Human Retinal Epithelial Cell Line Research Articles

A Vicious NGF-p75NTR Positive Feedback Loop Exacerbates the Toxic Effects of Oxidative Damage in the Human Retinal Epithelial Cell Line ARPE-19.

In spite of its variety of biological activities, the clinical exploitation of human NGF (hNGF) is currently limited to ocular pathologies. It is therefore interesting to test the effects of hNGF in preclinical models that may predict their efficacy and safety in the clinical setting of ocular disorders and compare the effects of hNGF with those of its analogs. We used a human retinal pigment cell line, ARPE-19 cells, to investigate the effects of hNGF and its analogs, mouse NGF (mNGF) and painless NGF (pNGF), on cell viability under basal conditions and after exposure to oxidative stimuli, i.e., hydrogen peroxide (H2O2) and ultraviolet (UV)-A rays. The effects of hNGF and pNGF were also tested on the gene expression and protein synthesis of the two NGF receptor subtypes, p75 neurotrophic receptors (p75NTR) and tyrosine kinase A (TrkA) receptors. We drew the following conclusions: (i) the exposure of ARPE-19 cells to H2O2 or UV-A causes a dose-dependent decrease in the number of viable cells; (ii) under baseline conditions, hNGF, but not pNGF, causes a concentration-dependent decrease in cell viability in the range of doses 1-100 ng/mL; (iii) hNGF, but not pNGF, significantly potentiates the toxic effects of H2O2 or of UV-A on ARPE-19 cells in the range of doses 1-100 ng/mL, while mNGF at the same doses presents an intermediate behavior; (iv) 100 ng/mL of hNGF triggers an increase in p75NTR expression in H2O2-treated ARPE-19 cells, while pNGF at the same dose does not; (v) pNGF, but not hNGF (both given at 100 ng/mL), increases the total cell fluorescence intensity for TrkA receptors in H2O2-treated ARPE-19 cells. The present findings suggest a vicious positive feedback loop through which NGF-mediated upregulation of p75NTR contributes to worsening the toxic effects of oxidative damage in the human retinal epithelial cell line ARPE-19. Looking at the possible clinical relevance of these findings, one can postulate that pNGF might show a better benefit/risk ratio than hNGF in the treatment of ocular disorders.

Cytotoxicity of Ruthenium(II) Arene Complexes Containing Functionalized Ferrocenyl β-Diketonate Ligands.

The synthesis and characterization of 24 ruthenium(II) arene complexes of the type [(p-cym)RuCl(Fc-acac)] (where p-cym = p-cymene and Fc-acac = functionalized ferrocenyl β-diketonate ligands) are reported, including single-crystal X-ray diffraction for 21 new complexes. Chemosensitivity studies have been conducted against human pancreatic carcinoma (MIA PaCa-2), human colorectal adenocarcinoma p53-wildtype (HCT116 p53+/+) and normal human retinal epithelial cell lines (APRE-19). The most active complex, which contains a 2-furan-substituted ligand (4), is 5x more cytotoxic than the analogs 3-furan complex (5) against MIA PaCa-2. Several complexes were screened under hypoxic conditions and at shorter-time incubations, and their ability to damage DNA was determined by the comet assay. Compounds were also screened for their potential to inhibit the growth of both bacterial and fungal strains.

Lycium barbarum polysaccharide protects ARPE‑19 cells against H2O2‑induced oxidative stress via the Nrf2/HO‑1 pathway.

Age-related macular degeneration (AMD) is a global health problem. Lycium barbarum polysaccharide (LBP), a traditional Chinese herbal medicine, has been proven to be effective against several eye diseases. However, only a few studies have investigated the effectiveness of LBP for AMD. In the present study, the human retinal epithelial cell line, ARPE-19, was pretreated with LBP for 24 h before exposure to H2O2 (500 µM). Cell viability was assessed, and a series of oxidative and antioxidant indicators were evaluated to determine the influence of LBP on H2O2-triggered oxidative stress. The present study also determined the apoptosis status, as well as the expression levels of apoptotic proteins and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway proteins. The present study aimed to determine the protective role for LBP pretreatment and its underlying molecular mechanism. The results of the present study suggest that pretreatment of ARPE-19 cells with LBP exhibit high efficacy at reducing oxidative damage and inhibiting cell apoptosis. Furthermore, LBP may modulate the expression of proteins involved in the apoptotic pathway and activate the Nrf2 signaling pathway.

