ARPE-19 Cells Research Articles

Thrombospondin 1 Mediates Autophagy Upon Inhibition of the Rho-Associated Protein Kinase Inhibitor

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Investigating p.Ala1035Val in NPC1: New Cellular Models for Niemann–Pick Type C Disease

Niemann–Pick type C (NPC) is a lysosomal storage disorder (LSD) caused by pathogenic variants in either the NPC1 or NPC2 genes, which encode proteins involved in the lysosomal export of unesterified cholesterol. In patients of Western European descent, the p.Ile1061Thr variant in NPC1 is especially prevalent. However, mounting evidence has positioned p.Ala1035Val as the most common variant in Portugal and the second most prevalent variant worldwide. By analyzing 10 Portuguese NPC patients homozygous for p.Ala1035Val, we found an SNP in cis on position 858 (p.Ile858Val), which we hypothesize could have a disease-modifying effect. To address this query, we created variant-specific in vitro models of NPC by stably transducing NPC1−/− ARPE-19 cells with constructs encoding different fluorescently-tagged variants of NPC1, which we used, alongside patient-derived skin fibroblasts, to investigate lysosomal positioning and the trafficking routes elicited by p.Ile1061Thr and p.Ala1035Val (with and without the p.Ile858Val SNP in cis). Our results corroborate the previously described decrease in p.Ile1061Thr-NPC1 trafficking to the lysosome and suggest a similar, if not worse, scenario for the p.Ala1035Val variant, especially when in cis with p.Ile858Val. This is the first reported functional study addressing the impact of the p.Ala1035Val variant at the cellular level, paving the way for novel therapeutic options.

M6A-methylase METTL3 promotes retinal angiogenesis through modulation of metabolic reprogramming in RPE cells

Retinal neovascularization (RNV) disease is one of the leading causes of blindness, yet the molecular underpinnings of this condition are not well understood. To delve into the critical aspects of cell-mediated angiogenesis, we analyzed our previously published single-cell data. Our analysis revealed that retinal pigment epithelium (RPE) cells serve a crucial promotional function in angiogenesis. RPE cells were regulated by N6-methyladenosine (m6A). Next, we detected several critical m6A methylase in hypoxic ARPE-19 cells and in oxygen-induced retinopathy (OIR) mice, our results revealed a significant decrease in the level of methyltransferase like 3 (METTL3). METTL3 specific inhibitor STM2457 intravitreal injection or METTL3 conditional knockout mice both showed a significantly reduced neovascularization area of retina. Additionally, the angiogenesis-related abilities of human retinal endothelial cells (HRECs) were diminished after co-cultured with ARPE-19 treated with STM2457 or sh-METTL3 in vitro. Furthermore, through the integration of Methylated RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing, we discovered that the metabolic enzyme quinolinate phosphoribosyltransferase (QPRT) was directly modified by METTL3 and recognized by the YTH N6-methyladenosine RNA binding protein C1 (YTHDC1). Moreover, after over-expressing QPRT, the angiogenic abilities of HRECs were improved through the phosphorylated phosphatidylinositol-3-kinase (p-PI3K)/ phosphorylated threonine kinase (p-AKT) pathway. Collectively, our study provided a novel therapeutic target for retinal angiogenesis.

PHB2 alleviates retinal pigment epithelium cell fibrosis by suppressing the AGE-RAGE pathway.

Fibrosis is the primary cause of retinal detachment and visual decline. Here, we investigated the role of Prohibitin 2 (PHB2) in modulating fibrosis in ARPE-19 cells stimulated by transforming growth factor (TGF)-β2. The proliferation, migration, and apoptosis of ARPE-19 cells were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and flow cytometry assays, and levels of fibrosis-associated and pathway-related proteins were determined by performing western blotting. To examine the mechanisms underlying ARPE-19 cell fibrosis, we performed RNA sequencing, protein-protein interaction network, and enrichment analyses. We detected increases in the expression of the fibrosis-related proteins fibronectin and collagen I in response to TGF-β2 treatment, whereas the expression of PHB2 was downregulated. PHB2 overexpression suppressed the proliferation and migration of TGF-β2-stimulated ARPE-19 cells, promoted apoptosis, and inhibited fibrosis and Smad and non-Smad pathways. PHB2 overexpression inhibited the advanced glycation end-product (AGE)-receptor of advanced glycation end-product (RAGE) pathway activated by TGF-β2 treatment, which contributed to enhancing the effects of PHB2 on cellular processes, fibrosis, and Smad and non-Smad pathways. Conversely, exogenous application of AGE counteracted the effects of PHB2 overexpression. We conclude that by suppressing the AGE-RAGE pathway, PHB2 exerts an inhibitory effect on TGF-β2-induced fibrosis in ARPE-19 cells.

