Biological role of EET in induction of vascular endotberial growth factor underr hypoxia

细胞色素P450(P450)催化依赖NADPH的花生四烯酸氧化生成环氧二十碳三烯酸(EET)，EET有4种异构体：5，6-EET、8，9-EET、11，12-EET和14，15-EET。由于环氧合酶具有很强的生物活性(调节血管张力和抗炎活性等)，人们对环氧合酶反应特别感兴趣。归因于外星人。我着重研究了EETs在低氧条件下诱导血管内皮生长因子(VEGF)的一种新的生物学功能。本研究以人肝癌细胞、人肝癌细胞株Hep3B和人脐动脉内皮细胞(HUAEC)为实验材料，分别在常氧和低氧条件下进行细胞培养。质膜提供花生四烯酸的磷脂酶A2抑制剂MAFP可抑制缺氧条件下HUAEC对血管内皮生长因子的诱导。NADPH-P450还原酶(NPR)为P450提供电子，是P450氧化花生四烯酸所必需的。NPR的抑制剂DPIC可抑制血管内皮生长因子的诱导。这些结果提示花生四烯酸和P450在缺氧条件下诱导HUAEC血管内皮生长因子的表达中起重要作用。对Hep3B细胞也观察到同样的作用。在Hep3B细胞中，P450抑制剂、磺胺苯唑(CYP2C8和CYP2C9的抑制剂)和酮康唑(CYP3A的抑制剂)能有效地抑制低氧反应(促红细胞生成素的诱导)。缺氧条件下加入11，12-EET或14，15-EET可诱导血管内皮细胞血管内皮生长因子生成，但水化产物11，12-DHETE和14，15-DHETE不能诱导血管内皮细胞生成血管内皮生长因子。低氧诱导因子(HIF-1)是低氧诱导血管内皮细胞生长因子(VEGF)产生所必需的。11，12-EET和14，15-EET不改变HIF-1的水平。综上所述，P450产生的EETs在细胞的缺氧反应中起重要作用。EETs诱导的血管内皮生长因子不依赖于HIF-1，而HIF-1是缺氧反应的重要因素。

Cytochrome P450 (P450) catalyze the NADPH-dependent oxidation of arachidonic acid to several unique eicosanoids such as epoxyeicosatrienoic acid (EET) which has four isomers, 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET. A particular interest in the epoxygenase reaction has developed because of the potent biological activities (modulation of vascular tone and anti-inflammatory activity etc.) attributed to the EETs. I focused on a new biological function of EETs which induce vascular endotherial growth factor (VEGF) under hypoxia. In this study, human hepatoma cell, Hep3B and human umbilical artery endotherial cell (HUAEC) were used under normoxic and hypoxic conditions. An inhibitor for phospholipase A2 which supplies arachidonic acid from plasma membrane, MAFP, inhibited the VEGF induction in HUAEC under hypoxia. NADPH-P450 reductase (NPR) supplies electrons to P450 and is necessary for P450 to oxidize arachidonic acid. An inhibitor for NPR, DPIC, inhibited the VEGF induction. These results suggest that arachidonic acid and P450 are important for the VEGF induction in HUAEC under hypoxia. The same effects on Hep3B cells were also observed. In Hep3B cells, inhibitors for P450s, sulfaphenazol (an inhibitor for CYP2C8 and CYP2C9) and ketoconazole (an inhibitor for CYP3A) inhibited hypoxia response (erythropoietin induction) efficiently. Addition of 11,12-EET or 14,15-EET induced VEGF in HUAEC under hypoxia but hydration products, 11,12-DHETE and 14,15-DHETE did not induce VEGF. Hypoxia-inducible factor (HIF-1) is necessary for induction of VEGF under hypoxia. 11,12-EET and 14,15-EET did not change the levels of HIF-1. In conclusion, EETs produced by P450s play an important role in hypoxic response of cells. EETs induced VEGF independent on HIF-1 which is an important factor of hypoxic response.