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Crystallins, the highly abundant proteins of the ocular lens, are essential determinants of the transparency and refractivity required for lens function. Initially thought to be lens-specific and to have evolved as lens proteins, it is now clear that crystallins were recruited to the lens from proteins that existed before lenses evolved. Crystallins are expressed outside of the lens and most have been shown to have cellular functions distinct from their roles as structural elements in the lens. For one major crystallin group, the β/γ-crystallin superfamily, no such functions have yet been established. We have explored possible functions for the polypeptides (βA3-and βA1-crystallins) encoded by Cryba1 , one of the 6 β-crystallin genes, using a spontaneous rat mutant and genetically engineered mouse models. βA3-and βA1-crystallins are expressed in retinal astrocytes and retinal pigment epithelial (RPE) cells. In both cell types, these proteins appear to be required for the proper acidification of the lysosomes. In RPE cells, elevated pH in the lysosomes is shown to impair the critical processes of phagocytosis and autophagy, leading to accumulation of undigested cargo in (auto) phagolysosomes. We postulate that this accumulation may cause pathological changes in the cells resembling some of those characteristic of age-related macular degeneration (AMD). Our studies suggest an important regulatory function of βA3/A1-crystallin in astrocytes. We provide evidence that the cellular function of βA3/A1-crystallin involves its interaction with V-ATPase, the proton pump responsible for acidification of the endolysosomal system. [ABSTRACT FROM AUTHOR]

Background: Oxidative stress causes the defects of retinal pigment epithelial (RPE) cells that contribute to age-related macular degeneration (AMD). This study was conducted to determine whether wogonin could prevent H2O2-induced oxidative stress in RPE cells. Methods: A RPE cell line, ARPE-19, was obtained for the cell model. ARPE-19 cells were pre-treated with various concentrations of wogonin for 24 h before being exposed to H2O2 for 2 h to induce oxidative stress. Cell metabolic activity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was quantified by the flow cytometry. Protein level was assed by western blot. Results: The RPE cells exposed to to 200 mM H2O2 demonstrated a significant depression in the cell viability; whereas pre-treatment with 50 and 100 mmol/l wogonin could significantly improve the cell viability in a dose-dependent manner. The proportion of PI-positive cells was increased significantly in RPE cells treated with H2O2 alone; whereas pretreatment with 100 mM wogonin significantly reduced H2O2 -induced RPE cell death rate. In protein level, the wogonin use could reduce the level of p-Akt significantly and this is the possible mechanism of the antioxidant effect of wogonin. Conclusions: Our study showed that wogonin pre-treatment can protect RPE cells from H2O2-induced apoptosis. This suggests potential effect of wogonin in the prevention of retinal diseases associated with H2O2-induced oxidative stress such as AMD. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of the overexpression of miRNA-9 to the ratio of pro- and anti-angiogenic isoforms of vascular endothelial growth factor (VEGF) in human retinal pigment cells (ARPE-19). Methods: Oxidative stress was induced to ARPE-19 cells by 4-hydroxynonenal (4-HNE), tert-butyl hydroperoxide (t-BH), and hypoxia chamber with 1 % O. Expression patterns of miRNAs were validated by qPCR. Relative mRNA levels of VEGF and PEDF were measured by semi-quantitative PCR. After the transfection of miR-9 mimic and inhibitor, transcriptional levels of VEGF165a, VEGF 165b, and SRPK-1 were measured by qPCR. Results: We demonstrated that miR-9 expression is decreased in ARPE-19 human retinal pigment cells under hypoxic stress induced by 4-HNE, a lipid peroxidation end-product. We observed that miR-9 mimic transfection of ARPE-19 inhibited one of its targets, serine-arginine protein kinase-1 (SRPK-1), modulating the transcriptional level of VEGF165b. Transfection of miR-9 reduced the alternative splicing of VEGF165a mRNA in ARPE-19 cells under hypoxic conditions, suggesting that miR-mediated regulation of alternative splicing could be a potential therapeutic target in neovascular pathologies. Conclusions: Hypoxic stress decreased the miR-9 level in ARPE-19 cells, which increased the transcriptional level of SRPK-1, resulting in alternative splicing shift to pro-angiogenic isoforms of VEGF165 in human retinal pigment epithelial cells [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of Ginkgo biloba extract (GBE) on expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells under chemical hypoxia. Materials and Methods: RPE cells (ARPE-19) were cultured in either untreated media (control group), media treated with 200 μM cobalt chloride (hypoxia group) or media treated with both 200 μM cobalt chloride and 100 μg/ml GBE (hypoxia + GBE group) for various amounts of time. HIF-1α and VEGF expression were compared between groups. HIF-1α and VEGF messenger RNA (mRNA) were quantified using real-time polymerase chain reaction (PCR). HIF-1α of extracted nuclei and VEGF of the media were quantified using enzyme-linked immunosorbent assay (ELISA). The expression of HIF-1α was also assessed with Western blot and immunocytochemistry. Results: HIF-1α mRNA, VEGF mRNA, HIF-1α and VEGF levels were higher in the hypoxia group compared with the control group; however, real-time PCR revealed decreased expression of HIF-1α mRNA and VEGF mRNA in the hypoxia + GBE group compared with the hypoxia group. In the ELISA, the HIF-1α and VEGF protein concentrations also decreased with GBE treatment. Western blot and immunostaining showed that the intensity of HIF-1α signals in the hypoxia + GBE group was lower than that of the hypoxia group. Conclusion: GBE reduced HIF-1α and VEGF expression in RPE cells cultured under chemical hypoxia in vitro. [ABSTRACT FROM AUTHOR]