Early silicone oil opacification in the absence of emulsification is caused by an increased level of silicone dioxide.

In October 2023, other cases with early silicone oil opacification within hours after intraocular implantation in the absence of emulsification occurred. While multiple, small series of this phenomenon are now documented during the last decade, the cause was never unmasked. In this study, we analyzed explanted patient samples, unused samples of the affected, and a control batch of the same silicone oil. The oils were examined on their optical and viscosimetric properties, interfacial tension and for presence of particles. Contamination was investigated using Inductively Coupled Plasma - Mass Spectrometry, thermogravimetric analyses, and Raman-spectroscopy. Cytotoxicity examination in ARPE-19 cells was performed. The oil's effect on pH-changes of aqueous solutions and the oil's defoaming ability were analyzed. The affected batch passed all regulatory limits. However, the affected batch showed an elevated turbidity already before implantation. 3000-fold magnification revealed dispersed particles only in the affected batch. Contamination analyses revealed the absence of other elements indicating a silicon-containing contamination. The affected batch showed no in-vitro cytotoxicity but altered viscosimetric properties and a lower pH of the hydrophilic phase after inducing emulsification. Both batches did not show relevant defoaming properties. The results obtained combined with information from suppliers and the manufacturer suggest an elevated level of silica particles as the cause of the opacification. Based on these results, the manufacturer and suppliers of the latest affected batch adjusted cut-off values for turbidity measurements and introduced turbidity measurements of the raw material prior to distillation. Other manufacturers should follow these preventive measures.

Targeting the VEGFR2 signaling pathway for angiogenesis and fibrosis regulation in neovascular age-related macular degeneration

Neovascular age-related macular degeneration (nAMD) is characterized by abnormal blood vessel growth from the choroid, leading to complications and eventual blindness. Despite anti-VEGF therapy, subretinal fibrosis remains a major concern, as VEGF/VEGF receptor-2 (VEGFR2) signaling can contribute to both angiogenesis and fibrosis. For the identification of the aqueous humor proteome, we performed liquid chromatography with tandem mass spectrometry analysis. To investigate the potential therapeutic effects of targeting the VEGF signaling pathway using apatinib, a highly selective VEGFR2 tyrosine kinase inhibitor, this study employed in vitro (THP-1 conditioned media-treated ARPE-19 cells) and in vivo (laser-induced choroidal neovascularization mouse) models of nAMD. This study revealed elevated VEGFR2 protein levels in the aqueous humor of nAMD patients, suggesting a potential target to mitigate neovascularization and fibrosis in nAMD. Apatinib effectively reduced VEGFA and αSMA levels in both in vitro and in vivo models. Moreover, apatinib showed improvement in laser-induced subretinal hyper-reflective lesions. The action mechanism was linked to the inhibition of VEGFR2 activation, leading to the suppression of both angiogenesis and fibrosis through the downregulation of STAT3 phosphorylation. Therefore, the VEGFR2 signaling pathway appears to play a central role in the development of nAMD by regulating both angiogenesis and fibrosis.

Adiponectin-induced activation of ERK1/2 drives fibrosis in retinal pigment epithelial cells.

