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

描述（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划分。这种生理性血管分带在角膜损伤和黄斑变性中受损。本项目将重点研究角膜和视网膜下空间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隔化的分子信号、介质和调节因子。我们之前已经证明，角膜的无血管性依赖于可溶性VEGFR-1 （sFlt-1）。Morpholinos（合成的基于morpholine的寡核苷酸）上调sFlt-1抑制缝合线诱导的角膜新生血管和激光诱导的脉络膜新生血管，提高角膜移植存活率。在令人兴奋的新研究中，我们发现内源性核调节蛋白Raver2通过调节RNA加工将Flt-1剪接向可溶性sFlt-1异构体转移。这是第一个描述的Raver2的功能，也是第一个确定的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，对光感受器无血管特权至关重要。我们将在这些发现的基础上描述sFlt-1和Raver2在角膜和视网膜下空间中的调节。我们的主要假设是sFlt-1对光感受器无血管特权至关重要，而Raver2敲低会损害眼部血管划分。我们的具体目标是：1。目的：确定sFlt-1在角膜和RPE中表达的分子调控因子。我们将确定调节sFlt-1表达的核因子，验证其在体内和体外血管生成试验中的相关性，并确定其RNA结合序列。我们将确定Raver2的作用机制，以及眼部发育的主要调节因子Pax6是否与Raver2启动子结合。2. 为了验证Raver2上调sFlt-1可以改善AMD模型角膜移植存活和恢复光受体无血管特权的预测，我们将确定Raver2是否可以改善角膜移植存活（通过减少角膜新生血管和相关的免疫浸润）。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。3. 目的：确定基因消融Raver2是否诱导角膜或脉络膜新生血管形成。利用现有的靶向条件胚胎干细胞（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性的）。我们将评估Raver2敲除对出生前后角膜无血管和视网膜血管划分发展的影响，确定敲除中Raver2的截断转录本和表达动力学的存在，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.