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Molecules (Basel, Switzerland) Dec 2022Esculetin is a coumarin-derived compound with antioxidant and anti-inflammatory properties. The current study aims to evaluate the therapeutic implications of esculetin...
Esculetin is a coumarin-derived compound with antioxidant and anti-inflammatory properties. The current study aims to evaluate the therapeutic implications of esculetin on retinal dysfunction and uncover the underlying mechanisms. -butyl hydroperoxide (-BHP) at a concentration of 300 μM was used to induce oxidative stress in human retinal pigment epithelial cell line (ARPE-19) cells. Esculetin at concentrations below 250 μM did not cause cytotoxicity to ARPE-19 cells. Cell viability analysis confirmed that -BHP induced oxidative injury of ARPE-19 cells. However, ARPE-19 cells were protected from -BHP-induced oxidative injury by esculetin in a concentration-dependent manner. As a result of the TUNEL assay to confirm apoptosis, esculetin treatment reduced the number of TUNEL-positive cells. Esculetin down-regulated the expression levels of Bax, Caspase-3, and PARP and up-regulated the expression level of Bcl2. Collectively, this study demonstrates that esculetin exerts potent antioxidant properties in ARPE-19 cells, inhibiting -BHP-induced apoptosis under the regulation of apoptotic factors.
Topics: Humans; Antioxidants; tert-Butylhydroperoxide; Oxidative Stress; Apoptosis; Epithelial Cells; Retinal Pigments; Retinal Pigment Epithelium; Cell Survival
PubMed: 36558102
DOI: 10.3390/molecules27248970 -
Stem Cell Research & Therapy Sep 2022Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-derived RPE) are a promising source for cell-replacement therapy to treat retinal degenerative...
BACKGROUND
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-derived RPE) are a promising source for cell-replacement therapy to treat retinal degenerative diseases, but research on RPE cryopreservation is limited. This study aimed to determine the best phase for RPE cryopreservation to preserve the post-thaw function and uncover the mechanism underlying RPE freezing tolerance.
METHODS
hESC-derived RPE cells were cryopreserved at various time points after seeding. After thawing, the survival and attachment rates, RPE marker gene expression, apical-basal polarity, PEDF secretion, transepithelial resistance, and phagocytotic ability of post-thaw RPE cells were evaluated. RNA sequencing was performed on RPE cells at three-time points, differentially expressed genes were identified, and gene ontology, Kyoto encyclopedia of genes and genomes, and protein-protein interaction analyses were used to investigate the key pathways or molecules associated with RPE cell freezing tolerance.
RESULTS
RPE frozen at passage 2 day 5 (P2D5) had the highest cell viability and attachment after thawing. They also retained properly localized expression of RPE marker genes and biological functions such as PEDF secretion, high transepithelial resistance, and phagocytic ability. The RNA-sequencing analysis revealed that RPE cells at P2D5 expressed high levels of cell cycle/DNA replication and ECM binding associated genes, as well as THBS1, which may serve as a possible hub gene involved in freezing tolerance. We also confirmed that the RPE cells at P2D5 were in the exponential stage with active DNA replication.
CONCLUSIONS
We propose that freezing hESC-derived RPE cells during their exponential phase results in the best post-thawing outcome in terms of cell viability and preservation of RPE cell properties and functions. The high expression levels of the cell cycle and ECM binding associated genes, particularly THBS1, may contribute to better cell recovery at this stage.
Topics: Cell Differentiation; Cryopreservation; Epithelial Cells; Human Embryonic Stem Cells; Humans; Retinal Pigment Epithelium; Retinal Pigments
PubMed: 36064625
DOI: 10.1186/s13287-022-03141-2 -
Investigative Ophthalmology & Visual... Dec 2023Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of...
PURPOSE
Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles.
METHODS
A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods.
RESULTS
In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin.
CONCLUSIONS
Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.
Topics: Humans; Young Adult; Epithelial Cells; Organelles; Retina; Retinal Pigment Epithelium; Retinal Pigments; Male
PubMed: 38133501
DOI: 10.1167/iovs.64.15.35 -
Investigative Ophthalmology & Visual... Aug 2022To identify key retinal pigment epithelium (RPE) genes linked to the induction of myopia in guinea pigs.
