Human Retinal Pigment Epithelial Physiology

人类视网膜色素上皮生理学

• 批准号: 8339776
• 负责人: Sheldon Miller
• 金额: $ 103.25万
• 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/8339776
• 关键词: AQP1 gene; Acidosis; Adult; Affect; Age related macular degeneration; Apical; Bathing; Bicarbonates; Binding; Blindness; CSF1R gene; CTF1 gene; Calcium; Carbon Dioxide; Cell Culture Techniques; Cell physiology; Cells; Cellular Structures; Ciliary Neurotrophic Factor; Computer Simulation; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Dark Adaptation; Data; Diabetes Mellitus; Diffusion; Dose; Electric Capacitance; Electron Microscopy; Epidermal Growth Factor; Epithelial; Epithelial Physiology; Extracellular Signal Regulated Kinases; Eye; Eye Injuries; Eye diseases; Failure; Fibroblast Growth Factor 2; Functional disorder; Gene Expression; Generations; Genes; Goals; Health; Human; Human body; IRF1 gene; Image; Imaging Techniques; Immunohistochemistry; In Vitro; Inflammatory; Inflammatory Response; Interferons; Ion Transport; Ions; JAK2 gene; Lactic acid; Lead; Light; Liquid substance; MAP Kinase Gene; MAPK14 gene; Measurement; Measures; Mediating; Membrane Proteins; Metabolic; Methods; MicroRNAs; Microarray Analysis; Microelectrodes; Modeling; Molecular; Morphology; Muller' s cell; Neurons; Oxygen Consumption; Pathology; Pathway Analysis; Pathway interactions; Phosphorylation; Photoreceptors; Physiological; Physiology; Pigments; Platelet-Derived Growth Factor; Process; Production; Property; Proteins; Rattus; Receptor Activation; Resistance; Respiration; Retina; Retinal; Retinal Degeneration; Retinal Detachment; Retinal Diseases; Retinal Pigments; Role; STAT1 gene; STAT3 gene; Second Messenger Systems; Services; Side; Signal Pathway; Signal Transduction; Source; Structure of retinal pigment epithelium; Surface; Techniques; Time; Tissues; Transfection; Vascular blood supply; Water; Zinc; absorption; apical membrane; basolateral membrane; cytokine; fetal; fluid flow; in vivo; mRNA Expression; migration; monolayer; neurotrophic factor; protein complex; receptor; research study; response; second messenger; solute; therapeutic target; voltage

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 been able to produce confluent pigmented RPE cell cultures with classic epithelial morphology, transepithelial potential of 1 - 3mV, and transepithelial resistance greater than 200 Ohms*cm2. In the present experiments we further characterized these cultures using electron-microscopy and immunohistochemistry to identify cellular structures, localize apical and basolateral membrane proteins, and intercellular junctional complex proteins. ELISAs were used to confirm the polarity of secretion of selected cytokines. Intracellular microelectrodes were used to characterize receptor-mediated second messenger pathways and their downstream electrophysiological properties at the apical and basolateral membranes. The capacitance probe technique was used to measure net transepithelial fluid transport. Gene signature of RPE was defined. We also localized functionally active IFNg receptors to the basolateral membrane of human fetal retinal pigment epithelium (hfRPE). Activation of these receptors inhibits 5% FBS, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) induced RPE proliferation and migration. Addition of IFNg to the basal, but not the apical bath, significantly increased fluid transport (JV) across the hfRPE monolayer from the apical to basal side. We conclude that IFNg inhibits RPE migration and proliferation, activates CFTR-dependent fluid absorption across RPE in vitro and in vivo, and that JAK/STAT1/IRF-1, P38 MAPK and NO, PKA are all involved in mediating these responses. These finding suggest several therapeutic targets for treating proliferative retinal diseases and removing the fluid accumulation in the subretinal space that occurs following many retinal pathologies. 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), identified as in silico targets of miR-155, were critical for ocular function. Many eye injuries and degenerative pathologies trigger compensatory release of neurotrophic factors. In other sets of experiments we investigated CNTF, a well-known neurotrophic factor, and its ability to regulate RPE physiology. Gene expression of CNTF, CT1, OSM and their receptor subunits were analyzed on human RPE. Binding of CNTF, CT1 and OsM to their receptors activate the JAK/STAT3 signaling pathway in primary culture of hfRPE and adult RPE (ARPE-19). While OsM significantly activated P44/P42 (ERK) MAP kinase pathway, both CNTF and CT1 has no apparent effects on the phosphorylation of ERK. CNTF has small but significant stimulatory effect on hfRPE proliferation (P < 0.05). CT1 show dose response stimulatory effect on RPE proliferation and the maximum stimulatory effect (25%) was observed at 100 ng/ml; OsM show dose-dependent inhibitory effect on hfRPE proliferation from 10-80 ng/ml. Furthermore, CNTF significantly increased fluid transport (JV) across RPE from 8.7 0.7 to 20.7 3.3 uL*cm-2*hr-1 (n= 3; P < 0.05). 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 membrane is 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. The inner retina is a significant source of lactic acid, consistent with the high lactate concentration (3.8 - 13 mM) at the SRS even in light-adapted eyes. Lactic acid is a byproduct of anaerobic respiration, and is released in high quantities in the dark adapted eye. Accumulation of lactic acid within the subretinal space is detrimental to the function of the photoreceptors. This study uses pHi-imaging techniques, combined with the more traditional electrophysiological methods of epithelial voltage and resistance measurements to study the mechanisms involved in lactate transport in the RPE. 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. In another set of experiments, we also show that these K- and Cl- channels were not directly activated by the apical lactate induced acidification. These experiments suggest that the lactate induced activation of K- and Cl- channels may be mediated by allosteric interactions with monocarboxylates. Preliminary experiments have also showed that apical lactate caused a decrease in intracellular calcium concentration. This may have other effects on cell physiology that will be investigated further.

