Viability Of ARPE-19 Cells Research Articles

Centella asiatica extract prevents visual impairment by promoting the production of rhodopsin in the retina.

BACKGROUND/OBJECTIVECentella asiatica, also known as Gotu kola, is a tropical medicinal plant native to Madagascar, Southeast Asia, and South Africa. It is well known to have biological activities, including wound healing, anti-inflammatory, antidiabetic, cytotoxic, and antioxidant effects. The purpose of this study was to determine the efficacy of extracts of C. asiatica against age-related eye degeneration and to examine their physiological activities.MATERIALS/METHODSTo determine the effects of CA-HE50 (C. asiatica 50% EtOH extract) on retinal pigment cells, we assessed the cytotoxicity of CoCl2 and oxidized-A2E in ARPE-19 cells and observed the protective effects of CA-HE50 against N-methyl-N-nitrosourea (MNU)-induced retinal damage in C57BL/6 mice. In particular, we measured factors related to apoptosis and anti-oxidation and the protein levels of rhodopsin/opsin. We also measured glucose uptake to characterize glucose metabolism, a major factor in cell protection.RESULTSInduction of cytotoxicity with CoCl2 and oxidized-A2E inhibited decreases in the viability of ARPE-19 cells when CA-HE50 was administered, and promoted glucose uptake under normal conditions (P < 0.05). In addition, CA-HE50 inhibited degeneration/apoptosis of the retina in the context of MNU-induced toxicity (P < 0.05). In particular, CA-HE50 at 200 mg/kg inhibited the cleavage of pro-caspase-3 and pro-poly (ADP-ribose)-polymerase and maintained the expressions of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 similar to normal control levels. Rhodopsin/opsin expression was maintained at a higher level than in normal controls.CONCLUSIONA series of experiments confirmed that CA-HE50 was effective for inhibiting or preventing age-related eye damage/degeneration. Based on these results, we believe it is worthwhile to develop drugs or functional foods related to age-related eye degeneration using CA-HE50.

C1q/TNF-related Protein 9 Inhibits High Glucose–Induced Oxidative Stress and Apoptosis in Retinal Pigment Epithelial Cells Through the Activation of AMPK/Nrf2 Signaling Pathway

Diabetic retinopathy (DR) is one of the common complications of diabetes mellitus. C1q/TNF-related protein 9 (CTRP9) has been demonstrated to be associated with the progression of diabetes and relative complications. However, its role in DR and underlying action of mechanism are not yet well understood. In the present study, human retinal pigment epithelial ARPE-19 cells were cultured under high concentration of glucose to simulate hyperglycemia condition in vitro. Our results showed that the expression of CTRP9 was significantly decreased in high glucose (HG)–stimulated ARPE-19 cells. CTRP9 overexpression improved HG-caused reduction in cell viability of ARPE-19 cells. CTRP9 overexpression significantly attenuated HG-induced oxidative stress, as proved by decreased levels of reactive oxygen species and malondialdehyde, and increased superoxide dismutase activity. Moreover, CTRP9 also prevented apoptosis in ARPE-19 cells in response to HG stimulation with decreased caspse-3 activity and bax expression, as well as increased bcl-2 expression. In contrast, knockdown of CTRP9 aggravated HG-induced oxidative stress and apoptosis. Furthermore, CTRP9 significantly induced the activation of AMPK/Nrf2 pathway in HG-induced ARPE-19 cells. Notably, inhibiting AMPK or Nrf2 blocked the protective effect of CTRP9 on ARPE-19 cells exposed to HG stimulation. Taken together, our findings suggested a protective effect of CTRP9 on HG-induced ARPE-19 cells and a putative mechanism involving the activation of AMPK/Nrf2 signaling pathway.

Role of Long Non-Coding RNA (LncRNA) LINC-PINT Downregulation in Cardiomyopathy and Retinopathy Progression Among Patients with Type 2 Diabetes.

