Effects of systemic disease on corneal epithelial pathophysiology

项目摘要 糖尿病引起的角膜并发症很常见，通常非常痛苦，会对生活质量产生负面影响， 导致永久性视力损伤。由于大量的分子途径在细胞中被改变， 对于糖尿病角膜，由于异常的细胞反应和角膜基质的丧失，传统的治疗通常是不够的。 来自角膜神经的营养支持。 我们先前的研究表明，胰岛素样生长因子（IGF）系统在糖尿病中发生改变。这 包括在糖尿病患者中多效性分泌蛋白IGF-结合蛋白-3（IGFBP-3）的显著增加， 与角膜基底下神经丛缺失相关的人类泪液。而角膜上皮细胞不 需要胰岛素来摄取葡萄糖，我们的实验室发现，相反，胰岛素在 介导IGF 1型受体（IGF-1 R）、胰岛素受体（INSR）和IGFBP-3的表达。进一步 对这些通路的研究导致了角膜上皮细胞中新的令人兴奋的发现 （CECs）构成了当前提案的基础。与这项提议高度相关的是，我们所有的发现 暗示IGF-1系统作为线粒体功能和稳定性的介体。这具有临床意义 由于线粒体损伤是糖尿病和其它疾病发病率主要潜在原因 全身性疾病。 基于这些发现，我们提出了两个中心假设：（1）胰岛素和IGFBP-3调节 线粒体质量控制和稳定性通过协调细胞核串扰的反应 （2）电压依赖性阴离子通道VDAC 1、IGF-1 R和 INSR介导线粒体生物发生和稳定性。我们将在以下目标中检验这些假设。 目标1。比较IGF相关配体、受体和IGFBP-3对信号传导、增殖和细胞周期的影响。 正常血糖与高血糖CEC体外培养和糖尿病小鼠角膜中的代谢 体内上皮。目的2：比较IGF相关配体、受体和IGFBP-3对线粒体膜电位的影响。 体外CEC中的生物发生、通过与VDAC 1相互作用的稳定性和动力学（裂变/融合）， 糖尿病小鼠角膜上皮的体内研究。目的3：比较胰岛素样生长因子相关配体、受体和 IGFBP-3对体外CEC和体内糖尿病小鼠角膜上皮细胞的线粒体自噬和凋亡的影响。 众所周知，线粒体调节驱动能量产生和细胞存活的信号，并且它们的功能是通过调节细胞内的线粒体来实现的。 功能障碍与多种疾病有关。这些研究将首次提供 详细描述IGF家族在介导线粒体稳定性和功能中的作用的综合图片， 健康的角膜和疾病的病理学。这些结果可能代表着一个重大的范式转变 在我们对糖尿病角膜上皮和其他角膜上皮的理解和未来的治疗中， 线粒体功能改变的疾病和营养不良。

PROJECT SUMMARY Corneal complications from diabetes are common, often very painful, can negatively impact quality of life, and lead to permanent visual impairment. Owing to the vast array of molecular pathways that are altered in the diabetic cornea, traditional therapies are often not sufficient, due to abnormal cellular responses and the loss of trophic support from corneal nerves. Our prior studies have shown that the insulin-like growth factor (IGF) system is altered in diabetes. This includes a significant increase in the pleiotropic secretory protein, IGF-binding protein-3 (IGFBP-3) in diabetic human tears that correlates with loss of the corneal subbasal nerve plexus. While corneal epithelial cells do not require insulin for glucose uptake, our laboratory has found instead that insulin has an essential role in mediating expression of the IGF type 1 receptor (IGF-1R), insulin receptor (INSR), and IGFBP-3. Further interrogation of these pathways led to the generation of novel and exciting findings in corneal epithelial cells (CECs) that form the basis for the current proposal. Of high relevance to this proposal, all of our findings implicate the IGF-1 system as a mediator of mitochondrial function and stability. This is clinically significant since mitochondrial damage is a major underlying cause of disease morbidity in diabetes and other systemic diseases. Based on these findings, we propose to test two central hypotheses: (1) that insulin and IGFBP-3 regulate mitochondrial quality control and stability by coordinating mitochondrial-nuclear crosstalk in response to stress; and, (2) that interactions between the voltage dependent anion channel VDAC1, IGF-1R, and INSR mediate mitochondrial biogenesis and stability. We will test these hypotheses in the following aims. Aim 1. Compare the effects of IGF-related ligands, receptors, and IGFBP-3 on signaling, proliferation, and metabolism in normoglycemic versus hyperglycemic CEC culture in vitro and the diabetic mouse corneal epithelium in vivo. Aim 2: Compare the effects of IGF-related ligands, receptors, and IGFBP-3 on mitochondrial biogenesis, stability through interactions with VDAC1, and dynamics (fission/fusion) in CECs in vitro and the diabetic mouse corneal epithelium in vivo. Aim 3: Compare the effects of IGF-related ligands, receptors, and IGFBP-3 on mitophagy and apoptosis in CECs in vitro and the diabetic mouse corneal epithelium in vivo. Mitochondria are well known to regulate signals that drive energy production and cell survival, and their dysfunction has been implicated in a wide range of diseases. These studies will provide the first comprehensive picture detailing the role of the IGF family in mediating mitochondrial stability and function in the healthy cornea and in the pathobiology of disease. The outcomes could represent a major paradigm shift in our understanding and future treatment of the diabetic corneal epithelium and other corneal diseases and dystrophies where mitochondrial function is altered.