RPE Exosomes in Age-related Macular Degeneration

摘要 老年性黄斑变性(AMD)是导致老年人不可逆性失明的主要原因 在发达国家，治疗选择有限。最早的临床体征是脂肪和蛋白质。 视网膜色素上皮(RPE)和邻近的视网膜之间形成的称为玻璃体的沉积物 五板层胶原性细胞外基质，Bruch膜。RPE的功能是维护外部 血-视网膜屏障和支持光感受器功能，包括视觉色素的再生和 外部管段的周转。视网膜色素上皮功能障碍是导致AMD的病理基础。人们认为，在 视网膜色素上皮细胞的这种失调至少在一定程度上是导致玻璃体形成的原因。目前， 玻璃疣形成的确切机制尚不清楚。一种潜在的检测AMD早期病理的方法 生物学上的变化是通过分离外切体来实现的，外切体是可以从 生物体液(如血浆和尿液)。由于RPE释放的外切体和其他细胞外小泡是必不可少的 内体途径的一部分，我假设从应激的RPE细胞释放的外切体是 不同于那些从非应激的RPE细胞释放的，这些外切体参与了 AMD早期和晚期的病理性沉积形成和细胞外基质改变。通过 评估两个互补的体外和体内AMD模型，我们这个项目的目标是确定 在与AMD相关的条件下，亚RPE沉积形成中的外体分泌和ECM的变化， 无论是药物治疗还是基因治疗/生物治疗干预都是可能的。在第一个 目的：对亚RPE沉积物、ECM和基底RPE的蛋白质和脂肪成分进行表征和定量。 RPE中的侧外切体受到与AMD(氧化应激， 补体调节失调和年龄)，同时通过药物和遗传调节外切体的释放 接近了。在第二个目标中，我将对RPE衍生的外切体进行分子标记、分离和鉴定 早期AMD特征良好的小鼠模型的血液。RPE派生的详细分析 正常和疾病环境中的外切体代表了新的研究，将提供对机制的洞察 这是AMD病理生物学变化的基础。

Abstract Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly in developed countries, and there are limited therapeutic options. The earliest clinical signs are lipid and protein deposits known as drusen, that form between the retinal pigmented epithelium (RPE) and the adjacent pentalaminar collagenous extracellular matrix, Bruch’s membrane. The RPE functions to maintain the outer blood-retinal barrier and to support photoreceptor function, including regeneration of visual pigment and turnover of outer segments. Dysfunction of the RPE underlies pathology leading to AMD. It is thought that in AMD this dysregulation in RPE cells is at least in part responsible for the formation of drusen. At present, the exact mechanisms for drusen formation are unknown. A potential approach to detect early AMD patho- biological changes is through the isolation of exosomes, which are nanovesicles that can be purified from biofluids (e.g. plasma and urine). Since RPE-released exosomes and other extracellular vesicles are essential parts of the endosomal pathway, I 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. By evaluating two complementary in vitro and in vivo AMD models, our goal for this project is to determine the role of exosome secretion in sub-RPE deposit formation and in ECM changes under conditions relevant to AMD, and whether pharmacological or gene therapy/biological therapeutic interventions are possible. In the first aim, I will characterize and quantify the protein and lipid composition of sub-RPE deposits, ECM and basal- side exosomes in RPE stressed by pathophysiological conditions implicated in AMD (oxidative stress, complement dysregulation, and age), while modulating exosome release by both pharmacological and genetic approaches. In the second aim, I will molecularly tag, isolate and characterize RPE-derived exosomes from the blood of a well characterized mouse model of early-stage AMD. Detailed profiling of RPE-derived exosomes in normal and diseased settings represent novel studies that will provide insight into mechanisms that underlie pathobiological changes in AMD.