RPE Cell Bystander Effects Contribute to AMD Pathology

 描述（由申请人提供）： 视网膜相关性黄斑变性（AMD）是一种涉及遗传因素和环境损伤的缓慢进展的多因素疾病。AMD是60岁以上美国人失明的主要原因。随着人口老龄化，AMD的患病率将在75岁时达到约30%的最大风险率。由于吸烟增加了AMD的风险，退伍军人的吸烟发生率比一般美国平民高20%，VA系统将不得不为潜在的≥ 700万AMD病例提供护理。AMD的当前概念认识到慢性氧化应激和炎症（包括补体激活）可触发RPE、布鲁赫膜（Bruch’s membrane，BrM）和脉络膜的病理变化。然而，该方案没有考虑的是AMD的“区域性”。损害不会从一个位置开始蔓延，而是在许多不同的位置触发。这表明损伤发生在敏感区域，而在更有弹性的区域（保留中央凹）延迟损伤。我们的研究是基于这样一个前提，即RPE是衰老和疾病的一个部位。RPE是视网膜的基本支持细胞;它是玻璃疣和BrM中发现的碎片的来源;并且RPE细胞形成通过间隙连接（GJ）连接的细胞网络。因此，RPE是扩展或限制后极内的应激反应的主要候选者。信息的传播或旁观者效应可以通过两种不同的机制介导：将分泌的信号转移到受体（外泌体中含有的单个分子或信号）;或通过经由GJ的通信方式传播信息。在这里，我们将以我们的总体假设为指导，即RPE单层中的外泌体和GJ介导的通讯有助于旁观者效应并介导RPE损伤的区域性。单个细胞对旁观者效应的敏感性被认为是由它们的代谢特征决定的。因此，我们进一步假设，网络中单个细胞的静息状态将决定其对压力的敏感性。[[这些问题将在来自ARPE-19细胞的RPE网络以及来自AMD的诱导多能干细胞衍生的RPE细胞和具有AMD的低和高遗传风险因素的对照患者中进行研究]]。在目的1中，我们将确定外泌体中包含的信使在RPE细胞中执行旁观者效应中的作用。我们已经证明，由氧化应激产生的外泌体可以作为信号载体，以补体依赖性方式在供体和受体细胞之间进行通信。将进一步探索这种机制，以确定外泌体形成的极性，外泌体对整体和单细胞反应的活性，以及外泌体传递信息的过程。目的2是确定GJ沟通在实施旁观者效应中的作用。我们的初步数据表明，供体细胞中的局部氧化应激可以触发信息的传播，导致有限数量的连接受体细胞中线粒体稳态的变化。这种观察将进一步扩展到确定由局部应激引起的供体和受体细胞中的信号传导事件以及受体细胞中与对来自供体细胞的信息的响应敏感性相关的代谢基线参数，并检查介导GJ通信的潜在信使。

 DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is a slowly progressing multifactorial disease involving genetic ab- normalities and environmental insults. AMD is the leading cause of blindness for Americans over age 60. As the population ages, the prevalence of AMD will reach a maximum risk rate of ~30% at age 75. As smoking increases the risk of AMD and there is a 20% higher incidence of smoking in veterans than in the general US civilian population, the VA system will have to provide care for potentially ≥7 million AMD cases. The current concepts of AMD recognize that chronic oxidative stress and inflammation (including complement activation) can trigger pathological changes in RPE, Bruch's membrane (BrM) and choroid. However, what this scheme does not consider is the "regionality" of AMD. Damage does not start in one location to spread from there, but is triggered in many different locations. This suggests that damage occurs in susceptible areas, while delaying it in more resilient areas (sparing of the fovea). Our research is based on the premise that the RPE is a site of aging and disease. The RPE is an essential support cell of the retina; it is a source for the debris found in drusen and BrM; and th RPE cells form a network of cells, connected via gap junctions (GJ). Hence the RPE is a prime candidate to either spread or limit the stress-response within the posterior pole. Spreading of infor- mation, or the bystander effect, can be mediated by two different mechanisms: transfer of a secreted signal to the recipient (individual molecules or signals contained in exosomes); or the spread of information by means of communication via GJ. Here we will be guided by our overall hypothesis that exosome and GJ-mediated com- munication in RPE monolayers contribute of the bystander effect and mediate regionality of RPE damage. The susceptibility of individual cells to the bystander effects is thought to be determined by their metabolic profile. Hence we further hypothesize that the resting state of the individual cell within a network will determine is sus- ceptibility to stress. [[These questions will be investigated in RPE networks derived from ARPE-19 cells as well as induced pluripotent stem cell-derived RPE cells from AMD and control patients with low and high genetic risk factors for AMD]]. In Aim 1, we will determine the effects of messengers contained in exosomes in execut- ing the bystander effect in RPE cells. We have demonstrated that exosomes generated by oxidative stress can serve as signaling vectors for communication between donor and recipient cells in a complement-dependent manner. This mechanism will be explored further to determine the polarity of exosome formation, the activity of exosomes on global and single cell responses, and the process by which exosomes transfer information. Aim 2 is designed to determine the effects of GJ communication in executing the bystander effect. Our preliminary data has shown that local oxidative stress in a donor cell can trigger spread of information leading to changes in mitochondrial homeostasis in a limited number of connected recipient cells. This observation will be further extended to determine the signaling events in donor and recipient cells elicited by local stress as well as the metabolic baseline parameters in recipient cells that correlate with susceptibility to responding to information from a donor cell, and to examine potential messengers that mediate GJ communication.