Pigment Epithelial Cell Line ARPE-19 Research Articles

Paper Withdrawn: Quercetin attenuates high glucose-induced injury in human retinal pigment epithelial cell line ARPE-19 by up-regulation of miR-29b.

Paper Withdrawn: Quercetin attenuates high glucose-induced injury in human retinal pigment epithelial cell line ARPE-19 by up-regulation of miR-29b.

Effects of FTMT Expression by Retinal Pigment Epithelial Cells on Features of Angiogenesis.

Aberrant angiogenesis is a pathological feature of a number of diseases and arises from the uncoordinated expression of angiogenic factors as response to different cellular stresses. Age-related macular degeneration (AMD), a leading cause of vision loss, can result from pathological angiogenesis. As a mutation in the mitochondrial ferritin (FTMT) gene has been associated with AMD, its possible role in modulating angiogenic factors and angiogenesis was investigated. FTMT is an iron-sequestering protein primarily expressed in metabolically active cells and tissues with high oxygen demand, including retina. In this study, we utilized the human retinal pigment epithelial cell line ARPE-19, both as undifferentiated and differentiated cells. The effects of proinflammatory cytokines, FTMT knockdown, and transient and stable overexpression of FTMT were investigated on expression of pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial-derived factor (PEDF). Proinflammatory cytokines induced FTMT and VEGF expression, while NF-κB inhibition significantly reduced FTMT expression. VEGF protein and mRNA expression were significantly increased in FTMT-silenced ARPE-19 cells. Using an in vitro angiogenesis assay with endothelial cells, we showed that conditioned media from FTMT-overexpressing cells had significant antiangiogenic effects. Collectively, our findings indicate that increased levels of FTMT inhibit angiogenesis, possibly by reducing levels of VEGF and increasing PEDF expression. The cellular models developed can be used to investigate if increased FTMT may be protective in angiogenic diseases, such as AMD.

Quercetin attenuates high glucose-induced injury in human retinal pigment epithelial cell line ARPE-19 by up-regulation of miR-29b.

Quercetin is a kind of distinctive bioactive flavonoid that has potent anti-oxidant, anti-inflammatory and anti-diabetic properties. The present article was designed to check the effect of quercetin on diabetic retinopathy. Adult retinal pigment epithelial cell line (ARPE)-19 cells were pre-treated with quercetin and then stimulated by high glucose. Cell damage was evaluated by CCK-8 assay, flow cytometer, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, 2,7-dichlorofluorescein diacetate probe and western blot. The association between quercetin and miR-29b expression as well as the downstream pathways was studied by qRT-PCR and western blot. Pre-treating ARPE-19 cells with quercetin clearly attenuated high glucose-induced viability loss, apoptosis, MCP-1 and IL-6 overproduction and reactive oxygen species (ROS) generation. Quercetin down-regulated p53, Bax and cleaved-caspase-3 expression, while up-regulated CyclinD1, CDK4 and Bcl-2. miR-29b was low expressed in high glucose-treated cell, but quercetin elevated its expression. Moreover, the protective action of quercetin towards ARPE-19 cells was attenuated when miR-29b was suppressed. Also, quercetin promoted PTEN/AKT pathway, while inhibited NF-κB pathway via a miR-29b-dependent way. These data illustrated quercetin possibly possess the anti-diabetic retinopathy potential, as quercetin clearly attenuated high glucose-evoked damage in ARPE-19 cells. The protective action of quercetin may due to its regulation on miR-29b expression as well as PTEN/AKT and NF-κB pathways.

Combination of Dark-Field and Confocal Microscopy for the Optical Detection of Silver and Titanium Nanoparticles in Mammalian Cells.

