Pigment Epithelial Cells Research Articles

Elovl4b knockout zebrafish as a model for ocular very-long-chain PUFA deficiency

Very-long-chain PUFAs (VLC-PUFAs) are a group of lipids with chain lengths >24 carbons, and the ELOVL4 (elongation of very-long-chain FA-4) enzyme is responsible for vertebrate VLC-PUFA biosynthesis. Studies on the role of VLC-PUFAs in vision have been hindered because of the need for adequate animal models to capture the global loss of VLC-PUFAs. Since homozygous Elovl4 ablation is lethal in neonatal mice because of catastrophic drying from the loss of their protective skin barrier, we established a zebrafish (Danio rerio) model of Elovl4 ablation. We generated Elovl4b KO zebrafish by creating a 56-bp deletion mutation in exon 2 of the Elovl4b gene using CRISPR-Cas9. We used GC-MS and LC-MS/MS to analyze the VLC-PUFA and lipid profiles from wild-type and Elovl4b KO fish eyes. We also performed histology and visual-behavioral tests. We found that heterozygous and homozygous Elovl4b KO zebrafish eyes had altered lipid profiles and a significantly lower C30 to C36 VLC-PUFA abundance than wild-type fish. Moreover, Elovl4b+/− and Elovl4b−/− KO larvae had significantly lower motor activity in response to light-dark cycles than their age-matched controls. Elovl4b−/− adult fish showed no obvious differences in gross retinal morphology and lamination compared with wild type, except for the presence of lipid droplets within the retinal pigment epithelial cell layer of Elovl4b−/− fish. Our data indicate that the loss of Elovl4b in zebrafish changes ocular lipid profiles and leads to visual abnormalities and subtle retinal changes. These findings highlight the use of zebrafish as a model for VLC-PUFA depletion and ELOVL4-related dysfunction.

TNFSF15 inhibits progression of diabetic retinopathy by blocking pyroptosis via interacting with GSDME

Diabetic retinopathy is a common microvascular complication of diabetes and a leading cause of blindness. Pyroptosis has emerged as a mechanism of cell death involved in diabetic retinopathy pathology. This study explored the role of GSDME-mediated pyroptosis and its regulation by TNFSF15 in diabetic retinopathy. We found GSDME was upregulated in the progression of diabetic retinopathy. High glucose promoted GSDME-induced pyroptosis in retinal endothelial cells and retinal pigment epithelial cells, attributed to the activation of caspase-3 which cleaves GSDME to generate the pyroptosis-executing N-terminal fragment. TNFSF15 was identified as a binding partner and inhibitor of GSDME-mediated pyroptosis. TNFSF15 expression was increased by high glucose but suppressed by the caspase-3 activator Raptinal. Moreover, TNFSF15 protein inhibited high glucose- and Raptinal-induced pyroptosis by interacting with GSDME in retinal cells. Collectively, our results demonstrate TNFSF15 inhibits diabetic retinopathy progression by blocking GSDME-dependent pyroptosis of retinal cells, suggesting the TNFSF15-GSDME interaction as a promising therapeutic target for diabetic retinopathy.

Resveratrol and quercetin protect from Benzo(a)pyrene-induced autophagy in retinal pigment epithelial cells.

