Human Retinal Pigment Epithelium Research Articles

TNF-alpha promotes cilia elongation via mixed lineage kinases signaling in mouse fibroblasts and human RPE-1 cells.

The primary cilium is a characteristic feature of most non-immune cells and functions as an environmental signal transduction sensor. The defects in primary cilium have profound effects on the developmental program, including the maturation of retinal epithelium. The ciliary length is tightly regulated during ciliogenesis, but the impact of inflammation on ciliary length remains elusive. The current study investigates the outcome of inflammatory stimuli for the primary cilium length in retinal epithelium cells and mouse embryonic fibroblasts. Here, we report that exposure to the pro-inflammatory cytokine TNF-alpha elongates cilia in a mixed-lineage kinase (MLK)-dependent manner. Pro-inflammatory stimuli such as bacterial LPS and interferon-gamma have similar effects on ciliary length. In contrast, febrile condition-mimicking heat stress dramatically reduced the number of ciliated cells regardless of TNF-alpha exposure but did not shorten TNF-induced elongation, suggesting distinct but rapid effects of inflammatory stresses on ciliogenesis.

VEGF-R2/CAV-1 Interaction Induced by Resveratrol/Eicosapentaenoic Acid/Docosahexaenoic Acid-Enriched Formulation through Functional Detergent-Resistant Membranes Is Associated with Decreased VEGF-A Release in ARPE-19 Cells.

Omega-3 fatty acids (O3FAs) and resveratrol (RSV) are known to be beneficial for certain eye diseases, such as age-related macular degeneration (AMD). Neovascular AMD is characterized by abnormal blood vessel formation due to the excessive synthesis of vascular endothelial growth factor (VEGF) by retinal pigment epithelium (RPE) cells. The study investigates whether a formulation based on eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and RSV is capable of counteracting VEGF-A secretion, and elucidates the molecular mechanism. The study finds, using ELISA, that O3FAs/RSV reduces VEGF-A secretion in human RPE cells. This phenomenon is related to the increased interaction between VEGF-receptor 2 (VEGF-R2) and caveolin-1 (CAV-1), a protein of detergent-resistant membranes (DRMs), as determined by co-immunoprecipitation and proximity ligation assay. Using microscale thermophoresis, the study confirms that O3FAs/RSV causes a high-affinity interaction. Isolation and analysis of DRMs reveal that this interaction is concomitant with VEGF-R2 relocalization in DRMs. The depletion of DRMs by a cholesterol-chelating agent blocks the VEGF-R2/CAV-1 interaction and EPA/DHA/RSV-mediated impairment of VEGF production. This specific interaction can provide a new strategy for countering VEGF-A production in human RPE cells and, consequently, reducing neovascularization in AMD. Further preclinical studies involving O3FAs and polyphenols are warranted.

Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci

BackgroundVision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space.ResultsTo understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4.ConclusionsThrough comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.Graphical

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

Pigmentation level of human iPSC-derived RPE does not indicate a specific gene expression profile

Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

Tricetin suppresses the cell migration and BMP-6 expression through p38 signaling pathways in human retinal pigment epithelium cells.

Proliferative vitreoretinopathy (PVR) is a visual-threatening disease, which cause from the migration of retinal pigment epithelium (RPE). Tricetin, a family of flavonoids, can inhibit the metastasis of several cancers. Herein, we aim to evaluate the possible effect of tricetin on inhibiting ARPE-19 cells migration. The Boyden chamber assay, wound healing assay, RNA sequencing, and Western blot analysis were applied in our experiment. The results revealed that tricetin inhibited the cell migration abilities of ARPE-19 cells. Moreover, using RNA sequencing technology, we revealed that tricetin repressed bone morphogenetic protein-6 (BMP-6) gene expressions in ARPE-19 cells. Overexpression of BMP-6 resulted in significant restoration of cell migration capabilities of tricetin-treated ARPE-19 cells. Furthermore, tricetin suppressed the phosphorylation of the p38 signaling pathway. Moreover, blocking the p38 pathway also inhibits BMP-6 expression and migration in the ARPE-19 cells. In conclusion, this study revealed that tricetin inhibits the ARPE-19 cell migration mainly via the suppression of BMP-6 expression and p38 signaling pathway.

