Dragan Milenkovic

Hesperidin contributes to the vascular protective effects of orange juice: a randomized crossover study in healthy volunteers

BACKGROUND Although numerous human studies have shown consistent effects of some polyphenol-rich foods on several intermediate markers for cardiovascular diseases, it is still unknown whether their action could be specifically related to polyphenols. OBJECTIVE We investigated the effect of orange juice and its major flavonoid, hesperidin, on microvascular reactivity, blood pressure, and cardiovascular risk biomarkers through both postprandial and chronic intervention studies. DESIGN Twenty-four healthy, overweight men (age 50-65 y) were included in a randomized, controlled, crossover study. Throughout the three 4-wk periods, volunteers daily consumed 500 mL orange juice, 500 mL control drink plus hesperidin (CDH), or 500 mL control drink plus placebo (CDP). All measurements and blood collections were performed in overnight-fasted subjects before and after the 4-wk treatment periods. The postprandial study was conducted at the beginning of each experimental period. RESULTS Diastolic blood pressure (DBP) was significantly lower after 4 wk consumption of orange juice or CDH than after consumption of CDP (P = 0.02), whereas microvascular endothelium-related reactivity was not significantly affected when measured after an overnight fast. However, both orange juice and CDH ingestion significantly improved postprandial microvascular endothelial reactivity compared with CDP (P < 0.05) when measured at the peak of plasma hesperetin concentration. CONCLUSIONS In healthy, middle-aged, moderately overweight men, orange juice decreases DBP when regularly consumed and postprandially increases endothelium-dependent microvascular reactivity. Our study suggests that hesperidin could be causally linked to the beneficial effect of orange juice. This trial is registered at clinicaltrials.gov as NCT00983086.

Citrus Flavanones: What Is Their Role in Cardiovascular Protection?

Flavanones, including hesperidin and naringin, are polyphenolic compounds highly and almost exclusively present in citrus. Epidemiological studies reported an inverse relationship between their intake and the risk of cardiovascular diseases. Clinical and experimental data further showed their antihypertensive, lipid-lowering, insulin-sensitizing, antioxidative, and anti-inflammatory properties, which could explain their antiatherogenic action in animal models. Although flavanones may be promising compounds that are particularly active in cardiovascular disease prevention, clinical data are still scarce and most in vitro data have been obtained under nonphysiologically relevant conditions. Moreover, the mechanisms responsible for flavanone action are not fully elucidated. Therefore, further research is needed to better evaluate and understand the protective effects of flavanones in cardiovascular diseases.

Modulation of miRNA Expression by Dietary Polyphenols in apoE Deficient Mice: A New Mechanism of the Action of Polyphenols

Background Polyphenols are the most abundant antioxidants in the human diet and are widespread constituents of fruits and beverages, such as tea, coffee or wine. Epidemiological, clinical and animal studies support a role of polyphenols in the prevention of various diseases, such as cardiovascular diseases, cancers or neurodegenerative diseases. Recent findings suggest that polyphenols could interact with cellular signaling cascades regulating the activity of transcription factors and consequently affecting the expression of genes. However, the impact of polyphenol on the expression of microRNA, small non-coding RNAs, has not yet been studied. The aim of this study was to investigate the impact of dietary supplementation with polyphenols at nutritional doses on miRNA expression in the livers of apolipoprotein E-deficient mice (apoE−/−) jointly with mRNA expression profiling. Methodology/Principal Findings Using microarrays, we measured the global miRNA expression in the livers of wild-type (C57B6/J) mice or apoE−/− mice fed diets supplemented with one of nine different polyphenols or a control diet. This analysis revealed that knock-out of the apoE gene induced significant modulation in the expression of miRNA. Moreover, changes in miRNA expression were observed after polyphenol supplementation, and five miRNAs (mmu-miR-291b-5p, mmu-miR-296-5p, mmu-miR-30c-1*, mmu-miR-467b* and mmu-miR-374*) were identified as being commonly modulated by these polyphenols. We also observed that these polyphenols counteracted the modulation of miRNA expression induced by apoE mutation. Pathway analyses on these five miRNA-target genes revealed common pathways, some of which were also identified from a pathway analysis on mRNA profiles. Conclusion This in vivo study demonstrated for the first time that polyphenols at nutritional doses modulate the expression of miRNA in the liver. Even if structurally different, all polyphenols induced a similar miRNA expression profile. Common pathways were identified from both miRNA-target and mRNA analysis, revealing cellular functions that could be regulated by polyphenols at both the miRNA and mRNA level.

Cytogenetic localization of 136 genes in the horse: comparative mapping with the human genome

The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clones. For the first time, genes were mapped to ECA13p, ECA29, and probably ECA30. A total of 284 genes are now FISH mapped on the horse chromosomes. Comparison with the human map defines 113 conserved segments that include new homologous segments not identified by Zoo-FISH on ECA7 and ECA13p.

