Cytochrome P450 Eicosanoids and Myocardial Injury

细胞色素 P450 类二十烷酸与心肌损伤

• 批准号: 7388473
• 负责人: GARRETT John GROSS
• 金额: $ 40.59万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7388473
• 关键词: CYP2C9 gene; Cardiac Myocytes; Cell Hypoxia; Cytochrome P450; Data; Eicosanoids; Epidermal Growth Factor Receptor; Feedback; Glycogen Synthase Kinases; Hypoxia; Injury; Isomerism; Lead; MAPK14 gene; Mitochondria; Mus; Myocardial; Myocardial Ischemia; Nuclear; Pathway interactions; Phosphorylation; Potassium Channel; Production; Protein Isoforms; Protein Overexpression; Rat-1; Reactive Oxygen Species; Receptor Activation; Research; Role; Signal Pathway; Stream; System; Testing; Tissues; Transactivation; Western Blotting; cell type; hypoxia inducible factor 1; knockout gene; mitochondrial permeability transition pore; protein kinase C epsilon

Several recent studies demonstrated that hypoxia induces the expression of CYP epoxygenases, particularly CYP2C9, and EET production in several cell types. Our exciting preliminary results suggest several lines of evidence for important roles of CYP-derived EETs and possible signajing pathways in eliciting cardioprotection. In cells, hypoxia induces the production of EETs. Preliminary data suggest that exogenously administered EETs increased the activity (phosphorylation) of Epidermal Growth Factor Receptor (EGFR). Furthermore, EETs increased hypoxia-inducible factor-1 (a subunit, HIF-1a), as detected by Western blots in the nuclear fraction. These results suggest several signaling pathways that are important in cardioprotection. For example, EETs may transactivate the EGFR. The activation of HIF-1a by EETs indicates a possible positive feedback of hypoxia-induced CYP-derived EETs, and in turn EETs upregulate HIF-1a which leads to cardioprotection. Other evidence from our studies which suggest mechanisms that might be involved in CYP epoxygenase and EET-induced cardioprotection include reactive oxygen species (ROS), Protein Kinase C-epsilon, the PI3K/Akt signaling pathway, glycogen synthase kinase-beta (GSK3- beta) and the sarcolemmal and mitochondria! ATP-regulated K channels and the mitochondrial permeability transition pore (MPTP). Specifically, we will test the following aims: Specific Aim 1: Demonstrate whether CYP epoxygenases and EETs are upregulated by hypoxia in cardiomyocytes. We will determine whether hypoxia induces expression of CYP epoxygenases (isoforms) and production of EETs (isomers). We will detect CYP epoxygenases in hypoxic cardiomyocytes and ischemic heart tissue. Specific Aim 2: Demonstrate the functions of EETs in cardiomyocytes using pharmacological agents and exogenous EETs. We will test the effects of exogenous EETs (11,12- and 14,15-EET) to increase the activity of EGFRandHIF-1a. Specific Aim 3: Elucidate the EET signaling pathways that lead to cardioprotection. We will investigate the transactivation of EGFR by EETs and the role of ROS in activating EGFR. Furthermore, we will investigate the down stream signaling pathways of EGFR activation by EETs, including the PKC/Akt/TK, p38, or ERK1/2 pathways. We will also investigate whether exogenous EETs activate HIF-1a, and whether it is through the activation of ROS and EGFR. Thus, in the ongoing research plan, we will investigate the mechanistic actions of EETs in cellular systems such as cardiomyocytes (HL-1 cells, rat cardiomyocytes) and in gene knockout or overexpressing mice.

最近的几项研究表明，缺氧诱导的表氧化酶的表达，特别是 CYP 2C 9和几种细胞类型中的EET产生。我们令人兴奋的初步结果表明， CYP源性Ehrs和可能的信号转导途径在诱导 心脏保护在细胞中，缺氧诱导产生雌二醇。初步数据显示， 外源性给予雌二醇可增加表皮生长因子的活性（磷酸化） 受体（EGFR）。此外，如检测到的那样，雌二醇增加缺氧诱导因子-1（a亚基，HIF-1a）， 通过Western印迹法在核组分中检测到。这些结果提示了几种重要的信号通路 心脏保护例如，雌二醇可以反式激活EGFR。雌二醇对缺氧诱导因子-1 α的激活作用 表明缺氧诱导的CYP衍生的Ehrs可能存在正反馈，反过来Ehrs上调 HIF-1a导致心脏保护。我们研究中的其他证据表明， EET诱导的心肌保护作用包括活性氧 (ROS)、蛋白激酶C-Akt、PI 3 K/Akt信号通路、糖原合成酶激酶-β（GSK 3- 以及肌膜和线粒体！ATP调节的钾通道与线粒体通透性 过渡孔（MPTP）。具体而言，我们将测试以下目标： 具体目标1：证明在缺氧条件下， 心肌细胞我们将确定缺氧是否会诱导CYP表氧化酶（亚型）的表达 和生产异构体。我们将检测缺氧心肌细胞中的β-环氧合酶， 缺血性心脏组织 具体目标2：使用药理学试剂证明心肌细胞中的Ekaryotic功能， 外源性雌二醇我们将测试外源性EET（11，12-和14，15-EET）对提高活性的作用 EGF和HIF-1a。 具体目标3：阐明导致心脏保护的EET信号通路。我们将调查 EGFR被Ehrs反式激活和ROS在激活EGFR中的作用。此外，我们将调查 EGFR被Ehrs激活的下游信号通路，包括PKC/Akt/TK、p38或ERK 1/2 途径。我们还将研究外源性E3是否激活HIF-1a，以及它是否是通过 ROS和EGFR的激活。因此，在正在进行的研究计划中，我们将研究机械作用 在细胞系统如心肌细胞（HL-1细胞，大鼠心肌细胞）和基因敲除中的E2受体的表达 或过度表达的小鼠。