Search Results (76)

Galectin-3 is a multifunctional protein that is associated with diseases of the chorioretinal interface, in which the retinal pigment epithelium (RPE) plays a central role in disease development and progression. Since galectin-3 can function extracellularly as well as intracellularly via different mechanisms, we developed an immortalized human RPE cell line (ARPE-19) with a knockdown for galectin-3 expression (ARPE-19/LGALS3^+/−) using a sgRNA/Cas9 all-in-one expression vector. By Western blot analysis, a reduced galectin-3 expression of approximately 48 to 60% in heterozygous ARPE-19/LGALS3^+/− cells was observed when compared to native controls. Furthermore, ARPE-19/LGALS3^+/− cells displayed a flattened, elongated phenotype with decreased E-cadherin as well as enhanced N-cadherin and α-smooth muscle actin mRNA expression, indicating an epithelial–mesenchymal transition of the cells. Compared to wildtype controls, ARPE-19/LGALS3^+/− cells had significantly reduced metabolic activity to 86% and a substantially decreased proliferation to 73%. Furthermore, an enhanced cell adhesion and a diminished migration of immortalized galectin-3 knockdown RPE cells was observed compared to native ARPE-19 cells. Finally, by Western blot analysis, reduced pAKT, pERK1/2, and β-catenin signaling were detected in ARPE-19/LGALS3^+/− cells when compared to wildtype controls. In summary, in RPE cells, endogenous galectin-3 appears to be essential for maintaining the epithelial phenotype as well as cell biological functions such as metabolism, proliferation, or migration, effects that might be mediated via a decreased activity of the AKT, ERK1/2, and β-catenin signaling pathways. Full article

Saffron, branded as Repron^®, is effective in slowing the progression of several neurodegenerative diseases. Its production, however, requires specific cultivation techniques and procedures that, together with low yields, make it expensive. To address this challenge, hydroponic cultivation has been adopted. Previous studies have shown that hydroponically cultivated saffron and Repron^® share comparable chemical compositions and neuroprotective effects under oxidative stress conditions. In this study, we evaluated the protective properties of extracts derived from Crocus sativus L. waste, compared with those of saffron derived from stigmas. Human retinal pigment epithelium (ARPE-19) cells were pre-treated with extracts of various plant waste fractions before being subjected to three stress conditions: H₂O₂-induced oxidative stress (500 μM, 3 h), lipopolysaccharide (LPS; 0.25 mg/mL, 24 h), and hyperglycemia (25 mM glucose, 96 h). Saffron Repron^® served as a positive control. The results revealed that the extract derived from C. sativus waste had superior protective effects against oxidative stress and inflammation by preserving the state of the mitochondria and tight junctions (ZO-1); conversely, the tepal extract alone was more effective under hyperglycemic conditions by also modulating acrolein levels. These results suggest that different plant fractions contain bioactive compounds with specific protective actions, which together lead to increased cell survival. Full article

Lipid metabolism and oxidative stress are major contributors to ocular diseases, including drusen formation and photoreceptor damage. Aster-B, encoded by GRAMD1B, mediates the non-vesicular transport of cholesterol and carotenoids and is highly expressed in the human eye, though its specific ocular functions remain unknown. We investigated Aster-B’s role in ARPE-19 cells, a model of the retinal pigment epithelium (RPE), using CRISPR/dCas9 to generate an Aster-B-expressing cell line. Aster-B expression significantly improved cell survival under oxidative stress induced by hydrogen peroxide (H₂O₂) and was associated with the activation of the p53 and TGFβ signaling pathways, indicating a role in modulating stress responses. To confirm its lipid transport activity, we treated cholesterol-depleted cells with carotenoids and tracked their localization. In Aster-B-expressing cells, carotenoids accumulated in mitochondria, while in control cells, they remained in other cellular compartments. Under oxidative stress, mitochondrial carotenoid levels declined in Aster-B-expressing cells but not in control cells. Interestingly, carotenoids enhanced survival in control cells exposed to H₂O₂ but had a detrimental effect in Aster-B-expressing cells, suggesting that carotenoid function is context and location dependent. These findings highlight Aster-B’s role in coordinating lipid transport and stress responses in the RPE, with implications for oxidative stress-related eye diseases. Full article