This study aims to investigate the role of Resveratrol (RES) and quercetin (QR) treatments against Benzo(a)pyrene (B(a)p)-induced autophagy in retinal pigment epithelial cells. The IC50 doses of B(a)p, RES and QR in retinal pigment epithelial cells were determined by MTT assay and the relevant agents were administered singly or in combinations to ARPE-19 cells for 24h. Occurrence of autophagy in the cells was verified by detection of autophagosomes using fluorescence microscope. Also, the mRNA expression levels of LC3 and Beclin 1 genes were analyzed by RT-PCR to collect further data on autophagy. Caspase-3 and IL-1β levels in lysed cells were analyzed by ELISA. Autophagosomes were detected in B(a)p-treated ARPE-19 cell lines, as well as a 1.787-fold increase in LC3 mRNA expression levels. No autophagosome occurred in RES and QR treatments, and a significant decrease in their percentage amounts were observed in B(a)p + RES and B(a)p + QR. The mRNA expression levels of LC3 and Beclin 1 also supported these findings. B(a)p had no effect on Caspase-3 levels in ARPE-19 cells, but combined with RES and QR, it increased Caspase-3 levels significantly.IL-1β levels were higher in B(a)p, B(a)p + QR, B(a)p + RES, RES and QR than control group. This rise in IL-1β levels was correlated with suppression of mRNA expression levels of Beclin 1. B(a)p exposure caused autophagy in ARPE-19 cells, but did not induce apoptosis. RES and QR treatments prevented B(a)p-induced autophagy. Therefore, RES and QR treatments showed protective effect against potential degenerative diseases caused by chronic exposure to B(a)p.

IFT27 regulates the long-term maintenance of photoreceptor outer segments in zebrafish

Approximately a quarter of Retinitis Pigmentosa (RP) is caused by mutations in transport-related genes in cilia. IFT27 (Intraflagellar Transport 27), a core component of the ciliary intraflagellar transport (IFT) system, has been implicated as a significant pathogenic gene in RP. The pathogenic mechanisms and subsequent pathology related to IFT27 mutations in RP are largely obscure. Here, we utilized TALEN technology to create an ift27 knockout (ift27-/-) zebrafish model. Electroretinography (ERG) detection showed impaired vision in this model. Histopathological examinations disclosed that ift27 mutations cause progressive degeneration of photoreceptors in zebrafish, and this degeneration was late-onset. Immunofluorescence labeling of outer segments showed that rods degenerated before cones, aligning with the conventional characterization of RP. In cultured human retinal pigment epithelial cells, we found that IFT27 was involved in maintaining ciliary morphology. Furthermore, decreased IFT27 expression resulted in the inhibition of the Hedgehog (Hh) signaling pathway, including decreased expression of key factors in the Hh pathway and abnormal localization of the ciliary mediator Gli2. In summary, we generated an ift27-/- zebrafish line with retinal degeneration which mimicked the symptoms of RP patients, highlighting IFT27′s integral role in the long-term maintenance of cilia via the Hh signaling pathway. This work may furnish new insights into the treatment or delay of RP caused by IFT27 mutations.

Exploring the Regenerative Potential of Stem Cells for Treating Eye Diseases: A Review of the New Findings

The escalating prevalence of vision loss due to eye diseases has instigated a quest for innovative therapies, given that conventional approaches often fall short in repairing and regenerating damaged eye tissues, particularly the retina. Stem cell-based interventions have emerged as a promising avenue, with numerous studies in animal models and human trials exploring their potential to enhance visual acuity. Beyond addressing conditions like age-related macular degeneration (AMD) and diabetic retinopathy (DR), stem cell therapies demonstrate efficacy in treating genetic disorders such as retinitis pigmentosa (RP). In severe eye damage necessitating regeneration, stem cells play a pivotal role, leveraging their regenerative capabilities. Noteworthy is the transplantation of retinal pigment epithelial (RPE) cells derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), showcasing promising results in preclinical models and clinical studies, leading to improved retinal function without severe side effects. Mesenchymal stem cells (MSCs) have successfully treated optic neuropathy, RP, DR, and glaucoma, yielding positive clinical outcomes. The safety of adult stem cells, particularly MSCs derived from adipose tissue or bone marrow, has been firmly established. This review highlights significant advancements in utilizing human ESC-derived retinal pigmented epithelium and iPSCs for treating eye injuries. While cell-based therapy is relatively nascent, with numerous clinical trials pending review, stem cells' regenerative potential and clinical applications in addressing eye diseases offer substantial promise. This study aims to comprehensively examine the applications of stem cells in the context of eye diseases and their potential role in regenerative medicine.

