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

项目摘要/摘要 Fuchs内皮营养不良(FECD)是内源性角膜内皮最常见的原因 病因不明的退化症。角膜移植是目前唯一可用的 恢复失明视力的措施。越来越多的证据表明，氧化应激导致角膜损伤。 FECD中的内皮细胞。我们的初步研究已经确定抗氧化反应元素减少。 (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 FECD血管内皮细胞线粒体DNA氧化水平。由于表达不足的抗氧化剂具有 共同启动子区，抗氧化反应元件(ARE)，我们调查了主要ARE的水平。 结合转录因子，核因子-E2相关因子-2(Nrf2)。我们检测到Nrf2蛋白的减少 在FECD内皮细胞中的水平。然而，人们对慢性氧化应激是如何引起的了解有限 易感的人角膜内皮细胞的分子和细胞损伤及其关键途径 特异性地导致进行性内皮细胞凋亡和变性。重要的是调查 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，可以产生 对这种致盲情况的治疗产生了重大影响。拟议研究的目标是确定 可被操纵以逆转内皮细胞退化的特定细胞机制。这些研究 之所以意义重大，是因为氧化-抗氧化失衡的关键调节因子的修饰以及由此导致的细胞 损伤可能改善内皮细胞对应激诱导的凋亡细胞死亡的敏感性 FECD。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 正常人和正常人的蛋白质组和基因组差异以及遗传和药物治疗操作 FECD内皮细胞系。我们的具体目标是：目标1：确定氧化剂-抗氧化剂的效果 FECD中细胞外和应激相关蛋白表达的失衡 营养不良和血管内皮细胞凋亡。目标2：确定Nrf2调节的防御能力如何减弱 FECD角膜内皮细胞通过1)比较Nrf2的表达导致氧化-抗氧化失衡 正常和FECD内皮之间的通路成分，以及2)决定是否增强 通过质粒法和细胞内稳定剂，如~3H-1，2-二硫杂环-3-硫醚-3- 硫酮(D3T)和叔丁基对苯二酚(TBHQ)，可增强ARE驱动的抗氧化表达和 改善和逆转氧化剂对病变角膜内皮的损伤。目标3：调查 DNA氧化损伤对内皮细胞变性和凋亡的影响 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，以及3)确定线粒体靶向抗氧化剂对其能力的影响 改善FECD中氧化应激诱导的细胞损伤。

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.