CORNEAL ARACHIDONATE METABOLITES VIA CYTOCHROME P450

通过细胞色素 P450 测定角膜花生四烯酸代谢物

• 批准号: 2710922
• 负责人: Michal Laniado Schwartzman
• 金额: $ 30.72万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2710922
• 关键词: angiogenesis; arachidonate; biological signal transduction; cornea; cornea disorder; corneal epithelium; cytochrome P450; eicosanoid metabolism; enzyme activity; enzyme inhibitors; enzyme linked immunosorbent assay; eye injury; gas chromatography mass spectrometry; gel mobility shift assay; high performance liquid chromatography; inflammation; intraocular aqueous flow; isozymes; laboratory rabbit; molecular cloning; northern blottings; scintillation spectrometry; tissue /cell culture; transcription factor; western blottings

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.

这是一项检验角膜损伤假设的提案 增加上皮细胞色素 P450 (CYP) 同工酶的活性 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) 包括人类在内的多个物种的角膜上皮都具有 CYP 能够将 AA 立体特异性代谢为 12(R)- 的单加氧酶 HETE 和 12(R)-HETrE； 2）闭眼时角膜上皮损伤 佩戴隐形眼镜会导致 CYP-AA 的时间依赖性形成 与原位炎症密切相关的代谢物 回复; 3) 显着抑制该模型中的 CYP-AA 代谢 减少原位炎症反应，表明因果关系 CYP-AA代谢与炎症的关系； 4) 12(R)-HETrE, 具有有效的体外和体内生物活性 促炎因子（例如，血管舒张、毛细血管扩张 通透性、中性粒细胞趋化性和血管生成）； 5) 金额 受损角膜上皮产生的 12(R)HETrE 足以 其促炎特性的表达表明它是一种主要的 眼睛中此类反应的病理生理介质。具体的 目标分为两个研究领域：（A）生物化学和分子 角膜中 CYP-AA 代谢酶的鉴定 上皮处于控制和发炎状态。在实现这一目标的过程中， CYP酶活性和代谢物的内源水平将 在 CYP 诱导/抑制下对正常和受伤的角膜进行评估 状况。这种类型的表征将为 与接下来的研究进行比较，其中蛋白质和 mRNA 水平 几种 CYP 同工型将在相同条件下进行测量。这 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）的亚型 与损伤后 CYP-AA 活性和代谢物水平相关。我们 然后将继续进行该亚型的分子克隆。 (B) 的 阐明亲细胞和分子机制 12(R)-HETrE 的炎症特性，特别是其血管生成 活动。这将包括其细胞受体的表征 角膜缘内皮细胞和信号通路，包括激活 转录因子、早期直接基因和至关重要的基因 对于血管生成的过程。这将使我们充分了解 该途径及其代谢物的病理生理学后果 可以深入了解角膜上皮和角膜之间的相互作用 角膜上皮周围的角膜缘微血管 受伤。了解这个新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 开发旨在抑制前体合成的治疗方法 炎症介质（代谢抑制剂）以及预防其 治疗炎症的作用（受体/功能拮抗剂） 与角膜损伤、感染和手术有关。