TGF-ß Isoforms and Local Environments Greatly Modulate Biological Nature of Human Retinal Pigment Epithelium Cells.

To characterize transforming growth factor-ß (TGF-ß) isoform (TGF-ß1~3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including collagen 1 (COL1), COL4, and COL6; α-smooth muscle actin (αSMA); hypoxia-inducible factor 1α (HIF1α); and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α), a master regulator for mitochondrial respiration;, tight junction-related molecules, Zonula occludens-1 (ZO1) and E-cadherin; and vascular endothelial growth factor (VEGF); (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-ß isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-ß1 and TGF-ß3 caused a marked increase in TEER values, and TGF-ß2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-ß isoforms, among which TGF-ß1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-ß2 and TGF-ß3 or by TGF-ß1 and TGF-ß3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-ß isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-ß-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-ß isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.

Metabolic Imaging and Molecular Biology Reveal the Interplay between Lipid Metabolism and DHA-Induced Modulation of Redox Homeostasis in RPE Cells.

Diabetes-induced oxidative stress induces the development of vascular complications, which are significant causes of morbidity and mortality in diabetic patients. Among these, diabetic retinopathy (DR) is often caused by functional changes in the blood-retinal barrier (BRB) due to harmful oxidative stress events in lipids, proteins, and DNA. Docosahexaenoic acid (DHA) has a potential therapeutic effect against hyperglycemia-induced oxidative damage and apoptotic pathways in the main constituents of BRB, retinal pigment epithelium cells (ARPE-19). Effective antioxidant response elicited by DHA is driven by the activation of the Nrf2/Nqo1 signaling cascade, which leads to the formation of NADH, a reductive agent found in the cytoplasm. Nrf2 also induces the expression of genes encoding enzymes involved in lipid metabolism. This study, therefore, aims at investigating the modulation of lipid metabolism induced by high-glucose (HG) on ARPE-19 cells through the integration of metabolic imaging and molecular biology to provide a comprehensive functional and molecular characterization of the mechanisms activated in the disease, as well the therapeutic role of DHA. This study shows that HG augments RPE metabolic processes by enhancing lipid metabolism, from fatty acid uptake and turnover to lipid biosynthesis and ß-oxidation. DHA exerts its beneficial effect by ameliorating lipid metabolism and reducing the increased ROS production under HG conditions. This investigation may provide novel insight for formulating novel treatments for DR by targeting lipid metabolism pathways.

Investigation of DHA-Induced Regulation of Redox Homeostasis in Retinal Pigment Epithelium Cells through the Combination of Metabolic Imaging and Molecular Biology.

Diabetes-induced oxidative stress leads to the onset of vascular complications, which are major causes of disability and death in diabetic patients. Among these, diabetic retinopathy (DR) often arises from functional alterations of the blood-retinal barrier (BRB) due to damaging oxidative stress reactions in lipids, proteins, and DNA. This study aimed to investigate the impact of the ω3-polyunsaturated docosahexaenoic acid (DHA) on the regulation of redox homeostasis in the human retinal pigment epithelial (RPE) cell line (ARPE-19) under hyperglycemic-like conditions. The present results show that the treatment with DHA under high-glucose conditions activated erythroid 2-related factor Nrf2, which orchestrates the activation of cellular antioxidant pathways and ultimately inhibits apoptosis. This process was accompanied by a marked increase in the expression of NADH (Nicotinamide Adenine Dinucleotide plus Hydrogen) Quinone Oxidoreductase 1 (Nqo1), which is correlated with a contextual modulation and intracellular re-organization of the NAD+/NADH redox balance. This investigation of the mechanisms underlying the impairment induced by high levels of glucose on redox homeostasis of the BRB and the subsequent recovery provided by DHA provides both a powerful indicator for the detection of RPE cell impairment as well as a potential metabolic therapeutic target for the early intervention in its treatment.

