A new pathogenic mechanism for diabetic retinopathy

糖尿病视网膜病变的新发病机制

• 批准号: 7565534
• 负责人: Jian-Xing Ma
• 金额: $ 36.63万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7565534
• 关键词: Abbreviations; Adenovirus Vector; Adenoviruses; Age; Age related macular degeneration; Angiography; Animal Model; Animals; Antibodies; Antioxidants; Attenuated; Binding; Biological Assay; Blindness; Blood Vessels; Capillary Endothelial Cell; Cell Culture Techniques; Cells; Choroidal Neovascularization; Chronic; Complex; Complications of Diabetes Mellitus; Cysteine; Diabetes Mellitus; Diabetic Retinopathy; Drug Delivery Systems; Endothelial Cells; Enzymes; Extravasation; Gene Targeting; Generations; Glucose; Glycogen Synthase Kinases; Glycoproteins; Goals; Human; Hypoxia; Immunohistochemistry; Inflammation; Inflammatory; Intercellular adhesion molecule 1; Intervention; Ischemia; Knock-out; LDL-Receptor Related Protein 1; LDL-Receptor Related Proteins; Low-Density Lipoproteins; Measures; Mediating; Messenger RNA; Modeling; Molecular; Molecular Target; Muller' s cell; Mus; Newborn Infant; Nuclear Translocation; Oxidative Stress; Oxygen; PTGS2 gene; Pathogenesis; Pathologic Processes; Pathway interactions; Pharmaceutical Preparations; Photoreceptors; Physiological; Plasminogen; Plasminogen Activator Inhibitor 1; Play; Population; Proteins; RNA Interference; Rattus; Reporting; Retina; Retinal; Retinal Diseases; Retinal Neovascularization; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Stream; Streptozocin; Structure of retinal pigment epithelium; TCF Transcription Factor; Testing; Transgenic Mice; Umbilical vein; Up-Regulation; VLDL receptor; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factors; Vascular Permeabilities; Western Blotting; Work; angiogenesis; base; cell type; diabetic; diabetic rat; hypoxia inducible factor 1; inhibitor/antagonist; innovation; insight; knock-down; lipoprotein receptor related protein 5; macular edema; mutant; neovascular; neovascularization; novel; prevent; public health relevance; receptor; vascular inflammation

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.

描述（由申请人提供）：视网膜新血管形成（NV）和糖尿病黄斑水肿是负责糖尿病性视网膜病变视力丧失的主要病理特征。积累的证据表明，视网膜中的慢性炎症和氧化应激在糖尿病性视网膜病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，与卷曲（FZ）受体结合或与由FZ和低密度脂蛋白受体相关蛋白5或6（LRP5/6）组成的共受体复合物（LRP5/6）组成，并调节包括血管生成和炎症在内的多个生理和病理学。但是，Wnt信号在糖尿病性视网膜病中的作用尚未报道。我们的初步研究提供了以下证据，表明Wnt途径在糖尿病性视网膜病变中的致病作用：1）LRP5/6表达在链霉菌素（STZ）诱发的糖尿病大鼠的视网膜中上调，并用氧气诱导的视网膜病（OIR）上调。 2）在STZ糖尿病大鼠和OIR大鼠的视网膜中积累了Wnt途径的下游效应子，2-catenin，并在培养的内皮细胞（EC）中被缺氧上调。 3）在培养的EC和小鼠视网膜中，仅WNT途径的激活会导致VEGF过表达。 4）DKK1是Wnt途径的特定抑制剂，阻止了VEGF表达并降低了OIR模型中的视网膜NV，并减少了STZ-糖尿病大鼠的视网膜血管渗漏。基于这些结果，我们假设Wnt途径的激活在视网膜炎症，血管泄漏和NV中起致病作用。为了检验这一假设，我们提出了以下研究：1）确定Wnt信号在糖尿病性视网膜病中的致病作用。我们将首先识别糖尿病中Wnt途径激活的视网膜细胞类型。为了进一步确定Wnt途径在糖尿病性视网膜病中的作用，我们将使用2-Catenin的组成性活性突变体激活Wnt途径，并研究单独激活WNT途径是否会导致视网膜炎症，正常动物中的血管泄漏和NV。此外，我们将使用Wnt途径的特异性抑制剂DKK1阻止Wnt途径，以确定它是否减弱了STZ糖尿病大鼠和Akita小鼠的视网膜炎症和血管渗漏，并减轻OIR大鼠的视网膜NV。 2）阐明Wnt途径介导糖尿病性视网膜病的分子机制。我们将首先确定Wnt，LRP5或LRP6的共受体，这对于诱导糖尿病性视网膜病至关重要。我们将使用培养的EC中使用特定的siRNA击倒LRP5和LRP6的表达，以确定哪些siRNA阻止了缺氧诱导的VEGF过表达。我们将在转基因小鼠的视网膜中表达LRP5或LRP6的组成性活性突变体，以确定哪些突变体引起视网膜炎症，血管泄漏和NV。此外，我们将与Akita小鼠穿越LRP5 - / - 小鼠，以确定LRP5敲除（KO）是否会减轻糖尿病引起的血管渗漏和视网膜炎症。为了研究LRP5在视网膜NV中的作用，新生儿LRP5 - / - 小鼠将受到OIR的影响，以揭示LRP5 KO是否阻止或减轻缺血诱导的视网膜NV。接下来，我们将研究Wnt途径诱导的VEGF表达是否通过缺氧诱导因子1（HIF-1）。我们将在HIF-11 Floxed小鼠中培养的EC中淘汰HIF-11，并研究缺乏HIF-11是否会减弱WNT信号传导引起的VEGF过表达。我们还将研究VEGF是否对于介导Wnt信号在糖尿病性视网膜病中的致病作用至关重要。为此，我们将与表达LRP5或LRP6的组成型活性突变体的转基因小鼠跨越Muller细胞特异性的有条件VEGF - / - 小鼠，以激活VEGF - / - 小鼠中的Wnt途径并确定VEGF KO在vegf KO中阻止了视网膜血管泄漏，炎症，NV是否通过WNT诱导WNT pation pation pation pation pation pation pation。 3）阐明在糖尿病中激活Wnt途径的机制。我们假设氧化应激是糖尿病性视网膜病中Wnt途径激活的原因。我们将在糖尿病模型视网膜中高葡萄糖培养基或表达抗氧化酶中培养的原代EC中使用抗氧化剂，以确定抑制的ROS产生是否可以抑制Wnt途径激活。该项目代表了一种探索糖尿病性视网膜病的发病机理的创新方法。这些研究将在糖尿病性视网膜病变中将WNT途径确立为一种新的致病途径。该项目产生的信息不仅将为糖尿病并发症的发病机理提供新的见解，而且还揭示了糖尿病性视网膜病毒药物干预的新靶标。公共卫生相关性：糖尿病性视网膜病是糖尿病的常见并发症，是在工作年龄人群中失明的主要原因。该项目旨在探索一种新型的致病机制，即Wnt信号途径的激活，用于糖尿病性视网膜病，并揭示其治疗的新药物。