Retinal Pigment Epithelium Dysfunction Research Articles

Selective Ablation of Dehydrodolichyl Diphosphate Synthase in Murine Retinal Pigment Epithelium (RPE) Causes RPE Atrophy and Retinal Degeneration.

Patients with certain defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene (RP59; OMIM #613861) exhibit classic symptoms of retinitis pigmentosa, as well as macular changes, suggestive of retinal pigment epithelium (RPE) involvement. The DHDDS enzyme is ubiquitously required for several pathways of protein glycosylation. We wish to understand the basis for selective ocular pathology associated with certain DHDDS mutations and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. To circumvent embryonic lethality associated with Dhdds knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). We used targeted Cre expression to study the importance of the enzyme in the RPE. Structural alterations of the RPE and retina including reduction in outer retinal thickness, cell layer disruption, and increased RPE hyper-reflectivity were apparent at one postnatal month. At three months, RPE and photoreceptor disruption was observed non-uniformly across the retina as well as RPE transmigration into the photoreceptor layer, external limiting membrane descent towards the RPE, and patchy loss of photoreceptors. Functional loss measured by electroretinography was consistent with structural loss showing scotopic a- and b-wave reductions of 83% and 77%, respectively, at three months. These results indicate that RPE dysfunction contributes to DHDDS mutation-mediated pathology and suggests a more complicated disease mechanism than simply disruption of glycosylation.

LINC00167 Regulates RPE Differentiation by Targeting the miR-203a-3p/SOCS3 Axis

Increasing evidence has indicated that long non-coding RNAs (lncRNAs) play significant roles in various diseases; however, their roles in age-related macular degeneration (AMD) remain unclear. Dedifferentiation and dysfunction of retinal pigment epithelium (RPE) cells have been shown to contribute to AMD etiology in several studies. Herein, we found that lncRNA LINC00167 was downregulated in RPE-choroid samples of AMD patients and dysfunctional RPE cells, and it was consistently upregulated along with RPE differentiation. In vitro study indicated that reduced endogenous LINC00167 expression resulted in RPE dedifferentiation, which was typified by attenuated expression of RPE markers, reduced vascular endothelial growth factor A secretion, accumulation of mitochondrial reactive oxygen species, and interrupted phagocytic ability. Mechanistically, LINC00167 functioned as a sponge for microRNA miR-203a-3p to restore the expression of the suppressor of cytokine signaling 3 (SOCS3), which further inhibited the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. Taken together, our study demonstrated that LINC00167 showed a protective role in AMD by maintaining RPE differentiation through the LINC00167/miR-203a-3p/SOCS3 axis and might be a potential therapeutic target for AMD.

Circular Noncoding RNA NR3C1 Acts as a miR-382-5p Sponge to Protect RPE Functions via Regulating PTEN/AKT/mTOR Signaling Pathway

Age-related macular degeneration (AMD) is a universal leading cause for irreversible blindness in the elderly population. Dedifferentiation of retinal pigment epithelium (RPE) cells initiates early pathological events in atrophic AMD. Herein, we aim to investigate effects of a circular RNA derived from the NR3C1 gene (circNR3C1) on regulating RPE function and AMD pathogenesis. circNR3C1 expression was consistently upregulated along with RPE differentiation and was downregulated in dysfunctional RPE and blood serum of AMD patients. Silencing of circNR3C1 reduced RPE characteristic transcripts and proteins, interrupted phagocytosis, accelerated intracellular reactive oxygen species (ROS) generation, and promoted RPE proliferation invitro. circN3C1 silencing also decreased expressions of RPE characteristic markers and disturbed the ultrastructure of RPE invivo, as shown by a thickened RPE with twisted basal infoldings and outer segments. Mechanistically, circNR3C1 acted as an endogenous microRNA-382-5p (miR-382-5p) sponge to sequester its activity, which increased phosphatase and tensin homolog on chromosome 10 (PTEN) expression and inhibited the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway. miR-382-5p overexpression and PTEN silencing mimicked effects of circNR3C1 silencing on RPE phenotypes invivo and invitro. In conclusion, circNR3C1 prevents AMD progression and protects RPE by directly sponging miR-382-5p to block its interaction with PTEN and subsequently blocks the AKT/mTOR pathway. Pharmacological circNR3C1 supplementations are promising therapeutic options for atrophic AMD.

