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Experimental Eye Research Jul 2022A serious form of ocular fibrotic disease is proliferative vitreoretinopathy (PVR) that can ultimately lead to blindness. While the pathogenesis of PVR is known to be...
A serious form of ocular fibrotic disease is proliferative vitreoretinopathy (PVR) that can ultimately lead to blindness. While the pathogenesis of PVR is known to be closely tied to retinal pigment epithelial (RPE) cell epithelial-mesenchymal transition (EMT) characterized by E-cadherin downregulation and N-cadherin upregulation. Herein, we developed a model of transforming growth factor-β1 (TGF-β1)-induced EMT using human RPE (hRPE) cells as a tool for exploring the mechanistic basis for E-cadherin to N-cadherin switching. This analysis revealed that the loss of E-cadherin led to the separation of β-catenin from the catenin-cadherin complex whereupon it underwent nuclear entry to activate zinc finger E-box binding homeobox 1 (ZEB1), in turn promoting N-cadherin upregulation in this biological context. E-cadherin overexpression was sufficient to inhibit this EMT process and proliferation in RPE cells, further constraining their TGF-β1-induced apoptosis.
Topics: Antigens, CD; Cadherins; Epithelial-Mesenchymal Transition; Humans; Retinal Pigment Epithelium; Retinal Pigments; Transforming Growth Factor beta1; Vitreoretinopathy, Proliferative
PubMed: 35500674
DOI: 10.1016/j.exer.2022.109085 -
Biochemistry May 2012As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following...
As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated release of retinal from a short-wavelength-sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t(1/2)) of release of retinal from VCOP was 7.1 s, 250-fold faster than that of rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t(1/2) decreasing from 23 to 4 s with the pH decreasing from 4.1 to 8, respectively. However, the Arrhenius activation energy (E(a)) for VCOP derived from kinetic measurements between 4 and 20 °C was 17.4 kcal/mol, similar to the value of 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D(2)O) effect in VCOP, but this effect was smaller than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOP(D108A)) produced a pigment with an unprotonated chromophore (λ(max) = 360 nm) and dramatically slowed (t(1/2) ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D and D108A) was designed to move the counterion one α-helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (λ(max) = 420 nm). Moreover, the VCOP(S85D/D108A) mutant had retinal release kinetics (t(1/2) = 7 s) and an E(a) (18 kcal/mol) similar to those of the native pigment exhibiting no pH dependence. By contrast, the single mutant VCOP(S85D) had an ~3-fold decreased retinal release rate compared to that of the native pigment. Photoactivated VCOP(D108A) had kinetics comparable to those of a rhodopsin counterion mutant, Rho(E113Q), both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis but rather from differences in the properties of noncovalent binding of the retinal chromophore to the protein.
Topics: Humans; Hydrogen-Ion Concentration; Photoreceptor Cells, Vertebrate; Retinal Pigments; Retinaldehyde; Rhodopsin; Schiff Bases; Spectrophotometry, Ultraviolet
PubMed: 22217337
DOI: 10.1021/bi201522h -
Acta Ophthalmologica May 2024
Topics: Humans; Oxidative Stress; Macular Degeneration; Epithelial Cells; Retinal Pigments; Retinal Pigment Epithelium
PubMed: 38415914
DOI: 10.1111/aos.16664 -
Toxicologic Pathology Aug 2015Incidental findings in the rat eye are not uncommon in acute and long-term toxicological studies. These findings can be associated with a number of causes unrelated to...
Incidental findings in the rat eye are not uncommon in acute and long-term toxicological studies. These findings can be associated with a number of causes unrelated to treatment with the test article, including congenital malformation, trauma, infection, metabolic disease, genetic predisposition, and age-related changes. The occurrence of pigment deposition in the retina of Wistar Hannover (Crl:WI (Han)) rats in a 4-week toxicity study is reported in this communication. The microscopic examination of the eyes in the 4-week toxicity study revealed focal yellow-brown pigment deposits in the retina, mainly located in the ganglion cell layer. The retinal pigment deposits were randomly distributed in the control and treated groups and were considered incidental. The deposits were clearly positive for ferric iron in the Perls' stain but not for lipofuscin by the Schmorl's and Long Ziehl-Neelsen methods. The iron-containing pigment is likely to represent hemosiderin accumulation after retinal micro-hemorrhage or could be indicative of the normal intraretinal iron transport and turnover.