Identification of Age-associated Proteins and Functional Alterations in Human Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) has essential functions, such as nourishing and supporting the neural retina, and is of vital importance in the pathogenesis of age-related retinal degeneration. However, the exact molecular changes of RPE during aging remain poorly understood. Here, we isolated human primary RPE (hRPE) cells from 18 eye donors distributed over a wide age range (10-67 years old). A quantitative proteomic analysis was performed to analyze changes in their intracellular and secreted proteins. Age-group related subtypes and age-associated proteins were revealed and potential age-associated mechanisms were validated in ARPE-19 and hRPE cells. The results of proteomic data analysis and verifications suggest that RNF123- and RNF149-related protein ubiquitination plays an important role in protecting hRPE cells from oxidative damage during aging. In older hRPE cells, apoptotic signaling-related pathways were up-regulated, and endoplasmic reticulum organization was down-regulated both in the intracellular and secreted proteomes. Our work paints a detailed molecular picture of hRPE cells during the aging process and provides new insights into the molecular characteristics of RPE during aging and under other related clinical retinal conditions.

Hypoxia Differently Affects TGF-ß2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells.

The hypoxia associated with the transforming growth factor-ß2 (TGF-ß2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O2 gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-ß2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-ß2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-ß2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-ß2 increased proton leakage. The findings reported herein indicate that the TGF-ß2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.

Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476 (4-(4-(2,3-dihydrobenzo [1,4] dioxin-6-yl)-5-pyridin-2-yl-1H-imidazole-2-yl) benzamide), a specific casein kinase 1 inhibitor, alleviate CQ-induced retinopathy in adult retinal pigment epithelial (ARPE-19) cells. Cultured ARPE-19 cells were exposed to CQ with or without D4476 and cell death was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To examine autophagy flux, ARPE-19 cells were transfected with green fluorescence protein light chain 3 (GFP-LC3)-red fluorescence protein (RFP)-LC3ΔG plasmid DNA and co-stained with the lysosomal-associated membrane protein (LAMP)-1 antibody. Western blotting and fluorescence-activated cell sorting (FACS) showed apoptosis, whereas the fluorescence intensity of 2'-7'-dichlorofluorescein diacetate revealed levels of cellular oxidative stress. We then confirmed the effect of D4476 on the interaction between Beclin 1 and B-cell lymphoma-2 (Bcl-2) through immunoprecipitation with an anti-Bcl-2 antibody. Following CQ exposure, ARPE-19 cells accumulated autophagosomes because of defective lysosomal degradation. Furthermore, CQ trapped Beclin 1 with Bcl-2, disturbing autophagy initiation and autolysosome formation. However, D4476 alleviated CQ-induced effects by rescuing ARPE-19 cells from CQ-induced toxicity by modulating the association between Beclin 1 and Bcl-2. Therefore, D4476 controls autophagy and apoptosis simultaneously by upregulating autophagy flux, decreasing ROS formation, and triggering the expression of anti-apoptotic proteins through inhibition of mTOR, JNK, and p38 MAPK signals. We conclude that D4476 is a promising treatment strategy for CQ-mediated retinopathy.

Ameliorating hydroxychloroquine induced retinal toxicity through cerium oxide nanoparticle treatments.

The present study investigated the potential protective effects of cerium oxide nanoparticles (CNP) on human retinal pigment epithelium (ARPE-19) cells damaged by hydroxychloroquine (HCQ). Toxicity of HCQ on the ARPE-19 cells was explored with a dose response trial. CNP rescue both a pre-treatment protocol, where CNP were applied 24 hours prior to HCQ application and a simultaneous treatment protocol where both CNP and HCQ were applied together, were used. In the dose response trial, 250 µM HCQ showed 51.84% cell viability after 24 hours and 32.75% after 48 hours time period. This was selected as model HCQ dose for rescue trials. The simultaneous treatment trials did not show a significant increase in viability compared to model toxic dose. The CNP pre-treatment trials showed a significant increase in cellular viability compared to model toxic dose with 68.03% ± 3.27 viability (p = 4.56E-05) at 24 hours and 51.85% ± 4.96 (p = 1.18E-05) at 48 hours time period. CNP pre-treatment showed significant protection of cells from HCQ induced toxicity. The difference in efficacy of simultaneous and pre-treatment is hypothesized to lie in the cellular localization of CNP. Furthermore, including the reactive oxygen species (ROS) scavenging properties of CNP seems to be responsible for protection, the effect of CNP on autophagosome and lysosome colocalization are also hypothesized to play a significant role.

