Cilostazol Induces PGI(2) Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

BACKGROUND: Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I(2) (PGI(2)) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI(2) synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI(2) stimulation in endothelial cells. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI(2) accumulation in the culture media was measured. Cilostazol increased PGI(2) synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI(2) synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI(2) synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI(2) synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI(2) synthesis. CONCLUSIONS: The PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI(2) synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI(2) synthesis.