Azoramide ameliorates cadmium-induced cytotoxicity by inhibiting endoplasmic reticulum stress and suppressing oxidative stress.

Cadmium (Cd) is hazardous to human health because of its cytotoxicity and long biological half-life. Azoramide is a small molecular agent that targets the endoplasmic reticulum (ER) and moderates the unfolded protein response. However, its role in Cd-induced cytotoxicity remains unclear. This study was performed to investigate the protective effect of azoramide against Cd-induced cytotoxicity and elucidate its underlying mechanisms. Inductively coupled plasma‒mass spectrometry was used to measure Cd concentrations in each tissue of ICR male mice. The human proximal tubule epithelial cell line HK-2 and the human retinal pigment epithelial cell line ARPE-19 were used in the in vitro study. Cell apoptosis was determined by DAPI staining, JC-1 staining, and annexin V/propidium iodide double staining. Intracellular oxidative stress was detected by MitoSOX red staining, western blot, and quantitative real-time PCR. Moreover, ER stress signaling, MAPK cascades, and autophagy signaling were analyzed by western blot. The present data showed that Cd accumulated in various organs of ICR mice, and the concentrations of Cd in the studied organs, from high to low, were as follows: liver > kidney > testis > lung > spleen > eye. Our study demonstrated that azoramide inhibited ER stress by promoting BiP expression and suppressing the PERK-eIF2α-CHOP pathway. Additionally, we also found that azoramide significantly decreased ER stress-associated radical oxidative species production, attenuated p38 MAPK and JNK signaling, and inhibited autophagy, thus suppressing apoptosis in HK-2 and ARPE-19 cells. Our study investigated the effect of azoramide on Cd-induced cytotoxicity and revealed that azoramide may be a therapeutic drug for Cd poisoning.

Molecular Network Mechanism Analysis of Urine Stem Cells Against Retinal Aging.

To investigate the effect and potential mechanism of human-derived urine stem cells (hUSCs) in inhibiting retinal aging by using experimental and bioinformatics. Retinal pigment epithelial cells cultured in vitro, which were randomly divided into normal group, aging group and supernatant of hUSCs group. Cell counting kit-8 detection, senescence-related β-galactosidase, and Annexin V/PI staining were performed to detect cell viability, senescence, and apoptosis. Subsequently, bioinformatics methods were used to explore the underlying mechanisms, in which, targets both hUSCs and aging retina-related targets were obtained from GeneCards. Then, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and protein-protein interaction network were analysis, and the expressional level of hub gene was validated by q-PCR. Supernatant addition of hUSCs promoted markedly cellular proliferation, improved viability and inhibited senescence and apoptosis in vitro. A total of 1476 hUSCs-related targets (Relevance score > 20), 692 retinal disease-related targets, and 732 targets related to disease of aging were selected from GeneCards database, and 289 common targets of hUSCs against aging retina were confirmed through Venn analysis. Enrichment analysis demonstrated that hUSCs might exert its anti-apoptosis efficacy in multiple biological processes, including oxidative stress, inflammation and apoptosis, and core targets were associated with HIF-1, MAPK and PI3K-Akt signal. hUSCs inhibited retinal senescence by regulating multiply targets and signaling pathways, of these, HIF-1, MAPK, and PI3K may be important candidates.

DEL-1: a promising treatment for AMD-associated ER stress in retinal pigment epithelial cells

