ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

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-1ß, 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.

Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-ß2 stimulation in retinal pigment epithelial cells through modulation of the p38 and JNK MAPK pathways.

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-ß2 (TGF-ß2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-ß2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-ß2. The treatment of RPE cells with TGF-ß2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-ß2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-ß2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-ß2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.

Oxidized-LDL Induces Metabolic Dysfunction in Retinal Pigment Epithelial Cells.

Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid ß-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.

Gigantol protects retinal pigment epithelial cells against high glucose-induced apoptosis, oxidative stress and inflammation by inhibiting MTDH-mediated NF-kB signaling pathway.

OBJECTIVE: As a frequent complication of diabetes mellitus (DM), diabetic retinopathy (DR) is now one of the major causes of blindness. Recent reports have shown that retinal pigment epithelial cell (RPEC) damage plays an essential part in DR development and progression. This work intended to explore the potential effects of Gigantol on high glucose (HG)-stimulated RPEC damage and identify potential mechanisms. METHODS: Cell viability, cell damage, and cell apoptosis were evaluated by CCK-8, lactate dehydrogenase (LDH) and flow cytometry assays. The levels of oxidative stress biomarkers and pro-inflammatory cytokines were assessed using corresponding commercial kits and ELISA. Additionally, the levels of MTDH and NF-kB signaling pathway-related proteins were detected by western blotting. RESULTS: Gigantol dose-dependently enhanced cell viability and decreased apoptosis in HG-challenged ARPE-19 cells. Also, Gigantol notably relieved oxidative stress and inflammatory responses in ARPE-19 cells under HG conditions. Gigantol dose-dependently suppressed MTDH expression. In addition, MTDH restoration partially counteracted the protective effects of Gigantol on ARPE-19 cells subject to HG treatment. Mechanically, Gigantol inactivated the NF-kB signaling pathway, which was partly restored after MTDH overexpression. CONCLUSION: Our findings suggested that Gigantol protected against HG-induced RPEC damage by inactivating the NF-kB signaling via MTDH inhibition, offering a potent therapeutic drug for DR treatment.

Ascorbic acid protects retinal pigment epithelial cells from high glucose by inhibiting the NF-κB signal pathway through MALAT1/IGF2BP3 axis.

BACKGROUND: Diabetic retinopathy (DR) is a common complication of diabetes with nocuous effects on patients' eye health, typically accompanies by excessive inflammation and oxidative stress. Insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) was engaged with inflammation, whereas its precise role in the DR process was unclear. And enhanced lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and decreased ascorbic acid (AA) were also found in DR. This study was to explore the regulatory role and mechanism of IGF2BP3, MALAT1 and AA in the high glucose (HG)-induced retinal pigment epithelial (RPE) cell injury. METHODS: ARPE-19 cells were treated with HG to establish the in vitro RPE cell injury model. The mRNA and protein levels of the gene were evaluated by qRT-PCR or Western blot. Immunofluorescence detected the translocation condition of the p65 protein. Inflammatory factor levels were detected by ELISA assays. Apoptosis was detected by flow cytometry. The binding interaction of IGF2BP3 and MALAT1 was validated by RIP-qPCR assays. RESULTS: In HG-induced RPE cell injury, IGF2BP3 expression, inflammatory response and apoptosis were enhanced. Next, the IGF2BP3 activated the NF-κB signalling to promote the RPE cell injury development. MALAT1 could directly bind with IGF2BP3 and up-regulate its expression. In addition, AA ameliorated the HG-induced RPE cell injury through the regulation of MALAT1. CONCLUSION: Ascorbic acid ameliorated HG-induced RPE cell injury by repressing the NF-κB signalling pathway via modulating the MALAT1/IGF2BP3 axis.

Effects of galectin-3 protein on UVA-induced damage in retinal pigment epithelial cells.