Amino acid limitation regulates the expression of genes involved in several specific biological processes through GCN2-dependent and GCN2-independent pathways

Evidence has accumulated that amino acids play an important role in controlling gene expression. Nevertheless, two components of the amino acid control of gene expression are not yet completely understood in mammals: (a) the target genes and biological processes regulated by amino acid availability, and (b) the signaling pathways that mediate the amino acid response. Using large‐scale analysis of gene expression, the objective of this study was to gain a better understanding of the control of gene expression by amino acid limitation. We found that a 6 h period of leucine starvation regulated the expression of a specific set of genes: 420 genes were up‐regulated by more than 1.8‐fold and 311 genes were down‐regulated. These genes were involved in the control of several biological processes, such as amino acid metabolism, lipid metabolism and signal regulation. Using GCN2−/− cells and rapamycin treatment, we checked for the role of mGCN2 and mTORC1 kinases in this regulation. We found that (a) the GCN2 pathway was the major, but not unique, signaling pathway involved in the up‐ and down‐regulation of gene expression in response to amino acid starvation, and (b) that rapamycin regulates the expression of a set of genes that only partially overlaps with the set of genes regulated by leucine starvation.

Naringin, the major grapefruit flavonoid, specifically affects atherosclerosis development in diet-induced hypercholesterolemia in mice

Naringin (NAR) from grapefruit has exhibited potential protective effects against atherosclerosis development. However, specific mechanisms responsible for such effects are poorly understood. Thus, we aimed to investigate the antiatherogenic effects of NAR in different mouse models of hypercholesterolemia and decipher its molecular targets in the aorta using transcriptomic approach. Two mouse models of hypercholesterolemia, wild-type mice fed a high-fat/high-cholesterol diet and apolipoprotein E-deficient mice fed a semisynthetic diet, were studied. Mice were fed a respective control diets supplemented or not for 18 weeks with 0.02% of NAR, that is, nutritional supplementation. NAR supplementation reduced plaque progression only in wild-type mice fed the high-fat/high-cholesterol diet (-41%). Consistent with this protective effect, NAR reduced plasma non-high-density lipoprotein cholesterol concentrations as well as biomarkers of endothelial dysfunction. Microarray studies performed on aortas demonstrated differentially expressed genes encoding proteins involved in cell adhesion, actin cytoskeleton organization and cell division. Thus, the changes in gene expression induced by NAR could suggest a limited atherosclerosis progression by preventing immune cell adhesion and infiltration in the intima of vascular wall, as well as smooth muscle cell proliferation. Furthermore, this hypothesis was strengthened by in vitro experiments, which showed the ability of naringenin to reduce monocyte adhesion to endothelial cells and smooth muscle cell proliferation. In conclusion, this study revealed the antiatherogenic effect of NAR supplemented at a nutritionally achievable dose, specifically toward diet-induced atherosclerosis, and depicted its multitarget mode of action at the vascular level.

Catechin reduces atherosclerotic lesion development in apo E-deficient mice: A transcriptomic study

Much experimental evidence supports a protective role of dietary flavonoids against cardiovascular diseases. The aim of the present study was to investigate the anti-atherosclerotic effects of catechin supplemented in the diet of apoE deficient mice at a low nutritional level and to explore the mechanisms of action by a transcriptomic approach. After 6 weeks of supplementation, atherosclerotic lesions were assessed by histomorphometry and several markers of lipid, inflammation and oxidative stress status were evaluated. Analysis of the global gene expression in the aorta was carried out using pangenomic arrays. Catechin supplementation reduced the mean atherosclerotic lesion area by 32% but had no effect on total cholesterol and triacylglycerol levels in the plasma and the liver. The plasma antioxidant capacity (FRAP) and inflammatory status (serum amyloid A) were unchanged. The expression of 450 genes was significantly modified by catechin supplementation. Some of the most significantly down-regulated genes included genes coding for adhesion molecules such as CD34 and PSGL-1 known to play a key role in leukocyte adhesion to the endothelium. Other genes involved in energy metabolism, lipid metabolism and lipids trafficking such as FABP4, LPL and SCARA5 were down-regulated and may contribute to the atheroprotective effect of catechin. This work shows that transcriptomic allows characterizing the biological effects of low doses of flavonoids where common markers were not significantly affected.

