Characterization of Retinoid-Binding Protein 3 (RBP3): A Protective Factor Against Diabetic Retinopathy Identified in People with Extreme Diabetes Duration

类视黄醇结合蛋白 3 (RBP3) 的表征：在患有极度糖尿病病程的人群中发现的针对糖尿病视网膜病变的保护因子

• 批准号: 10543746
• 负责人: GEORGE L KING
• 金额: $ 47.98万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543746
• 关键词: Adverse effects; Affect; Antibodies; Autopsy; Binding; Biological Assay; Blindness; Blood Vessels; Blood capillaries; Cell Line; Cell Surface Proteins; Cells; Chronic; Clinical Research Protocols; Clinical Trials; Data; Developed Countries; Development; Diabetes Mellitus; Diabetic Retinopathy; Down-Regulation; Electroretinography; Embryo; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Eye; FRAP1 gene; Finland; Gene Targeting; Glucose; Glycosylated hemoglobin A; Grant; Hyperglycemia; Individual; Inflammatory; Injections; Insulin-Dependent Diabetes Mellitus; Interleukin-6; Intervention; Intervention Studies; KDR gene; Length; Lentivirus; Mass Spectrum Analysis; Medicine; Microvascular Dysfunction; Modality; Muller' s cell; Nuclear; Paper; Pathway interactions; Patients; Persons; Photoreceptors; Plasma; Population Study; Protein Isoforms; Proteins; Proteomics; Protocols documentation; Recombinants; Regulation; Reporting; Retina; Retinal Photoreceptors; Retinol Binding Proteins; Rhodopsin; Rodent; SLC2A1 gene; Science; Serum; Severities; Structure; Structure-Activity Relationship; Therapeutic Agents; Therapeutic Intervention; Tissues; Toxic effect; Transgenes; Translational Research; Validation; Vascular Endothelial Growth Factors; Vascular Permeabilities; cohort; diabetic; diabetic rat; glucose uptake; glycemic control; in vivo; inhibitor; interstitial retinol-binding protein; laser photocoagulation; mRNA Expression; macular edema; new therapeutic target; non-diabetic; novel; novel therapeutic intervention; novel therapeutics; overexpression; potential biomarker; prevent; proliferative diabetic retinopathy; promoter; prospective; protective factors; protein activation; protein expression; protein kinase C-delta; subretinal injection; therapeutic target; transcription factor; translational medicine; type I and type II diabetes

PROJECT SUMMARY/ABSTRACT Although treatment exists for late-stage diabetic retinopathy (DR) and macular edema, interventions to inhibit DR onset and worsening, other than glycemic control, have generally not been successful. To identify novel DR therapeutic targets, we studied Joslin 50-Year Medalists (N=1019), all of whom have type 1 diabetes (T1D) for 50-87 years. The presence of DR protective factors is supported by a bimodal distribution of DR in this cohort; 41% of Medalists have no-mild DR and 47% have quiescent proliferative DR (QPDR) despite no significant difference in glycemic control. Longitudinal data for up to 60 years shows that Medalists protected from proliferative DR (PDR) did not experience DR worsening after their first 17 years of diabetes. Mass spectrometry of post-mortem retina and vitreous found a novel protective factor, interphotoreceptor retinol- binding protein 3 (RBP3), to be elevated in Medalists with no-mild DR despite poor glycemic control. Our paper in Science Transl. Medicine (2019) confirmed that RBP3 is elevated in the retina and vitreous of Medalists with no-mild DR versus Medalists and non-Medalists with QPDR, and that RBP3 in the retina and vitreous of diabetic individuals is lower than in non-diabetic controls. RBP3 overexpression in in vivo studies by lentivirus subretinal injection, embryonically by transgene targeting photoreceptors or intravitreous injection of recombinant RBP3, inhibited retinal VEGF and IL-6 expression and normalized vascular permeability, electroretinogram changes and acellular capillaries in diabetic rodents. Mechanistic studies showed that in Muller and endothelial cells, RBP3 binds to cell surface proteins including GLUT-1 to decrease glucose uptake and glycolytic flux, neutralizing adverse actions of hyperglycemia. We developed a sensitive and specific ELISA assay that showed RBP3 levels in the vitreous and serum (at 1/1000 of vitreous levels) were correlated with each other and with DR severity, and inversely correlated with vitreous VEGF. RBP3 expression in photoreceptor cells was reduced by high glucose, possibly due to protein kinase C (PKC) δ activation and inhibition of serum reactive factor (SRF) transcription factor via the Akt pathway. Preliminary studies of RBP3 subdomains show structure-function activities for inhibiting glucose uptake by binding to GLUT-1 transporters and reducing VEGF and IL-6 expression in Muller cells. The specific aims proposed are: Sp. Aim 1: To characterize and compare RBP3 levels in the retina, vitreous and serum as a potential biomarker for DR in T1D and T2D patients at the Joslin Diabetes Center, with validation in the Finland FinnDiane T1D cohort and DRCR Protocol T (T2D) cohort. Sp. Aim 2: To determine the mechanism for hyperglycemia-induced downregulation of RBP3 expression in photoreceptors in vivo and in a photoreceptor cell line by activation of PKCδ and deactivation of the Akt/mTOR/S6K pathway and SRF transcription factor. Sp. Aim 3: To define structure-function relationships between the RBP3 full length protein and its subdomains with regard to interaction with GLUT-1 and glucose uptake in Müller and retinal endothelial cells.