Several inflammatory molecules have been suggested as biomarkers of age-related macular degeneration (AMD). Galectin-3 (Gal-3), which has been shown to have a protective role in corneal injury by promoting epithelial cells adhesion and migration to the extracellular matrix, is also highly expressed in the retinal pigment epithelium (RPE) of patients with AMD. This study evaluated the role of Gal-3 in an in vitro model of UVA-induced RPE damage, as a proof-of-concept. ARPE-19 cells (human RPE cell line), were incubated with Gal-3 at 0.5-2.5 µg/mL concentrations prior to UVA irradiation for 15, 30, and 45 min, which resulted in accumulated doses of 2.5, 5, and 7.5 J/cm2, respectively. After 24 h incubation, MTT and LDH assays, immunofluorescence, and ELISA were performed. UVA irradiation for 15, 30, and 45 min proved to reduce viability in 83%, 46%, and 11%, respectively. Based on the latter results, we chose the intermediate dose (5-J/cm2) for further analysis. Pretreatment with Gal-3 at concentrations > 1.5 µg/mL showed to increase the viability of UVA-irradiated cells (~ 75%) compared to untreated cells (64%). Increased levels of cleaved caspase 3, a marker of cell death, were detected in the ARPE cells after UVA irradiation with or without addition of exogenous Gal-3. The inhibitory effect of Gal-3 on UVA-induced cell damage was characterized by decreased ROS levels and increased p38 activation, as detected by fluorescence analysis. In conclusion, our study suggests a photoprotective effect of Gal-3 on RPE by reducing oxidative stress and increasing p38 activation.

Particulate matter 2.5 exposure induces epithelial-mesenchymal transition via PI3K/AKT/mTOR pathway in human retinal pigment epithelial ARPE-19 cells.

Exposure to particulate matter 2.5 (PM2.5) has been linked to ocular surface diseases, yet knowledge of the molecular mechanism impacted on retina pathogenesis is limited. Therefore, the purpose of this study was to explore the effects and involved factors of PM2.5 exposure in human retinal pigment epithelial APRE-19 cells. Our data revealed a decreased cell viability and an increased migratory ability in APRE-19 cells after PM2.5 stimulation. The MMP-2 and MMP-9 protein levels were markedly increased while the MMPs regulators TIMP-1 and TIMP-2 were significantly reduced in PM2.5-exposed APRE-19 cells. PM2.5 also increased pro-MMP-2 expression in the cell culture supernatants. Additionally, PM2.5 promoted the EMT markers through the activation of PI3K/AKT/mTOR pathway. Moreover, the ICAM-1 production was also remarkably increased by PM2.5 but reduced by PI3K/AKT inhibitor LY294002 in APRE-19 cells. Taken together, these results suggest that PM2.5 promotes EMT in a PI3K/AKT/mTOR-dependent manner in the retinal pigment epithelium.

The modulation of cAMP/PKA pathway by asiaticoside ameliorates high glucose-induced inflammation and apoptosis of retinal pigment epithelial cells.

Asiaticoside, the major bioactive constituent purified from Centella asiatica, is a pentacyclic triterpene saponin with sugar moieties (glucose-glucose-rhamnose). Its biological activities including anti-inflammation and antioxidant have been widely reported. This study aimed to investigate the role of asiaticoside in diabetic retinopathy (DR). Human retinal pigment epithelium (RPE) cells ARPE-19 were induced by high glucose. Then, cell survival rate, expression of inflammatory factors, oxidative stress, and apoptosis were measured by MTT method, western blot, oxidative stress detection kits and TUNEL respectively. To uncover the underlying mechanism, the levels of cyclic AMP (cAMP) and protein kinase A (PKA) were measured by Enzyme linked immunosorbent assay (ELISA) and PKA activities were detected by the Kemptide phosphorylation assay. Furthermore, cAMP inhibitor SQ22536 was also used to validate the mechanism. Asiaticoside suppressed the inflammation and apoptosis of ARPE-19 cells, and the activities of cAMP and PKA were inhibited upon HG induction while again released after further administration of asiaticoside. However, these effects were all abolished by SQ22536. In conclusion, we have demonstrated in this paper that asiaticoside ameliorates high glucose-induced inflammation and apoptosis of RPE cells by activating cAMP/PKA signaling pathway. asiaticoside-mediated activation of cAMP/PKA signaling pathway may serve as a potential target for the management of DR.