Adiponectin (APN), a vasoactive cytokine produced by adipocytes, has emerged as a critical player in retinal diseases. Renowned for its antioxidant, anti-angiogenic, and anti-inflammatory properties, APN levels are closely linked to metabolic disorders, such as insulin resistance, obesity, and diabetic retinopathy (DR). Our previous work demonstrated that APN is similar in efficiency as Avastin in limiting neovascularization in retinal endothelial cells. In this study, we analyzed the effect of APN on retinal epithelial cells to understand its potential impact on eye-related pathologies. Overexpression of APN in ARPE-19 cells predominantly yielded the MMW-APN form, accompanied by increased expression of pro-fibrotic markers and decreased levels of tight junction (TJ) proteins, ZO-1, and Occludin. Further, confocal imaging revealed impaired TJ assembly and the integrity of TJ was also compromised as evidenced by the higher paracellular permeability and lower TEER. Besides, rAPN treatment in ARPE-19 cells as well triggered increased expression of pro-fibrotic markers, pro-MMP2, and enhanced cell migration and proliferation. Mechanistically, these pro-fibrotic effects were mediated by APN-induced phosphorylation of ERK1/2, causing RPE cell transdifferentiation. Furthermore, we identified that MMW-APN was the most prevalent form detected in the vitreous humor of proliferative diabetic retinopathy (PDR) patients, emphasizing the clinical relevance of our findings. Overall, our data suggest that APN, particularly its MMW form, induces epithelial-mesenchymal transition (EMT) and fibrosis in RPE cells, potentially driving the angio-fibrotic shift observed in PDR via ERK1/2 activation.

Extracellular Vesicles Derived from Adipose-Derived Mesenchymal Stem Cells Alleviate Apoptosis and Oxidative Stress of Retinal Pigment Epithelial Cells Through Activation of Nrf2 Signaling Pathway.

Purpose: To examine the potential protective effects of adipose-derived mesenchymal stem cell-derived extracellular vesicles (ASC-EVs) on ARPE-19 cells exposed to hydrogen peroxide (H2O2) stress and to evaluate their ability to delay retinal degeneration in Royal College of Surgeons (RCS) rats. Methods: ARPE-19 cells were pre-treated with ASC-EVs for 24 h, followed by exposure to 200 μM H2O2 for an additional 24 h. RCS rats received an intravitreal injection of phosphate-buffered saline in one eye and ASC-EVs in the other eye. Results: ASC-EV pretreatment significantly protected against H2O2 in the Cell Counting Kit-8 assay and was also effective in the lactate dehydrogenase-release assay. It notably reduced early apoptosis (Annexin V-fluorescein isothiocyanate/propidium iodide assay) and late apoptosis (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling assay), while significantly decreasing intracellular reactive oxygen species, glutathione levels, and superoxide dismutase activity. NFE2L2, HMOX1, and NQO1 mRNA levels, along with Nrf2, HO-1, and NQO1 protein levels, were significantly elevated with ASC-EV pretreatment. Compared with ARPE-19-derived EVs, 11 miRNAs were upregulated and 34 were downregulated in ASC-EVs. In RCS rats, intravitreal injections of ASC-EVs led to significant preservation of the outer nuclear layer and photoreceptor segments, along with increased nuclear Nrf2 expression and elevated HO-1 and NQO1 levels in the inner retina. Eyes that received intravitreal injections of ASC-EVs demonstrated significantly preserved electroretinography a- and b-wave amplitudes at 1 week post-injection, though this effect faded by 2 weeks. Conclusions: ASC-EVs mitigated apoptosis and oxidative stress in ARPE-19 cells subjected to H2O2 exposure and temporarily slowed retinal degeneration in RCS rats via Nrf2 pathway activation by miRNAs.

Metabolomic signature in ocular dosing: Exploring the metabolic impacts of sublethal high-dose naringenin on ARPE-19 cells

IntroductionNaringenin (NRG), a flavanone polyphenol found in citrus fruits, has been increasingly recognized for its potential therapeutic effects in ocular disorders. This study aimed to assess the impact of high-dose NRG on human retinal pigment epithelial cells (ARPE-19) cells through metabolomic analysis. MethodsCell viability was evaluated using the thiazolyl blue tetrazolium bromide (MTT) assay, demonstrating non-cytotoxicity at concentrations ranging from 3 to 100 μM within a 24-h exposure period. High-dose (100 μM) NRG was selected for further investigation based on its non-cytotoxic nature. Chromatographic analyses were performed using Liquid Chromatography Quadropole Time-of-Flight Mass Spectrometry (LC-Q-ToF-MS). ResultsMetabolomic analysis revealed subtle metabolic changes, with similar profiles between control and test groups, emphasizing the nuanced effects of NRG. Multivariate analysis, including Principal Component Analysis (PCA), illustrated distinct clustering of control and test groups, indicating consistent metabolic adjustments within each group. Despite maintaining cell viability, stress responses were identified in ARPE-19 cells, as evidenced by reduced glucose-6-phosphate levels and alterations in pyrimidine/purine pathways. This highlights the importance of exploring cellular responses using metabolomics beyond traditional viability metrics. ConclusionIn the broader context, this study contributes data on the interplay between NRG and retinal pigmented epithelial. As NRG is utilized in routine ocular applications, particularly as an eye drop, our findings suggest careful dosage selection, considering both non-irritability and subtle metabolic changes. Further research is needed to refine dosage strategies and comprehensively assess the safety profile of NRG in ocular applications.

