Investigation into the use of gamma irradiated Cytodex-1 microcarriers to produce a human cytomegalovirus (HCMV) vaccine candidate in epithelial cells.

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a "ready-to-use" product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.

Regulation of oxidative stress and inflammatory responses in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is an eye disease, which causes impaired vision that can lead to blindness. The incidence of AMD increases with age. Retinal pigment epithelial (RPE) cells maintain retinal homeostasis and support the functionality of photoreceptors. In the pathogenesis of AMD, the degeneration of the RPE cells precedes photoreceptor cell death. RPE cells are susceptible to oxidative stress, and chronic inflammation involving nucleotide-binding domain, leucine-rich repeat and pyrin domain 3 (NLRP3) inflammasome activation and impaired autophagy are challenges faced by aged RPE cells in AMD. There are two types of AMD, dry (85-90%) and wet (10-15%) disease forms. Choroidal neovascularization is typical for wet AMD, and anti-vascular endothelial growth factor (anti-VEGF) injections are used to prevent the progression of the disease but there is no curative treatment. There is no cure for the dry disease form, but antioxidants have been proposed as a potential treatment option. Ageing is the most important risk factor of AMD, and tobacco smoke is the most important environmental risk factor that can be controlled. Hydroquinone is a cytotoxic, immunotoxic, carcinogenic and pro-oxidative component of tobacco smoke. The aim of this PhD thesis was to study hydroquinone-induced oxidative stress and NLRP3 inflammasome activation in human RPE cells (ARPE-19 cells). An age-related eye disease study (AREDS) formulation (incl. omega-3 fatty acids, vitamin C and E, copper, zinc, lutein and zeaxanthin), which is clinically investigated p.o. dosing combination of dietary supplements for AMD patients, has been evaluated as a possible treatment and restraining option for AMD. Resvega (4.1.1, Table 2) is a similar kind of product to AREDS with added resveratrol, and many of the components incorporated within Resvega can be considered as belonging to the normal antioxidative defence system of the retina. Another aim was to evaluate the effects of Resvega on hydroquinone-induced oxidative stress or NLRP3 inflammasome activation induced by impaired protein clearance. The results of this study reveal that hydroquinone elevated the activity of NADPH oxidase which subsequently mediated the production of reactive oxygen species (ROS) and predisposed RPE cells to degeneration by reducing levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Hydroquinone induced an NLRP3-independent IL-18 release and NLRP3 accumulation inside the IL-1α-primed cells. Resvega treatment reduced the extent of hydroquinone-induced ROS production and NLRP3 inflammasome activation evoked by impaired protein clearance. Thus, Resvega alleviated hydroquinone- and impaired protein clearance-induced stress in human RPE cells, but more studies are needed, for example, to reveal the most optimal route of administration for targeting the cells in the retina, since both oxidative stress and NLRP3 inflammasome activation are important contributors to the development of AMD and represent significant treatment targets.

Ultrahigh Pressure Facilitates the Acylation of Malvidin and Chlorogenic Acid to Increase the Stability and Protective Effect of Malvidin Derivatives on H2O2-Induced ARPE-19 Cells.

We explored the effects of ultrahigh-pressure technology and chlorogenic acid on the color stability and structure-activity relationship of malvidin (MV). Experimental conditions were optimized through single-factor experiments and response surface analysis at a pressure of 300 MPa, mass ratio of MV to chlorogenic acid of 1:3.64 (w/w), and time of 5 min. Compared with MV, MV derivatives showed higher stability and in vitro antioxidant activity. X-ray diffraction analysis, UV-vis spectroscopy, Fourier transform infrared spectroscopy, high-performance liquid chromatography, and mass spectrometry were conducted to determine the structures of MV derivatives for the first time. Ultrahigh pressure facilitated acylation of chlorogenic acid and MV and produced four new MV derivatives. Analysis of the effect of malvidin-3-O-6-(acrylic acid-(2-hydroxy, 4-carboxy-cyclohexanol) ester)-guaiacol (Mv3ACEC) on ARPE-19 cells exposed to H2O2 by RNA transcriptome sequencing showed that Mv3ACEC simultaneously inhibited various inflammatory and apoptotic signal transduction pathways, exerted a synergistic effect, and partly inhibited cell apoptosis through the MAPK signaling pathway. Therefore, the results show that ultrahigh pressure will cause acylation of chlorogenic acid and MV to produce four new MV derivatives, and MV derivatives protect ARPE-19 cells from H2O2-induced oxidative stress.

