International Journal of Cardiology 181 (2015) 160–165

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Letter to the Editor

Oleuropein prevents angiotensin II-mediated Human vascular progenitor cell depletion Sung Hyun Choi a,1, Hong Beom Joo a,1, Seon Jin Lee a, He Yun Choi a, Ji Hye Park a, Sang Hong Baek b,⁎, Sang Mo Kwon a,⁎⁎ a b

Convergence Stem Cell Research Center, Department of Physiology, Pusan National University School of Medicine, South Korea Division of Cardiovascular Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea School of Medicine, South Korea

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Article history: Received 3 November 2014 Accepted 23 November 2014 Available online 25 November 2014 Keywords: Angiotensin II Oleuropein Reactive oxygen species Vascular progenitor cell Peroxiredoxin

Dear Editor Although vascular progenitor cells (VPCs) have great potential for use in stem cell-based therapy to regenerate damaged blood vessels, pathological conditions, including hypertension, accelerate reactive oxygen species (ROS)-mediated VPC depletion [1]. ROS have a doubleedged effect, regulating cell signal transduction at non-toxic levels, while accelerating cellular dysfunction when generated in excess [2,3]. Thus, in order for stem cell-based cell therapy for cardiovascular regeneration to succeed, ROS-mediated VPC depletion must be overcome by the development of novel stem cell priming technologies. Mammalian cells have unique defense mechanisms against oxidative stress, including peroxidases (e.g., catalase, glutathione peroxidase, and superoxide dismutase). Accumulating evidence suggests that anti-hypertensive agents [4,5], antioxidants [6], and natural compounds [7] can reduce oxidative stress by regulating ⁎ Correspondence to: S.H. Baek, Division of Cardiovascular Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea School of Medicine, 505, Banpo-dong, Seocho-gu, Seoul 137-070, South Korea. ⁎⁎ Correspondence to: S.M. Kwon, Department of Physiology, Pusan National University School of Medicine, Mulgeum-eup, Beomeo-ri, YangSan, Gyeongsangnam-do, South Korea. E-mail addresses: [email protected] (S.H. Baek), [email protected] (S.M. Kwon). 1 First Two Authors equally contributed to this study.

http://dx.doi.org/10.1016/j.ijcard.2014.11.174 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

antioxidant enzymes. Peroxiredoxins (Prdx) are a new type of peroxidases that play an important role as antioxidants, because Prdx is abundant in the cell cytosol, mitochondria, peroxisome, and plasma membrane [8]; and they have a high binding affinity for oxygen-derived radicals [9]. Oleuropein (OLP, Fig. 1A), an olive oil extract, has great potential as a VPC primer because of its high antioxidant activity and bioavailability [10]. The mechanisms by which OLP regulates Prdx 1/2 expression and angiotensin II (AngII)-mediated VPC depletion are unknown. In this study, we have examined OLP treatment reduced AngII-mediated oxidative stress and the regulation of Prdx-1/2 expression in human VPCs (hVPCs). hVPCs were isolated from human umbilical cord blood, supplied by Pusan National University YangSan Hospital, as previously described [11]. The Ethical Review Board of the Pusan National University YangSan Hospital, Gyeongsangnam-do, South Korea, approved the protocols. The experimental study was conducted in accordance with the Declaration of Helsinki. The ability of AngII (AngII, Sigma-Aldrich, St. Louis, MO) and OLP (Extrasynthese, Genay Cedex, France) to affect the viability of isolated hVPCs was examined by MTS assay (Ez cytox, Dail Lab, Seoul, South Korea). Cellular oxidative stress was examined by carboxylH2DFFDA (Molecular Probes, Carlsbad, CA) and dehydroethidium (DHE; Molecular Probes) staining. The expression of endogenous antioxidant enzymes was analyzed by western blot. hVPC viability was significantly reduced after treatment with 10 μM of AngII (p b 0.01, Fig. 1B), but not at lower concentrations (1 μM, 100 nM, or 10 nM). OLP had no significant effect on cell viability (Fig. 1C). Based on these results, we used AngII at a concentration of 100 nM in further experiments. The fluorescence intensity of the staining for cellular superoxide anion and total reactive oxygen species were measured (ImageJ, Free software, http://rsb.info.nih/gob/ij/). Superoxide anion levels (red fluorescence) in hVPCs (Fig. 2A) were significantly increased after treatment with 100 nM AngII (p b 0.01, Fig. 2B). This increase in fluorescence intensity was significantly reduced after treatment with OLP (p b 0.01, Fig. 2B). The patterns of carboxyl-H2DFFDAand DHE fluorescence were similar (Fig. 2C,D). Thus, treatment with OLP reduced AngII-mediated cellular oxidative stress in hVPCs. Next, we examined whether OLP regulated endogenous anti-oxidant enzymes such as SOD-1, Prdx-1, and Prdx-2. OLP did not affect the SOD1 expression pattern (p N 0.05, Fig. 3A). Prdx-1 and -2 expressions were