BackgroundAge-related macular degeneration (AMD) is an irreversible eye disease that can cause blurred vision. Regular exercise has been suggested as a therapeutic strategy for treating AMD, but how exercise improves AMD is not yet understood. This study investigated the protective effects of developmental endothelial locus-1 (DEL-1), a myokine upregulated during exercise, on endoplasmic reticulum (ER) stress-induced injury in retinal pigment epithelial cells.MethodsWe evaluated the levels of AMPK phosphorylation, autophagy markers, and ER stress markers in DEL-1-treated human retinal pigment epithelial cells (hRPE) using Western blotting. We also performed cell viability, caspase 3 activity assays, and autophagosome staining.ResultsOur findings showed that treatment with recombinant DEL-1 dose-dependently reduced the impairment of cell viability and caspase 3 activity in tunicamycin-treated hRPE cells. DEL-1 treatment also alleviated tunicamycin-induced ER stress markers and VEGF expression. Moreover, AMPK phosphorylation and autophagy markers were increased in hRPE cells in the presence of DEL-1. However, the effects of DEL-1 on ER stress, VEGF expression, and apoptosis in tunicamycin-treated hRPE cells were reduced by AMPK siRNA or 3-methyladenine (3-MA), an autophagy inhibitor.ConclusionsOur study suggests that DEL-1, a myokine, may have potential as a treatment strategy for AMD by attenuating ER stress-induced injury in retinal pigment epithelial cells.

SB431542 partially inhibits high glucose-induced EMT by restoring mitochondrial homeostasis in RPE cells

BackgroundThe epithelial–mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells participated in the development of retinal fibrosis. SB431542 is a small molecule inhibitor with inhibitory effects on the ALK4, ALK5 and ALK7. Our study aimed to explore the effect of SB431542 on the EMT of RPE cells and to provide new ideas for the treatment of retinal fibrosis.MethodsWe performed fundus fluorescein angiography, optical coherence tomography and hematoxylin–eosin staining in vivo to observe the effect of SB431542 on choroidal neovascularization (CNV)-induced retinopathy. The proliferation, migration, cytoskeleton, adhesion, reactive oxygen species (ROS), mitochondrial morphology and membrane potential of RPE cells were observed in vitro through fluorescein diacetate staining, Cell Counting Kit-8 experiment, wound healing assay, phalloidin staining, immunofluorescence, MitoSOX, DCFH-DA, MitoTracker and JC-10 staining. Western blot, reverse transcription quantitative and immunofluorescence were used to detect the expression of EMT–related markers, pERK1/2, pGSK3β and β-catenin.ResultsSB431542 significantly alleviated retinopathy in the CNV model. The proliferation, migration and adhesion in RPE cells decreased to a certain extent in SB431542 treatment. SB431542 partially normalized the structure of RPE cells. The expression levels of E-cadherin increased, while the expression levels of laminin and N-cadherin decreased with SB431542 treatment. SB431542 reduced the production of total ROS, mitochondrial SOX and recovered the mitochondrial membrane potential to a certain degree. In addition, our study showed that SB431542 downregulated the phosphorylation of ERK1/2, GSK3β and the expression of β-catenin.ConclusionSB431542 improved EMT in RPE cells by maintaining mitochondrial homeostasis via the ERK1/2 and GSK3β/β-catenin pathways.1HzEs7TmoETCuNYu8FcEmbVideo Graphical SB431542 inhibits EMT in RPE cells under high glucose conditions.

RNA fusion in human retinal development.

Chimeric RNAs have been found in both cancerous and healthy human cells. They have regulatory effects on human stem/progenitor cell differentiation, stemness maintenance, and central nervous system development. However, whether they are present in human retinal cells and their physiological functions in the retinal development remain unknown. Based on the human embryonic stem cell-derived retinal organoids (ROs) spanning from days 0 to 120, we present the expression atlas of chimeric RNAs throughout the developing ROs. We confirmed the existence of some common chimeric RNAs and also discovered many novel chimeric RNAs during retinal development. We focused on CTNNBIP1-CLSTN1 (CTCL) whose downregulation caused precocious neuronal differentiation and a marked reduction of neural progenitors in human cerebral organoids. CTCL is universally present in human retinas, ROs, and retinal cell lines, and its loss-of-function biases the progenitor cells toward retinal pigment epithelial cell fate at the expense of retinal cells. Together, this work provides a landscape of chimeric RNAs and reveals evidence for their critical role in human retinal development.

Sestrin2 ameliorates diabetic retinopathy by regulating autophagy and ferroptosis.

