Vitamin C alleviates hyperglycemic stress in retinal pigment epithelial cells.

BACKGROUND: Retinal pigment epithelial cells (RPECs) are a type of retinal cells that structurally and physiologically support photoreceptors. However, hyperglycemia has been shown to play a critical role in the progression of diabetic retinopathy (DR), which is one of the leading causes of vision impairment. In the diabetic eye, the high glucose environment damages RPECs via the induction of oxidative stress, leading to the release of excess reactive oxygen species (ROS) and triggering apoptosis. In this study, we aim to investigate the antioxidant mechanism of Vitamin C in reducing hyperglycemia-induced stress and whether this mechanism can preserve the function of RPECs. METHODS AND RESULTS: ARPE-19 cells were treated with high glucose in the presence or absence of Vitamin C. Cell viability was measured by MTT assay. Cleaved poly ADP-ribose polymerase (PARP) was used to identify apoptosis in the cells. ROS were detected by the DCFH-DA reaction. The accumulation of sorbitol in the aldose reductase (AR) polyol pathway was determined using the sorbitol detection assay. Primary mouse RPECs were isolated from adult mice and identified by Rpe65 expression. The mitochondrial damage was measured by mitochondrial membrane depolarization. Our results showed that high glucose conditions reduce cell viability in RPECs while Vitamin C can restore cell viability, compared to the vehicle treatment. We also demonstrated that Vitamin C reduces hyperglycemia-induced ROS production and prevents cell apoptosis in RPECs in an AR-independent pathway. CONCLUSIONS: These results suggest that Vitamin C is not only a nutritional necessity but also an adjuvant that can be combined with AR inhibitors for alleviating hyperglycemic stress in RPECs.

IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stage streptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

Both hemoglobin and hemin cause damage to retinal pigment epithelium through the iron ion accumulation.

Subretinal hemorrhages result in poor vision and visual field defects. During hemorrhage, several potentially toxic substances are released from iron-based hemoglobin and hemin, inducing cellular damage, the detailed mechanisms of which remain unknown. We examined the effects of excess intracellular iron on retinal pigment epithelial (RPE) cells. A Fe2+ probe, SiRhoNox-1 was used to investigate Fe2+ accumulation after treatment with hemoglobin or hemin in the human RPE cell line ARPE-19. We also evaluated the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, the protective effect of-an iron chelator, 2,2'-bipyridyl (BP), and ferrostatin-1 (Fer-1) on the cell damage, was evaluated. Fe2+ accumulation increased in the hemoglobin- or hemin-treated groups, as well as intracellular ROS production and lipid peroxidation. In contrast, BP treatment suppressed RPE cell death, ROS production, and lipid peroxidation. Pretreatment with Fer-1 ameliorated cell death in a concentration-dependent manner and suppressed ROS production and lipid peroxidation. Taken together, these findings indicate that hemoglobin and hemin, as well as subretinal hemorrhage, may induce RPE cell damage and visual dysfunction via intracellular iron accumulation.

Tamoxifen protects photoreceptors in the sodium iodate model.

Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO3) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO3. RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics.

Artemisinin Confers Cytoprotection toward Hydrogen Peroxide-Induced Cell Apoptosis in Retinal Pigment Epithelial Cells in Correlation with the Increased Acetylation of Histone H4 at Lysine 8.

Increased oxidative stress is one of the critical pathologies inducing age-related macular degeneration (AMD), characterized by retinal pigment epithelial (RPE) cell damage and death. The unbalanced acetylation and deacetylation of histones have been implicated in AMD pathogenesis or hydrogen peroxide (H2O2)-induced cell damage. Therefore, strategies aimed at controlling the balance between acetylation and deacetylation may effectively protect RPE cells from oxidative damage. Artemisinin is an antimalarial lactone drug derived from Artemisia annua, with antioxidant activity known to modulate histone acetylation in the brain, but its effect on the retina is unknown. In this study, we aimed to investigate whether Artemisinin exerts a cytoprotective effect on oxidative stress-induced apoptosis in RPE cells by regulating histone acetylation. We hypothesized that Artemisinin confers cytoprotection toward H2O2-induced apoptosis in RPE cells through this mechanism. In the present study, we found that Artemisinin at a sub-clinic dosage of 20 µM inhibited the H2O2-induced cell viability decrease and B-cell lymphoma 2 (Bcl-2) protein level decrease and attenuated the H2O2-induced decrease in the histone H4 lysine (Lys) 8 acetylation [Acetyl-H4 (Lys 8)] level in the retinal RPE cell line D407. As expected, histone deacetylase inhibitor Trichostatin A at the concentration of 250 nM increased the Acetyl-H4 (Lys 8) level in D407 cells and attenuated the H2O2-induced cell viability decrease and apoptosis. Similar findings were obtained using adult RPE (ARPE)19 cells, another human RPE cell line, and primary human RPE cell cultures. In conclusion, these results confirmed our hypothesis and indicated that Artemisinin attenuated H2O2-induced apoptosis in apparent correlation with the increase in the Acetyl-H4 (Lys 8) level, which is associated with gene transcription and cell survival. By modulating histone acetylation, Artemisinin may restore the balance between acetylation and deacetylation and enhance the resistance and survival of RPE cells under oxidative stress. Our study provides novel mechanistic insights into the effect of Artemisinin on histone acetylation and apoptosis in RPE cells and supports the potential application of Artemisinin in the prevention and/or treatment of AMD.