项目概要/摘要 尽管存在针对晚期糖尿病视网膜病变 (DR) 和黄斑水肿的治疗方法，但仍需采取干预措施来抑制 除了血糖控制之外，DR 的发生和恶化通常尚未成功进行新的鉴定。 DR 治疗目标，我们研究了 Joslin 50 年奖章获得者 (N=1019)，他们都患有 1 型糖尿病 (T1D) 50-87 年 DR 的双峰分布支持 DR 保护因素的存在。 该队列中，41% 的获奖者没有轻度 DR，47% 的获奖者有静态增殖性 DR (QPDR)，尽管没有 长达 60 年的血糖控制的显着差异表明奖牌获得者受到保护。 增殖性 DR (PDR) 患者在患糖尿病 17 年后并未出现 DR 恶化的情况。 死后视网膜和玻璃体的光谱测定发现了一种新的保护因子，光感受器间视黄醇- 尽管血糖控制不佳，但在非轻度 DR 的获奖者中，结合蛋白 3 (RBP3) 仍会升高。 Science Transl. Medicine (2019) 上的论文证实 RBP3 在视网膜和玻璃体中升高 患有非轻度 DR 的奖牌获得者与患有 QPDR 的奖牌获得者和非奖牌获得者，以及视网膜中的 RBP3 和 在体内研究中，糖尿病个体的玻璃体中 RBP3 的过度表达低于非糖尿病对照。 通过慢病毒视网膜下注射，胚胎通过靶向光感受器的转基因或玻璃体内 注射重组RBP3，抑制视网膜VEGF和IL-6表达，使血管正常化 糖尿病啮齿动物的渗透性、视网膜电图变化和脱细胞毛细血管的机制研究。 研究表明，在 Muller 和内皮细胞中，RBP3 与包括 GLUT-1 在内的细胞表面蛋白结合，以减少 葡萄糖摄取和糖酵解通量，中和高血糖的不利作用我们开发了一种敏感的药物。 以及显示玻璃体和血清中 RBP3 水平的特异性 ELISA 测定（玻璃体水平的 1/1000） 彼此相关并与 DR 严重程度相关，与玻璃体 VEGF 呈负相关。 感光细胞中的表达因高葡萄糖而降低，可能是由于蛋白激酶 C (PKC) δ 通过 Akt 途径激活和抑制血清反应因子 (SRF) 转录因子。 RBP3 子结构域的研究表明，通过结合来抑制葡萄糖摄取的结构功能活性 GLUT-1 转运蛋白和降低 Muller 细胞中 VEGF 和 IL-6 表达的具体目标。 目标 1：表征和比较视网膜、玻璃体和血清中的 RBP3 水平作为潜在的 Joslin 糖尿病中心针对 T1D 和 T2D 患者的 DR 生物标志物，已在芬兰进行验证 FinnDiane T1D 队列和 DRCR Protocol T (T2D) Sp 的机制。 高血糖诱导的体内光感受器和光感受器中 RBP3 表达的下调 通过激活 PKCδ 并失活 Akt/mTOR/S6K 通路和 SRF 转录因子来抑制细胞系。 Sp 目标 3：定义 RBP3 全长蛋白及其之间的结构-功能关系。 与 Müller 和视网膜内皮细胞中 GLUT-1 相互作用以及葡萄糖摄取有关的子域。