We describe here two optical microscopy techniques-dark-field confocal light scanning microscopy (DF-CLSM) and dark-field wide-field confocal microscopy (DF-WFCM), that can be used to study interaction between nanoparticles and cells in 3D space. Dark field microscopy can detect very small structures below the diffraction limit of conventional light microscopes, while a confocal setup provides vertical sectioning capabilities to render specimens in 3D. The use of DF-WFCM instead of DF-CLSM allows faster sample processing but yields lower resolution. We used a retinal pigment epithelial cell line ARPE-19 to illustrate different optical and lighting conditions necessary for optimal imaging of metal and metal oxide nanoparticles (TiO2 and Ag). Our experimental setup primarily involved an E-800 Nikon and Nikon Ni upright microscopes and a Nikon Ti2 microscope connected to a xenon light source along with special dark-field objectives. For confocal studies we used either Leica and Nikon inverted confocal microscopes. For microscopic analyses, ARPE-19 cells were fixed in situ in cultured chamber slides or collected from T-25 flasks and then fixed in suspension. At the lowest concentrations of TiO2 or Ag tested (0.1-0.3μg/mL), we were able to detect as few as 5-10 nanoparticles per cell due to intense light scattering by the particles. The degree of brightness detected indicated that the uptake of nanoparticles within ARPE-19 cells could be monitored using dark-field microscopy. Here we describe how to use wide-field microscopy to follow nanoparticle uptake by cells and how to assess some aspects of cellular health in in vitro cell cultures exposed to nanoparticles.

Structure based modification of chalcone analogue activates Nrf2 in the human retinal pigment epithelial cell line ARPE-19

Oxidative stress-induced degeneration of retinal pigment epithelial (RPE) cells is known to be a key contributor to the development of age-related macular degeneration (AMD). Activation of the nuclear factor-(erythroid-derived 2)-related factor-2 (Nrf2)-mediated cellular defense system is believed to be a valid therapeutic approach. In the present study, we designed and synthesized a novel chalcone analogue, 1-(2,3,4-trimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-acrylketone (Tak), as a Nrf2 activator. The potency of Tak was measured in RPE cells by the induction of the Nrf2-dependent antioxidant genes HO-1, NQO-1, GCLc, and GCLm, which were regulated through the Erk pathway. We also showed that Tak could protect RPE cells against oxidative stress-induced cell death and mitochondrial dysfunction. Furthermore, by modifying the α, β unsaturated carbonyl entity in Tak, we showed that the induction of antioxidant genes was abolished, indicating that this unique feature in Tak was responsible for the Nrf2 activation. These results suggest that Tak is a potential candidate for clinical application against AMD.

RETRACTED: Schizandrin A Protects Human Retinal Pigment Epithelial Cell Line ARPE-19 against HG-Induced Cell Injury by Regulation of miR-145

Diabetic retinopathy (DR) is a serious complication of diabetes, which is the main cause of blindness among adults. Traditional Chinese medicines (TCMs) have been proven to delay the development of DR. Nonetheless, the effect of Schizandrin A (SchA) on DR remains uninvestigated. The present study aimed to probe the protective effect of SchA on high-glucose (HG)-induced injury in ARPE-19 cells. We observed that SchA accelerated cell proliferation, prohibited apoptosis, and restrained pro-inflammatory cytokines (monocyte chemoattractant protein-1 [MCP-1], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) and reactive oxygen species (ROS) level in HG-stimulated cells. Additionally, miR-145 expression was upregulated in HG and SchA co-treated cells, and miR-145 inhibition reversed the protective effect of SchA on HG-managed ARPE-19 cells. Interestingly, downregulated myeloid differentiation factor 88 (MyD88) was found in HG and SchA co-treated cells, and upregulation of MyD88 was observed in miR-145 inhibitor-transfected cells. Additionally, SchA hindered nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in HG-treated ARPE-19 cells. The findings validated that SchA could protect ARPE-19 cells from HG-induced cell injury by regulation of miR-145.

Dysregulation of miR-204-3p Driven by the Viability and Motility of Retinoblastoma via Wnt/β-catenin Pathway In Vitro and In Vivo.