Study on the apoptosis of human retinal pigment epithelium cells induced by EGCG / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the effect of epigallocatechin gallate(EGCG)on the apoptosis of human retinal pigment epithelium(ARPE-19)cells and its mechanism. METHODS:The ARPE-19 cells were cultured in vitro and treated with 0,40,80 and 160 μg/mL EGCG, respectively. At the proposed time of treatment the morphological changes were detected by hoechst 33258 staining. The apoptosis rate was detected by flow cytometry. The expression of apoptosis-related factors B lymphocytoma-2 gene(bcl-2), BCL2-Associated X protein(Bax),caspase-3 and p53 were detected by quantitative RT-PCR and Western blotting.RESULTS: Hoechst 33258 staining showed that the ARPE-19 cells with the increase of EGCG drug concentration, the number of apoptotic cells gradually increased and the apoptotic bodies were observed. Flow cytometry showed that the apoptosis rate increased gradually with the increase of EGCG drug concentration. The apoptosis rates at 40, 80 and 160 μg/mL were 4.95%±0.071%, 11.75%±0.075% and 21.25%±0.919% respectively, which was significantly different compared with the control group(2.8%±1.556%)(P<0.01), presented with a drug concentration-dependent. The results of quantitative PCR and Western blotting showed that EGCG could significantly up-regulate the expression of apoptosis-promoting factors Bax, caspase-3 and the mRNA and protein expression of p53, and down-regulate the apoptosis-inhibiting factor bcl-2, all of these showed concentration-dependent effects.CONCLUSION:EGCG can obviously induce the apoptosis of ARPE-19 cells. The mechanism is related with the inhibition of bcl-2 and increase the expression of Bax, caspase-3 and p53.

Long-Term, Noninvasive In Vivo Tracking of Progenitor Cells Using Multimodality Photoacoustic, Optical Coherence Tomography, and Fluorescence Imaging.

Stem cell regenerative medicine therapies have emerged as promising treatments for currently incurable diseases. A remaining challenge for cell therapies is the ability to track the migration and distribution of the transplanted cells in a long-term, noninvasive manner in vivo to assess their efficacy. This study develops a noninvasive, and high spatial resolution photoacoustic microscopy (PAM) and optical coherence tomography (OCT) imaging system for in vivo tracking of subretinally injected progenitor human retinal pigment epithelium cells (ARPE-19) labeled with chainlike gold nanoparticle (CGNP) clusters in RPE damage. CGNP provided significant PAM, OCT, and fluorescence signals to selectively track the migration of ARPE-19 cells in living rabbit eyes for 3 months. PAM and OCT imaging allow accurate anatomical information to determine the exact retinal layer in which the transplanted ARPE-19 cells are located which was confirmed by histology. This presents an efficient and advanced technology to visualize fundamental biological processes of cell therapies in complex in vivo environments in real time.

Protective Effect of Quercetin on Sodium Iodate-Induced Retinal Apoptosis through the Reactive Oxygen Species-Mediated Mitochondrion-Dependent Pathway.