BackgroundDespite the acknowledgement that LncRNA LINC-PINT may inhibit tumor cell invasion in human cancers, it is not yet determined when it comes to diabetes and its related complications.Material/MethodsThere were 244 patients with T2D and 126 healthy volunteers were admitted to People’s Hospital of Xinjiang Uygur Autonomous Region Hospital. Fasting blood (5 mL) was obtained from the patients and controls a day after admission. The diabetes patients’ fasting blood was extracted once every 6 months during follow-up. The total RNA was extracted and then used for detecting the expression of LINC-PINT.ResultsA comparison was made in this study, where LINC-PINT did not experience significant downregulation level in the majority of those suffering diabetes complications when in contrast to healthy controls, while LINC-PINT expression was found in diabetics. The follow-up study showed that LINC-PINT was downregulated in patients who developed cardiomyopathy and retinopathy or both but not in patients who developed other complications. Treatment with high glucose limited the extent of LINC-PINT expression in the ARPE-19 and AC16 cells. While the overexpression of LINC-PINT increased the viability of ARPE-19 and AC16 cells, siRNA-mediated silencing of LINC-PINT elicited the opposite effect.ConclusionsHence, we concluded that the overexpression of LINC-PINT may exhibit inhibitory effects on the progression of cardiomyopathy and retinopathy among patients with type 2 diabetes.

MicroRNA-411 plays a protective role in diabetic retinopathy through targeted regulating Robo4.

The aim of this study was to explore the role of microRNA-411 in diabetic retinopathy (DR) and to further understand its mechanism of action. A rat model of diabetes (20 in the DM group) and a normal control group (20 in the control group) was established. The changes in blood glucose and body weight were compared between the two groups. At the same time, the expression changes in microRNA-411 and Roundabout 4 (ROBO4) in the two groups of rats were detected. The biological prediction of the potential binding sites of ROBO4 and microRNA-411 was verified by the Dual-Luciferase reporter gene assay, and the regulatory relationship of microRNA-411 to ROBO4 was verified in human retinal pigment epithelial cells (ARPE-19). Meanwhile, we investigated the effects of high glucose and hypoxia on the viability of ARPE-19 cells and explored whether microRNA-411 has a regulatory role in the effects. Finally, a cell reverse experiment was designed to verify whether microRNA-411 functions through ROBO4. Compared with the NC group, the blood glucose of the DM group was significantly increased while the body weight was reduced. In addition, the expression level of microRNA-411 was markedly decreased in diabetic rats, and the mRNA and protein levels of ROBO4 were notably increased, which were all dependent on time. Biological prediction revealed that ROBO4 might be a potential target gene of microRNA-411, and the results of the Dual-Luciferase reporter gene assay confirmed that there is a binding relationship between them; meanwhile, microRNA-411 was proved to be able to inhibit the expression level of ROBO4 in vivo and in vitro. Both high glucose and hypoxia could inhibit the proliferation of ARPE-19 cells and increase the monolayer permeability. Additionally, up-regulation of microRNA-411 or down-regulation of ROBO4 could partially reverse this phenomenon. Cell reverse experiment showed that overexpression of ROBO4 partially reversed the protective effect of microRNA-411 on DR. MicroRNA-411 was down-regulated in the rat model of diabetic retinopathy and played a protective role in this disease, which might be achieved by negatively regulating the expression level of ROBO4.

Effects of microRNA‑217 on high glucose‑induced inflammation and apoptosis of human retinal pigment epithelial cells (ARPE‑19) and its underlying mechanism.