Diabetic retinopathy (DR) is a serious microvascular complication of diabetes. The aim of this study was to explore the effect of Sestrin2 on DR through the regulation of autophagy and ferroptosis levels and its mechanism. In vitro and in vivo DR models were established by high glucose (HG) and streptozotocin (STZ) induction of ARPE-19 human retinal pigment epithelial cells and C57BL/6 mice, respectively. In this study, we demonstrated that after HG treatment, the activity of ARPE-19 cells was decreased, the apoptosis rate was increased, endoplasmic reticulum (ER) stress was activated, autophagy levels were decreased, and ferroptosis levels were increased. Overexpression of Sestrin2 enhanced cell viability, reduced apoptosis and ferroptosis, and enhanced autophagy. However, the effect of overexpression of Sestrin2 was attenuated after the addition of the STAT3 phosphorylation activator Colivelin TFA (C-TFA), the mTOR pathway activator MHY1485 or the autophagy inhibitor 3-methyladenine (3-MA). In addition, the effect of Sestrin2 knockdown on cells was opposite to the effect of overexpression of Sestrin2, while the effect of Sestrin2 knockdown was attenuated after treatment with the ER stress inhibitor 4-phenylbutyric acid (4-PBA). Animal experiments also confirmed the results of cell experiments and attenuated the effects of overexpression of Sestrin2 after injection of the ferroptosis activators erastin or 3-MA. Our study revealed that Sestrin2 inhibits ferroptosis by inhibiting STAT3 phosphorylation and ER stress and promoting autophagy levels, thereby alleviating DR.

A Bispecific Modeling Framework Enables the Prediction of Efficacy, Toxicity, and Optimal Molecular Design of Bispecific Antibodies Targeting MerTK

Inhibiting MerTK on macrophages is a promising therapeutic strategy for augmenting anti-tumor immunity. However, blocking MerTK on retinal pigment epithelial cells (RPEs) results in retinal toxicity. Bispecific antibodies (bsAbs) containing an anti-MerTK therapeutic and anti-PD-L1 targeting arm were developed to reduce drug binding to MerTK on RPEs, since PD-L1 is overexpressed on macrophages but not RPEs. In this study, we present a modeling framework using in vitro receptor occupancy (RO) and pharmacokinetics (PK) data to predict efficacy, toxicity, and therapeutic index (TI) of anti-MerTK bsAbs. We first used simulations and in vitro RO data of anti-MerTK monospecific antibody (msAb) to estimate the required MerTK RO for in vivo efficacy and toxicity. Using these estimated RO thresholds, we employed our model to predict the efficacious and toxic doses for anti-MerTK bsAbs with varying affinities for MerTK. Our model predicted the highest TI for the anti-MerTK/PD-L1 bsAb with an attenuated MerTK binding arm, which was consistent with in vivo efficacy and toxicity observations. Subsequently, we used the model, in combination with sensitivity analysis and parameter scans, to suggest an optimal molecular design of anti-MerTK bsAb with the highest predicted TI in humans. Our prediction revealed that this optimized anti-MerTK bsAb should contain a MerTK therapeutic arm with relatively low affinity, along with a high affinity targeting arm that can bind to a low abundance target with slow turnover rate. Overall, these results demonstrated that our modeling framework can guide the rational design of bsAbs.Graphical

Exosomes-based dual drug-loaded nanocarrier for targeted and multiple proliferative vitreoretinopathy therapy.