Topics: Animals; Coloring Agents; Eye; Female; Ferric Compounds; Hemosiderin; Lipofuscin; Male; Rats; Rats, Wistar; Retina; Retinal Ganglion Cells; Retinal Hemorrhage; Retinal Pigment Epithelium; Retinal Pigments; Tissue Fixation
PubMed: 25717081
DOI: 10.1177/0192623315571135 -
Bioengineered Apr 2022Diabetic retinopathy is one of the most characteristic complications of diabetes mellitus, and pyroptosis plays acrucial role in the onset and development of diabetic...
Diabetic retinopathy is one of the most characteristic complications of diabetes mellitus, and pyroptosis plays acrucial role in the onset and development of diabetic retinopathy. Although microRNA-192 (miR-192) has been demonstrated to be involved in diabetic retinopathy progression, to the best of our knowledge, its potential and mechanism in cell pyroptosis in diabetic retinopathy have not been studied. The present study demonstrated that high glucose (HG) contributes to the pyroptosis of retinal pigment epithelial (RPE) cells in a dose-dependent manner. The results revealed that miR-192 was weakly expressed in HG-induced RPE cells. Furthermore, overexpression of miR-192 abrogated the role of HG in RPE cell pyroptosis. Based on the bioinformatics analysis, a dual-luciferase reporter assay, and an RNA pull-down assay, FTO α-ketoglutarate-dependent dioxygenase (FTO) was demonstrated to be a direct target of miR-192. Additionally, upregulation of FTO abolished the effects of miR-192 on RPE cells treated with HG. Nucleotide-binding domain leucine-rich repeat family protein 3 (NLRP3) inflammasome activation is vital for cell pyroptosis, and FTO functions as a pivotal modulator in the N-methyladenosine modifications of various genes. Mechanistically, FTO enhanced NLRP3 expression by facilitating demethylation of NLRP3. In conclusion, the present results demonstrate that miR-192 represses RPE cell pyroptosis triggered by HG via regulation of the FTO/NLRP3 signaling pathway.
Topics: Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Diabetic Retinopathy; Epithelial Cells; Glucose; Humans; Inflammasomes; MicroRNAs; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Retinal Pigments
PubMed: 35441575
DOI: 10.1080/21655979.2022.2044734 -
FASEB Journal : Official Publication of... Mar 2024The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative...
The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 μM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1β, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.
Topics: Humans; NF-kappa B; Diabetic Retinopathy; NF-KappaB Inhibitor alpha; Tumor Necrosis Factor-alpha; Macular Edema; Signal Transduction; Retinal Pigment Epithelium; Blood-Retinal Barrier; Tight Junctions; Epithelial Cells; Retinal Pigments; Hydroxamic Acids
PubMed: 38430220
DOI: 10.1096/fj.202301592R -
Journal of Visualized Experiments : JoVE Feb 2023Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and...
Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.
Topics: Humans; Kinetics; Energy Metabolism; Glycolysis; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Epithelial Cells; Retinal Pigments; Retinal Pigment Epithelium
PubMed: 36804905
DOI: 10.3791/64572 -
Medicina (Kaunas, Lithuania) Aug 2022Age-related macular degeneration is a slow-progressing disease in which lipofuscin accumulates in the retina, causing inflammation and apoptosis of retinal pigment...
Age-related macular degeneration is a slow-progressing disease in which lipofuscin accumulates in the retina, causing inflammation and apoptosis of retinal pigment epithelial (RPE) cells. This study aimed to identify -methyl-D-aspartate (NMDA) signaling as a novel mechanism for scavenging -retinylidene--retinylethanolamine (A2E), a component of ocular lipofuscin, in human RPE cells. A2E degradation assays were performed in ARPE-19 cells using fluorescently labeled A2E. The autophagic activity in ARPE-19 cells was measured upon blue light (BL) exposure, after A2E treatment. Autophagy flux was determined by measuring LC3-II formation using immunoblotting and confocal microscopy. To determine whether autophagy via the NMDA receptor is involved in A2E clearance, ATG5-deficient cells were used. Ro 25-6981, an NR2B-selective NMDA receptor antagonist, effectively cleared A2E. Ro 25-6981 reduced A2E accumulation in the lysosomes of ARPE-19 cells at sub-cytotoxic concentrations, while increasing the formation of LC3-II and decreasing p62 protein levels in a concentration-dependent manner. The autophagic flux monitored by RFP-GFP-LC3 and bafilomycin A1 assays was significantly increased by Ro 25-6981. A2E clearance by Ro 25-6981 was abolished in ATG5-depleted ARPE-19 cells, suggesting that A2E degradation by Ro 25-6981 was mediated by autophagy. Furthermore, treatment with other NMDA receptor antagonists, CP-101,606 and AZD6765, showed similar effects on autophagy activation and A2E degradation in ARPE-19 cells. In contrast, glutamate, an NMDA receptor agonist, exhibited a contrasting effect, suggesting that both the activation of autophagy and the degradation of A2E by Ro 25-6981 in ARPE-19 cells occur through inhibition of the NMDA receptor pathway. This study demonstrates that NMDA receptor antagonists degrade lipofuscin via autophagy in human RPE cells and suggests that NMDA receptor antagonists could be promising new therapeutics for retinal degenerative diseases.
