Anthocyanins and their gut metabolites attenuate monocyte adhesion and transendothelial migration through nutrigenomic mechanisms regulating endothelial cell permeability

Accumulating evidence suggests that the cardioprotective effects of dietary anthocyanins are partly attributed to their ability to maintain endothelial function. However, the underlying mechanisms of action are not fully understood. The aim of this study was to investigate the effect of anthocyanins and their gut metabolites, at physiologically-relevant concentration and time of exposition, on endothelial cell function and to decipher using the integrated omic approaches the underlying molecular mechanisms of action. Primary human umbilical vein endothelial cells were treated with a mixture of cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside and 4-hydroxybenzaldehyde (mix A) at a concentration of 0.1M for anthocyanins, and 0.5M for 4-hydroxybenzaldehyde for 3 hours. Endothelial cells were also exposed to a mixture of anthocyanin gut metabolites: protocatechuic, vanillic, ferulic and hippuric acid (mix B) at 0.2M, 2M, 1M, 2M concentration, respectively for 18 hours. In a third experimental condition, endothelial cells were successively treated with both mixtures, mix A for 3 hours then mix B for 18 hours, to mimic the anthocyanins pharmacokinetics following the consumption of anthocyanin-rich foods (mix A+B). Following the cell treatments, endothelial cell dysfunction was induced using TNF (1ng/ml). The effect of pre-exposure of endothelial cells to mixtures on monocyte adhesion and their transendothelial migration was then evaluated. Furthermore, gene and miRNA expression, cell-signaling protein phosphorylation and in silico docking analysis were performed to identify the underlying molecular mechanism. Pre-exposure of endothelial cells to mix A, mix B and mix A+B significantly decreased monocyte adhesion to TNF-activated endothelial cells by 26.4%, 16.4% and 30.6%, respectively. These mixtures also significantly attenuated transmigration of monocytes through endothelial monolayer by 43.9%, 50.5% and 41.1%, respectively. Gene expression analysis using TLDA arrays showed that mixtures could modulate the expression of genes involved in the regulation of cell-cell adhesion, cytoskeleton organization or focal adhesion. Bioinformatic analyses were performed to identify potential transcription factors involved in the observed nutrigenomic effects and cell signaling proteins regulating their activity. Using Western blot analysis we observed that anthocyanins modulated phosphorylation of NfB-p65 or p38 signaling proteins. Using docking analyses, we identified 25 out of 63 potential cell signaling proteins to which these bioactives can bind to and initiate the observed nutrigenomic effect, including one member of Dwarfin/SMAD family (SMAD2) and seven members of protein kinases superfamily belonging to five different families: AGC Ser/Thr protein kinase family (AKT3 and ROCK2), STE Ser/Thr protein kinase family (ASK1 and MEK2), Tyr protein kinase family (FAK1), Ser/Thr protein kinase family (IKKα) and CMGC Ser/Thr protein kinase family (JNK1). Nutrigenomic study also showed that anthocyanins and their metabolites modulated expression of 13 to 14 miRNAs. Bioinformatic analysis showed that over half of these miRNAs are involved in regulation of endothelial cell permeability, contributing to the observed changes in endothelial cell functions. In conclusion, integration of these results revealed endothelial-protective properties of anthocyanins and their gut metabolites and deciphered new underlying multi-target and multi-layered mode of action.