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

项目摘要 视网膜相关性黄斑变性（AMD）是西方世界不可逆失明的主要原因。 虽然AMD的病因是多因素的，但脉络膜血管系统的功能障碍，其提供了治疗AMD的方法。 大多数氧向视网膜光感受器的释放已经被认为是AMD中最早可检测的事件。 导致脉络膜血管功能障碍的一个重要因素是补体激活。补体是 一种先天免疫系统的多蛋白成分，可导致已知的溶解结构的形成 膜攻击复合物（MAC）虽然体循环血管床仍然没有MAC形成， 称为脉络膜毛细血管的浅表脉络膜毛细血管系统独特地积累MAC， 年龄增长。这种MAC的积累被认为是导致脉络膜毛细血管内皮细胞溶解性损伤的原因 和AMD的下游后遗症。然而，导致脉络膜毛细血管MAC的机制 积累是未知的。我们假设独特的转录网络驱动脉络膜毛细血管 发育，这使得这些内皮细胞特别容易受到年龄相关的分子变化， 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形成特别敏感的分子特征。我们将 从人诱导多能干细胞中分化脉络膜毛细血管、动脉和静脉内皮细胞 并确定脉络膜毛细血管细胞命运的转录组驱动因素。在单细胞RNA测序后，我们将 功能性验证影响MAC体外形成的脉络膜毛细血管富集基因。 2.确定年龄和AMD中增加脉络膜毛细血管MAC积累和MAC- 介导的损害。我们将鉴定年轻人、成年人和成年人脉络膜内皮细胞基因表达的差异， (5th- 第6个十年）、老年人（第8 - 9个十年）和AMD人类供体。我们将测试 衰老脉络膜毛细血管中差异表达的靶点影响体外MAC的形成。 3.确定RGCC在健康和AMD中对MAC的响应作用。我们将定量RGCC， 最近发现在人脉络膜毛细血管中高度表达，并与MAC介导的 内皮功能障碍，在RNA和蛋白质水平使用来自健康和AMD供体的人脉络膜。 我们还将在功能上评估RGCC介导内皮细胞死亡和功能障碍的程度。 脉络膜毛细血管和非眼内皮细胞系。 如果成功，这些研究将确定导致MAC积累的新分子特征， 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累和响应MAC 可能导致AMD的新治疗靶点，支持NEI的使命、目标和目的。

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.