Human Fetal Retinal Pigment Epithelium Cells Research Articles

Multi‐omic study of a cellular model of the retinal pigment epithelium producing extracellular deposits

Aims/Purpose: To study transcriptomic and metabolomic changes during a 6‐month follow‐up of an in vitro model of the retinal pigment epithelium (RPE) producing extracellular deposits.Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts for 6 months and harvested at 4, 12, 17 and 25 weeks. Progressive accumulation of extracellular material was determined by apolipoprotein E (APOE) and hydroxyapatite (HAP) localization. Transcriptome was analysed by RNA sequencing (RNAseq) and differential expression analysis was conducted using the platform Dr. Tom. Untargeted metabolomics was carried out by high‐resolution mass spectrometry (LC‐IM‐QTOF) and bioinformatic analysis was performed using Mass Professional Profiler software.Results: RPE cells formed a fully differentiated monolayer at 4 weeks in culture, but tight junctions and electrical resistance decreased from 12 weeks onwards, coinciding with the formation of extracellular deposits containing APOE and HAP. RNAseq showed alterations of cell migration, adhesion and differentiation, and oxidation–reduction, lipid metabolic and apoptotic processes during RPE ageing (4 vs 25 weeks, Q‐value < 0.001). Untargeted metabolomics provided 350 non‐redundant compounds, and principal component analysis showed a clear separation between time points and good reliability within each group (55% of cumulative variance). Moderated t‐test (Benjamin‐Hochberg correction) provided 103 compounds differentially altered during RPE follow‐up (p‐value < 0.001, fold‐change > 2), belonging to trans‐sulfuration, one carbon metabolism, glutathione metabolism, Kennedy pathway for sphingolipids and cysteine and methionine catabolism.Conclusions: Time‐dependent changes were observed in the cell culture model, including progressive sub‐RPE accumulation of APOE and HAP, and alterations in cell adhesion and migration, lipid metabolism and sphingolipids pathway contributing to photoreceptor protection and calcium mobilization.

Study of the metallome and transcriptome of a degenerating cellular model of the retinal pigment epithelium

Aims/Purpose: To study the changes on the metallome and transcriptome of a cellular model of the retinal pigment epithelium (RPE) during its in vitro degeneration after 4 months in culture.Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts and cultured for 4 months. Cell pigmentation and barrier function was followed up using optical microscopy and measurement of the transepithelial electrical resistance (TEER). Samples were collected at 21, 91 and 133 days in culture and analysed for gene expression, metal homeostasis of Ca, Na, P, Mg and P, and extracellular deposit formation through RNA sequencing (RNA‐Seq), elemental mass spectrometry (ICP‐MS) and immunocytochemistry assays, respectively.Results: RPE cells formed a differentiated monolayer at 21 days in culture, expressing specific genes and proteins of RPE and giving increasing TEER values. However, after 133 days in culture, loss of tight junction proteins (i.e. CLDN19 and BEST1) and electrical resistance was seen, together with loss of pigmentation and increase of apolipoprotein E deposition in the extracellular space. Gene expression analysis showed an enrichment on apoptotic processes, biomolecule phosphorylation, and signs of epithelial‐to‐mesenchymal transition (EMT). Degeneration of RPE cells concurred with an increase of Na and P content in the cytosol and decrease of Mg, while a decrease of Mg, P and Cu was determined in the insoluble fraction of cells. Time‐dependent changes of metals were accompanied with the alteration of metal transporters and antioxidant enzymes using metals as cofactors.Conclusions: Degeneration of RPE cells in culture was characterized by depigmentation of cells, improper barrier function, downregulation of antioxidant protection, loss of epithelial phenotype, and changes on the levels of Na, P, Mg and Cu, as well as of the expression of their homeostatic proteins.

Modulation of Hypoxia-Inducible Factors and Vascular Endothelial Growth Factor Expressions by Superfood Camu-Camu (Myrciaria dubia) Treatment in ARPE-19 and Fetal Human RPE Cells.