Diabetic retinopathy is a major complication of diabetes. Increasing evidence has indicated that microRNAs (miRs) serves an important role in diabetic retinopathy. However, the expression and mechanism of miR-217 in high glucose-induced human retinal pigment epithelial cells ARPE-19 is still unclear. Therefore, the aim of this study was to investigate the role of miR-217 in high glucose-induced retinal epithelial cell damage, and further to explore the molecular mechanisms. In our study, we found that compared with control group, miR-217 was upregulated in high glucose-induced ARPE-19 cells. In addition, TargetScan and a dual-luciferase reporter gene assay showed that Sirtuin 1 (SIRT1) was a direct target of miR-217. Then, we performed reverse transcription-quantitative polymerase chain reaction assay and western blot assay to explore the expression of SIRT1 in high glucose-induced ARPE-19 cells. Our results demonstrated that SIRT1 was downregulated at the mRNA and protein levels in high glucose-induced ARPE-19 cells. Then, ARPE-19 cells were transfected with inhibitor control, miR-217 inhibitor or miR-217 inhibitor + SIRT1-small interfering RNA for 6 h, and then the cells were treated with 50 mM D-glucose for 24 h. We then investigated the effects of miR-217 inhibitor on ARPE-19 cell viability and apoptosis. An MTT assay revealed that miR-217 inhibitor significantly increased the viability and decreased the apoptosis of high glucose-induced ARPE-19 cells. ELISA indicated that miR-217 inhibitor significantly reduced the expression of inflammatory factors, such as interleukin (IL)-1β, tumor necrosis factor-α, and IL-6 in high glucose-treated ARPE-19 cells. Additionally, a western blot assay demonstrated that miR-217 inhibitor suppressed the expression of p-p65. The effects of miR-217 inhibitor on high glucose-treated ARPE-19 cells were significantly reversed by the silencing the SIRT1 gene. Therefore, our findings suggested that miR-217 inhibitor protected against retinal epithelial cell damage caused by high glucose via targeting SIRT1, thereby playing a protective role in diabetic retinopathy. Targeting miR-217 may have therapeutic potential in the treatment of diabetic retinopathy.

CTRP3 inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells

Diabetic retinopathy (DR) is one of the most common diabetic complications and remains the leading cause of vision loss among adults. C1q/TNF-related protein 3 (CTRP3) is a member of CTRP family that has been found to be involved in the progression of diabetes mellitus and diabetic complications. However, the role of CTRP3 in DR has not been fully understood. In the present study, the results showed that CTRP3 expression was significantly decreased in DR patients compared with controls. In vitro investigations proved that overexpression of CTRP3 improved cell viability of ARPE-19 cells in response to high glucose (HG) stimulation. CTRP3 also attenuated HG-induced oxidative stress in ARPE-19 cells with decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and increased superoxide dismutase (SOD) activity. Apoptotic rate was significantly decreased in CTRP3 overexpressing ARPE-19 cells. Besides, bcl-2 expression was increased, while bax expression was decreased by CTRP3 overexpression. Moreover, overexpression of CTRP3 enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1) pathway in HG-stimulated ARPE-19 cells, and Nrf2 knockdown reversed CTRP3-mediated oxidative stress and apoptosis. These findings suggested that CTRP3 attenuated HG-stimulated oxidative stress and apoptosis in ARPE-19 cells, which were mediated by activation of Nrf2/HO-1 pathway.

An in vitro Assessment of Thermo-Reversible Gel Formulation Containing Sunitinib Nanoparticles for Neovascular Age-Related Macular Degeneration.

Anti-vascular endothelial growth factor agents have been widely used to treat several eye diseases including age-related macular degeneration (AMD). An approach to maximize the local concentration of drug at the target site and minimize systemic exposure is to be sought. Sunitinib malate, a multiple receptor tyrosine kinase inhibitor was encapsulated in poly(lactic-co-glycolic acid) nanoparticles to impart sustained release. The residence time in vitreal fluid was further increased by incorporating nanoparticles in thermo-reversible gel. Nanoparticles were characterized using TEM, DSC, FTIR, and in vitro drug release profile. The cytotoxicity of the formulation was assessed on ARPE-19 cells using the MTT assay. The cellular uptake, wound scratch assay, and VEGF expression levels were determined in in vitro settings. The optimized formulation had a particle size of 164.5nm and zeta potential of - 18.27mV. The entrapment efficiency of 72.0% ± 3.5% and percent drug loading of 9.1 ± 0.7% were achieved. The viability of ARPE-19 cells was greater than 90% for gel loaded, as such and blank nanoparticles at 10μM and 20μM concentration tested, whereas for drug solution viability was found to be 83% and 71% respectively at above concentration. The cell viability results suggest the compatibility of the developed formulation. Evaluation of cellular uptake, wound scratch assay, and VEGF expression levels for the developed formulations indicated that the formulation had higher uptake, superior anti-angiogenic potential, and prolonged inhibition of VEGF activity compared with drug solution. The results showed successful development of sunitinib-loaded nanoparticle-based thermo-reversible gel which may be used for the treatment of neovascular AMD.

