Human Retinal Pigment Epithelial Physiology

人类视网膜色素上皮生理学

• 批准号: 10266881
• 负责人: David Schneeweis
• 金额: $ 113.19万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10266881
• 关键词: AQP1 gene; Acidosis; Affect; Age related macular degeneration; Agreement; Apical; Area; Atrophic; Autologous; Bathing; Bicarbonates; Biological Assay; Blindness; CLC-2 protein; CSF1R gene; Calcium; Carbon Dioxide; Cell Culture Techniques; Cell Death; Cells; Cellular Structures; Characteristics; Choroid; Communities; Critical Pathways; Cystic Fibrosis Transmembrane Conductance Regulator; Dark Adaptation; Data; Development; Diabetes Mellitus; Diffusion; Electric Capacitance; Electron Microscopy; Electrophysiology (science); Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelial; Epithelial Physiology; Epithelium; Europe; Eye diseases; FDA approved; FGF2 gene; Failure; Functional disorder; Gene Expression Profile; Generations; Genes; Goals; Gold; Health; Human; Human body; Image; Immunohistochemistry; Inflammatory; Inflammatory Response; Intercellular Junctions; Interferons; Ion Transport; Ions; JAK2 gene; Laboratories; Lead; Liquid substance; Measurement; Measures; Mediating; Membrane Proteins; Metabolic; Methods; MicroRNAs; Microarray Analysis; Microelectrodes; Modeling; Molecular; Morphology; Muller' s cell; Neurons; Nonexudative age-related macular degeneration; Oxygen Consumption; Pathology; Pathway Analysis; Pathway interactions; Patients; Peripheral; Phagocytes; Photoreceptors; Physiological; Physiology; Platelet-Derived Growth Factor; Process; Production; Property; Publishing; Rattus; Receptor Activation; Reproducibility; Resistance; Retina; Retinal Degeneration; Retinal Detachment; Retinal Pigments; Role; Second Messenger Systems; Services; Side; Signal Pathway; Signal Transduction; Structure; Structure of retinal pigment epithelium; Surface; Techniques; Tight Junctions; Time; Tissues; Transfection; Transplantation; Vascular blood supply; Vision; Water; Zinc; absorption; adult stem cell; apical membrane; basolateral membrane; cytokine; experimental study; fluid flow; genetic signature; human fetal retinal pigment epithelial cell; improved; in silico; mRNA Expression; macula; metabolic rate; migration; monolayer; prevent; protein complex; receptor; response; solute; vector

