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

项目摘要/摘要 虽然有治疗晚期糖尿病视网膜病变(DR)和黄斑水肿的方法，但干预措施抑制了 除了血糖控制外，DR的发病和恶化通常都不成功。辨别小说 DR治疗靶点，我们研究了Joslin 50年奖牌获得者(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保护因子的存在 41%的奖牌获得者患有非轻度糖尿病视网膜病变，47%的患者患有静止性增殖性糖尿病视网膜病变(QPDR)，尽管没有 在血糖控制方面有显著差异。长达60年的纵向数据显示，奖牌获得者 来自增生性DR(PDR)的患者在糖尿病的前17年没有经历DR的恶化。质量 死后视网膜和玻璃体的光谱分析发现了一种新的保护因子，光感受器间视黄醇- 结合蛋白3(RBP3)，在患有非轻度DR的奖牌获得者中升高，尽管血糖控制不佳。我们的 《科学》杂志论文翻译。医学(2019)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相比，视网膜和 糖尿病患者的玻璃体厚度低于非糖尿病对照组。活体研究中RBP3的过表达 通过慢病毒视网膜下注射，胚胎通过转基因靶向光感受器或玻璃体内注射 注射重组RBP3抑制视网膜血管内皮生长因子和IL-6的表达并使血管正常化 糖尿病啮齿动物的通透性、视网膜电信号改变和无细胞毛细血管。机械学研究 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减少 葡萄糖摄取和糖酵解通量，中和高血糖的不良反应。我们开发了一种敏感的 和特异的ELISA法，显示玻璃体和血清中的RBP3水平(玻璃体水平的1/1000) 与视网膜病变严重程度呈正相关，与玻璃体血管内皮生长因子呈负相关。RBP3 高糖降低光感受器细胞的表达，可能与蛋白激酶C有关 通过Akt途径激活和抑制血清反应因子(SRF)转录因子。初步 研究表明RBP3亚区通过结合抑制葡萄糖摄取的结构-功能活性 GLUT-1转运体和降低MULER细胞中VEGF和IL-6的表达。建议的具体目标 它们是：Sp.目的1：表征和比较视网膜、玻璃体和血清中RBP3的水平作为潜在的 Joslin糖尿病中心T1D和T2D患者DR的生物标记物，并在芬兰进行验证 FinnDiane T1D队列和DRCR议定书T(T2D)队列。SP.目标2：确定疾病发生的机制 高血糖诱导体内和感光器RBP3表达下调 通过激活蛋白激酶C，失活Akt/mTor/S6K通路和SRF转录因子。 SP.目的3：确定RBP3全长蛋白与其结构与功能的关系 亚区与GLUT-1的相互作用和葡萄糖摄取在Müler和视网膜内皮细胞。

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.