Characterization And Functional Significance Of P450 Arachidonate Epoxygenases

细胞色素P450将花生四烯酸(AA)代谢成环氧二十碳三烯酸(EETs)，对心血管和肾脏功能有重要影响。EETs被可溶性环氧化物水解酶(SEH)代谢成相应的二醇(DHETs)。目前的研究包括：(1)在生物化学和分子水平上对细胞色素P450和细胞色素P450亚家族进行表征；(2)评价细胞色素P450产物在心血管和肾脏生理中的功能作用；(3)在选定的人类疾病(缺血性心脏病、高血压、动脉粥样硬化、癌症)动物模型中检测这一通路。我们已经发现了一些哺乳动物的CYP2Js，尽管我们主要集中在人类的CYP2J2和小鼠的CYP2J5上。人CYP2J2是人类主要在心脏和血管系统表达的P450，定位于心肌细胞和内皮细胞，在AA到EETs的代谢中起活跃作用。CYP2J2来源的EET是一种血管扩张剂，可抑制细胞因子诱导的内皮细胞黏附分子表达，诱导组织纤溶酶原激活剂基因表达，抑制血管平滑肌细胞迁移，保护内皮细胞免受缺氧-复氧损伤和细胞凋亡，上调内皮细胞一氧化氮生物合成，影响心脏电生理，保护心脏免受缺血损伤。建立了CYP2J2转基因小鼠(α-肌球蛋白重链启动子驱动的心脏特异性表达)，以研究EETs增加对体内心脏功能的影响。这些小鼠的基础心脏解剖和功能正常，缺血后左心功能改善，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，sEH基因缺失的小鼠表现出EET水解率降低，改善了缺血后的功能恢复。我们还建立了在血管内皮细胞(Tie2启动子驱动)中仅表达CYP2J2、CYP2C8或sEH的转基因小鼠，以检测这些酶及其产物对血管功能的作用。这些小鼠的表型目前正在评估中。人类细胞色素P450 2J2基因已被克隆、测序和鉴定。我们已经确定了几个功能上相关的CYP2J2多态变异，其中一个与CYP2J2表达降低相关，并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，并表明在美国的一个大型多民族队列中，它们与心血管疾病风险有关。CYP2J5是一种主要的小鼠P450花生四烯酸环氧合酶，在肾脏表达，定位于近端小管。为了评估这个P450和它的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基因缺失的小鼠有自发性高血压，在高盐和低盐饮食中都持续存在。高血压在女性中更为严重，与循环中的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基因缺失的雌性小鼠近端肾小管转运速率增加，血管对血管紧张素II和内皮素的反应性增强，生育力降低。这一新的动物模型将使人们更好地理解肾脏P450、性激素、肾脏二十烷类化合物和血压调节之间的复杂相互关系。与这些发现一致的是，我们已经证明，在田纳西州的一个队列中，CYP2J2多态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.