LEUKOTRIENES AND DIABETIC RETINOPATHY

描述(申请人提供)：糖尿病视网膜病变是导致失明的主要原因。先前的研究表明，在疾病过程的早期，慢性炎症会损害视网膜的毛细血管。这会导致视网膜缺血，多年来，新血管会大量涌入玻璃体。新血管的出血限制了视力。临床上非常需要治疗方法来预防或减缓疾病的进展，早期干预以减少炎症可能是有益的。白三烯是花生四烯酸的代谢产物，在从哮喘到动脉粥样硬化的多种疾病中都是有效的炎症介质。在小鼠中，白三烯的缺乏可以保护视网膜免受毛细血管退化的影响。该提案的目的是研究FDA批准的白三烯抑制剂齐留通和孟鲁司特在治疗小鼠糖尿病视网膜病变方面的疗效。在具体目标1中，糖尿病小鼠服用齐留通或孟鲁司特2个月，评估糖尿病引起的典型视网膜白细胞粘附性、超氧化物产生、NFkB介导性炎症基因转录和血管通透性增加，以及9个月视网膜组织病理学评估。具体目的2将探讨白三烯调节糖尿病视网膜慢性炎症的三种机制：1)自分泌：白细胞可通过自身放大产生更多的超氧化物和白三烯B4；2)旁分泌：白细胞可分泌白三烯B4，诱导视网膜微血管内皮细胞表达ICAM1和额外的超氧化物生成；3)跨细胞代谢：视网膜微血管内皮细胞和神经胶质细胞在白三烯A4转移后合成白三烯，由白细胞产生，这可能进一步放大视网膜内部的炎症信号。检测细胞死亡、超氧化物和炎症标志物的分析将在白细胞和视网膜微血管内皮细胞共培养中进行。在共培养体系中，将使用选择性白三烯抑制剂或靶向siRNA转基因来确定白三烯的作用机制，这将进一步指导治疗干预。在特定目标3中，将从不同血糖控制的糖尿病受试者中分离出白细胞。在使用齐留通或孟鲁司特治疗3个月之前和之后，将进行超氧化物和白三烯B4生成的测量。对临床批准的白三烯抑制剂预防小鼠糖尿病视网膜病变的有效性和作用机制的研究，以及用于人类受试者的潜在筛查工具，将为将这项工作转化为患者护理提供必要的信息。

DESCRIPTION (provided by applicant): Diabetic retinopathy is a leading cause of blindness. Prior studies implicate that, early in the disease process; chronic inflammation damages the capillaries in the retina. This leads to retinal ischemia and, over many years, proliferation of new blood vessels into the vitreous. Bleeding from the new vessels limits vision. There is great clinical need for therapies to prevent or slow the progression of the disease, and early intervention to reduce the inflammation may be beneficial. Leukotrienes, metabolites of arachidonic acid, are potent mediators of inflammation in several diseases from asthma to atherosclerosis. In mice, deficiency of leukotrienes protects the retina from capillary degeneration. The goal of this proposal is to investigate the FDA-approved leukotriene inhibitors, zileuton and montelukast, for efficacy in the treatment of diabetic retinopathy in mice. In Specific Aim 1, diabetic mice will be administered zileuton or montelukast for 2 months with assessment of the typical diabetes-induced increases in retinal leukocyte adherence, superoxide production, NFkB-mediated inflammatory gene transcription, and vascular permeability, and for 9 months with evaluation of retinal histopathology. Specific Aim 2 will investigate three mechanisms by which leukotrienes may regulate chronic inflammation in the diabetic retina: 1) autocrine: leukocytes may generate increased superoxide and leukotriene B4 through autoamplification, 2) paracrine: leukocytes may secrete leukotriene B4 which induces retinal micro vascular endothelial cell expression of ICAM1 and additional superoxide generation, and 3) transcellular metabolism: retinal microvascular endothelial cells and glial cells synthesize leukotrienes following transfer of leukotriene A4, generated by the leukocyte, which may further amplify the inflammatory signal within the retina itself. Assays to detect cell death, superoxide, and inflammatory markers will be performed on leukocyte and retinal microvascular endothelial cells in co-culture. To the co-culture system, selective leukotriene inhibitors or targeted siRNA transfections will be employed to determine the mechanism of leukotriene action, which will further guide therapeutic intervention. In Specific Aim 3, leukocytes will be isolated from human subjects with diabetes mellitus of varying glycemic control. Measurements of superoxide and leukotriene B4 generation will be performed before and after treatment with zileuton or montelukast for 3 months. The study of the efficacy and mechanism of action of clinically approved leukotriene inhibitors to prevent diabetic retinopathy in the mouse coupled with a potential screening tool for use in human subjects will provide the necessary information for translation of this work to patient care.