Transforming growth factor-beta (TGF-β) is one of the main epithelial–mesenchymal transition (EMT)-inducing factors. In general, TGF-β-induced EMT promotes cell migration and invasion. TGF-β also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPAR's interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-β and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-β2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-β2 and/or the inhibitor of uPA (u-PA-STOP®) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-β2 or u-PA-STOP® suppressed cell proliferation. Combination treatment of TGF-β2 and u-PA-STOP® enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-β2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-β inhibitor SB431542 suppressed TGF-β2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-β2 alone or with TGF-β2 and u-PA-STOP®. TGF-β2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-β2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with TGF-β2 alone. Furthermore, the interaction between uPA and ARPE-19 cells was analyzed using a surface plasmon biosensor system. The binding of uPA to ARPE-19 cells was observed. In addition, TGF-β2 significantly promoted the binding activity of uPA to ARPE-19 cells in a time-dependent or cell-number-dependent fashion. These results indicate that TGF-β-induced EMT-associated phenotype changes in ARPE-19 cells and the invasiveness of ARPE-19 cells into a collagen gel matrix are mediated, at least in part, by uPA. [ABSTRACT FROM AUTHOR]

Background: In non-excitable cells, one major route for calcium entry is through store-operated calcium (SOC) channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ store. STIM1 and Orai1 are major regulators of SOC channels. In this study, we explored the functions of STIM1 and Orai1 in epidermal growth factor (EGF)-induced cell proliferation and migration in retinal pigment epithelial cells (ARPE-19 cell line). Results: EGF triggers cell proliferation and migration in ARPE-19 cells. Cell proliferation and migration involve STIM1 and Orai1, as well as phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, and Akt. Pharmacological inhibitors of SOC channels and siRNA of Orai1 and STIM1 suppress cell proliferation and migration. Pre-treatment of mitogen-activated protein kinase kinase (MEK) inhibitors and a phosphatidylinositol 3 kinases (PI3K) inhibitor attenuated cell proliferation and migration. However, inhibition of the SOC channels failed to prevent EGF-mediated ERK 1/2 and Akt phosphorylation. Conclusions: Our results showed that STIM1, Orai1, ERK 1/2, and Akt are key determinants of EGF-mediated cell growth in ARPE-19 cells. EGF is a potent growth molecule that has been linked to the development of PVR, and therefore, STIM1, Orai1, as well as the MEK/ERK 1/2 and PI3K/Akt pathways, might be potential therapeutic targets for drugs aimed at treating such disorders. [ABSTRACT FROM AUTHOR]

. Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR). It is widely accepted that the progression of DR is mainly due to a local imbalance of pro- versus anti-angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithelial (RPE) cells produced pro-angiogenic factors under high glucose (HG) conditions in vitro. Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium from retinal pigment epithelium cells (ARPE-19) grown in HG medium and assessed for tube formation. Based on the expression profiles of ARPE-19, we investigated whether ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions using cultured human RE cells as the test system for experiments with recombinant protein, conditioned medium from ARPE-19 and RNA interference (RNAi). Results: The conditioned medium from ARPE-19 cultured under HG conditions promoted tube formation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all of the elements of angiogenesis in human RE cells in vitro. The results of experiments using conditioned medium from ARPE-19 combined with RNAi demonstrated that ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions. Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angiogenic activity on RE cells. Our results are unique and may potentially add a new candidate to the long list of molecules involved in diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Abstract: Age-related macular degeneration is the most common cause of vision loss in the elderly, which starts from aging processes of retinal pigment epithelial cells. Among variable risk factors in occurrence and progression of age-related macular degeneration, oxidized low density lipoprotein could be causally involved in pathobiological changes of RPE cells. Herein we showed that oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood–retinal barrier dysfunction. Under sub-lethal concentration, oxidized low density lipoprotein could promote advanced senescence of retinal pigment epithelial cells. Interestingly expression of CRALBP and RPE 65, indicators of retinal pigment epithelial cell differentiation, was decreased by oxidized low density lipoprotein. In addition, oxidized low density lipoprotein induced reactive oxygen species production and up-regulated inflammatory factors such as tumor necrosis factor-α and vascular endothelial growth factor, when β-catenin, a critical mediator of the canonical Wnt pathway, was also elevated. Oxidized low density lipoprotein increased paracellular permeability of retinal pigment epithelial cells, when zonula occludens-1 at intercellular junctions markedly decreased as well. Furthermore, in retinal pigment epithelial cells and choriocapillaris of human apolipoprotein E2 transgenic mouse eye, increased vascular endothelial growth factor and decreased zonula occludens-1 expression was observed. Therefore, our results suggest that oxidized low density lipoprotein could promote senescence of retinal pigment epithelial cells which leads to induce outer blood–retinal barrier dysfunction as an early pathogenesis of age-related macular degeneration. [Copyright &y& Elsevier]

