Role of Oxidative Stress in Pathogenesis of Fuchs Endothelial Corneal Dystrophy

项目摘要/摘要 Fuchs内皮性皮质营养不良(FECD)是一种常见的年龄相关性营养不良，在 女性，病因不明。在FECD中，角膜内皮细胞(CE)的丢失伴随着异常 细胞外基质(ECM)以牙胶形式沉积。我们的实验室是第一个将氧化DNA FECD发病机制中的损伤和线粒体功能障碍。具体地说，我们表明DNA损伤， 在紫外线A(UVA)光诱导下，引起小鼠FECD。此外，UVA还可诱导CE细胞周期停滞。 G2/M期与细胞衰老。我们确定了关键雌激素--细胞色素P1B1的新参与。 代谢酶，在CE对UVA的易感性上存在性别差异；并检测到更大的 雌性小鼠线粒体DNA(MtDNA)损伤。然而，观察到的机制更大 雌性小鼠对UVA诱导的DNA损伤的敏感性尚不清楚。根据我们之前的发现， 我们建议研究紫外光诱导的氧化-抗氧化失衡是否导致衰老和细胞外基质 通过导致G2/M细胞周期停滞而沉积；如果这种不平衡导致CYP1B1转位到 线粒体触发雌激素对女性线粒体DNA的更大损伤。基因阵列分析揭示 FECD中DNA修复基因的下调；因此，我们将确定这是否会导致 在G2/M细胞周期停滞期间进行DNA损伤修复，触发细胞衰老或 细胞凋亡。我们的研究很有意义，因为研究性别依赖的机制涉及氧化 应激诱导的细胞损伤将为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基于CE的生理结果的非遗传性FECD小鼠模型 长波紫外线与永生化人CE细胞系、人房水和体外培养的敏感性 FECD供者的基因分型标本。我们的具体目标是：目标1：研究UVA辐射在 FECD中G2/M期细胞周期阻滞及诱导细胞衰老和ECM沉积。这一目标是基于 DNA损伤导致G2/M期停滞并诱导细胞衰老的假说，进而 导致细胞外基质在FECD中以牙胶的形式异常沉积。目的2：确定UVA辐射是否 激活CYP1B1并诱导雌激素代谢，对女性造成更大的DNA损伤。 这一目标是基于这样一种假设，即女性FECD的发病率和严重性更高是由于UVA- 诱导CYP1B1移位到线粒体，从而触发反应性雌激素代谢物的形成， 造成线粒体DNA损伤。目的3：确定DNA损伤反应和DNA修复在UVA过程中的作用 诱导FECD细胞周期停滞。这一目标是基于这样一种假设：在G2/M期，DNA修复缺陷 细胞周期停滞决定了FECD中的细胞是衰老还是凋亡性死亡。

Project Summary/Abstract Fuchs Endothelial Cornel 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 DNA damage and mitochondrial dysfunction in FECD pathogenesis. Specifically, we showed that DNA damage, induced by ultraviolet-A (UVA) light, causes FECD in mice. Moreover, the UVA induced CE cell cycle arrest in G2/M phase and cellular senescence. We identified the novel involvement of CYP1B1, the key estrogen- metabolizing enzyme, in sex-dependent differences in CE susceptibility to UVA; and detected greater mitochondrial DNA (mtDNA) damage in female mice. However, the mechanism of the observed greater susceptibility of female mice to UVA-induced DNA damage is unknown. Building upon our previous findings, we propose to investigate if UV light–induced oxidant-antioxidant imbalance leads to senescence and ECM deposition by causing G2/M cell cycle arrest; and if this imbalance causes translocation of CYP1B1 into mitochondria triggering greater estrogen-induced mtDNA damage in females. Gene array analysis revealed downregulation of DNA repair genes in FECD; therefore, we will determine whether this leads to decreased DNA damage repair during G2/M cell cycle arrest, triggering the cells to undergo either senescence or apoptosis. Our study is significant, as the investigation sex-dependent mechanisms involved in oxidative stress-induced cellular damage will provide new treatment targets for FECD. In order to achieve these aims, we will use our newly developed non-genetic mouse model of FECD based on physiologic outcome of CE susceptibility to UVA along with immortalized human CE cell lines, human aqueous fluid, and ex vivo specimens of genotyped FECD donors. Our Specific Aims are: Aim 1: Investigate the role of UVA irradiation in G2/M cell cycle arrest and induction of cellular senescence and ECM deposition in FECD. This aim is based on the hypothesis that DNA damage leads to G2/M phase arrest and induces cellular senescence, which in turn leads to aberrant ECM deposition in the form of guttae in FECD. Aim 2: Determine whether UVA irradiation activates CYP1B1 and induces estrogen metabolism causing preferentially greater DNA damage in females. This aim is based on the hypothesis that higher incidence and severity of FECD in women occurs due to UVA- induced translocation of CYP1B1 into mitochondria, which triggers formation of reactive estrogen metabolites, causing mtDNA damage. Aim 3: Determine the role of DNA damage response and DNA repair during UVA- induced cell cycle arrest in FECD. This aim is based on the hypothesis that DNA repair deficiency during G2/M cell cycle arrest determines whether cells undergo senescence or apoptotic cell death in FECD.