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Age-related macular degeneration (AMD) is the most frequent cause of blindness in developed countries. The replacement of dysfunctional human retinal pigment epithelium (hRPE) cells by the transplantation of in vitro-cultivated hRPE cells to the affected area emerges as a feasible strategy for regenerative therapy. Synthetic biomimetic membranes arise as powerful hRPE cell carriers, but as biodegradability is a requirement, it also poses a challenge due to its limited durability. hRPE cells exhibit several characteristics that putatively respond to the type of membrane carrier, and they can be used as biomarkers to evaluate and further optimize such membranes. Here, we analyze the pigmentation, transepithelial resistance, genome integrity, and maturation markers of hRPE cells plated on commercial polycarbonate (PC) versus in-house electrospun polylactide-based (PLA) membranes, both enabling separate apical/basolateral compartments. Our results show that PLA is superior to PC-based membranes for the cultivation of hRPEs, and the BEST1/RPE65 maturation markers emerge as the best biomarkers for addressing the quality of hRPE cultivated in vitro. The stability of the cultures was observed to be affected by PLA aging, which is an effect that could be partially palliated by the coating of the PLA membranes. [ABSTRACT FROM AUTHOR]

Background: Subretinal macular hemorrhage (SRMH) secondary to age-related macular degeneration (AMD) is a relatively rare condition in ophthalmology characterized by blood collection between the neurosensory retina and the retinal pigment epithelium (RPE). Without prompt treatment, visual prognosis is poor. A plethora of treatment approaches have been tried over the past years ranging from intravitreal anti-vascular endothelial growth factor (anti-VEGF) monotherapy to direct subretinal surgery, with no conclusive superiority of one over the other. Materials and Methods: We conducted a systematic review of the outcomes and treatment modalities of SRMH from inception to 14 June 2022, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA). The level of evidence was assessed for all included articles according to the quality of evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. Results: A total of 2745 articles were initially extracted, out of which 1654 articles were obtained after duplicates were removed and their abstracts screened. A total of 155 articles were included for full-text review. Finally, 81 articles remained that fulfilled the inclusion criteria. Conclusions: Even though there are solid results supporting a variety of treatments for SRMH, the best treatment modality has still not been conclusively demonstrated and further research is needed. [ABSTRACT FROM AUTHOR]

Vitreous substitutes are indispensable tools in vitreoretinal surgery. The two crucial functions of these substitutes are their ability to displace intravitreal fluid from the retinal surface and to allow the retina to adhere to the retinal pigment epithelium. Today, vitreoretinal surgeons can choose among a plethora of vitreous tamponades, and the tamponade of choice might be difficult to determine in the ever-expanding range of possibilities for a favorable outcome. The currently available vitreous substitutes have disadvantages that need to be addressed to improve the surgical outcome achievable today. Herein, the fundamental physical and chemical proprieties of all vitreous substitutes are reported, and their use and clinical applications are described alongside some surgical techniques of intra-operative manipulation. The major upcoming developments in vitreous substitutes are extensively discussed, keeping a translational perspective throughout. Conclusions on future perspectives are derived through an in-depth analysis of what is lacking today in terms of desired outcomes and biomaterials technology. [ABSTRACT FROM AUTHOR]

Synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy is a non-destructive and chemically sensitive technique for the rapid detection of changes in the different components of the cell's biomacromolecular profile. Reactive oxygen species and oxidative stress may cause damage to the DNA, RNA, and proteins in the retinal pigment epithelium (RPE), which can further lead to age-related macular degeneration (AMD) and visual loss in the elderly. In this study, human primary RPEs (hRPEs) were used to study AMD pathogenesis by using an established in vitro cellular model of the disease. Autophagy—a mechanism of intracellular degradation, which is altered during AMD, was studied in the hRPEs by using the autophagy inducer rapamycin and treated with the autophagy inhibitor bafilomycin A1. In addition, oxidative stress was induced by the hydrogen peroxide (H2O2) treatment of hRPEs. By using SR-FTIR microspectroscopy and multivariate analyses, the changes in the phosphate groups of nucleic acids, Amide I and II of the proteins, the carbonyl groups, and the lipid status in the hRPEs showed a significantly different pattern under oxidative stress/autophagy induction and inhibition. This biomolecular fingerprint can be evaluated in future drug discovery studies affecting autophagy and oxidative stress in AMD. [ABSTRACT FROM AUTHOR]

The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of the basic biology, including the genetics and molecular pathways of RPE development. It encompasses basic developmental biology and stem cell-based developmental studies to uncover RPE differentiation. Knowledge of the in utero developmental cues provides an inclusive methodology required for deriving RPEs using stem cells. [ABSTRACT FROM AUTHOR]

