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Molecular regulation of angioblast migration during cornea development

角膜发育过程中成血管细胞迁移的分子调控

基本信息

批准号：
8618905
负责人：
Peter Y Lwigale
金额：
$ 36.26万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8618905
关键词：
Ablation Address Adult Angioblast Angiogenic Factor Behavior Binding Biological Assay Birds Blindness Blood Vessels CXC Chemokines Cardiac Cells Chemotactic Factors Complication Contact Lenses Cornea Coupled Defect Development Disease Embryo Endothelial Cells Endothelium Equilibrium Eye Eye Development Fibroblast Growth Factor Genes Genetic Immigration Immunohistochemistry In Situ Hybridization In Vitro Infection Knockout Mice Ligands Maintenance Malignant Neoplasms Migration Assay Mitogens Molecular Mus Mutant Strains Mice Neural Crest Neural Crest Cell Ophthalmology Pattern Peptides Play Quail RNA Interference Regulation Role Signal Transduction Testing Therapeutic Time Tissues Transgenic Organisms Trauma Vascular Endothelial Growth Factors Vascularization Vision Visual Acuity Work angiogenesis antiangiogenesis therapy base cytokine embryo tissue gain of function in vivo insight lens loss of function migration mutant neovascular neovascularization overexpression precursor cell receptor research study spatiotemporal tumor tumor growth vasculogenesis

项目摘要

DESCRIPTION (provided by applicant): Vascularization of the cornea is a vision-threatening complication. The molecular mechanisms underlying corneal avascularity are still not well understood and comparatively little is known about the development of corneal avascularity. We propose to study the molecular regulation of migratory blood vessel precursor cells (angioblasts) during development of corneal avascularity. Previously, we showed that periocular angioblasts express Neuropilin1 (Nrp1) a dual receptor for the angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic guidance molecule Semaphorin3A (Sema3A). Our ongoing studies show that periocular angioblasts also express PlexinD1 a receptor for anti-angiogenic Sema3E during cornea development. We also show that the anti-angiogenic cytokine CXCL14 is strongly expressed by stromal keratocytes shortly after differentiation. Based on these observations we hypothesize that a tightly regulated balance between pro- and anti-angiogenic factors controls angioblast migration and vascular patterning during development of corneal avascularity. To test this hypothesis, we will take advantage of mouse genetics and the ease of manipulating avian eyes to explore the function of inhibitory genes associated with vasculogenesis during corneal development. We will examine the behavior of migratory angioblasts during eye development to determine where and when they segregate from other migratory cells that give rise to the cornea stroma and endothelium. We will determine the spatiotemporal expression of guidance genes and their receptors during cornea development by qPCR, in situ hybridization, and immunohistochemistry. We will perform loss- and gain-of-function experiments in Tie1:H2B-eYFP transgenic quail embryos with fluorescent blood vessels to elucidate the function of inhibitory molecules and their receptors during periocular angioblast migration. Knockout mice lacking the function of each inhibitory gene or receptor will be examined in detail for defects in angioblast migration and vascularization of the cornea. In vivo experiments will be coupled with in vitro migration assays to directly test the effect of inhibitory molecules on periocular angioblast migration in isolation from other ocular tissues. The following Specific Aims will test our hypothesis: 1. Determine the role of Nrp1 and its ligands VEGF and Sema3A in angioblast migration during development of corneal avascularity. 2. Identify the role of PlexinD1 during development of corneal avascularity. 3. Determine the functional significance of CXCL14 expression during cornea development. Successful completion of our proposed study will provide insight into the mechanisms that regulate angioblast migration and vasculogenesis leading to development of an avascular cornea. Similar mechanisms may inhibit neovascularization of the normal adult cornea and thus pave the way for discovery of potential therapeutic anti-angiogenesis agents for treating vascularized adult corneas and targeting angiogenesis due to cancer.

描述(申请人提供)：角膜血管化是一种威胁视力的并发症。角膜无血管形成的分子机制目前尚不清楚，对角膜无血管的发生发展也知之甚少。我们建议研究迁移血管前体细胞(血管母细胞)在角膜无血管形成过程中的分子调控。先前，我们发现眼周血管母细胞表达神经粘蛋白1(Nrp1)和抗血管生成导向分子Semaphorin3A(Sema3A)，Nrp1是血管生成血管内皮生长因子(VEGF)的双重受体。我们正在进行的研究表明，在角膜发育过程中，眼周血管母细胞也表达PlexinD1，一种抗血管生成的Sema3E受体。我们还发现，抗血管生成细胞因子CXCL14在基质角质细胞分化后不久强烈表达。基于这些观察，我们假设，在角膜无血管的发展过程中，促血管生成因子和抗血管生成因子之间的严格调控平衡控制着血管母细胞的迁移和血管构型。为了验证这一假设，我们将利用小鼠的遗传学和鸟眼的易操作性来探索角膜发育过程中与血管生成相关的抑制基因的功能。我们将研究眼睛发育过程中迁移血管母细胞的行为，以确定它们何时何地与其他形成角膜基质和内皮的迁移细胞分离。我们将通过定量聚合酶链式反应、原位杂交和免疫组织化学的方法来确定引导基因及其受体在角膜发育过程中的时空表达。我们将在带有荧光血管的Tie1：H2B-EYFP转基因鹌鹑胚胎上进行功能丧失和功能获得的实验，以阐明抑制分子及其受体在眼周血管母细胞迁移过程中的功能。缺乏每个抑制基因或受体功能的基因敲除小鼠将被详细检查血管母细胞迁移和角膜血管形成方面的缺陷。体内实验将与体外迁移试验相结合，从其他眼组织中分离出来，直接测试抑制分子对眼周血管母细胞迁移的影响。1.确定Nrp1及其配体VEGF和Sema3A在角膜无血管形成过程中血管母细胞迁移中的作用。2.明确PlexinD1在角膜无血管形成中的作用。3.确定CXCL14在角膜发育过程中的功能意义。我们建议的研究的成功完成将为调节血管母细胞迁移和血管生成导致无血管角膜发育的机制提供洞察力。类似的机制可能会抑制正常成人角膜的新生血管，从而为发现潜在的治疗血管化成人角膜和靶向肿瘤血管生成的抗血管生成药物铺平道路。