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: 10320034
• 负责人: GEORGE L KING
• 金额: $ 46.54万
• 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/10320034
• 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; Glucose; Glycosylated hemoglobin A; Grant; Hyperglycemia; Individual; Inflammatory; Injections; Institutes; Insulin-Dependent Diabetes Mellitus; Interleukin-6; Intervention; Intervention Studies; KDR gene; Length; Lentivirus; Mass Spectrum Analysis; Medicine; Microvascular Dysfunction; Muller' s cell; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Paper; Pathway interactions; Patients; Persons; Photoreceptors; Plasma; Population Study; Protein Isoforms; Protein Kinase C; Proteins; Proteomics; 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; subretinal injection; therapeutic target; transcription factor; translational medicine

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)  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 PKC 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.

项目摘要/摘要 尽管存在晚期糖尿病性视网膜病（DR）和黄斑水肿的治疗 除血糖控制以外，博士发作和担心通常没有成功。识别小说 DR理论目标，我们研究了Joslin 50年的奖牌获得者（n = 1019），所有这些都有1型糖尿病 （T1D）持续50 - 87年。 DR在DR的双峰分布中支持DR保护因子的存在 这个队列； 41％的奖牌获得者没有MILD DR，而47％的奖牌获得者具有静态增殖者DR（QPDR）dospite no 血糖控制的显着差异。长达60年的纵向数据表明，获得奖章的人受到保护 在糖尿病的头17年后，从增殖的DR（PDR）中没有经历过奇迹。大量的 事后视网膜和玻璃体的光谱法发现了一种新型的保护因子，photoreceptor-betrotector- 结合蛋白3（RBP3），将在没有损害的奖章中提高dod Desire desure deaste desiongemic对照。我们的 科学翻译中的论文。 Medicine（2019）证实RBP3在视网膜中升高 拥有QPDR的奖牌获得者与奖章和非医学家的奖牌获得者，以及在视网膜中的RBP3 糖尿病个体的玻璃体低于非糖尿病对照。 RBP3在体内研究中的过表达 通过慢病毒下注射，通过转化靶向感受器或静脉内的胚胎 注射重组RBP3，抑制视网膜VEGF和IL-6表达并标准化血管 糖尿病啮齿动物中的渗透性，电子图的变化和细胞毛细血管。机械研究 表明在穆勒和内皮细胞中，RBP3与包括GLUT-1的细胞表面蛋白结合以减少 葡萄糖摄取和糖酵解通量，中和高血糖的不良作用。我们发展了一个敏感的 以及在玻璃体和血清中显示RBP3水平的特定ELISA分析（在玻璃体水平的1/1000） 相互关联，与DR的严重程度相关，并与玻璃体VEGF成反比。 rbp3 由于蛋白激酶C（PKC）可通过高葡萄糖来减少感光细胞中的表达。 通过AKT途径激活和抑制血清反应因子（SRF）转录因子。初步的 RBP3亚域的研究表明结构功能活性通过结合到抑制葡萄糖的摄取 GLUT-1转运蛋白并降低Muller细胞中的VEGF和IL-6表达。提出的具体目的 是：sp。目标1：要表征和比较视网膜中的RBP3水平，玻璃体和血清作为潜力 乔斯林糖尿病中心的T1D和T2D患者的DR生物标志物，在芬兰进行验证 Finndiane T1D队列和DRCR协议t（T2D）队列。 sp。目标2：确定机制 高血糖诱导的体内光感受器和感光体中RBP3表达的下调 通过激活PKC和Akt/MTOR/S6K途径和SRF转录因子的电池线。 sp。目标3：定义RBP3全长蛋白与其之间的结构功能关系 亚域与Müller和更多内皮细胞中与Glut-1和葡萄糖摄取的相互作用有关。