Retinoblastoma (RB) is a malignant intraocular tumor that frequently occurs in infants and toddlers. Although the most of RB patients in the developed countries could survival from this cancer, the patients in undeveloped areas are still suffering. The human retinal pigment epithelial cell line ARPE-19 and human retinoblastoma (RB) cell lines HXO-RB44, Y79, and WERI-Rb1 were cultured. The mRNA levels of BANCR and miR-204-3p in these cell lines were measured by qRT-PCR. After transfection with sh-BANCR or treatment with miR-204-3p inhibitor in Y79 cells, the cell proliferation rate, growth, invasion, migration, apoptosis and Wnt/β-catenin signaling pathway activity were measured. The regular Y79 and Y79 cells stably expressed sh-BANCR were injected subcutaneously into nude mice, respectively. The volumes and pathohistological futures of tumors were compared. The biochemical features similar to the cell culture were detected and compered. The mRNA measurements showed that BANCR negatively modulate miR-204-3p expression via directly integration with it. Besides, miR-204-3p and Wnt/β-catenin signalling pathway were found to participate in the oncogenic effects of BANCR on RB cell line by Hoechst staining, cell Counting Kit-8 (CCK-8) assay, wound healing assay, transwell assay, and Western blot analysis in vitro. In addition, an in vivo tumorigenesis experiment in nude mice injected with Y79 cells stably expressed sh-BANCR conformed in the effects of BANCR on RB. Taken together, the knockdown of BANCR inhibited cell proliferation, apoptosis, invasion, and migration in RB via targeting miR-204-3p, the mechanism may involve inhibiting Wnt/β-catenin signaling pathway.

RETRACTED ARTICLE: Salidroside alleviates high-glucose-induced injury in retinal pigment epithelial cell line ARPE-19 by down-regulation of miR-138

ABSTRACTDiabetic retinopathy (DR) is a complication of diabetes leading cause of blindness in adults. Salidroside (SAL) is a main ingredient from Rhodiola rosea L., has been reported to have a beneficial protection on vascular function. However, whether SAL is a suitable treatment for DR remains unreported. The study aimed to investigate the effect of SAL on high-glucose (HG)-induced injury in ARPE-19 cells. ARPE-19 cells were managed with diverse concentrations of glucose, and constructed a model of HG-induced ARPE-19 cells injury. Then, SAL was employed to stimulate ARPE-19 cells, and cell viability, apoptosis, apoptosis-associated factors, the pro-inflammatory cytokines, and ROS levels were determined. The correlation between miR-138 and SIRT1 was predicated by bioinformatics software of TargetScan (http://www.targetscan.org/) and Dual luciferase reporter assay. MiR-138 mimic, inhibitor and NCs were transfected into ARPE-19 cells, and the impacts of miR-138 on HG-induced cell injury were investigated. PI3K/AKT and AMPK signalling pathways were examined to explore the underlying mechanism. The results disclosed that HG inhibited cell viability, promoted apoptosis, up-regulated IL-6 and TNF-α, as well as increased ROS level in ARPE-19 cells. But, SAL obviously alleviated HG-induced ARPE-19 cells injury. Repressed miR-138 was triggered by SAL, and SIRT1 was predicated as a direct target of miR-138. Overexpressed miR-138 declined the protective effect of SAL on HG-injured ARPE-19 cells. Besides, SAL activated PI3K/AKT and AMPK pathways by adjusting miR-138. In conclusions, SAL flattened HG-induced injury in ARPE-19 cells by repression of miR-138 and activating PI3K/AKT and AMPK pathways.

MiR-361-5p inhibits cell proliferation and induces cell apoptosis in retinoblastoma by negatively regulating CLDN8.

