Retinal Pigment Epithelium Damage Research Articles

Fenofibrate Ameliorates Retinal Pigment Epithelium Injury Induced by Excessive Fat Through Upregulation of PI3K/AKT Signaling.

This study aimed to determine the effect and its mechanism of fenofibrate on retinal pigment epithelium (RPE) injury induced by excessive fat in vitro and in vivo. ARPE-19 cells were co-incubated with palmitic acid (PA) and fenofibric acid (the active form of fenofibrate after metabolism in vivo) and mice fed with high-fat diet (HFD) were supplemented with fenofibrate. The following methods were used: Western blot and immunofluorescent staining to determine expressions of reactive oxygen species (ROS)-associated factors and proinflammatory cytokines; electroretinogram (ERG) c-wave to evaluate RPE function; TUNEL staining to detect the apoptotic cell in RPE tissue. Additionally, ARPE19 cells were treated with PI3K/AKT inhibitor or agonist to investigate the mechanism of fenofibric acid inhibiting PA-induced RPE damage. We found that the application of PA inhibited RPE cell viability in a dose-dependent manner, and increased the levels of NAPDH oxidase 4 (NOX4), 3-nitrotyrosin (3-NT), intracellular adhesion molecule-1(ICAM1), tumor necrosis factor alpha (TNFα) and vascular endothelial growth factor (VEGF) at 400μM. The application of fenofibric acid resulted in the inhibition of NOX4, 3-NT, TNFα, ICAM1 and VEGF expression in ARPE-19 cells treated with PA. Moreover, wortmannin, as a selective inhibitor of PI3K/AKT pathway, abolished the effects of fenofibrate on the oxidative stress and inflammation in ARPE-19 cells. In addition, 740Y-P, a selective agonist of PI3K/AKT pathway, enhanced the protective action of fenofibrate. Meanwhile, in vivo dosing of fenofibrate ameliorated the downregulated amplitudes of ERG c-wave in HFD-fed mice and suppressed the HFD-induced oxidative injury and inflammatory response in RPE tissues. Our results suggested that fenofibrate ameliorated RPE cell damage induced by excessive fat in vitro and in vivo, in part, through activation of the PI3K/AKT signaling pathway.

A versatile laser-induced porcine model of outer retinal and choroidal degeneration for preclinical testing.

Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2). We developed a versatile pig model to mimic different types and stages of retinal degeneration. Using an adjustable power micropulse laser, we generated varying degrees of RPE, PR, and CC damage and confirmed the damage by longitudinal analysis of clinically relevant outcomes, including analyses by adaptive optics and optical coherence tomography/angiography, along with automated image analysis. By imparting a tunable yet targeted damage to the porcine CC and visual streak - with a structure similar to the human macula - this model is optimal for testing cell and gene therapies for outer retinal diseases including AMD, retinitis pigmentosa, Stargardt, and choroideremia. The amenability of this model to clinically relevant imaging outcomes will facilitate faster translation to patients.

Investigation of the Influence of Pulse Duration and Application Mode on Microsecond Laser Microsurgery of the Retinal Pigment Epithelium.

Optical microsurgery confined to the retinal pigment epithelium (RPE) requires locally optimized laser parameters and reliable real-time feedback dosimetry (RFD) to prevent unwanted neuroretinal overexposure. This study aimed to compare pulses of different durations and application modes (single, ramp, burst). Moreover, optical coherence tomography (OCT)-based RFD was investigated in an ex vivo experiment, utilizing nine porcine eyes that were exposed to laser pulses of 8, 12, 16 and 20 µs duration (wavelength: 532 nm, exposure area: 90 × 90 µm2, radiant exposure: 247 to 1975 mJ/µm2). Simultaneously, time-resolved OCT M-scans were recorded (central wavelength: 870 nm, scan rate: 85 kHz) for RFD. Post irradiation, retinal changes were assessed with color fundus photography (CFP) and cross-sectional OCT B-scans. RPE cell damage was quantified via fluorescence-based cell viability assay and compared to the OCT dosimetry feedback. Our experiments indicate cumulative RPE damage for pulse bursts of 16 µs and 20 µs, whereas no cumulative effects were found for pulse durations of 8 µs and 12 µs applied in ramp mode. According to statistical analysis, OCT-RFD correctly detected RPE cell damage with 96% sensitivity and 97% specificity using pulses of 8 µs duration in ramp mode.

