CORNEAL ARACHIDONATE METABOLITES VIA CYTOCHROME P450

这项提议是为了检验对角膜的损伤 提高上皮细胞色素P450(CYP)同工酶的活性(S) 它将花生四烯酸(AA)代谢成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)和12(R)-HETrE直接作用于相邻的 角膜缘血管内皮细胞促进角膜新生血管的研究 眼角膜。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都具有细胞色素P。 能立体特异性代谢AA为12(R)-的单加氧酶(S) HETE和12(R)-HETRE；2)闭眼对角膜上皮的损伤- 隐形眼镜配戴导致CYP-AA的时间依赖性形成 与原位炎症密切相关的代谢物 反应；3)显著抑制CYP-AA代谢 减少表明因果关系的原位炎症反应 CYP-AA代谢与炎症的关系4)12(R)-HETrE， 在体外和体内具有很强的生物活性，表明 促炎因子(如血管扩张、毛细血管增多 渗透性、中性粒细胞趋化和血管生成)；以及5)数量 由损伤的角膜上皮产生的12(R)HETrE足以 其促炎特性的表达表明它是一种主要的 这种反应在眼睛中的病理生理介体。具体的 AIMS分为两个研究领域：(A)生化和分子 角膜中细胞色素P-AA代谢酶(S)的鉴定 上皮细胞处于可控和炎症状态。为了实现这一目标， CYP酶活性和内源代谢物水平将 在CYP诱导/抑制下对正常和损伤角膜的评估 条件。这种类型的表征将为 与接下来的研究相比，在这些研究中， 在相同的条件下，将测量几种CYP亚型。这个 这两项研究的结果都应该提供大量的信息 关于其亚型(S)的表达(蛋白质和信使核糖核酸水平) 与损伤后CYP-AA活性和代谢物水平相关。我们 然后进行这种异构体的分子克隆。(B) 阐明了细胞和分子机制的基础前- 12(R)-HETrE的炎症性质，特别是其血管生成 活动。这将包括对其细胞受体的表征 角膜缘内皮细胞和信号通路，包括激活 转录因子、早期即刻基因和至关重要的基因 在血管生成的过程中。这将使我们能够充分了解 该途径及其代谢物的病理生理分支和 可能有助于深入了解角膜上皮和角膜之间的相互作用。 角膜上皮细胞周围的角膜缘微血管构筑 受伤。了解这一新的参与者在慢性粒细胞白血病发病机制中的作用 角膜炎症反应和新生血管将允许 以抑制前体合成为靶点的治疗药物的发展 炎性介质(代谢抑制物)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。

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.