Anti-vascular endothelial growth factor (anti-VEGF) therapy via intravitreal injection is an effective treatment for patients with abnormal ocular neovascularization, such as age-related macular degeneration (AMD) and diabetic macular edema (DME). However, prolonged and frequent anti-VEGF treatment is associated with a risk of local and systemic adverse events, including geographic atrophy, cerebrovascular disease, and death. Furthermore, some patients do not adequately respond to anti-VEGF therapy. Hypoxia-inducible factor (HIF) is a transcription factor that controls the expression of hypoxia-responsive genes involved in angiogenesis, inflammation, and metabolism. The HIF/VEGF pathway plays an important role in neovascularization, and the inhibition of HIF activation could be an effective biomolecular target for neovascular diseases. The demand for disease prevention or treatment using functional foods such as superfoods has increased in recent years. Few reports to date have focused on the antineovascular effects of superfoods in the retinal pigment epithelium (RPE). In light of the growing demand for functional foods, we aimed to find novel HIF inhibitors from superfoods worked in RPE cells, which could be an adjuvant for anti-VEGF therapy. Seven superfoods were examined to identify novel HIF inhibitor candidates using luciferase assay screening. We used the human RPE cell line ARPE-19 and fetal human RPE (fhRPE) to investigate the biomolecular actions of novel HIF inhibitors using quantitative PCR and western blotting. Under CoCl2-induced pseudohypoxic condition and 1% oxygen hypoxic incubation, camu-camu (Myrciaria dubia) showed HIF inhibitory effects determined by luciferase assays. Camu-camu downregulated HIF-1α and VEGFA mRNA expressions in a concentration-dependent manner. Camu-camu also inhibited HIF-1α protein expressions, and its inhibitory effect was greater than that of vitamin C, which is present at high levels in camu-camu. The camu-camu extract suppressed the activation of HIF and VEGF in RPE cells. This could assist anti-VEGF therapy in patients with abnormal ocular neovascularization.

Divergent Metabolomic Signatures of TGFβ2 and TNFα in the Induction of Retinal Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a dedifferentiation program in which polarized, differentiated epithelial cells lose their cell-cell adhesions and transform into matrix-producing mesenchymal cells. EMT of retinal pigment epithelial (RPE) cells plays a crucial role in many retinal diseases, including age-related macular degeneration, proliferative vitreoretinopathy, and diabetic retinopathy. This dynamic process requires complex metabolic reprogramming to accommodate the demands of this dramatic cellular transformation. Both transforming growth factor-beta 2 (TGFβ2) and tumor necrosis factor-alpha (TNFα) have the capacity to induce EMT in RPE cells; however, little is known about their impact on the RPE metabolome. Untargeted metabolomics using high-resolution mass spectrometry was performed to reveal the metabolomic signatures of cellular and secreted metabolites of primary human fetal RPE cells treated with either TGFβ2 or TNFα for 5 days. A total of 638 metabolites were detected in both samples; 188 were annotated as primary metabolites. Metabolomics profiling showed distinct metabolomic signatures associated with TGFβ2 and TNFα treatment. Enrichment pathway network analysis revealed alterations in the pentose phosphate pathway, galactose metabolism, nucleotide and pyrimidine metabolism, purine metabolism, and arginine and proline metabolism in TNFα-treated cells compared to untreated control cells, whereas TGFβ2 treatment induced perturbations in fatty acid biosynthesis metabolism, the linoleic acid pathway, and the Notch signaling pathway. These results provide a broad metabolic understanding of the bioenergetic rewiring processes governing TGFβ2- and TNFα-dependent induction of EMT. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences in promoting EMT of RPE will enable the identification of novel biomarkers for diagnosis, management, and tailored drug development for retinal fibrotic diseases.

Vitamin B3, nicotinamide, enhances mitochondrial metabolism to promote differentiation of the retinal pigment epithelium

In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception.

Key Role for CRB2 in the Maintenance of Apicobasal Polarity in Retinal Pigment Epithelial Cells.

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization in vitro, and in mature murine RPE cells in vivo. After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells. We found that CRB2 plays a key role in tight junction maintenance as well as in cell cycle arrest. In addition, our studies in vivo show that the knockdown of CRB2 in the RPE affects to the distribution of different apical polarity proteins and results in perturbed retinal homeostasis, manifested by the invasion of activated microglial cells into the subretinal space. Together our results demonstrate that CRB2 is a key protein for the development and maintenance of a polarized epithelium.

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells in vitro

ABSTRACT Purpose Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE). Methods ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After 7 days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin’s effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content. Results Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported. Conclusions This is the first study to demonstrate that supplementing the culture media with sericin promotes the viability of iPSC-RPE and ARPE-19. Sericin’s viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

An in vitro model of chronic wounding and its implication for age-related macular degeneration.

