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

描述（由申请方提供）：Fuchs内皮营养不良（FECD）是内源性角膜内皮变性的最常见原因，其主要病因不明。角膜移植是目前唯一可用的恢复丧失视力的措施。有越来越多的证据表明，氧化应激诱导损伤角膜内皮细胞在FECD。我们的初步研究已经确定了减少的抗氧化反应元件（ARE）驱动的抗氧化剂，细胞外和应激相关蛋白的过度表达，并在FECD内皮细胞的氧化线粒体DNA的水平增加。由于低表达的抗氧化剂具有共同的启动子区域，抗氧化剂反应元件（ARE），我们研究了主要ARE结合转录因子，核因子E2相关因子2（Nrf 2）的水平。我们检测到FECD内皮中Nrf 2蛋白水平的降低。然而，对于慢性氧化应激如何导致易感的人角膜内皮细胞的分子和细胞损伤以及哪些关键途径特别导致进行性内皮细胞凋亡和变性的理解有限。重要的是要调查的作用，氧化应激的发病机制FECD，因为它打开了一个新的研究途径，可以产生显着的影响，治疗这种盲目的条件。拟议研究的目标是确定可用于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和由此产生的细胞损伤可以改善内皮细胞对应激诱导的细胞凋亡的敏感性，这是FECD的特征。研究将使用来自FECD患者和正常尸体的天然样本进一步表征蛋白质组和基因组差异，以及正常和FECD内皮细胞系的遗传和药理学操作。我们的具体目标是：目标1：确定FECD中观察到的氧化剂-抗氧化剂失衡对细胞外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发生特征性改变。目标二：通过1）比较正常和FECD内皮之间Nrf 2途径组分的表达，和2）确定通过质粒转染和细胞内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））是否增强细胞内Nrf 2水平，可以增强ARE驱动的抗氧化剂表达，改善和逆转病变角膜内皮中氧化剂诱导的损伤。目标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相关联，3）确定靶向抗氧化剂对改善FECD中观察到的氧化应激诱导的细胞损伤的能力的影响，研究氧化DNA损伤对FECD中观察到的内皮细胞变性和细胞凋亡的贡献。 公共卫生相关性：Fuchs内皮营养不良（FECD）是角膜内皮细胞死亡导致进行性失明的主要原因，也是老年人角膜移植的第二大常见原因在FECD中，角膜内皮是一层细胞，其主要功能是维持角膜透明度，被假设对导致进行性细胞死亡的氧化应激的有害作用具有高度的易感性。本申请中提出的研究将提供氧化应激如何在易感FECD内皮中引起分子和细胞损伤的新信息。了解抗氧化防御和氧化应激诱导的细胞损伤的关键调节因子可能有助于开发FECD患者的药物治疗。

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.