Dietary Ellagitannin Metabolites Exert Antiatherosclerotic Effects in Vitro

Reduction of the prevalence of atherosclerotic cardiovascular disease through the consumption of polyphenol-enriched diets has been established in several epidemiological studies. Ellagitannins are polyphenols mainly found in berries, walnuts and pomegranate. These compounds are poorly absorbed after oral ingestion and are metabolized by the colonic microbiota to form a series of catabolites called urolithins. We hypothesized that these circulating metabolites may be, at least in part, the molecules responsible for the beneficial effects against atherosclerosis attributed to ellagitannin-containing foods. In the present study we investigated the activity of urolithin A,B,C,D and B glucoronide in vitro on processes involved in atherogenesis. Cell cholesterol uptake was quantified by a fluorimetric assay in THP-1 macrophages, incubated with urolithins 1-10µM in presence of human serum or acetylated LDL as cholesterol source. Cholesterol efflux was evaluated by a radioisotope-based assay in THP-1 macrophages exposed to urolithins 10µM and human HDL as cholesterol acceptors. Monocyte adhesion to endothelial cells was investigated in human umbilical vein endothelial cells (HUVECs) treated with urolithins 1-10µM for different times, stimulated with TNF-α, and exposed to THP-1 monocytes. Monocyte adhesion to extracellular matrix was performed in THP-1 monocytes treated with urolithins 10µM and successively transferred to fibronectin-coated wells. In these assays, adherent cells were quantified by a fluorimetric or spectrophotometric count respectively. Urolithin C and D reduced cholesterol accumulation induced by human serum in THP-1 macrophages by 25% (p<0.001 for both), whereas cholesterol efflux was not affected by any compound. Urolithin C inhibited THP-1 adhesion to HUVECs after 6h (-44%; p<0.001) and to a lesser extent after 24h of treatment (-29%; p<0.05), whereas no effects were observed after shorter treatment. The co-incubation of urolithin A and B for 6 hours significantly decreased THP-1 monocyte adhesion to fibronectin (-41%; p<0.05). Longer times of treatment resulted in the loss of this effect. Urolithins may retard the formation of atherosclerotic plaque by inhibiting monocyte adhesion to extracellular matrix and to HUVECs and by contrasting macrophage foam cell formation. The present study identifies the key players and describes the mode of action through which the ellagitannin-containing foods elicit their protecting effects against cardiovascular diseases.