Elsevier

Atherosclerosis

Volume 189, Issue 2, December 2006, Pages 350-357
Atherosclerosis

Activation of endothelial nitric oxide synthase by cilostazol via a cAMP/protein kinase A- and phosphatidylinositol 3-kinase/Akt-dependent mechanism

https://doi.org/10.1016/j.atherosclerosis.2006.01.022Get rights and content

Abstract

We investigated the effect of cilostazol on nitric oxide (NO) production in human aortic endothelial cells (HAEC). Cilostazol increased NO production in a concentration-dependent manner, and NO production was also increased by other cyclic-AMP (cAMP)-elevating agents (forskolin, cilostamide, and rolipram). Cilostazol increased intracellular cAMP level, and that effect was enhanced in the presence of forskolin. In Western blot analysis, cilostazol increased phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 and of Akt at Ser473 and dephosphorylation of eNOS at Thr495. Cilostazol's regulation of eNOS phosphorylation was reversed by protein kinase A inhibitor peptide (PKAI) and by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Moreover, the cilostazol-induced increase in NO production was inhibited by PKAI, LY294002, and NG-nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor. In an in vitro model of angiogenesis, cilostazol-enhanced endothelial tube formation, an effect that was completely attenuated by inhibitors of PKA, PI3K, and NOS. These results suggest that cilostazol induces NO production by eNOS activation via a cAMP/PKA- and PI3K/Akt-dependent mechanism and that this effect is involved in capillary-like tube formation in HAEC.

Introduction

Hypercholesterolemia, hypertension, diabetes, and smoking all accelerate the development and progression of atherosclerosis, which is associated with endothelial dysfunction [1]. Endothelial-derived nitric oxide (NO) production is often used as a representative marker of endothelial function, as NO is not only a mediator of endothelium-dependent vasodilation, but also has anti-inflammatory and anti-thrombotic effects, as well as an impact on atherosclerotic lesion formation [2]. Therefore, the improvement of endothelial dysfunction may be one therapeutic strategy for preventing atherogenesis.

Cilostazol is a selective inhibitor of phosphodiesterase 3 (PDE3) and accordingly it increases intracellular cAMP content and activates protein kinase A (PKA), resulting in anti-platelet aggregation and peripheral vasodilation [3]. Cilostazol has therefore been used as a vasodilating anti-platelet drug for the treatment of ischemic symptoms in chronic peripheral arterial obstruction or intermittent claudication and for preventing recurrence of cerebral infarction [3], [4]. At the same time, in preclinical studies, cilostazol was shown to protect endothelial cells from apoptosis induced by serum deprivation, high d-glucose, and lipopolysaccharide (LPS) [5], [6]. Moreover, it was reported that cilostazol attenuated the expression of cell adhesion molecules and monocyte chemoattractant protein-1 (MCP-1), and as a result, prevented monocyte adhesion to endothelial cells [7], [8], [9]. Furthermore, it was suggested that cilostazol-induced nitric oxide (NO) release is involved in endothelium-dependent relaxation in the rat aorta and the inhibition of high glucose-mediated endothelial–neutrophil adhesion in human endothelial cells [10], [11]. Based on these findings, in the present study, we clarified the increasing effect of cilostazol on NO production and the underlying mechanism for that effect by analyzing phosphorylation of endothelial nitric oxide synthase (eNOS) and Akt in human aortic endothelial cells. Furthermore, we examined the effects of cilostazol on endothelial tube formation as a NO-mediated downstream event, since it has been established that endothelium-derived NO plays a critical role in regulation of angiogenesis [12].

Section snippets

Materials

Cilostazol was synthesized by Otsuka Pharmaceutical. Forskolin, rolipram, VEGF, and NG-nitro-l-arginine methyl ester hydrochloride (l-NAME) were purchased from Sigma–Aldrich (St. Louis, MO). LY294002 was obtained from Alexis Biochemicals (San Diego, CA), myristoylated cell-permeable PKA inhibitor peptide sequence (14–22) amide (PKAI) was obtained from Calbiochem (EMD Biosciences, Darmstadt, Germany), and cilostamide and Griess-Romijn nitrite reagent were obtained from Wako Pure Chemical

Nitric oxide production

NO production significantly increased in a concentration-dependent manner when HAEC were treated with cilostazol for 1 h at 10 and 30 μM (2.7- and 4.6-fold versus the vehicle, respectively, Fig. 1A). The other cAMP-elevating agents forskolin, an activator of adenylate cyclase, cilostamide, a PDE3 inhibitor, and rolipram, a PDE4 inhibitor, also significantly increased nitrite production in a concentration-dependent manner (Fig. 1B).

Cyclic-AMP production

When HAEC were treated with cilostazol alone, cAMP level was

Discussion

In the present study, we demonstrated that cilostazol-induced NO production by eNOS activation via a cAMP/PKA- and PI3K/Akt-dependent mechanism and that this effect was involved in capillary-like tube formation in HAEC. This study is the first time that cilostazol-induced NO production has been confirmed using cultured endothelial cells, although it was previously confirmed in the porcine thoracic aorta by an ESR technique [10] and in clinical practice by endothelial-dependent vasodilation [15]

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