PurposeTo investigate the role of Ca2+ in lipofuscin formation in human retinal pigment epithelial (RPE) cells that phagocytize bovine photoreceptor outer segments (POSs).MethodsCultured human RPE cells fed with 2 × 107 per l bovine POS were treated with flunarizine, an antagonist of Ca2+ channel, or/and centrophenoxine, a lipofuscin scavenger. The Ca2+ changes and lipofuscin formation were measured with fluoresence dye Fluo-3/AM ester, laser scanning confocal microscopy (LSCM) and flow cytometry (FCM). The activity of RPE cells was measured by methyl thiazolyl tetrazolium (MTT) assay and argyrophilic nucleolar organizer regions (AgNORs) assay.ResultsThe Ca2+ fluorescence intensity (CFI) of RPE cells fed with POS was significantly increased compared with the controls (165.36±29.92 U). It reached a peak with 777.33±63.86 U (P<0.01) at 12 h, and then decreased but still maintained a high level of 316.90±36.07 U (P<0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca2+ overload to 227.18±14.00 U at 12 h and 211.06±20.45 U at 4 days. FCM confirmed these changes. The drugs also showed an inhibitory effect on the lipofuscin formation. The proliferation rate of the cells fed with POS increased significantly. Both drugs had inhibitory effects on the activity of the cultured cells. This tendency was confirmed by AgNORs assay.ConclusionsThe Ca2+ inflow initiated lipofuscin accumulation in RPE cells fed with POS. Flunarizine and centrophenoxine can decrease Ca2+ overload and lipofuscin formation in RPE cells, accompanied by maintaining cellular vitality. [ABSTRACT FROM AUTHOR]

Abstract: Chronic exposure to oxidative stress causes damage to retinal pigment epithelial cells which may lead to the development of age-related macular degeneration, the major cause of vision loss in humans. Anti-oxidants provide a natural defense against retinal cell damage. The present study was designed to evaluate the potential anti-oxidant activity and protective effect of two diarylheptanoids isolated from a medicinal herb Curcuma comosa; 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound A), and 1,7-diphenyl-4(E),6(E)-heptadien-3-ol (compound B) against oxidative stress (H2O2)-induced human retinal pigment epithelial (APRE-19) cell death. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay indicated that the anti-oxidant activity (IC50) of compound A was similar to that of vitamin C. Pre-treatment of ARPE-19 cells with 20μM compound A for 4h afforded greater protection against the insult from 500μM H2O2, compared to a similar protection period for compound B. Compound A lowered H2O2-induced lipid peroxidation, malondialdehyde formation and intracellular reactive oxygen species. Furthermore, compound A ameliorated the H2O2-induced decrease in anti-oxidant enzyme activities and subsequent apoptotic cell death in ARPE-19 cells in a dose and time-dependent manner. These results suggest that compound A protects ARPE-19 cells against oxidative stress, in part, by enhancing several anti-oxidant defense mechanisms. Therefore, compound A may have therapeutic potential for diseases associated with oxidative stress, particularly degenerative retinal diseases. [ABSTRACT FROM AUTHOR]

Rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR) are eye diseases that are characterized by mechanical stress involving stretching of the retinal pigment epithelial (RPE) cells by the vitreous or the hyperplastic membranes. Here, we assessed whether mechanical force could change the expression of matrix metalloproteinases (MMPs) in RPE cells via the mitogen-activated protein kinase (MAPK) pathway. Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured RPE cells at focal adhesions. Activation of the MAPK, including extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 were determined over a time course from 5 to 30 min by Western-blot analysis. Activation of p38 was also tested using immunofluorescence staining. The mRNA levels of MMP-2, MMP-9, tissue inhibitor of MMP (TIMP)-2 and fibronectin (FN) were analyzed by RT-PCR. Active MMP-2 and MMP-9 were demonstrated by zymography. MMP-2 secretion was evaluated by enzyme immunoassay. Stimulation of RPE cells with mechanical stress did not change the total protein expression of the MAPK proteins ERK, JNK, and p38. However, of the three kinases, only active p38 showed an increased protein expression which was also shown by a 2.8-fold increase in immunofluorescence staining at 5 min following mechanical stress stimulation. This increase in active p38 expression was blocked by treating the cells with the p38 inhibitor SB203580. FN mRNA increased 2.4-fold at 15 min and MMP-2 mRNA increased 2.1-fold at 4 h. MMP-2 secretion increased 1.5-fold at 4 h and 1.9-fold at 12 h. The expression of MMP-2 and FN, and the activation and secretion of MMP-2, were inhibited in the presence of SB203580. The mRNA expression of MMP-9 and TIMP-2 did not change throughout. This study shows that mechanical stress upregulates MMP-2 and FN expression through activation of the p38 pathway. The increase in MMP-2 levels evoked by mechanical force may contribute to the remodeling of the extracellular matrix around RPE cells, weakening the interlinkage and membrane attachment between RPE cells, and facilitate cellular migration. [ABSTRACT FROM AUTHOR]

Abstract: Retinal pigment epithelial (RPE) cells play a dominant role in the development of proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal reattachment surgery. Several studies have shown that platelet-derived growth factor (PDGF) exhibits chemotaxis and proliferation effects on RPE cells in PVR. In this study, the inhibitory effect of lycopene on PDGF-BB-induced ARPE19 cell migration is examined. In electric cell–substrate impedance sensing (ECIS) and Transwell migration assays, significant suppression of PDGF-BB-induced ARPE19 cell migration by lycopene is observed. Cell viability assays show no cytotoxicity of lycopene on RPE cells. Lycopene shows no effect on ARPE19 cell adhesion and is found to inhibit PDGF-BB-induced tyrosine phosphorylation and the underlying signaling pathways of PI3K, Akt, ERK and p38 activation. However, PDGF-BB and lycopene show no effects on JNK activation. Taken together, our results demonstrate that lycopene inhibits PDGF-BB-induced ARPE19 cell migration through inhibition of PI3K/Akt, ERK and p38 activation. [Copyright &y& Elsevier]

