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：
8424240
负责人：
Peter Y Lwigale
金额：
$ 35.18万
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/8424240
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（由申请人提供）：角膜的血管化是一种威胁性的并发症。角膜血管性的分子机制仍然没有很好地了解，并且对角膜血管的发展知之甚少。我们建议研究角膜血管性发育过程中迁移血管前体细胞（血管细胞）的分子调节。以前，我们表明眼周血管细胞表达神经蛋白酶（NRP1）是血管生成血管内皮生长因子（VEGF）和抗血管生成指导分子Smaphorin3a（SEMA3A）的双重受体。我们正在进行的研究表明，眼周血管细胞在角膜发育过程中也表达抗血管生成SEMA3E的Plexind1 A受体。我们还表明，抗血管生成细胞因子CXCL14在分化后不久由基质角膜细胞强烈表达。基于这些观察结果，我们假设促和抗血管生成因子之间的严格调节平衡控制角膜血管性发育过程中血管细胞迁移和血管图案。为了检验这一假设，我们将利用小鼠遗传学和操纵鸟类眼睛的易于探索在角膜发育过程中与血管生成相关的抑制基因的功能。我们将检查眼睛发育过程中迁移血管细胞的行为，以确定它们与其他迁移细胞的分离，从而导致角膜基质和内皮。我们将通过qPCR，原位杂交和免疫组织化学确定引导基因及其受体的时空表达。我们将在TIE1：H2B-EYFP转基因鹌鹑胚胎中使用荧光血管进行损失和功能获得实验，以阐明眼部血管生成细胞迁移期间抑制性分子及其受体的功能。缺乏每个抑制基因或受体功能的敲除小鼠将详细检查血管细胞迁移和角膜血管的缺陷。体内实验将与体外迁移测定法结合，以直接测试抑制性分子对眼周血管细胞迁移的影响，从其他眼组织分离。以下具体目的将检验我们的假设：1。确定NRP1及其配体VEGF和SEMA3A在角膜血管性发育过程中的血管细胞迁移中的作用。 2。确定Plexind1在角膜血管发展中的作用。 3。确定角膜发育过程中CXCL14表达的功能意义。我们提出的研究的成功完成将洞悉调节血管细胞迁移和血管生成的机制，从而导致血管性角膜的发展。类似的机制可能会抑制正常成年角膜的新血管形成，从而为发现潜在的治疗性抗血管生成剂铺平了道路，用于治疗血管化的成年角膜和靶向因癌症而靶向血管生成。