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.

眼病如老年性黄斑变性或糖尿病会影响RPE功能，导致视网膜变性、视力丧失和失明。为了研究RPE的功能、生理和病理，我们培养了人类RPE作为天然组织的更容易获得的替代品。我们能够产生具有经典上皮形态的融合色素RPE细胞培养物，经皮电位为1 - 3mV，经皮电阻大于200欧姆*cm2。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了这些培养物，以确定细胞结构，定位根尖和基底外侧膜蛋白，以及细胞间连接复合物蛋白。采用elisa法确定所选细胞因子的分泌极性。细胞内微电极用于表征受体介导的第二信使通路及其在根尖和基底膜上的下游电生理特性。电容探针技术用于测量净上皮液体传输。定义了RPE的基因特征。我们还将功能活跃的IFNg受体定位在人胎儿视网膜色素上皮（hfRPE）的基底外膜上。激活这些受体可抑制5% FBS、碱性成纤维细胞生长因子（bFGF）、血小板衍生生长因子（PDGF）和表皮生长因子（EGF）诱导的RPE增殖和迁移。将IFNg添加到基底层，而不添加到根尖浴中，显著增加了hfRPE单层从根尖向基侧的流体输送（JV）。