Demethoxycurcumin inhibits the cell migration and MMP-2 expression in human retinal pigment epithelial cells by targeting the STAT-3 pathway.

Proliferative vitreoretinopathy (PVR) involves retinal pigment epithelium (RPE) cell proliferation and migration and leads to tractional retinal detachment. Demethoxycurcumin (DMC), a curcuminoid, has anti-inflammatory and anti-tumour properties. However, whether DMC affects the migration of RPE cells and the molecular mechanism of human PVR remains unclear. The aim of the current study was to investigate the effects of DMC on the inhibition of migration and proteinase expression of human ARPE-19 cells. Herein, we provided molecular evidence associated with PVR prevention through DMC by inhibiting ARPE-19 cell migration. We performed gelatin zymography, Western blot and RT-PCR and respectively found that DMC is sufficient to reduce matrix metalloproteinase-2 (MMP-2) activity, protein level and mRNA expression. DMC suppressed the nuclear levels of transcriptional factors specificity protein 1 and c-Fos, which are involved in the modulation of the transcriptional activation of the MMP-2 gene. DMC also inhibited STAT-3 phosphorylation in ARPE-19 cells. Selective STAT-3 induction by a STAT-3 activator, colivelin, reverted MMP activity and protein expression and cell migration, which were reduced in response to DMC. The results proved the inhibitory effect of DMC on RPE cell migration and MMP-2 expression by the down-regulation of the STAT-3 signalling pathway.

Quantitative differences between cyclin-dependent kinases underlie the unique functions of CDK1 in human cells.

One of the most intriguing features of cell-cycle control is that, although there are multiple cyclin-dependent kinases (CDKs) in higher eukaryotes, a single CDK is responsible for both G1-S and G2-M in yeasts. By leveraging a rapid conditional silencing system in human cell lines, we confirm that CDK1 assumes the role of G1-S CDK in the absence of CDK2. Unexpectedly, CDK1 deficiency does not prevent mitotic entry. Nonetheless, inadequate phosphorylation of mitotic substrates by noncanonical cyclin B-CDK2 complexes does not allow progression beyond metaphase and underscores deleterious late mitotic events, including the uncoupling of anaphase A and B and cytokinesis. Elevation of CDK2 to a level similar to CDK1 overcomes the mitotic defects caused by CDK1 deficiency, indicating that the relatively low concentration of CDK2 accounts for the defective anaphase. Collectively, these results reveal that the difference between G2-M and G1-S CDKs in human cells is essentially quantitative.

Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK.

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE.

Sulforaphane inhibits blue light-induced inflammation and apoptosis by upregulating the SIRT1/PGC-1α/Nrf2 pathway and autophagy in retinal pigment epithelial cells.

The present study elucidated mechanisms through which sulforaphane (SFN) protects retinal pigment epithelial (RPE) cells from blue light-induced impairment. SFN could activate the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increase the expression of the heme oxygenease-1 (HO-1) gene and production of glutathione. SFN reduced blue light-induced oxidative stress, and effectively activated cytoprotective components including Nrf-2, HO-1, thioredoxin-1, and glutathione. The protective effect of SFN on blue light-induced injury was blocked by the Nrf2 inhibitor ML385, suggesting that the SFN-induced Nrf2 pathway is involved in the cytoprotective effect of SFN. SFN inhibited intercellular adhesion molecule-1 expression induced by TNF-α or blue light, suggesting the anti-inflammatory activity of SFN. The inhibitory effect of SFN was associated with the blocking of NF-κB p65 nuclear translocation in blue light-exposed RPE cells. SFN protected RPE cells from blue light-induced interruption of the mitochondrial membrane potential and reduction of the Bcl-2/Bax ratio and cleaved caspase-3 and PARP-1 expression, suggesting the antiapoptotic activity of SFN. SFN alone or together with blue light exposure increased the expression of the autophagy-related proteins LC3BII and p62. An autophagy inhibitor, 3-MA, inhibited the protective effect of SFN on blue light-induced cell damage. SFN increased sirtuin-1 (SIRT1) expression; however, treatment with blue light induced peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) expression. Our study results demonstrated that SFN exerts its protective effect under blue light exposure by maintaining the Nrf2-related redox state and upregulating SIRT1 and PGC-1α expression and autophagy.

