Ebselen inhibits tumor necrosis factor-α-induced c-jun N-terminal kinase activation and adhesion molecule expression in endothelial cells

Tumor necrosis factor-alpha (TNF-alpha) stimulates expression of endothelial cell (EC) genes that may promote atherosclerosis in part by an activation of mitogen-activated protein (MAP) kinases. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a selenoorganic compound, is effective for acute ischemic stroke; however, its effect on EC has not yet been elucidated. We examined the effect of ebselen on TNF-alpha-induced MAP kinase activation and adhesion molecule expression in cultured human umbilical vein endothelial cells (HUVEC). Extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 were rapidly and significantly activated by TNF-alpha in HUVEC. TNF-alpha-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 were not affected. Apoptosis signal-regulated kinase 1 (ASK1) was suggested to be involved in TNF-alpha-induced JNK activation because transfection of kinase-inactive ASK1 inhibited TNF-alpha-induced JNK activation. Ebselen inhibited TNF-alpha-induced TNF receptor-associated factor 2 (TRAF2)-ASK1 complex formation and phosphorylation of stress-activated protein kinase ERK kinase 1 (SEK1), which is an upstream signaling molecule of JNK. Finally, TNF-alpha-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation and resultant intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions were inhibited by ebselen. Specific inhibitors for JNK and NF-kappaB also inhibited TNF-alpha-induced ICAM-1 and VCAM-1 expressions in HUVEC. These findings suggest that ebselen prevents TNF-alpha-induced EC activation through the inhibition of TRAF2-ASK1-SEK1 signaling pathway, which leads to JNK activation. Inhibition of JNK by ebselen may imply its usefulness for the prevention of atherosclerosis relevant to EC activation.


Ebsalen inhibits p38MAP kinase-mediated endothelial cell death by hydrogen peroxide.

Ebselen (2-phenyl-1, 2-benzisoselenazol-3[2H]-one) is a seleno-organic compound exhibiting both glutathione peroxidase and antioxidant activity. Although it has been reported that ebselen is effective against hydrogen peroxide (H(2)O(2))-induced cell death in several cell types, its effect on endothelial cell damage has not yet been elucidated. In the present study, we examined the effect of ebselen on H(2)O(2)-induced human umbilical vein endothelial cells (HUVECs) death, and its intracellular mechanism. Our findings showed that pretreatment of HUVECs with ebselen resulted in a significant recovery from H(2)O(2)-induced cell death in a concentration-dependent manner. In addition to the inhibition of lactate dehydrogenase (LDH) leakage, ebselen inhibited H(2)O(2)-induced cytochrome c release and caspase-3 activation and the resultant apoptosis in HUVECs. Moreover, it was observed that H(2)O(2) significantly stimulated activation of mitogen-activated protein (MAP) kinases, i.e., p38 MAP kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2). Ebselen inhibited H(2)O(2)-induced p38 MAP kinase, but not JNK or ERK1/2 activation. Furthermore, SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]-1H-imidazole), a specific p38 MAP kinase inhibitor, inhibited H(2)O(2)-induced p38 MAP kinase phosphorylation, cytochrome c release, caspase-3 activation, as well as cell death in HUVECs. These findings suggest that ebselen attenuates H(2)O(2)-induced endothelial cell death through the inhibition of signaling pathways mediated by p38 MAP kinase, caspase-3, and cytochrome c release. Thus, inhibition of p38 MAP kinase by ebselen may imply its usefulness for prevention and/or treatment of endothelial cell dysfunction, which was suggested to be the first step in the development of atherosclerosis.


Lysophosphatidylcholine(LPC)transactivates vascular endothelial growth factor(VEGF) receptor via c-Src in HUVEC

ヒト臍帯静脈内皮細胞(HUVEC)における Lysophosphatidylcholine(LPC)刺激によるVEGFレセプターのトランスアクチベーション

One of the major lipid components of oxidized low density lipoprotein, lysophosphatidylcholine(LPC)is involved in numerous biological processes as a bioactive lipid molecule and has beenshown to be involved in the progression of atherosclerosis. As counter-ligands, G2A and GPR4wereidentified with high binding affinity for LPC that are belonging to orphan G-protein-coupledreceptors(GPCRs)at plasma membranes. Although several GPCR ligands transactivate receptortyrosine kinases (RTKs), such as epidermal growth factor receptor, transactivation of RTK byLPC has not yet been reported. Here we observed for the first time that LPC treatment inducestyrosyl phosphorylation of vascular endothelial growth factor(VEGF)receptor2(fetal liverkinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)in human umbilical vein endothelialcells(HUVEC). VEGF receptor tyrosine kinase inhibitors, SU1498and VTKi inhibited Flk-1/KDRtransactivation by LPC. Furthermore, we examined the effect of the Src family kinases inhibitors,Herbimycin A and PP2 on LPC-induced Flk-1/KDR transactivation. Herbimycin A and PP2inhibited Flk-1/KDR transactivation in HUVEC, suggesting that c-Src is involved in LPC-inducedFlk-1/KDR transactivation. Kinase-inactive(KI)Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, LPC activated extracellular signal-regulated kinase1/2and Akt,which are downstream effectors of Flk-1/KDR, and these were inhibited by SU1498，VTKi,Herbimycin A, PP2and KI Src transfection in HUVEC. LPC-mediated HUVEC proliferation wasshown to be secondary to transactivation because it was suppressed by SU1498，VTKi, HerbimycinA, PP2and KI Src transfection. It is concluded that c-Src-mediated Flk-1/KDR transactivation byLPC may have important implications for the progression of atherosclerosis.