Topics: Autophagy; Epithelial Cells; Humans; Lipofuscin; Receptors, N-Methyl-D-Aspartate; Retinal Pigment Epithelium; Retinal Pigments; Retinoids
PubMed: 36013596
DOI: 10.3390/medicina58081129 -
Experimental Eye Research Apr 2022The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of...
The retinal pigment epithelium is a pigmented monolayer of cells that help maintain a healthy retina. Loss of this essential cell layer is implicated in a number of visual disorders, including age-related macular degeneration (AMD). Utilizing primary RPE cultures to investigate disease is an important step in understanding disease mechanisms. However, the use of primary RPE cultures presents a number of challenges, including the limited number of cells available and the presence of auto-fluorescent pigment that interferes with quantifying fluorescent probes. Additionally, primary RPE are difficult to transfect with exogenous nucleic acids traditionally used for fluorescent imaging. To overcome these challenges, we used an adeno-associated viral (AAV) vector to express a pH sensitive fluorescent protein, mKeima, fused to the mitochondrial targeting sequence of cytochrome oxidase subunit 8A (mKeima-mito). mKeima-mito allows for quantification of mitochondrial autophagy (mitophagy) in live-cell time-lapse imaging experiments. We also developed an image analysis pipeline to selectively quantify mKeima-mito while removing the signal of auto-fluorescent pigment from the dataset by utilizing information from the mKeima fluorescent channels. These techniques are demonstrated in primary RPE cultures expressing mKeima-mito treated with 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP), an uncoupler that depolarizes the mitochondrial membrane and leads to mitochondrial fragmentation and mitophagy. The techniques outlined provide a roadmap for investigating disease mechanisms or the effect of treatments utilizing fluorescent probes in an important cell culture model.
Topics: Cells, Cultured; Epithelial Cells; Fluorescent Dyes; Humans; Mitophagy; Retinal Pigment Epithelium; Retinal Pigments
PubMed: 35167864
DOI: 10.1016/j.exer.2022.108981 -
Cells Jun 2023Marmosets have emerged as a valuable primate model in ophthalmic research due to their similarity to the human visual system and their potential for generating...
Marmosets have emerged as a valuable primate model in ophthalmic research due to their similarity to the human visual system and their potential for generating transgenic models to advance the development of therapies. In this study, we isolated and cultured primary retinal pigment epithelium (RPE) cells from marmosets to investigate the mechanisms underlying RPE dysfunction in aging and age-related macular degeneration (AMD). We confirmed that our culture conditions and materials supported the formation of RPE monolayers with functional tight junctions that closely resembled the in vivo RPE. Since serum has been shown to induce epithelial-mesenchymal transition (EMT) in RPE cells, we compared the effects of fetal bovine serum (FBS) with serum-free supplements B27 on transepithelial electrical resistance (TER), cell proliferation, and morphological characteristics. Additionally, we assessed the age-related morphological changes of in vivo and primary RPE cells. Our results indicate that primary marmoset RPE cells exhibit in vivo-like characteristics, while cells obtained from an older donor show evidence of aging, including a failure to form a polarized monolayer, low TER, and delayed cell cycle. In conclusion, our primary marmoset RPE cells provide a reliable in vitro model for developing novel therapeutics for visual-threatening disorders such as AMD, which can be used before animal experiments using marmosets.
Topics: Animals; Humans; Callithrix; Retinal Pigment Epithelium; Cells, Cultured; Macular Degeneration; Epithelial Cells; Retinal Pigments
PubMed: 37371114
DOI: 10.3390/cells12121644