EFFECT OF FLAVONOIDS ON OXIDATIVE STRESS, APOPTOSIS, AND CELL MARKERS OF PERIPHERAL BLOOD-DERIVED ENDOTHELIAL PROGENITOR CELLS: AN IN VITRO STUDY
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.
2. Werner N, Wassmann S, Ahlers P, Schiegl T, Kosiol S, Link A, et al. Endothelial progenitor cells correlate with endothelial function in patients with coronary artery disease. Basic Res Cardiol 2007;102:565–71.
3. Imanishi T, Tsujioka H, Akasaka T. Endothelial progenitor cells dysfunction and senescence: contribution to oxidative stress. Curr Cardiol Rev 2008;4:275-86.
4. Kirton JP, Xu Q. Endothelial precursors in vascular repair. Microvasc Res 2010;79:193-9.
5. Green DR, Llambi F. Cell death signalling. Cold Spring Harb Perspect Biol 2015;7:1-24.
6. Pizzino G, Irrera N, Cucinotta M, Pallio G, Mannino F, Arcoraci V, et al. Oxidative stress: harms and benefits for human health. Oxid Med Cell Longev 2017:1-13. DOI:10.1155/2017/8416763
7. Van Deursen JM. The role of senescent cells in ageing. Nature 2014:509:439-46.
8. Widowati W, Janeva BW, Nadya S, Amalia A, Arumwardana S, Kusuma HS, et al. Antioxidant and antiaging activities of Jasminum sambac extract, and its compounds. J Rep Pharm Sci 2018;7:270-85.
9. Widowati W, Darsono L, Suherman J, Afifah E, Rizal R, Arinta Y, et al. Mangosteen peel extract (Garcinia mangostana L.) and its constituents to lower lipid content on adipogenesis cells model (3T3-L1). J Nat Remed 2018;18:41–8.
10. Widowati W, Darsono L, Suherman J, Fauziah N, Maesaroh M, Erawijantari PP. Anti-inflammatory effect of mangosteen (Garcinia mangostana L.) peel extract and its compounds in LPS-induced RAW264.7 cells. Nat Prod Sci 2016;22:147-53.
11. Feliciano RP, Pritzel S, Heiss C, Rodriguez Mateos A. Flavonoid intake and cardiovascular disease risk. Curr Opin Food Sci 2015;2:92–9.
12. McCullough ML, Peterson JJ, Patel R, Jacques PF, Shah R, Dwyer JT. Flavonoid intake and cardiovascular disease mortality in a prospective cohort of US adults. Am J Clin Nutr 2012;95:454-64.
13. Asamina K, Gary W. At the interface of antioxidant signalling and cellular function: key polyphenol effects. Mol Nutr Food Res 2016;60:1770-88.
14. Widowati W, Widyanto RM, Husin W, Ratnawati H, Laksmitawati DR, Setiawan B, et al. Green tea extract protects endothelial progenitor cells from oxidative insult through reduction of intracellular reactive oxygen species activity. Iran J Basic Med Sci 2014;17:702-9.
15. Widowati W, Wijaya L, Laksmitawati DR, Widyanto RM, Erawijantari PP, Fauziah N, et al. Tea flavonoids induced differentiation of peripheral blood-derived mononuclear cells into peripheral blood-derived endothelial progenitor cells and suppressed intracellular reactive oxygen species level of peripheral blood-derived endothelial progenitor cells. Nat Prod Sci 2016;22:87-92.
16. Zhao LR, Du YJ, Chen L, Liu ZG, Pan YH, Liu JF, et al. Quercetin protects against high glucose-induced damage in bone marrow-derived endothelial progenitor cells. Int J Mol Med 2014;34:1025–31.
17. Hagiwara H, Higashibata A, Ogawa S, Kanazawa S, Mizuno H, Tanaka R. Effectiveness of endothelial progenitor cell culture under microgravity for improved angiogenic potential. Sci Rep 2018;8:1-10.
18. Wlodkowic D, Skommer J, Darzynkiewicz Z. Flow cytometry-based apoptosis detection. Methods Mol Biol 2009;559:19-32.
19. Henneberg R, Otuki MF, Furman AEF, Hermann P, do Nascimento AJ, Leonart MSS. Protective effect of flavonoids against reactive oxygen species production in sickle cell anemia patients treated with hydroxyurea. Rev Bras Hematol Hemoter 2012:35:52-5.
20. Fleissner F, Thum T. Critical role of the nitric oxide/reactive oxygen species balance in endothelial progenitor dysfunction. Antioxid Redox Signal 2011;15:933–48.
21. Si H, Yu J, Jiang H, Lum H, Liu D. Phytoestrogen genistein up-regulates endothelial nitric oxide synthase expression via activation of cAMP response element-binding protein in human aortic endothelial cells. Endocrinol 2012;153:3190-8.
22. Ahn HY, Kim CH. Epigallocatechin-3-gallate regulates inducible nitric oxide synthase expression in human umbilical vein endothelial cells. Lab Anim Res 2011;27:85-90.
23. Horie K, Nanashima N, Maeda H. Phytoestrogenic effects of blackcurrant anthocyanins increased endothelial nitric oxide synthase (eNOS) expression in human endothelial cells and ovariectomized rats. Molecules 2019;24:1259-70.
24. Forte M, Conti V, Damato A, Ambrosio M, Puca AA, Sciarretta S, et al. Targeting nitric oxide with natural derived compounds as a therapeutic strategy in vascular diseases. Oxid Med Cell Longev 2016:1-20. DOI:10.1155/2016/7364138
25. Huang PH, Chen YH, Tsai HY, Chen JS, Wu TC, Lin FY, et al. Intake of red wine increases the number and functional capacity of circulating endothelial progenitor cells by enhancing nitric oxide bioavailability. Arterioscler Thromb Vasc Biol 2010;30:869–77.
26. Li PG, Sun L, Han X, Ling S, Gan W, Xu JW. Quercetin induces rapid eNOS phosphorylation and vasodilation by an Akt-Independent and PKA-dependent mechanism. Pharmacology 2012;89:220-8.
27. Wen HJ, Liu GF, Xiao LZ, Wu YG. Involvement of endothelial nitric oxide synthase pathway in IGF?1 protects endothelial progenitor cells against injury from oxidized LDLs. Mol Med Rep 2018;19:660-6.
28. Hyun PW. The effects of exogenous H2O2 on cell death, reactive oxygen species and glutathione levels in calf pulmonary artery and human umbilical vein endothelial cells. Int J Mol Med 2012;31:47-6.
29. Zhang HW, Hu JJ, Fu RQ, Liu X, Zhang YH, Li J, et al. Flavonoids inhibit cell proliferation and induce apoptosis and autophagy through downregulation of PI3K? mediated PI3K/AKT/mTOR/p70S6K/ULK signaling pathway in human breast cancer cells. Nat Sci Rep 2018;8:1-13.
30. Herrmann M, Binder A, Menzel U, Zeiter S, Alini M, Verrier S. CD34/CD133 enriched bone marrow progenitor cells promote neovascularization of tissue-engineered constructs in vivo. Stem Cell Res 2014;13:465–77.
31. Decano JL, Moran AM, Giordano N, Ruiz-Opazo N, Herrera VL. Analysis of CD45-[CD34+/KDR+] endothelial progenitor cells as juvenile protective factors in a rat model of ischemic-hemorrhagic stroke. PLoS One 2013;8:e55222.
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