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

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，但抑制DR和黄斑水肿的干预措施仍然存在。 除血糖控制外，DR发作和恶化通常不成功。鉴定新 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保护因子的存在， 41%的Medalists患有非轻度DR，47%的Medalists患有静止期增殖性DR（QPDR）， 血糖控制有显著差异。长达60年的纵向数据显示， 增殖性DR（PDR）患者在糖尿病的前17年后没有经历DR恶化。质量 死后视网膜和玻璃体的光谱发现了一种新的保护因子，感光细胞间视黄醇， 结合蛋白3（RBP3），尽管血糖控制不佳，但在非轻度DR的奖牌获得者中升高。我们 发表在Science transl. Medicine（2019）证实，RBP3在视网膜和玻璃体中升高， 非轻度DR的奖牌获得者与QPDR的奖牌获得者和非奖牌获得者相比，视网膜和 糖尿病个体的玻璃体低于非糖尿病对照。体内研究中的RBP3过表达 通过视网膜下注射慢病毒，通过胚胎靶向光感受器的转基因或玻璃体内注射 注射重组RBP3，抑制视网膜VEGF和IL-6的表达，并使血管正常化。 糖尿病啮齿类动物的视网膜通透性、视网膜电图变化和脱细胞毛细血管。机制研究 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， 葡萄糖摄取和糖酵解通量，中和高血糖症的不利作用。我们开发了一种敏感的 和特异性ELISA测定，显示玻璃体和血清中的RBP3水平（玻璃体水平的1/1000） 与DR严重程度相关，与玻璃体VEGF呈负相关。RBP3 高浓度葡萄糖降低了感光细胞中的表达，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。初步 RBP3亚结构域的研究显示了通过结合 GLUT-1转运蛋白和减少Muller细胞中VEGF和IL-6的表达。提出的具体目标 目的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方案T（T2D）队列。目的2：确定 高血糖诱导的视网膜色素P3在体内和光感受器中表达的下调 通过激活PKC δ和失活Akt/mTOR/S6K途径和SRF转录因子来诱导细胞系的凋亡。 sp.目的3：确定RBP3全长蛋白与其结构之间的结构-功能关系。 Müller和视网膜内皮细胞中GLUT-1和葡萄糖摄取的相互作用。

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.