Eye diseases such as age-related macular degeneration or diabetes affect RPE function and lead to retinal degeneration, vision loss, and blindness. To study RPE function, physiology, and pathology, we have cultured human RPE as a more accessible alternative to the native tissue. We have been able to produce confluent pigmented RPE cell cultures with classic epithelial morphology, transepithelial potential of 1 - 3mV, and transepithelial resistance greater than 200 ohmscm2. In ongoing experiments we continue to further characterize these cultures using electron-microscopy and immunohistochemistry to identify cellular structures, localize apical and basolateral membrane proteins, and intercellular junctional complex proteins. ELISAs are used to confirm the polarity of secretion of selected cytokines. Intracellular microelectrodes are used to characterize receptor-mediated second messenger pathways and their downstream electrophysiological properties at the apical and basolateral membranes of the human RPE. The capacitance probe technique was used to measure net transepithelial fluid transport. We have also defined the gene signature of human RPE. Physiologically, we have localized functionally active IFN receptors to the basolateral membrane of human fetal retinal pigment epithelium (hfRPE). Activation of these receptors inhibits RPE proliferation and migration induced by 5% FBS, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). Addition of IFN to the basal, but not the apical bath, significantly increased fluid transport (JV) across the hfRPE monolayer from the apical to basal side. These human RPE monolayers are continually shared with colleagues across US and Europe (by now more than 38 laboratories) to continue the in depth characterization of human RPE for better understanding of their function and pathophysiology. To better understand mechanisms regulating inflammatory response we used the Asuragen DiscovArray miRNA Expression Service which measures the expression levels of 13,000 confirmed and putative miRNAs. Only miR-155 was significantly increased by ICM. Transfection of a miR-155 mimic into intact monolayers of hfRPE significantly decreased TER to 60% of control; a similar result was previously obtained by addition of ICM. This result strongly suggests that the effects of pro-inflammatory cytokines are in part determined by miR-155. Using Ingenuity Pathway Analysis (IPA), we identified components of several canonical signaling pathways (IFN and NFkB) that are expected to be involved in ICM signaling and a subset of genes (e.g., APC, CLCN5, CSF1R, LRAT, PCDHB5, SLC13A3, JAK2, SOSC1), initially identified as in silico targets of miR-155, were critical for ocular function. The photoreceptor is the most metabolically active neuronal cell in the human body; oxygen consumption at the inner segment of the photoreceptors increases upon dark adaptation, mainly because of the increased ATP requirements needed to maintain the dark current. Since the oxygen consumption at the inner segment of the photoreceptor increases approximately 1.5 - 3 times upon dark adaptation, we expect a proportionate increase in CO2 generation and the subsequent increase in CO2 at the subretinal space. The accumulation of CO2 within the subretinal space (SRS) causes acidosis that is detrimental to the health of surrounding cells (i.e., Muller cells, photoreceptors, and RPE), thus metabolic CO2 must be quickly dissipated from the SRS. We hypothesize that a large fraction of this CO2 load is dissipated by diffusion to the choroidal blood supply, and that this process is mediated by the RPE. In this study, we describe the transport of CO2 across the RPE, which involves multiple ion-transport mechanisms that consequently increase fluid-absorption across the RPE. We investigated the possibility that CO2-flux across the apical membraneis mediated by aquaporin 1, which has high mRNA expression levels in hfRPE cultures and is found at the apical membrane of rat RPE. However, pH-imaging experiments showed that this was not the case in the hfRPE. We showed that CO2 affects multiple ion-transporters that ultimately increase net Na, Cl, and HCO3 absorption across the RPE. Since fluid flows with an osmotic gradient, the increase in solute transport would enhance the steady-state fluid absorption across the RPE. The CO2-induced increase in fluid-absorption may have an important physiological role because the rate of metabolic water production at the retina is approximately 10% of the steady state fluid absorption across the human RPE. Therefore failure to remove water from the subretinal space can potentially cause retinal detachment. Cl-efflux at the basolateral membrane is known to be mediated mainly by the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+ - activated Cl channels. However, current experiments suggest that the apical lactate induced TEP response was not caused by the activation of either of these two channels. In current experiments, we show that ClC-2 proteins are highly expressed in RPE. In addition, microarray analysis also showed high mRNA expression for the ClC-2 protein. More importantly, basal application of zinc reduced the apical lactate induced TEP response by 30-50%. In contrast, apical application of zinc to the apical surface did not reduce the apical lactate induced TEP response. Collectively, our data suggests that ClC-2 is expressed at the basolateral membrane and mediates, in part, the apical lactate - induced TEP response. Currently no FDA-approved treatments exist for an advanced AMD stage called dry AMD where RPE cells atrophy leads to photoreceptor cell death. Multiple ongoing efforts utilize pluripotent or adult stem cells to generate healthy RPE cells as potential replacement for damaged/atrophied RPE monolayer with the goal to prevent photoreceptors loss. These efforts are founded on successful earlier studies which demonstrated that autologous RPE-choroid graft translocated from an unaffected peripheral area to the macula could lead to improved vision in AMD patients. For transplantation, however, there has been no acknowledged gold standard for what constitutes the defining characteristics of an authentically derived RPE or agreement as to how those cells can best be evaluated and selected for transplantation. In recent years, RPE physiology data collected in the lab have provided a unique set of functional markers that are critical for validating normal RPE function. In addition to the well-established practice of verifying the typical RPE markers, gene expression profile, tight junction formation, and phagocytic ability, several key functional assays (calcium imaging, electrophysiological measurements, and vectorial fluid transport) including a purinergic signaling pathway are critical checkpoints to verify the structure, functional intactness and integrity of whole RPE monolayer rather than single RPE cells. In the last two years (Miyagishima et al., 2017) we published a set of gold standards for defining well-functioning RPE and established methods of modifying physiological assays and techniques to make them more accessible to the scientific community worldwide for use in the development of new RPE transplantation techniques.

