Inhibition of hypoxia-inducible factors suppresses subretinal fibrosis.

Age-related macular degeneration (AMD) is a common cause of vision loss. The aggressive form of AMD is associated with ocular neovascularization and subretinal fibrosis, representing a responsive outcome against neovascularization mediated by epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells. A failure of the current treatment (anti-vascular endothelial growth factor therapy) has also been attributed to the progression of subretinal fibrosis. Hypoxia-inducible factors (HIFs) increase gene expressions to promote fibrosis and neovascularization. HIFs act as a central pathway in the pathogenesis of AMD. HIF inhibitors may suppress ocular neovascularization. Nonetheless, further investigation is required to unravel the aspects of subretinal fibrosis. In this study, we used RPE-specific HIFs or von Hippel-Lindau (VHL, a regulator of HIFs) conditional knockout (cKO) mice, along with pharmacological HIF inhibitors, to demonstrate the suppression of subretinal fibrosis. Fibrosis was suppressed by treatments of HIF inhibitors, and similar suppressive effects were detected in RPE-specific Hif1a/Hif2a- and Hif1a-cKO mice. Promotive effects were observed in RPE-specific Vhl-cKO mice, where fibrosis-mediated pathologic processes were evident. Marine products' extracts and their component taurine suppressed fibrosis as HIF inhibitors. Our study shows critical roles of HIFs in the progression of fibrosis, linking them to the potential development of therapeutics for AMD.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.

The Total Macular Burden of Hyperreflective Foci and the Onset of Persistent Choroidal Hypertransmission Defects in Intermediate AMD.

PURPOSE: The association between the total macular burden of hyperreflective foci (HRF) in eyes with intermediate AMD (iAMD) and the onset of persistent choroidal hypertransmission defects (hyperTDs) was studied using swept-source optical coherence tomography (SS-OCT). DESIGN: Post hoc subgroup analysis of a prospective study. METHODS: A retrospective review of iAMD eyes from subjects enrolled in a prospective SS-OCT study was performed. All eyes underwent 6×6 mm SS-OCT angiography (SS-OCTA) imaging at baseline and follow-up visits. En face sub-retinal pigment epithelium (subRPE) slabs with segmentation boundaries positioned 64-400 µm beneath Bruch's membrane (BM) were used to identify persistent choroidal hyperTDs. None of the eyes had persistent hyperTDs at baseline. The same subRPE slab was used to identify choroidal hypotransmission defects (hypoTDs) attributable to HRF located either intraretinally (iHRF) or along the RPE (rpeHRF) based on corresponding B-scans. A semiautomated algorithm was used by two independent graders to validate and refine the HRF outlines. The HRF area and the drusen volume within a 5mm fovea-centered circle were measured at each visit. RESULTS: The median follow-up time for the 171 eyes from 121 patients included in this study was 59.1 months (95%CI: 52.0-67.8 months). Of these, 149 eyes (87%) had HRF, and 82 (48%) developed at least one persistent hyperTD during the follow-up. Although univariable Cox regression analyses showed that both drusen volume and total HRF area were associated with the onset of the first persistent hyperTD, multivariable analysis showed that the area of total HRF was the sole significant predictor for the onset of hyperTDs (P<0.001). ROC analysis identified an HRF area ≥ 0.07 mm² to predict the onset of persistent hyperTDs within one year with an area under the curve (AUC) of 0.661 (0.570-0.753), corresponding to a sensitivity of 55% and a specificity of 74% (P<0.001). CONCLUSIONS: The total macular burden of HRF, which includes both the HRF along the RPE and within the retina, is an important predictor of disease progression from iAMD to the onset of persistent hyperTDs and should serve as a key OCT biomarker to select iAMD patients at high-risk for disease progression in future clinical trials.

TGF-ß Isoforms and Local Environments Greatly Modulate Biological Nature of Human Retinal Pigment Epithelium Cells.