Age-related reduction in the functional properties of adult stem cells located in the peripheral region of human retinal pigment epithelium.

Adult stem cells (SCs) with self-renewal and multilineage potential have been reported upon culturing human retinal pigment epithelial (RPE) cells. The current study aimed to identify the location of SCs in human RPE and to elucidate the age-related changes. Peripheral, equatorial, and central RPE cells from donors of three age groups were analyzed for their sphere-forming, clonal, and label-retaining cell properties. Furthermore, native human RPE flatmounts were immunostained for SC and proliferating cell markers. Cells with higher sphere-forming and clonal ability were identified only in young donors (<30 years) and were restricted to the periphery. Upon culturing, cells from peripheral and equatorial regions had the label-retaining cell (LRC) property. With aging, the LRCs were restricted to the periphery and were reduced. In young donors, Ki67+ proliferating cells were not observed in native RPE. However, such cells were observed in the peripheral RPE of older donors correlating with the need for regeneration. The native RPE cells were negative for SC marker expression. The above findings highlighted the presence of SCs with the ability to proliferate in the peripheral RPE and a reduction in these functional properties of SCs with aging.

CD200R promotes high glucose-induced oxidative stress and damage in human retinal pigment epithelial cells by activating the mTOR signaling pathway

Diabetic retinopathy (DR) is established as the primary cause of visual impairment and preventable blindness, posing significant social and economic burdens on healthcare systems worldwide. Oxidative stress has been identified as a major contributor to DR, yet the precise role of the transmembrane glycoprotein CD200R in this context remains elusive. We studied human retinal pigment epithelia ARPE-19 cells to investigate the role of CD200R in high-glucose (HG) induced oxidative stress. Under HG conditions, we found a significant increase in CD200R expression in a time-dependent pattern. Conversely, knockdown of CD200R effectively alleviated oxidative stress and restored cell viability in HG-treated ARPE-19 cells, a phenomenon corroborated by the addition of a reactive oxygen species (ROS) scavenger. Exploration of the AKT/mTOR signaling pathway confirmed its mediating role regarding CD200R knockdown suppression of the expression of key proteins induced by HG conditions. Additionally, we found that the inhibition of mTOR signaling with Rapamycin effectively countered HG-induced oxidative stress in ARPE-19 cells, suggesting a promising therapeutic target against oxidative stress in the context of DR. This study establishes the crucial role of CD200R in HG-induced oxidative stress and identifies potential therapeutic avenues for the treatment of DR.

Metabolic reprogramming of the retinal pigment epithelium by cytokines associated with age-related macular degeneration.

The complex metabolic relationship between the retinal pigment epithelium (RPE) and photoreceptors is essential for maintaining retinal health. Recent evidence indicates the RPE acts as an adjacent lactate sink, suppressing glycolysis in the epithelium in order to maximize glycolysis in the photoreceptors. Dysregulated metabolism within the RPE has been implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of vision loss. In the present study, we investigate the effects of four cytokines associated with AMD, TNFα, TGF-β2, IL-6, and IL-1β, as well as a cocktail containing all four cytokines, on RPE metabolism using ARPE-19 cells, primary human RPE cells, and ex vivo rat eyecups. Strikingly, we found cytokine-specific changes in numerous metabolic markers including lactate production, glucose consumption, extracellular acidification rate, and oxygen consumption rate accompanied by increases in total mitochondrial volume and ATP production. Together, all four cytokines could potently override the constitutive suppression of glycolysis in the RPE, through a mechanism independent of PI3K/AKT, MEK/ERK, or NF-κB. Finally, we observed changes in glycolytic gene expression with cytokine treatment, including in lactate dehydrogenase subunit and glucose transporter expression. Our findings provide new insights into the metabolic changes in the RPE under inflammatory conditions and highlight potential therapeutic targets for AMD.

Artemisinin Confers Cytoprotection toward Hydrogen Peroxide-Induced Cell Apoptosis in Retinal Pigment Epithelial Cells in Correlation with the Increased Acetylation of Histone H4 at Lysine 8.

