Early Mueller cell changes may play pivotal role in diabetic retinopathy

早期穆勒细胞变化可能在糖尿病视网膜病变中发挥关键作用

• 批准号: 7294191
• 负责人: Ernest Clarence Steele
• 金额: $ 17.5万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294191
• 关键词: Acute; Animal Model; Blindness; Blood Vessels; Blood-Retinal Barrier; Calcium; Calcium Signaling; Cell Line; Cell physiology; Cells; Clinic; Clinical Trials; Communication; Complex; Complications of Diabetes Mellitus; Condition; Culture Media; Cultured Cells; Data; Detection; Development; Diabetes Mellitus; Diabetes prevention; Diabetic Retinopathy; Disease; Disease Progression; Drug usage; Early Intervention; Endothelial Cells; Environment; Exhibits; Functional disorder; Fura-2; Future; Glial Fibrillary Acidic Protein; Glucose; Goals; Hemorrhage; Human; Hyperglycemia; Insulin; Intervention; Lead; Mediating; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Molecular Profiling; Molecular Target; Muller' s cell; Neuronal Dysfunction; Neurons; P2X-receptor; Patients; Pharmaceutical Preparations; Physiological; Play; Population Study; Prevention; Prevention strategy; Property; Proteins; Purines; Purinoceptor; Rattus; Reagent; Reporter; Research; Retina; Retinal; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Staging; Streptozocin; Stress; Testing; Therapeutic; Time; Treatment Protocols; Vision; Visual; Visual Acuity; Western Blotting; cellular targeting; diabetic; diabetic rat; improved; innovation; neovascularization; neuron loss; non-diabetic; novel; novel therapeutics; optical imaging; prevent; purine; receptor; receptor expression; response; retinal damage

DESCRIPTION (provided by applicant): The long-term goal of this project is to test an innovative hypothesis regarding the role of macroglial M¿ller cells in the early development and progression of diabetic retinopathy. We hypothesize that M¿ller cells are a primary cellular target of diabetic insult and may play a pivotal role in the early stages of development and progression of diabetic retinopathy via alterations of purinergic receptor expression and function. Because M¿ller cells play a central role in coordinating the activity of neuronal cells and endothelial cells via purinergic signaling, such alterations within the M¿ller cells would predispose the retina toward neuronal dysfunction and loss as well as breakdown of the blood-retinal barrier and alterations in endothelial cell function. We will test this hypothesis by comparing the M¿ller cell profiles of P2X and P2Y purinergic receptor expression and functional profiles in nondiabetic and streptozotocin-induced diabetic rats at various time points following the onset of hyperglycemia which precede the time required for neuronal and vascular complications to develop in this animal model. Immunohistochemical analysis of retinal sections and Western blot analyses of retinal protein lysates will be used to compare protein levels, while RT-PCR analyses will be used to compare mRNA levels. We will also use the fluorescent calcium reporter fura-2 in optical imaging studies to compare the P2X- and P2Y receptor-mediated intracellular calcium dynamics of M¿ller cells from diabetic and nondiabetic rats at these same early time points. If alterations are observed in the P2X and/or P2Y receptors at these early time points, it will establish the M¿ller cell as a cellular target and the purinergic receptors as a molecular target for developing novel intervention and prevention strategies to combat the widespread loss of vision due to irreversible loss of neurons and breakdown of the blood-retinal barrier in late stages of diabetic retinopathy. We also hypothesize that the diabetes-induced changes in M¿ller cell purinergic receptor expression and function reflect a direct response of the M¿ller cells to diabetic conditions and are not merely consequences of other subtle diabetes-induced changes in the functioning of the retinal circuit. To test this hypothesis, we will employ an immortalized rat M¿ller (rMC-1) cell line to compare the expression and functional profiles of P2X and P2Y receptors in rMC-1 cells cultured in nondiabetic (low glucose and high insulin levels) with those of cells cultured in diabetic (high glucose and low insulin levels) culture media. Recapitulation of the changes noted in the diabetic rat model will establish the rMC-1 cell line as a useful model to determine the molecular mechanisms underlying the changes and to test the ability of both existing and novel therapeutic reagents to reverse or prevent these alterations. In summary, the combined proposed studies promise to provide a novel and rapid path toward identifying earlier intervention and prevention of blinding retinopathy in diabetic patients. Retinal disease often develops and progresses to blindness in many diabetic patients. While great strides have been made using drugs to slow down the progression of the later retinal complications of diabetes, less progress has been made toward identifying effective strategies for early intervention and prevention of diabetes-induced retinal damage. The proposed research will rapidly provide informative data regarding novel potential targets for early intervention and prevention of irreversible retinal damage caused by diabetes.

描述（应用程序提供）：该项目的长期目标是测试有关大型细胞在糖尿病性视网膜病的早期发育和进展中的作用的创新假设。我们假设M ller细胞是糖尿病侮辱的主要细胞靶标，并且可能在糖尿病性视网膜病变的早期阶段起关键作用，这是通过改变嘌呤能受体表达和功能的改变。由于m ller细胞在通过嘌呤能信号传导协调神经元细胞和内皮细胞的活性方面起着核心作用，因此在M ller细胞内的这种改变会使视网膜倾向于倾向于神经元功能障碍和神经元障碍以及血液 - 视网膜屏障的损失和衰减。我们将通过比较p2x和P2Y嘌呤能受体表达的M ller细胞谱以及在高血糖发作后的各个时间点的非糖尿病和链蛋白酶诱导的糖尿病大鼠的糖尿病诱导的大鼠的功能特征，这是在这种动物模型中开发神经元和血管并发症所需的时间。残留切片和残留蛋白水平的蛋白质印迹分析的免疫组织化学分析将用于比较蛋白质水平，而RT-PCR分析将用于比较mRNA水平。我们还将在光学成像研究中使用荧光钙报告基因FURA-2比较了来自糖尿病和非糖尿病大鼠的P2X和P2Y受体介导的细胞内细胞的细胞内钙动力学。如果在这些早期时间点在P2X和/或P2Y受体中观察到改变，它将建立M ller细胞作为细胞靶标和嘌呤能受体，作为开发新型干预和预防策略的分子靶标，以应对由于神经元的不可逆性丧失和衰减的遗传性而导致的视力丧失，因此血液中的遗传性不可逆转。我们还假设糖尿病诱导的M ller细胞嘌呤能受体表达和功能的变化反映了M ller细胞对糖尿病条件的直接反应，而不仅仅是其他微妙的糖尿病诱导的视网膜电路功能变化的后果。为了检验这一假设，我们将采用永生的大鼠M ller（RMC-1）细胞系来比较在非糖尿病（低葡萄糖和高胰岛素水平）中培养的RMC-1细胞中P2X和P2Y受体的表达和功能分布与在糖尿病（高葡萄糖和低葡萄糖和低胰岛素水平）中培养的细胞中培养的细胞的表达和功能谱。糖尿病大鼠模型中指出的变化的概括将建立RMC-1细胞系作为一个有用的模型，以确定变化的分子机制，并测试现有和新型治疗剂逆转或防止这些改变的能力。总而言之，拟议的研究有望为鉴定糖尿病患者的早期干预和预防视网膜病变提供新颖而快速的途径。在许多糖尿病患者中，视网膜疾病经常发展并发展为失明。尽管已经使用药物取得了长足的进步，以减慢后来的糖尿病的视网膜并发症的进展，但在确定早期干预和预防糖尿病引起的视网膜损害方面的有效策略方面取得了较少的进步。拟议的研究将迅速提供有关早期干预和预防糖尿病引起的不可逆视网膜损害的新型潜在目标的信息信息。