The Role of Oxidative Stress in the Pathogenesis of Fuchs Endothelial Corneal Dys

氧化应激在福克斯内皮角膜病变发病机制中的作用

• 批准号: 8474767
• 负责人: Ula V. Jurkunas
• 金额: $ 39.81万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8474767
• 关键词: 2-tert-butylhydroquinone; Accounting; Antioxidants; Apoptosis; Apoptotic; Binding; Biological Assay; Blindness; Cadaver; Cell Death; Cell Line; Cells; Characteristics; Chronic; Clinical; Collagen; Cornea; Corneal Endothelium; Corneal dystrophy; Critical Pathways; DNA; DNA Damage; Development; Disease; Elderly; Endothelial Cells; Endothelium; Etiology; Fuchs' Endothelial Dystrophy; Gene Expression; Genetic; Genomics; Human; In Vitro; Keratoplasty; Lead; Measures; Mitochondria; Mitochondrial DNA; Modification; Molecular; Nuclear; Oxidants; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Plasmids; Polymerase Chain Reaction; Predisposition; Production; Program Development; Promoter Regions; Proteins; Proteomics; Reactive Oxygen Species; Research; Response Elements; Role; SECTM1 gene; Sampling; Scientist; Specimen; Stress; System; Thiones; Transfection; Vision; abstracting; base; extracellular; human old age (65+); mitochondrial dysfunction; overexpression; oxidative DNA damage; response; sulfated glycoprotein 2; transcription factor

Project Summary/Abstract Fuchs endothelial dystrophy (FECD) is the most common cause of endogenous corneal endothelial degeneration whose primary etiology is unknown. Corneal transplantation is the only currently available measure to restore lost vision. There is mounting evidence that oxidative stress induces damage to corneal endothelium in FECD. Our preliminary studies have identified a decrease in the antioxidant response element (ARE)-driven antioxidants, overexpression of extracellular and stress-related proteins, and an increase in the levels of oxidized mitochondrial DNA in FECD endothelium. Since the underexpressed antioxidants have the common promoter region, antioxidant response element (ARE), we investigated levels of the main ARE- binding transcription factor, nuclear factor-E2-related factor-2 (Nrf2). We detected a decrease in Nrf2 protein level in FECD endothelium. There is, however, limited understanding of how chronic oxidative stress causes molecular and cellular damage in susceptible human corneal endothelial cells and which critical pathways specifically lead to progressive endothelial cell apoptosis and degeneration. It is important to investigate the role of oxidative stress in the pathogenesis of FECD since it opens a new avenue of study that can produce a significant impact on treatment of this blinding condition. The GOAL of the proposed studies is to determine specific cellular mechanisms that can be manipulated to reverse endothelial cell degeneration. These studies are significant because modification of key regulators of oxidant-antioxidant imbalance and resulting cellular damage may ameliorate endothelial cell susceptibility to stress-induced apoptotic cell death that characterizes FECD. Studies will use native samples from FECD patients and normal cadavers for further characterization of the proteomic and genomic differences, and genetic and pharmacotherapeutic manipulation of normal and FECD endothelial cell lines. Our Specific Aims are: Aim 1: Determine the effects of oxidant-antioxidant imbalance seen in FECD on expression of extracellular and stress-related proteins characteristically altered in the dystrophy and endothelial cell apoptosis. Aim 2: Determine how diminished Nrf2-regulated defense in FECD corneal endothelium leads to oxidant-antioxidant imbalance by 1) comparing the expression of Nrf2 pathway components between normal and FECD endothelium, and 2) determining whether enhancement of intracellular Nrf2 levels by plasmid transfection and by intracellular Nrf2 stabilizers, such 3H-1,2-dithiole-3- thione (D3T) and tert-butylhydroquinone (tBHQ), can enhance ARE-driven antioxidant expression and ameliorate and reverse the oxidant-induced damage in diseased corneal endothelium. Aim 3: Investigate the contribution of oxidative DNA damage on endothelial cell degeneration and apoptosis seen in FECD by 1) comparing levels of mitochondrial and nuclear DNA oxidative damage, 2) correlating the damage to mitochondrial dysfunction, and 3) determining the effects of mitochondria-targeted antioxidants in their ability to ameliorate the oxidative stress-induced cellular damage seen in FECD.

项目总结/摘要 Fuchs内皮营养不良（FECD）是内源性角膜内皮营养不良的最常见原因。 主要病因不明的变性。角膜移植是目前唯一可行的 恢复失去的视力。越来越多的证据表明，氧化应激会导致角膜损伤， FECD中的内皮细胞。我们的初步研究已经确定了抗氧化反应元件的减少 （ARE）驱动的抗氧化剂，细胞外和应激相关蛋白的过度表达，以及细胞外基质的增加。 FECD内皮细胞中的氧化线粒体DNA水平。由于低表达的抗氧化剂具有 共同启动子区，抗氧化反应元件（ARE），我们研究了主要的ARE- 结合转录因子，核因子E2相关因子-2（Nrf 2）。我们检测到Nrf 2蛋白的减少 FECD内皮水平。然而，对于慢性氧化应激如何导致 易感人角膜内皮细胞的分子和细胞损伤， 特别是导致进行性内皮细胞凋亡和变性。重要的是要调查 氧化应激在FECD发病机制中的作用，因为它开辟了一条新的研究途径， 对该设盲状况的治疗有显著影响。拟议研究的目标是确定 可以操纵以逆转内皮细胞变性的特定细胞机制。这些研究 是重要的，因为氧化剂-抗氧化剂不平衡的关键调节剂的修饰， 损伤可以改善内皮细胞对应激诱导的凋亡性细胞死亡的敏感性， FECD。研究将使用来自FECD患者和正常尸体的天然样本，以进一步表征 蛋白质组学和基因组学差异，以及正常和 内皮细胞系。我们的具体目标是：目标1：确定氧化剂-抗氧化剂的影响 在FECD中观察到细胞外和应激相关蛋白表达的不平衡， 营养不良和内皮细胞凋亡。目的2：确定Nrf 2调节的防御在 FECD角膜内皮通过以下方式导致氧化剂-抗氧化剂失衡：1）比较Nrf 2 正常和FECD内皮之间的通路成分，和2）确定是否增强 通过质粒转染和通过细胞内Nrf 2稳定剂，如3 H-1，2-二硫杂环戊烯-3-基， D3 T和叔丁基对苯二酚（tBHQ）可以增强ARE驱动的抗氧化剂表达， 改善和逆转氧化剂诱导的病变角膜内皮损伤。目标3：调查 氧化性DNA损伤对FECD中观察到的内皮细胞变性和凋亡的贡献1） 比较线粒体和核DNA氧化损伤的水平，2）将损伤与 线粒体功能障碍，和3）确定靶向抗氧化剂的作用，在他们的能力， 改善FECD中观察到的氧化应激诱导的细胞损伤。