Purpose: The migration of retinal pigment epithelium (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). We investigated the expression of connective tissue growth factor (CTGF) in an in vitro model of wound healing and effects of recombinant human CTGF (rhCTGF) on modulating migration and Ca2+ signaling in RPE cells. Methods: Cultured human RPE monolayers were used to establish a wound-healing model. Western blot and in situ hybridization were used to detect the CTGF expression in RPE cells. Migration of RPE cells was measured under the stimulation of rhCTGF alone or in combination with dexamethasone (DEX) or 8-Br-cAMP. To determine the concentration of cytoplasmic-free Ca2+ ([Ca2+]i) responding to CTGF, the fluo-3/AM-loaded RPE cells were observed with a laser scanning confocal microscope. Results: The CTGF expression first increased after being wounded in RPE cells, then reached a peak and maintained at a high level. The positive expression was mainly at the edge of scrape and in motile RPE cells. rhCTGF-stimulated RPE cells migrated in a dose-dependent manner, and both DEX and 8-Br-cAMP could significantly inhibit the CTGF-induced migrations. CTGF induced a (Ca2+)i elevation in RPE cells in a concentration-dependent manner. Moreover, stimulation of RPE cells with CTGF and DEX or 8-Br-cAMP counteracted the elevation of (Ca2+)i induced by CTGF. Conclusions: The CTGF expression could be induced by an in vitro model of scrape wounding. rhCTGF stimulated the migration and Ca2+ signal pathway in RPE cells in a dose-dependent manner, and DEX and 8-Br-cAMP suppressed this effect. Our results indicate that CTGF is involved in the wound-healing process and plays an important role in the pathogenesis of intraocular proliferative diseases. [ABSTRACT FROM AUTHOR]

Deficiency in docosahexaenoic acid (DHA) is associated with impaired visual and neurological postnatal development, cognitive decline, macular degeneration, and other neurodegenerative diseases. DHA is an omega-3 polyunsaturated fatty acyl chain concentrated in phospholipids of brain and retina, with photoreceptor cells displaying the highest content of DHA of all cell membranes. The identification and characterization of neuroprotectin D1 (NPD1, 10R, 17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) contributes in understanding the biological significance of DHA. In oxidative stress-challenged human retinal pigment epithelial (RPE) cells, human brain cells, or rat brains undergoing ischemia-reperfusion, NPD1 synthesis is enhanced as a response for sustaining homeostasis. Thus, neurotrophins, Aβ peptide 42 (Aβ42), calcium ionophore A23187, interleukin (IL)-1β, or DHA supply enhances NPD1 synthesis. NPD1, in turn, up-regulates the antiapoptotic proteins of the Bcl-2 family and decreases the expression of proapoptotic Bcl-2 family members. Moreover, NPD1 inhibits IL-1β-stimulated expression of cyclooxygenase-2 (COX-2). Because both RPE and photoreceptors are damaged and then die in retinal degenerations, elucidating how NPD1 signaling contributes to retinal cell survival may lead to a new understanding of disease mechanisms. In human neural cells, DHA attenuates amyloid-β (Aβ) secretion, resulting in concomitant formation of NPD1. NPD1 was found to be reduced in the Alzheimer''s disease (AD) cornu ammonis region 1 (CA1) hippocampal region, but not in other areas of the brain. The expression of key enzymes for NPD1 biosynthesis, cytosolic phospholipase A2 (cPLA2), and 15-lipoxygenase (15-LOX) was found altered in the AD hippocampal CA1 region. NPD1 repressed Aβ42-triggered activation of pro-inflammatory genes and upregulated the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1) in human brain cells in culture. Overall, these results support the concept that NPD1 promotes brain and retina cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Aβ42-induced neurotoxicity and other forms of cell injury, which in turn fosters homeostasis during development in aging, as well as during the initiation and progression of neurodegenerative diseases. [Copyright &y& Elsevier]

Purpose: To observe the effects of electric fields (EFs) on the migration of human retinal pigment epithelial (RPE) cells and to explore possible related mechanisms. Methods: Cultured human RPE cells were exposed to EFs, and images of the cells were obtained at regular intervals to evaluate the cell migration and viability. Distribution of F-actin and β 1 integrin was measured by immunohistochemistry. Expression of β 1 integrin was determined by PCR and Western blotting. Results: Exposure to EFs resulted in a cathodal-directed migration of RPE cells and a cathodal accumulation of F-actin and β 1 integrin in the cells. EF stimulation increased expression of β 1 integrin both in mRNA and protein levels. These effects could be inhibited by cytochalasin B. Conclusion: EFs induce directed migration of RPE cells, and this effect is, to some extent, regulated by the interaction between cytoskeleton and integrins. [ABSTRACT FROM AUTHOR]

