The wnt signaling pathway in choroidal neovascularization

描述（由申请人提供）：脉络膜新生血管（CNV）是年龄相关性黄斑变性（AMD）的严重并发症。人类遗传学研究最近揭示了极低密度脂蛋白受体（VLDLR）和wnt辅助受体低密度脂蛋白受体相关蛋白6（LRP 6）基因与人类患者中的AMD的显著关联。我们最近的研究表明，VLDLR基因敲除小鼠发生了典型的CNV，并表现出人类AMD的大多数异常，如视网膜炎症、血管渗漏、视锥ERG受损和视锥变性。Wnt是一组分泌的富含胱氨酸的糖蛋白，其结合卷曲（Fz）受体或由Fz和LRP 5或LRP 6（LRP 5/6）组成的共受体复合物，并调节靶基因（如VEGF）的表达。已知wnt信号传导介导多种生物学功能，包括血管生成。然而，wnt通路在CNV中的作用尚未研究。我们的初步研究提供了以下证据，表明VLDLR和wnt通路在CNV中的致病作用：1）在VLDLR-/-眼杯中LRP 5/6表达显著上调。2)wnt途径的下游效应物糖原合成酶激酶-32（GSK- 32）和2-连环蛋白都在Vldlr-/-眼杯中被激活。3)VEGF表达在Vldlr-/-眼杯中上调。4)在培养的内皮细胞中，通过siRNA下调VLDLR激活wnt信号传导和VEGF过表达。5)Wnt通路的特异性抑制剂DKK 1阻断VLDLR siRNA诱导的VEGF过表达和Vldlr-/-小鼠RPE中的VEGF过表达。该项目的中心假设是VLDLR作为CNV的负调节剂发挥作用，并且VLDLR的调节作用是通过wnt途径。在这个项目中，我们将首先确定Vldlr-/-小鼠中RPE衍生的VEGF过表达是否对CNV的发展至关重要。我们将通过将RPE特异性VEGF条件性KO小鼠与Vldlr-/-小鼠杂交来产生VLDLR/VEGF双敲除（KO）小鼠，并确定RPE中的VEGF KO是否减弱Vldlr-/-小鼠中的CNV。我们还将使用RPE特异性HIF-11/VLDLR双KO小鼠研究VLDLR KO诱导的VEGF过表达是否通过HIF-1。第二，我们将检验Vldlr-/-小鼠中的CNV由wnt途径介导的假设。将在来自Vldlr-/-小鼠的原代RPE和视网膜内皮细胞中测定β 2-连环蛋白的活化。我们将研究通过DKK 1抑制wnt通路是否会预防或减轻Vldlr-/-小鼠中的CNV。此外，LRP 5和LRP 6的组成型活性突变体将在转基因小鼠的RPE中表达，以确定单独的wnt途径的过度活化是否会诱导CNV。我们将通过产生LRP 5/VLDLR双KO小鼠来确定LRP 5或LRP 6是否介导VLDLR-/-小鼠中的CNV。第三，我们将研究VLDLR如何调节LRP 5/6基因表达。将测试两种假定的机制：1）VLDLR和LRP 5/6可竞争与相同配体的结合。当VLDLR缺乏时，更多的配体可以与LRP 5/6结合并诱导受体的表达。2)VLDLR可能与细胞内信号通路相互作用，通过该信号通路VLDLR下调LRP 5/6基因转录。我们将删除VLDLR的胞内结构域和胞外配体结合结构域，并确定配体结合结构域或胞内结构域与信号通路相互作用是否对其对LRP 5/6表达的调节作用至关重要。如果配体结合结构域是必需的，我们将鉴定与VLDLR和LRP两者结合的配体。另一方面，如果胞内结构域是必需的，我们将确定可能与VLDLR相互作用的信号通路。VLDL诱导因子-1和DAB-1/Src激酶途径的作用将作为第一候选者进行研究。本研究将进一步明确VLDLR作为wnt信号通路的新成员，并探讨VLDLR和wnt信号通路在CNV发病机制中的作用。这些研究有可能揭示CNV的新的致病机制，并为CNV在AMD中的治疗提供新的药物靶点。公共卫生相关性：脉络膜新生血管是老年性黄斑变性的严重并发症，也是老年人致盲的主要原因。本课题旨在探索脉络膜新生血管的新的发病机制，建立一种新的脉络膜新生血管动物模型。

DESCRIPTION (provided by applicant): Choroidal neovascularization (CNV) is a severe complication of age-related macular degeneration (AMD). Human genetic studies have recently revealed significant association of the very low-density lipoprotein receptor (VLDLR) and a wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6) genes with AMD in human patients. Our recent studies showed that VLDLR knockout mice develop typical CNV, and manifest most abnormalities of human AMD such as retinal inflammation, vascular leakage, and impaired cone ERG and cone degeneration. Wnts are a group of secreted, cystine-rich glycoproteins which bind to frizzled (Fz) receptors or to a co-receptor complex consisting of Fz and LRP5 or LRP6 (LRP5/6) and regulate expression of target genes, such as VEGF. The wnt signaling is known to mediate multiple biological functions including angiogenesis. However, the role of the wnt pathway in CNV has not been investigated. Our preliminary studies have provided the following evidence suggesting a pathogenic role of VLDLR and the wnt pathway in CNV: 1) LRP5/6 expression is significantly up-regulated in Vldlr-/- eyecups. 2) The down-stream effectors of the wnt pathway, glycogen synthase kinase-32 (GSK- 32) and 2-catenin, are both activated in Vldlr-/- eyecups. 3) VEGF expression is up-regulated in Vldlr-/- eyecups. 4) In cultured endothelial cells, down-regulation of VLDLR by siRNA activates the wnt signaling and VEGF over-expression. 