S.H. Choi et al. / International Journal of Cardiology 181 (2015) 160–165

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Fig. 1. Chemical structure of oleuropein (OLP) and cell viability assay. Representative chemical structure of OLP (A). Cell viability depends after treatment with angiotensin II (AngII, B), and oleuropein (OLP, C) using the MTS assay.

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regulated by ERK1/2 phosphorylation. The expression of phosphorylated-ERK1/2 (p-ERK1/2) was significantly reduced in the AngII-treated group (p b 0.01, Fig. 3D). The reduced p-ERK1/2 expression was significantly increased after treatment with OLP (p b 0.01, Fig. 3D). Akt and eNOS phosphorylation were significantly reduced after treatment with AngII (p b 0.05, Fig. 3E), and the down-regulated Akt and eNOS phosphorylation were significantly increased after treatment with OLP (p b 0.01, Fig. 3E). These results show that OLP reduces oxidative stress through the ERK1/2-Prdx signaling pathways, and that OLP may increase Akt/eNOS signaling. In this study, we first demonstrated that treatment with OLP regulates AngII-mediated oxidative stress through regulation of endogenous Prdx-1 and -2 expressions. Excessive generation of ROS is associated with diverse human diseases, including cardiovascular disease [12]. Superoxide anion (O− 2 ) is a highly active oxygen radical that accelerates the onset of oxidative stress by tissue oxidation. In hypertension, O− 2 is generated by NADPH oxidase subunit activation through AngII receptor antagonism [13]. We observed that intracellular O− 2 levels in hVPCs were significantly increased after treatment with AngII, and increased O− 2 levels were significantly reduced after treatment with OLP. Cellular O− 2 is converted to hydrogen peroxide (H2O2) by superoxide dismutase1 (SOD-1). However, treatment with OLP did not affect SOD-1 expression (Fig. 3A). This result indicates that treatment with OLP could directly scavenge O− 2 [14]. H2O2 converted to hydroxyl radicals by Fenton's reaction, causes cellular damage by lipid peroxidation [15]. However, H2O2 can be removed though endogenous peroxidase including Prdx [16]. Our results show that intracellular H2O2 levels, shown by carboxyl-H2DFFDA staining, were significantly increased after treatment with AngII. However, treatment with OLP reduced intracellular H2O2 levels. Prdx-1 and -2 expression levels were similarly reduced after treatment with AngII and recovered after treatment with OLP. These results show that treatment with OLP reduces cellular H2O2 levels by increasing endogenous Prdx-1 and -2 expressions. Cellular Prdx-1 and -2 expressions are regulated by the ERK1/2mediated signaling axis [17]. Furthermore, our previous research suggested that specific ERK1/2 inhibition could reduce Prdx-1 and -2 expressions and rescue cellular senescence in human cardiac stem cells [18]. Here we have shown that treatment with AngII reduces ERK1/2 phosphorylation in hVPCs, and this reduced phosphorylation was reversed after treatment with OLP. The Akt/eNOS signaling axis plays an important role in hVPC function, including angiogenesis ability [19]. In our studies, reduced Akt/eNOS phosphorylation signaling was recovered by treatment with OLP, indicating that OLP could improve AngII-mediated hVPC dysfunction. Based on the above results, we concluded that treatment with OLP, a biologically safe natural compound, could attenuate AngII-mediated oxidative stress and VPC depletion via regulation of ERK1/2-Prdx and Akt/ eNOS signaling. Thus, OLP could have beneficial effects in the development of phytomedicinal drugs for vascular dysfunction and offers a novel cellular priming technology for improvement of stem cell function.

