The calcineurin/NFAT signaling axis in diabetic retinopathy pathogenesis

糖尿病视网膜病变发病机制中的钙调神经磷酸酶/NFAT信号轴

基本信息

批准号：
8829273
负责人：
JOHN S. PENN
金额：
$ 38.47万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8829273
关键词：
Address Adhesions Animal Model Background Diabetic Retinopathy Binding Biochemical Biological Assay Blood Vessels Blood capillaries CXCL2 gene Calcineurin Calcineurin inhibitor Calcium Cells Code Complex Cues Data Development Diabetes Mellitus Diabetic Retinopathy Endothelial Cells Event Experimental Models Familiarity Funding Gene Targeting Generations Genetic Transcription Glucose Goals Growth Factor Health Homeostasis Human Hyperglycemia IL8 gene In Situ In Vitro Inflammation Mediators Inflammatory Intercellular adhesion molecule 1 Interleukin-6 Intracellular Second Messenger Knockout Mice Lead Leukocyte Rolling Leukocytes Leukostasis Link Luciferases Mediating Messenger RNA Modeling Molecular Monitor Mus Nuclear Translocation PTGS2 gene Pathogenesis Pathologic Pathologic Processes Pathology Pathway interactions Permeability Play Production Protein Isoforms Proteins Publishing Retinal Retinal Diseases Retinal Edemas Rodent Model Role Second Messenger Systems Signal Pathway Signal Transduction Small Interfering RNA Staging Streptozocin T-Cell Activation Testing Therapeutic Tissues Transgenic Mice Treatment Efficacy Vascular Cell Adhesion Molecule-1 Vascular Diseases Vascular Endothelial Growth Factors Vascular Permeabilities Vision Work base capillary cell type cytokine diabetic expectation in vivo inhibitor/antagonist insight monolayer nuclear factors of activated T-cells promoter release of sequestered calcium ion into cytoplasm research study response temporal measurement therapeutic target transcription factor vascular inflammation

项目摘要

DESCRIPTION (provided by applicant): Disruption of cellular calcium homeostasis is a recognized consequence of diabetes-induced hyperglycemia. In certain retinal cell types, elevated intracellular calcium stimulates pathogenic responses that result in retinal leukostasis and vascular hyperpermeability - two vision-threatening hallmarks of non-proliferative diabetic retinopathy (DR). Thus, as a critical intracellular second messenger, Ca2+ may play an important role in high glucose-induced vascular pathology in DR, but the downstream signaling pathways through which pathogenic Ca2+ signals are mediated remain unknown. Recent evidence points to a critical role for Nuclear Factor of Activated T Cells (NFAT) in the pathway linking hyperglycemia-induced Ca2+ flux to these pathologic cellular responses. Elevated intracellular calcium triggers calcineurin (CN)-mediated activation of NFAT, leading to its nuclear translocation. NFAT integrates hyperglycemia-induced calcium flux with other biochemical signals by heterodimeric binding with other transcription factors. These heterodimeric complexes stimulate the transcription of target genes in a manner specific to multiple biochemical cues. For example, in retinal Müller cells, activation of the NFAT pathway leads to the generation of mRNA coding for HIF-1a, COX-2, and VEGF, resulting in elevated retinal VEGF levels, which induces vascular hyperpermeability and retinal edema. Our preliminary data indicate that the majority of high glucose induction of VEGF is CN-dependent. Likewise, in retinal microvascular endothelial cells (RMEC), NFAT transcriptional activity leads to the production of mRNA coding for the inflammatory cytokines CXCL2, VCAM-1, ICAM-1, IL-6, and IL-8, which promote leukostasis and hyperpermeability. Our preliminary data support the links between Ca2+, CN, NFAT, and production and secretion of inflammatory mediators. In order to understand the role of CN/NFAT signaling in DR, its signaling pathways must be characterized in the specific retinal cells involved in the diabetic response: Müller cells produce growth factor and cytokines in response to increases in intracellular calcium, and RMEC both produce and respond to these factors. Using primary cultures of these two cell types, the complicated interactions between hyperglycemia-induced Ca2+ flux, CN activation, NFAT activation, and consequent cell responses will be studied systematically. These studies will lead to the identification of signaling intermediates that may serve as appropriate therapeutic targets. We propose the following studies, aimed at a better understanding of the role of CN/NFAT signaling in retinal vascular disease: 1) comparisons of the effects of high glucose and/or VEGF in the presence of highly selective CN and NFAT inhibitors on cells isolated from mouse or human retinal tissues; 2) comparisons of the relevant effects of high glucose on cells isolated from wild type and NFAT-/- mice or of the effects of high glucose and/or VEGF on NFAT isoform-directed siRNA- treated cells; and 3) determination of the therapeutic efficacy of highly specific CN and NFAT inhibitors in STZ- treated mice. The STZ model has been selected for its specific relevance to non-proliferative DR.

描述（由申请人提供）：细胞钙稳态的破坏是糖尿病引起的高血糖的公认后果。在某些视网膜细胞类型中，细胞内钙升高会刺激导致视网膜白细胞停滞和血管通透性过高的致病反应——这是非糖尿病的两个威胁视力的标志。 - 增殖性糖尿病视网膜病变 (DR) 因此，作为关键的细胞内第二信使，Ca2+ 可能在高浓度糖尿病视网膜病变中发挥重要作用。 DR 中葡萄糖诱导的血管病理学，但介导致病性 Ca2+ 信号的下游信号通路仍然未知。这些病理性细胞反应会触发钙调神经磷酸酶 (CN) 介导的 NFAT 激活，从而导致 NFAT 整合高血糖诱导的钙流。这些异二聚体复合物以多种生化信号特异的方式刺激靶基因的转录，例如，在视网膜穆勒细胞中，NFAT 通路的激活导致 mRNA 编码的产生。 HIF-1a、COX-2 和 VEGF，导致视网膜 VEGF 水平升高，从而诱发血管通透性过高和视网膜水肿。 VEGF 的高葡萄糖诱导是 CN 依赖性的，在视网膜微血管内皮细胞 (RMEC) 中，NFAT 转录活性导致编码炎症细胞因子 CXCL2、VCAM-1、ICAM-1、IL-6 和 IL-6 的 mRNA 的产生。 IL-8 可促进白细胞停滞和通透性过高。我们的初步数据支持 Ca2+、CN、NFAT 与炎症介质的产生和分泌之间的联系。为了了解 CN/NFAT 信号在 DR 中的作用，必须在参与糖尿病反应的特定视网膜细胞中表征其信号通路：Müller 细胞响应细胞内钙的增加而产生生长因子和细胞因子，而 RMEC 既产生又响应使用这两种细胞类型的原代培养物，将系统地研究高血糖诱导的 Ca2+ 通量、CN 激活、NFAT 激活以及随后的细胞反应之间的复杂相互作用。为了鉴定可作为适当治疗靶点的信号传导中间体，我们提出以下研究，旨在更好地了解 CN/NFAT 信号传导在视网膜血管疾病中的作用：1）比较高葡萄糖和/或高血糖的作用。在高选择性 CN 和 NFAT 抑制剂存在下，对从小鼠或人视网膜组织中分离的细胞进行 VEGF 检测；2) 高葡萄糖对从野生动物中分离的细胞的相关影响的比较；类型和NFAT-/-小鼠或高葡萄糖和/或VEGF对NFAT亚型导向的siRNA处理的细胞的影响；以及3)确定高度特异性CN和NFAT抑制剂在STZ处理的小鼠中的治疗功效。模型因其与非增殖性 DR 的特定相关性而被选中。