Cyanidin-3-glucoside phenolic metabolites, protocatechuic acid and vanillic acid prevent TNF-α induced endothelial dysfunction in vitro: Implications for anthocyanins and vascular health

Anthocyanins are a subgroup of flavonoid polyphenols, derived from many fruits and vegetables, previously reported to improve cardiovascular health. However, their poor bioavailability in vivo raises the question of whether the observed biological activity is due to their metabolites. It is known that elderberry (EB) has a high content of cyanidin-3-glucoside (C3G), which is known to induce cardiovascular protective properties by improving endothelial function. After the consumption of C3G, it is rapidly degraded to many metabolites although mainly in the form of protocatechuic acid (PCA), which can be methylated to form vanillic acid (VA). Both have been shown to reach higher plasma concentrations than its original C3G form. However, their potential positive effect on endothelial health is yet to be investigated in inflammatory conditions. An understanding of their action will help to better define the in vivo vascular health benefits of anthocyanin rich foods such as EB. Purpose: The main aim of this thesis was to investigate the effects of PCA and VA on endothelial function at physiological concentrations in the presence of TNF-α, a pro-inflammatory mediator. Moreover, it was aimed to determine whether metabolites could be identified as the bioactive compounds responsible for the observed effect of anthocyanins, specifically focusing on C3G as a representative anthocyanin compound. Methods: Primary human umbilical vein endothelial cells (HUVEC) were pre-treated with either EB (50 μg/ml) or C3G, PCA and VA at various concentrations including 1,5 or 10 μM at various timepoints stimulated with or without TNF-α (10 or 20 ng/ml) as stated. Cell viability, apoptosis, oxidative stress; Akt and eNOS at the mRNA level and protein level as well as NF-κB, Nrf2, ERK1/2, NOX-4 protein level was measured. Additionally, a co-culture model between endothelial cells and monocytes to mimic the initial stages of atherosclerosis were implemented within this thesis. For monocyte adherence protocol, fluorescently labelled monocytes (THP-1) were added to HUVECs for 1 h after TNF-α stimulation and measured using a fluorescence plate reader. Supernatant was removed pre- and post-monocyte adherence to measure the concentration of cytokine release including IL-6, MCP-1, and IL-1β. Adhesion molecules on the surface of endothelial cells including VCAM-1 and ICAM-1 were also measured. Results: HUVECs pre-treated with EB, C3G, PCA and VA all prevented TNF-α induced monocyte adherence to endothelial cells (p < 0.03). This effect may have been a result of the treatments showing a reduction in TNF-α induced oxidative stress and inflammation (p < 0.05), as well as improving endothelial cell viability (p <0.05). Phenolic metabolites PCA and VA only increased eNOS activity in response to TNF-α (p <0.05), moreover VA showed increased activity by reducing NOX-4 and increasing HO-1 expression at physiological relevant concentrations. Discussion: The current thesis provides evidence that pre-exposing endothelial cells to phenolic metabolites, such as PCA and VA, enhances endothelial function by regulating multiple molecular pathways associated with endothelial function/dysfunction. Moreover, phenolic metabolites reduce the adhesion of monocytes to endothelial cells, which is a crucial initial stage in the progression of atherosclerosis. Importantly, these effects are observed at concentrations that are physiologically relevant. This was also associated with all treatments displaying improvements in cell viability, reduction in TNF-α induced apoptosis and reduction in oxidative stress. It is likely that the induction of inflammation is a pre-requisite for phenolic metabolites to induce its protective properties. Further research should explore these findings as mixture of metabolites or in more in vivo like models. The findings of this thesis could help further research with understanding which compounds have cardio-protective properties in polyphenol rich foods.