Bilberry anthocyanin-rich extract alters expression of genes related to atherosclerosis development in aorta of apo E-deficient mice ☆

Intake of anthocyanin-rich foods has been associated with a reduced risk of cardiovascular diseases. We recently reported that a nutritional supplementation with a bilberry anthocyanin-rich extract (BE) attenuates atherosclerotic lesion development in apolipoprotein E-deficient (apoE⁻/⁻) mice. However, the mechanism(s) of their preventive action are not completely understood. Anthocyanins may alter mRNA levels of genes related to atherosclerosis in cultured macrophages and endothelial cells, but in vivo studies remain scarce. The aim of the present study was to explore the in vivo mechanisms of action of the same bilberry extract, administered by supplementation at a nutritional level, in the aorta of apo E⁻/⁻ mice using a global transcriptomic approach. This study focused on the early stage of atherosclerosis development for better assessment of BE action on initiation mechanisms of this pathology. After a two week period, plasma lipid and antioxidant capacity were evaluated and the global genomic analysis was carried out using pangenomic microarrays. BE supplementation significantly improved hypercholesterolemia whereas the plasmatic antioxidant status remained unchanged. Nutrigenomic analysis identified 1261 genes which expression was modulated by BE in the aorta. Bioinformatic analysis revealed that these genes are implicated in different cellular processes such as oxidative stress, inflammation, transendothelial migration and angiogenesis, processes associated with atherosclerosis development/protection. Some of the most significantly down-regulated genes included genes coding for AOX1, CYP2E1 or TXNIP implicated in the regulation of oxidative stress, JAM-A coding for adhesion molecules or VEGFR2 implicate in regulation of angiogenesis. Other genes were up-regulated, such as CRB3, CLDN14 or CDH4 potentially associated with increased cell-cell adhesion and decreased paracellular permeability. These results provide a global integrated view of the mechanisms involved in the preventive action of bilberry anthocyanin-rich extract against atherosclerosis.

Hesperidin Displays Relevant Role in the Nutrigenomic Effect of Orange Juice on Blood Leukocytes in Human Volunteers: A Randomized Controlled Cross-Over Study

Background We previously showed, in healthy, middle-aged, moderately overweight men, that orange juice decreases diastolic blood pressure and significantly improves postprandial microvascular endothelial reactivity and that hesperidin could be causally linked to the observed beneficial effect of orange juice. The objective was to determine the effect of chronic consumption of orange juice on the gene expression profile of leukocytes in healthy volunteers and to assess to what extent hesperidin is involved in the effect of orange juice. Methodology/Principal Findings Volunteers were included in a randomized, controlled, crossover study. Throughout three 4-week periods, volunteers consumed daily: 500 ml orange juice, 500 ml control drink plus hesperidin or 500 ml control drink and placebo. Blood samplings were performed on 10 overnight-fasted subjects after the 4-week treatment period. Global gene expression profiles were determined using human whole genome cDNA microarrays. Both orange juice and hesperidin consumption significantly affected leukocyte gene expression. Orange juice consumption induced changes in expression of, 3,422 genes, while hesperidin intake modulated the expression of 1,819 genes. Between the orange juice and hesperidin consumption groups, 1,582 regulated genes were in common. Many of these genes are implicated in chemotaxis, adhesion, infiltration and lipid transport, which is suggestive of lower recruitment and infiltration of circulating cells to vascular wall and lower lipid accumulation. Conclusions This study shows that regular consumption of orange juice for 4 weeks alters leukocyte gene expression to an anti-inflammatory and anti-atherogenic profile, and hesperidin displays a relevant role in the genomic effect of this beverage. Trial Registration ClinicalTrials.gov NCT 00983086

Apple polyphenols and fibers attenuate atherosclerosis in apolipoprotein E-deficient mice.

Atherosclerosis, which is closely linked to nutritional habits, is a major cause of mortality in Western countries. Most of the previous investigations carried out on health effects of apples have been focused on their capacity to lower lipid concentration as well as on their antioxidant effects. The aim of the present study was to investigate the antiatherosclerotic effects of apple polyphenols and fibers. A crude apple polyphenol extract and low-viscosity apple fibers isolated from cider apples were administered separately or in association with the diet of apo E-deficient mice. After 4 months of supplementation, lipemia and oxidative stress biomarkers were measured and atheroslerotic lesions assessed by histomorphometry. Total plasmatic cholesterol and triacylgycerol levels were not affected by supplementation, and hepatic cholesterol level was lower in the group supplemented with both fibers and polyphenols. Uric acid concentrations and antioxidant capacity (FRAP) in plasma were reduced in all groups supplemented with polyphenols or fibers. The mean lesion area was reduced by 17, 38, and 38%, respectively, for the polyphenol, fiber, and polyphenol + fiber groups. Apple constituents supplied at nutritional doses therefore limit the development of atherosclerotic lesions in the aorta of apo E-deficient mice. On the basis of the results, we hypothesize that apple fibers and polyphenols may play a role in preventing atherosclerosis disease by decreasing uric acid plasma level.