RPE Cell Bystander Effects Contribute to AMD Pathology

RPE 细胞旁观者效应有助于 AMD 病理学

• 批准号: 10293593
• 负责人: Baerbel Rohrer
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10293593
• 关键词: Affect; Age; Age related macular degeneration; American; Biology; Blindness; Bruch' s basal membrane structure; Bystander Effect; Calcium; Caring; Cells; Cellular Stress; Cessation of life; Choroid; Choroidal Neovascularization; Chronic; Communication; Complement; Complement 3a; Complement Activation; Complement Inactivators; Complex; Complication; DNA; Development; Disease; Drusen; Elastases; Epigenetic Process; Family suidae; Funding; Future; Gap Junctions; Genetic; Goals; HDAC6 gene; Haplotypes; Homeostasis; Hybrids; Incidence; Individual; Inflammation; Knowledge; Life; Location; Mediating; Mediator of activation protein; Melanins; Membrane Potentials; Metabolic; Metabolism; Mitochondria; Mitochondrial DNA; Molecular Abnormality; Normalcy; Organelles; Oxidative Stress; Pathologic; Pathology; Population; Prevalence; Production; Receptor Signaling; Research; Retinal Photoreceptors; Risk; Role; Severities; Signal Transduction; Smoking; Stress; Structure of retinal pigment epithelium; System; Testing; Therapeutic; Veterans; base; cell injury; complement C3a receptor; complement system; design; exosome; experimental study; geographic atrophy; induced pluripotent stem cell; mitochondrial dysfunction; mitochondrial membrane; mitochondrial metabolism; monolayer; receptor; response; risk variant; uptake

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 sixty. Since smoking, a major cause of oxidative stress, significantly increases the risk of AMD and there is a 20% higher incidence of smoking in veterans than in the general U.S. civilian population, the VA system will have to provide care for potentially >7 million AMD cases. The current concepts of AMD recognize that chronic oxida- tive stress and inflammation (including complement activation) can trigger pathological changes in RPE, Bruch’s membrane (BrM) and choroid. Major efforts to develop new AMD therapeutics have been focused on complement inhibitors. However, to date, all targets within the complement cascade have failed. We pose that one complication not yet considered is the potential issue of intracellular complement signaling. The overarching goal is to determine how damage signals leading to mitochondrial dysfunction are spread throughout the RPE monolayer, and to elucidate the contribution of intracellular complement signaling. Using highly differentiated RPE cells we have made significant progress demonstrating that oxidative stress changes the cargo in exosomes that are released from cells and that those exosomes induce short- and long-term changes in recipient RPE. Importantly, we found that long-term changes triggered by exosomes included changes in mitochondrial homeostasis and have identified potential mediators. Likewise, we showed that oxidative stress triggers mitochondrial membrane potential and calcium changes in an individual stressed cell that is then propagated through gap junctions to receptive cells. A receptive cell was identified as a cell with high baseline calcium, less negative membrane potential and low melanin levels. Based on the new knowledge identified in the previous funding period, we wish to expand on the following hypotheses. 1) As it is now accepted that an overactive complement system is tied to AMD incidence, we propose to investi- gate the contribution of intracellular complement signaling in mitochondrial damage and ensuing pathology. And 2) as mitochondria are the organelles responsible for energy production and making life/death decisions in a cell, we wish to determine the contribution of exosomes to the transfer of information to recipient cells. Two aims were designed to answers those questions. Aim 1 tests the prediction that intracellular calcium and complement signaling contribute to mitochondrial stress in the RPE network; Aim 2 tests the prediction that exosomal content alters intracellular milieu resulting in changes in mitochondrial homeostasis in the RPE network. We will test questions in three sets of cells with different features: ARPE-19, which can easily be manipulated; second, we will use J and H mitochondrial cybrids, in which the J haplotype is associated with increased risk for AMD; and third, we will examine questions in iPSC-RPE cells with Chr1 versus Chr10 risk alleles and their isogenic controls. These studies will have important implication in AMD biology. First, knowledge about the role of intracellular complement signaling in mitochondrial metabolism, and second, elu- cidating how endogenous signaling transmitted via exosomes can alter mitochondrial homeostasis within the RPE, together will have important consequences for the future development of AMD therapeutics.

视网膜相关性黄斑变性（AMD）是一种缓慢进展的多因素疾病， 环境污染和侮辱。AMD是导致60岁以上美国人失明的主要原因。 由于吸烟，氧化应激的主要原因，显着增加AMD的风险，并且有20%的人患有AMD。 退伍军人吸烟的发生率高于一般美国平民，退伍军人管理局系统将不得不 为可能超过700万例AMD病例提供护理。目前AMD的概念认识到慢性氧化损伤- 应激和炎症（包括补体激活）可引发RPE的病理变化， Bruch膜（BrM）和脉络膜。开发新的AMD疗法的主要努力集中在 补体抑制剂。然而，迄今为止，补体级联中的所有靶标都失败了。我们提出 一个尚未考虑的并发症是细胞内补体信号传导的潜在问题。的 首要目标是确定导致线粒体功能障碍的损伤信号如何传播 整个RPE单层，并阐明细胞内补体信号传导的贡献。使用 高度分化的RPE细胞，我们已经取得了重大进展，证明氧化应激， 改变从细胞释放的外泌体中的货物，并且这些外泌体诱导短-和 接受者RPE的长期变化。重要的是，我们发现由外来体引发的长期变化 包括线粒体内稳态的变化，并确定了潜在的介质。同样我们 表明氧化应激会触发个体的线粒体膜电位和钙的变化 应激细胞然后通过缝隙连接繁殖到感受性细胞。鉴定出一个感受细胞 作为具有高基线钙、较少负膜电位和低黑色素水平的细胞。基于 鉴于上一个供资期间发现的新知识，我们希望扩展以下假设。第一章 由于现在公认过度活跃的补体系统与AMD发病率有关，我们建议研究 门控细胞内补体信号传导在线粒体损伤和随后的病理学中的作用。 2）由于线粒体是负责能量产生和做出生死决定的细胞器 在细胞中，我们希望确定外泌体对向受体细胞传递信息的贡献。 为回答这些问题，设计了两个目标。目的1检验细胞内钙离子 和补体信号有助于RPE网络中的线粒体应激; Aim 2测试了预测 外泌体内容物改变细胞内环境，导致线粒体内稳态的变化， RPE网络。我们将在三组具有不同特征的细胞中测试问题：ARPE-19，它可以很容易地 第二，我们将使用J和H线粒体胞质杂交体，其中J单倍型是相关的。 与AMD的风险增加;第三，我们将检查iPSC-RPE细胞与Chr 1和Chr 10的问题 风险等位基因及其同基因控制。这些研究将对AMD生物学研究具有重要意义。第一、 了解细胞内补体信号在线粒体代谢中的作用，第二，elu- 通过外泌体传递的内源性信号如何改变线粒体内的稳态， RPE一起将对AMD治疗的未来发展产生重要影响。