Cilostazol inhibits cytokine-induced nuclear factor-kappaB activation via AMP-activated protein kinase activation in vascular endothelial cells.

AIMS: Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cyclic AMP (cAMP) levels and activates protein kinase A, thereby inhibiting platelet aggregation and inducing peripheral vasodilation. We hypothesized that cilostazol may prevent inflammatory cytokine induced-nuclear factor (NF)-kappaB activation by activating AMP-activated protein kinase (AMPK) in vascular endothelial cells. METHODS AND RESULTS: Cilostazol was observed to activate AMPK and its downstream target, acetyl-CoA carboxylase, in human umbilical vein endothelial cells (HUVEC). Phosphorylation of AMPK with cilostazol was not affected by co-treatment with an adenylate cyclase inhibitor, SQ 22536, and a cell-permeable cAMP analogue, pCTP-cAMP, did not induce AMPK phosphorylation and had no effect on cilostazol-induced AMPK phosphorylation, suggesting that cilostazol-induced AMPK activation occurs through a signalling pathway independent of cyclic AMP. Cilostazol also dose-dependently inhibited tumour necrosis factor alpha (TNFalpha)-induced NF-kappaB activation and TNFalpha-induced I kappa B kinase activity. Furthermore, cilostazol attenuated the TNFalpha-induced gene expression of various pro-inflammatory and cell adhesion molecules, such as vascular cell adhesion molecule-1, E-selectin, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 (MCP-1), and PECAM-1 in HUVEC. RNA interference of AMPK alpha 1 or the AMPK inhibitor compound C attenuated cilostazol-induced inhibition of NF-kappaB activation by TNFalpha. CONCLUSION: In the light of these findings, we suggest that cilostazol might attenuate the cytokine-induced expression of adhesion molecule genes by inhibiting NF-kappaB following AMPK activation.

PPARalpha activators upregulate eNOS activity and inhibit cytokine-induced NF-kappaB activation through AMP-activated protein kinase activation.

Endothelium-derived NO is an important mediator of vascular protection and adhesion molecule expression on the endothelial cell surface is critical for leukocyte recruitment to atherosclerotic lesions. We hypothesized that AMP-activated protein kinase (AMPK) activity is a down-stream mediator of the beneficial effects of PPARalpha activators on vascular endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with fenofibrate or WY14643 resulted in transient activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase. Fenofibrate caused phosphorylation of Akt and eNOS, leading to increased production of NO, and also caused inhibition of cytokine-induced NF-kappaB activation, leading to suppression of expression of adhesion molecule genes. Significant decreases in eNOS activity and NO production in response to fenofibrate were observed in cells treated with AMPK siRNA or with AraA, a pharmacological inhibitor of AMPK. The attenuation of fenofibrate-induced inhibition of NF-kappaB activation was observed in mouse endothelial (SVEC4) cells treated with AMPK siRNA or with AraA. We demonstrated that TNFalpha stimulates IkappaB-alpha phosphorylation through induction of IKK activity, and that fenofibrate inhibits IKK activity and TNFalpha-induced IkappaB-alpha phosphorylation. Our findings suggest that the beneficial effects of PPARalpha activators on endothelial cells such as inhibition of diabetic microangiopathy might be attributed to the induction of AMPK activation beyond its lipid-lowering actions.

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells.

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.