A new pathogenic mechanism for diabetic retinopathy

描述（由申请人提供）：视网膜新生血管（NV）和糖尿病性黄斑水肿是导致糖尿病性视网膜病变视力丧失的主要病理特征。越来越多的证据表明，视网膜的慢性炎症和氧化应激在糖尿病视网膜病变中起重要的致病作用。Wnt是一组分泌的富含半胱氨酸的糖蛋白，其结合卷曲（Fz）受体或由Fz和低密度脂蛋白受体相关蛋白5或6（LRP 5/6）组成的共受体复合物，并调节多种生理和病理过程，包括血管生成和炎症。然而，wnt信号通路在糖尿病视网膜病变中的作用还未见报道。我们的初步研究提供了以下证据表明wnt通路在糖尿病视网膜病变中的致病作用：1）LRP 5/6在链脲佐菌素（STZ）诱导的糖尿病大鼠和氧诱导的视网膜病变（OIR）大鼠的视网膜中表达上调。2)Wnt通路的下游效应物2-连环蛋白在STZ-糖尿病大鼠和OIR大鼠的视网膜中积累，并且在培养的内皮细胞（EC）中通过缺氧上调。3)在培养的EC和小鼠视网膜中，单独激活wnt通路诱导VEGF过表达。4)Wnt通路的特异性抑制剂DKK 1在OIR模型中阻断VEGF表达并减少视网膜NV，并减少STZ糖尿病大鼠中的视网膜血管渗漏。基于这些结果，我们假设wnt通路的激活在糖尿病视网膜病变的视网膜炎症、血管渗漏和NV中起致病作用。为了验证这一假设，我们提出了以下研究：1）建立wnt信号在糖尿病视网膜病变中的致病作用。我们将首先确定在糖尿病中发生wnt通路激活的视网膜细胞类型。为了进一步确定wnt通路在糖尿病视网膜病变中的作用，我们将使用2-连环蛋白的组成型活性突变体激活wnt通路，并研究单独激活wnt通路是否会导致正常动物的视网膜炎症、血管渗漏和NV。此外，我们将使用Wnt通路的特异性抑制剂DKK 1阻断Wnt通路，以确定其是否减轻STZ糖尿病大鼠和秋田小鼠中的视网膜炎症和血管渗漏，以及减轻OIR大鼠中的视网膜NV。2)阐明wnt通路介导糖尿病视网膜病变的分子机制。我们将首先鉴定Wnt的共受体，LRP 5或LRP 6，其对于诱导糖尿病视网膜病变至关重要。我们将在培养的EC中使用特异性siRNA敲低LRP 5和LRP 6的表达，以确定哪种siRNA阻断缺氧诱导的VEGF过表达。我们将在转基因小鼠的视网膜中表达LRP 5或LRP 6的组成型活性突变体，以确定哪种突变体引起视网膜炎症、血管渗漏和NV。此外，我们将LRP 5-/-小鼠与秋田小鼠杂交，以确定LRP 5敲除（KO）是否减弱糖尿病诱导的血管渗漏和视网膜炎症。为了研究LRP 5在视网膜NV中的作用，新生LRP 5-/-小鼠将经受OIR以揭示LRP 5 KO是否预防或减轻缺血诱导的视网膜NV。接下来，我们将研究wnt通路诱导的VEGF表达是否通过缺氧诱导因子-1（HIF-1）。我们将在从HIF-11基因敲除的小鼠培养的EC中敲除HIF-11，并研究HIF-11的缺乏是否减弱由wnt信号传导诱导的VEGF过表达。我们还将研究VEGF是否对介导wnt信号在糖尿病视网膜病变中的致病作用至关重要。为此，我们将Muller细胞特异性条件性VEGF-/-小鼠与表达LRP 5或LRP 6的组成型活性突变体的转基因小鼠杂交，以激活VEGF-/-小鼠中的wnt途径，并确定VEGF KO是否阻断由wnt途径激活诱导的视网膜血管渗漏、炎症和NV。3)阐明糖尿病中wnt通路被激活的机制。我们推测氧化应激是糖尿病视网膜病变中wnt通路激活的原因。我们将在高糖培养基中培养的原代EC中使用抗氧化剂或在糖尿病模型的视网膜中表达抗氧化酶，以确定受抑制的ROS产生是否可以抑制wnt通路的激活。该项目代表了探索糖尿病视网膜病变发病机制的创新方法。这些研究将确立wnt通路作为糖尿病视网膜病变的一种新的致病通路。本项目所产生的信息不仅将为糖尿病并发症的发病机制提供新的见解，而且还将为糖尿病视网膜病变的药物干预提供新的靶点。公共卫生相关性：糖尿病视网膜病变是糖尿病的常见并发症，也是工作年龄人群失明的主要原因。本项目旨在探索糖尿病视网膜病变的一种新的致病机制，即wnt信号通路的激活，并为糖尿病视网膜病变的治疗提供一个新的药物靶点。

DESCRIPTION (provided by applicant): Retinal neovascularization (NV) and diabetic macular edema are the major pathological features responsible for vision loss in diabetic retinopathy. Accumulating evidence suggests that chronic inflammation and oxidative stress in the retina play important pathogenic roles in diabetic retinopathy. Wnts are a group of secreted, cysteine-rich glycoproteins which bind to the frizzled (Fz) receptors or to the co-receptor complex consisting of Fz and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) and regulate multiple physiological and pathological processes including angiogenesis and inflammation. However, the role of the wnt signaling in diabetic retinopathy has not been reported. Our preliminary studies have provided the following evidence suggesting a pathogenic role of the wnt pathway in diabetic retinopathy: 1) LRP5/6 expression is up-regulated in the retina of streptozotocin (STZ)-induced diabetic rats and of rats with oxygen-induced retinopathy (OIR). 2) A down-stream effector of the wnt pathway, 2-catenin, is accumulated in the retina of STZ-diabetic rats and OIR rats, and is up-regulated by hypoxia in cultured endothelial cells (EC). 3) In cultured EC and in the mouse retina, activation of the wnt pathway alone induces VEGF over-expression. 