PSAT1 is upregulated by METTL3 to attenuate high glucose-induced retinal pigment epithelial cell apoptosis and oxidative stress

BackgroundDiabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported.MethodsHigh-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay.ResultsPSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation.ConclusionMETTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR.

Splice Variant of Retinal G-Protein-Coupled Receptor Deletion-Mediated Dysregulation of Autophagy Increases the Susceptibility to Age-Related Macular Degeneration-Like Defects

Introduction: The splice variant of retinal G-protein-coupled receptor deletion (RGR-d) is a persistent component of drusen and may be involved in the pathogenesis of dry age-related macular degeneration (AMD). Increasing evidence has demonstrated the critical role of autophagy in AMD. In this study, we investigated whether RGR-d disrupts autophagy in early dry AMD in vivo and in vitro. Methods: Fundus imaging and fluoroscopy were performed on RGR-d mice created by multiplex gene editing. The retina microstructure was evaluated by performing hematoxylin and eosin (H&E) staining as well as transmission electron microscopy (TEM). Retinal function was assessed by full-field electroretinography (ERG). After lentivirus transfection and stimulation, the permeability, phagocytosis, and tight junctions of ARPE-19 cells were evaluated. Western blotting of ATG5, Beclin-1, LC3II/I, and P62 was performed to detect the changes in autophagy pathways. Results: Atrophy and patchy penetrating hyperfluorescent foci, consistent with early AMD-like defects, were observed in the fundus of 12-month-old RGR-d mice. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. H&E staining of retinal tissues indicated thinning of each layer of the retinal structure. TEM analysis showed some diffuse granular deposits. And the morphology of choroidal microvascular endothelial cells was degraded and distorted. The morphology of the photoreceptor outer segments showed structural damage, and Bruch’s membrane was thickened. ERG indicated that the photoreceptor of RGR-d mice were dysfunctional. Changes in autophagy-related protein expression were observed in the retinal pigment epithelium and retinal neurepithelium, and autophagy regulation was decreased. Palmitic acid (PA) stimulation caused permeability, phagocytosis, and tight junction dysfunction in cells overexpressing RGR-d. Beclin-1 and LC3II/I expression levels were significantly decreased and that of P62 was elevated in RGR-d cells after PA stimulation. Conclusion: RGR-d disrupts the autophagy pathway, causing the development of an early AMD-like pathophysiology.

Tropisetron attenuates high glucose-induced oxidative stress and inflammation in ARPE-19 cells in vitro via regulating SIRT1/ROCK1 signaling.