Age-related macular degeneration (AMD) leads to gradual central vision loss and is the third leading cause of irreversible blindness worldwide. The underlying mechanisms for this progressive neurodegenerative disease remain unclear and there is currently no preventive treatment for dry AMD. Sodium iodate (NaIO3) has been reported to induce AMD-like retinal pathology in mice. We established a mouse model for AMD to evaluate the effects of quercetin on NaIO3-induced retinal apoptosis, and to investigate the pertinent underlying mechanisms. Our in vitro results indicated that quercetin protected human retinal pigment epithelium (ARPE-19) cells from NaIO3-induced apoptosis by inhibiting reactive oxygen species production and loss of mitochondrial membrane potential as detected by Annexin V-FITC/PI flow cytometry. We also evaluated the relative expression of proteins in the apoptosis pathway. Quercetin downregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP and upregulated the expression of Bcl-2 through reduced PI3K and pAKT expressions. Furthermore, our in vivo results indicated that quercetin improved retinal deformation and increased the thickness of both the outer nuclear layer and inner nuclear layer, whereas the expression of caspase-3 was inhibited. Taken together, these results demonstrate that quercetin could protect retinal pigment epithelium and the retina from NaIO3-induced cell apoptosis via reactive oxygen species-mediated mitochondrial dysfunction, involving the PI3K/AKT signaling pathway. This suggests that quercetin has the potential to prevent and delay AMD and other retinal diseases involving NaIO3-mediated apoptosis.

Involvement of endoplasmic reticulum stress response and IRE1-mediated ASK1/JNK/Mcl-1 pathways in silver nanoparticle-induced apoptosis of human retinal pigment epithelial cells.

Silver nanoparticles (AgNPs) have cytotoxic effects on various human cell types. The endoplasmic reticulum (ER) is very sensitive to cytotoxic damage. Retina tissue is easily affected by internal and external stimuli. However, the effect of AgNPs on human retinal cells is not known. This study examined the effect of AgNPs on ER stress induction and their mechanism of action in human retinal pigment epithelium (RPE) ARPE-19 cells. We found that AgNPs significantly increased ARPE-19 cell cytotoxicity and stimulated caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage, as well as mitochondrial membrane potential (MMP) depolarization, in ARPE-19 cells in a dose-dependent manner (0.2-5 µg/mL for 18 h). AgNPs (5 µg/mL for 18 h) induced several signature ER stress markers, as indicated by the upregulated expressions of CCAAT/enhancer-binding protein-homologous protein (CHOP), phosphorylated protein kinase RNA-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), and inositol-requiring protein 1 (IRE1), and cleaved activating transcription factor 6 (ATF6). AgNPs also activated ASK1 and JNK in ARPE-19 cells, and induced increases in Bax and Puma expressions, as well as a decrease in Mcl-1 expression. However, inhibition of the ER stress response by pretreatment with 4-PBA included apparently and dose-dependently reduced levels of p-PERK, p-IRE1, CHOP, cleaved ATF6, p-ASK1, p-JNK, cleaved caspase-3, procaspase-12, and MMP depolarization in AgNP-treated ARPE-19 cells; it also led to significantly increased Mcl-1 protein levels in a dose-dependent manner in ARPE-19 cells. Pretreatment with JNK inhibitor SP600125 significantly attenuated caspase-3 cleavage and MMP depolarization and increased Mcl-1 protein levels in AgNPs-treated ARPE-19 cells in a dose-dependent manner. Hence, our study demonstrated that AgNPs induced apoptosis in human RPE ARPE-19 cells by ER stress response and ER stress-dependent mitochondrial apoptosis via the IRE1/ASK1/JNK/Mcl-1 pathways.

Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy.

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.

Disease-causing mutations associated with bestrophinopathies promote apoptosis in retinal pigment epithelium cells.