PURPOSE
To identify key retinal pigment epithelium (RPE) genes linked to the induction of myopia in guinea pigs.
METHODS
To induce myopia, two-week-old pigmented guinea pigs (New Zealand strain, n = 5) wore -10 diopter (D) rigid gas-permeable contact lenses (CLs), for one day; fellow eyes were left without CLs and served as controls. Spherical equivalent refractive errors (SE) and axial length (AL) were measured at baseline and one day after initiation of CL wear. RNA sequencing was applied to RPE collected from both treated and fellow (control) eyes after one day of CL-wear to identify related gene expression changes. Additional RPE-RNA samples from treated and fellow eyes were subjected to quantitative real-time PCR (qRT-PCR) analysis for validation purposes.
RESULTS
The CLs induced myopia. The change from baseline values in SE was significantly different (P = 0.016), whereas there was no significant difference in the change in AL (P = 0.10). RNA sequencing revealed significant interocular differences in the expression in RPE of 13 genes: eight genes were significantly upregulated in treated eyes relative to their fellows, and five genes, including bone morphogenetic protein 2 (Bmp2), were significantly downregulated. The latter result was also confirmed by qRT-PCR. Additional analysis of differentially expressed genes revealed significant enrichment for bone morphogenetic protein (BMP) and TGF-β signaling pathways.
CONCLUSIONS
The results of this RPE gene expression study provide further supporting evidence for an important role of BMP2 in eye growth regulation, here from a guinea pig myopia model.
Topics: Animals; Contact Lenses; Disease Models, Animal; Guinea Pigs; Myopia; Retinal Pigment Epithelium; Retinal Pigments; Transcriptome
PubMed: 36006019
DOI: 10.1167/iovs.63.9.25 -
Translational Vision Science &... May 2020This study investigated the effects of esterification and increased lipophilicity on cellular penetration, accumulation and retention in ARPE-19-nic cells using ester...
PURPOSE
This study investigated the effects of esterification and increased lipophilicity on cellular penetration, accumulation and retention in ARPE-19-nic cells using ester functionalized rhodamine B dyes.
METHODS
Rhodamine B was esterified to generate four dyes with increasing lipophilicity. Cellular uptake, retention and mitochondrial localization were investigated in vitro using ARPE-19-nic cells using direct intracellular and extracellular and mitochondrial fluorescence quantitation, confocal and high-resolution live cell imaging and co-localization with Mito-GFP.
RESULTS
Cellular penetrance, mitochondrial accumulation, and retention of the esterified dyes were increased in ARPE-19-nic cells compared with the nonesterified parent dye by direct fluorescence quantitation. Imaging demonstrated intracellular accumulation was confined to mitochondria as confirmed by colocalization with Mito-GFP.
CONCLUSIONS
Esterification is an effective way to increase lipophilicity of a dye to improve cellular penetration of chemical entities. These observations may be key to improving retinal drug delivery for retinal pigment epithelium-based diseases.
TRANSLATIONAL RELEVANCE
Understanding the intracellular distribution of drugs into retinal pigment epithelium cells is a critical component for identifying potential therapies for retinal pigment epithelium-based diseases.
Topics: Biological Transport; Esterification; Retinal Pigment Epithelium; Retinal Pigments; Rhodamines
PubMed: 32821515
DOI: 10.1167/tvst.9.6.18 -
Cells Oct 2022Retinal degenerative diseases are frequently caused by the loss of retinal neural cells such as photoreceptors. Cell replacement is regarded as one of the most promising...
Retinal degenerative diseases are frequently caused by the loss of retinal neural cells such as photoreceptors. Cell replacement is regarded as one of the most promising therapies. Multiple types of stem and somatic cells have been tested for photoreceptor conversion. However, current induction efficiencies are still low and the molecular mechanisms underlying reprogramming remain to be clarified. In this work, by combining treatment with small molecules, we directly reprogrammed human fetal retinal pigment epithelial (RPE) cells into chemically induced photoreceptor-like cells (CiPCs) in vitro. Bulk and single-cell RNA sequencing, as well as methylation sequencing, were performed to understand the transcriptional and epigenetic changes during CiPCs conversion. A multi-omics analysis showed that the direct reprogramming process partly resembled events of early retina development. We also found that the efficiency of CiPCs conversion from RPE is much better than that from human dermal fibroblasts (HDF). The small molecules effectively induced RPE cells into CiPCs via suppression of the epithelial-to-mesenchymal transition (EMT). Among the signaling pathways involved in CiPCs conversion, glutamate receptor activation is prominent. In summary, RPE cells can be efficiently reprogrammed into photoreceptor-like cells through defined pharmacological modulations, providing a useful cell source for photoreceptor generation in cell replacement therapy for retinal degenerative diseases.