Fucoidans from Laminaria hyperborea (LH) can be antioxidative, antiangiogenic, and anti-inflammatory. In this study, a very high-molecular weight (3700 kDa) fucoidan from LH, FucBB04, was tested regarding its bioactivity in age-related macular degeneration (AMD) models in vitro. Primary retinal pigment epithelium (RPE) from pig eyes, human uveal melanoma cell line OMM-1, and RPE cell line ARPE-19 were used. Substituents of the extract were determined with chemical analysis. Cell viability was tested with tetrazolium assay (MTT), oxidative stress was induced by H₂O₂ or erastin, respectively. Secreted vascular endothelial growth factor A (VEGF-A) was assessed with ELISA. Retinal pigment epithelium 65 kDa protein (RPE65) and protectin (CD59) protein expression were tested in Western blot. Cell barrier was assessed by measuring trans-epithelial electrical resistance (TEER), phagocytic ability by a fluorescence assay. Gene expression and secretion of interleukin 6 (IL-6) and interleukin 8 (IL-8) were tested in real-time PCR and ELISA. FucBB04 displayed no oxidative stress protective effects. Its effect on VEGF was inconsistent, with VEGF secretion reduced in primary RPE, but not in ARPE-19. On the other hand, Lipopolysaccharide (LPS) and polyinosinic/polycytidylic acid (PIC)-induced IL-6 or IL-8 secretion was reduced by FucBB04, while complement inhibiting protein CD59 was not affected. In addition, FucBB04 did not influence the gene expression of IL-6 or IL-8. Visual cycle protein RPE65 expression, phagocytic ability, and barrier function were reduced by FucBB04. Very high-molecular weight fucoidan from LH shows bioactivities against AMD-related pathological pathways, but adverse effects on RPE function may limit its suitability as a therapeutic compound. Smaller high-molecular weight fucoidans are recommended for further research. Full article

High glucose levels may cause vascular alterations in patients with diabetes, which can lead to complications such as diabetic retinopathy—an abnormal growth of retinal blood vessels. The micro-RNA miR-205-5p is known to regulate angiogenesis by modulating the expression of the vascular endothelial growth factor (VEGFA) in different systems. This study investigates the role of miR-205-5p in controlling VEGFA expression both in vitro and in the eye under hyperglycemic conditions. An alloxan-induced diabetic mouse model and retinal pigment epithelium human cell line (ARPE-19) were exposed to high glucose and treated with an ectopic miR-205-5p mimic. VEGFA mRNA and protein levels were assessed using qRT-PCR, Western blot, and immunocytochemistry. Additionally, human umbilical vein endothelial cells (HUVECs) were employed to evaluate angiogenesis. Our results show that high glucose significantly reduced miR-205-5p levels while upregulating VEGFA expression in both ARPE-19 cells and diabetic mice. The ectopic administration of miR-205-5p (via transfection or intravitreal injection) restored VEGFA levels and inhibited angiogenesis in HUVEC cultures. Based on these preliminary data, we suggest a potential therapeutic strategy against VEGFA involving miR-205-5p in proliferative eye-related vascular disorders. Full article

Yeast TIM8 was initially identified as a homolog of human TIMM8A/DDP1, which is associated with human deafness–dystonia syndrome. Tim8p is located in the mitochondrial intermembrane space and forms a hetero-oligomeric complex with Tim13p to facilitate protein transport through the TIM22 translocation system. Previous research has indicated that TIM8 is not essential for yeast survival but does affect the import of Tim23p in the absence of the Tim8-Tim13 complex. Previous research on TIM8 has focused mainly on its involvement in the mitochondrial protein transport pathway, and the precise biological function of TIM8 remains incompletely understood. In this study, we provide the first report that yeast TIM8 is associated with the endoplasmic reticulum (ER) stress response and chronological senescence. We found that deletion of TIM8 leads to both oxidative stress and ER stress in yeast cells while increasing resistance to the ER stress inducer tunicamycin (TM), which is accompanied by an enhanced basic unfolded protein response (UPR). More importantly, TIM8 deficiency can lead to a shortened chronological lifespan (CLS) but does not affect the replicative lifespan (RLS). Moreover, we found that improving the antioxidant capacity further increased TM resistance in the tim8Δ strain. Importantly, we provide evidence that the knockdown of TIMM8A in ARPE-19 human retinal pigment epithelium cells can also induce ER stress, suggesting the potential function of the TIM8 gene in ER stress is conserved from budding yeast to higher eukaryotes. In summary, these results suggest novel roles for TIM8 in maintaining ER homeostasis and CLS maintenance. Full article

The retinal pigment epithelium (RPE) contributes to retinal homeostasis, and its metabolic dysfunction is implied in the development of retinal degenerative disease. The isoform M2 of pyruvate kinase (PKM2) is a key factor in cell metabolism, and its function may be affected by insulin-like growth factor 1 (IGF-1). This study aims to investigate the effect of IGF-1 on PKM2 modulation of RPE cells and whether co-treatment with klotho may preserve it. ARPE-19 cells, an ex vivo model of human pigmented epithelium, were exposed to IGF-1. Then, we evaluated PKM2 expression, dimerization and subcellular localization, energy metabolism, and redox balance, and whether pre-treatment with Klotho may antagonize the effects of IGF-1. The results show that IGF-1 favors PKM2 dimerization, thus reducing the activity of PKM2 and leading to an altered cellular energy status coupled with reduced oxidative stress. In conclusion, PKM2 plays a pivotal role in the modulation of RPE metabolism and redox balance and could explain the mechanisms through which IGF-1 participates in the pathogenesis of some retinal diseases. Klotho may exert protective effects by mitigating the IGF-1 signal and its effect on mitochondrial function. Full article

