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

Abstract

Cardioprotective effects of dietary anthocyanins are partly attributed to their ability to maintain endothelial function. However, the underlying cellular and molecular mechanisms of action are not fully understood. This study aimed to evaluate the effect of anthocyanins and their gut metabolites, at physiologically-relevant conditions, on endothelial cell (EC) function and decipher the underlying molecular mechanisms of action using integrated omics approaches. Primary EC were treated with a mixture of 0.1 mu M cyanidin-3-arabinoside, 0.1 mu M cyanidin-3-galactoside, 0.1 mu M cyanidin-3-glucoside, 0.1 mu M delphinidin-3-glucoside, 0.1 mu M peonidin-3-glucoside and 0.5 mu M 4-hydroxybenzaldehyde for 3 h or a mixture of gut metabolites: 0.2 mu M protocatechuic, 2 mu M vanillic, 1 mu M ferulic and 2 mu M hippuric acids for 18 h. Also, successive exposure of EC to both mixtures was performed to mimic anthocyanin pharmacokinetics following their intake. Inflammatory stress was induced using TNF alpha and monocytes added to assess adhesion and transmigration. Effects of these mixtures on gene, miRNA expression and their potential interaction with cell signalling were investigated. Anthocyanins and their gut metabolites significantly reduced monocyte adhesion and transendothelial migration. Gene expression analysis, using macroarrays, showed that tested compounds modulated the expression of genes involved in cell-cell adhesion, cytoskeleton organisation or focal adhesion. Bioinformatics analyses of gene expression data identified potential transcription factors involved in the observed nutrigenomic effects and signalling proteins regulating their activity. Molecular docking revealed cell signalling proteins to which these bioactives may bind to and potentially affect their activity and the activation of downstream signalling, effects that were in agreement with the results of Western blot analyses. Microarray analysis showed that anthocyanins and their gut metabolites affected miRNA expression in EC, especially those involved in regulation of EC permeability, contributing to the observed changes in EC function. 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.