Toxicity of blue led light and A2E is associated to mitochondrial dynamics impairment in ARPE-19 cells: implications for age-related macular degeneration.

Age-related macular degeneration (AMD) is a multifactorial retinal disease characterized by a progressive loss of central vision. Retinal pigment epithelium (RPE) degeneration is a critical event in AMD. It has been associated to A2E accumulation, which sensitizes RPE to blue light photodamage. Mitochondrial quality control mechanisms have evolved to ensure mitochondrial integrity and preserve cellular homeostasis. Particularly, mitochondrial dynamics involve the regulation of mitochondrial fission and fusion to preserve a healthy mitochondrial network. The present study aims to clarify the cellular and molecular mechanisms underlying photodamage-induced RPE cell death with particular focus on the involvement of defective mitochondrial dynamics. Light-emitting diodes irradiation (445 ± 18nm; 4.43mW/cm2) significantly reduced the viability of both unloaded and A2E-loaded human ARPE-19 cells and increased reactive oxygen species production. A2E along with blue light, triggered apoptosis measured by MC540/PI-flow cytometry and activated caspase-3. Blue light induced mitochondrial fusion/fission imbalance towards mitochondrial fragmentation in both non-loaded and A2E-loaded cells which correlated with the deregulation of mitochondria-shaping proteins level (OPA1, DRP1 and OMA1). To our knowledge, this is the first work reporting that photodamage causes mitochondrial dynamics deregulation in RPE cells. This process could possibly contribute to AMD pathology. Our findings suggest that the regulation of mitochondrial dynamics may be a valuable strategy for treating retinal degeneration diseases, such as AMD.

RETRACTED: ANRIL acts as onco-lncRNA by regulation of microRNA-24/c-Myc, MEK/ERK and Wnt/β-catenin pathway in retinoblastoma.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors. Current research findings in the authors' laboratory are in conflict with previous experimental results published in this paper. It was found that the data reported in figures 4 and 5 were not able to be replicated and so therefore the authors have decided that it is best for the scientific record to retract this paper. All the authors agree with the retraction. The authors apologise for any confusion this paper might have caused readers.

Scutellarin protects human retinal pigment epithelial cells against hydrogen peroxide (H2O2)-induced oxidative damage

BackgroundProliferative vitreoretinopathy (PVR) is a severe blinding complication of retinal detachment surgery. Increasing evidence demonstrate that PVR is associated with oxidative stress. Scutellarin is a natural flavone compound that has been reported to have anti-oxidative activity. However, the effect of scutellarin on PVR remains unknown. In the current study, we assessed the effect of scutellarin on hydrogen peroxide (H2O2)-induced oxidative injury in human retinal pigment epithelium cells (ARPE-19).MethodsARPE-19 cells were pretreated with different concentrations of scutellarin for 2 h, and then challenged with H2O2 (1 mM) for 24 h. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) and glutathione (GSH) activity were measured to assess the level of oxidative stress. Flow cytometry was performed to detect the apoptosis rate of ARPE-19 cells. Expression levels of bcl-2, bax, cleaved-caspase-3, p-JAK2, JAK2, p-STAT3, and STAT3 were measured using western blot.ResultsOur results revealed that scutellarin improved the cell viability of H2O2-induced ARPE-19 cells. Scutellarin alleviated the H2O2-induced oxidative stress in ARPE-19 cells, which was illustrated by reduced levels of ROS and MDA, accompanied by increased SOD activity and GSH level. The increased apoptosis rate of ARPE-19 cells caused by H2O2 induction was significantly decreased after scutellarin treatment. H2O2 treatment resulted in significant increase in bax expression and decrease in bcl-2 expression, while the changes in the expressions of bax and bcl-2 were reversed by scutellarin treatment. In addition, scutellarin promoted the activation of JAK2/STAT3 signaling pathway in H2O2-induced ARPE-19 cells. Suppression of JAK2/STAT3 signaling pathway abolished the protective effects of scutellarin on H2O2-induced ARPE-19 cells.ConclusionThese findings suggested that scutellarin was capable for alleviating H2O2-induced oxidative damage in ARPE-19 cells, which might be ascribed to the activation of JAK2/STAT3 signaling pathway.

