Corneal Arachidonate Metabolites Cytochrome P-450

描述(申请人提供)：角膜表面损伤引起 炎症反应释放花生四烯酸(AA)导致产生 不同的二十烷类化合物，其中一些被认为是通过 环氧合酶、脂氧合酶和细胞色素P450单加氧酶(CYP)途径。 我们是第一个在眼睛中发现依赖CYP的AA代谢的人 建立兔原发角膜上皮炎性通路 眼表炎症模型。这种角膜上皮细胞色素P代谢 对两个主要的12-羟基二十烷：12(R)-HETE和12(R)-HETrE 强效的炎症和血管生成特性。这两种代谢物是 眼表炎症的关键组织衍生介质是强有力的 由我们实验室的研究证明：1)他们 在体外和体内损伤后合成/水平增加；2)其 水平与原位炎症反应呈正相关；3) 抑制它们的合成可减轻体内眼表炎症 暗示一种潜在的因果关系；4)它们的生物学 体外和体内的活性，特别是12(R)-HETrE的活性 强效炎症介质的特点(包括血管扩张、 中性粒细胞趋化和血管生成)；以及5)这两种代谢物 在人类的眼泪中存在，更重要的是， 眼睛发炎的受试者的泪水。 细胞色素P450表达和功能的调控机制及其在细胞周期调控中的意义 眼表炎症还有待研究。这个项目的总体目标是 建议鉴定CYP-AA异构体，以阐明其作用 及其在损伤诱导的炎症中的表达和作用机制 眼表。我们假设角膜损伤会增加眼球的活动 上皮型CYP亚型(S)代谢AA为12(R)-HETE和 12(R)-HETRE和那个12(R)-他Tre，一个角膜！上皮源性血管生成 因子，直接作用于邻近的角膜缘血管内皮细胞 促进角膜新生血管的形成。我们建议：(A)识别 CYP-AA在角膜上皮细胞中的广泛表达 强调调节其表达增加的细胞/分子机制 以及受伤后的活动。(B)调查强权者的作用 血管生成代谢物12(R)-HETrE在炎症中的作用 细胞/分子作用机制。对病理生理的理解 这一途径及其代谢物的分支将为深入了解 角膜上皮与周围角膜缘之间的相互作用 角膜上皮损伤后的微血管形成。它还将有助于 以抑制α-氨基丁酸合成为靶点的治疗药物研究进展 促炎介质(代谢抑制物或分子探针)或 阻止其活性(受体/功能拮抗剂)治疗 与角膜损伤、感染和手术相关的炎症。

DESCRIPTION (provided by applicant): Corneal surface injury evokes an inflammatory reaction releasing arachidonic acid (AA) leading to the production of various eicosanoids some of which are thought to be proinflammatory via the cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase (CYP) pathways. We were the first to identify CYP-dependent AA metabolism in the eye and establish it as a primary corneal epithelial inflammatory pathway in rabbit models of ocular surface inflammation. This corneal epithelial CYP metabolizes AA to two major 12-hydroxyeicosanoids: 12(R)-HETE and 12(R)-HETrE which exhibit potent inflammatory and angiogenic properties. That these two metabolites are critical tissue-derived mediators of ocular surface inflammation is strongly supported by studies from our laboratory demonstrating that: 1) their synthesis/levels are increased following injury in vitro and in vivo; 2) their levels positively correlate with the in situ inflammatory response; 3) inhibition of their synthesis attenuates ocular surface inflammation in vivo suggesting a potential cause-effect relationship; 4) their biological activities, in particular those of 12(R)-HETrE, in vitro and in vivo, are characteristic of potent inflammatory mediators (including vasodilation, neutrophil chemotaxis, and angiogenesis); and 5) these two metabolites are present in human tears and, more significantly, the levels are much higher in tears from subjects with ocular inflammation. The mechanisms that regulate CYP expression and function and its importance to ocular surface inflammation are yet to be explored. The overall goal of this proposal is to identify the CYP-AA isoform in order to elucidate both its role and mechanisms of expression and function in injury-induced inflammation of the ocular surface. We hypothesize that injury to the cornea increases the activity of an epithelial CYP isoform(s) which metabolizes AA to 12(R)-HETE and 12(R)-HETrE and that 12(R)-HE TrE, a cornea! epithelial-derived angiogenic factor, acts directly on the adjacent limbal vessel's endothelial cells to promote neovascularization of the cornea. We propose: (A) the identification and extensive characterization of the CYP-AA in the corneal epithelium emphasizing cellular/molecular mechanisms regulating its increased expression and activity following injury. (B) investigation into the role of the potent angiogenic metabolite 12(R)-HETrE in inflammation with emphasis on cellular/molecular mechanisms of action. Understanding the pathophysiologic ramifications of this pathway and its metabolites will offer insight into the interplay between the corneal epithelium and the surrounding limbal microvasculature following corneal epithelial injury. It will also aid in the development of therapeutics targeted at inhibiting the synthesis of a pro-inflammatory mediator (metabolic inhibitors or molecular probes) or preventing its activity (receptor/functional antagonists) for the treatment of inflammation associated with corneal injury, infection and surgery.