Retinal Pigment Epithelium Dysfunction Research Articles

Persistent Serous Choroidal and Retinal Detachment After Ab Interno Trabeculotomy for Glaucoma.

We describe a case of serous retinal detachment (SRD) with ciliochoroidal detachment (CCD) that persisted for 2 years and 7 months after minimally invasive glaucoma surgery (MIGS). A 71-year-old woman with primary open-angle glaucoma and cataracts had a central corneal thickness of 489 μm/492 μm and an ocular axis length of 24.05 mm/24.30 mm. She underwent phacoemulsification and intraocular lens implantation in the right eye (OD), along with goniosynechialysis and microhook ab interno trabeculotomy. Postoperative intraocular pressure was 4-6 mmHg in the OD. Five months later, SRD was observed temporally and inferiorly to the macula, with increased choroidal thickness. Best-corrected visual acuity at 5 months was (1.2)/(1.2) (right eye [OD]/left eye [OS]), and intraocular pressure was 6 mmHg/13 mmHg. CCD in the OD was accompanied by choroidal vessel dilation and choroidal vascular hyperpermeability. Two years and 7 months post-surgery, intraocular pressure spiked to 50-54 mmHg but settled at 12 mmHg 1 week later. CCD resolved, and choroidal folds and SRD disappeared, with decreased choroid thickness. Two years and 10 months postoperatively, there was no SRD recurrence at 10 mmHg on two antiglaucoma eye drops, and best-corrected visual acuity remained stable at (1.0)/(1.0). This case suggests that SRD may result from increased choroidal vessel permeability and retinal pigment epithelium dysfunction secondary to prolonged CCD/low IOP after MIGS. The prolonged disease course may be attributed to the balance between aqueous humor excretion and absorption, influenced by the limited size of the cyclodialysis cleft caused by MIGS.

Aberrant Lipid Metabolism and Complement Activation in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) stands as a leading cause of severe visual impairment and blindness among the elderly globally. As a multifactorial disease, AMD's pathogenesis is influenced by genetic, environmental, and age-related factors, with lipid metabolism abnormalities and complement system dysregulation playing critical roles. This review delves into recent advancements in understanding the intricate interaction between these two crucial pathways, highlighting their contribution to the disease's progression through chronic inflammation, drusen formation, and retinal pigment epithelium dysfunction. Importantly, emerging evidence points to dysregulated lipid profiles, particularly alterations in high-density lipoprotein levels, oxidized lipid deposits, and intracellular lipofuscin accumulation, as exacerbating factors that enhance complement activation and subsequently amplify tissue damage in AMD. Furthermore, genetic studies have revealed significant associations between AMD and specific genes involved in lipid transport and complement regulation, shedding light on disease susceptibility and underlying mechanisms. The review further explores the clinical implications of these findings, advocating for a novel therapeutic approach that integrates lipid metabolism modulators with complement inhibitors. By concurrently targeting these pathways, the dual-targeted approach holds promise in significantly improving outcomes for AMD patients, heralding a new horizon in AMD management and treatment.

Clustered ARPE-19 cells distinct in mitochondrial membrane potential may play a pivotal role in cell differentiation

Age-related macular degeneration (AMD) is associated with the dysfunction and degeneration of retinal pigment epithelium (RPE) cells. Here, we examined how the formation and expansions of cell clusters are regulated by the differentiation of the RPE cells. In this study, ARPE-19 cells were cultivated in standard or differentiation media, i.e., without or with nicotinamide, to evaluate the spreading of cell clusters specified with differentiated cell phenotypes. Mitochondria membrane potential (MMP) and the distribution of the RPE cell clusters was also monitored with or without rotenone, a mitochondrial electron transport chain (ETC) complex I inhibitor. Cultured ARPE-19 cells generated scattered cell clusters composed mostly of smaller size cells expressing the differentiation markers mouse anti-cellular retinaldehyde–binding protein (CRALBP) and Bestrophin only in differentiation medium. After the increase of the number of clusters, the clusters appeared to paracellularly merge, resulting in expansion of the area occupied by the clusters. Of note, the cells within the clusters selectively had high MMP and were in accordance with the expression of RPE differentiation markers. Rotenone repressed the formation of the clusters and decreased intracellular MMP. The above results suggest that clustering of RPE cells with functional mitochondria plays a pivotal role in RPE cell differentiation process and the ETC complex I inhibition greatly influences the composition of RPE cells that are degenerated or differentiation disposed.

