Function of Nephronectin in the corneal ECM during development, homeostasis, and wound healing

肾连蛋白在角膜 ECM 中的发育、稳态和伤口愈合过程中的功能

• 批准号: 10393587
• 负责人: Peter Y Lwigale
• 金额: $ 35.99万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393587
• 关键词: Adult; Affect; Anterior; Basement membrane; Biological Assay; Birds; Blindness; Cell Proliferation; Cell physiology; Cell-Matrix Junction; Cells; Chick; Congenital Abnormality; Cornea; Corneal Diseases; Corneal Endothelium; Corneal Injury; Corneal Stroma; Coupled; Data; Defect; Descemet' s membrane; Development; Developmental Process; Disease; EGF gene; Embryo; Embryonic Eye; Endothelial Cells; Endothelium; Environment; Epithelial; Extracellular Matrix; Extracellular Matrix Proteins; Eye; Eye Development; Fibronectins; Goals; Homeostasis; Human; In Vitro; Knock-out; Knockout Mice; Laminin; Maintenance; Mediating; Membrane Proteins; Mesenchyme; Micromanipulation; Migration Assay; Modeling; Molecular; Molecular Biology Techniques; Morbidity - disease rate; Morphogenesis; Mus; Neoplasm Metastasis; Neural Crest Cell; Pathologic; Pattern; Pharmacology; Play; Population; Process; Proteins; Proteomics; Regulation; Role; Signal Transduction; Speed; Tenascin; Testing; Thinness; Time; Trauma; Viral; Virus; Vision; Work; base; blastomere structure; cell motility; confocal imaging; corneal epithelial wound healing; corneal epithelium; corneal repair; design; experimental study; extracellular; gain of function; gene function; in vivo; knock-down; migration; mouse genetics; multipotent cell; nephronectin; novel; overexpression; perlecan; receptor; repaired; response; small hairpin RNA; spatiotemporal; wound; wound healing

Dysgenesis of the anterior eye together with corneal diseases and injuries are major causes of ocular defects and loss of vision. The periocular neural crest cells (pNC) are multipotent embryonic cell population that provide crucial signals and contribute to the cellular and extracellular components of the cornea, but the molecular mechanisms underlying these processes are still not well understood. We have identified novel expression of a recently discovered extracellular matrix (ECM) protein, nephronectin (Npnt), during corneal development. Although Npnt has been shown to function in various developmental processes, and identified in pathological conditions including cancer metastasis, it has not been studied in the cornea. This project seeks to understand the mechanisms of Npnt function in the cornea by testing the hypothesis that Npnt promotes cell migration and attachment during corneal development, homeostasis and wound healing. Our ongoing studies have identified that Npnt is expressed in the ECM of the presumptive cornea whereas its major receptor Itgα8 is expressed by the pNC prior to and during migration. In addition, we observed spatial differences in the localization of Npnt to the chick epithelial and mouse endothelial basement membranes, and that it is maintained throughout adulthood in mice. Based on these observations, we will take advantage of mouse genetics and the ease of manipulating avian eyes, combined the spatiotemporal differences in Npnt expression in the two models, to provide a comprehensive understanding of the function of Npnt in the cornea. Our preliminary studies of Npnt knockdown and overexpression in chick show corneal thinning and thickening, respectively. In addition, we show that knockdown of Npnt causes corneal epithelial defects and that Npnt/Itgα8 signaling augments pNC migration in vitro. We will further examine the function of Npnt during pNC migration and in the corneal epithelial basement membrane and embryonic corneal wound healing. Analysis of Npnt knockout mice will indicate the function of Npnt during a different pattern of pNC migration, formation and maintenance of the Descemet's membrane, and function in adult corneal wound healing. We will also perform proteomic analysis to identify Npnt interacting partners and determine how the absence of Npnt affects the corneal ECM and basement membrane proteins. All proposed studies are supplemented with micromanipulation of chick embryonic eyes, in vitro culture and molecular biology techniques, and pharmacological inhibition of gene function. The following Specific Aims will test our hypothesis: (1) Determine the role of nephronectin during migration of pNC into the cornea. (2) Investigate the role of Npnt in the corneal epithelial and endothelial basement membranes. (3) Determine the function of Npnt during embryonic and adult corneal wound healing. The proposed studies will reveal novel functions of Npnt in cellular processes that are required for normal development, function, and repair of the cornea.

前眼发育不良与角膜疾病和损伤是眼缺损的主要原因 和视力丧失眼周神经嵴细胞（pNC）是多能胚胎细胞群， 提供关键的信号，并有助于角膜的细胞和细胞外成分，但 这些过程背后的分子机制仍然没有很好地理解。我们发现了一种新的 最近发现的细胞外基质（ECM）蛋白，肾连蛋白（Npnt）的表达， 发展尽管Npnt已被证明在各种发育过程中起作用，并且在哺乳动物中被鉴定为 包括癌症转移在内的病理状况，但尚未在角膜中进行研究。本项目谋求 通过测试Npnt促进细胞增殖的假设，了解Npnt在角膜中的作用机制。 在角膜发育、体内平衡和伤口愈合期间的迁移和附着。我们正在进行的 研究已经确定Npnt在假定角膜的ECM中表达，而其主要受体 Itgα8在迁移之前和迁移期间由pNC表达。此外，我们还观察到， Npnt定位于鸡上皮细胞和小鼠内皮细胞基底膜， 在小鼠的整个成年期内保持。基于这些观察，我们将利用鼠标 遗传学和容易操纵鸟类眼睛，结合Npnt表达的时空差异， 在这两种模型中，提供对Npnt在角膜中的功能的全面理解。我们 在鸡中Npnt敲低和过表达的初步研究显示角膜变薄和增厚， 分别此外，我们发现敲除Npnt会导致角膜上皮缺损， Npnt/Itgα8信号转导增强pNC的体外迁移我们将进一步研究Npnt的功能， pNC迁移并在角膜上皮基底膜和胚胎角膜伤口愈合。 对Npnt敲除小鼠的分析将表明Npnt在不同模式的pNC迁移过程中的功能， 后弹力层的形成和维持，以及在成人角膜伤口愈合中的功能。我们 还将进行蛋白质组学分析，以确定Npnt相互作用的伙伴，并确定如何缺乏 Npnt影响角膜ECM和基底膜蛋白。所有拟议的研究都补充了 鸡胚眼的显微操作，体外培养和分子生物学技术， 基因功能的药理学抑制。以下具体目标将检验我们的假设： (1)确定pNC迁移至角膜过程中肾连蛋白的作用。 (2)探讨Npnt在角膜上皮和内皮基底膜中的作用。 (3)确定胚胎和成人角膜伤口愈合过程中Npnt的功能。 这些研究将揭示Npnt在细胞过程中的新功能， 角膜的发育、功能和修复。