Abstract
Objective: Circulating EPCs (endothelial progenitor cells) play a role in neovascularization and vascular repair. Oxidative stress impairs endothelial progenitor. Flavonoid is a phytochemical compound for antioxidant activity. Flavonoid effects toward oxidative stress, apoptosis, and expression of the cell markers on EPCs are not fully understood. This study was aimed to elucidate the effects of quercetin, kaempferol, and myricetin toward oxidative stress, apoptosis, and cell markers of peripheral blood-derived-EPCs.
 Methods: EPCs (endothelial progenitor cells) were isolated from peripheral blood mononuclear cells (PBMNCs) using cultivation under EPCs spesific media. Oxidative stress in EPCs was induced by H2O2 and then treated by quercetin, kaempferol, and myricetin. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, while intracellular reactive oxygen species (ROS), apoptosis and characterization of cells, which expressed CD133 and KDR, was measured using flow cytometry.
 Results: Quercetin, kaempferol, and myricetin at concentration 12.50 µmol/l were not toxic on EPCs as the cells viability were 96.11±4.03%, 95.42±7.75%, and 94.22±9.49%, respectively. Flavonoids decreased intracellular ROS level in EPCs (quercetin: 14.38±1.47%, kaempferol: 20.21±6.25%, and myricetin: 13.88±4.02%) compared to EPCs treated with H2O2 (30.70%±1.04). Percetage of EPCs apoptosis was not significantly different among each treatment. Immunophenotyping showed the increasing of CD133 and KDR expression in EPCs treated with flavonoids.
 Conclusion: Quercetin, kaempferol, and myricetin were safe for EPCs, decreased ROS levels, and increased CD133 and KDR expression. However, the flavonoids did not significantly affect EPCs apoptosis.
Highlights
Endothelial progenitor cells (EPCs) are bone marrow-derived cells that can be found in peripheral blood
Flavonoids decreased intracellular reactive oxygen species (ROS) level in EPCs compared to EPCs treated with H2O2 (30.70%±1.04)
Immunophenotyping showed the increasing of CD133 and KDR expression in EPCs treated with flavonoids
Summary
Human blood samples (n=3) were provided by healthy human volunteers. All volunteers signed an informed consent prior to blood collection. The nucleus staining was done using 2'-7'dichlorofluorescein diacetate (DCF-DA) and 4',6-diamidino-2phenylindole (DAPI) The cells both positive FITC-UEA-I and Dil-ac LDL were characterized as EPCs [14, 15]. The EPCs were treated using different concentrations (100; 50; 25; 12.5 μmol/l) of quercetin, kaempferol, and myricetin that were diluted in dimethyl sulfoxide (DMSO). EPCs (5 × 105) were inoculated with serum-free medium in 6-well plates incubated at 37 °C, humidified, and CO2 5%. EPCs were treated with quercetin, kaempferol, and myricetin (12.5 μmol/l) diluted in DMSO for 24 h. EPCs were harvested and stained with DCF-DA (10 μmol/l) at 37 °C, humidified, and CO2 5% for 30 min. EPCs were treated with, kaempferol, quercetin, and myricetin (12.5 μmol/l) diluted in DMSO and incubated for 24 h. The result is considered as significant if the Pvalue was lower than 0.05
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