Conflict of interest significantly reduced after treatment with AngII (p b 0.01, Fig. 3B,C). The reduced Prdx-1 and -2 expressions were significantly increased after treatment with OLP in a concentration-dependent manner (p b 0.01, Fig. 3B,C). Furthermore, the expression of Prdx-SO3 in the AngII-treated group was significantly higher than the control group, and was significantly reduced after treatment with OLP (Fig. 3C). These results indicate that treatment with OLP reduces cellular oxidative stress via regulation of Prdx-1 and -2 expressions. We also examined whether treatment with OLP could regulate ERK1/2 phosphorylation, because Prdx-1 and -2 expressions are

The authors report no relationships that could be construed as a conflict of interest.

Acknowledgments This study was supported by a Grant from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI13C1256).

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[10] A. Parzonko, M.E. Czerwinska, A.K. Kiss, M. Naruszewicz, Oleuropein and oleacein may restore biological functions of endothelial progenitor cells impaired by angiotensin II via activation of Nrf2/heme oxygenase-1 pathway, Phytomedicine 20 (2013) 1088–1094. [11] J.H. Lee, S.H. Lee, S.Y. Yoo, T. Asahara, S.M. Kwon, CD34 hybrid cells promote endothelial colony-forming cell bioactivity and therapeutic potential for ischemic diseases, Arterioscler. Thromb. Vasc. Biol. 33 (2013) 1622–1634. [12] K. Sugamura, J.F. Keaney Jr., Reactive oxygen species in cardiovascular disease, Free Radic. Biol. Med. 51 (2011) 978–992. [13] A.M. Garrido, K.K. Griendling, NADPH oxidases and angiotensin II receptor signaling, Mol. Cell. Endocrinol. 302 (2009) 148–158. [14] F. Visioli, G. Bellomo, C. Galli, Free radical-scavenging properties of olive oil polyphenols, Biochem. Biophys. Res. Commun. 247 (1998) 60–64. [15] L.J. Marnett, Lipid peroxidation-DNA damage by malondialdehyde, Mutat. Res. 424 (1999) 83–95. [16] S.G. Rhee, S.W. Kang, W. Jeong, T.S. Chang, K.S. Yang, H.A. Woo, Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins, Curr. Opin. Cell Biol. 17 (2005) 183–189. [17] Y.H. Han, J.H. Kwon, D.Y. Yu, E.Y. Moon, Inhibitory effect of peroxiredoxin II (Prx II) on Ras-ERK-NFkappaB pathway in mouse embryonic fibroblast (MEF) senescence, Free Radic. Res. 40 (2006) 1182–1189. [18] S.H. Choi, S.Y. Jung, S.Y. Yoo, S.M. Yoo, Y. Kim da, S. Kang, et al., Regulation of ROS-independent ERK signaling rescues replicative cellular senescence in ex vivo expanded human c-kit-positive cardiac progenitor cells, Int. J. Cardiol. 169 (2013) 73–82. [19] J. Yu, Q. Wang, H. Wang, W. Lu, W. Li, Z. Qin, et al., Activation of liver X receptor enhances the proliferation and migration of endothelial progenitor cells and promotes vascular repair through PI3K/Akt/eNOS signaling pathway activation, Vasc. Pharmacol. 62 (2014) 150–161.

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Fig. 2. Cellular ROS level analysis. Cellular superoxide anion level was analyzed by dehydroethidium stain (A). Quantification of the results is presented in B. Cellular total reactive oxygen species levels were analyzed by carboxyl-H2DFFDA stain (C), and the quantified results are presented in D. * — p b 0.01; vs. control, # — p b 0.01; vs. AngII-treated. Scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Protein expression analysis after treatment with OLP in AngII-induced VPCs. Analysis of expression pattern of SOD-1 (A), Prdx-1/Prdx-SO3 (B), Prdx-2 (C), ERK (D), and Akt/eNOS (E). * — p b 0.05, vs. control, ** — p b 0.01 vs. control, # — p b 0.05 vs. AngII-treated, ## — p b 0.01 vs. AngII-treated.

Oleuropein prevents angiotensin II-mediated: Human vascular progenitor cell depletion.

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