Purpose: Retinal ischemia (RI) and progressive neuronal death are sight-threatening conditions. Mitochondrial (mt) dysfunction and fusion/fission processes have been suggested to play a role in the pathophysiology of RI. This study focuses on changes in the mt parameters of the neuroretina, retinal pigment epithelium (RPE) and choroid in a porcine high intraocular pressure (IOP)-induced RI minipig model. Methods: In one eye, an acute IOP elevation was induced in minipigs and compared to the other control eye. Activity and amount of respiratory chain complexes (RCC) were analyzed by spectrophotometry and Western blot, respectively. The coenzyme Q10 (CoQ10) content was measured using HPLC, and the ultrastructure of the mt was studied via transmission electron microscopy. The expression of selected mt-pathway genes was determined by RT-PCR. Results: At a functional level, increased RCC I activity and decreased total CoQ10 content were found in RPE cells. At a protein level, CORE2, a subunit of RCC III, and DRP1, was significantly decreased in the neuroretina. Drp1 and Opa1, protein-encoding genes responsible for mt quality control, were decreased in most of the samples from the RPE and neuroretina. Conclusions: The eyes of the minipig can be considered a potential RI model to study mt dysfunction in this disease. Strategies targeting mt protection may provide a promising way to delay the acute damage and onset of RI. [ABSTRACT FROM AUTHOR]

Purpose: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. Methods: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L‐lactide‐co‐DL‐lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. Results: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real‐time qPCR (RT‐qPCR) and electron microscopy. RT‐qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde‐binding protein 1), RPE65 (retinal pigment epithelium‐specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia‐associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+/K+ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α‐SMA proved the stability of the RPE polarization state and the absence of epithelial‐to‐mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. Conclusions: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long‐term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA‐cultured RPEs are a plausible source for the replacement of non‐functioning RPEs during cell therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: Retinal diseases are closely associated with both decreased oxygenation and increased inflammation. It is not known if hypoxia-induced vascular endothelial growth factor (VEGF) expression in the retina itself evokes inflammation, or whether inflammation is a prerequisite for the development of neovascularization. Methods: Human ARPE-19 cell line and primary human retinal pigment epithelium (RPE) cells were used. ARPE-19 cells were kept either under normoxic (24 h or 48 h) or hypoxic conditions (1% O, 24 h). Part of the cells were re-oxygenated (24 h). Some ARPE-19 cells were additionally pre-treated with bacterial lipopolysaccharide (LPS). The levels of IL-6, IL-8, IL-1β, and IL-18 were determined from medium samples by an enzyme-linked immunosorbent assay (ELISA) method. Primary human RPE cells were exposed to hypoxia for 24 h, and the subsequent release of IL-6 and IL-8 was measured with ELISA. VEGF secretion from ARPE-19 cells was determined up to 24 h. Results: Hypoxia induced significant ( P < 0.01) increases in the levels of both IL-6 and IL-8 in ARPE-19 cells, and LPS pre-treatment further enhanced these responses. Hypoxia exposure did not affect the IL-1β or IL-18 release irrespective of LPS pre-treatment. If primary RPE cells were incubated for 4 h in hypoxic conditions, IL-6 and IL-8 concentrations were increased by 7 and 8-fold respectively. Hypoxia increased the VEGF secretion from ARPE-19 cells in a similar manner with or without pre-treatment with LPS. Conclusions: Hypoxia causes an inflammatory reaction in RPE cells that is potentiated by pre-treatment with the Toll-like receptor-activating agent, LPS. The secretion of VEGF from these cells is regulated directly by hypoxia and is not mediated by inflammation. [ABSTRACT FROM AUTHOR]