Effect of Sirt3 on retinal pigment epithelial cells in high glucose through Foxo3a/ PINK1-Parkin pathway mediated mitophagy.

Sirt3 is closely associated with mitophagy. This study aimed to investigate the effect and potential mechanism of Sirt3 on mitophagy in retinal pigment epithelium (RPE) in a high glucose environment. The expression levels of Sirt3, Foxo3a, PINK1, Parkin and LC3B in RPE subjected to high-glucose (HG, 30 mM D-glucose) conditions were detected by RT-PCR and western blotting. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to detect the level of reactive oxygen species (ROS) in RPE treated with HG. MitoTracker and LysoTracker probes were used to label mitochondria and lysosomes, respectively, to observe the occurrence of autophagy. Sirt3-dependent regulation of mitophagy through the Foxo3a/PINK1-Parkin pathway was further investigated by virus transfection-mediated Sirt3 overexpression and PINK1 silencing. The effect of Sirt3 overexpression on apoptosis was detected by flow cytometry. The Sirt3 expression was decreased, the Foxo3a/PINK1-Parkin pathway was inhibited, intracellular ROS level was increased, and mitophagy was attenuated in RPE under HG condition. Sirt3 overexpression activated the Foxo3a/PINK1-Parkin signaling pathway and mitophagy, and inhibited cell apoptosis. Silencing PINK1 inhibited the effect of Sirt3 overexpression on mitophagy. In summary, Sirt3 can activate mitophagy through the Foxo3a/PINK1-Parkin pathway and reduce HG-induced apoptosis of RPE. This study provides a new direction to understand the pathogenesis and develop a potential therapeutic target for diabetic retinopathy.

Elimination of senescent cells inhibits epithelial-mesenchymal transition of retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is one of the most common leading causes of irreversible blindness, and there is no effective treatment for it. It has been reported that aging is the greatest risk factor for AMD, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells plays an important role in the pathogenesis of AMD. To clarify the relationship between senescence and EMT in RPE cells, we used the replicative senescence model, H2O2- and/or Nutlin3a-induced senescence model, and low-density and/or TGF-ß-induced EMT model to detect the expression of senescence-, RPE- and EMT-related genes, and assessed the motility of cells by using a scratch wound migration assay. The results showed that replicative senescence of RPE cells was accompanied by increased expression of EMT markers. However, senescent RPE cells themselves did not undergo EMT, as the H2O2and Nutlin3a treated cells showed no increase in EMT characteristics, including unchanged or decreased expression of EMT markers and decreased motility. Furthermore, conditioned medium (CM) from senescent cells induced EMT in presenescent RPE cells, and EMT accelerated the process of senescence. Importantly, dasatinib plus quercetin, which selectively eliminates senescent cells, inhibited low-density-induced EMT in RPE cells. These findings provide a better understanding of the interconnection between senescence and EMT in RPE cells. Removal of senescent cells by certain methods such as senolytics, might be a promising potential approach to prevent or delay the progression of RPE-EMT-related retinal diseases such as AMD.

Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476 (4-(4-(2,3-dihydrobenzo [1,4] dioxin-6-yl)-5-pyridin-2-yl-1H-imidazole-2-yl) benzamide), a specific casein kinase 1 inhibitor, alleviate CQ-induced retinopathy in adult retinal pigment epithelial (ARPE-19) cells. Cultured ARPE-19 cells were exposed to CQ with or without D4476 and cell death was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To examine autophagy flux, ARPE-19 cells were transfected with green fluorescence protein light chain 3 (GFP-LC3)-red fluorescence protein (RFP)-LC3ΔG plasmid DNA and co-stained with the lysosomal-associated membrane protein (LAMP)-1 antibody. Western blotting and fluorescence-activated cell sorting (FACS) showed apoptosis, whereas the fluorescence intensity of 2'-7'-dichlorofluorescein diacetate revealed levels of cellular oxidative stress. We then confirmed the effect of D4476 on the interaction between Beclin 1 and B-cell lymphoma-2 (Bcl-2) through immunoprecipitation with an anti-Bcl-2 antibody. Following CQ exposure, ARPE-19 cells accumulated autophagosomes because of defective lysosomal degradation. Furthermore, CQ trapped Beclin 1 with Bcl-2, disturbing autophagy initiation and autolysosome formation. However, D4476 alleviated CQ-induced effects by rescuing ARPE-19 cells from CQ-induced toxicity by modulating the association between Beclin 1 and Bcl-2. Therefore, D4476 controls autophagy and apoptosis simultaneously by upregulating autophagy flux, decreasing ROS formation, and triggering the expression of anti-apoptotic proteins through inhibition of mTOR, JNK, and p38 MAPK signals. We conclude that D4476 is a promising treatment strategy for CQ-mediated retinopathy.

CircFAT1 regulates retinal pigment epithelial cell pyroptosis and autophagy via mediating m6A reader protein YTHDF2 expression in diabetic retinopathy.

Diabetic retinopathy (DR) is a serious blinding complication of diabetes. At present, the therapeutic intervention effect is limited. We aimed to investigate the circRNA expression profiles in retinal proliferative fibrovascular membranes of patients with DR and explore the effect of circFAT1 on pyroptosis and autophagy of high glucose (HG)-induced retinal pigment epithelial (RPE) cells and its molecualr mechanism. In this study, circRNA sequencing was performed to determine the expression profiles of circRNAs in DR patients. The expression of circFAT1 was measured by qRT-PCR. Cell counting kit-8, transmission electron microscope, western blot, immunofluorescence and enzyme-linked immunosorbent assay were conducted to explore the roles of HG and circFAT1 in RPE cell pyroptosis and autophagy. RNA pull down was used to determine the binding protein of circFAT1. Our data showed that HG significantly reduced the viability of RPE cells, inhibited cell autophagy and contributed to cell pyroptosis. In addition, a total of 189 differentially expressed circRNAs (DEcircRNAs) were identified between DR patients and non-DR patients, including 93 upregulated and 96 downregulated DEcircRNAs in the retinal proliferative fibrovascular membranes of DR patients. Pathway analysis showed that DEcircRNAs were mainly involved in MAPK signaling pathway, TGF-beta signaling pathway and adherens junction. Moreover, circFAT1 was significantly downregulated in retinal proliferative fibrovascular membranes of DR patients and HG-induced RPE cells. CircFAT1 overexpression remarkably enhanced the expression of LC3B, while reduced the expression of GSDMD in HG-induced RPE cells. RNA pull down combined with western blot analysis indicated that circFAT1 bound to m6A reader YTHDF2. YTHDF2 overexpression significantly increased the protein expression of LC3B in HG-induced RPE cells. In summary, circFAT1 promoted autophagy and inhibited pyroptosis of RPE cells induced by HG, and could combine with YTHDF2. This study provides new ideas for DR prevention and treatment.

NMDA Receptor Antagonists Degrade Lipofuscin via Autophagy in Human Retinal Pigment Epithelial Cells.

Background and Objectives: 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 N-methyl-D-aspartate (NMDA) signaling as a novel mechanism for scavenging N-retinylidene-N-retinylethanolamine (A2E), a component of ocular lipofuscin, in human RPE cells. Materials and Methods: 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. Results: 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. Conclusions: 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.

Method for measuring extracellular flux from intact polarized epithelial monolayers.