Profiles of Cytokines Secreted by ARPE-19 Cells Exposed to Light and Incubated with Anti-VEGF Antibody.

The retinal pigment epithelium (RPE) is the major source of cytokines in the retina regulating the intraocular immune environment, and a primary target of photodamage. Here, we examined 27 types of cytokines secreted by ARPE-19 cells exposed to visible light and incubated with aflibercept or ranibizumab, which are two anti-vascular endothelial growth factor (VEGF) antibodies. The cells were cultured for 24 h in the dark or under 2000 lux irradiation from a daylight-colored fluorescent lamp, and cytokine levels in the culture supernatant were measured. In the light-irradiated culture, the levels of IL-9, IL-17A and bFGF were higher, and the levels of IL-6, IL-7, IL-8 and MCP-1 were lower than those in the dark culture, while there was no significant difference with the VEGF-A level. In subgroup analyses of the light-irradiated culture, the bFGF level under 250 to 2000 lux irradiation was elevated in a light intensity-dependent manner. In culture exposed to blue, green or red light, the bFGF level was elevated by blue light and was high compared to that by green or red light. In culture with aflibercept or ranibizumab in the dark, the levels of IL-6, IL-8, bFGF and MCP-1 were increased, and the IL-12 level decreased synchronously with a reduction in the VEGF-A level. Our findings indicate that continuous irradiation of visible light and VEGF suppression may be an influential factor in expression patterns of inflammatory cytokines secreted by human RPE cells.

DABCO-Customized Nanoemulsions: Characterization, Cell Viability and Genotoxicity in Retinal Pigmented Epithelium and Microglia Cells.

Quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and of quinuclidine surfactants were used to develop oil-in-water nanoemulsions with the purpose of selecting the best long-term stable nanoemulsion for the ocular administration of triamcinolone acetonide (TA). The combination of the best physicochemical properties (i.e., mean droplet size, polydispersity index, zeta potential, osmolality, viscoelastic properties, surface tension) was considered, together with the cell viability assays in ARPE-19 and HMC3 cell lines. Surfactants with cationic properties have been used to tailor the nanoemulsions' surface for site-specific delivery of drugs to the ocular structure for the delivery of TA. They are tailored for the eye because they have cationic properties that interact with the anionic surface of the eye.

Protective effects of upregulated HO-1 gene against the apoptosis of human retinal pigment epithelial cells in vitro.

AIM: To investigate the protective effect of heme oxygenase-1 (HO-1) against H2O2-induced apoptosis in human ARPE-19 cells. METHODS: The lentiviral vector expressing HO-1 was prepared and transfected into apoptotic ARPE-19 cells induced by H2O2. Functional experiments including cell counting kit-8 (CCK-8) assay, flow cytometry (FCM) and mitochondrial membrane potential assay were conducted. RESULTS: The ultrastructure of ARPE-19 cells was observed using transmission electron microscope (TEM). It was found that exogenous HO-1 significantly ameliorated H2O2-induced loss of cell viability, apoptosis and intracellular levels of reactive oxygen species (ROS) in ARPE-19 cells. The overexpression of HO-1 facilitated the transfer of nuclear factor erythroid-2-related factor 2 (Nrf2) from cytoplasm to nucleus, which in turn upregualted expressions HO-1 and B-cell lymphoma-2 (Bcl-2). Furthermore, HO-1 upregulation further inhibited H2O2-induced release of cysteinyl aspartate specific proteinase-3 (caspase-3). CONCLUSION: Exogenous HO-1 protect ARPE-19 cells against H2O2-induced oxidative stress by regulating the expressions of Nrf2, HO-1, Bcl-2, and caspase-3.