PURPOSE: Best vitelliform macular dystrophy (BVMD) and autosomal recessive bestrophinopathy (ARB) are two kinds of bestrophinopathies which are caused by BEST1 mutations and characterized by accumulation of lipofuscin-like materials on the retinal pigment epithelium cell-photoreceptor interface. In the past two decades, research about the pathogenesis of bestrophinopathies was mainly focused on the anion channel and intracellular Ca2+ signaling, but seldom concentrated on the function of retinal pigment epithelium (RPE) cells. In this study, we explored the possible effect of the three BEST1 mutations p.V143F, p.S142G, and p.A146T on the apoptosis in human fetal RPE cells. METHODS: Wild-type plasmid and mutant plasmids BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T were transfected to human fetal RPE cells. The molecules caspase-3, phospho-Bcl-2, BAX, PARP, and AIF associated with apoptosis were determined by quantitative PCR and Western blot. Apoptotic rate and active Caspase-3 staining were analyzed by flow cytometry. RESULTS: Caspase-3 and PARP expression were significantly increased in BEST1-pcDNA3.1 p.S142G and p.A146T group. Flow cytometry showed that the apoptosis rates were significantly increased in the BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T group compared with the wild-type group. CONCLUSIONS: For the first time, we found that the three mutations promoted RPE cell apoptosis. Furthermore, the results indicated that BEST1 mutations p.S142G and p.A146T may contribute apoptosis of RPE cells by targeting Caspase 3. Our observations suggested that the apoptosis of RPE cells may be one of the mechanisms in bestrophinopathies, which may provide a new potential therapeutic target for the treatment of this disease.

Silver Nanoparticle-Induced Apoptosis in ARPE-19 Cells Is Inhibited by Toxoplasma gondii Pre-Infection Through Suppression of NOX4-Dependent ROS Generation.

PURPOSE: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity. MATERIALS AND METHODS: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4-/ - mice. RESULTS: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed. CONCLUSION: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.

Glucose-induced microRNA-218 suppresses the proliferation and promotes the apoptosis of human retinal pigment epithelium cells by targeting RUNX2.

OBJECTIVE: MicroRNA-218 (miR-218) critical for preventing the progression of numerous diseases, including diseases of the retinal pigment epithelium (RPE). However, the mechanism by which miR-218 regulates the PRE in humans remains largely unknown. Our study investigated the effects of glucose-induced miR-218 expression on human RPE cells (ARPE-19), as well as its targeted regulatory effect. METHODS: The levels of miR-218 and runt-related transcription factor 2 (RUNX2) expression were investigated by RT-qPCR or Western blot assays. Cell viability and apoptosis were assessed by CCK-8 assays, flow cytometry, and Hoechst staining. A luciferase reporter assay was performed to determine whether Runx2 is a target gene of miR-218. RESULTS: Our results showed that glucose up-regulated miR-218 expression, suppressed proliferation, and induced the apoptosis of ARPE-19 cells. We verified that miR-218 could inhibit the proliferation and facilitate the apoptosis of ARPE-19 cells, while inhibition of miR-218 expression produced the opposite effects. In terms of mechanism, we demonstrated that RUNX2 was a direct target of miR-218. Functional experiments showed that Runx2 served as a miR-218 target to help inhibit the proliferation and induction of apoptosis in ARPE-19 cells. CONCLUSION: Our findings suggest the miR-218/Runx2 axis as a potential target for treating diabetic retinopathy (DR).

Exenatide modulates expression of metalloproteinases and their tissue inhibitors in TNF-α stimulated human retinal pigment epithelial cells.

BACKGROUND: Diabetic retinopathy (DR) is one of the most common complications of diabetes and the leading cause of acquired blindness in adults. In diabetic patients hyperglycemia induces complex metabolic abnormalities affecting retinal homeostasis, and promotes retinal inflammation and angiogenesis. Incretin mimetic drugs such exenatide, are a relatively new group of drugs used in the treatment of diabetes. We investigated the potential direct effects of exenatide on human retinal pigment epithelium (HRPE). METHODS: cAMP production was measured after stimulation of HRPE cells with GLP-1 and exenatide. Intracellular signaling pathways were also examined. HRPE cells were stimulated with TNF-α and subsequently incubated with exenatide. The concentration of metalloproteinases, MMP-1, MMP-2 and MMP-9, and tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and TIMP-3 were evaluated. Viability, cytotoxicity and caspase 3/7 activation were determined. Activity of dipeptidyl peptidase-4 (DPP-4), an enzyme involved in GLP-1 inactivation, was also determined. RESULTS: Both GLP-1 and exenatide stimulation in HRPE cells caused no effect in cAMP levels suggesting alternative signaling pathways. Signaling pathway analysis showed that exenatide reduced phosphorylation of Akt-Ser473, PRAS40, SAPK/JNK, Bad, and S6 proteins but not Akt-Thr308. Exenatide also decreased MMP-1, MMP-9, and TIMP-2 protein levels whereas MMP-2 level in HRPE cells was increased. Finally, we show that exenatide decreased the activity of DPP-4 in TNF-α stimulated HRPE cells. CONCLUSIONS: These findings indicate that exenatide modulates regulation of extracellular matrix components involved in retinal remodeling.

