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