Induced Pluripotent Stem Cell Modeling of Best Disease and Autosomal Recessive Bestrophinopathy.

PurposeTo understand the pathophysiology of Best disease (BD) and autosomal recessive bestrophinopathy (ARB) by establishing an in vitro model using human induced pluripotent stem cell (iPSC).Materials and MethodsHuman iPSC lines were generated from mononuclear cells in peripheral blood of one ARB patient, one autosomal dominant BD patient, and two normal controls. Immunocytochemistry and reverse transcriptase polymerase chain reaction in iPSC lines were conducted to demonstrate the pluripotent markers. After the differentiation of iPSC into functional retinal pigment epithelium (RPE), morphological characteristics of the RPE were evaluated using confocal microscopy and immunocytochemistry. The rates of fluid flow across iPSC-RPE monolayer were measured to compare apical to basal fluid transports by RPE. RNA sequencing was performed on iPSC-RPE to identify the differences in gene expression profiles, and specific gene sets were tested using Gene Set Enrichment Analysis.ResultsMorphological characteristics, gene expression, and epithelial integrity of ARB iPSC were comparable to those of BD patient or normal control. Fluid transport from apical to basal was significantly decreased in ARB iPSC-RPE compared with BD iPSC-RPE or control iPSC-RPE. Gene Set Enrichment Analysis confirmed that ARB iPSC-RPE exhibited significant enrichments of epithelial-mesenchymal transition gene set and TNF-α signaling via NF-κB gene set compared to control iPSC-RPE or BD iPSC-RPE.ConclusionA human iPSC model of ARB showed a functional deficiency rather than anatomical defects. ARB may be caused by RPE dysfunction following BEST1 mutation.

Repeat exposure to polyinosinic:polycytidylic acid induces TLR3 expression via JAK-STAT signaling and synergistically potentiates NFκB-RelA signaling in ARPE-19 cells

Dry age-related macular degeneration (AMD), accounting for approximately 90% of AMD cases, is characterized by photoreceptor death, retinal pigment epithelium (RPE) dysfunction and, ultimately, geographic atrophy – the localized death of RPE leading to loss of the center of the visual field. The pathological etiology of AMD is multifactorial, but innate immune signaling and inflammation are involved in early stages of the disease. Although numerous single-nucleotide polymorphisms in innate immune genes are associated with dry AMD, no single gene appears to cause dry AMD.Here, we hypothesized that activation of TLR3 potentiates expression of TLR3 itself and the NFκB-p65 (RelA) subunit as part of pro-inflammatory RPE signaling. Furthermore, we hypothesized that TLR3 activation can ‘prime’ cells to future RelA stimulation, leading to enhanced, persistent RelA expression and signaling following a second TLR3 activation. We used the human RPE-derived cell line ARPE-19 as a model system for RPE signaling and measured NFκB expression and activity in response to TLR3 stimulation with its ligand, polyinosinic:polycytidylic acid (pI:C).Activation of TLR3 with pI:C led to increased TLR3 and RelA expression that was sustained for at least 24 h. Cells exposed for a second time to pI:C after an initial pI:C exposure displayed elevated RelA expression and RelA nuclear translocation above the level generated by individual primary or secondary exposures alone. Such an elevated response could also not be generated by a single application of higher concentrations of the agonist pI:C. Additionally, we determined the mechanism for TLR3 mediated TLR3 and RelA expression by using inhibitors of canonical TLR3-TBK1-IKKε and JAK-STAT signaling pathways.These data suggest that initial exposure of ARPE-19 cells to pI:C upregulates TLR3 and RelA signaling, leading to potentiated and persistent RelA signaling potentially generated by a positive feedback loop that may cause exacerbated inflammation in AMD. Furthermore, inhibition of JAK-STAT signaling may be a possible therapeutic treatment to prevent induction of TLR3 expression subsequent to pI:C exposure. Our results identify possible therapeutic targets to reduce the TLR3 positive feedback loop and subsequent overproduction of pro-inflammatory cytokines in RPE cells.