4) DKK1, a specific inhibitor of the wnt pathway, blocks VEGF expression and decreases retinal NV in the OIR model, and reduces retinal vascular leakage in STZ- diabetic rats. Based on these results, we hypothesize that activation of the wnt pathway plays a pathogenic role in retinal inflammation, vascular leakage and NV in diabetic retinopathy. To test this hypothesis, we propose the following studies: 1) To establish the causative role of the wnt signaling in diabetic retinopathy. We will first identify retinal cell types in which the wnt pathway activation occurs in diabetes. To further establish the role of the wnt pathway in diabetic retinopathy, we will activate the wnt pathway using a constitutively active mutant of 2-catenin and to investigate if activation of the wnt pathway alone causes retinal inflammation, vascular leakage and NV in normal animals. Further, we will block the wnt pathway using DKK1, a specific inhibitor of the wnt pathway, to determine if it attenuates the retinal inflammation and vascular leakage in STZ-diabetic rats and Akita mice, and mitigates retinal NV in OIR rats. 2) To elucidate the molecular mechanism by which the wnt pathway mediates diabetic retinopathy. We will first identify the co-receptor of the wnt, LRP5 or LRP6, essential for induction of diabetic retinopathy. We will knock down the expression of LRP5 and LRP6 using specific siRNAs in cultured EC, to determine which siRNA blocks the VEGF over-expression induced by hypoxia. We will express a constitutively active mutant of LRP5 or LRP6 in the retina of transgenic mice to determine which mutant causes retinal inflammation, vascular leakage and NV. Further, we will cross LRP5-/- mice with Akita mice to determine if LRP5 knockout (KO) attenuates vascular leakage and retinal inflammation induced by diabetes. To investigate the role of LRP5 in retinal NV, newborn LRP5-/- mice will be subjected to OIR to reveal if LRP5 KO prevents or mitigates ischemia-induced retinal NV. Next, we will investigate if the VEGF expression induced by the wnt pathway is through hypoxia-inducible factor-1 (HIF-1). We will knock out HIF-11 in EC cultured from HIF-11 floxed mice, and investigate if the lack of HIF-11 attenuates VEGF over-expression induced by the wnt signaling. We will also investigate if VEGF is essential for mediating the pathogenic role of the wnt signaling in diabetic retinopathy. For this purpose, we will cross Muller cell-specific conditional VEGF-/- mice with the transgenic mice expressing the constitutively active mutant of LRP5 or LRP6 to activate the wnt pathway in VEGF-/- mice and determine if VEGF KO blocks the retinal vascular leakage, inflammation and NV induced by the wnt pathway activation. 3) To elucidate the mechanism by which the wnt pathway is activated in diabetes. We hypothesize that oxidative stress is responsible for the wnt pathway activation in diabetic retinopathy. We will use anti-oxidants in primary EC cultured in high glucose medium or express anti-oxidant enzymes in the retina of the diabetes models to determine if the suppressed ROS generation can inhibit the wnt pathway activation. This project represents an innovative approach to explore the pathogenesis of diabetic retinopathy. These studies will establish the wnt pathway as a novel pathogenic pathway in diabetic retinopathy. The information generated from this project will not only provide new insights into the pathogenesis of diabetic complications, but also reveal a new target for drug intervention of diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Diabetic retinopathy is a common complication of diabetes and a major cause of blindness in working age population. This project aims to explore a novel pathogenic mechanism, i.e, activation of the wnt signal pathway, for diabetic retinopathy and to reveal a new drug target for its treatment.