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

项目总结 干眼病(DED)是最常见的视觉疾病之一，随着年龄的增长和 对女性的影响不成比例。DED扰乱视力，经常是痛苦的，并对生活质量产生负面影响 对于那些受影响的人。目前的治疗方法疗效有限，而且没有治愈方法。 泪液渗透压升高是DED的主要特征。高渗透压引发线粒体功能障碍 这最终导致caspase的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。分泌物 IGFBP-3的表达下调，以响应高渗透压。对这项提议非常重要的是，增加 重组IGFBP-3对高渗条件下培养的CECs的抑制作用 线粒体呼吸减少。我们进一步提供了新的数据，表明IGFBP-3可能在 应激期间内皮细胞的自噬通量。这项提议的目的是调查一个潜在的角色 IGFBP-3在DED线粒体代谢、有丝分裂吞噬和大自噬(自噬)中的作用 基于这些集体发现，我们提出了IGFBP-3介导细胞 高渗应激下内皮细胞的动态平衡及其与线粒体代谢的关系 自噬机制(S)。这一假说将使用体外细胞培养和体内实验相结合的方法进行验证。 活体动物研究以确定IGFBP-3与线粒体动态平衡和代谢的关系。 在目标1中，我们将重点研究IGFBP-3在不同水平的高渗透压下对CECs的代谢影响。 体外应激及IGFBP-3在干燥应激小鼠体内的表达特征。在目标2中， 我们将重点研究IGFBP-3在高渗培养的CECs有丝分裂吞噬和自噬中的作用。 体外和体内使用CEC系和稳定表达自噬体标记物GFP-LC3和 PH值敏感的荧光团，凯马山。 这项拟议的研究将首次探索IGFBP-3在线粒体中的病理生理学作用 高渗应激下CECs的呼吸、动态平衡、有丝分裂吞噬和自噬。 阐明IGFBP-3在DED中的作用可能有助于开发治疗和缓解DED的新疗法 疾病。德克萨斯大学西南分校卓越的研究环境与集体 来自我的多学科指导团队的专业知识将提供特殊的培训和坚实的 作为未来眼科和视觉科学领域的内科医生和科学家的平台。

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.