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

描述(申请人提供)：角膜和视网膜光感受器层的光学透明度取决于严格的血管划分。这种生理血管分区在角膜损伤和黄斑变性中受到损害。该项目将通过重点研究角膜和视网膜下空间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Rver2，来阐明维持眼部血管分区的分子信号、介体和调节因子。我们先前证明角膜无血管依赖于可溶性VEGFR-1(sFlt-1)。合成吗啉寡核苷酸上调sFlt-1抑制缝线诱导的角膜新生血管和激光诱导的脉络膜新生血管，提高角膜移植的存活率。在令人兴奋的新研究中，我们发现内源性核调节蛋白Rver2通过调节RNA的加工将Flt-1的剪接转移到可溶性的sFlt-1亚型。这是River2的第一个被描述的功能，也是第一个被发现的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，表征sFlt-1和Rver2在角膜和视网膜下空间的调节。我们的中心假设是，sflt-1对光感受器无血管特权至关重要，并且River2基因敲除将损害眼部血管的划分。我们的具体目标是：1.确定sFlt-1在角膜和RPE中表达的分子调控。我们将确定调控sFlt-1表达的核因子，验证它们在体内和体外血管生成试验中的相关性，并确定它们的RNA结合序列。我们将确定Rver2的作用机制，以及眼发育的主要调节因子Pax6是否与River2启动子结合。2.为了验证Rver2上调sFlt-1可以提高AMD模型角膜移植存活率和恢复光感受器无血管特权的预测，我们将确定Rver2是否可以提高角膜移植存活率(通过减少角膜新生血管和相关的免疫渗透)。接下来，我们将确定River2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.确定Rver2基因消融是否可诱导角膜或脉络膜新生血管。利用可用的靶向条件ES细胞(Rver2tm1e(KOMP)WTSI)，我们将开发出Rver2基因敲除小鼠(如果该基因敲除是胚胎致死的，则为Rver2-lox/loxP动物)。我们将评估River2基因敲除在出生前和出生后对角膜无血管和视网膜血管分界的影响，确定Rver2基因敲除中是否存在截断的转录本和表达动力学，以及Rver2基因敲除是否影响抗血管生成因子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.