Protective Effect of β-Glucogallin on Damaged Cataract Against Methylglyoxal Induced Oxidative Stress in Cultured Lens Epithelial Cells.

Backgroundβ-glucogallin (GG) is one of the major plant polyphenolic antioxidants that have been associated with positive effects on human health and are crucial in the developing defense mechanism against the risk of diseases. However, reports on the protective mechanism of GG in lens epithelial cells are limited.Material/MethodsARPE-19 cells (a human retinal epithelial cell line) were exposed to methylglyoxal (MG) with or without GG to illuminate the protective role of GG in counteracting the cataract signaling.ResultsCells predisposed to MG demonstrated an increase in oxidative stress with augmented (P<0.01) inflammatory cytokines such as cyclooxygenase (COX)-2, chemokine receptor CXCR4, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule 1 (ICAM-1) genes. In addition, the expression of aldose reductase (AR) was increased to 2-fold with accumulated sorbitol in MG exposed cells compared to control. On the other hand, cells exposed to MG evidenced a 3-fold increase in RAGE (receptor for advanced glycation end products) and a 2-fold increase in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) expression compared to control cells. Intriguingly, lens epithelial cells pre-treated with GG attenuated the reactive oxygen species levels with improved antioxidant enzymes. Simultaneously, the levels of AR and other inflammatory cytokines were observed in the levels closer to control cells in GG pre-treated cells.ConclusionsThus, the results of the present investigation show that GG may be a potential drug for the prevention of cataract development and progression.

Characterization of Magnesium Orotate‐Loaded Chitosan Polymer Nanoparticles for a Drug Delivery System

AbstractThe drug release properties of magnesium orotate (MgOr) encapsulated in the chitosan (CS) cavity and the complexation behavior between MgOr and CS were investigated. The MgOr‐loaded CS nanoparticles (MgOrCSNPs) were characterized by differential scanning calorimetry, Fourier transform infrared spectroscopy, X‐ray diffraction, transmission electron microscopy, and scanning electron microscopy with energy‐dispersive X‐ray spectroscopy. MgOr was successfully encapsulated into the CS cavity. Results with 3‐(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyl tetrazolium bromide indicated that MgOrCSNPs retained their cytotoxic activity against the liver cancer cell line (HepG2) and breast cancer cell line (MCF‐7), and low toxicity against the human cell line (3T3) and human retinal epithelial cell line (ARPE‐19).

Splitting Up: Finding a New Way to Monitor Mitochondrial CaMKII Using SplitGFP

Important signaling events are specified by subcellular targeting, but current approaches may be inadequate to measure these events in living cells or in vivo. CaMKII (calcium/calmodulin-dependent protein kinase II) is an important mediator of cellular signaling, calcium homeostasis, heart failure, and lethal cardiac arrhythmias. There are two major CaMKII isoforms in the heart: γ and δ. Despite the broad functionality CaMKII has in regulating the cell, the availability of sensitive and specific tools to dynamically monitor CaMKII in subcellular compartments is limited. Recently, CaMKII was reported to reside in mitochondria where it mediates calcium homoeostasis and redox status in the organelle. Current methods of mitochondrial CaMKII detection rely on antibody based detection that cannot reliably distinguish between isoforms, and in some cases lack specificity. Here we developed a genetic reporter using SplitGFP to track mitochondrial CaMKII (mitoCaMKII) accumulation. Our strategy ‘splits’ self-complementing GFP, comprised of 11 β-strands, into two parts. First, GFP strands 1-10 were fused with a mitochondria localization sequence (MTS) and mCherry tag. Second, the GFP strand 11 was fused with CaMKIId. When both components are localized in the mitochondria the GFP self-complements and fluorescence reconstitutes. Using the excitable human retinal epithelial cell line (RPE1) we found that our CaMKIId-SplitGFP reporter is a sensitive marker of mitoCaMKII. Further, our reporter is capable of detecting dynamic increases of mitoCaMKII in response to β-adrenergic stress. These data suggest that CaMKII-SplitGFP is a useful tool in monitoring the dynamics and accumulation of mitoCaMKII, expanding the previously limited repertoire of CaMKII detection methods.

Quantification of somatic mutation flow across individual cell division events by lineage sequencing.

Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon (POLE) proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.

Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability.