Role of mitochondrial DNA in human retinal pigment epithelium cells apoptosis induced by blue light / 中华实验眼科杂志

Objective To research the role of mitochondrial DNA mediate the cultured human retinal pigment epithelium (hRPE) cell apoptosis induced by blue light and the relationship with time.Methods Established the blue light damage model of cultured hRPE cells in vitro with light emitting diode (LED) blue light density of (4.0-±0.5)mW/cm2 adjusted by FL-1D blue light illumination meter,and the illumination time was set as 0,0.5,1,2,4,6,12 and 24 hours,then the cells were grouped according to the illumination time.Immunofluorescence were used to identify the cells;the expressions of caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,bax and bcl-2 were detected with Western blot.Quantitative PCR was used to detect the copy number of mitochondrial DNA and PCR was used to detect mitochondrial DNA 4977bp common deletion.Results Immunofluorescence results showed that the RPE65 protein was expressed in the cytoplasm.The expressions of bax were upregulated after illumination for 1 hour,cleaved caspase-3 were upregulated after illumination for 2 hours,caspase-3,caspase-9,cleaved caspase-9 were upregulated after illumination for 4 hours,while the expression of bcl-2 was downregulated after illuminated for 2 hours,with significant differences compared to the normal control group (all at P＜0.05).The copy number of mitochondrial DNA in 0.5,1,2,4,6,12 and 24 hours groups was downregulated,with significant differences compared to the normal control group (all at P＜0.05).The expressions of 4977bp common deletion in 0.5,1,2,4,6,12 and 24 hours groups were increased,with significant differences compared with the normal control group (all at P＜0.05).Conclusions Blue light can cause cell apoptosis,especially mitochondrial apoptosis,in hRPE probably motivated by mitochondrial DNA damage.

Protection of Mcc950 against H2O2-induced inflammatory injury in ARPE-19 cells / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To research the protection of Mcc950, the inhibitor of NLRP3, against the inflammatory injury to human retinal pigment epithelium cell line ARPE-19. <p>METHODS: Cultured cell line ARPE-19 was divided into control group, H₂O₂ treating group, Mcc950 treating group and Mcc950+H₂O₂ treating group. Different concentrations of H₂O₂ and Mcc950 were used to treat the cells. Cell activity was detected by using CKK8 and proper experimental concentration of H₂O₂ and Mcc950 were determined. After the treatment, the concentration of IL-1β were detected by using ELISA. The change of NLRP3 related proteins were detected by Western blot. And cell apoptosis was examined by TUNEL stain. <p>RESULTS: Cell ability was gradually decreased along with the increasing treating concentrations of H₂O₂. Cell ability showed statistical difference when the concentration of H₂O₂ arrived 400μmol/L. With the concentration of 0.1 and 1μmol/L, Mcc950 had no effect on cell ability. So we chose 400μmol/L H₂O₂ and 1μmol/L Mcc950 as the experimental concentrations. Compared with the normal control group, the cell viability in the H₂O₂ treating group was significantly reduced, the IL-1β in the supernatant was significantly increased, and the apoptosis rate was significantly increased, with statistically significant differences(<i>P</i><0.05). In Mcc950+H₂O₂ treating group, cell viability was significantly increased, the IL-1β in the supernatant and the apoptosis rate were significantly decreased(<i>P</i><0.05). By Western blot, after treated with 400μmol/L H₂O₂, the IL-1β, NLRP3, pro-caspase1 and caspase1 were obviously increased compared to control group. After treated with Mcc950, the NLRP3 and pro-caspase1 still were at high level, the expression of caspase1 was suppressed, which indicated that Mcc950 effectively inhibited the activation of NLRP3 inflammasome consequently disturbed the formation of caspase1.<p>CONCLUSION: Mcc950 can inhibits the function of NLRP3, leading to increasing of the cell ability and decreasing of the cell apoptosis.