MiR-361-5p has been reported to act as tumor suppressor in several types of cancers. Retinoblastoma (RB) is the most common ocular tumor in childhood. The current study aimed to investigate the expression pattern and biological function of miR-361-5p in RB. Quantitative real time was utilized to determine and compare the expression of miR-361-5p in RB cells and normal retinal pigment epithelial cell line ARPE-19. CCK-8 and Edu assay were performed to assess cell proliferation. Cell apoptosis was evaluated using flow cytometry assay. Bioinformatics databases and luciferase reporter assay were applied to predict and confirm the target gene of miR-361-5p in RB cells. Here, we found miR-361-5p was significantly downregulated in RB cells compared with normal retinal pigment epithelial cell line ARPE-19. MiR-361-5p overexpression significantly inhibited or silencing promoted cell proliferation in Y79 and SO-RB50 cells, respectively. Flow cytometry assay showed a significantly decreased cell apoptosis in miR-361-5p silencing Y79 cells and increased cell apoptosis in miR-361-5p overexpressing SO-RB50 cells. Moreover, miR-361-5p directly bound to the 3' untranslated region of claudin 8 (CLDN8) and inhibited the expression of CLDN8. Furthermore, we found knockdown of CLDN8 photocopied the effect of miR-361-5p on cell proliferation and apoptosis in RB cells. These results indicated that overexpression of miR-361-5p might act as a suppressor in RB by targeting CLDN8 to inhibit the cellular function.

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 improves diabetic retinopathy.

Diabetes induced a serious of complications including diabetic retinopathy. Our study aimed to investigate the role of Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in diabetic retinopathy. A mice model of diabetic retinopathy was established, and expression of SDF-1 and CXCR4 in retina was examined by Real-time quantitative PCR (qRT-PCR). Cells of human retinal pigment epithelial cell line ARPE-19 were treated with CXCR4 siRNAs and expression vector, and cell viability was detected by MTT assay. We found that expression of SDF-1 and CXCR4 in retina was significantly downregulated in mice with diabetic retinopathy than in normal healthy mice. High glucose treatment downregulated the expression of SDF-1 and CXCR4 in ARPE-19 cells at both mRNA and protein levels. Transfection with CXCR4 siRNAs decreased, while transfection with CXCR4 expression vector increased cell viability under high glucose treatment. We concluded that SDF-1/CXCR4 pathway improved diabetic retinopathy possibly by increasing cell viability. Abbreviations: SDF-1: Stromal cell-derived factor 1; CXCL12: C-X-C motif chemokine 12; qRT-PCR: Real-time quantitative PCR.

Long Non-Coding RNA BANCR Is Overexpressed in Patients with Diabetic Retinopathy and Promotes Apoptosis of Retinal Pigment Epithelial Cells.

BackgroundlncRNA BANCR participates in the pathogenesis of various types of human diseases; however, its involvement in diabetic retinopathy is unknown.Material/MethodsIn this study, the expression of lncRNA BANCR in plasma of patients with diabetic retinopathy, diabetic patients without complications, and healthy controls was analyzed by qRT-PCR. The accuracy plasma BANCR in diagnosing diabetic retinopathy was analyzed by ROC curve analysis. lncRNA BANCR expression vector and siRNA were transfected into the human retinal pigment epithelial cell line ARPE-19, and cell apoptosis was analyzed by cell apoptosis assay.ResultsWe found that lncRNA BANCR was significantly upregulated in patients with diabetic retinopathy compared to diabetic patients without complications and healthy controls. Upregulation of lncRNA BANCR effectively distinguished patients with diabetic retinopathy from diabetic patients without complications and healthy controls. High-glucose treatment led to upregulated expression of BANCR in the human retinal pigment epithelial cell line ARPE-19. Overexpression of BANCR promoted and siRNA silencing inhibited the apoptosis of cells in the human retinal pigment epithelial cell line ARPE-19 in a high-glucose environment.ConclusionslncRNA BANCR is overexpressed in patients with diabetic retinopathy and can promote apoptosis of retinal pigment epithelial cells.

Downregulation of lncRNA BANCR participates in the development of retinopathy among diabetic patients.