Proteome and Secretome Dynamics of Human Retinal Pigment Epithelium in Response to Reactive Oxygen Species

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, and is characterized by slow retinal degeneration linked to chronic reactive oxygen species (ROS) in the retinal pigmented epithelium (RPE). The molecular mechanisms leading to RPE dysfunction in response to ROS are unclear. Here, human stem cell-derived RPE samples were stressed with ROS for 1 or 3 weeks, and both intracellular and secreted proteomes were quantified by mass spectrometry. ROS increased glycolytic proteins but decreased mitochondrial complex I subunits, as well as membrane proteins required for endocytosis. RPE secreted over 1,000 proteins, many of which changed significantly due to ROS. Notably, secreted APOE is decreased 4-fold, and urotensin-II, the strongest known vasoconstrictor, doubled. Furthermore, secreted TGF-beta is increased, and its cognate signaler BMP1 decreased in the secretome. Together, our results paint a detailed molecular picture of the retinal stress response in space and time.

Auranofin Mediates Mitochondrial Dysregulation and Inflammatory Cell Death in Human Retinal Pigment Epithelial Cells: Implications of Retinal Neurodegenerative Diseases.

PurposePhotoreceptor degeneration occurs in various retinal diseases including age-related macular degeneration (AMD), Retinitis pigmentosa (RP), and diabetic retinopathy (DR). However, molecular mechanisms are not fully understood yet. The retinal pigment epithelium (RPE) forms the outer blood retinal barrier (oBRB) and supplies glucose, oxygen and nutrients from the fenestrated choriocapillaris to photoreceptors for visual function. Therefore, RPE dysfunction leads to photoreceptor injury/death and progression of blinding eye diseases. This study aims to understand the role of the thioredoxin (Trx) and its reductase (TrxR) redox signaling in human RPE dysfunction and cell death mechanism(s) in an in vitro system.MethodsA human RPE cell line (APRE-19) was cultured in DMEM/F12 medium and treated with auranofin (AF – 4 μM, an inhibitor of TrxR) for 4 and 24 h. Mitochondrial and lysosomal function, cellular oxidative stress and NLRP3 inflammasome activity were measured using cell assays, Western blotting, and confocal microscopy. Antioxidants and anti-inflammatory compounds were tested for blocking AF effects on RPE damage. Cell death mechanisms (LDH release to culture media) were determined using necroptosis, ferroptosis and pyroptosis inhibitors. P < 0.05 was considered significant in statistical analysis.ResultsAuranofin causes mitochondrial dysfunction (Δψm↓ and ATP↓), oxidative stress (H2O2↑) and mitophagic flux to lysosomes. Furthermore, the lysosomal enzyme (cathepsin L) activity is reduced while that of pro-inflammatory caspase-1 (NLRP3 inflammasome) is enhanced in ARPE-19. These effects of AF on ARPE-19 are inhibited by antioxidant N-acetylcysteine (5 mM, NAC) and significantly by a combination of SS31 (mitochondrial antioxidant) and anti-inflammatory drugs (amlexanox and tranilast). AF also causes cell death as measured by cytosolic LDH release/leakage, which is not inhibited by either ferrostatin-1 or necrostatin-1 (ferroptosis and necroptosis inhibitors, respectively). Conversely, AF-induced LDH release is significantly reduced by MCC950 and Ac-YVAD-cmk (NLRP3 and Caspase-1 inhibitors, respectively), suggesting a pro-inflammatory cell death by pyroptosis.ConclusionThe Trx/TrxR redox system is critical for RPE function and viability. We previously showed that thioredoxin-interacting protein (TXNIP) is strongly induced in DR inhibiting the Trx/TrxR system and RPE dysfunction. Therefore, our results suggest that the TXNIP-Trx-TrxR redox pathway may participate in RPE dysfunction in DR and other retinal neurodegenerative diseases.