Effect of adalimumab as an anti-inflammatory agent on gene expression of retinal pigment epithelial cells.

Adalimumab (ADA) is an anti-inflammatory antibody that has FDA approval as a systemic medication for treating noninfectious uveitis. It is also provisionally being investigated as an intravitreal injection for various retinal conditions. This study aimed to assess the effect of ADA on apoptotic, inflammatory, and fibrogenesis gene expression at mRNA and protein levels in retinal pigment epithelial (RPE) cells. RPEs were treated with serial concentrations of ADA (0.5x, x, 2x, and 4x; [x = 250 µg/mL]) for 24 hours. MTT assay was done and the mRNA and protein expressions were quantified using real-time PCR and ELISA assay, respectively. The mRNA levels of IL-1b and IL-6 were significantly increased in ADA-treated RPEs at 0.5x and x concentrations. However, the increase in cytokine secretion was observed only in IL-1b at x concentration. TGF-ß was significantly upregulated in the 0.5x and 4x doses of ADA both at mRNA and protein levels. MTT assay, along with an unchanged BCL-2/BAX ratio confirmed the safety of ADA on RPEs at all studied concentrations. In conclusion, despite its safety, the 2x concentration of ADA was the only dose that did not ignite the expression of any of the studied inflammatory and fibrogenesis genes. This dosage, which is roughly equal to 2 mg intravitreal dose in a clinical setting, might be referred to as a reference starting point for future in-vivo studies in ocular conditions.

Screening and evaluation of antioxidants for retinal pigment epithelial cell protection: L-ergothioneine as a novel therapeutic candidate through NRF2 activation.

The continual exposure of retinal tissues to oxidative stress leads to discernible anatomical and physiological alterations. Specifically, the onslaught of oxidative damage escalates the irreversible death of retinal pigmented epithelium (RPE) cells, pinpointed as the fundamental pathological event in dry age-related macular degeneration (AMD). There is a conspicuous lack of effective therapeutic strategies to counteract this degenerative process. This study screened a library of antioxidants for their ability to protect RPE cells against oxidative stress and identified L-ergothioneine (EGT) as a potent cytoprotective agent. L-ergothioneine provided efficient protection against oxidative stress-damaged RPE and maintained cell redox homeostasis and normal physiological functions. It maintained the normal structure of the retina in mice under oxidative stress conditions. Transcriptomic analysis revealed that EGT counteracted major gene expression changes induced by oxidative stress. It upregulated antioxidant gene expression and inhibited NRF2 translocation. The inhibition of NRF2 abolished EGT's protective effects, suggesting that NRF2 activation contributes to its mechanism of action. In conclusion, we identified EGT as a safe and effective small-molecule compound that is expected to be a novel antioxidative agent for treating AMD.

PEDF-derived peptide protects against Amyloid-ß toxicity in vitro and prevents retinal dysfunction in rats.

Amyloid-beta (Aß), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aß42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aß42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aß-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aß42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aß, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aß42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aß42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aß42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aß42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aß42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aß42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aß42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aß42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aß42 in the retina and suggest concepts on the molecular mechanism of Aß retinal pathogenicity.

Senescence-induced alteration of circadian phagocytic activity of retinal pigment epithelium cell line ARPE-19.