An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells.

DMEM/F12 nutrient mixture, a recommended media for ARPE-19 culture, contains glucose concentration of 17.5mM. But, several recent studies employed normal glucose media (5.5mM) that was shown to affect the growth and function of ARPE-19 cells. Here, we set a protocol to study the effect of hyperglycemia on intracellular oxidative stress and redox status in ARPE-19 using DMEM/F12 as control. The WST-1 assay was performed to analyze the viability of ARPE-19 upon glucose treatment. The intracellular oxidative stress was measured by a dichlorofluorescein assay. The mitochondrial membrane potential (MMP) was monitored by using a JC-10 MMP assay kit. The expression of antioxidant marker proteins was analyzed by western blotting. Exogenous addition of glucose (7.5 and 12.5mM) for 24 and 48h did not change the viability and morphology of ARPE-19 cells. Hyperglycemia increased intracellular ROS level and decreased MMP in a dose-dependent manner. High-glucose treatment for 24h down-regulated the protein expression of redox-specific transcription factors Nrf-2, XBP-1 and NF-κB, and subsequently decreased the expression of HO-1, catalase, and SOD-2. This study offers baseline information for the subsequent use of DMEM/F12 nutrient mixture to study glucose-mediated changes in intracellular oxidative stress and redox status of ARPE-19 without affecting its basic functions.

Cyanidin-3-glucoside attenuates 4-hydroxynonenal- and visible light-induced retinal damage in vitro and in vivo.

4-Hydroxynonenal (HNE) is a highly reactive end-product of lipid peroxidation reaction that leads to retinal pigment epithelial (RPE) cell damage. Cyanidin-3-glucoside (C3G), the most abundant anthocyanin in the edible parts of plants, is a nutritional supplement used for preventing retinal damage. However, the protective effect of C3G against HNE-induced RPE cell damage remains to be elucidated. The protective mechanisms of C3G on ARPE-19 cells after HNE exposure were investigated in this study. Results showed that compared with HNE-treated cells, the viability of ARPE-19 cells was significantly (P < 0.05) increased after 1 and 5 μM C3G treatment. C3G exhibited a significant (P < 0.05) inhibitory effect on the expression of senescence-associated β-galactosidase in ARPE-19 cells. VEGF levels in the C3G groups were significantly (P < 0.05) decreased relative to those of the HNE-treated group. C3G also regulated the release of two inflammatory mediators, namely monocyte chemoattractant protein 1 and interleukine-8, in ARPE-19 cells after HNE treatment. Furthermore, C3G attenuated retinal cell apoptosis in pigmented rabbits induced by visible light. Therefore, our data showed that C3G has efficient protective effects on HNE-induced apoptosis, angiogenesis, and dysregulated cytokine production in ARPE-19 cells.

Mst1 facilitates hyperglycemia-induced retinal pigmented epithelial cell apoptosis by evoking mitochondrial stress and activating the Smad2 signaling pathway.

