Cilostazol Inhibits Vascular Smooth Muscle Cell Proliferation and Reactive Oxygen Species Production through Activation of AMP-activated Protein Kinase Induced by Heme Oxygenase-1

Jung Eun Kim; Jin Young Sung; Chang-Hoon Woo; Young Jin Kang; Kwang Youn Lee; Hee Sun Kim; Woo Hyung Kwun; Hyoung Chul Choi

doi:10.4196/kjpp.2011.15.4.203

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Kim, Sung, Woo, Kang, Lee, Kim, Kwun, and Choi: Cilostazol Inhibits Vascular Smooth Muscle Cell Proliferation and Reactive Oxygen Species Production through Activation of AMP-activated Protein Kinase Induced by Heme Oxygenase-1

Original Article

Korean J Physiol Pharmacol 2011;15(4):203-210.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.4.203

Cilostazol Inhibits Vascular Smooth Muscle Cell Proliferation and Reactive Oxygen Species Production through Activation of AMP-activated Protein Kinase Induced by Heme Oxygenase-1

Jung Eun Kim^1,^2,^∗, Jin Young Sung^1,^2,^∗, Chang-Hoon Woo^1,², Young Jin Kang¹, Kwang Youn Lee¹, Hee Sun Kim³, Woo Hyung Kwun⁴, Hyoung Chul Choi^1,^2,

¹Department of Pharmacology, College of Medicine, Yeungnam University, Daegu 705-717, Korea

²Aging-associated Vascular Disease Research Center, College of Medicine, Yeungnam University, Daegu 705-717, Korea

³Department of Microbiology, College of Medicine, Yeungnam University, Daegu 705-717, Korea

⁴Department of Surgery, College of Medicine, Yeungnam University, Daegu 705-717, Korea

Corresponding to: Hyoung Chul Choi, Department of Pharmacology, College of Medicine, Yeungnam University, 317-1, Daemyung 5-dong, Nam-gu, Daegu 705-717, Korea. (Tel) 82-53-620-4353, (Fax) 82-53-656-7995, (E-mail) hcchoi@med.yu.ac.kr

^∗ These authors are equally contributed to this study.

Received 7 June 2011 Revised 14 July 2011 Accepted 16 July 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cAMP levels and activates protein kinase A, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMP-activated protein kinase (AMPK) activation induced by heme oxygenase-1 (HO-1) is a mediator of the beneficial effects of cilostazol and whether cilostazol may prevent cell proliferation and reactive oxygen species (ROS) production by activating AMPK in VSMC. In the present study, we investigated VSMC with various concentrations of cilostazol. Treatment with cilostazol increased HO-1 expression and phosphorylation of AMPK in a dose- and time-dependent manner. Cilostazol also significantly decreased platelet-derived growth factor (PDGF)-induced VSMC proliferation and ROS production by activating AMPK induced by HO-1. Pharmacological and genetic inhibition of HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and ROS production. These data suggest that cilostazol-induced HO-1 expression and AMPK activation might attenuate PDGF-induced VSMC proliferation and ROS production.

Keywords: Cilostazol, Proliferation, ROS, AMPK, HO-1

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Fig. 1.

Cilostazol strongly induces HO-1 as well as AMPK activation in VSMC. (A) Cells were exposed to different concentrations (1∼200 μM) of cilostazol for 24 hr and (C) treated with 100 μM of cilostazol for the indicated times. Expression of HO-1, p-AMPK and AMPK were analyzed by western blot. Each graph represents the densitometry analysis of HO-1 and p-AMPK (B, D). Data are represented as the mean±S.E.M (n=3). ^∗p-value<0.01 compared with control.

Fig. 2.

Cilostazol inhibits PDGF-stimulated VSMC proliferation via HO-1 and AMPK. Cell proliferation was determined by MTTassay (A) or BrdU incorporation assay (B) in the presence of indicated concentration of cilostazol for 24 hr. Dose-dependent inhibitory effect of cilostazol on percent change in VSMC proliferation is noted. (C) Protein expressions of HO-1 andp-AMPK were determined by western blot analysis. Data are represented as the mean±S.E.M (n=4). ^∗p-value<0.01 compared with control, ^†p-value<0.05 compared with PDGF.

Fig. 3.

Cilostazol suppresses ROS production generated by PDGF in VSMC. (A) ROS generated in viable cells produce a uniform bright green color in the cytoplasm and nuclei. For observation of intracellular ROS by fluorescence microscopy, cells were pretreated with cilostazol for 24 hr and then stimulated with PDGF (10 ng/ml) for 2 hr in the presence of 10 μM DCF-DA. (B) ROS production was assessed by FACS analysis. Data are represented as the mean±S.E.M (n=3). ^∗p-value<0.01 compared with control, ^†p-value<0.01 compared with PDGF. Cil, cilostazol.

Fig. 4.

Compound C and SNPP IX restore the cilostazol-induced inhibition of VSMC proliferation and ROS production. (A, B) Cells were pretreated with compound C or SNPP IX in the presence of cilostazol and then stimulated with PDGF. Cell proliferation was determined by the MTT assay and BrdU incorporation. (C) Protein expressions of HO-1 and p-AMPK were determined by western blot analysis. Representative blots from three independent experiments were shown. (D) For observation of intracellular ROS by fluorescence microscope, cells were pretreated with compound C or SNPP IX in the presence of cilostazol, and then stimulated with PDGF in the presence of 10 μM DCF-DA. Data are represented as the mean±S.E.M (n=4). ^∗p-value<0.01 compared with control, ^†p-value<0.01 compared with PDGF, ^#p-value<0.05 compared with cilostazol+PDGF. Cil, cilostazol; Comp C, compound C.

Fig. 5.

Genetic inhibition of HO-1 and AMPK restores the cilostazol-induced inhibition of VSMC proliferation and ROS production. (A, B) Cells were transfected with HO-1 or AMPK siRNA, and then stimulated with PDGF. Cell proliferation was assessed by the MTT assay and BrdU incorporation. (C) Protein expressions of HO-1 and p-AMPK were determined by western blot analysis. Representative blots from three independent experiments are shown. (D) For observation of intracellular ROS by fluorescence microscopy, cells were transfected with HO-1 siRNA or AMPK siRNA in the presence of cilostazol, and then stimulated with PDGF in the presence of 10 μM DCF-DA. Data are represented as the mean±S.E.M (n=4). ^∗p-value<0.01 compared with control, ^†p-value<0.01 compared with PDGF, ^#p-value<0.05 compared with cilostazol + PDGF. Cil, cilostazol.

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