Purpose: The Seahorse XFp platform is widely used for metabolic assessment of cultured cells. Current methods require replating of cells into specialized plates. This is problematic for certain cell types, such as primary human fetal RPE (hfRPE) cells, which must be cultured for months to become properly differentiated. Our goal was to overcome this limitation by devising a method for assaying intact cell monolayers with the Seahorse XFp, without the need for replating. Methods: Primary hfRPE cells were differentiated by prolonged culture on filter inserts. Triangular sections of filters with differentiated cells attached were excised, transferred to XFp cell culture miniplate wells, immobilized at the bottoms, and subjected to mitochondrial stress tests. Replated cells were measured for comparison. Differentiated hfRPE cells were challenged or not with bovine photoreceptor outer segments (POS), and mitochondrial stress tests were performed 3.5 h later, after filter excision and transfer to assay plates. Results: Differentiated hfRPE cells assayed following filter excision demonstrated increased maximal respiration, increased spare respiration capacity, and increased extracellular acidification rate (ECAR) relative to replated controls. hfRPE cells challenged with POS exhibited increased maximal respiration and spare capacity, with no apparent change in the ECAR, relative to untreated controls. Conclusions: We have developed a method to reproducibly assay intact, polarized monolayers of hfRPE cells with the Seahorse XFp platform and have shown that the method yields more robust metabolic measurements compared to standard methods and is suitable for assessing the consequences of prolonged perturbations of differentiated cells. We expect our approach to be useful for a variety of studies involving metabolic assessment of adherent cells cultured on filters.

Anthocyanins Inhibits Oxidative Injury in Human Retinal Pigment Epithelial ARPE-19 Cells via Activating Heme Oxygenase-1.

Anthocyanins belong to phenolic pigments and are known to have various pharmacological activities. This study aimed to investigate whether anthocyanins could inhibit hydrogen peroxide (H2O2)-induced oxidative damage in human retinal pigment epithelial ARPE-19 cells. Our results indicated that anthocyanins suppressed H2O2-induced genotoxicity, while inhibiting reactive oxygen species (ROS) production and preserving diminished glutathione. Anthocyanins also suppressed H2O2-induced apoptosis by reversing the Bcl-2/Bax ratio and inhibiting caspase-3 activation. Additionally, anthocyanins attenuated the release of cytochrome c into the cytosol, which was achieved by interfering with mitochondrial membrane disruption. Moreover, anthocyanins increased the expression of heme oxygenase-1 (HO-1) as well as its activity, which was correlated with the phosphorylation and nuclear translocation of nuclear factor-erythroid-2 related factor 2 (Nrf2). However, the cytoprotective and anti-apoptotic effects of anthocyanins were significantly attenuated by the HO-1 inhibitor, demonstrating that anthocyanins promoted Nrf2-induced HO-1 activity to prevent ARPE-19 cells from oxidative stress. Therefore, our findings suggest that anthocyanins, as Nrf2 activators, have potent ROS scavenging activity and may have the potential to protect ocular injury caused by oxidative stress.

Mitochondrial damage and clearance in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a devastating eye disease that causes permanent vision loss in the central part of the retina, known as the macula. Patients with such severe visual loss face a reduced quality of life and are at a 1.5 times greater risk of death compared to the general population. Currently, there is no cure for or effective treatment for dry AMD. There are several mechanisms thought to underlie the disease, for example, ageing-associated chronic oxidative stress, mitochondrial damage, harmful protein aggregation and inflammation. As a way of gaining a better understanding of the molecular mechanisms behind AMD and thus developing new therapies, we have created a peroxisome proliferator-activated receptor gamma coactivator 1-alpha and nuclear factor erythroid 2-related factor 2 (PGC1α/NFE2L2) double-knockout (dKO) mouse model that mimics many of the clinical features of dry AMD, including elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in retinal pigment epithelial cells (RPE). In addition, a human RPE cell-based model was established to examine the impact of non-functional intracellular clearance systems on inflammasome activation. In this study, we found that there was a disturbance in the autolysosomal machinery responsible for clearing mitochondria in the RPE cells of one-year-old PGC1α/NFE2L2-deficient mice. The confocal immunohistochemical analysis revealed an increase in autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as multiple mitophagy markers such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN), along with signs of damaged mitochondria. However, no increase in autolysosome formation was detected, nor was there a colocalization of the lysosomal marker LAMP2 or the mitochondrial marker, ATP synthase ß. There was an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells, together with autofluorescent aggregates. Additionally, we observed an increase in the numbers of Toll-like receptors 3 and 9, while those of NOD-like receptor 3 were decreased in PGC1α/NFE2L2 dKO retinal specimens compared to wild-type animals. There was a trend towards increased complement component C5a and increased involvement of the serine protease enzyme, thrombin, in enhancing the terminal pathway producing C5a, independent of C3. The levels of primary acute phase C-reactive protein and receptor for advanced glycation end products were also increased in the PGC1α/NFE2L2 dKO retina. Furthermore, selective proteasome inhibition with epoxomicin promoted both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial-mediated oxidative stress, leading to the release of mitochondrial DNA to the cytosol, resulting in potassium efflux-dependent activation of the absent in melanoma 2 (AIM2) inflammasome and the subsequent secretion of interleukin-1ß in ARPE-19 cells. In conclusion, the data suggest that there is at least a relative decrease in mitophagy, increases in the amounts of C5 and thrombin and decreased C3 levels in this dry AMD-like model. Moreover, selective proteasome inhibition evoked mitochondrial damage and AIM2 inflammasome activation in ARPE-19 cells.