Topoisomerase IIα represses transcription by enforcing promoter-proximal pausing.

Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.

Muscle Glycogen Phosphorylase and Its Functional Partners in Health and Disease.

Glycogen phosphorylase (PG) is a key enzyme taking part in the first step of glycogenolysis. Muscle glycogen phosphorylase (PYGM) differs from other PG isoforms in expression pattern and biochemical properties. The main role of PYGM is providing sufficient energy for muscle contraction. However, it is expressed in tissues other than muscle, such as the brain, lymphoid tissues, and blood. PYGM is important not only in glycogen metabolism, but also in such diverse processes as the insulin and glucagon signaling pathway, insulin resistance, necroptosis, immune response, and phototransduction. PYGM is implicated in several pathological states, such as muscle glycogen phosphorylase deficiency (McArdle disease), schizophrenia, and cancer. Here we attempt to analyze the available data regarding the protein partners of PYGM to shed light on its possible interactions and functions. We also underline the potential for zebrafish to become a convenient and applicable model to study PYGM functions, especially because of its unique features that can complement data obtained from other approaches.

Farrerol Enhances Nrf2-Mediated Defense Mechanisms against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial Cells by Activating Akt and MAPK.

Oxidative stress of the retinal pigment epithelium (RPE) is an essential element contributing to the progression of age-related macular degeneration (AMD). Notably, the activation of Nrf2 is regarded as an effective strategy for controlling oxidation. The novel 2,3-dihydroflavonoid compound farrerol, which is extracted from Rhododendron, possesses antioxidant properties. In this study, we investigated the mechanism by which farrerol protects against oxidative damage mediated by hydrogen peroxide (H2O2) in adult retinal pigment epithelial cell line 19 (ARPE-19) cells. Farrerol supplementation conspicuously reversed H2O2-related cell damage through declining the generation of intracellular reactive oxygen species (ROS) and MDA and increasing the concentrations of GSH and SOD. According to the results of the apoptosis assay, a farrerol pretreatment decreased the protein expression of the Bax/Bcl-2, cleaved caspase-3, PARP, caspase-8, and caspase-9 proteins. Furthermore, farrerol markedly activated Nrf2, thereby increasing the levels of antioxidant enzymes downstream of Nrf2, such as HO-1, NQO1, and GCLM. Knockdown of Nrf2 with a specific siRNA successfully suppressed farrerol-mediated HO-1 transcription and partially abolished the cytoprotective effect on ARPE-19 cells. Meanwhile, farrerol induced Akt and MAPK phosphorylation in a dose-related way. However, inhibiting Akt and MAPK substantially blocked the cytoprotective functions of farrerol. Therefore, farrerol enhanced Nrf2-mediated cytoprotection of oxidative damage caused by H2O2, which may be inseparable from the activation of Akt and MAPK.

SIRT3 deficiency increases mitochondrial oxidative stress and promotes migration of retinal pigment epithelial cells.