Hyperglycemia induces retinal pigmented epithelial cell apoptosis and mitochondrial stress via poorly understood mechanisms. The goal of our current study is to explore whether mammalian sterile 20-like kinase 1 (Mst1) is involved in the pathogenesis of hyperglycemia-mediated retinal pigmented epithelial cell apoptosis by triggering mitochondrial abnormalities and activating the Smad2 signaling pathway. Retinal pigmented epithelial ARPE-19 cells were presented with a high-glucose challenge in vitro. Cell viability and apoptosis were measured via western blotting, ELISAs, and immunofluorescence assays. Mitochondrial function was detected via JC-1 staining, mitochondrial ROS flow cytometry, western blotting, and ELISAs. Loss- and gain-of-function assays were performed via cell transfection and transduction with Mst1 siRNA and Smad2 adenovirus, respectively. The results indicated that hyperglycemia treatment upregulated the levels of Mst1, an effect that was accompanied by an increase in ARPE-19 cell apoptosis. Loss of Mst1 attenuated hyperglycemia-induced cell apoptosis, and this effect seemed to be associated with mitochondrial protection. In response to hyperglycemia stimulus, mitochondrial stress was noted in ARPE-19 cells, including mitochondrial ROS overproduction, mitochondrial respiratory metabolism dysfunction, mitochondrial fission/fusion imbalance, and mitochondrial apoptosis activation. Further, we provided evidence to support the crucial role played by Smad2 in promoting Mst1-mediated cell apoptosis and mitochondrial stress. Overexpression of Smad2 abrogated the beneficial effects of Mst1 deletion on ARPE-19 cell viability and mitochondrial protection. Altogether, our results identified Mst1 as a novel mediator controlling the fate of retinal pigmented epithelial cells and mitochondrial homeostasis via the Smad2 signaling pathway. Based on this finding, strategies to repress Mst1 upregulation and block Smad2 activation are vital to alleviate hyperglycemia-mediated retinal pigmented epithelial cell damage.

Long Non-Coding RNA of Myocardial Infarction Associated Transcript (LncRNA-MIAT) Promotes Diabetic Retinopathy by Upregulating Transforming Growth Factor-β1 (TGF-β1) Signaling.

BackgroundLong non-coding RNA of myocardial infarction associated transcript (lncRNA-MIAT) has a reported role in microvascular dysfunction. This study aimed to investigate the role of lncRNA-MIAT and its effects on transforming growth factor-β1 (TGF-β1) signaling in patients with diabetic retinopathy and in ARPE-19 adult retinal pigment epithelial cells in vitro.Material/MethodsStudy participants provided plasma samples and included patients with non-proliferative diabetic retinopathy (n=52), patients with diabetes without diabetic retinopathy (n=63), and healthy controls (n=56). Plasma levels of lncRNA-MIAT and TGF-β1 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Pearson correlation analysis was performed on the plasma data, and the diagnostic relevance of plasma levels of lncRNA-MIAT for diabetic retinopathy was evaluated by receiver operating characteristic (ROC) curve analysis. Cells of the human retinal pigment epithelial cell line, ARPE-19, were cultured in high glucose with construction and transfection of a MIAT expression plasmid vector. Viability of ARPE-19 cells was detected by the MTT assay and Western blot measured the expression levels of TGF-β1.ResultsPlasma levels of lncRNA-MIAT were significantly increased in patients with diabetic retinopathy compared with patients with diabetes without diabetic retinopathy and with healthy controls. ARPE-19 cells cultured in a high glucose environment showed reduced cell viability and upregulation of lncRNA-MIAT expression.ConclusionsIncreased plasma levels of lncRNA-MIAT were significantly associated with the presence of diabetic retinopathy, and increased expression of lncRNA-MIAT reduced the viability of ARPE-19 cells in vitro by upregulating TGF-β1 signaling.

Essential Role of mTOR Signaling in Human Retinal Pigment Epithelial Cell Regeneration After Laser Photocoagulation.