Abstract: Complement activation is involved in the pathogenesis of age-related macular degeneration. How complement is activated in the retina is not known. Previously we have shown that complement factor H (CFH) is constitutively expressed by retinal pigment epithelial (RPE) cells and the production of CFH is negatively regulated by inflammatory cytokines and oxidative insults. Here we investigated the production and regulation of complement factor B (CFB) in RPE cells. Immunohistochemistry showed that CFB is expressed at low levels on the apical portion of the RPE cells in normal physiological conditions. With age, CFB expression increases and extends to the basal part of RPE cells. Confocal microscopy and real-time PCR of RPE cultures indicated that the production of CFB by RPE cells is positively regulated by TNF-α, IFN-γ and long-term (30days) photoreceptor outer segments treatments. Increased CFB expression in RPE cells in vivo is accompanied by the accumulation of complement C3 and C3a deposition at the Bruch''s membrane and the basal layer of RPE cells. Our results suggest that RPE cells play important roles in regulating complement activation in the retina. Increased complement activation in the aged retina may be important for retinal homeostasis in the context of accumulating photoreceptor waste products. [Copyright &y& Elsevier]

Abstract: The interaction of programmed death-1 (PD-1) expressed on T cells with its ligands B7H1 (PDL1) and B7DC (PDL2) is known to be a mechanism of T cell inhibition. In the present study, we examined whether human or murine retinal pigment epithelial (RPE) cells express B7H1 and B7DC, and if so, whether these molecules expressed on RPE cells play an inhibitory role via interaction with T cells. The transcriptional levels and surface expression of B7H1 and B7DC on human RPE cell line (ARPE-19), RPE cells freshly isolated from healthy human subjects, and murine RPE cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In addition, T cells from healthy subjects were cultured with ARPE-19 for 72h in the presence or absence of monoclonal antibody (mAb) to B7H1 or B7DC, and cytokine production (IFN-γ, IL-8, and MCP-1) was measured. Messenger RNA and cell surface protein expression of B7H1 and B7DC were demonstrated on non-stimulated ARPE-19 and freshly isolated human RPE cells, and the expression of these molecules was predominantly upregulated by treatment with IFN-γ. In murine RPE cells, B7H1 expression was detected only when stimulated with IFN-γ. IFN-γ, IL-8, and MCP-1 production by T cells co-cultured with IFN-γ-untreated or -treated ARPE-19 was significantly enhanced in the presence of anti-B7H1 mAb. These data suggest that B7H1 expressed on RPE cells plays an immunosuppressive role in ocular inflammation, which may contribute to immune privilege in the posterior segment of the eye. [Copyright &y& Elsevier]

Abstract: Retinal pigment epithelial (RPE) cells play critical roles in the maintenance of visual function, partly by secreting various biologically active factors that modulate the intraocular environment. Recent studies suggest involvement of Wnt proteins secreted by RPE cells in the pathogenesis of photoreceptor degeneration. In the present study, we examined, via the luciferase assay, the effect of media conditioned by RPE cells (RPE-CM) on activity of the canonical Wnt pathway in vitro. We isolated primary RPE cells from Long–Evans rats at P6–P9. In culture, these cells formed a monolayer with polygonal cell morphology and demonstrated repigmentation at confluency and immunoreactivity for ZO-1, a marker for tight junctions. To evaluate the effect of RPE-CM on the canonical Wnt pathway, we replaced the culture media of COS-7 cells transfected with (Tcf)7LUC, a multimeric Tcf-responsive element luciferase reporter construct, with RPE-CM and measured luciferase activity with or without Wnt3a or SB216763, a specific GSK3 inhibitor. RPE-CM did not enhance basal or Wnt3a-induced (Tcf)7LUC activity; instead, this activity decreased by 60%. RPE-CM also reduced SB216763-induced (Tcf)7LUC activity by 65%, which suggests that the inhibitory effect of RPE-CM is probably due to intracellular crosstalk rather than extracellular antagonism. RPE cells may thus be able to modulate the intraocular environment by regulating the canonical Wnt pathway. [Copyright &y& Elsevier]

This review aims to cover the transplant procedures that have been developed and investigated as potential treatments for age-related macular degeneration. The choice of transplant materials that will be discussed ranges from isolated cells (including stem cells, iris pigment epithelial cells and retinal pigment epithelial cells) to monolayers of cells on artificial or nonartificial substrates and also to whole patches of tissue (e.g., fetal retina, full-thickness patch grafts and macular relocation). Finally, we will address the current and future technologies and questions that need to be addressed in order that transplant procedures can have an effective role in the treatment of patients with age-related macular degeneration. [ABSTRACT FROM AUTHOR]

