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.
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