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. These human RPE monolayers are continually shared with colleagues across US and Europe (by now more than 38 laboratories) to continue in depth characterization of human RPE for better understanding they function and pathologies. 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. 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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAEY000419-10
Application #
8737635
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2013
Total Cost
$598,071
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Ferrer, Marc; Corneo, Barbara; Davis, Janine et al. (2014) A multiplex high-throughput gene expression assay to simultaneously detect disease and functional markers in induced pluripotent stem cell-derived retinal pigment epithelium. Stem Cells Transl Med 3:911-22
Doyle, Sarah L; Ozaki, Ema; Brennan, Kiva et al. (2014) IL-18 attenuates experimental choroidal neovascularization as a potential therapy for wet age-related macular degeneration. Sci Transl Med 6:230ra44
Ratnapriya, Rinki; Zhan, Xiaowei; Fariss, Robert N et al. (2014) Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration. Hum Mol Genet 23:5827-37
Kay, Paul; Yang, Yit C; Hiscott, Paul et al. (2014) Age-related changes of cystatin C expression and polarized secretion by retinal pigment epithelium: potential age-related macular degeneration links. Invest Ophthalmol Vis Sci 55:926-34
Bharti, Kapil; Miller, Sheldon S; Arnheiter, Heinz (2011) The new paradigm: retinal pigment epithelium cells generated from embryonic or induced pluripotent stem cells. Pigment Cell Melanoma Res 24:21-34
Shi, Guangpu; Maminishkis, Arvydas; Banzon, Tina et al. (2008) Control of chemokine gradients by the retinal pigment epithelium. Invest Ophthalmol Vis Sci 49:4620-30