To characterize transforming growth factor-ß (TGF-ß) isoform (TGF-ß1~3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including collagen 1 (COL1), COL4, and COL6; α-smooth muscle actin (αSMA); hypoxia-inducible factor 1α (HIF1α); and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α), a master regulator for mitochondrial respiration;, tight junction-related molecules, Zonula occludens-1 (ZO1) and E-cadherin; and vascular endothelial growth factor (VEGF); (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-ß isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-ß1 and TGF-ß3 caused a marked increase in TEER values, and TGF-ß2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-ß isoforms, among which TGF-ß1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-ß2 and TGF-ß3 or by TGF-ß1 and TGF-ß3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-ß isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-ß-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-ß isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.

Role of inflammation in a rat model of radiation retinopathy.

Radiation retinopathy (RR) is a major side effect of ocular tumor treatment by plaque brachytherapy or proton beam therapy. RR manifests as delayed and progressive microvasculopathy, ischemia and macular edema, ultimately leading to vision loss, neovascular glaucoma, and, in extreme cases, secondary enucleation. Intravitreal anti-VEGF agents, steroids and laser photocoagulation have limited effects on RR. The role of retinal inflammation and its contribution to the microvascular damage occurring in RR remain incompletely understood. To explore cellular and vascular events after irradiation, we analyzed their time course at 1 week, 1 month and 6 months after rat eyes received 45 Gy X-beam photons. Müller glial cells, astrocytes and microglia were rapidly activated, and these markers of retinal inflammation persisted for 6 months after irradiation. This was accompanied by early cell death in the outer retina, which persisted at later time points, leading to retinal thinning. A delayed loss of small retinal capillaries and retinal hypoxia were observed after 6 months, indicating inner blood‒retinal barrier (BRB) alteration but without cell death in the inner retina. Moreover, activated microglial cells invaded the entire retina and surrounded retinal vessels, suggesting the role of inflammation in vascular alteration and in retinal cell death. Radiation also triggered early and persistent invasion of the retinal pigment epithelium by microglia and macrophages, contributing to outer BRB disruption. This study highlights the role of progressive and long-lasting inflammatory mechanisms in RR development and demonstrates the relevance of this rat model to investigate human pathology.

Alternative oxidase blunts pseudohypoxia and photoreceptor degeneration due to RPE mitochondrial dysfunction.

Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.

Lutein Modulates Cellular Functionalities and Regulates NLRP3 Inflammasome in a H2O2-Challenged Three-Dimensional Retinal Pigment Epithelium Model.

Excessive hydrogen peroxide (H2O2) generated during retinal cell metabolic activity could lead to oxidative degeneration of retinal pigment epithelium (RPE) tissue, a specific pathological process implicated in various retinal diseases resulting in blindness, which can be mitigated by taking dietary antioxidants to prevent inflammation and impaired cellular dysfunction. This study tested the hypothesis that damages induced by oxidative stresses can be mitigated by lutein in a H2O2-challenged model, which was based on an ARPE-19 cell monolayer cultured on three-dimensional (3D)-printed fibrous scaffolds. Pretreating these models with lutein (0.5 µM) for 24 h can significantly lower the oxidative stress and maintain phagocytosis and barrier function. Moreover, lutein can modulate the NLRP3 inflammasome, leading to a ∼40% decrease in the pro-inflammatory cytokine (IL-1ß and IL-18) levels. Collectively, this study suggests that the 3D RPE model is an effective tool to examine the capability of lutein to modulate cellular functionalities and regulate NLRP3 inflammation.

Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.

Potential role of CTSS in AMDImmune modulatory and anti-angiogenic effects of cathepsin S knockdown in ARPE-19 cells.