Pathological Neurovascular Unit Mapping onto Multimodal Imaging in Diabetic Macular Edema.

Diabetic retinopathy is a form of diabetic microangiopathy, and vascular hyperpermeability in the macula leads to retinal thickening and concomitant reduction of visual acuity in diabetic macular edema (DME). In this review, we discuss multimodal fundus imaging, comparing the pathogenesis and interventions. Clinicians diagnose DME using two major criteria, clinically significant macular edema by fundus examination and center-involving diabetic macular edema using optical coherence tomography (OCT), to determine the appropriate treatment. In addition to fundus photography, fluorescein angiography (FA) is a classical modality to evaluate morphological and functional changes in retinal capillaries, e.g., microaneurysms, capillary nonperfusion, and fluorescein leakage. Recently, optical coherence tomography angiography (OCTA) has allowed us to evaluate the three-dimensional structure of the retinal vasculature and newly demonstrated that lamellar capillary nonperfusion in the deep layer is associated with retinal edema. The clinical application of OCT has accelerated our understanding of various neuronal damages in DME. Retinal thickness measured by OCT enables us to quantitatively assess therapeutic effects. Sectional OCT images depict the deformation of neural tissues, e.g., cystoid macular edema, serous retinal detachment, and sponge-like retinal swelling. The disorganization of retinal inner layers (DRIL) and foveal photoreceptor damage, biomarkers of neurodegeneration, are associated with visual impairment. Fundus autofluorescence derives from the retinal pigment epithelium (RPE) and its qualitative and quantitative changes suggest that the RPE damage contributes to the neuronal changes in DME. These clinical findings on multimodal imaging help to elucidate the pathology in the neurovascular units and lead to the next generation of clinical and translational research in DME.

A case of choroidal melanocytosis observed by multimodal imaging with laser speckle flowgraphy

BackgroundChoroidal melanocytosis is characterized by congenital diffuse melanin pigmentation with extensive parenchymal infiltration of spindle cells in the choroid; however, little is known about the choroidal circulation and morphological changes. We herein report a case of choroidal melanocytosis observed by multimodal imaging with laser speckle flowgraphy (LSFG).Case PresentationA 56-year-old woman was referred to our hospital because of serous retinal detachment (SRD) in her left eye. At the initial examination, her best-corrected visual acuity (BCVA) was 1.5 oculus dexter (OD) and 0.8 oculus sinister (OS). An irregular, flat, brownish lesion was noted around the macula OS. Optical coherence tomography showed a choroidal structure with marked hyporeflectivity and SRD where the retinal thickness was preserved. Indocyanine green angiography demonstrated fluorescence blockade throughout. Fundus autofluorescence revealed enlarged macular hypofluorescence, suggesting chronic retinal pigment epithelium damage associated with prolonged SRD. B-mode echography showed no choroidal elevation. Based on the clinical findings, the left eye was diagnosed with choroidal melanocytosis. Four years and 10 months after the initial visit, her BCVA was 0.5 and SRD remained. During the entire period of observation, the mean blur rate (MBR) (mean ± standard deviation) of choroidal blood flow velocity on LSFG was 10.15 ± 0.72 arbitrary units (AU) OD and 1.31 ± 0.06 AU OS.ConclusionChoroidal melanocytosis presented with chronic minor circulatory disturbances due to melanocyte proliferation in the choroid, but the markedly low MBR values by LSFG were dissociated from her retinal thickness and visual function. The proliferation of melanocytes may be a cause of overestimating the cold-color signal of LSFG due to their pigmentation.

SIRT6 overexpression in the nucleus protects mouse retinal pigment epithelium from oxidative stress.

Retinal pigment epithelium (RPE) is essential for the survival of retinal photoreceptors. To study retinal degeneration, sodium iodate (NaIO3) has been used to cause oxidative stress-induced RPE death followed by photoreceptor degeneration. However, analyses of RPE damage itself are still limited. Here, we characterized NaIO3-induced RPE damage, which was divided into three regions: periphery with normal-shaped RPE, transitional zone with elongated cells, and center with severely damaged or lost RPE. Elongated cells in the transitional zone exhibited molecular characteristics of epithelial-mesenchymal transition. Central RPE was more susceptible to stresses than peripheral RPE. Under stresses, SIRT6, an NAD+-dependent protein deacylase, rapidly translocated from the nucleus to the cytoplasm and colocalized with stress granule factor G3BP1, leading to nuclear SIRT6 depletion. To overcome this SIRT6 depletion, SIRT6 overexpression was induced in the nucleus in transgenic mice, which protected RPE from NaIO3 and partially preserved catalase expression. These results demonstrate topological differences of mouse RPE and warrant further exploring SIRT6 as a potential target for protecting RPE from oxidative stress-induced damage.