Topics: Epigenesis, Genetic; Epithelial Cells; Humans; Retina; Retinal Degeneration; Retinal Pigment Epithelium; Retinal Pigments
PubMed: 36231108
DOI: 10.3390/cells11193146 -
In Vivo (Athens, Greece) 2022The role of epigenetic alterations in the pathogenesis of retinal degenerative diseases, such as macular degeneration is not well established. This study aimed to...
BACKGROUND
The role of epigenetic alterations in the pathogenesis of retinal degenerative diseases, such as macular degeneration is not well established. This study aimed to evaluate whether treatments with gamma-mangostin can rescue the hydrogen peroxide (HO)-induced cytotoxicity in human retinal pigment epithelial (ARPE-19) cells.
MATERIALS AND METHODS
ARPE-19 cells were treated with HO alone or with gamma-mangostin plus HO to investigate changes relating to cell viability, appearance of sub-G1 cells, antioxidant enzymes, and apoptotic-related proteins.
RESULTS
The data showed that under HO treatment of 400 μM, there was a significant decrease in cell viability and enhanced apoptosis, together with an increased expression of Bax, Bad, cleaved-caspase-3, -8, and -9 at the protein level. On the contrary, the protein expression levels of Bcl2 and Bcl-xl were decreased. Gamma-mangostin pre-treatments (2-16 μM) could effectively prevent all alterations.
CONCLUSION
Gamma-mangostin may conduct its eye-protective effects against HO-induced oxidative damage via anti- apoptotic and antioxidant mechanisms in ARPE-19 cells.
Topics: Antioxidants; Apoptosis; Cell Survival; Epithelial Cells; Humans; Hydrogen; Hydrogen Peroxide; Oxidative Stress; Reactive Oxygen Species; Retinal Pigments; Xanthones
PubMed: 35738618
DOI: 10.21873/invivo.12879 -
Photochemical & Photobiological... Jan 2023Several 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...
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 0.5-2.5 µg/mL concentrations prior to UVA irradiation for 15, 30, and 45 min, which resulted in accumulated doses of 2.5, 5, and 7.5 J/cm, respectively. After 24 h incubation, MTT and LDH assays, immunofluorescence, and ELISA were performed. UVA irradiation for 15, 30, and 45 min proved to reduce viability in 83%, 46%, and 11%, respectively. Based on the latter results, we chose the intermediate dose (5-J/cm) for further analysis. Pretreatment with Gal-3 at concentrations > 1.5 µg/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.
Topics: Humans; Galectin 3; Oxidative Stress; Cell Death; Retinal Pigment Epithelium; Epithelial Cells; Retinal Pigments; Reactive Oxygen Species
PubMed: 36036336
DOI: 10.1007/s43630-022-00294-0 -
Photochemistry and Photobiology 2010The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible...
The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm(-2) UVA, 0.07 and 0.9 J cm(-2) UVB, 20 nmβ-PDH (pigment dispersing hormone) or 10 μm SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo. Cultured cells were exposed to 250 μm L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or β-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and β-PDH. The same responses to UVA and UVB were observed in vivo. SIN-1 did not induce pigment dispersion in the cell cultures. L-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by β-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.
Topics: Animals; Brachyura; In Vitro Techniques; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Peptides; Photoreceptor Cells, Invertebrate; Retinal Pigments; Ultraviolet Rays
PubMed: 21091482
DOI: 10.1111/j.1751-1097.2010.00787.x -
Biological & Pharmaceutical Bulletin 2024Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD)....
Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid β-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.
Topics: Humans; Retinal Pigment Epithelium; Macular Degeneration; Lipoproteins, LDL; Oxidative Stress; Epithelial Cells; Retinal Pigments
PubMed: 38508744
DOI: 10.1248/bpb.b23-00849