the ability of their human phase-2 conjugate metabolites to inhibit VEGF signalling using HUVEC cells and measuring VEGFR2 phosphorylation by ELISA assay (PathScan Phospho-VEGFR2 (Try 1175) sandwich ELISA kit).These results have shown that in general, the metabolites are not as potent as their unconjugated (aglycone) forms in terms of inhibiting VEGF signalling.Finally, we have developed an advanced in vitro model using a combination of Caco-2 cells (to metabolise) and vascular endothelial cells (to assess pVEGFR2) to be used for a routine assessment of the attenuation of VEGF signalling following ingestion of polyphenols. Polyphenols showing potent inhibition of VEGF signalling and which are present in strawberry, wine and green tea were selected for testing in this model.In summary, the project findings reveal a novel molecular mechanism by which dietary polyphenols at sub-micromolar concentration can alter vascular endothelial cell function demonstrating how polyphenols in foods and beverages can influence human health that is consistent with their generally low bioavailability.The project results have provide new advances in the understanding of the potential health benefits of foods (polyphenol-enriched-foods, beverages and supplements), and in demonstrating a plausible physiological mechanism, have provided the potential for increased competitiveness of the European Union (EU) (e.g. by underpinning health claims for foods and/or the development of novel prophylactives for use as dietary disease-prevention agents. The present project has determined biological activity of food compounds directly implementable in reduction of CVD risk. This also benefits EU research by enhancing the knowledge on molecular mechanisms of interaction between the food molecules and biological targets which is a necessary component of the process of demonstrating the cause and effect principle that underpins the assessment of health claims in Europe. Additionally, the agreements that are now in place between IFR and an small to medium-sized enterprise (SME) will facilitate exploitation of this knowledge (as intellectual property) in the functional food and pharma domains. The sound underpinning science developed in this project will hugely increase the chances that the SME will be able to develop and successfully market new products and thus demonstrate direct economic impact of this research. The ultimate beneficiaries are therefore the public through the improvement of diet for reducing CVD mortality rates as well as the social and economic costs associated with the disease.