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

• Kim JE
• Sung JY
• Woo CH
• Kang YJ
• Lee KY
• Kim HS
• Kwun WH
• Choi HC

• KMID: 1448925
• DOI: http://doi.org/10.4196/kjpp.2011.15.4.203

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.

Keyword

Cilostazol; Proliferation; ROS; AMPK; HO-1

MeSH Terms

AMP-Activated Protein Kinases
Cell Proliferation
Cyclic AMP-Dependent Protein Kinases
Heme
Heme Oxygenase-1
Muscle, Smooth, Vascular
Phosphorylation
Platelet-Derived Growth Factor
Reactive Oxygen Species
Tetrazoles
AMP-Activated Protein Kinases
Cyclic AMP-Dependent Protein Kinases
Heme
Heme Oxygenase-1
Platelet-Derived Growth Factor
Reactive Oxygen Species
Tetrazoles

Figure

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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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