Renewal of retinal photoreceptor outer segments is conducted through daily shedding of distal photoreceptor outer segment tips and subsequent their phagocytosis by the adjacent retinal pigment epithelium (RPE) monolayer. Dysregulation of the diurnal clearance of photoreceptor outer segment tips has been implicated in age-related retinal degeneration, but it remains to be clarified how the circadian phagocytic activity of RPE cells is modulated by senescence. In this study, we used the human RPE cell line ARPE-19 to investigate whether hydrogen peroxide (H2O2)-induced senescence in ARPE-19 cells alters the circadian rhythm of their phagocytic activity. After synchronization of the cellular circadian clock by dexamethasone treatment, the phagocytic activity of normal ARPE-19 cells exhibited significant 24-h oscillation, but this oscillation was modulated by senescence. The phagocytic activity of senescent ARPE-19 cells increased constantly throughout the 24-h period, which still exhibited blunted circadian oscillation, accompanied by an alteration in the rhythmic expression of circadian clock genes and clock-controlled phagocytosis-related genes. The expression levels of REV-ERBα, a molecular component of the circadian clock, were constitutively increased in senescent ARPE-19 cells. Furthermore, pharmacological activation of REV-ERBα by its agonist SR9009 enhanced the phagocytic activity of normal ARPE-19 cells and increased the expression of clock-controlled phagocytosis-related genes. Our present findings extend to understand the role of circadian clock in the alteration of phagocytic activity in RPE during aging. Constitutive enhancement of phagocytic activity of senescent RPE may contribute to age-related retinal degeneration.

Vitamin B3, nicotinamide, enhances mitochondrial metabolism to promote differentiation of the retinal pigment epithelium.

In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception.

5-Lipoxygenase Inhibition Protects Retinal Pigment Epithelium from Sodium Iodate-Induced Ferroptosis and Prevents Retinal Degeneration.

Excessive reactive oxygen species (ROS) contribute to damage of retinal cells and the development of retinal diseases including age-related macular degeneration (AMD). ROS result in increased metabolites of lipoxygenases (LOXs), which react with ROS to induce lipid peroxidation and may lead to ferroptosis. In this study, the effect of 5-LOX inhibition on alleviating ROS-induced cell death was evaluated using sodium iodate (NaIO3) in the retinal pigment epithelium (RPE) cell line ARPE-19 and a mouse model investigating oxidative stress in AMD. We demonstrated that NaIO3 induced cell death in the RPE cells through mechanisms including ferroptosis. Inhibition of 5-LOX with specific inhibitor, Zileuton, or siRNA knockdown of ALXO5 mitigated NaIO3-induced lipid peroxidation, mitochondrial damage, DNA impairment, and cell death in ARPE-19 cells. Additionally, in the mouse model, pretreatment with Zileuton reduced the NaIO3-induced lipid peroxidation of RPE cells, cell death in the photoreceptor layer of the retina, inflammatory responses, and degeneration of both the neuroretina and RPE monolayer cells. Our results suggest that 5-LOX plays a crucial role in ROS-induced cell death in the RPE and that regulating 5-LOX activity could be a useful approach to control ROS and ferroptosis-induced damage, which promote degeneration in retinal diseases.

Late-stage rescue of visually guided behavior in the context of a significantly remodeled retinitis pigmentosa mouse model.

Patients with progressive neurodegenerative disorder retinitis pigmentosa (RP) are diagnosed in the midst of ongoing retinal degeneration and remodeling. Here, we used a Pde6b-deficient RP gene therapy mouse model to test whether treatment at late disease stages can halt photoreceptor degeneration and degradative remodeling, while sustaining constructive remodeling and restoring function. We demonstrated that when fewer than 13% of rods remain, our genetic rescue halts photoreceptor degeneration, electroretinography (ERG) functional decline and inner retinal remodeling. In addition, in a water maze test, the performance of mice treated at 16 weeks of age or earlier was indistinguishable from wild type. In contrast, no efficacy was apparent in mice treated at 24 weeks of age, suggesting the photoreceptors had reached a point of no return. Further, remodeling in the retinal pigment epithelium (RPE) and retinal vasculature was not halted at 16 or 24 weeks of age, although there appeared to be some slowing of blood vessel degradation. These data suggest a novel working model in which restoration of clinically significant visual function requires only modest threshold numbers of resilient photoreceptors, halting of destructive remodeling and sustained constructive remodeling. These novel findings define the potential and limitations of RP treatment and suggest possible nonphotoreceptor targets for gene therapy optimization.