To elucidate the unidentified roles of a selective peroxisome proliferator-activated receptor α (PPARα) agonist, pemafibrate (Pema), on the pathogenesis of retinal ischemic diseases (RID)s, the pharmacological effects of Pema on the retinal pigment epithelium (RPE), which is involved in the pathogenesis of RID, were compared with the pharmacological effects of the non-fibrate PPARα agonist GW7647 (GW). For this purpose, the human RPE cell line ARPE19 that was untreated (NT) or treated with Pema or GW was subjected to Seahorse cellular metabolic analysis and RNA sequencing analysis. Real-time cellular metabolic function analysis revealed that pharmacological effects of the PPARα agonist actions on essential metabolic functions in RPE cells were substantially different between Pema-treated cells and GW-treated cells. RNA sequencing analysis revealed the following differentially expressed genes (DEGs): (1) NT vs. Pema-treated cells, 37 substantially upregulated and 72 substantially downregulated DEGs; (2) NT vs. GW-treated cells, 32 substantially upregulated and 54 substantially downregulated DEGs; and (3) Pema vs. GW, 67 substantially upregulated and 51 markedly downregulated DEGs. Gene ontology (GO) analysis and ingenuity pathway analysis (IPA) showed several overlaps or differences in biological functions and pathways estimated by the DEGs between NT and Pema-treated cells and between NT and GW-treated cells, presumably due to common PPARα agonist actions or unspecific off-target effects to each. For further estimation, overlaps of DEGs among different pairs of comparisons (NT vs. Pema, NT vs. GW, and Pema vs. GW) were listed up. Angiopoietin-like 4 (ANGPTL4), which has been shown to cause deterioration of RID, was the only DEG identified as a common significantly upregulated DEG in all three pairs of comparisons, suggesting that ANGPTL4 was upregulated by the PPARα agonist action but that its levels were substantially lower in Pema-treated cells than in GW-treated cells. In qPCR analysis, such lower efficacy for upregulation of the mRNA expression of ANGPTL4 by Pema than by GW was confirmed, in addition to substantial upregulation of the mRNA expression of HIF1α by both agonists. However, different Pema and GW-induced effects on mRNA expression of HIF1α (Pema, no change; GW, significantly downregulated) and mRNA expression of ANGPTL4 (Pema, significantly upregulated; GW, significantly downregulated) were observed in HepG2 cells, a human hepatocyte cell line. The results of this study suggest that actions of the PPARα agonists Pema and GW are significantly organ-specific and that lower upregulation of mRNA expression of the DR-worsening factor ANGPTL4 by Pema than by GW in ARPE19 cells may minimize the risk for development of RID. Full article

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. Full article

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. Full article

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE. Full article

Mitochondrial malfunction, excessive production of reactive oxygen species (ROS), deficient autophagy/mitophagy, and chronic inflammation are hallmarks of age-related macular degeneration (AMD). Metformin has been shown to activate mitophagy, alleviate inflammation, and lower the odds of developing AMD. Here, we explored the ability of metformin to activate mitophagy and alleviate inflammation in retinal pigment epithelium (RPE) cells. Human ARPE-19 cells were pre-treated with metformin for 1 h prior to exposure to antimycin A (10 µM), which induced mitochondrial damage. Cell viability, ROS production, and inflammatory cytokine production were measured, while autophagy/mitophagy proteins were studied using Western blotting and immunocytochemistry. Metformin pre-treatment reduced the levels of proinflammatory cytokines IL-6 and IL-8 to 42% and 65% compared to ARPE-19 cells exposed to antimycin A alone. Metformin reduced the accumulation of the autophagy substrate SQSTM1/p62 (43.9%) and the levels of LC3 I and II (51.6% and 48.6%, respectively) after antimycin A exposure. Metformin also increased the colocalization of LC3 with TOM20 1.5-fold, suggesting active mitophagy. Antimycin A exposure increased the production of mitochondrial ROS (226%), which was reduced by the metformin pre-treatment (84.5%). Collectively, metformin showed anti-inflammatory and antioxidative potential with mitophagy induction in human RPE cells suffering from mitochondrial damage. Full article

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells. Full article

To characterize transforming growth factor-β (TGF-β) isoform (TGF-β1~3)-b’s biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including collagen 1 (COL1), COL4, and COL6; α-smooth muscle actin (αSMA); hypoxia-inducible factor 1α (HIF1α); and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α), a master regulator for mitochondrial respiration;, tight junction-related molecules, Zonula occludens-1 (ZO1) and E-cadherin; and vascular endothelial growth factor (VEGF); (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-β isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-β1 and TGF-β3 caused a marked increase in TEER values, and TGF-β2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-β isoforms, among which TGF-β1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-β2 and TGF-β3 or by TGF-β1 and TGF-β3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-β isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-β-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-β isoforms and oxygen levels, suggesting that pathological conditions including epithelial–mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors. Full article

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes’ transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4. Full article