Abstract: Glucosamine is an amine-containing sugar that exhibits immunosuppressive effects in vitro and in vivo, although its mechanism of action is unknown. We investigated whether glucosamine sulfate (GS) modulates the proinflammatory cytokine interleukin (IL)-1β-induced expression and production of intercellular adhesion molecule (ICAM)-1, the mechanism responsible for this effect, and whether GS inhibits leukocyte adhesion to the monolayer of retinal pigment epithelial (RPE) cells stimulated with various cytokines. We used flow cytometry and an ARPE-19 cell model to determine the effect of GS on the production of ICAM-1 in response to IL-1β, IL-6, tumor necrosis factor (TNF)-α plus IL-1β, TNF-α plus IL-6, and TNF-α plus interferon (IFN)-γ. We also used semiquantitative RT–PCR to determine the effect of GS on IL-1β-induced expression of the ICAM-1 gene, and immunocytochemistry and western blotting to measure the effect of GS on the activation and nuclear translocation of the nuclear factor NF-κB and the degradation of cytoplasmic IκB. The functionality of GS-modulated ICAM-1 on leukocyte adhesion was demonstrated in an RPE cell–neutrophil adherence assay. IL-1β increased the expression of ICAM-1 at the mRNA and protein levels in ARPE-19 cells. GS downregulated the production of ICAM-1 induced by IL-1β, IL-6, TNF-α, and IFN-γ at the protein level in a dose-dependent manner. GS also inhibited the nuclear translocation of NF-κB subunit p65 and partially prevented the degradation of cytoplasmic IκB in IL-1β-stimulated ARPE-19 cells. GS significantly decreased the number of neutrophils adhering to the RPE monolayer in response to cytokines IL-1β, IL-6, TNF-α, and IFN-γ. GS inhibits the expression of the ICAM-1 gene in ARPE-19 cells stimulated with IL-1β by blocking NF-κB subunit p65 translocation and by partially preventing IκB degradation. GS also decreases leukocyte adhesion to the monolayer of ARPE-19 cells stimulated with various cytokines by decreasing ICAM-1 production. Our study demonstrates a potentially important property of GS in reducing ICAM-1-mediated inflammatory mechanisms in the eye. [Copyright &y& Elsevier]

Abstract: We investigated the secretion and expression of VEGF-A and its receptors in human retinal pigment epithelial cells (RPE) under conditions of oxidative stress induced by glutathione (GSH) depletion. RPE cells were treated with 500μM dl-buthionine-(S,R)-sulfoximine (BSO) for varying times up to 24h. Cellular GSH levels, GSH:GSSG ratios, VEGF-A mRNA and protein expression, as well as VEGF-A secretion, and VEGFR-1 and VEGFR-2 receptor expression were determined. Treatment with BSO caused a significant decrease in intracellular GSH and in GSH/GSSG ratios. Treatment with BSO increased VEGF-A mRNA linearly with time which was significant at 24h (p <0.01 vs untreated controls). An increase was also found for VEGF-A secretion with BSO treatment; incubation of RPE with GSH monoethyl ester (GSH-MEE) caused an 84% decrease in VEGF-A secretion. Further, thiol depletion by BSO caused a significant induction of VEGFR-1 and VEGFR-2. Thus, our studies show that cellular redox status plays an important role in VEGF regulation in RPE cells. [Copyright &y& Elsevier]

Abstract: Reversible protein phosphorylation regulates the biological activities of many human proteins involved in crucial cellular processes, e.g., protein–protein interactions, cell signaling, gene transcription, cell growth, and death. A malfunction of cellular homeostasis in retinal pigment epithelial (RPE) cells is involved in the age-related retinal degeneration. In this study, we examined cytotoxicity in human RPE cells subjected to the protein phosphatase inhibitor, okadaic acid (OA). Moreover, the influence of Hsp90 inhibitor geldanamycin (GA), a benzoquinone ansamycin, in cytoprotection was assessed. Hsp70 protein levels were analyzed by Western blot. Cellular viability was determined by LDH and MTT assays. To study apoptotic cell death, caspase-3 enzyme activity was measured by assaying the cleavage of a fluorescent peptide substrate and Hoechst dye was used to visualize nuclear morphology. OA treatment caused morphological changes and induced cytotoxicity by caspase-3-independent manner in the RPE cells. No evidence of nuclear fragmentation was observed in response to OA. Interestingly, GA treatment accumulated Hsp70 protein and attenuated OA-induced cytotoxicity. This study suggests that Hsp70 and Hsp90 are closely related to cytoprotection of RPE cells in response to protein phosphatase inhibition. [Copyright &y& Elsevier]

Abstract: Emerging evidence indicates that the autofluorescent pigments that accumulate as lipofuscin in retinal pigment epithelial (RPE) cells may reach levels that contribute to a decline in cell function. Since recent findings with respect to the origin, composition and adverse effects of RPE lipofuscin have informed our view of this material, the goal of this article is to review our current understanding of these issues. [Copyright &y& Elsevier]

Purpose: To determine the effect of the calcium signaling modulating drug carboxyamido-triazole (CAI) on substeps of exudative age-related macular degeneration (AMD) in vitro. Materials and Methods: Zymography and ELISA determined the effect of CAI on MMP-2 production of choroidal endothelial cells (CECs) stimulated by bFGF and VEGF. The effects of CAI on attachment of retinal pigment endothelial (RPE) cells/CECs onto fibronectin, laminin, collagen IV, and migration toward fibronectin were investigated. Proliferation induced by serum and bFGF (10 μ g/ml) with and without CAI (0.1–10 μ M) was measured by cell counting and [3] H-uptake. Viability and apoptosis of the exposed cells was assessed by an MTT and an apoptosis assay. Results: CAI inhibited serum- and bFGF-induced proliferation, cell attachment onto fibronectin and collagen IV, but only CEC attachment onto laminin. Inhibition of MMP-2 production was observed (10 μ M CAI). CAI reduced the cellular viability by apoptosis induction. Conclusions: CAI inhibits substeps of exudative macular degeneration and may be of value for the treatment of the disease. [ABSTRACT FROM AUTHOR]