Increased oxidative stress is one of the critical pathologies inducing age-related macular degeneration (AMD), characterized by retinal pigment epithelial (RPE) cell damage and death. The unbalanced acetylation and deacetylation of histones have been implicated in AMD pathogenesis or hydrogen peroxide (H2O2)-induced cell damage. Therefore, strategies aimed at controlling the balance between acetylation and deacetylation may effectively protect RPE cells from oxidative damage. Artemisinin is an antimalarial lactone drug derived from Artemisia annua, with antioxidant activity known to modulate histone acetylation in the brain, but its effect on the retina is unknown. In this study, we aimed to investigate whether Artemisinin exerts a cytoprotective effect on oxidative stress-induced apoptosis in RPE cells by regulating histone acetylation. We hypothesized that Artemisinin confers cytoprotection toward H2O2-induced apoptosis in RPE cells through this mechanism. In the present study, we found that Artemisinin at a sub-clinic dosage of 20 μM inhibited the H2O2-induced cell viability decrease and B-cell lymphoma 2 (Bcl-2) protein level decrease and attenuated the H2O2-induced decrease in the histone H4 lysine (Lys) 8 acetylation [Acetyl-H4 (Lys 8)] level in the retinal RPE cell line D407. As expected, histone deacetylase inhibitor Trichostatin A at the concentration of 250 nM increased the Acetyl-H4 (Lys 8) level in D407 cells and attenuated the H2O2-induced cell viability decrease and apoptosis. Similar findings were obtained using adult RPE (ARPE)19 cells, another human RPE cell line, and primary human RPE cell cultures. In conclusion, these results confirmed our hypothesis and indicated that Artemisinin attenuated H2O2-induced apoptosis in apparent correlation with the increase in the Acetyl-H4 (Lys 8) level, which is associated with gene transcription and cell survival. By modulating histone acetylation, Artemisinin may restore the balance between acetylation and deacetylation and enhance the resistance and survival of RPE cells under oxidative stress. Our study provides novel mechanistic insights into the effect of Artemisinin on histone acetylation and apoptosis in RPE cells and supports the potential application of Artemisinin in the prevention and/or treatment of AMD.

Transcriptional patterns of human retinal pigment epithelial cells under protracted high glucose.

The retinal pigment epithelium (RPE) is essential for retinal homeostasis. Comprehensively exploring the transcriptional patterns of diabetic human RPEpromotes the understanding of diabetic retinopathy (DR). A total of 4125 differentially expressed genes (DEGs) were screened out from the human primary RPE cells subjected to prolonged high glucose (HG). The subsequent bioinformatics analysis is divided into 3 steps. In Step 1, 21 genes were revealed by intersecting the enriched genes from the KEGG, WIKI, and Reactome databases. In Step 2, WGCNA was applied and intersected with the DEGs. Further intersection based on the enrichments with the GO biological processes, GO cellular components, and GO molecular functions databases screened out 12 candidate genes. In Step 3, 13 genes were found to be simultaneously up-regulated in the DEGs and a GEO dataset involving human diabetic retinal tissues. VEGFA and ERN1 were the 2 starred genes finally screened out by overlapping the 3 Steps. In this study, multiple genes were identified as crucial in the pathological process of RPE under protracted HG, providing potentialcandidates for future researches on DR. The current study highlights the importance of RPE in DR pathogenesis.

Both hemoglobin and hemin cause damage to retinal pigment epithelium through the iron ion accumulation

Subretinal hemorrhages result in poor vision and visual field defects. During hemorrhage, several potentially toxic substances are released from iron-based hemoglobin and hemin, inducing cellular damage, the detailed mechanisms of which remain unknown. We examined the effects of excess intracellular iron on retinal pigment epithelial (RPE) cells. A Fe2+ probe, SiRhoNox-1 was used to investigate Fe2+ accumulation after treatment with hemoglobin or hemin in the human RPE cell line ARPE-19. We also evaluated the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, the protective effect of-an iron chelator, 2,2′-bipyridyl (BP), and ferrostatin-1 (Fer-1) on the cell damage, was evaluated. Fe2+ accumulation increased in the hemoglobin- or hemin-treated groups, as well as intracellular ROS production and lipid peroxidation. In contrast, BP treatment suppressed RPE cell death, ROS production, and lipid peroxidation. Pretreatment with Fer-1 ameliorated cell death in a concentration-dependent manner and suppressed ROS production and lipid peroxidation. Taken together, these findings indicate that hemoglobin and hemin, as well as subretinal hemorrhage, may induce RPE cell damage and visual dysfunction via intracellular iron accumulation.