Protective Effect of Chrysanthemum morifolium cv. Fubaiju Hot-Water Extracts Against ARPE-19 Cell Oxidative Damage by Activating PI3K/Akt-Mediated Nrf2/HO-1 Signaling Pathway.

Chrysanthemum morifolium cv. Fubaiju is a kind of widely consumed herb tea with multiple health benefits. The present study was aimed to evaluate the protective capacity of C. morifolium cv. Fubaiju hot-water extracts (CMs) against ARPE-19 cell oxidative damage. The results showed that pretreatment with 100 µg/mL CM could significantly reduce cell oxidative damage and apoptosis. Proapoptotic protein expression such as Bax, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) was significantly decreased after CM addition, while the expression level of antioxidant enzymes including catalase, glutamate-cysteine ligase catalytic subunit (GCLc), superoxide dismutase 2 (SOD2), and NAD(P)H:quinone oxidoreductase 1 (NQO-1) was significantly promoted. Meanwhile, CM treatment upregulated Akt phosphorylation, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, and the expression level of antioxidant gene heme oxygenase-1 (HO-1) in a dose-dependent manner under oxidative stress. Knockdown of Nrf2 by targeted small interfering RNA (siRNA) alleviated CM-mediated HO-1 transcription and almost abolished CM-mediated protection against hydrogen peroxide (H2O2)-induced cell damage. Correspondingly, the protective effect of CM was dramatically blocked after interference with phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor LY294002, indicating that the protective effect of CM on cell oxidative damage was attributed to PI3K/Akt-mediated Nrf2/HO-1 signaling pathway.

Effects of Resvega on Inflammasome Activation in Conjunction with Dysfunctional Intracellular Clearance in Retinal Pigment Epithelial (RPE) Cells.

Age-related macular degeneration (AMD) is an eye disease in which retinal pigment epithelium (RPE) cells play a crucial role in maintaining retinal homeostasis and photoreceptors' functionality. During disease progression, there is increased inflammation with nucleotide-binding domain, leucine-rich repeat, and Pyrin domain 3 (NLRP3) inflammasome activation, oxidative stress, and impaired autophagy in RPE cells. Previously, we have shown that the dietary supplement Resvega reduces reactive oxygen species (ROS) production and induces autophagy in RPE cells. Here, we investigated the ability of Resvega to prevent NLRP3 inflammasome activation with impaired protein clearance in human RPE cells. Cell viability was measured using the lactate dehydrogenase (LDH) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Enzyme-linked immunosorbent assays (ELISA) were utilized to determine the secretion of cytokines, NLRP3, and vascular endothelial growth factor (VEGF). Caspase-1 activity was measured with a fluorescent labeled inhibitor of caspase-1 (FLICA; FAM-YVAD-FMK) and detected microscopically. Resvega improved the cell membrane integrity, which was evident as reduced LDH leakage from cells. In addition, the caspase-1 activity and NLRP3 release were reduced, as was the secretion of two inflammatory cytokines, interleukin (IL)-1ß and IL-8, in IL-1α-primed ARPE-19 cells. According to our results, Resvega can potentially reduce NLRP3 inflammasome-mediated inflammation in RPE cells with impaired protein clearance.

Inhibition of PARP activity improves therapeutic effect of ARPE-19 transplantation in RCS rats through decreasing photoreceptor death.