Retinal pigment epithelial cells are closely associated with the pathogenesis of diabetic retinopathy. The mechanism by which diabetes impacts retinal pigment epithelial cell function is of significant interest. Sirtuins are an important class of proteins that primarily possess nicotinamide adenine dinucleotide-dependent deacetylases activity and involved in various cellular physiological and pathological processes. Here, we aimed to examine the role of sirtuins in the induction of diabetes-associated retinal pigment epithelial cell dysfunction. High glucose and platelet-derived growth factor (PDGF) treatment induced epithelial-mesenchymal transition and the migration of retinal pigment epithelial cells, and decreased sirtuin-3 expression. Sirtuin-3 knockdown using siRNA increased epithelial-mesenchymal transition and migration of retinal pigment epithelial cells. In contrast, sirtuin-3 overexpression attenuated the effects caused by high glucose and PDGF on epithelial-mesenchymal transition and migration of retinal pigment epithelial cells, suggesting that sirtuin-3 deficiency contributed to retinal pigment epithelial cell dysfunction induced by high glucose and PDGF. Mechanistically, sirtuin-3 deficiency induced retinal pigment epithelial cell dysfunction by the overproduction of mitochondrial reactive oxygen species. These results suggest that sirtuin-3 deficiency mediates the migration of retinal pigment epithelial cells, at least partially by increasing mitochondrial oxidative stress, and shed light on the importance of sirtuin-3 and mitochondrial reactive oxygen species as potential targets in diabetic retinopathy therapy.

Heparanase Deficiency Is Associated with Disruption, Detachment, and Folding of the Retinal Pigment Epithelium.

PURPOSE: Pentosan polysulfate sodium (PPS; Elmiron) is a FDA-approved heparanase inhibitor for the treatment of bladder pain and interstitial cystitis. The chronic use of PPS has been associated with a novel pigmentary maculopathy, associated with discrete vitelliform deposits that exhibit hyperfluorescence, macular hyper-pigmentary spots, and foci of nodular RPE enlargement. Therefore, this study aimed to investigate the retinal morphology of heparanase knockout mice. MATERIAL AND METHODS: The retinal morphology of heparanase knock-out and age-matched control wild type mice of 3-, 9- and 15-weeks old was characterized by means of histological evaluation. Immuno-histological stains for RPE65, F4/80 and Ki67 were performed for investigating the RPE, inflammatory and proliferating cells, respectively. RESULTS: Histological analysis showed no changes in age-matched wild-type controls, whereas the eyes of heparanase null mice were characterized by alterations in RPE and neural retina, as manifest by RPE folds and choroidal thickening, detached RPE cells, thickening of the photoreceptor layer and retinal disorganization. The presence of discrete hyperfluorescent foci, however, was absent. The prevalence of the RPE/choroidal changes or protrusions seemed to progress over time and were correlated with more RPE65 signal rather than influx of F4/80- or Ki67-positive cells. These results indicate that the subretinal alterations were mostly RPE driven, without influx of inflammatory or proliferating cells. CONCLUSIONS: Our results indicate that heparanase deficiency in the mice leads to RPE folds, choroidal thickening, and retinal disorganization. The presence of discrete hyperfluorescent foci, a key characteristic of the human disease, was not observed. However, it can be concluded that some of the observations in mice are similar to those seen after chronic use of PPS in humans. These findings indicate that the toxicity observed in the presence of heparanase inhibitors is target-related and will preclude the clinical use of heparanase inhibition as a therapeutic intervention.

Therapeutic Potential of Tpl2 (Tumor Progression Locus 2) Inhibition on Diabetic Vasculopathy Through the Blockage of the Inflammasome Complex.