Oxidative stress-mediated TXNIP loss causes RPE dysfunction

The disruption of the retinal pigment epithelium (RPE), for example, through oxidative damage, is a common factor underlying age-related macular degeneration (AMD). Aberrant autophagy also contributes to AMD pathology, as autophagy maintains RPE homeostasis to ensure blood–retinal barrier (BRB) integrity and protect photoreceptors. Thioredoxin-interacting protein (TXNIP) promotes cellular oxidative stress by inhibiting thioredoxin reducing capacity and is in turn inversely regulated by reactive oxygen species levels; however, its role in oxidative stress-induced RPE cell dysfunction and the mechanistic link between TXNIP and autophagy are largely unknown. Here, we observed that TXNIP expression was rapidly downregulated in RPE cells under oxidative stress and that RPE cell proliferation was decreased. TXNIP knockdown demonstrated that the suppression of proliferation resulted from TXNIP depletion-induced autophagic flux, causing increased p53 activation via nuclear localization, which in turn enhanced AMPK phosphorylation and activation. Moreover, TXNIP downregulation further negatively impacted BRB integrity by disrupting RPE cell tight junctions and enhancing cell motility by phosphorylating, and thereby activating, Src kinase. Finally, we also revealed that TXNIP knockdown upregulated HIF-1α, leading to the enhanced secretion of VEGF from RPE cells and the stimulation of angiogenesis in cocultured human retinal microvascular endothelial cells. This suggests that the exposure of RPE cells to sustained oxidative stress may promote choroidal neovascularization, another AMD pathology. Together, these findings reveal three distinct mechanisms by which TXNIP downregulation disrupts RPE cell function and thereby exacerbates AMD pathogenesis. Accordingly, reinforcing or restoring BRB integrity by targeting TXNIP may serve as an effective therapeutic strategy for preventing or attenuating photoreceptor damage in AMD.

MicroRNA-24 protects retina from degeneration in rats by down-regulating chitinase-3-like protein 1

MicroRNAs (miRNAs) have been shown to play critical roles in the pathogenesis and progression of degenerative retinal diseases like age-related macular degeneration (AMD). In this study, we first demonstrated that miR-24 plays an important role in maintaining retinal structure and visual function of rats by targeting chitinase-3-like protein 1 (CHI3L1). In the retinal pigment epithelial (RPE) cells of Royal College of Surgeons (RCS) rats, an animal model of genetic retinal degeneration (RD), miR-24 was found lower and CHI3L1 level was higher in comparison with those in Sprague-Dawley (SD) rats. Other changes in the eyes of RCS rats include activated AKT/mTOR and ERK pathways and abnormal autophagy in the RPE cells. Such roles of miR-24 and CHI3L1 were further confirmed in RCS rats by subretinal injection of agomiR-24, which decreased CHI3L1 level and preserved retinal structure and function. Upstream, NF-κB was identified as the regulator of miR-24 in the RPE cells of these rats. On the other hand, in SD rats, intraocular treatment of antagomiR-24 induced pathological changes similar to those in RCS rats. The results revealed the protective roles for miR-24 to RPE cells and a mechanism for RD in RCS rats was proposed: extracellular stress stimuli first activate the NF-κB signaling pathway, which lowers miR-24 expression so that CHI3L1 increased. CHI3L1 sequentially results in aberrant autophagy and RPE dysfunction by activating AKT/mTOR and ERK pathways. Taken together, although the possibility, that the therapeutic effects in RCS rats are caused by other transcriptional changes regulated by miR-24, cannot be excluded, these findings indicate that miR-24 protects rat retina by targeting CHI3L1. Thus, miR-24 and CHI3L1 might be the targets for developing more effective therapy for degenerative retinal diseases like AMD.