The Novel Application of EUK-134 in Retinal Degeneration: Preventing Mitochondrial Oxidative Stress-Triggered Retinal Pigment Epithelial Cell Apoptosis by Suppressing MAPK/p53 Signaling Pathway.

Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by mitochondrial dysfunction of retinal pigment epithelium (RPE) cells. EUK-134 is a mimetic of SOD2 and catalase, widely used for its antioxidant properties in models of light-induced damage or oxidative stress. However, its effects on the retina are not yet clear. Here, we investigated the capability of EUK-134 in averting AMD using sodium iodate (NaIO3)-induced Balb/c mouse and ARPE-19 cells (adult RPE cell line). In vivo, EUK-134 effectively antagonized NaIO3-induced retinal deformation and prevented outer and inner nuclear layer thinning. In addition, it was found that the EUK-134-treated group significantly down-regulated the expression of cleaved caspase-3 compared with the group treated with NaIO3 alone. Our results found that EUK-134 notably improved cell viability by preventing mitochondrial ROS accumulation-induced membrane potential depolarization-mediated apoptosis in NaIO3-inducted ARPE-19 cells. Furthermore, we found that EUK-134 could inhibit p-ERK, p-p38, p-JNK, p-p53, Bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP by increasing Bcl-2 protein expression. Additionally, we employed MAPK pathway inhibitors by SB203580 (a p38 inhibitor), U0126 (an ERK inhibitor), and SP600125 (a JNK inhibitor) to corroborate the aforementioned observation. The results support that EUK-134 may effectively prevent mitochondrial oxidative stress-mediated retinal apoptosis in NaIO3-induced retinopathy.

Sex-dependent regulation of retinal pigment epithelium and retinal function by Pgc-1α.

Age-related macular degeneration (AMD) is a major cause of blindness that affects people over 60. While aging is the prominent factor in AMD, studies have reported a higher prevalence of AMD in women compared to age-matched men. Higher levels of the innate immune response's effector proteins complement factor B and factor I were also found in females compared to males in intermediate AMD. However, the mechanisms underlying these differences remain elusive. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) is a key regulator of mitochondrial biogenesis and metabolic pathways. Previously, we showed that Pgc-1α repression and high-fat diet induce drastic AMD-like phenotypes in mice. Our recent data revealed that Pgc-1α repression alone can also induce retinal pigment epithelium (RPE) and retinal dysfunction in mice, and its inhibition in vitro results in lipid droplet accumulation in human RPE. Whether sex is a contributing factor in these phenotypes remains to be elucidated. Using electroretinography, we demonstrate that sex could influence RPE function during aging independent of Pgc-1α in wild-type (WT) mice. We further show that Pgc-1α repression exacerbates RPE and retinal dysfunction in females compared to aged-match male mice. Gene expression analyses revealed that Pgc-1α differentially regulates genes related to antioxidant enzymes and mitochondrial dynamics in males and females. RPE flat mounts immunolabeled with TOMM20 and DRP1 indicated a sex-dependent role for Pgc-1α in regulating mitochondrial fission. Analyses of mitochondrial network morphology suggested sex-dependent effects of Pgc-1α repression on mitochondrial dynamics. Together, our study demonstrates that inhibition of Pgc-1α induces a sex-dependent decline in RPE and retinal function in mice. These observations on the sex-dependent regulation of RPE and retinal function could offer novel insights into targeted therapeutic approaches for age-related RPE and retinal degeneration.

Sulindac prevents increased mitochondrial VDAC1 expression and cell surface mistargeting induced by pathological conditions in retinal cells

Diabetic retinopathy (DR) continues to be the primary cause of vision loss in poorly controlled diabetic subjects. The molecular mechanisms underlying retinal pigment epithelium (RPE) cell dysfunction in DR still remain elusive. We investigated the role of mitochondrial volt-age-dependent anion channel 1 (VDAC1) in RPE dysfunction under glucotoxic and inflammatory conditions. Our results demonstrate that both glucotoxicity and cytokine treatment reduces cellular viability accompanied by increased VDAC1 and inducible nitric oxide synthase (iNOS) expression, concomitant with decreased expression of mitochondrial VDAC2 and constitutively ex-pressed endothelial NOS (eNOS). Increased VDAC1 expression during these conditions leads to its mistargeting to the cell surface, leading to ATP loss. Additionally, VDAC1 upregulation by glucotoxicity and inflammatory cytokines induces leakage of mitochondrial DNA (mtDNA) into the cytosol. Sulindac, a nonsteroidal anti-inflammatory agent, mitigates the adverse effects associated with increased VDAC1 level under pathophysiological conditions, by suppressing VDAC1 expression. The effect of sulindac on restoring cell viability could be comparably achieved only with VDAC1 inhibitor (VBIT-4) or VDAC1-specific antibody and not with the iNOS inhibitor aminoguanidine.Our findings suggest that sulindac's beneficial effects on ARPE-19 cell function are mediated by prevention of increased VDAC1 expression under pathological conditions, thus preventing mtDNA leakage and ATP loss, which are the key steps in induction of cellular inflammatory responses involved in the development of DR.