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 Ohms*cm2 的跨上皮电阻。在本实验中，我们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途径及其在顶膜和基底外侧膜的下游电生理特性。电容探针技术用于测量净跨上皮液体转运。定义了 RPE 的基因特征。我们还将功能活跃的 IFNg 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 IFNg，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)。 我们得出的结论是，IFNg 抑制 RPE 迁移和增殖，在体外和体内激活 RPE 上的 CFTR 依赖性液体吸收，并且 JAK/STAT1/IRF-1、P38 MAPK 和 NO、PKA 都参与介导这些反应。这些发现提出了治疗增殖性视网膜疾病和消除许多视网膜病变后发生的视网膜下腔积液的几种治疗靶点。 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）被确定为 miR-155 的计算机靶标，对眼功能至关重要。 许多眼部损伤和退行性病变都会引发神经营养因子的代偿性释放。在其他组实验中，我们研究了 CNTF（一种众所周知的神经营养因子）及其调节 RPE 生理学的能力。 分析人 RPE 上 CNTF、CT1、OSM 及其受体亚基的基因表达。 CNTF、CT1 和 OsM 与其受体的结合可激活 hfRPE 和成人 RPE (ARPE-19) 原代培养物中的 JAK/STAT3 信号通路。虽然 OsM 显着激活 P44/P42 (ERK) MAP 激酶通路，但 CNTF 和 CT1 对 ERK 磷酸化没有明显影响。 CNTF对hfRPE增殖有微小但显着的刺激作用（P < 0.05）。 CT1显示对RPE增殖的剂量反应刺激作用，并且在100ng/ml时观察到最大刺激作用（25%）； OsM 在 10-80 ng/ml 范围内对 hfRPE 增殖表现出剂量依赖性抑制作用。此外，CNTF 显着增加了 RPE 上的液体转运 (JV)，从 8.7 ± 0.7 增加到 20.7 ± 3.3 uL*cm-2*hr-1 (n= 3；P < 0.05)。 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。 视网膜内层是乳酸的重要来源，即使在光适应的眼睛中，SRS 处的乳酸浓度也很高（3.8 - 13 mM）。乳酸是无氧呼吸的副产品，在适应黑暗的眼睛中大量释放。视网膜下腔内乳酸的积累不利于感光器的功能。本研究使用 pHi 成像技术，结合更传统的上皮电压和电阻测量的电生理学方法来研究 RPE 中乳酸转运的机制。 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。 在另一组实验中，我们还表明这些 K- 和 Cl- 通道并未直接被顶端乳酸诱导的酸化激活。这些实验表明，乳酸诱导的 K 通道和 Cl 通道激活可能是通过与单羧酸盐的变构相互作用介导的。初步实验还表明，顶端乳酸会导致细胞内钙浓度降低。这可能对细胞生理学产生其他影响，有待进一步研究。