Vitamin D Attenuates Oxidative Damage and Inflammation in Retinal Pigment Epithelial Cells

Age-related macular degeneration (AMD), the most common visual disorder in elderly people, is characterized by the formation of deposits beneath the retinal pigment epithelium (RPE) and by dysfunction of RPE and photoreceptor cells. The biologically active form of vitamin D, 1,25-(OH)2D3 (VITD), is categorized as a multifunctional steroid hormone that modulates many transcriptional processes of different genes and is involved in a broad range of cellular functions. Epidemiological and genetic association studies demonstrate that VITD may have a protective role in AMD, while single nucleotide polymorphisms in the vitamin D metabolism gene (CYP24A1) increase the risk of AMD. However, the functional mechanisms of VITD in AMD are not fully understood. In the current study, we investigated the impact of VITD on H2O2-induced oxidative stress and inflammation in human RPE cells. We demonstrate that exposure to H2O2 caused significantly reduced cell viability, increased production of reactive oxygen species (ROS), lowered expression of antioxidant enzymes and enhanced inflammation. VITD exposure notably counteracted the above H2O2-induced effects. Our data suggest that VITD protects the RPE from oxidative damage and elucidate molecular mechanisms of VITD deficiency in the development of AMD.

N-Acetyl-L-cysteine Protects Human Retinal Pigment Epithelial Cells from Oxidative Damage: Implications for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) involves the loss of retinal pigment epithelium (RPE) and photoreceptors and is one of the leading causes of blindness in the elderly. Oxidative damage to proteins, lipids, and DNA has been associated with RPE dysfunction and AMD. In this study, we evaluated oxidative stress in AMD and the efficacy of antioxidant, N-acetyl-L-cysteine (NAC), in protecting RPE from oxidative damage. To test this idea, primary cultures of RPE from human donors with AMD (n = 32) or without AMD (No AMD, n = 21) were examined for expression of NADPH oxidase (NOX) genes, a source of reactive oxygen species (ROS). Additionally, the cells were pretreated with NAC for 2 hours and then treated with either hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (t-BHP) to induce cellular oxidation. Twenty-four hours after treatment, ROS production, cell survival, the content of glutathione (GSH) and adenosine triphosphate (ATP), and cellular bioenergetics were measured. We found increased expression of p22phox, a NOX regulator, in AMD cells compared to No AMD cells (p = 0.02). In both AMD and No AMD cells, NAC pretreatment reduced t-BHP-induced ROS production and protected from H2O2-induced cell death and ATP depletion. In the absence of oxidation, NAC treatment improved mitochondrial function in both groups (p < 0.01). Conversely, the protective response exhibited by NAC was disease-dependent for some parameters. In the absence of oxidation, NAC significantly reduced ROS production (p < 0.001) and increased GSH content (p = 0.02) only in RPE from AMD donors. Additionally, NAC-mediated protection from H2O2-induced GSH depletion (p = 0.04) and mitochondrial dysfunction (p < 0.05) was more pronounced in AMD cells compared with No AMD cells. These results demonstrate the therapeutic benefit of NAC by mitigating oxidative damage in RPE. Additionally, the favorable outcomes observed for AMD RPE support NAC's relevance and the potential therapeutic value in treating AMD.

HMGB1 and Caveolin-1 related to RPE cell senescence in age-related macular degeneration.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is considered a major cause of RPE dysfunction and senescence in age-related macular degeneration (AMD), and N-retinylidene-N-retinylethanolamine (A2E) is the main fluorophore identified in lipofuscin from aged human eyes. Here, human-induced pluripotent stem cell (iPSC)-RPE was generated from healthy individuals to reveal proteomic changes associated with A2E-related RPE cell senescence. A novel RPE cell senescence-related protein, high-mobility group box 1 (HMGB1), was identified based on proteomic mass spectrometry measurements on iPSC-RPE with A2E treatment. Furthermore, HMGB1 upregulated Caveolin-1, which also was related RPE cell senescence. To investigate whether changes in HMGB1 and Caveolin-1 expression under A2E exposure contribute to RPE cell senescence, human ARPE-19 cells were stimulated with A2E; expression of HMGB1, Caveolin-1, tight junction proteins and senescent phenotypes were verified. HMGB1 inhibition alleviated A2E induced cell senescence. Migration of RPE cells was evaluated. Notably, A2E less than or equal to 10μM induced both HMGB1 and Caveolin-1 protein upregulation and HMGB1 translocation, while Caveolin-1 expression was downregulated when there was more than 10μM A2E. Our data indicate that A2E-induced upregulation of HMGB1、Caveolin-1 and HMGB1 release may relate to RPE cell senescence and play a role in the pathogenesis of AMD.