The long non-coding (lnc)RNA B-Raf proto-oncogene, serine/threonine kinase-activated non-protein coding RNA (BANCR) is a well-characterized oncogene, while its potential functions in other diseases remain elusive. In the present study, the possible association of BANCR with diabetic retinopathy was investigated. A total of 244 patients with diabetes were followed up every 6 months for 8 years to record the occurrence of retinopathy. A total of 38 patients developed diabetic retinopathy. During the follow-up, the plasma levels of lncRNA BANCR decreased in those patients with diabetic retinopathy but not in those with other complications or without any complications. The plasma levels of lncRNA BANCR at 12 months prior to the diagnosis of diabetic retinopathy are able to sufficiently distinguish diabetic retinopathy patients from healthy controls and diabetic patients without any obvious complications. In vitro, high-glucose treatment failed to affect the expression of lncRNA BANCR in the human retinal pigment epithelial cell line ARPE-19. However, lncRNA BANCR overexpression inhibited the apoptosis of ARPE-19 cells under high-glucose conditions. Therefore, it is indicated that lncRNA BANCR participates in the development of retinopathy in diabetic patients through its regulatory role in cell apoptosis, and may serve as a novel prognostic indicator and therapeutic target.

Characterization of CDNF-Secreting ARPE-19 Cell Clones for Encapsulated Cell Therapy

Cerebral dopamine neurotrophic factor (CDNF) shows beneficial effects in rodent models of Parkinson’s and Alzheimer’s disease. The brain is a challenging target for protein therapy due to its exclusive blood–brain barrier. Hence, the therapeutic protein should be delivered directly to the brain parenchyma. Implantation of encapsulated mammalian cells that constantly secrete CDNF is a potential approach for targeted and long-term protein delivery to the brain. In this study, we generated several CDNF-secreting cell clones derived from human retinal pigment epithelial cell line ARPE-19, and studied CDNF secretion from the clones maintained as monolayers and in polymeric microcapsules. The secretion of wild type (wt) CDNF transgene was low and the majority of the produced protein remained intracellular, locating mainly to the endoplasmic reticulum (ER). The secretion of wtCDNF decreased to even lower levels when the clones were in a non-dividing state, as in the microcapsules. Both codon optimization and deletion of the putative ER-retrieval signal (four last amino acids: KTEL) improved CDNF secretion. More importantly, the secretion of KTEL-deleted CDNF remained constant in the non-dividing clones. Thus, cells expressing KTEL-deleted CDNF, in contrast to wtCDNF, can be considered for cell encapsulation applications if the KTEL-deleted CDNF is proven to be biologically active in vivo.

Attachment of a 5-nitrofuroyl moiety to spirocyclic piperidines produces non-toxic nitrofurans that are efficacious in vitro against multidrug-resistant Mycobacterium tuberculosis

A selectively antimycobacterial compound belonging to the nitrofuran class of antimicrobials has been developed via conjugation of the nitrofuran moiety to a series of spirocyclic piperidines through an amide linkage. It proved to have comparable activity against drug-sensitive (H37Rv) strain as well as multidrug-resistant, patient-derived strains of Mycobacterium tuberculosis. The compound is druglike, showed no appreciable cytotoxicity toward human retinal pigment epithelial cell line ARPE-19 in concentrations up to 100 μM and displayed low toxicity when evaluated in mice.

LncRNA AK077216 is downregulated in diabetic retinopathy and inhibited the apoptosis of retinal pigment epithelial cells by downregulating miR-383.

LncRNA AK077216 is a novel lncRNA with critical role in osteoclastogenesis and bone resorption. Our preliminary RNA-seq data suggested its downregulated expression pattern in diabetic retinopathy (DR). We therefore further investigated the function of AK077216 in DR. Plasma samples used in this study were derived from blood, which was extracted from 60 diabetic retinopathy patients. AK077216 in plasma of DR patients, diabetic patients (DB) and controls (Control) was detected by RT-qPCR. ROC curve analysis was performed to evaluate the diagnostic value of plasma AK077216 for DR. It was found that plasma AK077216 was downregulated only in DR patients but not in diabetic patients (no obvious complications) comparing to healthy controls. Downregulation of AK077216 distinguished DR patients from diabetic patients and healthy controls. High glucose treatment failed to significantly affect AK077216 expression in cells of human retinal pigment epithelial cell line ARPE-19. MiR-383 was inversely correlated with AK077216 in DR patients. AK077216 overexpression caused miR-383 downregulation, but miR-383 overexpression did not significantly alter AK077216 expression. AK077216 overexpression inhibited ARPE-19 cell apoptosis, while miR-383 overexpression played an opposite role and attenuated the effects of AK077216 overexpression. Therefore, AK077216 is downregulated in diabetic retinopathy and inhibited the apoptosis of ARPE-19 cells by downregulating miR-383.

