Human Retinal Pigment Epithelial Physiology

人类视网膜色素上皮生理学

• 批准号: 10019995
• 负责人: Sheldon Miller
• 金额: $ 94.19万
• 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/10019995
• 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; Computer Simulation; 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; 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; 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.

年龄相关性黄斑变性或糖尿病等眼部疾病会影响 RPE 功能，导致视网膜变性、视力丧失和失明。为了研究 RPE 功能、生理学和病理学，我们培养了人类 RPE，作为天然组织更容易替代的选择。我们已经能够产生汇合的色素性 RPE 细胞培养物，具有经典的上皮形态、1 - 3mV 的跨上皮电位和大于 200 ohmscm2 的跨上皮电阻。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，以识别细胞结构、定位顶端和基底外侧膜蛋白以及细胞间连接复合物蛋白。 ELISA 用于确认所选细胞因子分泌的极性。细胞内微电极用于表征受体介导的第二信使通路及其在人类 RPE 顶膜和基底外侧膜处的下游电生理特性。电容探针技术用于测量净跨上皮液体转运。 我们还定义了人类 RPE 的基因特征。 生理学上，我们将功能活跃的 IFN 受体定位于人胎儿视网膜色素上皮 (hfRPE) 的基底外侧膜。这些受体的激活可抑制 5% FBS、碱性成纤维细胞生长因子 (bFGF)、血小板源性生长因子 (PDGF) 和表皮生长因子 (EGF) 诱导的 RPE 增殖和迁移。 在基底浴中添加 IFN，但不在顶端浴中添加，显着增加了从顶端到基底侧穿过 hfRPE 单层的液体转运 (JV)。这些人类 RPE 单层不断与美国和欧洲的同事（目前超过 38 个实验室）共享，以继续深入表征人类 RPE，以便更好地了解其功能和病理生理学。 为了更好地了解调节炎症反应的机制，我们使用 Asuragen DiscovArray miRNA 表达服务来测量 13,000 个已确认和推定的 miRNA 的表达水平。只有 miR-155 通过 ICM 显着增加。将 miR-155 模拟物转染到完整的 hfRPE 单层中，TER 显着降低至对照的 60%；之前通过添加 ICM 获得了类似的结果。这一结果强烈表明促炎细胞因子的作用部分是由 miR-155 决定的。使用 Ingenuity Pathway Analysis (IPA)，我们鉴定了预计参与 ICM 信号传导的几种典型信号传导通路（IFN 和 NFkB）的成分以及基因的子集（例如 APC、CLCN5、CSF1R、LRAT、PCDHB5、SLC13A3、 JAK2、SOSC1）最初被确定为 miR-155 的计算机靶标，对眼功能至关重要。 感光细胞是人体内代谢最活跃的神经细胞；光感受器内段的耗氧量在暗适应时增加，主要是因为维持暗电流所需的 ATP 需求增加。由于暗适应时感光器内段的耗氧量增加约 1.5 - 3 倍，因此我们预计 CO2 的产生量会成比例增加，随后视网膜下间隙的 CO2 也会相应增加。 CO2 在视网膜下腔 (SRS) 内积聚会导致酸中毒，不利于周围细胞（即 Muller 细胞、光感受器和 RPE）的健康，因此代谢 CO2 必须快速从 SRS 中消散。我们假设大部分二氧化碳负荷通过扩散到脉络膜血液供应而消散，并且该过程是由 RPE 介导的。在这项研究中，我们描述了 CO2 穿过 RPE 的传输，其中涉及多种离子传输机制，从而增加了 RPE 上的液体吸收。我们研究了穿过顶膜的 CO2 通量是由水通道蛋白 1 介导的可能性，水通道蛋白 1 在 hfRPE 培养物中具有高 mRNA 表达水平，并且在大鼠 RPE 的顶膜中发现。然而，pH 成像实验表明 hfRPE 的情况并非如此。我们发现，CO2 会影响多种离子转运蛋白，最终增加 RPE 上的 Na、Cl 和 HCO3 净吸收。由于流体以渗透梯度流动，溶质转运的增加将增强 RPE 上的稳态流体吸收。 CO2 引起的液体吸收增加可能具有重要的生理作用，因为视网膜处代谢水产生的速率约为人类 RPE 稳态液体吸收的 10%。因此，未能清除视网膜下腔的水分可能会导致视网膜脱离。 已知基底外侧膜的 Cl 流出主要由囊性纤维化跨膜电导调节器 (CFTR) 和 Ca2+ 激活的 Cl 通道介导。然而，目前的实验表明，顶端乳酸诱导的 TEP 反应并不是由这两个通道的激活引起的。在当前的实验中，我们表明 ClC-2 蛋白在 RPE 中高表达。此外，微阵列分析还显示ClC-2蛋白的mRNA高表达。更重要的是，基础施用锌可使顶端乳酸诱导的 TEP 反应降低 30-50%。相反，向顶端表面施用锌并没有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，并部分介导顶端乳酸诱导的 TEP 反应。 目前，对于称为干性 AMD 的晚期 AMD 阶段，RPE 细胞萎缩导致感光细胞死亡，尚无 FDA 批准的治疗方法。多项正在进行的工作利用多能干细胞或成体干细胞产生健康的 RPE 细胞，作为受损/萎缩的 RPE 单层的潜在替代品，目的是防止光感受器损失。这些努力建立在早期成功研究的基础上，这些研究表明，自体 RPE 脉络膜移植物从未受影响的周边区域转移到黄斑可以改善 AMD 患者的视力。然而，对于移植来说，对于真正衍生的 RPE 的定义特征的构成，还没有公认的黄金标准，也没有就如何最好地评估和选择这些细胞进行移植达成一致。近年来，实验室收集的 RPE 生理学数据提供了一组独特的功能标记，这对于验证正常 RPE 功能至关重要。除了验证典型 RPE 标记、基因表达谱、紧密连接形成和吞噬能力的成熟实践外，包括嘌呤能信号通路在内的几个关键功能测定（钙成像、电生理测量和矢量液体转运）也至关重要检查点来验证整个 RPE 单层而不是单个 RPE 细胞的结构、功能完整性和完整性。在过去两年中（Miyagishima 等人，2017），我们发布了一套定义功能良好的 RPE 的黄金标准，并建立了修改生理测定和技术的方法，使全世界的科学界更容易将其用于开发新的 RPE 移植技术。