The transcription factor MITF in RPE function and dysfunction.

Dysfunction and loss of the retinal pigment epithelium (RPE) are hallmarks of retinal degenerative diseases in mammals. A critical transcription factor for RPE development and function is the microphthalmia-associated transcription factor MITF and its germline mutations are associated with clinically distinct disorders, including albinism, microphthalmia, retinal degeneration, and increased risk of developing melanoma. Many studies have revealed new insights into central roles of MITF in RPE cell physiology, including melanogenesis, regulation of trophic factor expression, cell proliferation, anti-oxidant functions, and the visual cycle. In this review, we discuss the complex functional roles of MITF in RPE development, homeostasis, and retinal degeneration and touch upon key questions and challenges in neuroprotective strategies for retinal degenerative disorders associated with deficiencies in MITF or its many target genes.

Proline mediates metabolic communication between retinal pigment epithelial cells and the retina

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells between the choroid and the retina. RPE dysfunction underlies many retinal degenerative diseases, including age-related macular degeneration, the leading cause of age-related blindness. To perform its various functions in nutrient transport, phagocytosis of the outer segment, and cytokine secretion, the RPE relies on an active energy metabolism. We previously reported that human RPE cells prefer proline as a nutrient and transport proline-derived metabolites to the apical, or retinal, side. In this study, we investigated how RPE utilizes proline in vivo and why proline is a preferred substrate. By using [13C]proline labeling both ex vivo and in vivo, we found that the retina rarely uses proline directly, whereas the RPE utilizes it at a high rate, exporting proline-derived mitochondrial intermediates for use by the retina. We observed that in primary human RPE cell culture, proline is the only amino acid whose uptake increases with cellular maturity. In human RPE, proline was sufficient to stimulate de novo serine synthesis, increase reductive carboxylation, and protect against oxidative damage. Blocking proline catabolism in RPE impaired glucose metabolism and GSH production. Notably, in an acute model of RPE-induced retinal degeneration, dietary proline improved visual function. In conclusion, proline is an important nutrient that supports RPE metabolism and the metabolic demand of the retina.

Quercetin protect cigarette smoke extracts induced inflammation and apoptosis in RPE cells

Background Age-related macular degeneration (AMD) is the leading cause of blindness in elderly population in the developed world. Dysfunction of retinal pigment epithelium (RPE) likely triggers early AMD stages. The effect of Quercetin on the early AMD in-vitro model remained unclear. Methods The effect of Quercetin in the cell viability was detected with CCK8 methods in control, CSE treated, and CSE with Quercetin treatment group. The apoptotic status in each group was detected with tunnel assay. The oxidative and inflammation biomarkers were detected by ELISA. The expression levels of Keap1/Nrf2/ARE in RPE cells were measured by western blot after pretreatment of Quercetin followed by CSE treatment. Results It was found that Quercetin could improve the cell viability and decrease cellular apoptotic rate in the CSE treated RPE group. The expressions of inflammatory and apoptotic biomarkers were significantly decreased in Quercetin treatment group. Furthermore, Quercetin exerts protective effects via activation Keap1/Nrf2/ARE pathway in CSE treated RPE cells. Conclusions Quercetin demonstrated significant protective effects in an in-vitro model of early AMD and it might be a new therapeutic strategy for the management of early AMD.

Loss of PGC-1α in RPE induces mesenchymal transition and promotes retinal degeneration.

