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

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。角膜移植是目前唯一有效的恢复视力的方法。越来越多的证据表明，氧化应激可引起FECD患者角膜内皮细胞的损伤。我们的初步研究已经确定了抗氧化反应元件（ARE）驱动的抗氧化剂的减少，细胞外和应激相关蛋白的过度表达，以及FECD内皮中氧化线粒体DNA水平的增加。由于低表达的抗氧化剂具有共同的启动子区域，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 （Nrf2）的水平。我们检测到FECD内皮中Nrf2蛋白水平下降。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以及哪些关键途径特异性导致内皮细胞进行性凋亡和变性的了解有限。研究氧化应激在FECD发病机制中的作用是很重要的，因为它开辟了一条新的研究途径，可以对这种致盲疾病的治疗产生重大影响。拟议研究的目的是确定特定的细胞机制，可以操纵来逆转内皮细胞变性。这些研究意义重大，因为对氧化-抗氧化失衡的关键调节因子的修改和由此导致的细胞损伤可能改善内皮细胞对应激诱导的细胞凋亡的易感性，这是FECD的特征。研究将使用来自FECD患者和正常尸体的原生样本来进一步表征蛋白质组学和基因组差异，以及正常和FECD内皮细胞系的遗传和药物治疗操作。我们的具体目标是：目的1：确定feecd中氧化-抗氧化失衡对细胞外蛋白和应激相关蛋白表达的影响，这些蛋白在营养不良和内皮细胞凋亡中具有特征性改变。目标2:通过1)比较正常和FECD内皮中Nrf2通路组分的表达，以及2)通过质粒转染和细胞内Nrf2稳定剂(如3h -1,2-二硫基-3-硫酮（D3T）和叔丁基对苯二酚（tBHQ）是否增强细胞内Nrf2水平，确定Nrf2调控的FECD角膜内皮中防御功能的减弱如何导致氧化-抗氧化失衡。可以增强are驱动的抗氧化表达，改善和逆转病变角膜内皮中氧化引起的损伤。目的3：通过1)比较线粒体和核DNA氧化损伤水平，2)将损伤与线粒体功能障碍联系起来，以及3)确定线粒体靶向抗氧化剂在改善FECD中氧化应激诱导的细胞损伤方面的作用，研究氧化DNA损伤对FECD中内皮细胞退化和凋亡的贡献。

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.