Sub retinal pigment epithelium hyporeflective spaces preceding large drusen collapse.

To describe and study hyporeflective sub retinal pigment epithelium (RPE) spaces in large drusen and drusenoid pigment epithelial detachment prior to collapse. Retrospective longitudinal study which enrolled patients with large and very large drusen due to intermediate age-related macular degeneration (AMD). The following optical coherence tomography (OCT) parameters were assessed: Drusen size (maximum width and height), OCT biomarkers of RPE atrophy, presence of intraretinal and subretinal fluid (IRF, SRF), acquired vitelliform lesion and sub RPE regions of hyporeflectivity within the PED compartment. Of the 50 eyes from 41 patients (mean age of 77.1 ± 9 years, 78% women) with large and very large drusen, 16 eyes progressed to collapse. Eyes with sub RPE hyporeflective spaces (n=8 eyes, 50%) were associated with greater drusen width and height than eyes without sub RPE hyporeflective spaces. At the collapse visit, eyes with sub RPE hyporeflective spaces displayed poorer visual acuity and greater iRORA (incomplete RPE outer retinal atrophy) and cRORA (complete RORA) length than eyes without sub RPE hyporeflective spaces (p=0.004 and p=0.04, respectively). Sub RPE hyporeflective spaces are a novel OCT finding of large and very large drusen that collapse to atrophy. Progressive RPE dysfunction and failure may lead to reduced drusenoid material formation and progressive degenerative hydration of the large drusen prior to collapse, but this awaits confirmation with histopathological analysis.

Role of AMPK-regulated autophagy in retinal pigment epithelial cell homeostasis: A review.

The retinal pigment epithelium (RPE) is a regularly arranged monolayer of cells in the outermost layer of the retina. It is crucial for transporting nutrients and metabolic substances in the retina and maintaining the retinal barrier. RPE dysfunction causes diseases related to vision loss. Thus, understanding the mechanisms involved in normal RPE function is vital. Adenosine monophosphate-activated protein kinase (AMPK) is an RPE energy sensor regulating various signaling and metabolic pathways to maintain cellular energetic homeostasis. AMPK activation is involved in multiple signaling pathways regulated by autophagy in the RPE, thereby protecting the cells from oxidative stress and slowing RPE degeneration. In this review, we attempt to broaden the understanding of the pathogenesis of RPE dysfunction by focusing on the role and mechanism of AMPK regulation of autophagy in the RPE. The correlation between RPE cellular homeostasis and role of AMPK was determined by analyzing the structure and mechanism of AMPK and its signaling pathway in autophagy. The protective effect of AMPK-regulated autophagy on the RPE for gaining insights into the regulatory pathways of RPE dysfunction has been discussed.

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.

The microRNA Let-7f Induces Senescence and Exacerbates Oxidative Stress in Retinal Pigment Epithelial Cells.

This study aims to investigate the role of microRNA let-7f in the dysfunction and degeneration of retinal pigment epithelium (RPE) cells through the induction of senescence and oxidative stress. Furthermore, we explore whether let-7f inhibition can protect these cells against sodium iodate (SI)-induced oxidative stress. Oxidative stress and let-7f expression are reciprocally regulated in retinal pigment epithelial cells. Overexpression of let-7f in ARPE-19 cells induced oxidative stress as demonstrated by increased reactive oxygen species (ROS) production as well as senescence. Inhibition of let-7f successfully protected RPE cells from the detrimental effects induced by SI. In addition, let-7f overexpression induced RPE cellular dysfunction by diminishing their migratory capabilities and reducing the phagocytosis of porcine photoreceptor outer segments (POS). Results were further confirmed in vivo by intravitreal injections of SI and let-7f antagomir in C57BL/6 mice. Our results provide strong evidence that let-7f is implicated in the dysfunction of RPE cells through the induction of senescence and oxidative injury. These findings may help to uncover novel and relevant processes in the pathogenesis of dry AMD.