Degeneration of the retinal pigment epithelium (RPE) plays a central role in age-related macular degeneration (AMD). Throughout life, RPE cells are challenged by a variety of cytotoxic stressors, some of which are cumulative with age and may ultimately contribute to drusen and lipofuscin accumulation. Stressors such as these continually damage RPE cells resulting in a state of chronic wounding. Current cell-based platforms that model a state of chronic RPE cell wounding are limited, and the RPE cellular response is not entirely understood. Here, we used the electric cell-substrate impedance sensing (ECIS) system to induce a state of acute or chronic wounding on differentiated human fetal RPE cells to analyze changes in the wound repair response. RPE cells surrounding the lesioned area employ both cell migration and proliferation to repair wounds but fail to reestablish their original cell morphology or density after repetitive wounding. Chronically wounded RPE cells develop phenotypic AMD characteristics such as loss of cuboidal morphology, enlarged size, and multinucleation. Transcriptomic analysis suggests a systemic misregulation of RPE cell functions in bystander cells, which are not directly adjacent to the wound. Genes associated with the major RPE cell functions (LRAT, MITF, RDH11) significantly downregulate after wounding, in addition to differential expression of genes associated with the cell cycle (CDK1, CDC6, CDC20), inflammation (IL-18, CCL2), and apoptosis (FAS). Interestingly, repetitive wounding resulted in prolonged misregulation of genes, including FAS, LRAT, and PEDF. The use of ECIS to induce wounding resulted in an over-representation of AMD-associated genes among those dysregulated genes, particularly genes associated with advanced AMD. This simple system provides a new model for further investigation of RPE cell wound response in AMD pathogenesis.

Disease-causing mutations associated with bestrophinopathies promote apoptosis in retinal pigment epithelium cells.

Best vitelliform macular dystrophy (BVMD) and autosomal recessive bestrophinopathy (ARB) are two kinds of bestrophinopathies which are caused by BEST1 mutations and characterized by accumulation of lipofuscin-like materials on the retinal pigment epithelium cell-photoreceptor interface. In the past two decades, research about the pathogenesis of bestrophinopathies was mainly focused on the anion channel and intracellular Ca2+ signaling, but seldom concentrated on the function of retinal pigment epithelium (RPE) cells. In this study, we explored the possible effect of the three BEST1 mutations p.V143F, p.S142G, and p.A146T on the apoptosis in human fetal RPE cells. Wild-type plasmid and mutant plasmids BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T were transfected to human fetal RPE cells. The molecules caspase-3, phospho-Bcl-2, BAX, PARP, and AIF associated with apoptosis were determined by quantitative PCR and Western blot. Apoptotic rate and active Caspase-3 staining were analyzed by flow cytometry. Caspase-3 and PARP expression were significantly increased in BEST1-pcDNA3.1 p.S142G and p.A146T group. Flow cytometry showed that the apoptosis rates were significantly increased in the BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T group compared with the wild-type group. For the first time, we found that the three mutations promoted RPE cell apoptosis. Furthermore, the results indicated that BEST1 mutations p.S142G and p.A146T may contribute apoptosis of RPE cells by targeting Caspase 3. Our observations suggested that the apoptosis of RPE cells may be one of the mechanisms in bestrophinopathies, which may provide a new potential therapeutic target for the treatment of this disease.

Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.

Genetic analyses of human fetal retinal pigment epithelium gene expression suggest ocular disease mechanisms

The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis but has limited representation in large-scale functional genomics datasets. Understanding how common human genetic variants affect RPE gene expression could elucidate the sources of phenotypic variability in selected monogenic ocular diseases and pinpoint causal genes at genome-wide association study (GWAS) loci. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions and discovered hundreds of shared or condition-specific expression or splice quantitative trait loci (e/sQTLs). Co-localizations of fRPE e/sQTLs with age-related macular degeneration (AMD) and myopia GWAS data suggest new candidate genes, and mechanisms by which a common RDH5 allele contributes to both increased AMD risk and decreased myopia risk. Our study highlights the unique transcriptomic characteristics of fRPE and provides a resource to connect e/sQTLs in a critical ocular cell type to monogenic and complex eye disorders.

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration

BackgroundTo identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls.MethodsAfter uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups).ResultsIncreased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen.ConclusionsDifferential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Ascorbate Suppresses VEGF Expression in Retinal Pigment Epithelial Cells

PurposeTo investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells.MethodsDot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo−/− mice, which, like humans, cannot synthesize ascorbate de novo.ResultsTreatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome—3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo−/− mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor.ConclusionsAscorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD).

Changes in extracellular matrix cause RPE cells to make basal deposits and activate the alternative complement pathway.

The design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment epithelium (RPE) and Bruch's membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in an AMD, we generated abnormal extracellular matrix (ECM) using CRISPR-edited ARPE-19 cells. We introduced to these cells the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The abnormal ECM binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. Human fetal RPE (hfRPE) cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 activity. The local cleavage of C3 via convertase-independent mechanisms can be a new therapeutic target for early AMD.