Deletion of Specific Conserved Motifs from the N-Terminal Domain of αB-Crystallin Results in the Activation of Chaperone Functions.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54-61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54-61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21-28 residues from the activated αBΔ54-61 (to get αBΔ21-28, Δ54-61) on the structure-function of recombinant αBΔ21-28, Δ54-61. The αBΔ21-28, Δ54-61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21-28, ∆54-61 was found to prevent ß-amyloid (Aß1-42) fibril formation in vitro and suppressed Aß1-42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54-61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54-61 in oxidatively stressed cells. Our study shows that the residues 21-28 and 54-61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21-28 residues further potentiates the activated αBΔ54-61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21-28, Δ54-61.

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells.

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.

Chronic Systemic Dexamethasone Regulates the Mineralocorticoid/Glucocorticoid Pathways Balance in Rat Ocular Tissues.

Central serous chorioretinopathy (CSCR) is a retinal disease affecting the retinal pigment epithelium (RPE) and the choroid. This is a recognized side-effect of glucocorticoids (GCs), administered through nasal, articular, oral and dermal routes. However, CSCR does not occur after intraocular GCs administration, suggesting that a hypothalamic-pituitary-adrenal axis (HPA) brake could play a role in the mechanistic link between CSCR and GS. The aim of this study was to explore this hypothesis. To induce HPA brake, Lewis rats received a systemic injection of dexamethasone daily for five days. Control rats received saline injections. Baseline levels of corticosterone were measured by Elisa at baseline and at 5 days in the serum and the ocular media and dexamethasone levels were measured at 5 days in the serum and ocular media. The expression of genes encoding glucocorticoid receptor (GR), mineralocorticoid receptors (MR), and the 11 beta hydroxysteroid dehydrogenase (HSD) enzymes 1 and 2 were quantified in the neural retina and in RPE/ choroid. The expression of MR target genes was quantified in the retina (Scnn1A (encoding ENac-α, Kir4.1 and Aqp4) and in the RPE/choroid (Shroom 2, Ngal, Mmp9 and Omg, Ptx3, Plaur and Fosl-1). Only 10% of the corticosterone serum concentration was measured in the ocular media. Corticosterone levels in the serum and in the ocular media dropped after 5 days of dexamethasone systemic treatment, reflecting HPA axis brake. Whilst both GR and MR were downregulated in the retina without MR/GR imbalance, in the RPE/choroid, both MR/GR and 11ß-hsd2/11ß-hsd1 ratio increased, indicating MR pathway activation. MR-target genes were upregulated in the RPE/ choroid but not in the retina. The psychological stress induced by the repeated injection of saline also induced HPA axis brake with a trend towards MR pathway activation in RPE/ choroid. HPA axis brake causes an imbalance of corticoid receptors expression in the RPE/choroid towards overactivation of MR pathway, which could favor the occurrence of CSCR.

STING up-regulates VEGF expression in oxidative stress-induced senescence of retinal pigment epithelium via NF-κB/HIF-1α pathway.

AIM: Aging-related dysfunction of retinal pigment epithelium (RPE) is the main pathogenic factors for pathological angiogenesis due to dysregulated vascular endothelial growth factor (VEGF) in retinal vascular diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). However, the molecular mechanism behind the up-regulation of VEGF in senescent RPE is still blurred. MATERIALS AND METHODS: As oxidative damage is the key cause of RPE dysfunction, we employed a model of oxidative stress-induced premature senescence of ARPE-19 to explore the effect of senescent RPE on VEGF. KEY FINDINGS: We reported that senescent ARPE-19 up-regulated VEGF expression under both short-term and prolonged H2O2 treatment, accompanying with increased HIF-1α, the key mediator of VEGF. STING signaling, which could be activated by oxidative stress-damaged DNA, was also observed to be increased in senescent ARPE-19 treated with H2O2. And the inhibition of STING significantly reduced HIF-1α expression to alleviate the up-regulation of VEGF. NF-κB was also shown to be involved in the regulation of VEGF in senescent ARPE-19 in response to STING signaling. Furthermore, oxidative stress impaired the lysosomal clearance of damaged DNA to enhance STING signaling, thereby up-regulating VEGF expression in senescent RPE. SIGNIFICANCE: Our data provide evidence that STING plays an important role in VEGF regulation in senescent RPE induced by oxidative stress.

Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

Effect of Long-term Anti-VEGF Treatment on Viability and Function of RPE Cells.

