RPE Cell Bystander Effects Contribute to AMD Pathology

 描述(由申请人提供)： 年龄相关性黄斑变性(AMD)是一种进展缓慢的多因素疾病，涉及遗传异常和环境侮辱。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岁时达到最大风险率~30%。由于吸烟增加了患老年性黄斑变性的风险，而且退伍军人的吸烟率比普通美国平民高出20%，退伍军人管理局系统将不得不为潜在的≥700万老年性黄斑变性病例提供护理。目前AMD的概念认识到，慢性氧化应激和炎症(包括补体激活)可以触发RPE、Bruchs膜(BRM)和脉络膜的病理变化。然而，这一方案没有考虑到AMD的“地域性”。损害不是从一个位置开始扩散，而是在许多不同的位置触发。这表明损伤发生在敏感区域，而延迟发生在更有弹性的区域(保留中心凹)。我们的研究是基于这样一个前提，即RPE是衰老和疾病的场所。RPE是视网膜的重要支持细胞；它是玻璃体和BRM中碎片的来源；RPE细胞形成一个细胞网络，通过缝隙连接(GJ)连接。因此，RPE是传播或限制后极内应激反应的最佳候选者。信息的传播或旁观者效应可以通过两种不同的机制来调节：向接受者传递分泌信号(单个分子或外体中包含的信号)；或通过GJ通信来传播信息。在这里，我们将以我们的总体假设为指导，即外切体和GJ介导的RPE单层通讯有助于RPE损伤的旁观者效应和中介区域性。个别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，网络中单个细胞的休眠状态将决定对压力的敏感性。[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RPE细胞和具有AMD低和高遗传风险因素的对照患者中进行研究]]。在目标1中，我们将确定外体中包含的信使在RPE细胞中执行旁观者效应的作用。我们已经证明，氧化应激产生的外切体可以作为信号载体，以补体依赖的方式在供体细胞和受体细胞之间进行交流。这一机制将被进一步探索，以确定外切体形成的极性，外切体在整体和单细胞反应中的活性，以及外切体传递信息的过程。目标2旨在确定GJ沟通在执行旁观者效应中的效果。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。这一观察将进一步扩展，以确定局部应激在供体细胞和受体细胞中引发的信号事件，以及受体细胞中与对来自供体细胞的信息做出反应的敏感性相关的代谢基线参数，并检查介导GJ通讯的潜在信使。

 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.