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Oxidative stress is involved in the pathogenesis of age-related macular degeneration (AMD). Administration of conventional antioxidants has been shown to slow the progression of AMD and vision loss. Melatonin, an endogenous neurohormone produced by the pineal gland and retina, has been reported to be a potent antioxidant and free radical scavenger. In this study we tested whether melatonin can protect retinal pigment epithelial (RPE) cells against hydrogen peroxide (H2O2)-induced cell death. Since mitochondrial DNA (mtDNA) is preferentially susceptible to oxidative damage, we tested whether melatonin can reduce H2O2-induced mtDNA lesions. A human RPE cell line (ARPE-19) was cultured and exposed to H2O2 (100 and 200 μm) for 1 hr to induce cell death. Prior to H2O2 treatment, cells were treated with various concentrations (0·1–200 μm) of melatonin for 2, 24 or 72 hr. Control cells received either melatonin or ethanol alone. Cell viability, as determined by MTT assay, showed no significant (P>0·05) protection against H2O2 toxicity in cells receiving 2- and 24-hr pretreatment of melatonin at either concentration. However, when melatonin was administered diurnally for 3 consecutive days, this prolonged treatment markedly reduced H2O2-induced cell death (P<0·05). MtDNA damage, as assessed with quantitative PCR, was significantly decreased (P=0·031) in RPE cells pretreated with melatonin as compared to those without melatonin treatment. These results suggest that melatonin may play a role in protecting RPE cells from oxidative stress. [Copyright &y& Elsevier]

Nitric oxide (NO) has been implicated in many physiological and pathological conditions in the eyes. The induction of inducible NO synthase (iNOS) and NO production have been noted in immunostimulated retinal pigment epithelial (RPE) cells. Cellular NO production depends on the availability of arginine, a substrate for NOS. Arginine can be regenerated from citrulline, another product of the NOS reaction, by argininosuccinate synthetase and argininosuccinate lyase, forming the citrulline–NO cycle. When rat RPE-J cells were treated with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα) and lipopolysaccharide (LPS), and expression of the citrulline–NO cycle enzymes and related enzymes was analyzed, iNOS and argininosuccinate synthetase were highly induced at both mRNA and protein levels. On the other hand, argininosuccinate lyase was not induced. Among other related enzymes and transporters, mRNA for cationic amino acid transporter (CAT)-1 was weakly induced, whereas those for CAT-2, arginase I and II, ornithine aminotransferase and ornithine decarboxylase remained little changed. NO was produced by cells after stimulation with TNFα, IFNγ and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. Our findings indicate that in activated RPE-J cells citrulline–arginine recycling is important for NO production. [Copyright &y& Elsevier]

At the back of the eye, the outermost cell layer of the retina, the pigmented epithelium, lies against a basement membrane that is adjacent to the choroidal vessels that supply the outer sensory retina. During pathogenesis, these interfaces become damaged, and the homeostatic balance between the retinal pigment epithelium (RPE) and the choroidal vessels becomes disrupted, leading to choroidal neovascularization and blindness. To study the cell interactions at the back of the eye, we have used a coculture system in which a stable RPE monolayer has been cultured on a transwell insert and placed over a collagen gel sandwich into which choroidal endothelial cells (CECs) have been seeded. RPE cells have been stimulated by an inflammatory cytokine, interleukin-1 (IL-1β), and the ability of the underlying choroidal endothelium to form vascular tubes has been tested. IL-1β stimulation of the RPE insert increased the number of tubes formed by CECs in the gel as early as 3 d. By 7 d, tubes began to regress. Both IL-8 and monocyte chemotactic protein-1 (MCP-1) were found to be secreted in greater amounts in stimulated RPE. Because MCP-1 is also a chemokine for monocytes, which in turn secrete angiogenic factors, monocytes were added to the upper surface of the choroidal gel sandwich and then incubated with the stimulated RPE insert as above. By day 7, more tubes formed and there was no regression over the experimental time period. The versatility of this model has been illustrated in that both RPE and CECs can be cultured in a more natural construct and their molecular interactions tested by physiologically altering one cell type and not the other. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the changes in transepithelial electrical resistance (TEER) and ion concentrations, and their relationship in TGF-β-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells., Methods: RPE cell line ARPE-19 was employed and treated with 10 ng/ml TGF-β1 and TGF-β2 to establish the EMT model in vitro . The EMT markers fibronectin, N-cadherin, occludin, zona occludens 1(ZO-1) and claudin-19 were investigated by western blot and immunofluorescence. CellZscope system was used to monitor the TEER values. Fluorescent probe, flow cytometry and automatic microplate reader were employed to detect the changes of Ca 2+ , Mg 2+ , Zn 2+ , Na + and K + in ARPE-19 cells., Results: The TGF-β1-induced EMT of ARPE-19 cells was marked by the disruption of the distribution of occludin, ZO-1, and claudin-19. The development of TEER was significantly disturbed in both TGF-β1 and TGF-β2 treatment groups. Also, the time course of the maximum slope indicated that the fastest decrease in TEER values occurred after 36 hours. The concentrations of Ca 2+ , Mg 2+ , Zn 2+ , and K + increased in TGF-β1- and TGF-β2-treated ARPE-19 cells, while the concentration of Na + decreased. Significant inverse correlations were detected between the concentrations of Ca 2+ , Mg 2+ , Zn 2+ , and K + and TEER values in ARPE-19 cells treated with TGF-β1. The Na + concentration and TEER values showed a positive correlation. Similar results were observed in the TGF-β2 treatment group. The time-effect analysis showed that the concentrations of Ca 2+ , Mg 2+ , Zn 2+ and K + increased and peaked after 72, 72, 48, and 72 h, respectively, with the extension of TGF-β1 treatment time. In the TGF-β2 treatment group, the Ca 2+ , Mg 2+ , Zn 2+ , and K + concentrations were also upregulated and reached their highest after 72, 72, 72, and 36 h, respectively. In contrast, the concentration of Na + decreased and reached the lowest after 48 h in the TGF-β1 treatment group and after 72 h in the TGF-β2 treatment group., Conclusion: TGF-β1 and TGF-β2 disrupted the ARPE-19 cell monolayer, disturbed TJs integrity, downregulated TEER values, and changed intracellular ion permeability. These findings might help further understand the EMT of RPE cells during PVR., Competing Interests: None., (AJTR Copyright © 2022.)

