Characterization And Functional Significance Of P450 Arachidonate Epoxygenases

细胞色素P450将花生四烯酸（AA）代谢为环氧二碳三烯酸（EETs），对心血管和肾脏功能有重要影响。eet通过可溶性环氧化物水解酶（sEH）代谢成相应的二醇（DHETs）。目前的研究主要包括：(1)在生化和分子水平上对CYP2J和CYP2C亚家族p450进行表征；(2) CYP2J产物在心血管和肾脏生理中的功能作用评价；(3)在人类疾病（缺血性心脏病、高血压、动脉粥样硬化、癌症）的动物模型中对这一途径的研究。我们已经发现了许多哺乳动物的CYP2Js，尽管我们的大部分工作都集中在人类CYP2J2和小鼠CYP2J5上。人CYP2J2是主要表达于心脏和血管系统的P450蛋白，定位于心肌细胞和内皮细胞，在AA到eet的代谢中具有活性。cyp2j2衍生的eet具有血管扩张剂作用，抑制细胞因子诱导的内皮细胞粘附分子表达，诱导组织纤溶酶原激活物基因表达，抑制血管平滑肌细胞迁移，保护内皮细胞缺氧复氧损伤和凋亡，上调内皮一氧化氮生物合成，影响心脏电生理，保护心脏免受缺血性损伤。建立CYP2J2转基因小鼠（α -肌球蛋白重链启动子驱动心脏特异性表达），研究EETs增加对心脏功能的影响。这些小鼠的心脏基础解剖和功能正常，缺血后左心室功能改善，心脏动作电位缩短，心脏电生理改变，β -肾上腺素能受体反应性增强。同样，EET水解减少的sEH缺失小鼠具有改善的缺血后功能恢复。我们还开发了转基因小鼠，其中CYP2J2， CYP2C8或sEH仅在内皮细胞中表达（Tie2启动子驱动），以研究这些酶及其产物在血管功能中的作用。目前正在对这些小鼠的表型进行评估。人类CYP2J2基因已被克隆、测序和鉴定。我们已经确定了几个功能相关的CYP2J2多态性变异，其中一个与CYP2J2表达减少有关，并且在几个队列中与心血管疾病的风险相关。我们还发现了sEH基因的功能相关多态性，并在美国的一个大型多种族队列中表明它们与心血管疾病风险相关。CYP2J5是一种主要的小鼠P450花生四烯酸环氧化酶，在肾脏中表达并定位于近端小管。为了评估这种P450及其类二十烷类产物在肾功能和血压调节中的作用，我们通过同源重组破坏了Cyp2j5基因。CYP2J5缺失小鼠在高盐和低盐饮食中都存在自发性高血压。高血压在女性中更为严重，与循环雌二醇水平降低有关，并对补充雌激素有反应。与野生型小鼠相比，CYP2J5缺失的雌性小鼠近端小管运输率增加，血管对血管紧张素II和内皮素的反应性增强，生育能力降低。这种新的动物模型将有助于更好地理解肾p450、性激素、肾二十烷类化合物和血压调节之间复杂的相互关系。与这些发现一致，我们在田纳西州的一个队列中发现CYP2J2多态性变异与高血压之间存在关联。

Cytochromes P450 metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which have potent effects on cardiovascular and renal function. EETs are metabolized to corresponding diols (DHETs) by soluble epoxide hydrolase (sEH). Current research involves: (1) characterization of CYP2J and CYP2C subfamily P450s at the biochemical and molecular levels; (2) evaluation of the functional roles of CYP2J products in cardiovascular and renal physiology; and (3) examination of this pathway in selected animal models of human disease (ischemic heart disease, hypertension, atherosclerosis, cancer). We have discovered a number of mammalian CYP2Js, although we have focused most of our efforts on human CYP2J2 and mouse CYP2J5. Human CYP2J2 is the major human P450 expressed in heart and vasculature, where it is localized to cardiac myocytes and endothelial cells, and is active in the metabolism of AA to EETs. CYP2J2-derived EETs are vasodilators, inhibit cytokine-induced endothelial cell adhesion molecule expression, induce tissue plasminogen activator gene expression, inhibit vascular smooth muscle cell migration, protect endothelial cells against hypoxia-reoxygenation injury and apoptosis, upregulate endothelial nitric oxide biosynthesis, affect cardiac electrophysiology, and protect the heart from ischemic injury. CYP2J2 transgenic mice (alpha-myosin heavy chain promoter driven cardiac-specific expression) were developed to study the effects of increased EETs on cardiac function in vivo. These mice have normal basal heart anatomy and function, improved post-ischemic left ventricular function, shortened cardiac action potential, altered cardiac electrophysiology, and enhanced beta-adrenergic receptor responsiveness. Similarly, sEH null mice which exhibit reduced EET hydrolysis have improved postischemic functional recovery. We have also developed transgenic mice in which CYP2J2, CYP2C8 or sEH are expressed exclusively in endothelial cells (Tie2 promoter driven) to examine the role of these enzymes and their products on vascular function. The phenotype of these mice is currently being evaluated. The human CYP2J2 gene has been cloned, sequenced and characterized. We have identified several functionally relevant CYP2J2 polymorphic variants, one of which is associated with reduced CYP2J2 expression and is associated with risk of cardiovascular disease in several cohorts. We have also identified functionally relevant polymorphisms in the sEH gene and have shown that they are associated with cardiovascular disease risk in a large multiethnic cohort in the U.S. CYP2J5 is a major murine P450 arachidonic acid epoxygenase expressed in the kidney and localized to proximal tubules. To evaluate the role of this P450 and its eicosanoid products in renal function and blood pressure regulation, we disrupted the Cyp2j5 gene by homologous recombination. CYP2J5 null mice have spontaneous hypertension that persists on both high and low salt diets. The hypertension is much more severe in females, is associated with reduced circulating estradiol levels, and is responsive to estrogen supplementation. CYP2J5 null female mice also have increased proximal tubular transport rates, enhanced vascular responsiveness to angiotensin II and endothelin, and reduced fertility compared to wild type counterparts. This new animal model will lead to a better understanding of the complex interrelationship between renal P450s, sex hormones, renal eicosanoids and blood pressure regulation. Consistent with these findings, we have shown that there is an association between CYP2J2 polymorphic variants and hypertension in a cohort from Tennessee.