The Role of IGFBP-3 in Mitochondrial Homeostasis in the Corneal Epithelium

项目摘要 干眼症（DED）是最常见的视力状况之一，随着年龄的增长而增加， 会对女性产生不良影响。DED干扰视力，通常是痛苦的，并对生活质量产生负面影响 为那些受影响的人。目前的治疗方法疗效有限，无法治愈。 泪液渗透压的增加是DED的中心特征。高渗触发线粒体功能障碍 最终导致半胱天冬酶释放和随后的细胞凋亡。我们实验室的工作发现， 胰岛素样生长因子结合蛋白-3（IGFBP-3）由角膜上皮细胞（CEC）分泌。分泌 IGFBP-3的表达在高渗时下调。对这项建议非常重要的是， 重组IGFBP-3对高渗条件下培养的CEC的作用阻断了高渗诱导的 线粒体呼吸减少。我们进一步提供了新的数据，表明IGFBP-3可能介导 在应激期间CEC的自噬通量。本提案的目的是调查以下方面的潜在作用： IGFBP-3介导DED中的线粒体代谢、线粒体自噬和大自噬（自噬）。 基于这些共同的发现，我们提出了核心假设，即IGFBP-3介导细胞凋亡， CEC在高渗应激过程中通过控制线粒体代谢和 自噬机制。将使用体外细胞培养物和体内细胞培养物的组合来检验这一假设。 体内动物研究以建立IGFBP-3、线粒体稳态和代谢之间的关系。 在目标1中，我们将关注IGFBP-3在暴露于不同水平的高渗性胰岛素的CEC中的代谢作用。 体外应激和表征IGFBP-3在体内干燥应激小鼠模型中表达。在目标2中， 我们将关注IGFBP-3在高渗培养条件下CEC的线粒体自噬和自噬中的作用， 使用稳定表达自噬体标记物GFP-LC 3和自噬体标记物GFP-LC 3的CEC系和小鼠进行体外和体内研究。 pH敏感荧光团，mt-Keima。 拟议的研究将是第一个探索IGFBP-3在线粒体中的病理生理作用的研究 呼吸，稳态，线粒体自噬和自噬在CEC暴露于高渗应激。 阐明IGFBP-3在DED中的作用可能会导致开发新的治疗方法来治疗和减轻DED。 疾病UT西南大学杰出的研究环境与集体相结合， 我的多学科导师团队的专业知识将提供卓越的培训和坚实的 作为未来眼科和视觉科学领域的医生科学家，

PROJECT SUMMARY Dry eye disease (DED) is one of the most common visual conditions that increases with age and disproportionally affects women. DED disrupts vision, is often painful, and negatively impacts the quality of life for those affected. Current treatments have limited efficacy and there is no cure. An increase in tear osmolarity is a central feature of DED. Hyperosmolarity triggers mitochondrial dysfunction that ultimately results in caspase release and subsequent apoptosis. Work in our laboratory has found that the insulin-like growth factor binding protein-3 (IGFBP-3) is secreted from corneal epithelial cells (CEC). Secretion of IGFBP-3 is downregulated in response to hyperosmolarity. Of high importance to this proposal, the addition of recombinant IGFBP-3 to CECs cultured in hyperosmolar conditions blocks the hyperosmolar-induced decrease in mitochondrial respiration. We further provide novel data that suggests that IGFBP-3 may mediate autophagic flux in CECs during stress. The purpose of this proposal is to investigate a potential role for IGFBP-3 in mediating mitochondrial metabolism, mitophagy, and macroautophagy (autophagy) in DED. Based on these collective findings, we propose the central hypothesis that IGFBP-3 mediates cellular homeostasis in CECs during hyperosmolar stress through control of mitochondrial metabolism and autophagic mechanism(s). This hypothesis will be tested using a combination of in vitro cell cultures and in vivo animal studies to establish the relationship between IGFBP-3, mitochondrial homeostasis and metabolism. In Aim 1, we will focus on the metabolic effects of IGFBP-3 in CECs exposed to varying levels of hyperosmolar stress in vitro and characterize expression of IGFBP-3 in a desiccating stress mouse model in vivo. In Aim 2, we will focus on the role of IGFBP-3 in mitophagy and autophagy in CECs exposed to hyperosmolar culture in vitro and in vivo using CEC lines and mice that stably express the autophagosome marker, GFP-LC3, and the pH sensitive fluorophore, mt-Keima. The proposed studies will be the first to explore a pathophysiological role for IGFBP-3 in mitochondrial respiration, homeostasis, mitophagy and autophagy in CECs exposed to hyperosmolar stress. Elucidating the role of IGFBP-3 in DED may lead to the development of novel therapies to treat and mitigate disease. The outstanding research environment at UT Southwestern combined with the collective expertise from my multidisciplinary mentorship team will provide exceptional training and a solid platform to build upon as a future physician-scientist in Ophthalmology and Vision Science.