与年龄相关的黄斑变性或糖尿病等眼部疾病会影响RPE功能并导致视网膜变性，视力丧失和失明。为了研究RPE功能，生理和病理学，我们将人类RPE培养为对天然组织的更容易获得的替代方法。我们已经能够产生具有经典上皮形态，1-3mV的旋转电位和大于200 ohmscm2的旋转电位的经典上皮形态，1-3MV的旋转电位。在正在进行的实验中，我们继续使用电子显微镜和免疫组织化学进一步表征这些培养物，以鉴定细胞结构，定位顶端和基底外侧膜蛋白以及细胞间连接复合蛋白。 ELISA用于确认选定细胞因子分泌的极性。细胞内微电极用于表征人RPE的顶端和基底外侧膜上受体介导的第二信使途径及其下游电生理特性。电容探针技术用于测量净旋转液体运输。 我们还定义了人RPE的基因特征。 从生理上讲，我们在人类胎儿视网膜色素上皮（HFRPE）的基底外侧膜上具有局部功能活性的IFN受体。这些受体的激活抑制了5％FBS诱导的RPE增殖和迁移，基本成纤维细胞生长因子（BFGF），血小板衍生的生长因子（PDGF）和表皮生长因子（EGF）。 在基础浴中添加IFN，但不能在顶端浴中添加，从顶端到基础，横跨HFRPE单层的流体转运（JV）显着增加。这些人类RPE单层不断与我们和欧洲的同事（现在有38多个实验室）共享，以继续对人RPE进行深入的特征，以更好地理解其功能和病理生理学。 为了更好地理解调节炎症反应的机制，我们使用了Asuragen Discovarray miRNA表达服务，该服务测量了13,000个已确认和假定miRNA的表达水平。 ICM只有miR-155显着增加。将miR-155模拟于HFRPE的完整单层转染可显着降低到对照的60％。先前通过添加ICM获得了类似的结果。该结果强烈表明促炎细胞因子的作用部分由miR-155确定。 Using Ingenuity Pathway Analysis (IPA), we identified components of several canonical signaling pathways (IFN and NFkB) that are expected to be involved in ICM signaling and a subset of genes (e.g., APC, CLCN5, CSF1R, LRAT, PCDHB5, SLC13A3, JAK2, SOSC1), initially identified as in silico targets of miR-155, were critical对于眼功能。 感光体是人体中最活跃的神经元细胞。在暗适应后，光感受器内部段的氧气消耗增加，这主要是由于维持黑电流所需的ATP要求增加。由于在暗适应后，光感受器内部段的氧气消耗大约增加了1.5-3倍，因此我们预计二氧化碳生成比例增加，随后在视网膜下空间中二氧化碳的增加。二氧化碳在下视网膜下空间（SRS）中的积累导致酸中毒，这对周围细胞的健康有害（即muller细胞，光感受器和RPE），因此必须迅速从SRS中消除代谢CO2。我们假设，通过扩散到脉络膜血液供应，很大一部分二氧化碳负荷会消散，并且该过程是由RPE介导的。在这项研究中，我们描述了二氧化碳在RPE跨RPE的运输，该转移涉及多种离子传输机制，从而增加了整个RPE的流体吸收。我们研究了由Aquaporin 1介导的顶端膜的CO2-升 - 在HFRPE培养物中具有较高的mRNA表达水平，并且在大鼠RPE的顶端膜上发现。但是，pH模仿实验表明，在HFRPE中并非如此。我们表明，二氧化碳会影响多个离子转运蛋白，这些离子转运蛋白最终会增加整个RPE的Na，Cl和HCO3吸收。由于流体与渗透梯度流动，溶质转运的增加将增强整个RPE的稳态吸收。 