The retinal pigment epithelium (RPE) supports visual processing and photoreceptor homeostasis via energetically demanding cellular functions. Here, we describe the consequences of repressing peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α), a master regulator of mitochondrial function and biogenesis, on RPE epithelial integrity. The sustained silencing of PGC-1α in differentiating human RPE cells affected mitochondria/autophagy function, redox state, and impaired energy sensor activity ultimately inducing epithelial to mesenchymal transition (EMT). Adult conditional knockout of PGC-1 coactivators in mice resulted in rapid RPE dysfunction and transdifferentiation associated with severe photoreceptor degeneration. RPE anomalies were characteristic of autophagic defect and mesenchymal transition comparable with the ones observed in age-related macular degeneration. These findings demonstrate that PGC-1α is required to maintain the functional and phenotypic status of RPE by supporting the cells' oxidative metabolism and autophagy-mediated repression of EMT.

Mitochondrial oxidative stress in the retinal pigment epithelium (RPE) led to metabolic dysfunction in both the RPE and retinal photoreceptors

Age-related macular degeneration (AMD) is the leading cause of vision loss in the western world. Recent evidence suggests that RPE and photoreceptors have an interconnected metabolism and that mitochondrial damage in RPE is a trigger for degeneration in both RPE and photoreceptors in AMD. To test this hypothesis, this study was designed to induce mitochondrial damage in RPE in mice to determine whether this is sufficient to cause RPE and photoreceptor damage characteristic of AMD. In this study, we conditionally deleted the gene encoding the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD encoded by Sod2) in the retinal pigment epithelium (RPE) of albino BALB/cJ mice. VMD2-Cre;Sod2flox/flox BALB/cJ mice were housed in either 12-h dark, 12-h 200 lux white lighting (normal light), or 12-h dark, 12-h <10 lux red lighting (dim light). Electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT) were performed to assess retinal function and morphology. Immunofluorescence was used to examine protein expression; quantitative RT-PCR was used to measure gene expression. Sod2 knockout (KO) mice had reduced RPE function with age and increased oxidative stress compared to wild type (WT) controls as expected by the cell-specific deletion of Sod2. This was associated with alterations in RPE morphology and the structure and function of RPE mitochondria. In addition, data show a compensatory increase in RPE glycolytic metabolism. The metabolic shift in RPE correlated with severe disruption of photoreceptor mitochondria including a reduction in TOMM20 expression, mitochondrial fragmentation, and reduced COXIII/β-actin levels. These findings demonstrate that mitochondrial oxidative stress can lead to RPE dysfunction and metabolic reprogramming of RPE. Secondary to these changes, photoreceptors also undergo metabolic stress with increased mitochondrial damage. These data are consistent with the hypothesis of a linked metabolism between RPE and photoreceptors and suggest a mechanism of retinal degeneration in dry AMD.

Intravitreal Ets1 siRNA alleviates choroidal neovascularization in a mouse model of age-related macular degeneration.

Choroidal neovascularization (CNV) is the basic feature of neovascular age-related macular degeneration (AMD), the leading cause of blindness in elders. Macrophages and microglia promote CNV via producing pro-angiogenic factors and inflammatory cytokines. Transcription factor E26 transformation specific-1 (Ets1) plays a pro-angiogenic role via its pro-inflammatory function. In this study, Ets1 increased and localized in the macrophages and microglia of a mouse laser-induced CNV region. Ets1 siRNA intravitreal injection ameliorated the leakage and area of CNV, as well as inhibiting the dysfunction of retinal pigment epithelium (RPE) cells and the activation of macrophages/microglia. Taken together, we provide a new insight into the molecular mechanism of CNV progression, in which Ets1 can be a new therapeutic target.

Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation.

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch’s membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.

Clinical progress of cell therapy in treating age-related macular degeneration

Age-related macular degeneration is a retinal degenerative lesion caused by cell atrophy and dysfunction of retinal pigment epithelium. The degeneration of retinal pigment epithelium cells further leads to the apoptosis of photoreceptor cells, causes vision deterioration and eventually even blindness. Age-related macular degeneration is one of the top three retinal diseases causing blindness in the world. In recent years, exogenous retinal pigment epithelium cell therapy has emerged as a breakthrough treatment for age-related macular degeneration disease. In this review, we will briefly introduce the policies and regulations of cell therapy and discuss the clinical progress in the treatment of age-related macular degeneration using retinal pigment epithelium cells.