Photoreceptor (PR) dysfunction or death is the key pathological change in retinal degeneration (RD). The death of PRs might be due to a primary change in PRs themselves or secondary to the dysfunction of the retinal pigment epithelium (RPE). Poly(ADP-ribose) polymerase (PARP) was reported to be involved in primary PR death, but whether it plays a role in PR death secondary to RPE dysfunction has not been determined. To clarify this question and develop a new therapeutic approach, we studied the changes in PAR/PARP in the RCS rat, a RD model, and tested the effect of PARP intervention when given alone or in combination with RPE cell transplantation. The results showed that poly(ADP-ribosyl)ation of proteins was increased in PRs undergoing secondary death in RCS rats, and this result was confirmed by the observation of similar changes in sodium iodate (SI)-induced secondary RD in SD rats. The increase in PAR/PARP was highly associated with increased apoptotic PRs and decreased visual function, as represented by lowered b-wave amplitudes on electroretinogram (ERG). Then, as we expected, when the RCS rats were treated with subretinal injection of the PARP inhibitor PJ34, the RD process was delayed. Furthermore, when PJ34 was given simultaneously with subretinal ARPE-19 cell transplantation, the therapeutic effects were significantly improved and lasted longer than those of ARPE-19 or PJ34 treatment alone. These results provide a potential new approach for treating RD.

Protective effects of naringin phospholipid complex on oxidative injury in ARPE-19 cells associated with activation of Nrf2 pathway / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the protective effects of naringin(Nar)phospholipid complex(NPC)on oxidative injury in retinal pigment epithelium cells(ARPE-19 cells)induced by tert-butyl hydroperoxide(t-BHP)and elucidate the underlying mechanism.<p>METHODS:The NPC was prepared by solvent method. Experimental cells are divided into seven groups: control group \〖cultured with dimethylsulfoxide(DMSO)\〗, model group(intervention with 200μmol/L t-BHP), nuclear factor erythroid 2-related factor 2(Nrf2)-siRNA group(cell transfection for Nrf2 gene), naringin group(add 200μmol/L t-BHP after pretreatment with 200μmol/L naringin medium), NPC group(add 200μmol/L t-BHP after pretreatment with 200μmol/L NPC medium), Nrf2-siRNA+ naringin group(after 200μmol/L naringin pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP)and Nrf2-siRNA+ NPC group(after 200μmol/L NPC pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP). The intracellular levels of superoxide dismutase(SOD), malondialdehyde(MDA)and total antioxidant capacity(T-AOC)were detected, intracellular level of reactive oxygen species(ROS)was detected by DCFH-DA staining method. The mRNA and protein expressions of HO-1, NQO-1, GCL and Nrf2 were detected by real-time PCR and western blot, respectively. <p>RESULTS:NPC more significantly increased the levels of SOD and T-AOC, reduced the contents of ROS and MDA than naringin in t-BHP-treated ARPE-19 cells. After naringin and NPC pre-protected ARPE-19 cells, the relative expression and protein expression of Nrf2, HO-1, NQO-1 and GCL mRNA were higher than those of the model group and Nrf2-siRNA group. There were statistically significant differences in the relative expression of 4 genes and the expression levels of 4 proteins in the naringin group and the NPC group, the Nrf2-siRNA+naringin group and the Nrf2-siRNA+NPC group. The expression of Nrf2, HO-1 and NQO-1 protein in the Nrf2-siRNA+naringin group was not significantly different than that in the Nrf2-siRNA group. Compared with the Nrf2-siRNA group, the expression of 4 proteins in the Nrf2-siRNA+NPC group was statistically significant, and the effect of NPC was significantly stronger than that of naringin.<p>CONCLUSION: After naringin forms a phospholipid complex, it can significantly increase the antioxidant capacity in cells and reduce the oxidation level. It up-regulates the expression of Nrf2 and its downstream antioxidant enzymes and phase Ⅱ detoxification enzymes by activating the Nrf2/ARE antioxidative stress pathway to better protect ARPE-19 cells from oxidative damage.

Piceatannol Protects Human Retinal Pigment Epithelial Cells against Hydrogen Peroxide Induced Oxidative Stress and Apoptosis through Modulating PI3K/Akt Signaling Pathway.