CO2诱导的液体吸收吸收的增加可能具有重要的生理作用，因为视网膜的代谢水生产速率约为整个人RPE稳态吸收的10％。因此，未能从视网膜下空间去除水可能会导致视网膜脱离。 已知基底外侧膜处的Cl -Efflux主要是由囊性纤维化跨膜电导调节剂（CFTR）和Ca2+激活的CL通道介导的。但是，当前的实验表明，顶乳酸诱导的TEP反应不是由这两个通道中的任何一个激活引起的。在当前的实验中，我们表明CLC-2蛋白在RPE中高度表达。此外，微阵列分析还显示了CLC-2蛋白的高mRNA表达。更重要的是，锌的基础施用将顶乳酸诱导的TEP反应降低了30-50％。相比之下，锌在顶部表面的顶端应用并未减少顶端乳酸诱导的TEP反应。总的来说，我们的数据表明CLC -2在基底外侧膜上表达，并部分介导了顶端乳酸诱导的TEP反应。 目前，对于一个称为干AMD的晚期AMD阶段，尚无FDA批准的处理，其中RPE细胞萎缩会导致感光细胞死亡。多次正在进行的努力利用多能或成年干细胞产生健康的RPE细胞，以替代受损/萎缩的RPE单层，其目标是防止感光体损失。这些努力建立在成功的早期研究基础上，这些研究表明自体RPE-螺状移植物从未受影响的外围区域转移到黄斑可能会导致AMD患者的视力改善。然而，对于移植，尚未确认构成真实派生的RPE的定义特征或关于如何最好地评估和选择用于移植这些细胞的定义特征。近年来，在实验室中收集的RPE生理数据提供了一组独特的功能标记，这些功能标记对于验证正常RPE功能至关重要。除了验证典型的RPE标记，基因表达谱，紧密连接形成和吞噬能力的实践外，几种关键功能测定（钙成像，电生理学测量和媒介物流体运输），包括蠕虫信号通路，包括一个至关重要的检查点，是至关重要的检查单元格的一个单元和整体的结构。在过去的两年中（Miyagishima等，2017年），我们发布了一系列黄金标准，用于定义功能良好的RPE和既定方法，并建立了修改生理测定和技术的方法，以使它们更容易访问全球的科学社区，以便全世界用于开发新RPE移植技术。

项目成果

Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes.

• DOI: 10.1007/978-1-4939-7553-2_15
• 发表时间: 2018
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Khristov V;Wan Q;Sharma R;Lotfi M;Maminishkis A;Bharti K
• 通讯作者: Bharti K

Inflammasomes Induced by 7-Ketocholesterol and Other Stimuli in RPE and in Bone Marrow-Derived Cells Differ Markedly in Their Production of IL-1β and IL-18.

7-酮胆固醇和 RPE 和骨髓衍生细胞中的其他刺激物诱导的炎症体在产生 IL-1β 和 IL-18 方面存在显着差异。

• DOI: 10.1167/iovs.14-14557
• 发表时间: 2015
• 期刊: Investigative ophthalmology & visual science
• 影响因子: 4.4
• 作者: Shi,Guangpu;Chen,Siqi;Wandu,WambuiS;Ogbeifun,Osato;Nugent,LindseyF;Maminishkis,Arvydas;Hinshaw,SamuelJH;Rodriguez,IgnacioR;Gery,Igal
• 通讯作者: Gery,Igal