Proliferative vitreoretinopathy (PVR) is a common cause of vision loss after retinal reattachment surgery and ocular trauma. The key pathogenic mechanisms of PVR development include the proliferation, migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) activated by the growth factors and cytokines after surgery. Although some drugs have been tried in PVR treatments as basic investigations, the limited efficacy remains an obstacle, which may be due to the single pharmacological action and lack of targeting. Herein, the anti-proliferative Daunorubicin and anti-inflammatory Dexamethasone were co-loaded in the RPEs-derived exosomes (Exos), obtaining an Exos-based dual drug-loaded nanocarrier (Exos@D-D), and used for multiple PVR therapy. Owing to the advantages of homologous Exos and the dual drug loading, Exos@D-D showed good RPEs targeting as well as improved uptake efficiency, and could inhibit the proliferation, migration, as well as EMT of RPEs effectively. The animal studies have also demonstrated that Exos@D-D effectively inhibits the production of proliferative membranes and prevents the further development of inflammation, shows significant therapeutic effects on PVR and good biocompatibility. Such Exos-based dual drug-loaded nanocarrier investigation not only provides a promising approach for multifunctional exosome drug delivery systems construction, but also has great potential in PVR clinical therapy application.

Therapeutic potential of photobiomodulation on hydrogen peroxide‐induced oxidative damage in retinal pigment epithelial cells

Aims/Purpose: Photobiomodulation (PBM) is a cutting‐edge therapeutic method for restoring the functioning of injured mitochondria in experimental settings. Our goal was to find out how 780 nm PBM therapy affected the apoptosis and cell survival of hydrogen peroxide‐treated retinal pigment epithelial cells.Methods: After being treated with 150 μM hydrogen peroxide for 24 h, the retinal pigment epithelial (RPE) cells were exposed to continuous wave laser radiation with the following parameters: wavelength of 780 nm, power density of 140 mW/cm2, 65 mW/cm2 and 40 mW/cm2 and duration of 10 min and 30 min. Furthermore, light therapy was given to untreated control cells to check PBM effect on healthy group. After PBM therapy in a time‐dependent manner, cell survival and apoptosis were assessed by MTT assay and Annexin V/PI staining, respectively.Results: Based on MTT findings, PBM showed no obvious effect on cell viability in H2O2‐treated RPE cells, while after 48 h post‐PBM incubation, cell viability was dramatically improved. It was significantly increased at the power density of 140 mW/cm2 for 30 min application without unacceptable effect on control cells. RPE apoptosis was strikingly decreased in post‐PBM incubation group compared to H2O2‐treated group in a time‐dependent manner, as detected by flow cytometry.Conclusions: As a result, photobiomodulation supported its potential utility in the prevention and treatment of retinal disorders associated with oxidative stress by promoting cell survival and reducing apoptosis under oxidative stress.

ROCK inhibitor enhances mitochondrial transfer via tunneling nanotubes in retinal pigment epithelium.

Rationale: Tunnel nanotube (TNT)-mediated mitochondrial transport is crucial for the development and maintenance of multicellular organisms. Despite numerous studies highlighting the significance of this process in both physiological and pathological contexts, knowledge of the underlying mechanisms is still limited. This research focused on the role of the ROCK inhibitor Y-27632 in modulating TNT formation and mitochondrial transport in retinal pigment epithelial (RPE) cells. Methods: Two types of ARPE19 cells (a retinal pigment epithelial cell line) with distinct mitochondrial fluorescently labeled, were co-cultured and treated with ROCK inhibitor Y-27632. The formation of nanotubes and transport of mitochondria were assessed through cytoskeletal staining and live cell imaging. Mitochondrial dysfunction was induced by light damage to establish a model, while mitochondrial function was evaluated through measurement of oxygen consumption rate. The effects of Y-27632 on cytoskeletal and mitochondrial dynamics were further elucidated through detailed analysis. Results: Y-27632 treatment led to an increase in nanotube formation and enhanced mitochondrial transfer among ARPE19 cells, even following exposure to light-induced damage. Our analysis of cytoskeletal and mitochondrial distribution changes suggests that Y-27632 promotes nanotube-mediated mitochondrial transport by influencing cytoskeletal remodeling and mitochondrial movement. Conclusions: These results suggest that Y-27632 has the ability to enhance mitochondrial transfer via tunneling nanotubes in retinal pigment epithelium, and similarly predict that ROCK inhibitor can fulfill its therapeutic potential through promoting mitochondrial transport in the retinal pigment epithelium in the future.