This study investigated the protective effect and the molecular mechanism of piceatannol on hydrogen peroxide (H2O2)-induced retinal pigment epithelium cell (ARPE-19) damage. Piceatannol treatment significantly inhibited H2O2-induced RPE cell death and reactive oxygen species (ROS) generation by 64.4% and 75.0%, respectively. Results of flow cytometry showed that H2O2-induced ARPE-19 cells apoptosis was ameliorated by piceatannol supplementation, along with decreased relative protein expressions of Bax/Bcl-2, Cleave-Caspase-3, and Cleave-PARP. Moreover, piceatannol treatment induced NF-E2-related factor 2 (Nrf2) signaling activation, which was evidenced by increased transcription of anti-oxidant genes, glutamate-cysteine ligase catalytic subunit (GCLc), SOD, and HO-1. Knockdown of Nrf2 through targeted siRNA alleviated piceatannol-mediated HO-1 transcription, and significantly abolished piceatannol-mediated cytoprotection. LY294002 (PI3K inhibitor) dramatically blocked piceatannol-mediated increasing of Nrf2 nuclear translocation, HO-1 expression, and cytoprotective activity, indicating the involvement of PI3K/Akt pathway in the cytoprotective effect of piceatannol. The results from this suggest the potential of piceatannol in reducing the risk of age-related macular degeneration.

Effects of astaxanthin on antioxidant parameters in ARPE-19 cells on oxidative stress model.

AIM: To observe the protective effect of astaxanthin (AST) against hydroquinone (HQ) mediated cell death in the apoptotic cascade and evaluate intracellular Ca2+ release, caspase-3, and -9 activation, reactive oxygen species (ROS) production in ARPE-19 cells. METHODS: We cultured ARPE-19 cells in special mediums and performed MTT tests to determine protective effect of AST, before exposing the cells to HQ in an incubator. We analyzed intracellular Ca2+ release experiments, mitochondrial membrane depolarization, glutathione (GSH), glutathione peroxidase (GSH-Px) and ROS experiments, and apoptosis assay. RESULTS: ROS production ranges depend on the amount of cell death. We computed the correlation between ROS ranges and cell death by 20,70-dichlorofluorescein fluorescence, and Ca2+ levels by Fura-2-AM. HQ-induced cell death found out to rise ranges of caspase-3 and -9, and mitochondrial depolarization. These three steps were delayed by AST management. CONCLUSION: ARPE-19 cells are avoided from HQ-induced ROS production and caspase-3 and -9 activation by AST. AST may limit the range of caspase synthesis, Ca2+ release and excess production of ROS with antiapoptotic effect. This study proposes a new therapeutic approach for the treatment of age-related macular degeneration.

Dipenyleneiodonium Induces Growth Inhibition of Toxoplasma gondii through ROS Induction in ARPE-19 Cells.

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.

Dipenyleneiodonium Induces Growth Inhibition of Toxoplasma gondii through ROS Induction in ARPE-19 Cells

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.

Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway.

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2ß, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

The Retinal Pigment Epithelial Cell Line (ARPE-19) Displays Mosaic Structural Chromosomal Aberrations.

The retinal pigment epithelial cell line ARPE-19 was established in 1996 and remains widely used today for biomedical and in particular ophthalmology research. We have analyzed the chromosomes of the ARPE-19 cell line and found cultured cells exist as a heterogeneous mixture having both normal karyotypes and chromosomal rearrangements. In ARPE-19 cells, we observed metaphases with a single translocation t(15;19) and metaphases with two translocations t(5;15) and t(15;19) and a derivative chromosome 9. Aneuploidies have also been detected (monosomy: -16; trisomy: +11, +18). Multiple attempts to isolate clones with a normal karyotype from those with aberrant karyotypes failed due to senescence of cells of normal karyotypes. We could, however, isolate clones with the translocation t(15;19) and clones with two translocations t(5;15) and t(15;19). In continued cell culture after second subcloning for 30 passages, all clones maintained their cytogenetic integrity.We have further investigated the chromosomal profiles of the ARPE-19 cell line from another laboratory and observed cells with a normal karyotype as well as abnormalities in chromosomes 6p and 11q. The DNA profiles of the ARPE-19 cells from both labs were identical to the ATCC profiles, excluding contamination with other cell lines. Since chromosomal translocations in ARPE-19 cells differ from lab to lab and display a mosaicism for structural chromosomal aberrations, researchers dealing with ARPE-19 cells should screen their stocks for chromosomal aberrations and proceed with caution against misinterpretations during experimental manipulations with this cell line. This chapter describes in detail our laboratory methods for single cell cloning, karyotype analysis and fluorescence in situ hybridization (FISH), which we used for the identification and characterization of chromosomal translocations in the retinal pigment epithelial cell line ARPE-19.