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 6 -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.

Aim: To investigate the relationship between autophagy and apoptosis in photoinduced injuries in retinal pigment epithelium (RPE) cells and how Lycium barbarum polysaccharide (LBP) contributes to the increased of RPE cells to photoinduced autophagy., Methods: In vitro cultures of human RPE strains (ARPE-19) were prepared and randomly divided into the blank control, model, low-dose LBP, middle-dose LBP, high-dose LBP, and 3-methyladenine (3MA) groups. The viability of the RPE cells and apoptosis levels in each group were tested through cell counting kit-8 (CCK8) method with a flow cytometer (Annexin V/PI double staining technique). The expression levels of LC3II, LC3I, and P62 proteins were detected with the immunofluorescence method. The expression levels of beclin1, LC3, P62, PI3K, P-mTOR, mTOR, P-Akt, and Akt proteins were tested through Western blot., Results: LBP considerably strengthens cell viability and inhibits the apoptosis of RPE cells after photoinduction. The PI3K/Akt/mTOR signal pathway is activated because of the upregulation of the phosphorylation levels of Akt and mTOR proteins, and thus autophagy is inhibited., Conclusion: LBP can inhibit the excessive autophagy in RPE cells by activating the PI3K/Akt/mTOR signaling pathways and thereby protect RPE cells from photoinduced injuries., (International Journal of Ophthalmology Press.)

The purpose of this study was to investigate whether excessive extracellular matrix (ECM) deposition-induced mechanical matrix stiffness plays a key role in promoting retinal pigment epithelial (RPE) cell activation and the subsequent development of proliferative vitreoretinopathy (PVR). Human ARPE-19 cells were cultured on either 50 kappa (stiff) or 0.5 kappa (soft) gel-coated coverslips. Reverse and knockdown experiments were carried out to establish a model of matrix stiffness-induced activation in ARPE-19 cells in vitro. A PVR mouse model was established by the intravitreal injection of dispase. The effects of RhoA/YAP signalling blockade on matrix stiffness-induced ARPE-19 cell activation and PVR-induced retinal fibrosis were determined by using a combination of the Yes-associated protein (YAP) inhibitor verteporfin and the RhoA inhibitor C3 exoenzyme. Matrix stiffness stimulated YAP nuclear translocation and expression in ARPE-19 cells. The effect of YAP activation was dependent on F-actin cytoskeleton polymerization and RhoA activity, forming the RhoA/YAP signalling pathway. Upstream pharmacological blockade of RhoA by C3 exoenzyme or downstream blockade of YAP by verteporfin reduced the invasion, migration, and MMP expression of ARPE-19 cells and collagen gel contraction. Furthermore, blockade of RhoA/YAP signalling reduced PVR-induced retinal fibrogenesis and inhibited the TGF-β/Smad pathway in vivo. RhoA/YAP signalling modulates matrix stiffness-induced activation of ARPE-19 cells. Targeting this signalling pathway could alleviate PVR-induced retinal fibrosis and suggests attractive novel therapeutic strategies for intervening in the progression of PVR. • We firstly studied the role of mechanical matrix stiffness in promoting RPE cell activation and development of PVR. • Blockade of RhoA/YAP signaling reduced PVR-induced retinal fibrogenesis and inhibited the TGF-β/Smad pathway. • Targeting RhoA/YAP signaling could alleviate retinal fibrosis and suggest novel therapeutic strategy for intervening PVR. [ABSTRACT FROM AUTHOR]

The fine-structural features of pinealocytes of the white-footed mouse, Peromyscus leucopus, were examined. A single population of pineaocytes was observed in both superficial and deep components of the gland. Cells in both locations are characterized by the presence of an indented nucleus exhibiting a prominent nucleolus. The usual organelles in the perikaryon are the Golgi complexes, mitochondria, endoplasmic reticulum, polysemes, microtubules and dense-core vesicles. In addition the perikaryonal cytoplasm often contains a single myeloid body. These bodies are usually lenticular in shape, are formed of an array of flattened membranous cisternae, and are not bounded by a limiting membrane. This organelle usually lies in the vicinity of the nucleus and is infrequently associated with lipid bodies. Complex forms were also observed. The cisternae are continuous with elements of the endoplasmic reticulum at points along their expanded rims. The outer surface of the cisternal membrane exhibits a granularity or fuzziness. The cisternae may be fenestrated. Pinealocyte processes with an abundance of clear vesicles, and some dense-core vesicles and vesicle-crowned rodlets are present in the parenchyma. [ABSTRACT FROM AUTHOR]