Diabetic retinopathy (DR) is the leading cause of acquired blindness in diabetic patients. Tropisetron (TRO) exerts potent therapeutic effects against diabetic tissues. The present study aimed to investigate the effects of TRO on retinal injury under diabetic condition. Human retinal pigment epithelial cell line ARPE-19 was treated with high glucose (HG) for 48 h to mimic hyperglycemia-induced retinal damage and subsequently treated with multiple concentrations of TRO for therapeutic intervention. Cell viability and lactate dehydrogenase (LDH) release were detected to assess cell damage. The production of inflammatory cytokines and oxidative stress-related factors was evaluated by corresponding commercial kits. Cell apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The expression of inflammation-, apoptosis-, and SIRT1/ROCK1-related proteins was examined using western blot analysis. Additionally, ARPE-19 cells were transfected with over-express ROCK1 (Ov-ROCK1) or pretreatment with SIRT1 inhibitor EX527 to perform the rescue experiments. TRO alleviated cell damage in HG-induced ARPE-19 cells through elevating cell viability and reducing LDH release. HG-caused excessive production of TNF-α, IL-1β and IL-6, ROS, malondialdehyde and decreased superoxide dismutase activity were partly inhibited by TRO treatment. HG-induced cell apoptosis, accompanied with the upregulation of proapoptotic proteins and the downregulation of antiapoptotic proteins, was hindered by TRO treatment. HG led to the loss of SIRT1 and an elevation of ROCK1 in ARPE-19 cells, which was reversed following TRO treatment. Furthermore, pretreatment with EX527 or transfected with Ov-ROCK1 partially abolished the protective role of TRO against inflammation, oxidative stress and cell apoptosis in HG-challenged ARPE-19 cells. TRO exerted a protective role against HG-caused ARPE-19 cells inflammation, oxidative stress and cell apoptosis by regulating SIRT1/ROCK1 axis, suggesting that TRO might be therapeutic agent for alleviating retinal pigment epithelial cell damage in DR.

YTHDF1-regulated ALOX5 in retinal pigment epithelial cells under hypoxia enhances VEGF expression and promotes viability, migration, and angiogenesis of vascular endothelial cells

Upregulation of vascular endothelial growth factor (VEGF) and enhanced angiogenesis have been implicated in the severe progression of age-related macular degeneration (AMD). Abnormal arachidonate 5-lipoxygenase (ALOX5) is associated with AMD pathogenesis. However, no reports have shown the causal role of ALOX5 in angiogenesis during AMD. In the present study, ARPE-19 cells were exposed to hypoxia, an inducer of VEGF expression. Potential proteins implicated in AMD progression were predicted using bioinformatics. RNA affinity antisense purification-mass spectrometry (RAP-MS) was applied to identify the binding proteins of ALOX5 3′UTR. Expression of ALOX5 and YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) was detected by qRT-PCR and western blotting. VEGF expression and secretion were assessed by immunofluorescence and ELISA, respectively. The chicken embryo chorioallantoic membrane (CAM) was used to analyze the effect of ALOX5 on angiogenesis. RNA stability was assayed using the Actinomycin D assay. The results show that hypoxia promoted cell growth and increased VEGF expression in ARPE-19 cells. ALOX5 was associated with AMD progression, and hypoxia upregulated ALOX5 expression in ARPE-19 cells. ALOX5 silencing reduced VEGF expression induced by hypoxia in ARPE-19 cells. Moreover, the conditioned medium of ALOX5-silenced ARPE-19 cells could suppress the viability and migration of HUVECs and diminish angiogenesis in the CAM. Furthermore, YTHDF1 was validated to bind to ALOX5 3′UTR, and YTHDF1 promoted ALOX5 expression by elevating the stability of ALOX5 mRNA. In conclusion, our findings demonstrate that YTHDF1-regulated ALOX5 increases VEGF expression in hypoxia-exposed ARPE-19 cells and enhances the viability, migration, and angiogenesis of vascular endothelial cells.

Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro.

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods. We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.

E-cigarette flavoring chemicals and vehicles adversely impact the functions of pigmented human retinal ARPE-19 cells

Electronic cigarettes (ECs) have been shown to adversely impact the human eye's retinal pigment epithelium (RPE). Flavored e-liquids induced cytotoxicity in unpigmented human ARPE-19 cells independent of nicotine's presence in my previous study. In the current study, human ARPE-19 cells pigmented by sepia melanin were employed to examine the effects of four flavoring chemicals, vanillin, menthol, furanone, and cinnamaldehyde, and EC vehicles propylene glycol (PG)/vegetable glycerin (VG) ratios (0:100, 80:20, 100:0% v/v), on metabolic activity, membrane integrity, oxidative stress, and wound healing capacity of these cells. Results demonstrate that cinnamaldehyde was the most cytotoxic flavoring, and all vehicles showed marked cytotoxicity at the highest concentration of 10%. All four flavorings elicited a significant production of reactive oxygen species (ROS), while the three vehicles did not impact ROS levels. Vanillin significantly (p < 0.05) suppressed wound healing, while furanone and cinnamaldehyde had no effects, although menthol promoted wound healing at the lowest concentration. Moreover, the vehicles with two ratios of 0:100 PG/VG and 80:20 PG/VG suppressed wound healing. Together, these results suggest that vanillin and VG-containing vehicles exert the greatest adverse effects on ARPE-19 cells. These findings underscore the potential harm that exposure to ECs can cause to the human retina.