OBJECTIVE: Diabetic retinopathy, one of retinal vasculopathy, is characterized by retinal inflammation, vascular leakage, blood-retinal barrier breakdown, and neovascularization. However, the molecular mechanisms that contribute to diabetic retinopathy progression remain unclear. Approach and Results: Tpl2 (tumor progression locus 2) is a protein kinase implicated in inflammation and pathological vascular angiogenesis. Nε-carboxymethyllysine (CML) and inflammatory cytokines levels in human sera and in several diabetic murine models were detected by ELISA, whereas liquid chromatography-tandem mass spectrometry analysis was used for whole eye tissues. The CML and p-Tpl2 expressions on the human retinal pigment epithelium (RPE) cells were determined by immunofluorescence. Intravitreal injection of pharmacological inhibitor or NA (neutralizing antibody) was used in a diabetic rat model. Retinal leukostasis, optical coherence tomography, and H&E staining were used to observe pathological features. Sera of diabetic retinopathy patients had significantly increased CML levels that positively correlated with diabetic retinopathy severity and foveal thickness. CML and p-Tpl2 expressions also significantly increased in the RPE of both T1DM and T2DM diabetes animal models. Mechanistic studies on RPE revealed that CML-induced Tpl2 activation and NADPH oxidase, and inflammasome complex activation were all effectively attenuated by Tpl2 inhibition. Tpl2 inhibition by NA also effectively reduced inflammatory/angiogenic factors, retinal leukostasis in streptozotocin-induced diabetic rats, and RPE secretion of inflammatory cytokines. The attenuated release of angiogenic factors led to inhibited vascular abnormalities in the diabetic animal model. CONCLUSIONS: The inhibition of Tpl2 can block the inflammasome signaling pathway in RPE and has potential clinical and therapeutic implications in diabetes-associated retinal microvascular dysfunction.

Protective effects of PARP1-inhibitory compound in dry age-related macular degeneration.

Poly (ADP-ribose) polymerase 1 (PARP1)-dependent cell death in the retinal pigment epithelium (RPE) is implicated in dry age-related macular degeneration (AMD). Although PARP1 inhibitors are available for treating dry AMD, their delivery route is not ideal for patients. The aim of this study was to test the efficacy of a novel PARP1-inhibitory compound (PIC) in vitro and in vivo. This study presents PIC, a novel small molecule, with superior efficacy to PARP1 inhibitors in the market. PIC demonstrated a distinctive inhibitory profile against PARP isotypes than the FDA-approved PARP1 inhibitors. PIC inhibited PARP1 activation at an IC50 of 0.41 ± 0.15 nM in an enzyme-based assay in vitro and at IC50 and EC50 in ARPE-19 cells of 0.11 ± 0.02 nM and 0.22 ± 0.02 nM, respectively, upon H2O2 insult. PIC also moderated mitochondrial fission and depolarization and maintained cellular energy levels under oxidative stress in ARPE-19 cells. Furthermore, PIC demonstrated good corneal penetration in a rat model, presenting PIC as a promising candidate for eye drop therapeutics for dry AMD. When PIC was administered as an eye drop formulation, RPE morphology was preserved, maintaining the thickness of the outer nuclear layers under sodium iodate (SI) treatment in rats. In SI-treated rabbits, eye drop administration of PIC also retained the structural and functional integrity when analyzed using funduscopy and electroretinogram. Collectively, our data portray PIC as an attractive treatment measure for dry AMD.

Madecassoside protects retinal pigment epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis through the activation of Nrf2/HO-1 pathway.

Age-related macular degeneration (AMD) is a progressive and degenerative ocular disease associated with oxidative stress. Madecassoside (MADE) is a major bioactive triterpenoid saponin that possesses antioxidative activity. However, the role of MADE in AMD has never been investigated. In the current study, we aimed to evaluate the protective effect of MADE on retinal pigment epithelium (RPE) cells under oxidative stress condition. We used hydrogen peroxide (H2O2) to induce oxidative damage in human RPE cells (ARPE-19 cells). Our results showed that H2O2-caused significant decrease in cell viability and increase in lactate dehydrogenase (LDH) release were dose-dependently attenuated by MADE. MADE treatment also attenuated H2O2-induced reactive oxygen species (ROS) and malondialdehyde (MDA) production in RPE cells. The reduced glutathione (GSH) level and superoxide dismutase (SOD) activity in H2O2-induced ARPE-19 cells were elevated after MADE treatment. MADE also suppressed caspase-3 activity and bax expression, as well as increased bcl-2 expression. Furthermore, H2O2-induced increase in expression levels of HO-1 and nuclear Nrf2 were enhanced by MADE treatment. Finally, knockdown of Nrf2 reversed the protective effects of MADE on H2O2-induced ARPE-19 cells. In conclusion, these findings demonstrated that MADE protected ARPE-19 cells from H2O2-induced oxidative stress and apoptosis by inducing the activation of Nrf2/HO-1 signaling pathway.

