Corneal Arachidonate Metabolites Cytochrome P-450

描述（由申请人提供）：角膜表面损伤引起 炎症反应释放花生四烯酸（AA），导致生产 各种类花生酸，其中一些被认为是促炎通过 环氧合酶、脂氧合酶和细胞色素P450单加氧酶（CYP）途径。 我们是第一个确定CYP依赖性AA代谢的眼睛， 确立其为兔角膜上皮原发性炎症途径 眼表炎症模型。这种角膜上皮细胞代谢 AA转化为两种主要的12-羟基类二十烷酸：12（R）-HETE和12（R）-HETrE，其表现出 有效的炎症和血管生成特性。这两种代谢物 眼表炎症的关键组织源性介质强烈 我们实验室的研究表明：1） 合成/水平在体外和体内损伤后增加; 2）它们的 水平与原位炎症反应正相关; 3） 抑制其合成减轻体内眼表面炎症 提示潜在的因果关系; 4）它们的生物学特性 活性，特别是12（R）-HETrE的那些，在体外和体内， 其特征在于有效的炎症介质（包括血管舒张， 中性粒细胞趋化性和血管生成）;和5）这两种代谢物是 存在于人类的眼泪中，更重要的是， 眼部炎症受试者的眼泪。 调节p53表达和功能的机制及其对 眼表炎症仍有待研究。这个项目的总体目标是 一个建议是确定CYP-AA亚型，以阐明其作用， 以及在损伤诱导的炎症中的表达和功能机制。 眼表我们假设角膜损伤会增加 将AA代谢为12（R）-HETE的上皮细胞α-淀粉酶同种型， 12（R）-HETrE和12（R）-HETrE，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的内皮细胞， 促进角膜的新血管形成。我们建议：（一）识别 以及角膜上皮中CYP-AA的广泛表征 强调调节其表达增加的细胞/分子机制 和受伤后的活动。(B)调查的作用，有力的 血管生成代谢物12（R）-HETrE在炎症中的作用，重点是 细胞/分子作用机制。了解病理生理学 这一途径及其代谢产物的分支将提供深入了解 角膜上皮和周围角膜缘之间的相互作用 角膜上皮损伤后的微血管。它还将有助于 开发靶向抑制α-半乳糖苷合成的治疗剂 促炎介质（代谢抑制剂或分子探针）或 防止其治疗以下疾病的活性（受体/功能性拮抗剂） 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.