The Role of Soluble Flt-1 and Raver2 in Ocular Vascular Demarcations

描述（由申请人提供）：角膜和视网膜感光层的光学透明度取决于严格的血管分界。这种生理血管分区在角膜损伤和黄斑变性时受到损害。该项目将通过关注角膜和视网膜下腔中的内源性抗血管生成分子、可溶性 VEGFR-1（也称为 sFlt-1）及其核上调蛋白 Raver2，阐明维持眼部血管区室化的分子信号、介质和调节器。我们之前证明角膜无血管性依赖于可溶性 VEGFR-1 (sFlt-1)。 Morpholinos（基于吗啉的合成寡核苷酸）上调 sFlt-1 可抑制缝合诱导的角膜新生血管形成和激光诱导的脉络膜新生血管形成，并提高角膜移植存活率。在令人兴奋的新研究中，我们发现 Raver2（一种内源性核调节蛋白）通过调节 RNA 加工将 Flt-1 剪接转变为可溶性 sFlt-1 同工型。这是首次描述的 Raver2 功能，也是首次鉴定的 sFlt-1 产生的核启动子。在视网膜中，我们发现 sFlt-1 在视网膜色素上皮 (RPE) 中强烈表达，并且对于感光器无血管特权至关重要。我们将基于这些发现来描述角膜和视网膜下腔中 sFlt-1 和 Raver2 的调节。我们的中心假设是 sFlt-1 对于光感受器无血管特权至关重要，而 Raver2 敲低将损害眼部血管分界。我们的具体目标是： 1.确定角膜和RPE中sFlt-1表达的分子调节因子。我们将鉴定调节 sFlt-1 表达的核因子，验证它们在体内和体外血管生成测定中的相关性，并鉴定它们的 RNA 结合序列。我们将确定 Raver2 的作用机制以及眼睛发育的主要调节因子 Pax6 是否与 Raver2 启动子结合。 2.测试Raver2上调sFlt-1可以提高AMD模型中角膜移植存活率并恢复光感受器无血管特权的预测我们将确定Raver2是否可以提高角膜移植存活率（通过减少角膜新生血管形成和相关的免疫浸润）。接下来，我们将确定Raver2是否可以增加视网膜sFlt-1并抑制激光诱导的脉络膜新生血管形成。 3.确定Raver2的基因消融是否诱导角膜或脉络膜新生血管形成。使用现有的靶向条件 ES 细胞 (Raver2tm1e(KOMP)Wtsi)，我们将开发 Raver2 敲除小鼠（或 Raver2-lox/loxp 动物，如果敲除是胚胎致死的）。我们将评估 Raver2 敲除对产前和产后角膜无血管和视网膜血管分界发育的影响，确定敲除中是否存在截短的转录本和 Raver2 的表达动力学，以及 Raver2 敲除是否影响抗或促血管生成因子的表达。

DESCRIPTION (provided by applicant): Optical transparency in the cornea and retinal photoreceptor layer depends on strict vascular demarcation. This physiologic vascular zoning is compromised in corneal injury and macular degeneration. This project will elucidate the molecular signals, mediators and regulators that sustain ocular vascular compartmentalization, by focusing on the endogenous anti-angiogenic molecule, soluble VEGFR-1 (also known as sFlt-1), and its nuclear up-regulator protein Raver2, in the cornea and the subretinal space. We previously demonstrated that corneal avascularity is dependent on soluble VEGFR-1 (sFlt-1). Morpholinos (synthetic morpholine-based oligonucleotides) upregulating sFlt-1 suppressed suture-induced corneal neovascularization and laser-induced choroidal neovascularization, and improve cornea transplant survival. In exciting new studies, we have discovered that Raver2, an endogenous nuclear regulatory protein, shifts Flt-1 splicing towards the soluble sFlt-1 isoform by modulating RNA processing. This is the first described function of Raver2 and the first identified nuclear promoter of sFlt-1 production. In the retina, we found that sFlt-1 is strongly expressed by the retinal pigment epithelium (RPE) and is vital for photoreceptor avascular privilege. We will build on these findings to characterize the regulation of sFlt-1 and Raver2 in the cornea and the subretinal space. Our central hypotheses are that sFlt-1 is vital to photoreceptor avascular privilege and that Raver2 knockdown will compromise ocular vascular demarcations. Our specific aims are: 1. To determine the molecular regulators of sFlt-1 expression in the cornea and RPE. We will identify the nuclear factors that regulate sFlt-1 expression, validate their relevance in angiogenic assays in vivo and in vitro, and identify their RNA binding sequences. We will determine the mechanism of action of Raver2 and whether Pax6, a master regulator of ocular development, binds the Raver2 promoter. 2. To test the prediction that upregulation of sFlt-1 by Raver2 can improve corneal transplant survival and restore photoreceptor avascular privilege in models of AMD We will determine whether Raver2 can improve corneal transplant survival (by reducing corneal neovascularization and associated immune infiltration). Next, we will determine whether Raver2 can increase retinal sFlt-1 and inhibit laser induced choroidal neovascularization. 3. To determine whether genetic ablation of Raver2 induces corneal or choroidal neovascularization. Using available targeted conditional ES cells (Raver2tm1e(KOMP)Wtsi), we will develop a Raver2 knockout mouse (or Raver2-lox/loxp animal if the knockout is embryonically lethal). We will assess the impact of Raver2 knockout on development of corneal avascularity and retinal vascular demarcations pre- and post-natally, determine the presence of truncated transcripts and expression kinetics of Raver2 in the knockout, and whether Raver2 knockout affects expression of anti- or pro-angiogenic factors.