A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis.

Primary cilia are sensors of chemical and mechanical signals in the extracellular environment. The formation of primary cilia (i.e. ciliogenesis) requires dynamic membrane trafficking events, and several Rab small GTPases, key regulators of membrane trafficking, have recently been reported to participate in ciliogenesis. However, the precise mechanisms of Rab-mediated membrane trafficking during ciliogenesis remain largely unknown. In the present study, we used a collection of siRNAs against 62 human Rabs to perform a comprehensive knockdown screening for Rabs that regulate serum starvation-induced ciliogenesis in human telomerase reverse transcriptase retinal pigment epithelium 1 (hTERT-RPE1) cells and succeeded in identifying Rab34 as an essential Rab. Knockout (KO) of Rab34, but not of Rabs previously reported to regulate ciliogenesis (e.g. Rab8 and Rab10) in hTERT-RPE1 cells, drastically impaired serum starvation-induced ciliogenesis. Rab34 was also required for serum starvation-induced ciliogenesis in NIH/3T3 cells and MCF10A cells but not for ciliogenesis in Madin-Darby canine kidney (MDCK)-II cysts. We then attempted to identify a specific region(s) of Rab34 that is essential for ciliogenesis by performing deletion and mutation analyses of Rab34. Unexpectedly, instead of a specific sequence in the switch II region, which is generally important for recognizing effector proteins (e.g. Rab interacting lysosomal protein [RILP]), a unique long N-terminal region of Rab34 before the conserved GTPase domain was found to be essential. These findings suggest that Rab34 is an atypical Rab that regulates serum starvation-induced ciliogenesis through its unique N-terminal region.

Retinal Distribution and Extracellular Activity of Granzyme B: A Serine Protease That Degrades Retinal Pigment Epithelial Tight Junctions and Extracellular Matrix Proteins.

Granzymes are a family of serine proteases first shown to be intracellular initiators of immune-mediated cell death in target pathogenic cells. In addition to its intracellular role, Granzyme B (GzmB) has important extracellular functions in immune regulation and extracellular matrix (ECM) degradation. Verified substrates of extracellular GzmB activity include tight junctional and ECM proteins. Interestingly, little is known about the activity of GzmB in the outer human retina, a tissue in which the degradation of the tight junctional contacts of retinal pigment epithelial (RPE) cells and within the external limiting membrane, as well as remodeling of the ECM in Bruch's membrane, cause the breakdown of the blood-retinal barrier and slowing of metabolite transport between neuroretina and choroidal blood supply. Such pathological changes in outer retina signal early events in the development of age-related macular degeneration (AMD), a multifactorial, chronic inflammatory eye disease. This study is the first to focus on the distribution of GzmB in the outer retina of the healthy and diseased post-mortem human eye. Our results revealed that GzmB is present in RPE and choroidal mast cells. More immunoreactive cells are present in older (>65 years) compared to younger (<55 years) donor eyes, and choroidal immunoreactive cells are more numerous in eyes with choroidal neovascularization (CNV), while RPE immunoreactive cells are more numerous in eyes with soft drusen, an early AMD event. In vitro studies demonstrated that RPE-derived tight junctional and ECM proteins are cleaved by exogenous GzmB stimulation. These results suggest that the increased presence of GzmB immunoreactive cells in outer retina of older (healthy) eyes as well as in diseased eyes with CNV (from AMD) and eyes with soft drusen exacerbate ECM remodeling in the Bruch's membrane and degradation of the blood-retinal barrier. Currently there are no treatments that prevent remodeling of the Bruch's membrane and/or the loss of function of the outer blood-retinal barrier, known to promote early AMD changes, such as drusen deposition, RPE dysfunction and pro-inflammation. Specific inhibitors of GzmB, already in preclinical studies for non-ocular diseases, may provide new strategies to stop these early events associated with the development of AMD.

