Molecular regulation of angioblast migration during cornea development

描述（由申请人提供）：角膜血管化是一种威胁视力的并发症。角膜无血供的分子机制目前还不清楚，对角膜无血供的发展也知之甚少。我们建议研究角膜无血管形成过程中迁移性血管前体细胞（成血管细胞）的分子调控。以前，我们发现眼周成血管细胞表达神经纤毛蛋白1（Nrp 1），一种血管生成血管内皮生长因子（VEGF）和抗血管生成导向分子Semaphorin 3A（Sema 3A）的双重受体。我们正在进行的研究表明，眼周成血管细胞在角膜发育过程中也表达抗血管生成Sema 3E的受体PlexinD 1。我们还表明，抗血管生成细胞因子CXCL 14强烈表达的基质角膜细胞分化后不久。基于这些观察结果，我们推测，在角膜无血管性的发展过程中，促血管生成因子和抗血管生成因子之间的严格调节平衡控制着成血管细胞的迁移和血管图案化。为了验证这一假设，我们将利用小鼠遗传学和易于操纵的鸟类眼睛来探索角膜发育过程中与血管发生相关的抑制基因的功能。我们将研究眼发育过程中迁移性成血管细胞的行为，以确定它们在何处以及何时与其他迁移性细胞分离，从而产生角膜基质和内皮。我们将通过qPCR、原位杂交和免疫组织化学来确定角膜发育过程中引导基因及其受体的时空表达。我们将在具有荧光血管的Tie 1：H2 B-eYFP转基因鹌鹑胚胎中进行功能丧失和获得实验，以阐明眼周成血管细胞迁移过程中抑制分子及其受体的功能。将详细检查缺乏每种抑制基因或受体功能的敲除小鼠的角膜成血管细胞迁移和血管形成缺陷。体内实验将与体外迁移测定相结合，以直接测试抑制分子对与其他眼组织分离的眼周成血管细胞迁移的影响。以下具体目标将检验我们的假设：1。确定Nrp 1及其配体VEGF和Sema 3A在角膜无血管形成过程中成血管细胞迁移中的作用。2.确定丛蛋白D1在角膜无血管形成过程中的作用。3.确定角膜发育过程中CXCL 14表达的功能意义。成功完成本研究将有助于深入了解成血管细胞迁移和血管生成的调控机制，从而导致无血管角膜的形成。类似的机制可以抑制正常成人角膜的新血管形成，从而为发现用于治疗血管化成人角膜和靶向癌症引起的血管生成的潜在治疗性抗血管生成剂铺平道路。

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.