Several studies have investigated the protective functions of brain-derived neurotrophic factor (BDNF) in retinitis pigmentosa. However, a BDNF-based therapy for retinitis pigmentosa is not yet available. To develop an efficient treatment for fundus disease, an eukaryotic expression plasmid was generated and used to transfect human 293T cells to assess the expression and bioactivity of BDNF on acute retinal pigment epithelial-19 (ARPE-19) cells, a human retinal epithelial cell line. After 96 hours of co-culture in a Transwell chamber, ARPE-19 cells exposed to BDNF secreted by 293T cells were more viable than ARPE-19 cells not exposed to secreted BDNF. Western blot assay showed that Bax levels were downregulated and that Bcl-2 levels were upregulated in human ARPE-19 cells exposed to BDNF. Furthermore, 293T cells transfected with the BDNF gene steadily secreted the protein. The powerful anti-apoptotic function of this BDNF may be useful for the treatment of retinitis pigmentosa and other retinal degenerative diseases.

Intracellular Redox Status and Cell Death Induced by H2O2 in a Human Retinal Epithelial Cell Line (ARPE-19)

Hydrogen peroxide is a normal by-product of cellular metabolism that in higher concentrations can cause oxidative stress. Reactive oxygen species impair the physiological functions of retinal pigment epithelial (RPE) cells, which are known as one major cause of ocular pathologies. Most studies investigating the influence of H2O2 on cells in culture but H2O2 concentrations are not sustained in culture medium. Continuous generation using glucose oxidase (GOx) system allows application of relevant low H2O2 concentrations over physiologically relevant times periods (up to 24 h). Recent findings suggest that bolus and GOx treatments can lead to different cellular response, thus warranting a quantitative comparison between the two approaches. When added as a pulse H2O2 is rapidly depleted. Continuous generation of H2O2 produces different behavior in function of GOx activities. Cytotoxicity analyses show that cells can tolerate short exposure to high H2O2 doses delivered as a pulse but are susceptible to lower continuous doses. Application of hydrogen peroxide causes a concentration-dependent decrease in the intracellular glutathione (GSH) content that was accompanied by a matching decrease in the glutathione peroxide activity and reducing power (FRAP).

An Algorithm to Quantify Correlated Collective Cell Migration Behavior

Collective cell migration is an important process that determines cell reorganization in a number of biological events such as development and regeneration. Random cell reorganization within a confluent monolayer is a popular in vitro model system for understanding the mechanisms that underlie coordination between neighboring cells during collective motion. Here we describe a simple automated C++ algorithm to quantify the width of streams of correlated cells moving within monolayers. Our method is efficient and allows analysis of thousands of cells in under a minute; analysis of large data sets is therefore possible without limitations due to computational time, a common analysis bottleneck. Furthermore, our method allows characterization of the variability in correlated stream widths among a cell monolayer. We quantify stream width in the human retinal epithelial cell line ARPE-19 and the fibroblast cell line BJ, and find that for both cell types, stream widths within the monolayer vary in size significantly with a peak width of 40 µm, corresponding to a width of approximately two cells. Our algorithm provides a novel analytical tool to quantify and analyze correlated cell movement in confluent sheets at a population level and to assess factors that impact coordinated collective cell migration.

Mitochondrial DNA variants mediate energy production and expression levels for CFH, C3 and EFEMP1 genes: implications for age-related macular degeneration.

BackgroundMitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD).Methodology/Principal FindingsCybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.Conclusion/SignificanceOur findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

SEMA4A Mutations Lead to Susceptibility to Light Irradiation, Oxidative Stress, and ER Stress in Retinal Pigment Epithelial Cells

