花生四烯酸细胞色素P450表氧化酶代谢产物EET促进血管生成的研究

目的研究花生四烯酸细胞色素P450（cytochrome P450,CYP）表氧化酶代谢产物表氧二十碳三烯酸（epoxyeicosatrienoic acid,EET）对牛主动脉内皮细胞（bovine endothelial cells,BAEC）、对血管生成的影响及其机制.方法分离BAEC培养,给予外源性EET刺激、重组腺相关病毒介导的各种CYP表氧化酶（CYP2J2,CYP2C11,CYPF87V）转染后,采用细胞计数、噻唑蓝比色法检测细胞增殖改变,用流式细胞仪检测对细胞增殖周期的影响,同时检测细胞趋化移行的改变,比较对Matrigel中毛细血管样结构形成的影响,观察表氧化酶过度表达对鸡胚尿囊绒毛膜血管生成和大鼠缺血后肢毛细血管生成的影响.结果各种EET刺激或表氧化酶病毒转染均显著促进BAEC的增殖、趋化和移行,并使Matrigel中毛细血管样结构的形成明显增加,且EET呈剂量依赖性效应,而合用一氧化氮合酶抑制剂、丝裂原激活的蛋白激酶（mitogen-activated protein kinase,MAPK）抑制剂或磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase,PI3K）抑制剂均可显著抑制上述效应,另外表氧化酶病毒转染尚能明显促进CAM小血管和大鼠缺血后肢毛细血管的生成.结论花生四烯酸细胞色素P450（CYP）表氧化酶及其代谢产物EET可显著促进血管的生成,可改善局部组织的缺血,其作用由MAKP和PI3K介导,部分效应由其对一氧化氮的上调作用介导.

Arachidonic acid epoxygenases and their metabolites promote angiogenesis

Objective To investigate the angiogenenic effects of endogenous and exogenous epoxyeicosatrienoic acids (EET) and the relevant signaling mechanisms involved.Methods Bovine aortic endothelial cells (BAEC) were incubated with synthetic EETs or infected with recombinant adeno-associated viruses (rAAV) containing CYP2C11-CYPOR, CYP2J2 or CYP102 F87V mutant to increase endogenous expression levels of EETs. BAEC proliferation measured by cell counting and chromatometry, migration assessed by transwell analysis, and capillary formation determined by chicken embryo chorioallantoic membrane assays (CAM) and tube formation tests on matrigel and angiogenesis were analysed in vivo. The potential involvement of various signaling pathways were explored using selective inhibitors.Results Transfection with rAAV-2C11OR, rAAV-2J2 or rAAV-F87V promoted BAEC proliferation, migration, and capillary tubule formation. However, the effects of EETs on proliferation, migration and capillary tubule formation were attenuated by inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase)/Akt pathways, and partially attenuated by endothelial nitric oxide synthase (eNOS) inhibitor, but not by a protein kinase C inhibitor. In a rat ischemic hind limb model, rAAV-mediated epoxygenase transfection induced angiogenesis.Conclusions Arachidonic acid epoxygenase and its metabolites can promote angiogenesis through activating MAPK and PI3 kinase/Akt signaling pathways, and to some extent, the eNOS pathway, and the angiogenic effects may provide protection to ischemic tissues.