Protective effect of Ghrelin against oxidative stress induced by high glucose in human retinal pigment epithelium cells / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To investigate the effect of Ghrelin on oxidative stress induced by high glucose in human retinal pigment epithelium (RPE) cells. ·METHODS: RPE cells were cultured and divided into the negative control group, high sugar group, Ghrelin low dose group ( 10-9mol/L ) and high dose group (10-6mol/L). Cells survival rate were detected by CCK-8 colorimetry, cells oxidative damage were observed by oxygen sensitive fluorescence probe H2DCFDA staning, changes of intracellular reactive oxygen species ( ROS ) were detected by H2DCFDA staining, super oxide dismutase ( SOD) activity and malondialdehyde ( MDA) content were detected by spectrophotometer colorimetry. ·RESULTS: CCK-8 results showed that RPE cells survival rate increased to 54.79%±3.43% and 79.16%±3.29% after treated with 10-9mol/L, 10-6mol/L Ghrelin, the difference was statistically significant compared with high glucose group (41.65%±3.42%)(P<0.05). H2DCFDA fluorescent probe dying showed that Ghrelin reduced ROS generation in RPE cells and decreased oxidative damage cells. Spectrophotometer colorimetric method showed that according to the high sugar group, SOD activity increased and MDA content decreased in Ghrelin group. ·CONCLUSION: Ghrelin could inhibit high glucose - induced oxidative damage in human RPE cells, which has protective effect on the process of the occurrence and development of diabetic retinopathy.

Design, development and characterization of synthetic Bruch's membranes.

Age-related macular degeneration (AMD) is a leading cause of blindness, and dry AMD has no effective treatment. Retinal constructs comprising retinal pigment epithelium (RPE) cells supported by electrospun scaffolds have been investigated to treat dry AMD. However, electrospun scaffolds studied to-date do not mimic the structural microenvironment of human Bruch's membrane (BM), essential for native-like RPE monolayers. The aim of this study was to develop a structurally biomimetic scaffold designed to support a functional RPE monolayer, comprising porous, electrospun nanofibrous membranes (ENMs), coated with laminin, mimicking the inner collagenous layer (ICL) and basal RPE lamina respectively, the cell supporting layers of the BM. In vitro evaluation showed 70nm PLLA ENMs adsorbed high amounts of laminin and supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. 70nm PLLA ENMs were successfully implanted into the subretinal space of RCS-rdy+p+/LAV rats, also commonly know as rdy rats. At week 4, in the absence of immunosuppressants, implanted PLLA ENMs were surrounded by a significantly low number of activated microglial cells, compared to week 1, indicating no adverse long-term immune response. In conclusion, we successfully designed and tested ENMs emulating the RPE cell supporting layers of the BM, and found 70nm PLLA ENMs to be best suited as scaffolds for fabricating retinal constructs. STATEMENT OF SIGNIFICANCE: Age related macular degeneration (AMD) is a leading cause of vision loss in the developed world, with an increasing number of people suffering from blindness or severe visual impairment. Transplantation of retinal pigment epithelium (RPE) cells supported on a synthetic, biomimetic-like Bruch's membrane (BM) is considered a promising treatment. However, the synthetic scaffolds used do not mimic the microenvironment of the RPE cell supporting layers, required for the development of a functional RPE monolayer. This study indicated that porous, laminin coated, 70nm PLLA ENMs supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. These findings indicate the potential clinical use of porous, laminin coated, 70nm PLLA ENMs in fabricating retinal constructs aimed at treating dry AMD.