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

摘要 视网膜色素上皮(RPE)起着维持血-视网膜外部屏障和支持视网膜色素沉着的作用。 感受器功能，包括视觉色素的再生和外节的周转。失调症 RPE是导致老年性黄斑变性(AMD)发展的病理基础，AMD是 导致美国老年人失明的原因。鉴于RPE的主要功能是处理光接收- 对于视网膜外段，RPE内体通路的正常功能对视网膜健康很重要。多个- 大量证据表明，RPE功能障碍的主要罪魁祸首之一是 内体途径。据认为，在AMD中，RPE细胞的这种失调至少在一定程度上是 视网膜色素上皮和视网膜色素上皮基板之间形成玻璃体(富含蛋白质和脂肪的细胞外沉积)。 富含胶原蛋白和弹性蛋白的五层膜(BRM)。目前，针对这些问题的确切机制 玻璃灰岩的形成是未知的。由于RPE释放的外切体和其他细胞外小泡(EV)是 内体途径的重要部分，我们假设从应激的RPE细胞释放的外切体 与非应激的RPE细胞释放的那些不同，这些外切体参与了 早期和晚期的病原学沉积形成和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目标提供重要的见解。值得注意的是，人们对此知之甚少 关于RPE释放的外切体和其他电动汽车。通过评价两种互补的体外和体外AMD 模型，我们这个项目的总体目标是确定外体分泌在亚RPE沉积中的作用，以- 与AMD相关的条件下的信息和ECM的变化，无论是药理学的还是基因的 治疗/生物治疗干预是可能的。在第一个目标中，我们将表征和量化 病理胁迫下RPE亚RPE沉淀物、ECM和基底侧外体的蛋白质和脂肪组成 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切体的释放。在第二个目标中，我们将 正常人尸眼玻璃膜和视网膜基质中蛋白质和脂肪含量的特征 和AMD捐赠者，通过使用尖端仪器和 分析方法。这项工作作为一项临床前研究和原则证明，以探索潜在的 调节外切体分泌和靶向外切体组成作为治疗AMD的方法。

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.