PURPOSE/AIM OF THE STUDY: Vascular endothelial growth factor (VEGF)-antagonists are given over long time periods in the clinic, but the long-term effects on retinal pigment epithelium (RPE) cells are not fully investigated. This study aims to investigate these effects with two clinical relevant VEGF antagonists, bevacizumab and aflibercept, on the function of primary RPE cells. MATERIALS AND METHODS: All tests were conducted with primary porcine RPE. Cells were stimulated with bevacizumab or aflibercept (both 250 µg/ml) for 1 day, 7 days or 4 weeks. Cell viability was tested in MTT Assay. Secretion of TGF-ß was tested in ELISA, phagocytosis in a microscopic assay, migration in a scratch assay, and expression of RPE65 in Western blot. Barrier function was tested for bevacizumab in transwell-cultured cells by measuring transepithelial electrical resistance for up to 3 days. RESULTS: Viability was reduced by both antagonists at all time points tested. TGF-ß secretion was not altered by any treatment. Phagocytosis was not significantly reduced by any treatment. Wound healing ability was not significantly altered by any treatment. The expression of RPE65 was reduced by bevacizumab but not aflibercept after 4 weeks. Transepithelial electrical resistance was not altered. CONCLUSIONS: Long-term treatment with anti VEGF may affect viability of RPE cells, and treatment with bevacizumab may have effects on RPE function in long-term treatment.

Oleuropein Protects Human Retinal Pigment Epithelium Cells from IL-1ß-Induced Inflammation by Blocking MAPK/NF-κB Signaling Pathways.

Proinflammatory mediators such as interleukin (IL)-1ß cause retinal pigment epithelium (RPE) inflammation, which is related to visual deterioration, including age-related macular degeneration and diabetic retinopathy. Oleuropein is a polyphenol compound that shows potent anti-inflammatory, antioxidant, and anti-cancer activities, but its effects on IL-1ß-induced inflammation have not been examined in the adult RPE cell line ARPE-19. Here, we assessed the ability of oleuropein to attenuate this inflammation in ARPE-19 cells. IL-1ß induced secretion of the inflammatory cytokines IL-6, monocyte chemoattractant protein-1 (MCP)-1, and soluble intercellular adhesion molecule (sICAM)-1. As measured by enzyme-linked immunosorbent assay, oleuropein significantly inhibited levels of all three proteins and led to decreased monocyte adhesiveness to ARPE-19 cells. To clarify the underlying anti-inflammatory mechanisms, we used western blots to evaluate the effect of oleuropein on inactivation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. The results showed that oleuropein significantly decreased levels of the inflammatory mediator cyclooxygenase-2 and increased anti-inflammatory protein HO-1 expression. We next examined if the anti-inflammatory activity of oleuropein arises via inactivated NF-κB. We found that suppressing phosphorylation of the JNK1/2 and p38 MAPK signaling pathways inhibited IL-6, MCP-1, and sICAM-1 secretion, implicating these pathways and NF-κB suppression in the effects of oleuropein. These results indicate that oleuropein shows potential for the prevention and treatment of inflammatory diseases of the retina.

Inhibition of prolyl oligopeptidase: A promising pathway to prevent the progression of age-related macular degeneration.

Dry age-related macular degeneration (AMD) is a currently untreatable vision threatening disease. Impaired proteasomal clearance and autophagy in the retinal pigment epithelium (RPE) and subsequent photoreceptor damage are connected with dry AMD, but detailed pathophysiology is still unclear. In this paper, we discover inhibition of cytosolic protease, prolyl oligopeptidase (PREP), as a potential pathway to treat dry AMD. We showed that PREP inhibitor exposure induced autophagy in the RPE cells, shown by increased LC3-II levels and decreased p62 levels. PREP inhibitor treatment increased total levels of autophagic vacuoles in the RPE cells. Global proteomics was used to examine the phenotype of a commonly used cell model displaying AMD characteristics, oxidative stress and altered protein metabolism, in vitro. These RPE cells displayed induced protein aggregation and clear alterations in macromolecule metabolism, confirming the relevance of the cell model. Differences in intracellular target engagement of PREP inhibitors were observed with cellular thermal shift assay (CETSA). These differences were explained by intracellular drug exposure (the unbound cellular partition coefficient, Kpuu). Importantly, our data is in line with previous observations regarding the discrepancy between PREP's cleaving activity and outcomes in autophagy. This highlights the need to further explore PREP's role in autophagy so that more effective compounds can be designed to battle diseases in which autophagy induction is needed. The present work is the first report investigating the PREP pathway in the RPE and we predict that the PREP inhibitors can be further optimized for treatment of dry AMD.