Multiple Cellular Transport and Binding Processes of Unesterified Docosahexaenoic Acid in Outer Blood-Retinal Barrier Retinal Pigment Epithelial Cells.

Docosahexaenoic acid (DHA, 22 : 6) is an essential omega-3 long-chain polyunsaturated fatty acid that plays a pivotal role in vision. The purpose of this study was to clarify the cellular uptake and binding processes of free and protein-bound unesterified DHA in retinal pigment epithelial cell (RPE) line ARPE-19 as a model of the human outer blood-retinal barrier and isolated porcine RPE cell fractions. Uptake of free [14C]DHA by ARPE-19 cells was saturable with a Michaelis-Menten constant of 283 µM, and was significantly inhibited by eicosapentaenoic acid, arachidonic acid, and linoleic acid, but not by oleic acid. Further, the uptakes of [14C]DHA associated with retinol-binding protein ([14C]DHA-RBP), [14C]DHA associated with low-density lipoprotein ([14C]DHA-LDL) and [14C]DHA associated with bovine serum albumin ([14C]DHA-BSA) in ARPE-19 cells increased time-dependently at 37°C, and were significantly reduced at 4°C, suggesting the involvement of energy-dependent transport processes. [14C]DHA-LDL uptake by ARPE-19 cells was significantly inhibited by excess unlabeled LDL, but not by an inhibitor of scavenger receptor B type I. Fatty acid transport protein (FATP) 2 and 4 mRNAs were expressed in ARPE-19 cells, and [14C]DHA uptake was observed in FATP2- and FATP4-expressing Xenopus oocytes. Photo-reactive crosslinking and mass spectrometry analyses identified 65-kDa retinal pigment epithelium-specific protein (RPE65) as a DHA-binding protein in porcine RPE cell membrane fractions. Thus, RPE cells possess multiple cellular transport/binding processes for unesterified DHA, involving at least partly FATP2, FATP4, LDL, RBP, and RPE65.

Ocular Toxicity Profile of ST-162 and ST-168 as Novel Bifunctional MEK/PI3K Inhibitors.

ST-162 and ST-168 are small-molecule bifunctional inhibitors of MEK and PI3K signaling pathways that are being developed as novel antitumor agents. Previous small-molecule and biologic MEK inhibitors demonstrated ocular toxicity events that were dose limiting in clinical studies. We evaluated in vitro and in vivo ocular toxicity profiles of ST-162 and ST-168. Photoreceptor cell line 661W and adult retinal pigment epithelium cell line ARPE-19 were treated with increasing concentrations of bifunctional inhibitors. Western blots, cell viability, and caspase activity assays were performed to evaluate MEK and PI3K inhibition and dose-dependent in vitro toxicity, and compared with monotherapy. In vivo toxicity profile was assessed by intravitreal injection of ST-162 and ST-168 in Dutch-Belted rabbits, followed by ocular examination and histological analysis of enucleated eyes. Retinal cell lines treated with ST-162 or ST-168 exhibited dose-dependent inhibition of MEK and PI3K signaling. Compared with inhibition by monotherapies and their combinations, bifunctional inhibitors demonstrated reduced cell death and caspase activity. In vivo, both bifunctional inhibitors exhibited a more favorable toxicity profile when compared with MEK inhibitor PD0325901. Novel MEK and PI3K bifunctional inhibitors ST-162 and ST-168 demonstrate favorable in vitro and in vivo ocular toxicity profiles, supporting their further development as potential therapeutic agents targeting multiple aggressive tumors.