Protective effect of zinc against A2E-induced toxicity in ARPE-19 cells: Possible involvement of lysosomal acidification

A key pathogenic mechanism of dry age-related macular degeneration (AMD) is lysosomal dysfunction in retinal pigment epithelium (RPE) cells, which results in the accumulation of lipofuscins such as A2E (N-retinylidene-N-retinylethanolamine) that further compromises lysosomal function. This vicious cycle leads to cell death and poor visual acuity. Here, we established an in vitro model of AMD by treating a human RPE cell line (ARPE-19) with A2E and examined whether raising zinc levels confers protective effects against lysosomal dysfunction and cytotoxicity. MTT assay showed that A2E induced apoptosis in ARPE-19 cells. pHrodo™ Red fluorescence staining showed that lysosomal pH increased in A2E-treated ARPE-19 cells. Treatment with a zinc ionophore (clioquinol) reduced A2E accumulation, restored lysosomal pH to the acidic range, and reduced A2E-induced cell death, all of which were reversed by the addition of a zinc chelator (TPEN). Consistent with the in vitro results, subretinal injections of A2E in mouse eyes resulted in the death of RPE cells as well as lysosomal dysfunction, all of which were reversed by co-treatment with clioquinol. Our results suggest that restoring the levels of intracellular zinc, especially in lysosomes, would be helpful in mitigating A2E-induced cytotoxic changes including lysosomal dysfunction in RPE cells in the pathogenesis of AMD.

Survey of Dopamine Receptor D2 Antagonists as Retinal Antifibrotics.

Purpose: To evaluate the potency and efficacy of a library of dopamine receptor D2 (D2R) antagonists in the mitigation of fibrotic activation in retinal pigment epithelial (RPE) cells. Methods: ARPE-19 cells were cultured and treated with methotrexate or 27 district D2R antagonists using a fibronectin deposition assay. The most potent compounds were then further assessed in assays measuring cellular proliferation, cellular migration, and profibrotic gene expression. Results: The previously established antifibrotic D2R antagonist loxapine exerted a robust and dose-dependent inhibition of fibronectin deposition, whereas methotrexate exerted minimal inhibition. The most potent D2R antagonist identified, fluphenazine, effectively blocked in vitro models of fibrosis at 300-1,000 nM concentrations. Conclusions: Here we found multiple FDA-approved D2R antagonists that potently block RPE cell fibrogenesis. These findings further support the potential of D2R antagonism as a potential therapeutic for retinal fibrotic disease.

Intravitreal indocyanine green is toxic to the retinal cells

BackgroundIndocyanine green (ICG) is widely used to stain the epiretinal membranes and internal limiting membranes during the pars plana vitrectomy (PPV). This study aims to evaluate the effect of ICG on rat retinas and various retinal cell lines, including ARPE-19 cells, rMC-1 cells, BV2 cells, HRMECs and R28 cells. MethodsICG solutions were prepared and diluted with glucose solution (GS) according to the standard clinical protocols. The retinal cell lines, including ARPE-19 cells, rMC-1 cells, BV2 cells, HRMECs and R28 cells, were treated with the following solutions: normal glucose (NG, 5 mM), GS-1 (92.5 mM glucose), GS-2 (185.02 mM glucose), ICG-1 (92.5 mM glucose + 0.43 mM ICG), or ICG-2 (185.02 mM glucose + 0.86 mM ICG) for durations of 15 or 30 min. In vivo, the right eyes of the rats were intravitreally injected with ICG-1 or ICG-2 (2 μL), while the left eyes were intravitreally injected with GS-1 or GS-2, served as the osmotic controls, for 30 min or 60 min. The rats intravitreally injected with an equivalent volume of NG or 1x phosphate-buffered saline (1x PBS) were served as the normal control or vehicle control. The cell viability was measured with the Cell Counting Kit-8 (CCK-8), while the cell death in retinal cryosections was detected with the TUNEL assay. ResultsThe viabilities of the different retinal cell lines involved in this study were significantly reduced by both ICG-1 and ICG-2 treatments at both time points, with ICG-2 resulting in lower cell viability compared to the NG group and the osmotic control group. Additionally, GS-2 treatment also exhibited a decrease in retinal cell viabilities in vitro. To further confirm these results, intravitreal injection of ICG induced more apoptotic cell death in rat retinas as evidenced by the TUNEL assay. ConclusionsThe exposure of ICG leads to an augmented retinal cell death, which is directly proportional to the concentration and duration of exposure, both in vivo and in vitro. Caution should be exercised during vitrectomy procedures involving ICG administration during clinical practice. It is recommended to advocate for lower concentrations of ICG with reduced exposure time during ocular surgeries.