Anaphylatoxin C5a receptor signaling induces mitochondrial fusion and sensitizes retinal pigment epithelial cells to oxidative stress.

Mitochondrial dynamics is a morphological balance between fragmented and elongated shapes, reflecting mitochondrial metabolic status, cellular damage, and mitochondrial dysfunction. The anaphylatoxin C5a derived from complement component 5 cleavage, enhances cellular responses involved in pathological stimulation, innate immune responses, and host defense. However, the specific response of C5a and its receptor, C5a receptor (C5aR), in mitochondria is unclear. Here, we tested whether the C5a/C5aR signaling axis affects mitochondrial morphology in human-derived retinal pigment epithelial cell monolayers (ARPE-19). C5aR activation with the C5a polypeptide induced mitochondrial elongation. In contrast, oxidatively stressed cells (H2O2) responded to C5a with an enhancement of mitochondrial fragmentation and an increase in the number of pyknotic nuclei. C5a/C5aR signaling increased the expression of mitochondrial fusion-related protein, mitofusin-1 (MFN1) and - 2 (MFN2), as well as enhanced optic atrophy-1 (Opa1) cleavage, which are required for mitochondrial fusion events, whereas the mitochondrial fission protein, dynamin-related protein-1 (Drp1), and mitogen-activated protein kinase (MAPK)-dependent extracellular signal-regulated protein kinase (Erk1/2) phosphorylation were not affected. Moreover, C5aR activation increased the frequency of endoplasmic reticulum (ER)-mitochondria contacts. Finally, oxidative stress induced in a single cell within an RPE monolayer (488 nm blue laser spot stimulation) induced a bystander effect of mitochondrial fragmentation in adjacent surrounding cells only in C5a-treated monolayers. These results suggest that C5a/C5aR signaling produced an intermediate state, characterized by increased mitochondrial fusion and ER-mitochondrial contacts, that sensitizes cells to oxidative stress, leading to mitochondrial fragmentation and cell death.

The Bcl-2/Bcl-xL Inhibitor ABT-263 Attenuates Retinal Degeneration by Selectively Inducing Apoptosis in Senescent Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals. However, the currently used intravitreal injections of anti-vascular endothelial growth factor are invasive, and repetitive injections are also accompanied by a risk of intraocular infection. The pathogenic mechanism of AMD is still not completely understood, but a multifactorial mechanism that combines genetic predisposition and environmental factors, including cellular senescence, has been suggested. Cellular senescence refers to the accumulation of cells that stop dividing due to the presence of free radicals and DNA damage. Characteristics of senescent cells include nuclear hypertrophy, increased levels of cell cycle inhibitors such as p16 and p21, and resistance to apoptosis. Senolytic drugs remove senescent cells by targeting the main characteristics of these cells. One of the senolytic drugs, ABT-263, which inhibits the antiapoptotic functions of Bcl-2 and Bcl-xL, may be a new treatment for AMD patients because it targets senescent retinal pigment epithelium (RPE) cells. We proved that it selectively kills doxorubicin (Dox)-induced senescent ARPE-19 cells by activating apoptosis. By removing senescent cells, the expression of inflammatory cytokines was reduced, and the proliferation of the remaining cells was increased. When ABT-263 was orally administered to the mouse model of senescent RPE cells induced by Dox, we confirmed that senescent RPE cells were selectively removed and retinal degeneration was alleviated. Therefore, we suggest that ABT-263, which removes senescent RPE cells through its senolytic effect, has the potential to be the first orally administered senolytic drug for the treatment of AMD.