We aimed to determine the role of cathepsin S (CTSS) in modulating oxidative stress-induced immune and inflammatory reactions and angiogenesis in age-related macular degeneration. Human retinal pigment epithelium cells line ARPE-19 (immature) were maintained and treated with H2O2. The expression of CTSS, inflammatory cytokines, and complement factors induced by oxidative stress was compared between cells incubated without (control) and with CTSS knockdown (using small interfering ribonucleic acid; siRNA). To evaluate the role of CTSS in angiogenesis, we assayed tube formation using human umbilical vein endothelial cells and conditioned medium from ARPE-19 cells. We also used a mouse model of laser-induced choroidal neovascularization. CTSS levels were higher in ARPE-19 cells treated with H2O2 than in control cells. Oxidative stress-induced CTSS resulted in significantly elevated transcription of nuclear factor kappa B-dependent inflammatory cytokines, complement factors C3a and C5a, membrane attack complex (C5b-9), and C3a and C5a receptors. siRNA-mediated knockdown of CTSS reduced the number of inflammatory signals. Furthermore, oxidative stress-induced CTSS regulated the expression of peroxisome proliferator-activated receptor γ and vascular endothelial growth factor A/Akt serine/threonine kinase family signaling, which led to angiogenesis. Tube formation assays and mouse models of choroidal neovascularization revealed that CTSS knockdown ameliorated angiogenesis in vitro and in vivo. The present findings suggest that CTSS modulates the complement pathway, inflammatory reactions, and neovascularization, and that CTSS knockdown induces potent immunomodulatory effects. Hence, it could be a promising target for the prevention and treatment of early- and late-stage age-related macular degeneration.

RPE melanin and its influence on the progression of AMD.

OBJECTIVE: The aim of this review article is to summarize the latest findings and current understanding of the origin of melanin in the retinal pigment epithelium (RPE), its function within the RPE, its role in the pathogenesis of age-related macular degeneration (AMD), its effect on retinal development, and its potential therapeutic benefit in the treatment of AMD. METHODS: A comprehensive search of peer-reviewed journals was conducted using various combinations of key terms such as "melanin," "retinal pigment epithelium" or "RPE," "age-related macular degeneration" or AMD," "lipofuscin," "oxidative stress," and "albinism." Databases searched include PubMed, Scopus, Science Direct, and Google Scholar. 147 papers published between the years of 1957 and 2023 were considered with an emphasis on recent findings. SUMMARY OF FINDINGS: AMD is thought to result from chronic oxidative stress within the RPE that results in cellular dysfunction, metabolic dysregulation, inflammation, and lipofuscin accumulation. Melanin functions as a photoscreener, free radical scavenger, and metal cation binding reservoir within the RPE. RPE melanin does not regenerate, and it undergoes degradation over time in response to chronic light exposure and oxidative stress. RPE melanin is important for retinal development and RPE function, and in the aging eye, melanin loss is associated with increased lipid peroxidation, inflammation, and the accumulation of toxic oxidized cellular products. Therefore, melanin-based treatments may serve to preserve RPE and retinal function in AMD. CONCLUSIONS: The pathogenesis of AMD is not fully understood, but RPE dysfunction and melanin loss in response to chronic oxidative stress and inflammation are thought to be primary drivers of the disease. Due to melanin's antioxidative effects, melanin-based nanotechnology represents a promising avenue for the treatment of AMD.

Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres.

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway.

AIM: To explore the effect of epidermal growth factor receptor (EGFR) inhibition by erlotinib and EGFR siRNA on epidermal growth factor (EGF)-induced activation of retinal pigment epithelium (RPE) cells. METHODS: Human RPE cell line (ARPE-19 cells) was activated by 100 ng/mL EGF. Erlotinib and EGFR siRNA were used to intervene EGF treatment. Cellular viability, proliferation, and migration were detected by methyl thiazolyl tetrazolium (MTT) assay, bromodeoxyuridine (BrdU) staining assay and wound healing assay, respectively. EGFR/protein kinase B (AKT) pathway proteins and N-cadherin, α-smooth muscle actin (α-SMA), and vimentin were tested by Western blot assay. EGFR was also determined by immunofluorescence staining. RESULTS: EGF treatment for 24h induced a significant increase of ARPE-19 cells' viability, proliferation and migration, phosphorylation of EGFR/AKT proteins, and decreased total EGFR expression. Erlotinib suppressed ARPE-19 cells' viability, proliferation and migration through down regulating total EGFR and AKT protein expressions. Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin, α-SMA, and vimentin proteins. Similarly, EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation, viability, and migration, phosphorylation of EGFR/AKT proteins, and up-regulation of N-cadherin, α-SMA, and vimentin proteins. CONCLUSION: Erlotinib and EGFR-knockdown suppress EGF-induced cell viability, proliferation, and migration via EGFR/AKT pathway in RPE cells. EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy (PVR).

Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Impaired cathepsin D in retinal pigment epithelium cells mediates Stargardt disease pathogenesis.

Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4-/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4-/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.

Reassessing retinal pigment epithelial ketogenesis: Enzymatic assays for ketone body levels provide inaccurate results.

The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate (ß-HB). Prior work, based on detecting ß-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export ß-HB across the apical membrane. Here, we compare the accuracy of measuring ß-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of ß-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete ß-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming ß-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce ß-HB. Altogether, we substantiate ß-HB secretion in RPE but find that the secretion is equal apically and basally, RPE ß-HB can derive from ketogenic amino acids or fatty acids, and accurate ß-HB assessment requires mass spectrometric analysis.

PRDX1 exerts a photoprotection effect by inhibiting oxidative stress and regulating MAPK signaling on retinal pigment epithelium.

BACKGROUND: The purpose of this study was to investigate the photoprotection effect of peroxiredoxin 1 (PRDX1) protein in ultraviolet B (UVB) irradiation-induced damage of retinal pigment epithelium (RPE) and its possible molecular mechanism. METHODS: ARPE-19 cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the PRDX1 expression. The corresponding kits were employed to measure the levels or activities of lactate dehydrogenase (LDH), 8-hydroxy-2-deoxyguanosine (8-OHdG), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD). Western blotting was applied to examine PRDX1 expression and mitogen-activated protein kinase (MAPK) signaling pathway-related proteins. RESULTS: After exposure to 20 mJ/cm2 intensity of UVB irradiation for 24 h, ARPE-19 cells viability was decreased, the leakage degree of LDH and 8-OHdG were increased, and cell apoptosis was elevated. The expression of PRDX1 was significantly down-regulated in UVB-induced ARPE-19 cells. The low expression of PRDX1 was involved in high irradiation intensity. Overexpression of PRDX1 increased cell activity, decreased cell apoptosis, and LDH as well as 8-OHdG leakage in UVB-induced ARPE-19 cells. In addition to alleviating UVB-induced cell damage, PRDX1 overexpression also inhibited UVB-induced oxidative stress (down-regulation of ROS and MDA levels, up-regulation of GSH-Px and SOD activities) and the activation of MAPK signaling pathway in ARPE-19 cells. CONCLUSION: PRDX1 exerts a photoprotection effect on RPE by attenuating UVB-induced cell damage and inhibiting oxidative stress, which can be attributed to the inhibition of MAPK signaling pathway activation.

Multifocal Congenital Simple Hamartoma of the Retinal Pigment Epithelium: A Multimodal Imaging Case Study.

Congenital simple hamartoma of the retinal pigment epithelium (CSHRPE) is a rare benign tumor often detected incidentally during routine eye exams. We present a case of multifocal CSHRPE in a 32-year-old Hispanic woman, emphasizing the diagnostic challenges posed by its presentation and the pivotal role of multimodal imaging in accurate diagnosis. Despite initial difficulties due to a history of trauma and pigmented fundus, advanced imaging techniques, including optical coherence tomography (OCT), OCT angiography (OCTA), fluorescein angiography (FA), and indocyanine green angiography (ICGA), facilitated a precise diagnosis. Notably, OCTA revealed high signal intensity and flow at the largest nodule site while FA and ICGA exhibited characteristic blockage patterns. Moreover, smaller nodules exhibited OCT findings supporting the theory of islands of retinal pigment epithelium (RPE) cells proliferating ectopically within the retina. Our case underscores the importance of comprehensive imaging assessment in distinguishing CSHRPE from other lesions, contributing to a deeper understanding of this rare ocular condition.

Reduced Retinal Pigment Epithelial Autophagy Due to Loss of Rab12 Prenylation in a Human iPSC-RPE Model of Choroideremia.

Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in CHM, encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of CHM, the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited CHM-/- iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. CHM-/- iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in CHM-/- cells by transduction with gene replacement (ShH10-CMV-CHM) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.

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

BACKGROUND: Vision 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. RESULTS: To 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. CONCLUSIONS: Through 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.