Protective Effects of Purple Corn (Zea mays L.) Byproduct Extract on Blue Light-Induced Retinal Damage in A2E-Accumulated ARPE-19 Cells.

This study investigated the antioxidative characteristics of Zea mays L. purple corn cob and husk extract (PCHE) and its potential protective effects against blue light (BL)-induced damage in N-retinylidene-N-retinylethanolamine (A2E)-accumulated ARPE-19 retinal pigment epithelial cells. PCHE had a 2,2-diphenyl-1-picrylhydrazyl radical-scavenging capacity and Trolox equivalent antioxidant capacity of 1.28±0.43 mM Trolox equivalents (TE)/g and 2,545.41±34.13 mM TE/g, respectively. Total content of anthocyanins, polyphenols, and flavonoids in the PCHE was 11.13±0.10 mg cyanidin-3-glucoside equivalents/100 g, 227.90±7.38 mg gallic acid equivalents/g, and 117.75±2.46 mg catechin equivalents/g, respectively. PCHE suppressed the accumulation of A2E and the photooxidation caused by BL in a dose-dependent manner. After initial treatment with 25 µM/mL A2E and BL, ARPE-19 cells showed increased cell viability following additional treatment with 15 µg/mL PCHE while the expression of the p62 sequestosome 1 decreased, whereas that of heme oxygenase-1 protein increased compared with that in cells without PCHE treatment. This suggests that PCHE may slow the autophagy induced by BL exposure in A2E-accumulated retinal cells and protect them against oxidative stress.

Unexpected and Synergistical Effects of All-Trans Retinoic Acid and TGF-β2 on Biological Aspects of 2D and 3D Cultured ARPE19 Cells.

Objectives: To study the effects of all-trans retinoic acid (ATRA) on TGF-β2-induced effects of human retinal pigment epithelium cells under normoxia and hypoxia conditions. Methods: Two-dimensionally (2D) and three-dimensionally (3D) cultured ARPE19 cells were subjected to cellular functional analyses by transepithelial electrical resistance (TEER) and an extracellular flux assay (2D), measurement of levels of reactive oxygen species (ROS), gene expression analyses of COL1, αSMA, Zo-1, HIF1α, and PGC1α (2D), and physical property analyses (3D). Results: Under a normoxia condition, treatment with 100 nM ATRA substantially decreased barrier function regardless of the presence of 5 ng/mL TGF-β2 in 2D ARPE19 monolayer cells. Under a hypoxia condition, treatment with ATRA conversely increased barrier function, but the effect was masked by a marked increase in effects induced by TGF-β2. Although ATRA alone did not affect cellular metabolism and ROS levels in 2D ARPE cells, treatment with ATRA under a hypoxia condition did not affect ROS levels but shifted cellular metabolism from mitochondrial respiration to glycolysis. The changes of cellular metabolism and ROS levels were more pronounced with treatment of both ATRA and TGF-β2 independently of oxygen conditions. Changes in mRNA expressions of some of the above genes suggested the involvement of synergistical regulation of cellular functions by TGF-β2 and hypoxia. In 3D ARPE spheroids, the size was decreased and the stiffness was increased by either treatment with TGF-β2 or ATRA, but these changes were unexpectedly modulated by both ATRA and TGF-β2 treatment regardless of oxygen conditions. Conclusions: The findings reported herein indicate that TGF-β2 and hypoxia synergistically and differentially induce effects in 2D and 3D cultured ARPE19 cells and that their cellular properties are significantly altered by the presence of ATRA.