A CRISPR-Cas9-mediated F0 screen to identify pro-regenerative genes in the zebrafish retinal pigment epithelium

Ocular diseases resulting in death of the retinal pigment epithelium (RPE) lead to vision loss and blindness. There are currently no FDA-approved strategies to restore damaged RPE cells. Stimulating intrinsic regenerative responses within damaged tissues has gained traction as a possible mechanism for tissue repair. Zebrafish possess remarkable regenerative abilities, including within the RPE; however, our understanding of the underlying mechanisms remains limited. Here, we conducted an F0 in vivo CRISPR-Cas9-mediated screen of 27 candidate RPE regeneration genes. The screen involved injection of a ribonucleoprotein complex containing three highly mutagenic guide RNAs per target gene followed by PCR-based genotyping to identify large intragenic deletions and MATLAB-based automated quantification of RPE regeneration. Through this F0 screening pipeline, eight positive and seven negative regulators of RPE regeneration were identified. Further characterization of one candidate, cldn7b, revealed novel roles in regulating macrophage/microglia infiltration after RPE injury and in clearing RPE/pigment debris during late-phase RPE regeneration. Taken together, these data support the utility of targeted F0 screens for validating pro-regenerative factors and reveal novel factors that could regulate regenerative responses within the zebrafish RPE.

USH2A Gene Mutations in Rabbits Lead to Progressive Retinal Degeneration and Hearing Loss.

Mutations in USH2A gene are responsible for the greatest proportion of the Usher Syndrome (USH) population, among which more than 30% are frameshift mutations on exon 13. A clinically relevant animal model has been absent for USH2A-related vision loss. Here we sought to establish a rabbit model carrying USH2A frameshift mutation on exon 12 (human exon 13 equivalent). CRISPR/Cas9 reagents targeting the rabbit USH2A exon 12 were delivered into rabbit embryos to produce an USH2A mutant rabbit line. The USH2A knockout animals were subjected to a series of functional and morphological analyses, including acoustic auditory brainstem responses, electroretinography, optical coherence tomography, fundus photography, fundus autofluorescence, histology, and immunohistochemistry. The USH2A mutant rabbits exhibit hyper-autofluorescent signals on fundus autofluorescence and hyper-reflective signals on optical coherence tomography images as early as 4 months of age, which indicate retinal pigment epithelium damage. Auditory brainstem response measurement in these rabbits showed moderate to severe hearing loss. Electroretinography signals of both rod and cone function were decreased in the USH2A mutant rabbits starting from 7 months of age and further decreased at 15 to 22 months of age, indicating progressive photoreceptor degeneration, which is confirmed by histopathological examination. Disruption of USH2A gene in rabbits is sufficient to induce hearing loss and progressive photoreceptor degeneration, mimicking the USH2A clinical disease. To our knowledge, this study presents the first mammalian model of USH2 showing the phenotype of retinitis pigmentosa. This study supports the use of rabbits as a clinically relevant large animal model to understand the pathogenesis and to develop novel therapeutics for Usher syndrome.

Lycium barbarum Polysaccharides Regulating miR-181/Bcl-2 Decreased Autophagy of Retinal Pigment Epithelium with Oxidative Stress.

Disturbed structure and dysfunction of the retinal pigment epithelium (RPE) lead to degenerative diseases of the retina. Excessive accumulation of reactive oxygen species (ROS) in the RPE is thought to play an important role in RPE dysfunction and degeneration. Autophagy is a generally low-activity degradation process of cellular components that increases significantly when high levels of oxidative stress are present. Agents with antioxidant properties may decrease autophagy and provide protection against RPE dysfunction and damage caused by ROS. Lycium barbarum polysaccharide (LBP) has been widely studied as an antioxidant and cell-protective agent. Therefore, we designed this study to investigate the effects of LBP, which inhibits miR-181, on autophagy in retinal pigment epithelium (RPE) with oxidative stress in vitro and in vivo. In the current study, we found that the highly expressed miR-181 downregulated the expression of Bcl-2 in hydrogen peroxide- (H2O2-) induced ARPE-19 cells, resulting in an increase in ROS, apoptosis, and autophagy flux. LBP inhibited the expression of miR-181, decreased the levels of ROS, apoptosis, and autophagy flux, and increased cell viability in H2O2-induced ARPE-19 cells, suggesting that LBP provides protection against oxidative damage in ARPE-19 cells. We also found that LBP decreased RPE atrophy and autophagy flux in rd10 mice. Taken together, the results showed that LBP has a protective effect for RPE under oxidative stress by inhibiting miR-181 and affecting the Bcl-2/Beclin1 autophagy signaling pathway.