CFH Haploinsufficiency and Complement Alterations in Early-Onset Macular Degeneration.

Complement dysregulation is a key component in the pathogenesis of age-related macular degeneration (AMD) and related diseases such as early-onset macular drusen (EOMD). Although genetic variants of complement factor H (CFH) are associated with AMD risk, the impact of CFH and factor H-like protein 1 (FHL-1) expression on local complement activity in human retinal pigment epithelium (RPE) remains unclear. We identified a novel CFH variant in a family with EOMD and generated patient induced pluripotent stem cell (iPSC)-derived RPE cells. We assessed CFH and FHL-1 co-factor activity through C3b breakdown assays and measured complement activation by immunostaining for membrane attack complex (MAC) formation. Expression of CFH, FHL-1, local alternative pathway (AP) components, and regulators of complement activation (RCA) in EOMD RPE cells was determined by quantitative PCR, western blot, and immunostaining. Isogenic EOMD (cEOMD) RPE was generated using CRISPR/Cas9 gene editing. The CFH variant (c.351-2A>G) resulted in loss of CFH and FHL-1 expression and significantly reduced CFH and FHL-1 protein expression (∼50%) in EOMD iPSC RPE cells. These cells exhibited increased MAC deposition upon exposure to normal human serum. Under inflammatory or oxidative stress conditions, CFH and FHL-1 expression in EOMD RPE cells paralleled that of controls, whereas RCA expression, including MAC formation inhibitors, was elevated. CRISPR/Cas9 correction restored CFH/FHL-1 expression and mitigated alternative pathway complement activity in cEOMD RPE cells. Identification of a novel CFH variant in patients with EOMD resulting in reduced CFH and FHL-1 and increased local complement activity in EOMD iPSC RPE supports the involvement of CFH haploinsufficiency in EOMD pathogenesis.

Exploring the Impact of Saccharin on Neovascular Age-Related Macular Degeneration: A Comprehensive Study in Patients and Mice.

We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.

Influence of Substrate Stiffness on iPSC-Derived Retinal Pigmented Epithelial Cells.

Retinal degenerative diseases are a major cause of blindness involving the dysfunction of photoreceptors, retinal pigmented epithelium (RPE), or both. A promising treatment approach involves replacing these cells via surgical transplantation, and previous work has shown that cell delivery scaffolds are vital to ensure sufficient cell survival. Thus, identifying scaffold properties that are conducive to cell viability and maturation (such as suitable material and mechanical properties) is critical to ensuring a successful treatment approach. In this study, we investigated the effect of scaffold stiffness on human RPE attachment, survival, and differentiation, comparing immortalized (ARPE-19) and stem cell-derived RPE (iRPE) cells. Polydimethylsiloxane was used as a model polymer substrate, and varying stiffness (~12 to 800 kPa) was achieved by modulating the cross-link-to-base ratio. Post-attachment changes in gene and protein expression were assessed using qPCR and immunocytochemistry. We found that while ARPE-19 and iRPE exhibited significant differences in morphology and expression of RPE markers, substrate stiffness did not have a substantial impact on cell growth or maturation for either cell type. These results highlight the differences in expression between immortalized and iPSC-derived RPE cells, and also suggest that stiffnesses in this range (~12-800 kPa) may not result in significant differences in RPE growth and maturation, an important consideration in scaffold design.

Microfluidic Fabricated Liposomes for Nutlin-3a Ocular Delivery as Potential Candidate for Proliferative Vitreoretinal Diseases Treatment.

Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration. Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis. The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models. Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.