PGC‐1a inhibition promotes autophagy in induced pluripotent stem cell‐derived retinal pigment epithelial cells generated from age‐related macular degeneration patients

Aims/Purpose: Dysfunctional protein clearance, oxidative stress, mitochondrial damage, and chronic inflammation cause retinal pigment epithelial (RPE) cell damage and the development of age‐related macular degeneration (AMD). PGC‐1α can alleviate oxidative stress, is a key player in mitochondrial quality control and could be considered as a treatment target in AMD. Here, we assessed the effect of PGC‐1α inhibition on induced pluripotent stem cell (iPSC)‐derived RPE cells exposed to hydroquinone‐induced oxidative stress.Methods: iPSC‐derived RPE cells from two neovascular AMD patients or one healthy age‐matched control were cultured for 8 weeks on transwell cell culture inserts until mature. Cells were exposed to the PGC‐1α inhibitor SR‐18292 for 1 h before an exposure to hydroquinone for an additional 4 h. Lactate dehydrogenase (LDH) was measured from collected medium samples to assess cellular viability. Cells were lysed in 3x Laemmli sample buffer for analysis by western blotting or subjected to RNA extraction, followed by RT‐PCR analysis.Results: Hydroquinone caused a robust increase in LDH release to the medium, which was not modulated by PGC‐1α inhibition. In neovascular AMD‐derived iPSC‐RPE cells, hydroquinone caused an accumulation of p62 and LC3‐II, an effect that was reversed by SR‐18292 pre‐treatment. In control iPSC‐RPE cells, neither hydroquinone nor SR‐18292 affected autophagic markers p62 or LC3. RT‐PCR analysis showed an increase in p62 mRNA levels following hydroquinone exposure in both control‐ and AMD‐derived iPSC‐RPE cells while no change was observed in the RNA levels of LC3B or mTOR.Conclusions: Increased oxidative stress caused autophagy dysregulation in AMD‐derived iPSC‐RPE cells but not in healthy age‐matched control cells. Interestingly, PGC‐1α inhibition promoted autophagy and the clearance of p62, but further studies are necessary to explore the underlying pathways and the disease‐specific differences observed between healthy and disease‐derived RPE cells.

Comparative analysis of the effects of limbal and adipose‐derived mesenchymal stem cell conditioned media on oxidative stress‐induced damage in retinal pigment epithelial cells

Aims/Purpose: The purpose of this investigation was to evaluate the impact of adipose and limbal tissue‐derived mesenchymal stem cell‐conditioned media on survival, and inflammation in H2O2‐induced retinal pigment epithelial cells.Methods: Following national regulations and with the approval of the Koç University Ethics and Investigation Committee, human limbal (L) and adipose‐derived (Ad) mesenchymal stem cells (MSCs) were isolated from cadaveric corneaoscleral rims from donors that originated from the cornea bank of Ministry of Health in Turkey and adipose tissue from discarded human liposuction surgery specimens, respectively. Conditioned medium (CM) that obtained by culturing MSCs in absence of serum was concentrated by filtration. Following a 24‐h pre‐treatment with H2O2, retinal pigment epithelial (RPE) cells were treated with MSC‐CM. Groups were defined as follows: Control, H2O2‐treatment (OS), OS‐Basal Medium, OS‐AdMSC‐CM and OS‐LMSC‐CM. Cell viability was assessed using MTT assay. Gene expression levels of pro‐ and anti‐inflammatory cytokines were analysed by qRT‐PCR.Results: Based on MTT assay, H2O2 inhibited the growth of RPE cells with IC50 values of 150 μM at 24 h. We found that MSC‐CM attenuated H2O2‐induced loss of cell viability. In comparison to the OS group, the LMSC‐CM and AdMSC‐CM treatments demonstrated significantly decreased levels of IL‐6, IL‐8, IL‐10 and TGF‐β1 gene expression.Conclusions: MSC‐CM ameliorated H2O2‐induced retinal pigment epithelial cell damage by alleviating inflammation.

