Dragan Milenkovic, PhD

Dragan Milenkovic, PhD


Dr Dragan Milenkovic received his PhD from University of Versailles (France) in molecular genetics. He is currently research scientist at the French National Institute for Agricultural Research (INRA) at Clermont-Ferrand. The aim of his research is identification of molecular and cellular mechanisms underlying cardiovascular protective effect of bioactive plant compounds, particularly polyphenols. This research is based on nutrogenomic analyses of polyphenols in animal models of atherosclerosis as well in clinical trials in humans. Recently he has been studying the role of polyphenol metabolites on endothelial cell function and underlying mechanisms of action, including the research on miRNA and cell signalling pathways.


Bilberry anthocyanin-rich extract exerts atheroprotective property through complex molecular mechanism of action

Dragan Milenkovica, Aurelie Mauraya,b, Andrzej Mazura, Catherine Felginesb, Christine Moranda
a INRA, Human Nutrition Unit, UMR1019, INRA-Clermont Ferrand/Theix, 63122 Saint-Genès-Champanelle, France
b Laboratoire de Pharmacognosie, Université Clermont 1, UFR Pharmacie, 63001 Clermont-Ferrand, France

Anthocyanins are water-soluble plant pigments that belong to the large group of polyphenols and more specifically to the subclass of flavonoids. They are abundant in the human diet due to their wide occurrence in fruits, such as berries, and fruit-based beverages. Bilberry is one of the richest sources of anthocyanins, with an anthocyanin glycoside content of 300–600 mg/100 g of fresh weight. Once ingested, anthocyanin glycosides are rapidly absorbed in both the stomach and small intestine and appear in blood and urine as intact, methylated, glucurono- and/or sulfoconjugated forms. Dietary intake of anthocyanin-rich foods has been associated with a reduced risk of coronary heart disease in the Iowa Women’s Health Study, a prospective study of postmenopausal women (Mink et al., AJCN 2007.) Reduction of atherosclerotic lesions has been previously reported after supplementation of apolipoprotein E-deficient (apo E-/-) mice with anthocyanin-rich extracts from black rice and purple sweet potato (Miyazak et al., JAFC 2000). However, little is known about the molecular mechanisms underlying the cardiovascular protective effect of anthocyanins. In vitro experiments suggest that anthocyanins may affect the expression of genes in endothelial cells or macrophages, such as those encoding the cholesterol transporter ABCA-1, the pro-inflammatory enzyme COX-2 or the scavenger receptor CD36.

The aim of our studies was (1) to evaluate the effects of a bilberry anthocyanin-rich extract, when supplemented in the diet at a nutritional dose, on the development of atherosclerosis in apo E-/- mice and (2) to explore the in vivo mechanisms of action using a global transcriptomic approach. Dragan Milenkovic Ph.D. INRA/Centre de Recherche de Clemont – Ferrand/Theix, France

Male apo E-/- mice at 8 weeks of age received a diet supplemented with 0.02% of anthocyanin-rich extract from bilberry. The atherosclerotic plaque was quantified in the aortic sinus by histomorphometry. Total cholesterol, triglyceride and anti-oxidant capacity were measured in plasma and liver. Impact of anthocyanins on the expression of genes has been performed using pangenomic microarrays for both liver and aorta.

Bilberry extract is a purified anthocyanin-rich extract and the supplementation of the diet with 0.02% of this extract may correspond to an equivalent human intake of about 30 mg of anthocyanins per day; intake estimated to vary from 12.5 mg/day in the United States to 47 mg/day in Finland After a 16-week supplementation period, a significant reduction of atherosclerotic plaques was observed as compared to the control one (-15 %,). Consumption of the bilberry extracts did not modified the plasma antioxidant capacity, measured by the ORAC assay, nor the levels of markers of lipid oxidation (aortic F2-isoprostanes, hepatic TBARS). All these data support the hypothesis that the antiatherogenic effects of bilberry extracts are independent of their antioxidant capacity in this animal model.

Microarray analyses performed on the aortas of apoE -/- mice revealed that the bilberry extract-supplemented diet affected the expression of 1261 genes, with 554 genes up-regulated and 707 down-regulated. Bioinformatic analyses indicated that these differentially expressed genes are involved in the regulation of processes underlying atherosclerosis development, such as cell adhesion, migration, communication, inflammation, as well as angiogenesis and cell proliferation through vascular endothelial growth factor (VEGF) and WNT signalling pathways. The obtained gene expression profile could be related to increased inter-cellular adhesion, and decreased monocyte recruitment, cellular contractility and their regulating signalling pathways, thereby improving endothelial function and consequently lesser atherosclerosis development.

In the liver, the anthocyanin-rich extract affected the expression of 2,289 genes with 1,331 genes identified as up-regulated and 958 down-regulated. These genes are involved in various molecular and cellular pathways, such as cholesterol metabolism, VLDL removal, reverse cholesterol transport, but also inflammatory responses in the liver known to play a role in atherogenesis through the production of the pro-inflammatory cytokines.

The expression profile of these genes after bilberry extract supplementation could explain the observed reduction of plasma cholesterol level via an increased elimination as bile acids, and the reduction of triglycerides in the liver via a decrease in hepatic lipogenesis. Furthermore this gene expression profile suggests a lower inflammatory status via a decrease in the expression of pro-inflammatory genes.

Supplementation of the diet with bilberry anthocyanin-rich extract led to a significant inhibition of plaque development in apolipoprotein E deficient mice without an effect on oxidative stress parameters, suggesting the implication of other mechanisms of action. The use of holistic transcriptomic approach provided new data and a global integrative view of molecular mechanisms involved in the preventive action of bilberry anthocyanin-rich extract against atherosclerosis. Globally, bilberry extract induced changes in gene expression to an anti-inflammatory profile, which could be related to its anti-atherogenic properties.