Selective Ablation of Dehydrodolichyl Diphosphate Synthase in Murine Retinal Pigment Epithelium (RPE) Causes RPE Atrophy and Retinal Degeneration.

Patients with certain defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene (RP59; OMIM #613861) exhibit classic symptoms of retinitis pigmentosa, as well as macular changes, suggestive of retinal pigment epithelium (RPE) involvement. The DHDDS enzyme is ubiquitously required for several pathways of protein glycosylation. We wish to understand the basis for selective ocular pathology associated with certain DHDDS mutations and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. To circumvent embryonic lethality associated with Dhdds knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). We used targeted Cre expression to study the importance of the enzyme in the RPE. Structural alterations of the RPE and retina including reduction in outer retinal thickness, cell layer disruption, and increased RPE hyper-reflectivity were apparent at one postnatal month. At three months, RPE and photoreceptor disruption was observed non-uniformly across the retina as well as RPE transmigration into the photoreceptor layer, external limiting membrane descent towards the RPE, and patchy loss of photoreceptors. Functional loss measured by electroretinography was consistent with structural loss showing scotopic a- and b-wave reductions of 83% and 77%, respectively, at three months. These results indicate that RPE dysfunction contributes to DHDDS mutation-mediated pathology and suggests a more complicated disease mechanism than simply disruption of glycosylation.

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis.

Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.

APX-115A, a pan-NADPH Oxidase Inhibitor, Induces Caspase-dependent Cell Death by Suppressing NOX4-ROS Signaling in EBV-infected Retinal Epithelial Cells.

PURPOSE: Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells. METHODS: Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology. RESULTS: We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK. CONCLUSION: Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.

Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes.

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

Bisretinoids: More than Meets the Eye.

Bisretinoid fluorophores are the major constituents of the lipofuscin of retinal pigment epithelium (RPE) that accumulates with age and contributes to retina disease. Knowledge of the burden placed on the RPE cell by the accumulation of these phototoxic retinaldehyde-adducts depends on the identification and quantitation of the various bisretinoid species that constitute this family of fluorophores. Here we report a previously unidentified fluorescent bisretinoid by UPLC coupled to photodiode array detection, fluorescence, and electrospray ionization mass spectrometry (UPLC-PDA-FLR-ESI-MS) (Kim HJ, Sparrow JR, J Lipid Res 59:1620-1629, 2018). This novel bisretinoid is 1-octadecyl-2-lyso-sn-glycero A2PE (alkyl ether lysoA2PE). The structural assignment was based on molecular weight (m/z 998), UV-visible absorbance maxima (340, 440 nm), and retention time (73 minutes) and was corroborated by biomimetic synthesis using all-trans-retinal and glycerophosphoethanolamine analogues as starting materials. In mechanistic studies, A2PE was hydrolyzed by PLA2, and plasmalogen lysoA2PE was cleaved under acidic conditions. Unprecedented UPLC detection of the bisretinoid alkyl ether lysoA2PE in human RPE but not in neural retina indicates that the phospholipase A2 activity that generates the latter bisretinoid resides in RPE.

Role of the PNPLA2 Gene in the Regulation of Oxidative Stress Damage of RPE.

Oxidative stress-mediated injury of the retinal pigment epithelium (RPE) can precede progressive retinal degeneration and ultimately lead to blindness (e.g., age-related macular degeneration (AMD)). The RPE expresses the PNPLA2 gene and produces its protein product PEDF-R that exhibits lipase activity. We have shown that transient PNPLA2 overexpression decreases dead-cell proteolytic activity and that synthetic peptides derived from a central region of PEDF-R efficiently protect ARPE-19 and pig primary RPE cells from oxidative stress. This study aims to evaluate the effect of loss of PNPLA2 in RPE cells undergoing oxidative stress. Loss of PNPLA2 conferred increased resistance to cells when subjected to oxidative stress.