The Role of RPE-derived Exosomes in Deposit Formation and ECM Modulation

摘要 视网膜色素上皮细胞（RPE）的功能是维持外血视网膜屏障，并支持PHO。 受体功能，包括视色素的再生和外节的更新。功能障碍 RPE是导致年龄相关性黄斑变性（AMD）发展的病理基础，年龄相关性黄斑变性是主要的 导致美国老年人视力下降的原因。由于RPE的主要功能是处理光感受， 对于外节，RPE内体通路的正常功能对于视网膜健康是重要的。多- 多种证据表明，在RPE功能障碍中观察到的主要罪魁祸首之一是视网膜色素上皮细胞的调节异常。 内体途径据认为，在AMD中，RPE细胞中的这种失调至少部分地导致了AMD的发生。 在RPE的基底层之间形成玻璃疣（富含蛋白质和脂质的细胞外沉积物）， 富含胶原蛋白和弹性蛋白的五层Bruch膜（BrM）。目前， 玻璃疣的形成是未知的。由于RPE释放的外泌体和其他细胞外囊泡（EV）是 内体途径的重要部分，我们假设从应激RPE细胞释放的外泌体 与未应激的RPE细胞释放的外泌体不同，这些外泌体参与了 早期和晚期阶段的特征性存款形成和ECM变化 AMD.因此，表征这些囊泡并调节其释放的方法具有很高的潜力， 为疾病机制和新的治疗靶点提供重要见解。值得注意的是， 关于RPE释放的外泌体和其他EV。通过评估两种互补的体外和离体AMD， 模型，我们对这个项目的总体目标是确定外泌体分泌在亚RPE存款中的作用， 在与AMD相关的条件下，无论是药理学还是基因， 治疗/生物治疗干预是可能的。在第一个目标中，我们将描述和量化 蛋白质和脂质组成的亚RPE沉积物，ECM和基底侧外泌体在RPE应激的病理， 与AMD有关的生理条件（氧化应激、补体失调和年龄），而 通过药理学和遗传学方法调节外泌体释放。在第二个目标中， 对正常人死后眼玻璃疣和BrM中的蛋白质和脂质含量进行表征 和AMD供体，通过使用尖端仪器进行详尽的蛋白质组学和脂质组学分析， 分析方法。这项工作作为临床前研究和原理证明，以探索 调节外泌体分泌和靶向外泌体组合物作为治疗AMD的治疗方法。

Abstract The retinal pigmented epithelium (RPE) functions to maintain the outer blood-retinal barrier and to support pho- toreceptor function, including regeneration of visual pigment and turnover of outer segments. Dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among older Americans. Given that a major function of the RPE is to process photorecep- tor outer segments, the proper functioning of the RPE endosomal pathway is important for retinal health. Multi- ple lines of evidence indicate that one of the major culprits observed in RPE dysfunction is dysregulation in the endosomal pathway. It is thought that in AMD this dysregulation in RPE cells is at least in part responsible for the formation of drusen (protein- and lipid-rich extracellular deposits) between the basal lamina of the RPE and the pentalaminar collagen- and elastin-rich Bruch’s membrane (BrM). At present, the exact mechanisms for drusen formation are unknown. Since RPE-released exosomes and other extracellular vesicles (EVs) are essential parts of the endosomal pathway, we hypothesize that exosomes released from stressed RPE cells are distinct from those released from unstressed RPE cells, and that these exosomes are involved in the pathognomonic deposit formation and ECM changes that underlie the early and late stages of AMD. Accordingly, approaches to characterize these vesicles and modulate their release have high potential to give important insight to disease mechanisms and new treatment targets. Significantly, very little is known about RPE-released exosomes and other EVs. By evaluating two complementary in vitro and ex vivo AMD models, our overall goal for this project is to determine the role of exosome secretion in sub-RPE deposit for- mation and in ECM changes under conditions relevant to AMD, and whether pharmacological or gene therapy/biological therapeutic interventions are possible. In the first aim, we will characterize and quantify the protein and lipid composition of sub-RPE deposits, ECM and basal-side exosomes in RPE stressed by patho- physiological conditions implicated in AMD (oxidative stress, complement dysregulation, and age), while modulating exosome release by both pharmacological and genetic approaches. In the second aim we will characterize the protein and lipid content in drusen and BrM from human post-mortem eyes from normal aged and AMD donors, by exhaustive proteomic and lipidomic analyses with cutting-edge instrumentation and analysis methodologies. This work serves as a preclinical study and proof of principle to probe the potential of modulating exosome secretion and target exosome composition as a therapeutic approach for treating AMD.