Chrysin alleviates DNA damage to improve disturbed immune homeostasis and pro-angiogenic environment in laser-induced choroidal neovascularization

Choroidal neovascularization (CNV) is a devastating pathology of numerous ocular diseases, such as wet age-related macular degeneration (wAMD), which causes irreversible vision loss. Although anti-vascular endothelial growth factor (VEGF) therapy has been widely used, poor response or no response still exists in some cases, suggesting that there are other components involved in the angiogenic process. Therefore, the underlying mechanism needs to be clarified and new target of anti-angiogenic therapy is urgently needed. It has been demonstrated that damaged retinal pigment epithelium (RPE) cells can activate inflammasome, driving a degenerative tissue environment and an enhanced pro-angiogenic response, which implies that RPE dysfunction may be a hallmark of the pathogenesis. Previously, we have shown that DNA damage can induce RPE dysfunction, triggering senescence-associated secretory phenotype (SASP) and local inflammation. In this study, we identify that chrysin can reduce DNA damage, especially telomere erosion in vitro, thus compromise the dysfunction of RPE and the decreased expression of SASP factor. Importantly, we find that DNA damage of RPE cells is remarkable in laser-induced CNV lesion, resulting in inflammatory response, which can be ameliorated by chrysin, mainly through IL-17 signaling pathway and its downstream signal transducer and activator of transcription 3 (STAT3) activities. In summary, our results indicate the interplay between DNA damage, perturbed RPE homeostasis, inflammatory response and angiogenesis in laser-induced CNV, and more importantly, chrysin may be an effective therapeutic supplement for CNV.

Human iPSC‐derived RPE cells implantation into minipig eye: Two weeks follow‐up study

Aims/Purpose: Retinal pigment epithelium (RPE) replacement is a very promising cell‐based therapy for retinal degenerative diseases including age‐related macular degeneration (AMD). RPE dysfunction is the main cause of secondary damage to the photoreceptors. We hereby present RPE implantation into the minipig eye with a 2‐week follow‐up.Methods: Cultured human iPSC‐derived RPEs (iRPE) on nanofibrous membranes, with properties very similar to Bruch's membrane, were characterized for expression of differentiation and polarization markers using immunohistochemistry. The implantations of iRPE‐scaffolds were carried out into the minipig eyes using 3‐port pars plana vitrectomy. Due to the xenogeneic transplantation of human cells into minipig eyes, tacrolimus immunosuppression therapy was used. At the 2‐week follow‐up from transplantation, non‐invasive OCT and fundus camera examinations of the implanted iRPE‐scaffolds were performed. Consequently, euthanasia, histological and immunohistochemical investigations of the implanted retina were performed.Results: OCT and fundus camera examinations revealed successfully implanted iRPE‐scaffolds into the subretinal space with a healthy neuroretina above the implant, and healed retinotomy, without immune cell reaction against the implant. Histological and immunohistochemical investigation of the iRPE implants and adjacent retina showed structurally healthy iRPE‐scaffold implant and neuroretinal cells above the implant.Conclusions: RPE cell therapy for replacement of diseased RPEs by ex vivo cultured Ipsc–RPEs on highly permeable nanofibrous membranes supports the survival of adjacent photoreceptors and thus positively affects the structural changes seen in retinal neurodegeneration.

QUANTITATIVE AUTOFLUORESCENCE IN CENTRAL SEROUS CHORIORETINOPATHY.

