The role of the wnt signaling pathway in choroidal neovascularization

描述(申请人提供)：我们最近的研究表明，Wnt通路的异常激活在AMD和糖尿病视网膜病变的视网膜炎症、NV和纤维化的发病机制中发挥关键作用。一项遗传学研究报告，极低密度脂蛋白受体(VLDLR)的变异与AMD有关。我们最近报道，VLDLR是Wnt通路的负调节因子，因为VLDLR基因敲除导致Wnt通路过度激活，导致类似AMD的病理改变，如视网膜炎症、血管渗漏和视网膜下NV。关于VLDLR调控Wnt途径的机制，我们最近获得了以下初步数据：1)VLDLR及其可溶胞外域(VLDLRN)的表达抑制了Wnt信号传导。2)VLDLRN肽可降低总LRP6蛋白水平。3)免疫共沉淀实验表明，VLDLRN与LRP6结合，形成VLDLR-LRP6杂二聚体。4)VLDLRN多肽可阻断Wnt配体诱导的FZ-LRP6二聚化。基于这些观察，我们假设VLDLR与LRP6形成异二聚体，阻断Wnt配体诱导的LRP6聚集和LRP6信号体的形成。这种结合可能代表了VLDLR抑制WNT信号的机制。为了验证这一假设，我们将确定VLDLR与LRP6的结合是否阻止Wnt配体诱导的LRP6聚集和信号体的形成。我们还将确定VLDLR与LRP6的结合是否会影响LRP6的内吞作用和稳定性。此外，我们将使用缺失突变和免疫共沉淀分析来定义负责VLDLR和LRP6之间相互作用的序列结构域。通过评估VLDLR序列结构域对视网膜炎症、血管渗漏和NV的疗效，探讨其与LRP6结合并抑制Wnt信号转导的治疗潜力。最近，两项独立的大型临床试验报告，非诺贝特，一种PPAR1激动剂，降低循环中的VLDL水平，对2型糖尿病患者的视网膜血管渗漏和NV有治疗作用。我们的初步研究发现，非诺贝特抑制Wnt信号转导，上调VLDLR的表达及其启动子活性。因此，我们将验证这样一种假设，即非诺贝特通过上调VLDLR的表达来抑制Wnt信号是其有益于视网膜炎症、血管渗漏和NV的机制。我们将使用VLDLR KO小鼠、PPAR1 KO小鼠以及这些KO小鼠的原代RPE和内皮细胞来确定VLDLR和PPAR1是否在介导非诺贝特的Wnt抑制效应中起关键作用。这些研究将阐明VLDLR调节Wnt途径的机制，并确定这一重要途径的一种新的内源性调节机制。这些研究将揭示PPAR1与典型的Wnt通路之间的相互作用，并有助于AMD新疗法的开发。 公共卫生相关性：脉络膜新生血管是老年性黄斑变性(AMD)的严重并发症，也是老年人失明的主要原因。本项目旨在探索脉络膜新生血管的一种新的致病机制，即激活WNT信号通路。该项目还可能确定一种治疗AMD的临床药物。

DESCRIPTION (provided by applicant): Our recent studies have demonstrated that abnormal activation of the Wnt pathway plays key roles in pathogenesis of retinal inflammation, NV and fibrosis in both AMD and diabetic retinopathy. A genetic study has reported that variants of very low-density lipoprotein receptor (VLDLR) are associated with AMD. We have recently reported that VLDLR functions as a negative regulator of the Wnt pathway, as VLDLR knockout resulted in Wnt pathway over-activation, leading to AMD-like pathologies, such as retinal inflammation, vascular leakage and sub-retinal NV. Toward the mechanism by which VLDLR regulates the Wnt pathway, we have recently obtained the following preliminary data: 1) Expression of VLDLR and its soluble extracellular domain (VLDLRN) inhibits Wnt signaling. 2) The VLDLRN peptide decreases total LRP6 protein levels. 3) Co-immunoprecipitation assay showed that VLDLRN binds with LRP6, forming a VLDLR-LRP6 heterodimer. 4) VLDLRN peptide blocks Fz-LRP6 dimerization induced by Wnt ligand. Based on these observations, we hypothesize that VLDLR forms a heterodimer with LRP6, which blocks the Wnt ligand-induced LRP6 aggregation and LRP6 signalosome formation. This binding may represent a mechanism by which VLDLR inhibits Wnt signaling. To test this hypothesis, we will determine if binding of VLDLR to LRP6 blocks the Wnt ligand-induced LRP6 aggregation and formation of signalosomes. We will also determine if binding of VLDLR to LRP6 affects LRP6 endocytosis and stability. Further, we will define the sequence domains responsible for the interaction between VLDLR and LRP6 using deletion mutants and co-immunoprecipitation assays. The therapeutic potential of the sequence domain of VLDLR binding to LRP6 and inhibiting Wnt signaling will be explored by evaluating its efficacy on retinal inflammation, vascular leakage and NV. Recently, two independent large clinical trials reported that fenofibrate, a PPAR1 agonist which lowers VLDL levels in the circulation, has therapeutic effects on retinal vascular leakage and NV in type 2 diabetic patients. Our preliminary studies found that fenofibrate inhibits Wnt signaling and up- regulates VLDLR expression and its promoter activity. Therefore, we will test the hypothesis that inhibition of Wnt signaling through up-regulation of VLDLR expression by fenofibrate represents a mechanism for its beneficial effects on retinal inflammation, vascular leakage and NV. We will use VLDLR KO mice, PPAR1 KO mice and primary RPE and endothelial cells from these KO mice to determine if VLDLR and PPAR1 are essential for mediating the Wnt-inhibiting effect of fenofibrate. These studies will elucidate the mechanism by which VLDLR regulates the Wnt pathway and identify a novel, endogenous regulatory mechanism for this important pathway. The proposed studies will reveal the interactions between PPAR1 and the canonical Wnt pathway and contribute to the development of new treatment for AMD. PUBLIC HEALTH RELEVANCE: Choroidal neovascularization is a severe complication of age-related macular degeneration (AMD) and a major cause of blindness in aged population. This project aims to explore a novel pathogenic mechanism, i.e., activation of the wnt signal pathway, for choroidal neovascularization. This project may also identify a clinical drug for the treatment of AMD.