Oxidative stress in retinal pigment epithelium degeneration: from pathogenesis to therapeutic targets in dry age-related macular degeneration.

Age-related macular degeneration is a primary cause of blindness in the older adult population. Past decades of research in the pathophysiology of the disease have resulted in breakthroughs in the form of anti-vascular endothelial growth factor therapies against neovascular age-related macular degeneration; however, effective treatment is not yet available for geographical atrophy in dry age-related macular degeneration or for preventing the progression from early or mid to the late stage of age-related macular degeneration. Both clinical and experimental investigations involving human age-related macular degeneration retinas and animal models point towards the atrophic alterations in retinal pigment epithelium as a key feature in age-related macular degeneration progression. Retinal pigment epithelium cells are primarily responsible for cellular-structural maintenance and nutrition supply to keep photoreceptors healthy and functional. The retinal pigment epithelium constantly endures a highly oxidative environment that is balanced with a cascade of antioxidant enzyme systems regulated by nuclear factor erythroid-2-related factor 2 as a main redox sensing transcription factor. Aging and accumulated oxidative stress triggers retinal pigment epithelium dysfunction and eventually death. Exposure to both environmental and genetic factors aggravates oxidative stress damage in aging retinal pigment epithelium and accelerates retinal pigment epithelium degeneration in age-related macular degeneration pathophysiology. The present review summarizes the role of oxidative stress in retinal pigment epithelium degeneration, with potential impacts from both genetic and environmental factors in age-related macular degeneration development and progression. Potential strategies to counter retinal pigment epithelium damage and protect the retinal pigment epithelium through enhancing its antioxidant capacity are also discussed, focusing on existing antioxidant nutritional supplementation, and exploring nuclear factor erythroid-2-related factor 2 and its regulators including REV-ERBα as therapeutic targets to protect against age-related macular degeneration development and progression.

A cross-sectional study investigating the effects of argon laser retinal photocoagulation on lens clarity and corneal endothelial cells.

The mechanism of argon laser retinal photocoagulation (ALRP) treatment is to apply thermal-induced retinal pigment epithelium damage. Light passes through the anterior optical segments of the eye to reach the retina. Lens densitometry is a noninvasive and quantitative measurement providing information about corneal and lens clarity. This study aimed to investigate whether laser light affects lens clarity and corneal endothelial cells. This was a prospective, cross-sectional study. Lens densitometric (LD) analysis and specular microscopy were performed before, after, and 1 month after ALRP treatment, by an expert ophthalmologist, blinded to the medical status of the patients. LD analysis was performed using a Pentacam HR (Oculus, Wetzlar, Germany) and a Specular Microscope CEM-530 (Nidek, Japan) was used for endothelial cell analysis. The evaluation was made on 81 eyes of 41 patients, with a mean age of 54.46 ± 6.7 years. LD, after ALRP, was significantly more than before ALRP (p < 0.001). There was no statistically significant difference in LD, before ALRP, and 1 month after ALRP (p = 0.262). There was a statistically significant increase in LD after ALRP compared to before ALRP, but it decreased after 1 month. There was an increase in the coefficient of variance (CV) after ALRP compared to before ALRP but it was not statistically significant (p = 0.188). There was no statistically significant difference in CV between before ALRP and 1 month after ALRP (p = 1.000). There was no statistically significant difference in the cell density, the hexagonality percentage between before ALRP, after ALRP, and 1 month after ALRP (p = 0.993, and 0.863, respectively). ALRP may affect the lens densitometry temporarily during the procedure. Thermal damage may be the reason for increased lens densitometry.

Dimethyl Fumarate Protects Retinal Pigment Epithelium from Blue Light-Induced Oxidative Damage via the Nrf2 Pathway.