Central serous chorioretinopathy (CSC) is associated with pachychoroid and dysfunctional retinal pigment epithelium. Autofluorescence (AF) is typically altered. The authors performed this study to quantify these alterations using quantitative AF (qAF) in patients with CSC and in their fellow eye in comparison with a healthy control group. Patients with CSC and healthy controls were recruited prospectively. All patients received a full clinical examination including best-corrected visual acuity, enhanced depth imaging-optical coherence tomography, and qAF. Quantitative autofluorescence images were taken with a confocal scanning laser ophthalmoscope (Heidelberg Engineering). Quantitative autofluorescence values were assessed in specified regions of the inner eight and the middle ring of the Delori grid. In total, 141 eyes of 77 patients with CSC were included. Ninety eyes had a manifest CSC (group 1) while 51 fellow eyes (group 2) did not show signs of CSC. There were no significant differences of qAF values between these two groups: mean qAF values were 241.3 (inner eight) and 212.8 (middle ring) in group 1 and 235.9 (inner eight) and 210.0 (middle ring) in group 2 ( P = 1.0 and 1.0). We compared these eyes with healthy controls comprising 39 eyes. Quantitative autofluorescence signals (inner eight: 164.7; middle ring: 148.9) differed significantly compared with both CSC manifest ( P < 0.001) and fellow eyes ( P < 0.001). Our results show that patients with CSC have increased qAF values in both eyes with manifest CSC and asymptomatic, clinically unremarkable fellow eyes in comparison with healthy controls. This finding suggests that qAF alterations are present even before clinical signs can be observed.

Circ-CARD6 inhibits oxidative stress-induced apoptosis and autophagy in ARPE-19 cells via the miR-29b-3p/PRDX6/PI3K/Akt axis

BackgroundOxidative stress-induced damage and dysfunction of retinal pigment epithelium (RPE) cells are important pathogenetic factors of age-related macular degeneration (AMD) and hereditary retinopathy diseases (HRDs). This study aimed to elucidate the roles and mechanisms of circ-CARD6 and miR-29b-3p in oxidative stress-induced RPE and provide new ideas for the diagnosis and treatment of retinopathy disease (RD). MethodsA model of oxidative stress-induced RPE (ARPE-19) was established, and the level of malondialdehyde (MDA) and concentration of reactive oxygen species (ROS) were detected by a DCFH-DA fluorescent probe and MDA kit. The cell viability was measured by a CCK-8 assay. The expression of PRDX6/PI3K/Akt axis genes and proteins related to apoptosis and autophagy were determined by RT‒qPCR and Western blot analyses. The dual-luciferase reporter system confirmed the targeting relationship between miR-29b-3p and circ-CARD6 and between miR-29b-3p and PRDX6. ResultsIn H2O2-treated ARPE-19 cells, the expression of circ-CARD6 and PRDX6 was decreased, while the expression of miR-29b-3p was increased. The overexpression of circ-CARD6 inhibits oxidative stress-induced increases in ROS, apoptosis and autophagy in ARPE-19 cells. circ-CARD6 targets miR-29b-3p, miR-29b-3p targets PRDX6, and circ-CARD6 regulates PRDX6 via miR-29b-3p. Further studies showed that circ-CARD6 acts as a competitive endogenous RNA of miR-29b-3p to affect the expression of PRDX6, thereby inhibiting autophagy and apoptosis in ARPE-19 cells. Conclusioncirc-CARD6 can inhibit oxidative stress and apoptosis by regulating the miR-29b-3p/PRDX6/PI3K/Akt axis.

Value and Significance of Hypofluorescent Lesions Seen on Late-Phase Indocyanine Green Angiography

The hypofluorescence of fundus lesions observed during the late phase of indocyanine green angiography (ICGA) in various diseases has often been overlooked or misinterpreted. This article explores the significance of fundus lesions that are initially isofluorescent during the early phase of ICGA but become hypofluorescent later in the examination. Pathologies such as multiple evanescent white spot syndrome, acute posterior placoid syphilitic chorioretinitis, chronic central serous chorioretinopathy, choroidal hemangioma, and some fundus with drusen, present this phenomenon of late hypofluorescence. The interpretation of ICGA images and the role of indocyanine green (ICG) uptake by the retinal pigment epithelium (RPE) in late fundus fluorescence is debated. Experimental evidence suggests that ICG accumulates progressively in the RPE after intravenous injection of the dye or after direct contact invitro, making it a potential marker of RPE activity. Although the exact mechanisms of ICG diffusion through the choroid and its binding to the RPE require further investigation, the late hypofluorescence observed in certain ICGA diseases provides information on different modalities of RPE dysfunction. The author has no proprietary or commercial interest in any materials discussed in this article.

Kallistatin Deficiency Induces the Oxidative Stress-Related Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells: A Novel Protagonist in Age-Related Macular Degeneration.

