The Roles of Autophagy in Limbal/Corneal Epithelia.

自噬在角膜缘/角膜上皮中的作用。

• 批准号: 9910410
• 负责人: ROBERT M LAVKER
• 金额: $ 49.57万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910410
• 关键词: 3-Dimensional; Affect; Aging; Aniridia; Anterior; Attenuated; Autophagocytosis; BCL2 gene; Basal Cell; Biochemical; Biological; Biological Models; Biology; Catabolic Process; Cell Maintenance; Cell Nucleus; Cell Proliferation; Cells; Cellular Assay; Chemical Burns; Clinical; Complement; Comprehension; Conjunctival Pterygium; Connexin 43; Cornea; Corneal Diseases; Defect; Diabetes Mellitus; Disease; Environment; Epithelial; Epithelial Cells; Epithelium; Etiology; Excision; Eye; Family; Film; Fuchs' Endothelial Dystrophy; Genes; Goals; Homeostasis; Human; Impairment; Injury; Investigation; Keratin; Keratopathy; Knock-in Mouse; Knowledge; Label; Lead; Lighting; Lysosomes; Maintenance; Measures; Mediating; MicroRNAs; Microscopy; Modality; Modeling; Molecular; Monitor; Mucin 1 protein; Mus; Nuclear; Organelles; Patients; Pharmacology; Physiological; Plasminogen Activator Inhibitor 2; Play; Proteins; Receptor Protein-Tyrosine Kinases; Recurrence; Regulation; Reporting; Rest; Role; Signal Transduction; Starvation; Stevens-Johnson Syndrome; Stream; Stress; Structure; Surface; Techniques; Testing; Tissues; Transplantation; Vesicle; base; biomarker evaluation; clinical application; corneal epithelial wound healing; corneal epithelium; diabetic; diabetic patient; epithelial stem cell; gain of function; improved; in vivo; insight; involucrin; knock-down; limbal; loss of function; mouse model; negative affect; notch protein; novel; novel strategies; ocular surface; preservation; response; stem; stem cell homeostasis; stem cell proliferation; stem cells; wound; wound healing

PROJECT SUMMARY/ABSTRACT The anterior surface of the eye functions as a barrier to the external environment, protects the delicate underlying structures from injury, supports a tear film and maintains transparency. These functions are achieved, in part, through the corneal and limbal epithelia, which must maintain proper proliferation and differentiation. Autophagy, a catabolic process by which cells adapt to intrinsic and extrinsic stress-related situations, has been shown to also regulate proliferation and differentiation. Despite the many advances made in our understanding of corneal and limbal epithelial biology, autophagy remains largely unstudied. We have recently reported that: (i) under resting conditions, autophagy is greater in limbal epithelial basal compared with corneal epithelial basal cells; (ii) autophagy contributes to limbal epithelial proliferative capacity; and (iii) autophagy enables proper activation of transit amplifying (TA) cells following wounding. We also have preliminary evidence that: (i) NUS1 is a novel upstream regulator of autophagy in the limbal epithelium; (ii) blocking autophagy impairs corneal epithelial differentiation and the removal of nuclear material (nucelophagy); and (iii) the receptor tyrosine kinase, EphA2, positively affects corneal epithelial differentiation via regulating end stage autophagy. These observations lead us to hypothesize that autophagy is required to maintain limbal epithelial stem and TA cell homeostasis as well as insure proper corneal epithelial differentiation. We propose to test this hypothesis by focusing on how autophagy: (i) affects limbal epithelial stem cell proliferation; (ii) regulates differentiation in the corneal epithelium; and (iii) is regulated by EphA2 in the corneal epithelium. To accomplish these goals, we will capitalize on our ability to conduct gain- and loss-of-function studies of autophagy-related genes and their key up-stream regulators in complimentary model systems that include cultured human limbal (HLEKs) and corneal (HCEKs) epithelial cells, 3-D raft cultures of limbal and corneal epithelia, and mouse models of impaired autophagy and EphA2 signaling. We will assess the functional consequences of such modulations with a combination of morphogenetic (including Structured Illumination Microscopy and TEM), biochemical, molecular biological, cell biological and physiological approaches. By focusing on autophagy, our proposal represents a new approach to study the regulation of: (i) limbal stem and TA cell proliferation; and (ii) corneal epithelial differentiation and nucelophagy. Such knowledge has clinical applicability as sustaining proliferation is necessary for the proper expansion of cultured HLEKs used in ex vivo transplantation as well as in wound healing. The impact of autophagy and nucleophagy on corneal epithelial differentiation has translational relevance since aberrant differentiation is a feature of diseases of the corneal epithelium (e.g., aniridia, chemical burns) and modulation of autophagy-associated genes are potential treatment modalities, Understanding how autophagy is regulated in corneal epithelium is essential as this tissue is routinely subjected to stress, which requires a proper autophagic response to maintain homeostasis.

项目摘要/摘要 眼睛的前表面起到了阻挡外部环境的作用，保护脆弱的 防止损伤的底层结构，支持泪膜并保持透明度。这些函数是 部分是通过角膜和角膜缘上皮实现的，这必须保持适当的增殖和 差异化。自噬：细胞适应内在和外在压力的分解代谢过程。 已证明，在某些情况下，也可调节增殖和分化。尽管取得了许多进展 在我们对角膜和角膜缘上皮生物学的理解中，自噬在很大程度上仍未被研究。我们有 最近报道：(I)在静息状态下，角膜缘上皮基底部的自噬作用比 角膜上皮基底层细胞；(Ii)自噬有助于角膜缘上皮的增殖能力；以及(Iii) 自噬使创伤后的运输放大(TA)细胞得到适当的激活。我们还有 初步证据表明：(I)NUS1是一种新的自噬的上游调节因子；(Ii) 阻断自噬会损害角膜上皮的分化和核物质的去除(核吞噬)； 和(Iii)受体酪氨酸激酶EphA2通过调节角膜上皮细胞分化而促进角膜上皮细胞分化 末期自噬。这些观察结果使我们假设自噬是维持角膜缘所必需的。 上皮干细胞和TA细胞的动态平衡，以及确保适当的角膜上皮分化。我们建议 通过关注自噬如何影响角膜缘上皮干细胞增殖来检验这一假说； 调节角膜上皮的分化；和(Iii)受角膜上皮中的EphA2调节。至 为了实现这些目标，我们将利用我们进行功能得失研究的能力 免费模型系统中的自噬相关基因及其关键上游调控因子，包括 培养的人角膜缘(HLEK)和角膜(HCEK)上皮细胞，角膜缘和角膜的三维移植物培养 上皮细胞，自噬和EphA2信号受损的小鼠模型。我们将评估功能 这种调制与形态发生(包括结构照明)的组合的后果 显微镜和电子显微镜)、生化、分子生物学、细胞生物学和生理学方法。通过 着眼于自噬，我们的建议代表了一种新的方法来研究调节：(I)角膜缘干和 Ta细胞增殖；(Ii)角膜上皮细胞分化和吞核。这样的知识具有临床意义。 适用性因为维持增殖对于体外培养的HLEK细胞的适当扩增是必要的 移植以及在伤口愈合方面也是如此。自噬和吞核对角膜上皮细胞的影响 分化具有翻译相关性，因为异常分化是角膜疾病的一个特征 上皮细胞(如无虹膜、化学烧伤)和自噬相关基因的调节是可能的。 治疗方式，了解自噬是如何调节角膜上皮是至关重要的，因为这 组织经常受到压力，这需要适当的自噬反应来维持动态平衡。