The purpose of this study is to investigate the protective effect of dimethyl fumarate (DMF), the methyl-ester of fumaric acid, against blue-light (BL) exposure in retinal pigment epithelial (RPE) cells. ARPE-19 cells, a human RPE cell line, were cultured with DMF followed by exposure to BL. Reactive oxygen species (ROS) generation, cell viability, and cell death rate were determined. Real-time polymerase chain reaction and Western blotting were performed to determine the change in nuclear factor (erythroid-derived)-like 2 (NRF2) expression. Twenty-seven inflammatory cytokines in the supernatant of culture medium were measured. BL exposure induced ROS generation in ARPE-19 cells, which DMF alleviated in a concentration-dependent manner. BL exposure increased the ARPE-19 cell death rate, which DMF alleviated. BL exposure induced ARPE-19 cell apoptosis, again alleviated by DMF. Under BL exposure, DMF increased the NRF2 mRNA level and promoted NRF2 expression in the nucleus. BL also strongly increased interleukin (IL)-1β and fibroblast growth factor (FGF) expression. BL strongly induced RPE cell damage with apoptotic change while DMF mainly reduced inflammation in BL-induced RPE damage, resulting in blockade of cell death. DMF has a protective effect in RPE cells against BL exposure via activation of the NRF2 pathway.

Retinal Photoreceptor Protection in an AMD-Related Mouse Model by Selective Sigma-1 or Sigma-2 Receptor Modulation.

The structurally and genetically distinct sigma-1 receptor (S1R) and sigma-2 receptor (S2R) comprise a unique class of drug binding sites. Their alleles are associated with human diseases involving neuronal systems, such as age-related macular degeneration (AMD) characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. Previous studies have suggested neuroprotective benefits for the brain and retina from pharmacological modulation of S1R and/or S2R. However, the effect of such modulation on AMD pathology remains underexplored. Here, we evaluated S1R- or S2R-selective modulation in an AMD-related model of Abca4-/-Rdh8-/- mice with a disrupted visual cycle that predisposes RPE and photoreceptors to illumination-induced damage. For S1R modulation, we used (+)-pentazocine, which is a high-affinity S1R-selective drug. For S2R modulation, we chose CM398, a high-affinity and highly S2R-selective ligand with drug-like properties. Abca4-/-Rdh8-/- mice received a single i.p. injection of (+)-pentazocine or CM398 or vehicle 30 min before illumination. Pretreatment with (+)-pentazocine improved electroretinogram a- and b-waves compared to that with vehicle. Consistently, in another AMD-related mouse model induced by tail-vein injected NaIO3, S1R genetic ablation aggravated photoreceptor loss. In Abca4-/-Rdh8-/- mice, pretreatment with CM398 appeared to partially avert illumination-induced photoreceptor loss and autofluorescent granule formation that signals RPE damage, as revealed by optical coherence tomography. Thus, this study using AMD-related models provides evidence of photoreceptor protection afforded by selective modulation of S1R or S2R.

Retinal protective effect of curcumin metabolite hexahydrocurcumin against blue light-induced RPE damage

BackgroundAge-related macular degeneration (AMD) is a disease of retinal pigment epithelium (RPE) cells. We have previously demonstrated that blue light can damage RPE cells and their underlying mechanisms. We found that hexahydrocurcumin (HHC), a metabolite of curcumin, had better retinal protection than curcumin. However, the involved mechanisms remain unclear. MethodsBy exposing ARPE-19 human RPE cells and mouse primary RPE cells to blue light, the intracellular mechanisms of HHC in cells were investigated, including the proliferation of RPE cells and the effects of HHC on activating intracellular protective mechanisms and related factors. Next-generation sequencing (NGS) RNA sequencing revealed the underlying mechanisms involved in the induction and regulation of HHC treatment following blue light exposure. ResultsHHC promoted autophagy by enhancing autophagic flux, reduced oxidative stress and endoplasmic reticulum (ER) stress, and effectively reversed blue light-induced cell death. RNA sequencing-based bioinformatics approaches comprehensively analyze HHC-mediated cellular processes. ConclusionOur findings elucidate the mechanisms of HHC against blue light damage in RPE cells and are beneficial for the development of natural metabolite-based preventive drugs or functional foods.