Retinal pigment epithelium (RPE) dysfunction induced by oxidative stress-related epithelial-mesenchymal transition (EMT) of RPE is the primary underlying mechanism of age-related macular degeneration (AMD). Kallistatin (KAL) is a secreted protein with an antioxidative stress effect. However, the relationship between KAL and EMT in RPE has not been determined. Therefore we aimed to explore the impact and mechanism of KAL in oxidative stress-induced EMT of RPE. Sodium iodate (SI) was injected intraperitoneally to construct the AMD rat model and investigate the changes in RPE morphology and KAL expression. KAL knockout rats and KAL transgenic mice were used to explain the effects of KAL on EMT and oxidative stress. In addition, Snail overexpressed adenovirus and si-RNA transfected ARPE19 cells to verify the involvement of Snail in mediating KAL-suppressed EMT of RPE. AMD rats induced by SI expressed less KAL in the retina, and KAL knockout rats showed RPE dysfunction spontaneously where EMT and reactive oxygen species (ROS) production increased in RPE. In contrast, KAL overexpression attenuated EMT and ROS levels in RPE, even in TGF-β treatment. Mechanistically, Snail reversed the beneficial effect of KAL on EMT and ROS reduction. Moreover, KAL ameliorated SI-induced AMD-like pathological changes. Our findings demonstrated that KAL inhibits oxidative stress-induced EMT by downregulating the transcription factor Snail. Herein, KAL knockout rats may be an appropriate animal model for observing spontaneous RPE dysfunction for AMD-like retinopathy, and KAL may represent a novel therapeutic target for treating dry AMD.

TNFα induced by DNA-sensing in macrophage compromises retinal pigment epithelial (RPE) barrier function

Increasing evidence suggests that chronic inflammation plays an important role in the pathogenesis of age-related macular degeneration (AMD); however, the precise pathogenic stressors and sensors, and their impact on disease progression remain unclear. Several studies have demonstrated that type I interferon (IFN) response is activated in the retinal pigment epithelium (RPE) of AMD patients. Previously, we demonstrated that human RPE cells can initiate RNA-mediated type I IFN responses through RIG-I, yet are unable to directly sense and respond to DNA. In this study, we utilized a co-culture system combining primary human macrophage and iPS-derived RPE to study how each cell type responds to nucleic acids challenges and their effect on RPE barrier function in a homotypic and heterotypic manner. We find that DNA-induced macrophage activation induces an IFN response in the RPE, and compromises RPE barrier function via tight-junction remodeling. Investigation of the secreted cytokines responsible for RPE dysfunction following DNA-induced macrophages activation indicates that neutralization of macrophage-secreted TNFα, but not IFNβ, is sufficient to rescue RPE morphology and barrier function. Our data reveals a novel mechanism of intercellular communication by which DNA induces RPE dysfunction via macrophage-secreted TNFa, highlighting the complexity and potential pathological relevance of RPE and macrophage interactions.

Serous Maculopathy Due to Aspecific Choroidopathy (SMACH).

Serous Maculopathy Due to Aspecific Choroidopathy (SMACH).

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients' fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Downregulation of VEGF in the retinal pigment epithelium followed by choriocapillaris atrophy after NaIO3 treatment in mice

Sodium iodate (NaIO3) induces retinal pigment epithelium (RPE) dysfunction, which leads to photoreceptor degeneration. Previously, we used electron microscopy to show that the administration of NaIO3 resulted in the accumulation of cell debris in the subretinal space, which was thought to be caused by failed phagocytosis in the outer segment of the photoreceptor due to RPE dysfunction. We further analyzed the pathological changes in the retina and choroid of NaIO3-injected mice, and found that the expression of OTX2, an RPE marker, disappeared from central part of the RPE 1 day after NaIO3 administration. Furthermore, fenestrated capillaries (choriocapillaris, CC) adjacent to the RPE could not be identified only 2 days after NaIO3 administration. An examination of the expression of the CC-specific protein plasmalemma vesicle-associated protein (PLVAP), in sections and flat-mount retina/choroid specimens showed destruction of the CC, and complete disappearance of the PLVAP signal 7 days after NaIO3 administration. In contrast, CD31 flat-mount immunohistochemistry of the retina indicated no difference in retinal vessels between NaIO3-treated mice and controls. Electron microscopy showed that the fenestrated capillaries in the kidney and duodenum were morphologically indistinguishable between control and NaIO3-treated mice. We examined cytokine production in the retina and RPE, and found that the Vegfa transcript level in the RPE decreased starting 1 day after NaIO3 administration. Taken together, these observations show that NaIO3 reduces the CC in the early stages of the pathology, which is accompanied by a rapid decrease in Vegfa expression in the RPE.