Role of Oxidative Stress in Pathogenesis of Fuchs Endothelial Corneal Dystrophy

氧化应激在福克斯内皮性角膜营养不良发病机制中的作用

• 批准号: 10591213
• 负责人: Ula V. Jurkunas
• 金额: $ 7.4万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591213
• 关键词: Antioxidants; Apoptosis; Apoptotic; Blindness; CYP1B1 gene; Cell Aging; Cell Cycle Arrest; Cell Death; Cell Line; Cells; Cornea; Corneal Endothelium; DNA Damage; DNA Repair; DNA Repair Disorder; DNA Repair Gene; Deposition; Development; Down-Regulation; Elderly; Endothelial Cells; Endothelium; Enzymes; Estrogen Metabolism; Estrogens; Etiology; Extracellular Matrix; Female; Fuchs' Endothelial Dystrophy; Genes; Genotype; Human; Incidence; Investigation; Keratoplasty; Laboratories; Light; Link; Liquid substance; M Phase Arrest; M cell; Mitochondria; Mitochondrial DNA; Mitosis; Mus; Outcome; Oxidants; Oxidative Stress; Pathogenesis; Pathogenicity; Patients; Pharmacological Treatment; Physiological; Predisposition; Role; Severities; Sex Differences; Specimen; UV induced; UVA induced; Ultraviolet Rays; Woman; age related; aqueous; base; cell injury; human old age (65+); irradiation; mitochondrial dysfunction; mouse model; non-genetic; novel; oxidative DNA damage; repaired; response; senescence; sex; ultraviolet

Project Summary/Abstract Fuchs Endothelial Cornel Dystrophy (FECD), a common age-related dystrophy, which is more prevalent in women, is of unknown etiology. In FECD, corneal endothelial (CE) cell loss is accompanied by abnormal extracellular matrix (ECM) deposition in the form of guttae. Our laboratory was the first to link oxidative DNA damage and mitochondrial dysfunction in FECD pathogenesis. Specifically, we showed that DNA damage, induced by ultraviolet-A (UVA) light, causes FECD in mice. Moreover, the UVA induced CE cell cycle arrest in G2/M phase and cellular senescence. We identified the novel involvement of CYP1B1, the key estrogenmetabolizing enzyme, in sex-dependent differences in CE susceptibility to UVA; and detected greater mitochondrial DNA (mtDNA) damage in female mice. However, the mechanism of the observed greater susceptibility of female mice to UVA-induced DNA damage is unknown. Building upon our previous findings, we propose to investigate if UV light–induced oxidant-antioxidant imbalance leads to senescence and ECM deposition by causing G2/M cell cycle arrest; and if this imbalance causes translocation of CYP1B1 into mitochondria triggering greater estrogen-induced mtDNA damage in females. Gene array analysis revealed downregulation of DNA repair genes in FECD; therefore, we will determine whether this leads to decreased DNA damage repair during G2/M cell cycle arrest, triggering the cells to undergo either senescence or apoptosis. Our study is significant, as the investigation sex-dependent mechanisms involved in oxidative stress-induced cellular damage will provide new treatment targets for FECD. In order to achieve these aims, we will use our newly developed non-genetic mouse model of FECD based on physiologic outcome of CE susceptibility to UVA along with immortalized human CE cell lines, human aqueous fluid, and ex vivo specimens of genotyped FECD donors. Our Specific Aims are: Aim 1: Investigate the role of UVA irradiation in G2/M cell cycle arrest and induction of cellular senescence and ECM deposition in FECD. This aim is based on the hypothesis that DNA damage leads to G2/M phase arrest and induces cellular senescence, which in turn leads to aberrant ECM deposition in the form of guttae in FECD. Aim 2: Determine whether UVA irradiation activates CYP1B1 and induces estrogen metabolism causing preferentially greater DNA damage in females. This aim is based on the hypothesis that higher incidence and severity of FECD in women occurs due to UVAinduced translocation of CYP1B1 into mitochondria, which triggers formation of reactive estrogen metabolites, causing mtDNA damage. Aim 3: Determine the role of DNA damage response and DNA repair during UVAinduced cell cycle arrest in FECD. This aim is based on the hypothesis that DNA repair deficiency during G2/M cell cycle arrest determines whether cells undergo senescence or apoptotic cell death in FECD.

项目摘要/摘要 Fuchs内皮玉米骨营养不良（FECD），一种与年龄相关的营养不良，在 妇女，是未知的病因。在FECD中，角膜内皮（CE）细胞损失伴有异常 细胞外基质（ECM）的沉积，形式为guttae。我们的实验室是第一个连接氧化DNA的实验室 FECD发病机理中的损伤和线粒体功能障碍。具体来说，我们显示了DNA损伤， 由紫外线-A（UVA）诱导，导致小鼠的粪便。此外，UVA诱导CE细胞周期停滞 G2/m相和细胞感应。我们确定了CYP1B1（钥匙）的新颖参与 雌激素代谢酶在CE对UVA的敏感性方面存在性依赖性差异；并发现更大 雌性小鼠的线粒体DNA（mtDNA）损伤。但是，观察到的机制更大 雌性小鼠对UVA诱导的DNA损伤的敏感性尚不清楚。以我们以前的发现为基础 我们建议研究紫外线诱导的氧化剂 - 抗氧化剂是否会导致感应和ECM 通过引起G2/M细胞周期停滞来沉积；如果这种不平衡导致CYP1B1易位 线粒体触发女性雌激素诱导的mtDNA损伤更大。基因阵列分析显示 DNA修复基因在FECD中的下调；因此，我们将确定这是否导致下降 G2/M细胞周期停滞期间的DNA损伤修复，触发细胞受到感应或 凋亡。我们的研究很重要，因为与氧化有关的投资依赖性机制 应力诱导的细胞损伤将为FECD提供新的治疗靶标。为了实现这些目标， 我们将根据CE的生理结果使用新开发的非遗传小鼠模型 对UVA的敏感性以及永生的人CE细胞系，人水溶液和离体 基因分型的粪便供体的标本。我们的具体目的是：目标1：调查UVA辐照在 G2/M细胞周期停滞和FECD中细胞感应和ECM沉积的诱导。这个目标是基于 DNA损伤导致G2/M相阻滞并诱导细胞感应的假说，这又是 导致fecd中guttae形式的ECM沉积异常。目标2：确定UVA辐射是否 激活CYP1B1并诱导雌激素代谢，从而优先导致女性DNA损伤。 该目的是基于以下假设 UVAINDOD CYP1B1转移到线粒体上，这会触发反应性雌激素的形成 代谢物，导致mtDNA损伤。 AIM 3：确定DNA损伤响应和DNA修复的作用 在卵子导出的过程中，在FECD中停止了细胞周期。该目标是基于DNA修复缺陷的假设 在G2/M细胞周期停滞期间，确定细胞在FECD中是否经历感染或凋亡细胞死亡。