The importance of the polarity proteins CRB in the differentiation process of the RPE cells

SummaryApicobasal polarity is essential for epithelial cell function, and it is determined by the expression of three polarity protein complexes: Scribble, Par and Crumbs. Both the Par and the Crumbs complex, promote apical membrane identity. Crumbs complex is composed by PALS1, PATJ and the Crumbs homologs (CRB) proteins. We have recently described the expression of one of the Crumbs proteins, CRB2, in the retinal pigment epithelial (RPE) cells. To better understand this process, we have studied the role of CRB2 in human RPE cells during differentiation in vitro and in the mouse retina in vivo. To achieve this goal, we analyzed the temporal sequence of the establishment of the polarity proteins during human fetal RPE cells polarization and knocking‐down CRB2, we analyzed the establishment of junctional complexes and features involved in the acquisition of polarization compared to the control RPE cultures. We demonstrate that, in differentiating RPE cells, CRB2 functions, as the sole CRB protein, in the formation of the junctional complex and apicobasal polarity. Additionally, the absence of the CRB2 protein from these cells results in a delay in the formation of cell‐cell junctions and in an increase in cell proliferation in RPE cells.

Phagocytosis-dependent ketogenesis in retinal pigment epithelium

Daily, the retinal pigment epithelium (RPE) ingests a bolus of lipid and protein in the form of phagocytized photoreceptor outer segments (OS). The RPE, like the liver, expresses enzymes required for fatty acid oxidation and ketogenesis. This suggests that these pathways play a role in the disposal of lipids from ingested OS, as well as providing a mechanism for recycling metabolic intermediates back to the outer retina. In this study, we examined whether OS phagocytosis was linked to ketogenesis. We found increased levels of β-hydroxybutyrate (β-HB) in the apical medium following ingestion of OS by human fetal RPE and ARPE19 cells cultured on Transwell inserts. No increase in ketogenesis was observed following ingestion of oxidized OS or latex beads. Our studies further defined the connection between OS phagocytosis and ketogenesis in wild-type mice and mice with defects in phagosome maturation using a mouse RPE explant model. In explant studies, the levels of β-HB released were temporally correlated with OS phagocytic burst after light onset. In the Mreg-/- mouse where phagosome maturation is delayed, there was a temporal shift in the release of β-HB. An even more pronounced shift in maximal β-HB production was observed in the Abca4-/- RPE, in which loss of the ATP-binding cassette A4 transporter results in defective phagosome processing and accumulation of lipid debris. These studies suggest that FAO and ketogenesis are key to supporting the metabolism of the RPE and preventing the accumulation of lipids that lead to oxidative stress and mitochondrial dysfunction.

Oxytocin is involved in cellular signaling within the retina

BackgroundOxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G‐protein coupled receptor. We have recently localized OXTR to the retinal pigment epithelium (RPE) and OXT to the adjacent cone photoreceptors.ObjectiveWe sought to characterize the signaling mechanism of OXTR in the RPE and explore the downstream effects of OXT, focusing on the regulation of inwardly rectifying K+ channel Kir7.1.MethodsCa2+ response to OXT was measured in cultured human fetal RPE cells (hfRPE) using Fura‐2 AM in the presence of 2‐APB and nifedipine, pharmacological inhibitors of Ca2+ signaling pathways. HEK‐293 cells where used for the stable expression of human OXTR. Cellular signaling was visualized using live cell fluorescence imaging following transient expression of PH‐GFP and PKC‐GFP, monitors of GPCR metabolites PIP2 and DAG. Whole cell patch clamp electrophysiology was performed on HEK‐OXTR cells transfected with GFP‐fused Kir7.1 as well as freshly isolated mouse RPE cells to study Kir7.1 channel function.ResultsOXT treatment of RPE cells in culture resulted in a transient increase in cytoplasmic Ca2+ that was reduced by 95% in the presence of the IP3R antagonist, 2‐APB (P < 0.0001). Upon bathing the cells in Ca2+‐free extracellular solution or nifedipine, the Ca2+ response to OXT was not inhibited, although amplitude was reduced. Time to the rise in [Ca2+]i was not altered but recovery was slowed down with time constants (τ) of 0.35 ± 0.02 (r2 =.99), 0.34 ± 0.03 (r2 =.98), and 0.20 ± 0.005 (r2=.99) min for Ca2+‐free, nifedipine, and Ringer's solution, respectively. We also demonstrate that OXTR activation blunted Kir7.1 channel current. In isolated mouse RPE, we observed an average 61.81 ± 4.77 % decrease in K+ inward current amplitude at ‐160mV and an average 11.4 ± 3.2 mV depolarization in resting membrane potential by OXT.ConclusionWe propose that OXTR stimulates a mobilization of intracellular Ca2+, independent of extracellular Ca2+, through 2nd messengers coupled to OXTR/G‐protein in the RPE. This OXT‐OXTR signaling in the RPE cell also integrated the parallel modulation of the Kir7.1 channel. Kir7.1 channel is responsible for controlling RPE resting membrane potential and can influence retinal health and vision. We suggest that novel OXT‐OXTR signaling pathways in the outer retina will be of fundamental importance for eye development, health and visual function.Support or Funding InformationNIH EY024995, UnityPoint Health Meriter Foundation, Pediatrics, Graduate School, School of Medicine and Public Health

Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

A novel three-dimensional trilayer for studying early changes in age-related macular degeneration

Event Abstract Back to Event A novel three-dimensional trilayer for studying early changes in age-related macular degeneration Ali Shokoohmand1, 2, June E. Jeon1, Christina Theodoropoulos1, Dietmar W. Hutmacher1, 2, 3, 4 and Beatrix Feigl1, 5 1 Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation, Australia 2 QUT, Australian Prostate Cancer Research Centre – Queensland, Australia 3 Technical University of Munich (TUM), Institute for Advanced Study, Germany 4 Georgia Institute of Technology, George W Woodruff School of Mechanical Engineering, United States 5 Queensland Eye Institute, Australia Introduction: The understanding of the pathomechanisms of age-related macular degeneration (AMD) remains a challenge. Typical features of the disease such as drusen and the role of growth factors have been extensively studied[1]-[3], but it remains unclear what causes drusen formation and a growth factor imbalance. Research is directed towards animal models[4] and more recently towards stem cell based therapies which are still in their infancy[5]. Monolayer culture is a widely used alternative to animal studies, but is limited by the relative lack of sophistication when compared to the human retina, since the pathogenesis of AMD involves complex interplay between the retinal pigment epithelium (RPE), Bruch’s membrane and the choroid. It is becoming apparent that 3D culture systems are better suited for complex disease models to allow the study of cellular crosstalk within the microenvironment with advanced co-culture system[6]. Therefore, this study aimed at developing a novel humanized trilayer culture model by culturing human fetal RPE and primate choroidal cells on electrospun medical-grade polycaprolactone (PCL)-gelatin meshes to study the initial changes in AMD. Materials and Methods: Electrospun PCL-gelatin meshes were made by a custom solution electrospinning apparatus as described previously[7] (Figure 1). The overall architecture of the scaffolds, including the porosity and the surface area-to-volume ratio, was analyzed using the nano-computed tomography (nano-CT) system using a resolution of 1 µm, a voltage of 45 kVp and a current of 177 mA (n=4). The porous nature of the scaffold was an important consideration in studying the effect of co-culture and the possible crosstalk between the RPEs and the choroidal cells. The trilayer model was prepared by seeding the primate choroid endothelial cell line (RF/6A) on the bottom surface of the PCL-gelatin scaffold coated with human recombinant laminin followed by seeding human RPE cells on the top surface of the scaffold[8] (ratio 1:1; 104 cells/cm2), mimicking the retina in vivo microenvironment. Results and Discussion: Scanning electron microscopy images confirmed that PCL-gelatin electrospun scaffolds allowed excellent cell attachment and infiltration (Figure 2). Functional and morphological analysis of the human RPE cells showed that they were able to phagocytose vitronectin-coated microbeads and that they formed microvilli (Figure 2B). The quantitative analysis of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), factors critical in pathophysiological condition of AMD, indicated significant increases in production of both VEGF and PEDF in RPE/choroidal co-cultures compared to mono-cultures. Our findings indicate the functionality of the current model and provide a strong argument into the advantages of using a 3D trilayer in vitro culture model to study early changes involving the shift in the PEDF/VEGF equilibrium that occurs in AMD. Conclusion: In summary, we provide the initial evidence that a three dimensional in vitro model can be fabricated to exhibit functional characteristics typical for human RPE and choroidal cells. We believe that this novel bioengineered 3D construct can provide a platform for further studies in the pathomechanisms of AMD. This study was funded by National Health and Medical Research Council (NHMRC).

Enhanced Ca2+ response and stimulation of prostaglandin release by the bradykinin B2 receptor in human retinal pigment epithelial cells primed with proinflammatory cytokines

Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca2+ transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1β enhanced Ca2+ response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca2+ response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema.