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

描述(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可行的恢复失明视力的措施。越来越多的证据表明，氧化应激导致FECD的角膜内皮细胞损伤。我们的初步研究发现，抗氧化剂反应元件(ARE)驱动的抗氧化剂减少，细胞外和应激相关蛋白过度表达，FECD内皮细胞氧化线粒体DNA水平增加。由于表达不足的抗氧化剂具有共同的启动子区域，即抗氧化反应元件(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内皮细胞中Nrf2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定地导致进行性内皮细胞凋亡和退化，人们了解得很有限。研究氧化应激在FECD发病机制中的作用非常重要，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。这项拟议研究的目标是确定可以操纵以逆转内皮细胞退化的特定细胞机制。这些研究意义重大，因为氧化剂-抗氧化剂失衡的关键调节因素的修饰以及由此导致的细胞损伤可能会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患者和正常身体的本地样本来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物治疗操作。我们的具体目标是：目标1：确定FECD中氧化-抗氧化失衡对营养不良和内皮细胞凋亡中特有的细胞外和应激相关蛋白表达的影响。目的：通过1)比较正常和FECD角膜内皮细胞中Nrf2途径组分的表达，以及3 H-1，2-二硫醇-3-硫酮(D3T)和叔丁基对苯二酚(TBHQ)等细胞内Nrf2稳定剂对细胞内Nrf2水平的影响，以确定Nrf2在FECD角膜内皮细胞中的防御功能减弱是如何导致氧化-抗氧化失衡的，从而改善和逆转氧化诱导的角膜内皮损伤。目的：通过1)比较线粒体和核DNA氧化损伤的水平，2)线粒体损伤与线粒体功能障碍的相关性，以及3)确定线粒体靶向抗氧化剂在改善FECD氧化应激诱导的细胞损伤中的作用，以探讨氧化DNA损伤在FECD中的作用。 与公共卫生相关：Fuchs内皮细胞营养不良(FECD)是导致角膜内皮细胞死亡导致进行性失明的主要原因，也是美国老年人(65岁)进行角膜移植的第二大常见原因。在FECD中，角膜内皮是一层主要功能是维持角膜透明度的细胞层，被认为对氧化应激导致细胞进行性死亡的有害影响具有更高的易感性。这项申请中提出的研究将提供有关氧化应激如何导致易感FECD内皮细胞和分子损伤的新信息。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素可能有助于FECD患者药物治疗的发展。

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.