Anti-inflammatory potential of simvastatin and amfenac in ARPE-19 cells; insights in preventing re-detachment and proliferative vitreoretinopathy after rhegmatogenous retinal detachment surgery

PurposeRhegmatogenous retinal detachment is a severe vision-threatening complication that can result into proliferative vitreoretinopathy (PVR) and re-detachment of the retina if recovery from surgery fails. Inflammation and changes in retinal pigment epithelial (RPE) cells are important contributors to the disease. Here, we studied the effects of simvastatin and amfenac on ARPE-19 cells under inflammatory conditions.MethodsARPE-19 cells were pre-treated with simvastatin and/or amfenac for 24 h after which interleukin (IL)-1α or IL-1β was added for another 24 h. After treatments, lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) processing, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity, prostaglandin E2 (PGE2) level, and extracellular levels of IL-6, IL-8, monocytic chemoattractant protein (MCP-1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor, as well as the production of reactive oxygen species (ROS) were determined.ResultsPre-treatment of human ARPE-19 cells with simvastatin reduced the production of IL-6, IL-8, and MCP-1 cytokines, PGE2 levels, as well as NF-κB activity upon inflammation, whereas amfenac reduced IL-8 and MCP-1 release but increased ROS production. Together, simvastatin and amfenac reduced the release of IL-6, IL-8, and MCP-1 cytokines as well as NF-κB activity but increased the VEGF release upon inflammation in ARPE-19 cells.ConclusionOur present study supports the anti-inflammatory capacity of simvastatin as pre-treatment against inflammation in human RPE cells, and the addition of amfenac complements the effect. The early modulation of local conditions in the retina can prevent inflammation induced PVR formation and subsequent retinal re-detachment.

The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment

The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

In vivo evaluation of a Nano-enabled therapeutic vitreous substitute for the precise delivery of triamcinolone to the posterior segment of the eye.

This study focused on the design of a thermoresponsive, nano-enabled vitreous substitute for the treatment of retinal diseases. Synthesis of a hydrogel composed of hyaluronic acid and a poloxamer blend was undertaken. Poly(D,L-lactide-co-glycolide) acid nanoparticles encapsulating triamcinolone acetonide (TA) were synthesised with a spherical morphology and mean diameter of ~ 153nm. Hydrogel fabrication and nanoparticle loading within the hydrogel was confirmed via physicochemical analysis. Gelation studies indicated that hydrogels formed in nine minutes and 10min for the unloaded and nanoparticle-loaded hydrogels, respectively. The hydrogels displayed in situ gel formation properties, and rheometric viscoelastic studies indicated the unloaded and loaded hydrogels to have modulus values similar to those of the natural vitreous at 37°C. Administration of the hydrogels was possible via 26G needles allowing for clinical application and drug release of triamcinolone acetonide from the nanoparticle-loaded hydrogel, which provided sustained in vitro drug release over nine weeks. The hydrogels displayed minimal swelling, reaching equilibrium swelling within 12h for the unloaded hydrogel, and eight hours for the nanoparticle-loaded hydrogel. Biodegradation in simulated vitreous humour with lysozyme showed < 20% degradation within nine weeks. Biocompatibility of both unloaded and loaded hydrogels was shown with mouse fibroblast and human retinal pigment epithelium cell lines. Lastly, a pilot in vivo study in a New Zealand White rabbit model displayed minimal toxicity with precise, localised drug release behaviour, and ocular TA levels maintained within the therapeutic window for the 28-day investigation period, which supports the potential applicability of the unloaded and nanoparticle-loaded hydrogels as vitreous substitutes that function as drug delivery systems following vitrectomy surgery.

Phototoxicity of low doses of light and influence of the spectral composition on human RPE cells

Light is known to induce retinal damage affecting photoreceptors and retinal pigment epithelium. For polychromatic light, the blue part of the spectrum is thought to be the only responsible for photochemical damage, leading to the establishment of a phototoxicity threshold for blue light (445 nm). For humans it corresponds to a retinal dose of 22 J/cm2. Recent studies on rodents and non-human primates suggested that this value is overestimated. In this study, we aim at investigating the relevance of the current phototoxicity threshold and at providing new hints on the role of the different components of the white light spectrum on phototoxicity. We use an in vitro model of human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelial (iRPE) cells and exposed them to white, blue and red lights from LED devices at doses below 22 J/cm2. We show that exposure to white light at a dose of 3.6 J/cm2 induces an alteration of the global cellular structure, DNA damage and an activation of cellular stress pathways. The exposure to blue light triggers DNA damage and the activation of autophagy, while exposure to red light modulates the inflammatory response and inhibits autophagy.