The Membrane Attack Complex and the Choriocapillaris in Health and Age-Related Macular Degeneration

项目总结 老年性黄斑变性(AMD)是西方世界不可逆转失明的主要原因。 虽然AMD的病因是多因素的，但脉络膜血管功能障碍提供了 大多数氧到视网膜光感受器，已被认为是AMD最早可检测到的事件。 补体激活是脉络膜血管功能障碍的一个重要因素。补语是一种 先天免疫系统的多蛋白成分，可形成已知的裂解结构 作为膜攻击复合体(MAC)。虽然全身血管床仍未形成MAC，但 浅层脉络膜毛细血管系统，称为脉络膜毛细血管，独特地积聚MAC与 年事已高。这种MAC堆积被认为是导致脉络膜毛细血管内皮细胞裂解损伤的原因 以及AMD的下游后遗症。然而，导致绒毛膜毛细血管病变的机制 积累是人们普遍未知的。我们假设独特的转录网络驱动绒毛膜毛细血管 发育，使这些内皮细胞特别容易受到与年龄相关的分子变化的影响 Mac介导的细胞死亡。为了验证这一假设，我们提出了以下三个具体目标： 1.确定使脉络膜毛细血管对MAC形成具有独特易感性的分子特征。我们会 从人诱导的多能干细胞中分化出脉络膜毛细血管、动脉和静脉内皮细胞 并确定决定绒毛毛细血管细胞命运的转录驱动因素。在单细胞RNA测序之后，我们将 对影响体外MAC形成的绒毛膜毛细血管丰富的基因进行功能验证。 2.识别年龄和AMD的分子变化，这些变化增加了脉络膜毛细血管的MAC积累和MAC- 介入性损害。我们将确定年轻人和成年人脉络膜内皮细胞基因表达的差异 (第5-第6个十年)、老年人(第8-第9个十年)和AMD人类捐赠者。我们将测试在多大程度上 在体外，老化的脉络膜毛细血管的差异表达靶点影响MAC的形成。 3.确定RGCC在健康和AMD中应对MAC的作用。我们将量化RGCC，一种基因 最近发现在人绒毛膜毛细血管中高表达，并与MAC介导的 内皮功能障碍，在RNA和蛋白质水平上使用来自健康和AMD捐赠者的人类脉络膜。 我们还将从功能上评估RGCC在多大程度上介导血管内皮细胞死亡和功能障碍 脉络膜毛细血管和非眼内皮细胞系。 一旦成功，这些研究将确定导致MAC积累和 脉络膜毛细血管损伤。进一步了解脉络膜毛细血管细胞如何积聚和对MAC作出反应 可能导致AMD的新治疗目标，支持NEI的使命、目标和目标。

PROJECT SUMMARY Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the western world. While the etiology of AMD is multifactorial, dysfunction of the choroidal vasculature, which provides the majority of oxygen to retinal photoreceptors, has been implicated as the earliest detectable event in AMD. An important contributor to choroidal vascular dysfunction is complement activation. Complement is a multiprotein component of the innate immune system that results in the formation of a lytic structure known as the membrane attack complex (MAC). While systemic vascular beds remain free from MAC formation, the superficial choroidal capillary system known as the choriocapillaris uniquely accumulates the MAC with advancing age. Such MAC accumulation is proposed to cause lytic injury to choriocapillaris endothelial cells and the downstream sequelae of AMD. However, the mechanisms that lead to choriocapillaris MAC accumulation are widely unknown. We hypothesize that unique transcriptional networks drive choriocapillaris development, which make these endothelial cells particularly susceptible to age-related molecular changes and MAC-mediated cell death. To test this hypothesis, we propose the following three specific aims: 1. Identify molecular features that make the choriocapillaris uniquely susceptible to MAC formation. We will differentiate choriocapillaris, arterial, and venous endothelial cells from human induced pluripotent stem cells and identify transcriptomic drivers of the choriocapillaris cell fate. After single-cell RNA sequencing, we will functionally validate choriocapillaris-enriched genes that influence MAC formation in vitro. 2. Identify molecular changes in age and AMD that increase choriocapillaris MAC accumulation and MAC- mediated damage. We will identify choroidal endothelial cell gene expression differences between young, adult (5th - 6th decade), elderly (8th - 9th decade) and AMD human donors. We will test the degree to which differentially expressed targets in aging choriocapillaris influence formation of the MAC in vitro. 3. Determine the role of RGCC in response to the MAC in health and AMD. We will quantify RGCC, a gene recently identified to be highly expressed in human choriocapillaris and implicated in MAC-mediated endothelial dysfunction, at the RNA and protein level using human choroids from healthy and AMD donors. We will also functionally assess the degree to which RGCC mediates endothelial cell death and dysfunction in both choriocapillaris and non-ocular endothelial cell lines. When successful, these studies will identify novel molecular features that lead to MAC accumulation and choriocapillaris damage. Further understanding how choriocapillaris cells accumulate and respond to the MAC may lead to new therapeutic targets for AMD, supporting the Mission, Goals, and Objectives of the NEI.