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.

ROCK Inhibitor Y-27632 Promotes Human Retinal Pigment Epithelium Survival by Altering Cellular Biomechanical Properties.

Purpose: Dysfunction or death of retinal pigment epithelial (RPE) cells is a common pathogenesis of various types of retinal degenerative diseases. Recent reports indicated that ROCK pathway inhibitors regulate cell proliferation or apoptosis in a cell-type-dependent manner. Here, we aim to investigate the effect of ROCK inhibitor Y-27632 on the human retinal pigment epithelium (RPE) in vitro.Methods: Cell proliferation and apoptosis were analyzed by CCK-8 and flow cytometry respectively. Cell proliferation markers were detected by immunofluorescence and western blot. Cell morphology was evaluated using scanning electron microscopy. The topography and biomechanical properties of living cells were assessed using atomic force microscope (AFM). In addition, cytoskeleton and epithelial-mesenchymal transition (EMT) markers were detected by western blot and immunofluorescence.Results: 30μM Y-27632 significantly promoted cell proliferation and decreased apoptosis. Compared with control group, human retinal pigment epithelial cell line ARPE-19 cells treated with 30μM Y-27632 exhibited significantly decreased cytomembrane roughness (Ra: 41.04±1.63nm vs. 24.41±0.75nm, P<0.01; Rq: 51.56±2.03nm vs. 30.81±0.95nm, P<0.01) and increased elasticity modulus (16.66±0.83KPa vs. 32.55±1.48KPa, P<0.01). In addition, the inhibition of ROCK activity by Y-27632 caused cell elongation and reorganization of microfilaments and microtubules of cytoskeletons.Conclusion: Taken together, our data demonstrated that Y-27632 could alter biomechanical properties and reorganized cytoskeletons to promote RPE cell survival. These results are an important step toward the future application of Y-27632 in retinal degenerative diseases.

Effects of plasma kallikrein inhibitors in an in vitro RPE oxidative stress model

PurposePlasma Kallikrein (PK) Inhibitors (PKI) are a promising treatment for retinal diseases such as diabetic macular edema, where PK activity is implicated. Our aim was to set up an in vitro model of chronic oxidative stress in the retinal pigment epithelium cell line ARPE19 and use it to test the effect of 8 PKI on cell viability and proliferation.MethodsARPE19 cells were grown to a sub‐confluence stage, synchronized, and exposed to different dilutions of glucose oxidase (GOx) from Aspergillus niger, culture medium (− control) and benzalkonium chloride (+ control) for 24 hr. Cell viability and apoptosis were analyzed (MTT assay and caspase‐3/7 detection, respectively). The oxidative stressed cells were exposed to 8 different PKI developed by KalVista (Salisbury, UK) at 100 nm and 1 μm. Also, ARPE19 cells were exposed to PKI previously and simultaneously to GOx and cell viability and proliferation were assessed (alamarBlue® assays).ResultsExposure of ARPE19 to GOx induced dose‐dependent decreased cell viability and increased cell apoptosis. A sub‐lethal GOx dose reducing viability by 30% and increasing apoptosis by 59% was chosen for PKI exposure experiments. Exposure of GOx stressed cells to PKI for 24 hr did not further reduce cell viability after 5 days. No effect on cell proliferation was observed when GOx stressed cells were either pre‐treated or simultaneously exposed to PKI.ConclusionsGOx oxidative sub‐lethal stress model in ARPE19 cells may be used for screening new drugs with therapeutic potential for retinal diseases. The PKI did not reduce GOx‐induced cytotoxic effects in RPE cells suggesting plasma kallikrein has no role in this stress mechanism. Support: FP7‐PEOPLE‐2013‐IAPP (612218/3D‐NET) and Regional Junta de Castilla y León Scholarship/European Social Fund Program (Va040‐13).