Purpose: There are several retinal degenerative diseases including age‐related macular degeneration (AMD) where the retinal pigment epithelium (RPE) is affected primarily and photoreceptors secondarily. The RPE replacement based on changing of diseased RPE by ex vivo cultured iPSC‐derived RPE cells on nanofibrous membranes can prolong the survival of adjacent photoreceptors. Methods: The feeder‐free system of culturing and differentiation of human iPSCs was used. The phenotypic maturation was examined using immunolabelling for the differentiation and polarization markers. Scaffolds with confluent human iPSC‐derived RPE were implanted into the right eye of 7 minipigs using 3‐port pars plana vitrectomy. Because the human cells were transplanted into the porcine eye (xenogeneic transplantation), immunosuppression therapy with Tacrolimus was applied. At 1–4 weeks upon transplantation, noninvasive examinations of the implanted retina were performed by OCT and fundus camera. Results: The iPSC‐derived RPE cells showed typical cellular morphology and express the RPE markers such as ZO1, BEST, RPE65, and others, with no traces of pluripotent stem cells. Scaffolds with seeded RPE were successfully implanted into subretinal space of porcine eyes. OCT and fundus camera examinations were able to localize the implant in the retina and demonstrated a normal neuroretina in the place of the implant with healed retinotomy, without inflammatory reaction on the implant. Conclusions: In conclusion, we can state that the functional and morphological properties of human iPSC‐derived RPE cells cultured on nanomembranes confirmed the good quality of this epithelium including proper differentiation, confluency of the polarized monolayer, and ability to long‐term survival. The correct placing of implants in the subretinal space testified to the rightness of employed surgical techniques and functionality of the delivery instrumentarium. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the mechanism by which resveratrol acts upon retinal pigment epithelial (RPE) cells and to characterize its effect upon autophagy, survival, and inflammation, with consequent implications to treatment for age-related macular degeneration (AMD). Methods: Cultured ARPE-19 cells were exposed to 10 and 50 μM resveratrol. Cell survival/death was determined by annexin-FITC/propidium iodide using flow cytometry, while autophagy was studied by detecting autophagic vacuoles formation (acridine orange and transmission electron microscopy), as well as LC3II/I ratio and p62 expression by Western blot. In addition, time-lapse confocal microscopy of a pDENDRA-LC3 expression vector was performed to detect autophagy in transfected ARPE-19 cells under the different treatment conditions. Inhibition of proteasomal and autophagy-lysosomal fusion was carried out by MG-132 and chloroquine, respectively, while induction of autophagy was achieved by rapamycin treatment. Detection of secreted cytokines by ARPE-19 cells using Human XL Cytokine Array was performed under oxidative stress (H2O2) and resveratrol treatments, respectively. Results: Resveratrol induced autophagy in ARPE-19 cells as determined by augmented presence of autophagic vacuoles, increased LC3II/I ratio and decreased p62 expression, as well as time-lapse confocal microscopy using pDENDRA-LC3 expression vector. Resveratrol acted similarly to proteasomal inhibition and downstream of mammalian target of rapamycin (mTOR), since upstream inhibition of autophagy by 3-methyladenine could not inhibit autophagy in ARPE-19 cells. Co-treatmeant by rapamycin and/or proteasome inhibition showed no additive effect upon autophagy induction. ARPE-19 cells treated by resveratrol showed lower cell death rate compared to untreated controls. Resveratrol induced a specific anti-inflammatory response in ARPE-19 cells. Conclusions: Resveratrol can induce autophagy, pro-survival, and anti-inflammatory stimuli in ARPE-19 cells, properties which could be plausible to formulate future treatment modalities for AMD. [ABSTRACT FROM AUTHOR]

Inefficient removal of dying retinal pigment epithelial (RPE) cells by professional phagocytes can result in debris formation and development of age-related macular degeneration (AMD). Chronic oxidative stress and inflammation play an important role in AMD pathogenesis. Only a few well-established in vitro phagocytosis assay models exist. We propose human embryonic stem cell-derived-RPE cells as a new model for studying RPE cell removal by professional phagocytes. The characteristics of human embryonic stem cells-derived RPE (hESC-RPE) are similar to native RPEs based on their gene and protein expression profile, integrity, and barrier properties or regarding drug transport. However, no data exist about RPE death modalities and how efficiently dying hESC-RPEs are taken upby macrophages, and whether this process triggers an inflammatory responses. This study demonstrates hESC-RPEs can be induced to undergo anoikis or autophagy-associated cell death due to extracellular matrix detachment or serum deprivation and hydrogen-peroxide co-treatment, respectively, similar to primary human RPEs. Dying hESC-RPEs are efficiently engulfed by macrophages which results in high amounts of IL-6 and IL-8 cytokine release. These findings suggest that the clearance of anoikic and autophagy-associated dying hESC-RPEs can be used as a new model for investigating AMD pathogenesis or for testing the in vivo potential of these cells in stem cell therapy. [ABSTRACT FROM AUTHOR]

Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD. [ABSTRACT FROM AUTHOR]

Background The apopto-phagocytic gene expression patterns during clearance of dying cells in the retina and the effect of triamcinolone (TC) upon these processes have relevance to development of age-related macular degeneration (AMD). Methods ARPE-19 cells and primary human retinal pigment epithelium (hRPE) were induced to undergo cell death by anoikis and the clearance of these cells by living hRPE/ARPE-19 or human monocyte-derived macrophages (HMDMs) in the presence or absence of TC was quantified by flow cytometry. TaqMan low-density gene expression array determining known markers of phagocytosis and loss-of-function studies on selected apopto-phagocytic genes was carried out in HMDM engulfing anoikic cells. Results The glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDM engulfing anoikic ARPE-19 or hRPE cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and thrombospondin-1 (THSB-1). The key role of the MERTK could be demonstrated in HMDM engulfing dying cells using gene silencing as well as blocking antibodies. Similar pathways were found upregulated in living ARPE-19 engulfing anoikic ARPE-19 cells. Gas6 treatment enhanced phagocytosis in TC-treated HMDMs. Conclusions Specific agonists of the Mertk receptor may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy. General significance The use of Gas6 as enhancer of retinal phagocytosis via the MerTK receptor, alone or in combination with other specific ligands of the tyrosine kinase receptors' family may have a potential role in AMD therapy. [ABSTRACT FROM AUTHOR]