Management and diagnosis of subretinal and suprachoroidal haemorrhage

AbstractSubretinal and suprachoroidal haemorrhage, particularly when central macula is involved, represent challenging clinical entities since their natural course, if left untreated, often leads to severe, progressive and irreversible vision loss, due to photoreceptor and retinal pigment epithelium (RPE) damage. Significant variation in prognosis and treatment of different types of haemorrhage dictates the need to establish the level of haemorrhage, which is primarily based on fundus appearance, as haemorrhage in different levels tends to present with a distinct configuration. Additional modalities, such as ultra‐sonography, optical coherence tomography, fluorescein angiography and focal electroretinography, contribute to diagnosis the clinical evaluation.Despite the wide range of techniques described for managing both subretinal and suprachoroidal haemorrhage, there is still no consensus among vitreoretinal surgeons on optimal approach. Pneumatic displacement with or without the intraretinal use of tissue plasminogen activator (t‐PA), pars plana vitrectomy with subretinal t‐PA and gas tamponade, pars plana vitrectomy and retinectomy for clot extraction and co‐treatment with anti‐vascular endothelial growths factors (anti‐VEGFs) and pars plana vitrectomy with choroidal blood drainage, have all been described as effective management approaches. Over the last few years, a trend towards a more timely and less invasive approach has emerged. However, the preferred technique is often determined by the level, the extent and duration of haemorrhage as well as surgeon preference.

Microglial activation and retinal pigment epithelium alteration in a model of focal LED‐induced phototoxicity. Neuroprotection afforded by bFGF and minocycline

AbstractPurpose: To study the microglial reaction and degeneration of the retinal pigment epithelium (RPE) and its protection with basic fibroblast growth factor (bFGF) administered alone or combined with minocycline (MC) in a model of focal light‐emitting diode (LED)‐induced phototoxicity (LIP) in mice.Methods: In dark‐adapted adult C57 mice (20–25 g), the left eye was dilated and exposed for 45 seconds to a blue LED (400 nm; 500 lux) placed 2 mm perpendicular to the corneal apex (n = 71). Mice were treated with bFGF (0.5 μg) administered alone or in combination with MC (45 mg/kg). bFGF was administered in a single intravitreal injection just after LIP and the MC was daily injected intraperitoneally, starting the day before LIP (n = 10–13 per group). Vehicle or naïve groups were used as controls (n = 6–10 each group). Retinas were immunodetected with anti‐arrestin and anti‐Iba1 antibodies to study cone outer segments (a+OS) and microglial cells (Iba1+cells) and RPEs were immunodetected with anti‐ZO1 to study RPE cells, at 3 and 7 days after LIP. The number of cones and microglial cells was studied within a pre‐fixed area (PFA) of 0.9 mm diameter at the centre of the lesion.Results: Treatment with bFGF alone or in combination with MC was effective in protecting a+OS within the PFA compared to vehicles at 7 days after LIP (p = 0.034 and p = 0.001, respectively). The combined treatment of bFGF and MC was effective in reducing the number of Iba1+cells within the PFA in the outer plexiform layer at 7 days after LIP (p = 0.0003). However, the treatments had no restorative effect on RPE cell survival or morphological integrity at 7 days after LIP (p = 0.6762).Conclusions: Administration of bFGF alone or in combination with MC increased a+OS survival and reduces Iba1+cell activation but does not reduce RPE damage.

Inhibition of cellular senescence hallmarks by mitochondrial transplantation in senescence-induced ARPE-19 cells

Retinal pigment epithelium (RPE) damage is a major factor in age-related macular degeneration (AMD). The RPE in AMD shows mitochondrial dysfunction suggesting an association of AMD with mitochondrial function. Therefore, exogenous mitochondrial transplantation for restoring and replacing dysfunctional mitochondria may be an effective therapeutic strategy for AMD. Here, we investigated the effects of extrinsic mitochondrial transplantation on senescence-induced ARPE-19 cells. We demonstrated mitochondrial dysfunction in replicative senescence-induced ARPE-19 cells after repeated passage. Imbalanced mitophagy and mitochondrial dynamics resulted in increased mitochondrial numbers and elevated levels of mitochondrial and intracellular reactive oxygen species. Exogenous mitochondrial transplantation improved mitochondrial dysfunction and alleviated cellular senescence hallmarks, such as increased cell size, increased senescence-associated β-galactosidase activity, augmented NF-κB activity, increased inflammatory cytokines, and upregulated the cyclin-dependent kinase inhibitors p21 and p16. Further, cellular senescence properties were improved by exogenous mitochondrial transplantation in oxidative stress-induced senescent ARPE-19 cells. These results indicate that exogenous mitochondrial transplantation modulates cellular senescence and may be considered a novel therapeutic strategy for AMD.

PATTERN DYSTROPHY-LIKE CHANGES ASSOCIATED WITH A VERY HIGH SERUM FERRITIN LEVEL IN β-THALASSEMIA MAJOR

To report a rare case of pattern dystrophy-like retinal changes in a young β-thalassemia major patient with a very high serum ferritin level. Observational case report of a β-thalassemia major patient with bilateral pattern dystrophy-like retinal changes. An 18-year-old man experienced blurring of vision in both eyes. Patient had a very high serum ferritin level and had a history of using various iron-chelating drugs. Funduscopy revealed bilateral widespread retinal pigment epithelium changes with macular involvement, which resembled pattern dystrophy-like changes. Fundus autofluorescence imaging showed extensive area of decreased autofluorescence signal interspersed with increased autofluorescence signals, whereas optical coherence tomography demonstrated macular retinal pigment epithelium thinning and interdigitation zone disruption corresponding to retinal pigment epithelium damage and atrophy. Pattern dystrophy-like changes should be considered as a possible manifestation of retinal changes in patients with β-thalassemia major. Iron overload, which is indicated by a very high serum ferritin level, might have a role in the formation of this rare retinal manifestation in β-thalassemia major.

Effects of galectin-3 protein on UVA-induced damage in retinal pigment epithelial cells.

Several inflammatory molecules have been suggested as biomarkers of age-related macular degeneration (AMD). Galectin-3 (Gal-3), which has been shown to have a protective role in corneal injury by promoting epithelial cells adhesion and migration to the extracellular matrix, is also highly expressed in the retinal pigment epithelium (RPE) of patients with AMD. This study evaluated the role of Gal-3 in an in vitro model of UVA-induced RPE damage, as a proof-of-concept. ARPE-19 cells (human RPE cell line), were incubated with Gal-3 at 0.5-2.5µg/mL concentrations prior to UVA irradiation for 15, 30, and 45min, which resulted in accumulated doses of 2.5, 5, and 7.5J/cm2, respectively. After 24h incubation, MTT and LDH assays, immunofluorescence, and ELISA were performed. UVA irradiation for 15, 30, and 45min proved to reduce viability in 83%, 46%, and 11%, respectively. Based on the latter results, we chose the intermediate dose (5-J/cm2) for further analysis. Pretreatment with Gal-3 at concentrations > 1.5µg/mL showed to increase the viability of UVA-irradiated cells (~ 75%) compared to untreated cells (64%). Increased levels of cleaved caspase 3, a marker of cell death, were detected in the ARPE cells after UVA irradiation with or without addition of exogenous Gal-3. The inhibitory effect of Gal-3 on UVA-induced cell damage was characterized by decreased ROS levels and increased p38 activation, as detected by fluorescence analysis. In conclusion, our study suggests a photoprotective effect of Gal-3 on RPE by reducing oxidative stress and increasing p38 activation.

A Bioprinted Bruch's Membrane for Modeling Smoke-Induced Retinal Pigment Epithelium Degeneration via Hybrid Membrane Printing Technology.

The retinal pigment epithelium (RPE) not only forms the outer blood-retinal barrier (oBRB) but also plays a multifunctional role in the ocular system. The loss of this epithelium leads to serious diseases resulting in vision impairment. No effective treatment is available for the repair of RPE damage. A functional in vitro RPE model that allows the recapitulation of oBRB-related pathophysiological responses is lacking. Here, a hybrid membrane printing technology is developed to fabricate cellular monolayers on the basement membrane to mimic human Bruch's membrane (BM). Using this technology, in vitro oBRB model containing the RPE monolayer on the printed BM with stable mechanical properties and fibril diameter similar to that of natural BM is developed. Compared to traditional collagen bioink, BM-based bioink significantly promotes RPE functions in vitro. Finally, smoking-like conditions are exposed to the model to recapitulate the absorption of mainstream cigarette smoke which is known as one of the risk factors for the disease progression. RPE function is damaged due to oxidative stress. Furthermore, the versatility of the model as a drug-testing platform is confirmed by the suppression of oxidative stress via antioxidants. This technology shows potential for fabricating a functional oBRB model that reflects patient conditions.