5) DKK1, a specific inhibitor of the wnt pathway blocks the VEGF over- expression induced by the VLDLR siRNA and in the RPE of Vldlr-/- mice. The central hypothesis of this project is that VLDLR functions as a negative regulator of CNV, and the regulatory effect of VLDLR is through the wnt pathway. In this project, we will first determine if the RPE-derived VEGF over-expression in Vldlr-/- mice is essential for the development of CNV. We will generate VLDLR/VEGF double knockout (KO) mice by crossing the RPE-specific VEGF conditional KO mice with Vldlr-/- mice, and determine if the VEGF KO in the RPE attenuates CNV in Vldlr-/- mice. We will also investigate if the VEGF over-expression induced by VLDLR KO is through HIF-1 using RPE-specific HIF-11/VLDLR double KO mice. Second, we will test the hypothesis that the CNV in Vldlr-/- mice is mediated by the wnt pathway. The activation of the 2-catenin will be determined in primary RPE and retinal endothelial cells from Vldlr-/- mice. We will investigate if inhibition of the wnt pathway by DKK1 will prevent or alleviate CNV in Vldlr-/- mice. Further, constitutively active mutants of LRP5 and LRP6 will be expressed in the RPE of transgenic mice to determine if over-activation of the wnt pathway alone will induce CNV. We will determine whether LRP5 or LRP6 mediates the CNV in Vldlr-/- mice by generating LRP5/VLDLR double KO mice. Third, we will investigate how VLDLR regulates LRP5/6 gene expression. Two postulated mechanisms will be tested: 1) VLDLR and LRP5/6 may compete for binding with the same ligand. When VLDLR is deficient, more ligand could bind to LRP5/6 and induce the expression of the receptors. 2) VLDLR may interact with an intracellular signaling pathway, through which VLDLR down-regulates LRP5/6 gene transcription. We will delete the intracellular domain and the extracellular ligand-binding domain of VLDLR and determine if the ligand binding domain or the intracellular domain interacting with signaling pathways is essential for its regulatory effect on LRP5/6 expression. If the ligand binding domain is essential, we will identify the ligand binding to both VLDLR and LRP. On the other hand, if the intracellular domain is essential, we will identify the signaling pathway which could interact with VLDLR. The role of the VLDL-inducible factor-1 and DAB-1/Src kinase pathways will be investigated as the first candidates. This project will establish VLDLR as a new member of the wnt pathway and establish the roles of VLDLR and the wnt pathway in pathogenesis of CNV. These studies have potential to reveal a new pathogenic mechanism of CNV and a new drug target of future treatment of CNV in AMD. PUBLIC HEALTH RELEVANCE: Choroidal neovascularization is a severe complication of age-related macular degeneration and a major cause of blindness in aged population. This project aims to explore a novel pathogenic mechanism for choroidal neovascularization and to establish a new animal model of choroidal neovascularization.