This study assessed the role of mechanistic target of rapamycin (mTOR) pathway in the human adult retinal pigment epithelial (ARPE) cell response after laser photocoagulation (LP). The effect of mTOR inhibition on ARPE-19 cell was investigated by rapamycin treatment after LP. Cell viability and proliferation were explored using MTT and EdU assays, respectively. The expression of mTOR-related proteins and epithelial-mesenchymal transition (EMT) markers was verified by Western blot. Rapamycin retarded the LP area recovery in a dose-dependent manner by the 120h, while LP+DMSO vehicle-treated cells completely restored the lesion zone (P ≤ 0.01). ARPE-19 cell viability is significantly lower in LP + rapamycin 80 and 160ng/ml treated cultures compared to LP control at 120h (P ≤ 0.001). LP control group demonstrated significantly more proliferative cells compared to untreated cells at the 72 and 120h, whereas EdU-positive cell numbers in cultures treated with rapamycin at concentrations of 80 and 160ng/ml were similar to baseline values (P ≤ 0.01). mTOR pathway activation is essential for regulation of the RPE cell migration and proliferation after LP. mTOR inhibition with rapamycin effectively blocks the migration and proliferation of the RPE cells. Our results demonstrate that mTOR has an important role in ARPE-19 cell as a regulator of cell behavior under stress conditions, suggesting that mTOR could be a promising therapeutic target for numerous retinal diseases.

Photoreceptor Cell-Derived CAPN5 Regulates Retinal Pigment Epithelium Cell Proliferation Through Direct Regulation of SLIT2 Cleavage.

To identify the causative gene and investigate the corresponding mechanisms for an autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) family. Clinical examination and genetic analysis were performed in a Chinese ADNIV family. To dissect the molecular consequence, we used gene targeting to knock-in a patient's specific mutation in the mouse genome. Immunostaining and immunoprecipitation were harnessed to analyze the colocalization and interaction of CAPN5 with SLIT2 in photoreceptors. The purified SLIT2-N, SLIT2-C fragments, and the conditioned medium from 661W cells with the overexpression of CAPN5 were treated on ARPE-19 cells. The viability of ARPE-19 cells was determined by MTT assays. The activation of protein kinase A (PKA) was analyzed by immunofluorescence and Western blotting in 661W and ARPE-19 cells as well as in frozen retina tissue from wildtype (WT) and knock-in mice. We identified a novel CAPN5 mutation (p.R289W) in a Chinese family and generated the knock-in CAPN5R289W mouse. This mutation caused abnormal proliferative RPE in both humans and mice. CAPN5 directly cleaved WT SLIT2 in vitro, but not the mutant SLIT2 (p.R1113I). CAPN5 interacted with the SLIT2 in mouse retinal photoreceptors (661W cells) and increased cleavage and secretion of the SLIT2 fragments (SLIT2-N and SLIT2-C). Conditioned medium induced higher levels of secreted SLIT2 fragments, which promoted PKA activation and promoted proliferation of ARPE-19 cells. The novel CAPN5 mutation (p.R289W) is responsible for the present ADNIV family. The mutant CAPN5 stimulated secretion and cleavage of SLIT2 fragments that may act as a bystander to regulate abnormal RPE cell proliferation for ADNIV.

Acute retinal toxicity associated with a mixture of perfluorooctane and perfluorohexyloctane: failure of another indirect cytotoxicity analysis

AimsTo report new information related to acute retinal toxicity of Bio Octane Plus, a mixture of 90% perfluorooctane (PFO) and 10% perfluorohexyloctane.MethodsThis retrospective, descriptive case series reports the occurrence of...

Gac fruit extracts ameliorate proliferation and modulate angiogenic markers of human retinal pigment epithelial cells under high glucose conditions

