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: 8088101
• 负责人: Ula V. Jurkunas
• 金额: $ 41.9万
• 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/8088101
• 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; base; extracellular; human old age (65+); mitochondrial dysfunction; overexpression; oxidative DNA damage; public health relevance; response; sulfated glycoprotein 2; transcription factor

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Fuchs endothelial dystrophy (FECD) is the major cause of progressive blindness from corneal endothelial cell death and the second most common cause of corneal transplants done in the elderly (>65 years old) in the U.S. In FECD, corneal endothelium, a layer of cells whose primary function is to maintain corneal transparency, is hypothesized to have a heightened susceptibility to the deleterious effects of oxidative stress resulting in progressive cell death. The research proposed in this application will provide new information of how oxidative stress causes molecular and cellular damage in the susceptible FECD endothelium. Understanding the key regulators of antioxidant defense and oxidative stress-induced cellular damage may facilitate development of pharmacotherapeutic treatment for FECD patients.

描述（由申请人提供）：Fuchs内皮营养不良（FECD）是内源性角膜内皮变性的最常见原因，其主要病因尚不清楚。角膜移植是恢复失去视力的唯一可用措施。有越来越多的证据表明，氧化应激会诱导FECD中角膜内皮的损害。我们的初步研究已经确定了抗氧化剂反应元件（IS）驱动的抗氧化剂，细胞外和应激相关蛋白的过表达，以及在FECD内皮中氧化的线粒体DNA水平的增加。由于未经过压制的抗氧化剂具有共同启动子区域，因此抗氧化剂反应元件（AS），我们研究了主要的IAM结合转录因子，核因子-E2相关因子-2（NRF2）的水平。我们检测到FECD内皮中NRF2蛋白水平的降低。但是，对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的理解有限，以及这些关键途径专门导致渐进性内皮细胞凋亡和变性。重要的是要研究氧化应激在FECD发病机理中的作用，因为它开辟了一种新的研究途径，该研究可以对这种盲人的治疗产生重大影响。拟议的研究的目的是确定可以操纵以逆转内皮细胞变性的特定细胞机制。这些研究之所以重要，是因为修饰氧化剂抗氧化剂失衡和导致细胞损伤的关键调节剂可能会改善内皮细胞对压力诱导的凋亡细胞死亡的敏感性，这表征了FECD。研究将使用来自FECD患者和正常尸体的天然样本来进一步表征蛋白质组学和基因组差异，以及对正常和FECD内皮细胞系的遗传和药物治疗操作。我们的具体目的是：目标1：确定FECD中氧化剂与氧化剂失衡对细胞外和应激相关蛋白表达的影响，在营养不良和内皮细胞凋亡中特征改变了。目标2：确定粪便角膜内皮中NRF2调节的防御如何减少导致氧化剂 - 抗氧化剂不平衡1）通过比较1）比较正常内皮和FECD内皮之间的NRF2途径成分的表达，以及2）确定稳定性的NRF2水平是否通过质体经染料来增强质子质体的NRRFFFFFF。 3H-1,2-二硫代-3- thione（D3T）和叔丁基羟基喹酮（TBHQ）可以增强驱动的抗氧化剂表达，并改善并逆转氧化剂诱导的疾病角膜内皮内皮的损伤。目标3：研究氧化性DNA损伤对FECD中内皮细胞退化和凋亡的贡献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的损伤相关，以及3）确定氧化的氧化能力对抗毒性的影响，在抗氧化剂中的影响，并将其造成的抗氧化剂的抗性作用。粪 公共卫生相关性：Fuchs内皮营养不良（FECD）是角膜内皮细胞死亡的进行性失明的主要原因，也是美国老年人（> 65岁）在FECD，Corneal Endothium的老年人（> 65岁）进行的角膜移植的第二大最常见原因，其主要的作用是在其主要的作用中，其主要的作用是在角膜上造成的，以维持角膜透明度，是一种高度的透明度，是一种高度的透明度，是一种高度延伸的效果，导致进行性细胞死亡。在本应用中提出的研究将提供有关氧化应激如何在易感的FECD内皮中导致分子和细胞损伤的新信息。了解抗氧化剂防御和氧化应激诱导的细胞损伤的关键调节剂可能有助于开发FECD患者的药物治疗。