RP is a retinal degeneration disorder that is caused by mutations of various genes, including semaphorin-4A (SEMA4A). A number of retinal diseases, including RP, are associated with light exposure, oxidative stress, and endoplasmic reticulum (ER) stress. In this study, we investigated whether mutant SEMA4A causes retinal dysfunction via light exposure, oxidative stress, and ER stress. Mutant SEMA4A (D345H or F350C) was overexpressed in a human retinal epithelium cell line ARPE19. Intracellular localization of mutant SEMA4A was investigated using confocal laser scanning microscopy. The ARPE-19 cells were also irradiated with white light, and expression of 78 kDa glucose-regulated protein (GRP78), a marker of ER stress, and phagocytosis were measured. The cells were treated with an ER stress inducer, tunicamycin, or an oxidative stressor, H(2)O(2), and cell death was measured. Human SEMA4A mutants were expressed in zebrafish embryos with tunicamycin and mRNA of DNA damage-inducible transcript 3 (ddit3) was measured as an ER stress marker. Mutant SEMA4A was localized in the ER, whereas wild type (WT) SEMA4A was observed in cell membranes. The expression of GRP78 was increased by mutant SEMA4A following light irradiation, and phagocytosis was suppressed in mutant SEMA4A-transfected cells. Mutant SEMA4A induced susceptibility to ER stress and oxidative stress. In zebrafish, human mutant SEMA4A increased ddit3 mRNA compared with WT under the ER stress condition. Our results suggest that mutations in SEMA4A may cause susceptibility to light exposure, oxidative stress, and ER stress, which may be involved in the progression and pathology of RP.

Reversal of the Caspase-Dependent Apoptotic Cytotoxicity Pathway by Taurine fromLycium barbarum(Goji Berry) in Human Retinal Pigment Epithelial Cells: Potential Benefit in Diabetic Retinopathy

Diabetic retinopathy is a preventable microvascular diabetic complication and a leading cause of vision loss. Retinal pigment epithelial cell apoptosis is an early event in diabetic retinopathy. Taurine is reportedly beneficial for diabetic retinopathy and is abundant in the fruit of Lycium barbarum (LB). We have investigated the effect of pure taurine and an extract of LB rich in taurine on a model of diabetic retinopathy, the retinal ARPE-19 cell line exposed to high glucose. We demonstrate for the first time that LB extract and the active ligand, taurine, dose dependently enhance cell viability following high glucose treatment in the ARPE-19 retinal epithelial cell line. This cytoprotective effect was associated with the attenuation of high glucose-induced apoptosis, which was shown by characteristic morphological staining and the dose-dependent decrease in the number of apoptotic cells, determined by flow cytometry. Moreover, we have shown that LB extract and taurine dose dependently downregulate caspase-3 protein expression and the enzymatic activity of caspase-3. We conclude that taurine, a major component of LB, and the LB extract, have a cytoprotective effect against glucose exposure in a human retinal epithelial cell line and may provide useful approaches to delaying diabetic retinopathy progression.

Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids

Stargardt-like macular dystrophy (STGD3) is a dominantly inherited juvenile macular degeneration that eventually leads to loss of vision. Three independent mutations causing STGD3 have been identified in exon six of a gene named Elongation of very long chain fatty acids 4 (ELOVL4). The ELOVL4 protein was predicted to be involved in fatty acid elongation, although evidence for this and the specific step(s) it may catalyze have remained elusive. Here, using a gain-of-function approach, we provide direct and compelling evidence that ELOVL4 is required for the synthesis of C28 and C30 saturated fatty acids (VLC-FA) and of C28-C38 very long chain polyunsaturated fatty acids (VLC-PUFA), the latter being uniquely expressed in retina, sperm, and brain. Rat neonatal cardiomyocytes and a human retinal epithelium cell line (ARPE-19) were transduced with recombinant adenovirus type 5 carrying mouse Elovl4 and supplemented with 24:0, 20:5n3, or 22:5n3. The 24:0 was elongated to 28:0 and 30:0; 20:5n3 and 22:5n3 were elongated to a series of C28-C38 PUFA. Because retinal degeneration is the only known phenotype in STGD3 disease, we propose that reduced VLC-PUFA in the retinas of these patients may be the cause of photoreceptor cell death.

Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability

Vessel growth is often associated with ischemia. VEGF, a potent angiogenic factor, has been shown to be induced by low oxygen concentrations. These studies were conducted to investigate the molecular basis of the hypoxia-induced increase in VEGF mRNA. Run-on analysis of VEGF revealed a minimal increase in the rate of gene transcription in a human retinal epithelial cell line grown under hypoxic conditions. Examination of VEGF mRNA stability revealed that the half-life of VEGF transcripts under normoxia was short, 30–45 min, but was dramatically increased to 6–8 h in cells grown under hypoxia. Cobalt chloride, which elevates VEGF and has been suggested to be similar to hypoxia in its mechanism of action, had only a slight effect on decay rate. We postulate that hypoxia-induced increases in mRNA stability provide the sustained increases in VEGF mRNA levels necessary to support a neovascular response.