Objective: To investigate the impact of the extracts of Gac fruit parts (peel, pulp, seed, and aril) on the cell viability and angiogenesis markers of human retinal pigment epithelial (ARPE- 19) cells under high glucose conditions. Methods: The effect of the extracts of Gac fruit peel, pulp, seed and aril on the ARPE-19 cells was determined using MTT viability assay, Trypan blue dye and morphological changes were observed using light microscopy. Enzyme-linked immunosorbent-based assay was performed to evaluate the effect of Gac fruit parts on the reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and pigmented epithelium-derived factor (PEDF) secretions. Results: High glucose (HG) at 30 mmol/L increased ARPE-19 cell viability and ROS and VEGF secretions. While, the exposure of ARPE- 19 cells in high glucose condition to Gac fruit extracts led to inhibition of cell viability, induced morphological changes, decreased ROS and VEGF secretions, and increased PEDF level. Gac pulp, seed, and aril at 1 000 μg/mL showed significant inhibition activities [(7.5 ± 5.1)%, (2.7 ± 0.5)%, (3.2 ± 1.1)%, respectively] against HG-induced ARPE-19 cell viability. The findings also demonstrated that Gac aril at 250 μg/mL significantly decreased ROS and VEGF levels [(40.6 ± 3.3) pg/mL, (107.4 ± 48.3) pg/mL, respectively] compared to ROS [(71.7 ± 2.9) pg/ mL] and VEGF [(606.9 ± 81.1) pg/mL] in HG untreated cells. Moreover, 250 μg/mL of Gac peel dramatically increased PEDF level [(18.2 ± 0.3) ng/mL] compared to that in HG untreated cells [(0.48 ± 0.39) ng/mL]. Conclusions: This study indicates that the extracts of Gac peel, pulp, seed and aril reduced cell viability, minimized ROS generations and showed angiogenic activities. Therefore, our findings open new insights into the potentiality of Gac fruit against HG-related diabetic retinopathy disease.

Protective Effects of 17β-Estradiol on Benzo(e) pyrene[B(e)P]-induced Toxicity in ARPE-19 cells.

Purpose:The aim of this study was to examine the effect of 17β-estradiol on Benzo(e)pyrene [B(e)P]-induced toxicity in ARPE-19 cells.Methods:We pretreated ARPE-19 cells with 20 nM and 40 nM 17β-estradiol for 6 hours, followed by addition of 300 μM B(e)P for additional 24 hours. Cell viability was measured using Trypan blue dye-exclusion assay. JC-1 assay was performed to measure mitochondrial membrane potential (ΔΨm). For a quantitative estimation of cell death, apoptotic markers such as caspase-3/7, caspase-9, and caspase-12 were measured.Results:Our results demonstrated that when treated with B(e)P, the viability and ΔΨm of ARPE-19 cells declined by 25% and 63%, respectively (P < 0.05). However, pretreating with 17β-estradiol increased the viability of ARPE-19 cells by 21% (20 nM) and 10% (40 nM) (P < 0.05). Furthermore, the significantly reduced ΔΨm in βE+B(e)P treated cells ARPE-19 cells was restored by pre-treatment with 17β-estradiol- ΔΨm was increased by 177% (20 nM) and 158% (40 nM) (P < 0.05). We further observed a significant up-regulation in the activity of Caspases-3/7, -9, and -12 in B(e)P-treated ARPE-19 cells. However, 17β-estradiol treatment significantly reduced the activity of all apoptotic markers (P < 0.05).Conclusion:In conclusion, our results demonstrate that 17β-estradiol protects ARPE-19 cells against B(e)P-induced toxicity by decreasing apoptosis, preventing cell death, and restoring mitochondrial membrane potential.

Pigment Epithelium-derived Factor Protects Retinal Pigment Epithelial Cells Against Cytotoxicity "In Vitro".

Oxidative stress has been implicated in neurodegenerative diseases, such as age-related macular degeneration. Hydrogen peroxide and sodium iodate can mediate oxidative injury. Sodium iodate induces a selective retinal degeneration targeting the RPE. We describe a method of chronic sodium iodate-mediated injury on RPE cells that may serve to evaluate protective factors against oxidative stress. Cytotoxicity and cell viability curves of ARPE-19 cells with sodium iodate were generated. The antioxidant pigment epithelium-derived factor decreased sodium iodate-mediated cytotoxicity without affecting ARPE-19 cell viability. A cell culture system to evaluate protection against oxidative stress injury with PEDF is discussed.