Urban aerosol particulate matter promotes mitochondrial oxidative stress-induced cellular senescence in human retinal pigment epithelial ARPE-19 cells.

Environmental exposure to urban particulate matter (UPM) is a serious health concern worldwide. Although several studies have linked UPM to ocular diseases, no study has reported effects of UPM exposure on senescence in retinal cells. Therefore, this study aimed to investigate the effects of UPM on senescence and regulatory signaling in human retinal pigment epithelial ARPE-19 cells. Our study demonstrated that UPM significantly promoted senescence, with increased senescence-associated ß-galactosidase activity. Moreover, both mRNA and protein levels of senescence markers (p16 and p21) and the senescence-associated secretory phenotype, including IL-1ß, matrix metalloproteinase-1, and -3 were upregulated. Notably, UPM increased mitochondrial reactive oxygen species-dependent nuclear factor-kappa B (NF-κB) activation during senescence. In contrast, use of NF-κB inhibitor Bay 11-7082 reduced the level of senescence markers. Taken together, our results provide the first in vitro preliminary evidence that UPM induces senescence by promoting mitochondrial oxidative stress-mediated NF-κB activation in ARPE-19 cells.

Reduction of high glucose-induced oxidative injury in human retinal pigment epithelial cells by sarsasapogenin through inhibition of ROS generation and inactivation of NF-κB/NLRP3 inflammasome pathway.

BACKGROUND: Hyperglycemia-induced accumulation of reactive oxygen species (ROS) is a major risk factor for diabetic retinopathy (DR). Sarsasapogenin is a natural steroidal saponin that is known to have excellent antidiabetic effects and improve diabetic complications, but its potential efficacy and mechanism for DR are unknown. OBJECTIVES: The current study was designed to explore whether sarsasapogenin inhibits hyperglycemia-induced oxidative stress in human retinal pigment epithelial (RPE) ARPE-19 cells and to elucidate the molecular mechanisms. METHODS: To mimic hyperglycemic conditions, ARPE-19 cells were cultured in medium containing high glucose (HG). The suppressive effects of sarsasapogenin on HG-induced cell viability reduction, apoptosis and ROS production were investigated. In addition, the relevance of the nuclear factor-kappa B (NF-κB)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome signaling pathway was explored to investigate the mechanism of antioxidant and anti-inflammatory activity of sarsasapogenin. RESULTS: Sarsasapogenin significantly alleviated cytotoxicity and apoptosis in HG-treated ARPE-19 cells through inhibition of intracellular ROS generation. Sarsasapogenin also effectively attenuated HG-induced excess accumulation of mitochondrial superoxide, reduction of glutathione content, and inactivation of manganese superoxide dismutase and glutathione peroxidase. The HG condition markedly increased the expression and maturation of interleukin (IL)-1ß and IL-18 through the activation of the NF-kB signaling pathway, whereas sarsasapogenin reversed these effects. Moreover, although the expression of NLRP3 inflammasome multiprotein complex molecules was increased in ARPE-19 cells cultured under HG conditions, their levels remained similar to the control group in the presence of sarsasapogenin. CONCLUSION: Sarsasapogenin could protect RPE cells from HG-induced injury by inhibiting ROS generation and NF-κB/NLRP3 inflammasome pathway, suggesting its potential as a therapeutic agent to improve the symptoms of DR.