Cell and animal models of age‐related macular degeneration: Focus on oxidative stress and protein aggregation

Age‐related macular degeneration (AMD) is a multifactorial disease with a long asymptomatic development period. Due to its multifactorial feature and slow development, disease modelling capturing all the elements becomes difficult if not impossible. The disease has a strong genetic background in form of certain polymorphisms in the complement system and ARMS2/HTRA1 genes providing tools for studies regarding genetics. However, AMD is much more than genetics. At least prolonged oxidative stress and subsequent protein accumulation and aggregation together with local inflammation developing to chronic in retinal pigment epithelial (RPE) cells are the key elements of the cellular pathogenesis of AMD. The RPE cells are quiescent and responsible for the wellbeing of the entire retina functioning as the link between choroidal vasculature and other layers of the retina providing necessary support for normal retinal functions. Therefore, disturbance on the cellular homeostasis of the RPE compromise the function of the entire retina. Despite being such an integral part of the retina responsible for the vision, the RPE cells withstand one the highest levels of oxidative stress throughout a lifetime of an individual. This sets certain demands for the antioxidant system's capacity to eliminate harmful oxidant molecules and prevent the spread of oxidative injury inside the cell leading to oxidative stress. However, constant oxidative challenge leads to damages on the protective system and becomes more deleterious over time. Proteins are especially prone to oxidative damage resulting increased levels of dysfunctional or aggregating proteins challenging the repair/degradative systems of the cell. Modification of oxidative status or protein degradation systems have been shown to simulate the disease state in the RPE cells and leading to a disease resembling phenotype. In vitro models are efficient tools studying certain cell specific molecular pathways such as oxidative stress, function of antioxidant defence system, and repair/degradative pathways and discovering potential druggable targets. In vivo animal models offer information how these pathways function in a native environment and how the in vitro results are translated to a functional living organism. Also, animal models offer information about the potential administration routes as well as important pharmacological data, for example. The use of most common animals in research, rodents, needs genetic engineering, for example, to achieve desired disease modelling phenotype often lacking some critical features of the disease. However, the use of these models has greatly increased the understanding of AMD pathogenesis, lead to new discoveries on disease development later confirmed in patient population, and offered new treatment strategies.

De novo pigmentation of amelanotic retinal pigment epithelium – An ex vivo model

Purpose: Retinal Pigment Epithelial (RPE) cells perform critical functions in the visual cycle. Their melanin pigmentation, which is organized into specialized compartments ‐ melanosomes, is highly critical for proper vision. We hereby show an ex vivo model for de novo pigmentation of RPE cells.Methods: A chemical method to induce pigmentation in a non‐pigment model of ARPE‐19 cells was applied using L‐DOPA as a repurposed drug from the current treatment of Parkinson's disease.Results: L‐DOPA was optimized for its toxic effect on ARPE‐19 cells along with pigmentation development. We found 1000 μM L‐DOPA to start inducing pigmentation of RPEs by Day 3, and achieve full pigmentation by Day 5. The gene expression of RPE‐specific markers (Tyrosinase, CRALBP, PEDF) was significantly higher in L‐DOPA‐treated ARPE‐19 compared to non‐treated ones. Positive expression for Tyrosinase was confirmed by ICC at both Day 3 and Day 5 of L‐DOPA treatment. Transmission electron microscopy showed the de novo melanosome formation with ultrastructural features of various stages of maturity (Stage I to IV), apical‐basal polarity, and melanosomes localization on the apical side of the RPEs in the L‐DOPA treated cells.Conclusions: Our study showed that L‐DOPA treatment could induce de novo melanosome formation in amelanotic RPE cells.Keywords: RPE, Melanosomes, L‐DOPA, Pigmentation.

Survivin inhibition attenuates EGF-induced epithelial mesenchymal transformation of human RPE cells via the EGFR/MAPK pathway.

The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells. Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay. EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins. Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR.