CORNEAL ARACHIDONATE METABOLITES VIA CYTOCHROME P450

这是一个测试假设的提议， 增加上皮细胞色素P450（CYP1A1）同工酶的活性 其将花生四烯酸（AA）代谢为12（R）-羟基-5，8，10，14- 二十碳四烯酸（12（R）-HETE）和12（R）-羟基-5，8，14-二十碳三烯酸 酸（12（R）-HETrE），并且12（R）-HETrE直接作用于相邻的 角膜缘血管内皮细胞促进新生血管形成 角膜以下发现构成了我们假设的基础：1） 包括人类在内的几个物种的角膜上皮细胞都有一个 能够立体特异性地将AA代谢为12（R）- HETE和12（R）-HETrE; 2）通过闭眼对角膜上皮的损伤- 接触透镜配戴导致CYP-AA形成时间依赖性 与原位炎症反应密切相关的代谢物 反应; 3）在该模型中显著抑制CYP-AA代谢 减少原位炎症反应，表明因果关系 CYP-AA代谢和炎症之间的关系; 4）12（R）-HETrE， 在体外和体内具有有效的生物活性， 促炎因子（例如，血管舒张，毛细血管增加 渗透性、中性粒细胞趋化性和血管生成）;和5）量 由损伤的角膜上皮产生的12（R）HETrE足以 其促炎特性的表达暗示它是一种主要的 这是眼睛中这种反应的病理生理介质。具体 目标分为两个研究领域：（A）生物化学和分子生物学 角膜中CYP-AA代谢酶的鉴别 在控制和发炎条件下的上皮。为了实现这一目标， 代谢产物的酶活性和内源性水平将被 在CYP诱导/抑制的正常和受损角膜中进行评估 条件这种类型的表征将为以下方面提供基础： 与接下来的研究相比， 在相同的条件下测量几种β同工型。的 两项研究的结果应提供大量信息 关于其表达（蛋白质和mRNA水平） 与损伤后CYP-AA活性和代谢物水平相关。我们 将继续进行这种同种型的分子克隆。（B） 的细胞和分子机制的阐明， 12（R）-HETrE的炎性特性，特别是其血管生成特性， 活动这将包括表征其细胞受体， 角膜缘内皮细胞和信号通路，包括激活 转录因子，早期直接基因和关键基因 for the process处理of angiogenesis血管生成.这将使我们充分了解 该途径及其代谢产物的病理生理学分支， 可以提供深入了解角膜上皮和 角膜缘微血管 损伤了解这种新的参与者在发病机制中的作用， 角膜炎症反应和新生血管形成将允许 开发靶向抑制前体合成的治疗剂， 炎症介质（代谢抑制剂）以及防止其 用于治疗炎症的作用（受体/功能性拮抗剂） 与角膜损伤、感染和手术相关。

This is a proposal to test the hypothesis that injury to the cornea increases the activity of an epithelial cytochrome P450 (CYP) isozyme(s) which metabolizes arachidonic acid (AA) to 12(R)-hydroxy-5,8,10,14- eicosatetraenoic acid (12(R)-HETE) and 12(R)-hydroxy-5,8,14-eicosatrienoic acid (12(R)-HETrE) and that 12(R)-HETrE acts directly on the adjacent limbal vessel endothelial cells to promote neovascularization of the cornea. The following findings form the basis of our hypothesis: 1) Corneal epithelium from several species, including human, possesses a CYP monooxygenase(s) capable of metabolizing AA stereospecifically to 12(R)- HETE and 12(R)-HETrE; 2) injury to the corneal epithelium via closed eye- contact lens wear results in the time-dependent formation of CYP-AA metabolites which correlates strongly with the in situ inflammatory response; 3) inhibition of CYP-AA metabolism in this model dramatically reduces in situ inflammatory response indicating a cause-effect relationship between CYP-AA metabolism and inflammation; 4) 12(R)-HETrE, possesses potent biological activities in vitro and in vivo indicative of a pro-inflammatory factor (e.g., vasodilation, increased capillary permeability, neutrophil chemotaxis and angiogenesis); and 5) the amount of 12(R)HETrE produced by the injured corneal epithelium is sufficient for the expression of its proinflammatory properties implicating it as a major pathophysiological mediator of such responses in the eye. The specific aims fall into two research areas: (A) The biochemical and molecular identification of the CYP-AA metabolizing enzyme(s) in the corneal epithelium under control and inflamed conditions. In achieving this goal, CYP enzymatic activity and endogenous levels of metabolites will be assessed in normal and injured corneas under CYP-induced/suppressed conditions. This type of characterization will provide the basis for comparison with the next studies in which the protein and mRNA levels of several CYP isoforms will be measured under the same conditions. The results derived from both studies should provide substantial information with regard to the isoform(s) whose expression (protein and mRNA levels) correlates to CYP-AA activity and metabolite levels following injury. We will then proceed with the molecular cloning of this isoform. (B)The elucidation of the cellular and molecular mechanisms underlying the pro- inflammatory properties of 12(R)-HETrE, in particular, its angiogenic activity. This will include characterization of its cellular receptor in limbal endothelial cells and signaling pathways including activation of transcriptional factors, early immediate genes and genes that are crucial for the process of angiogenesis. This will allow us to fully understand the pathophysiologic ramifications of this pathway and its metabolite and may offer insight into the interplay between the corneal epithelium and the surrounding limbal microvasculature following corneal epithelial injury. Understanding the role of this new player in the pathogenesis of corneal inflammatory reaction and neovascularization will permit the development of therapeutics targeted at inhibiting the synthesis of a pro- inflammatory mediator (metabolic inhibitors) as well as preventing its action (receptor/functional antagonists) for the treatment of inflammation associated with corneal injury, infection and surgery.