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

基本信息

批准号：
10615668
负责人：
Peter Y Lwigale
金额：
$ 37.1万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10615668
关键词：
Adult Affect Anterior Basement membrane Biological Assay Birds Blindness Cell Proliferation Cell physiology Cell-Matrix Junction Cells Chick Chick Embryo Congenital Abnormality Cornea Corneal Diseases Corneal Endothelium Corneal Injury Corneal Stroma Coupled Data Defect Descemet&apos s membrane Development Developmental Process Disease EGF gene Embryo Embryonic Eye Endothelial Cells Endothelium Environment Epithelium Experimental Designs 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 Play Population Process Proliferating Proteins Proteomics Regulation Role Signal Transduction Speed Tenascin Testing Thinness Time Trauma Viral Virus Vision Work blastomere structure cell motility confocal imaging corneal epithelial wound healing corneal epithelium corneal repair design embryo cell experimental study extracellular gain of function gene function in vivo knock-down migration mouse genetics multipotent cell nephronectin novel overexpression perlecan pharmacologic receptor repaired response small hairpin RNA spatiotemporal wound wound healing

项目摘要

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.

前眼的失调与角膜疾病和损伤是眼部缺陷的主要原因和视力丧失。周期性神经rest细胞（PNC）是多能胚胎细胞群，提供关键信号，并有助于角膜的细胞和细胞外成分，但这些过程基础的分子机制仍然不太了解。我们已经确定了小说最近发现的细胞外基质（ECM）蛋白Nephronectin（NPNT）的表达尽管已经显示出NPNT在各种发展过程中起作用，但在包括癌症转移在内的病理状况，它尚未在角膜中研究。这个项目试图通过测试NPNT促进细胞的假设，了解角膜中NPNT功能的机制角膜发育，体内平衡和伤口愈合期间的迁移和依恋。我们正在进行的研究已经确定NPNT是在假定角膜的ECM中表达的，而其主要接收器 ITGα8在迁移之前和期间由PNC表达。此外，我们观察到了空间差异 NPNT将NPNT定位于鸡皮和小鼠内皮地下膜，并且是在整个成年期保持在小鼠中。根据这些观察，我们将利用鼠标遗传学和操纵鸟类眼睛的便利性，结合了NPNT表达的时空差异在这两个模型中，为了全面了解角膜中NPNT的功能。我们的 NPNT敲低和过表达的初步研究表明角膜变薄和增厚，此外，我们表明NPNT的敲低会导致角膜上皮缺陷，并且 NPNT/ITGα8信号传导在体外增强了PNC迁移。我们将进一步研究NPNT的功能 PNC迁移以及角膜上皮地下膜和胚胎角膜伤口愈合。 NPNT敲除小鼠的分析将指示NPNT在不同模式的PNC迁移模式中的功能，朝下膜的形成和维护，以及成人角膜伤口愈合的功能。我们还将执行蛋白质组学分析以识别NPNT相互作用伙伴并确定不存在 NPNT会影响角膜ECM和基底膜蛋白。所有提出的研究都补充了雏鸡胚胎眼，体外培养和分子生物学技术以及基因功能的药理抑制。以下具体目标将检验我们的假设：（1）确定肾膦素在PNC迁移到角膜中的作用。（2）研究NPNT在角膜上皮和内皮地下机制中的作用。（3）确定胚胎和成人角膜伤口愈合过程中NPNT的功能。拟议的研究将揭示NPNT在正常需要的细胞过程中的新功能角膜的开发，功能和修复。