Role of Oxidative Stress in Pathogenesis of Fuchs Endothelial Corneal Dystrophy

 描述（由申请方提供）：Fuchs角膜内皮营养不良（FECD）是一种常见的年龄相关性营养不良，在女性中更常见，病因不明。在FECD中，角膜内皮（CE）细胞损失伴随着以滴状物形式的异常细胞外基质（ECM）沉积。我们的实验室是第一个将氧化应激与FECD发病机制联系起来的实验室。具体来说，我们发现在FECD中，由于Nrf 2调节的次优抗氧化防御导致的氧化-抗氧化失衡，包括其转录靶点的下降， NQO 1导致DNA氧化损伤、线粒体功能障碍和细胞凋亡。然而，关于牙胶形成的机制和女性FECD发展的主导地位知之甚少。在FECD中细胞变性中观察到的DNA损伤的机制尚不清楚。基于我们先前的研究结果，我们建议调查FECD中的氧化剂-抗氧化剂失衡是否会导致内皮间质转化（EMT）伴ECM沉积，以及这种失衡是否会导致细胞毒性雌激素代谢物的形成，已知细胞毒性雌激素代谢物会在具有缺陷Nrf 2-NQO 1的组织中积累并导致DNA损伤，更常见于女性。由于我们的初步研究表明FECD中存在缺陷的DNA损伤反应（DDR），我们将确定它是否会导致DNA损伤修复减少，细胞衰老和凋亡。我们的建议旨在探索三种不同的疾病机制：EMT，雌激素遗传毒性和DDR，所有这些都可能单独或共同导致FECD的发展。我们的研究具有重要意义，因为对氧化应激诱导的细胞损伤机制的研究将为FECD提供新的治疗靶点。为了实现这些目标，我们将使用我们新开发的端粒酶永生化的人CE细胞和基于紫外线照射小鼠角膜的体内氧化应激模型。该模型与我们的研究特别相关，因为它在小鼠中复制了在FECD患者中典型观察到的CE形态学变化，包括肠线样病变和细胞凋亡。我们的具体目标是：目标1：确定FECD中观察到的氧化剂-抗氧化剂失衡是否激活EMT，从而导致滴液中观察到的异常ECM沉积。这一目的是基于以下假设：在FECD中，慢性氧化应激导致内皮细胞经历EMT和增强的ECM沉积，从而导致可以通过激活Nrf 2途径进行修饰的牙胶形成。目标二：研究反应性雌激素代谢产物在启动雌激素遗传毒性途径中的作用，这可能是女性FECD发生率和严重程度较高的原因。这一目的是基于这样的假设，即改变雌激素代谢酶的水平导致雌激素代谢失衡，并增加形成脱嘌呤雌激素-DNA加合物在FECD。目的3：探讨DDR在FECD氧化应激损伤中对线粒体和核DNA修复的作用。这一目标是基于以下假设：受损的DDR导致DNA修复缺陷，从而导致线粒体功能障碍、细胞衰老和凋亡。

 DESCRIPTION (provided by applicant): Fuchs Endothelial Corneal Dystrophy (FECD), a common age-related dystrophy, which is more prevalent in women, is of unknown etiology. In FECD, corneal endothelial (CE) cell loss is accompanied by abnormal extracellular matrix (ECM) deposition in the form of guttae. Our laboratory was the first to link oxidative stress with FECD pathogenesis. Specifically, we showed that in FECD, oxidant-antioxidant imbalance due to suboptimal Nrf2-regulated antioxidant defense, including a decline in its transcriptional target NQO1, leads to oxidative DNA damage, mitochondrial dysfunction, and apoptosis. However, little is known regarding the mechanism of guttae formation and the predominance of FECD development in women. The mechanism of the observed DNA damage in cellular degeneration in FECD is not known. Building upon our previous findings, we propose to investigate if oxidant-antioxidant imbalance in FECD leads to endothelial mesenchymal transition (EMT) with concomitant ECM deposition, and if this imbalance causes the formation of cytotoxic estrogen metabolites, which are known to accumulate in tissues with the defective Nrf2-NQO1 and cause DNA damage, more commonly in women. Since our preliminary studies indicate a defective DNA damage response (DDR) in FECD, we will determine whether it leads to decreased DNA damage repair, cellular senescence, and apoptosis. Our proposal seeks to explore three different disease mechanisms: EMT, estrogen genotoxicity, and DDR, all of which either independently or collectively may lead to the development of FECD. Our study is significant, as the investigation of the mechanisms involved in the oxidative stress-induced cellular damage will provide new treatment targets for FECD. In order to achieve these aims, we will use our newly developed telomerase-immortalized human CE cells and the in vivo oxidative stress model based on the ultraviolet-A irradiation of mouse corneas. This model is specifically relevant to our study since it replicates in mice the CE morphological changes with guttae-like lesions and apoptosis, characteristically seen in FECD patients. Our Specific Aims are: Aim 1: Determine whether oxidant-antioxidant imbalance seen in FECD activates EMT that leads to aberrant ECM deposition seen in guttae. This aim is based on the hypothesis that in FECD chronic oxidative stress causes endothelial cells to undergo EMT and enhanced ECM deposition leading to guttae formation that can be modified by activation of Nrf2 pathway. Aim 2: Investigate the role of reactive estrogen metabolites in initiating an estrogen genotoxic pathway that may account for higher incidence and severity of FECD in women. This aim is based on the hypothesis that altered levels of estrogen-metabolizing enzymes lead to imbalanced estrogen metabolism and increased formation of depurinating estrogen-DNA adducts in FECD. Aim 3: Investigate the role of DDR on mitochondrial and nuclear DNA repair during oxidative stress- induced damage of CE in FECD. This aim is based on the hypothesis that impaired DDR leads to deficient DNA repair that results in mitochondrial dysfunction, cellular senescence, and apoptosis.