Ultraviolet A radiation induces cortistatin overexpression and activation of somatostatin receptors in ARPE­19 cells.

Long-term exposure to ultraviolet (UV) radiation is associated with pathological alterations of the retinal pigment epithelium (RPE). It has been indicated that Cortistatin (CST) and somatostatin (SST) are able to inhibit the neurodegeneration of the RPE associated with diabetic retinopathy and retinal ischemia via activation of SST receptors (SSTRs). To the best of our knowledge, the present study indicated for the first time that treatment with UV­A (30 and 60 min) causes an increase of CST expression, rather than SST, which was linked with the upregulation of STTR3,4,5 subtype receptor gene expression levels. The study revealed that: i) SST and CST mRNA expression were both detected under basal conditions in a human retinal pigment epithelial cell line (Arpe­19); ii) SST expression remained constant from baseline to 1 h of UV­A treatment; iii) CST mRNA expression levels were 80 times increased compared with time 0 and after 30 min of exposition to ultraviolet irradiation; iv) SSTR1, SSTR2 mRNA and low levels of SSTR4 were expressed in basal conditions, whereas SSTR3 and SSTR5 mRNA were not detected under the same conditions; and v) only SSTR3, SSTR4 and SSTR5 were overexpressed after UV­A treatment, although in a different way. In conclusion, the findings provide reasonable evidence to support the pathophysiological role of the CST/SST/SSTRs system in the adaptive response of the RPE exposed to UV­A radiation.

Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation.

In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.

Photoprotective effects of cranberry juice and its various fractions against blue light-induced impairment in human retinal pigment epithelial cells.

CONTEXT: Cranberry has numerous biological activities, including antioxidation, anticancer, cardioprotection, as well as treatment of urinary tract infection (UTI), attributed to abundant phenolic contents. OBJECTIVE: The current study focused on the effect of cranberry juice (CJ) on blue light exposed human retinal pigment epithelial (ARPE-19) cells which mimic age-related macular degeneration (AMD). MATERIALS AND METHODS: Preliminary phytochemical and HPLC analysis, as well as total antioxidant capacity and scavenging activity of cranberry ethyl acetate extract and different CJ fractions (condensed tannins containing fraction), were evaluated. In cell line model, ARPE-19 were irradiated with blue light at 450 nm wavelength for 10 h (mimic AMD) and treated with different fractions of CJ extract at different doses (5-50 µg/mL) by assessing the cell viability or proliferation rate using MTT assay (repairing efficacy). RESULTS: Phytochemical and HPLC analysis reveals the presence of several phenolic compounds (flavonoids, proanthocyanidin, quercetin) in ethyl acetate extract and different fractions of CJ. However, the condensed tannin containing fraction of ethyl acetate extract of CJ displayed the greater (p < 0.05) scavenging activity especially at the dose of 1 mg/mL. Similarly, the condensed tannin containing fraction at 50 µg/mL presented better (p < 0.05) repairing ability (increased cell viability). Furthermore, the oligomeric condensed tannin containing fraction display the best (p < 0.05) repairing efficiency at 50 µg/mL. DISCUSSION AND CONCLUSION: In conclusion, this study distinctly proved that condensed tannin containing fraction of CJ probably exhibits better free radicals scavenging activity and thereby effectively protected the ARPE-19 cells and thus, hampers the progress of AMD.