Adult Retinal Pigment Epithelial Cells Research Articles

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.

Downregulation of Circular RNA PSEN1 ameliorates ferroptosis of the high glucose treated retinal pigment epithelial cells via miR-200b-3p/cofilin-2 axis

ABSTRACT Ferroptosis is a form of programmed cell death that participates in the progression of numerous diseases. Long noncoding RNAs (lncRNAs) are dysregulated in diabetic retinopathy (DR). However, the role of lncRNAs in DR-induced ferroptosis is unclear. Adult retinal pigment epithelial cell line-19 (ARPE19) cells were treated with a high concentration of glucose (high glucose, HG) to mimic DR in vitro. The intracellular contents of glutathione, malondialdehyde, and ferrous ions were analyzed using the corresponding kits. The MTT assay was performed to measure the cell survival rate, and cell death was determined using propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays. Western blotting was conducted to detect the protein levels of GPX4, SLC7A11, and TFR1. The targeting relationships were verified using luciferase reporter and RNA pull-down assays. circ-PSEN1 was upregulated in HG-treated ARPE19 cells and showed high resistance to RNase R and Act D. Inhibition of circ-PSEN1 in ARPE19 cells ameliorated the ferroptosis induced by HG was ameliorated, as evidenced by changes in the ferroptosis-related biomarkers/genes and decreased cell death. Subsequently, circ-PSEN1 acted as a sponge for miR-200b-3p. Inhibition of miR-200b-3p partially reversed the effects of circ-PSEN1 on ferroptosis. Furthermore, cofilin-2 (CFL2) was the target gene of miR-200b-3p, and it abrogated the inhibitory effect of miR-200b-3p on ferroptosis. Taken together, the findings indicate that knockdown of circ-PSEN1 can mitigate ferroptosis of ARPE19 cells induced by HG via the miR-200b-3p/CFL2 axis.

Exogenous administration of hydrogen sulfide alleviates homocysteine induced inflammation in ARPE-19 cells

Plasma homocysteine (Hcy) is an independent risk factor for Age related macular degeneration (AMD) and an inducer of inflammation. Homocysteine catabolism releases hydrogen sulfide (H2S). H2S has controversial effects on inflammation. In this study we have analysed the endogenous and exogenous H2S in modulating inflammation using adult retinal pigment epithelial (ARPE-19) cells as an in vitro model for AMD. ARPE-19 cells were treated with various concentrations of Hcy (15, 30 and 50 μM) for 3 h. Expression of Hcy transulfuration genes (CBS, CSE) by qPCR and western blot. H2S levels were measured using Free Radical Analyzer System (WPI, USA). The inflammatory markers (IL-6 and IL-8) were evaluated using real-time PCR and ELISA. Hcy exposure increased CBS protein expression, hydrogen sulfide levels and pro-inflammatory cytokines, modulating CBS by silencing did not alter H2S levels, but inhibition of CSE with PAG inhibited H2S production and decreased cytokine (IL-6 and IL-8) levels. On the contrary exogenous supply of hydrogen sulfide with NaHS and by compound 1c showed anti-inflammatory effects even in the presence of Hcy. This study shows that exogenous delivery of H2S decreases inflammation in retinal pigment epithelial cells on exposure to Hcy in ARPE-19 cells.

Polymorphism rs11200638 enhanced HtrA1 responsiveness and expression are associated with age-related macular degeneration.

To investigate the role of polymorphism rs11200638 of high-temperature requirement factor A-1 (HtrA1) gene in the pathogenesis of age-related macular degeneration (AMD). Cultured adult retinal pigment epithelial cells (ARPE-19) expressing HtrA1 gene were treated with H2O2 or lipopolysaccharides (LPS) and analysed using western blot and quantitative polymerase chain reaction to illustrate the effects of oxidative and inflammatory stress on HtrA1 gene expression. Luciferase reporter plasmid driven by HtrA1 promoter with either normal allele G or risk allele A at SNP rs11200638 was transfected to ARPE-19 cells to investigate the effect of the G/A variation on HtrA1 promoter activity. The effects of HtrA1 overexpression on ARPE-19 cells were analysed with respect to percentage of cell proliferation inhibition and cell apoptosis. HtrA1 expression was significantly increased with LPS or H2O2 stimulations (p < 0.05). In ARPE-19 cells, HtrA1 promoters (-1 to -2175 bp from translation starting point) with risk allele A or normal G at rs11200638 did not show statistically significant differences in their luciferase reporter expression (p = 0.054425173), however, both promoters showed a persistent trend of higher luciferase expressions after 100 ng/ml LPS treatment. The luciferase expression level was significantly greater in the promoter with risk A when compared to that with normal G. Overexpression of HtrA1 resulted in apoptosis of ARPE-19 cells with 53.8 ± 1.6% of proliferation inhibition (p < 0.01). Risk haplotype A at rs11200638 significantly increased the responsiveness of HtrA1 promoter to inflammation and subsequently enhanced HtrA1 expression. HtrA1 overexpression induced ARPE-19 apoptosis and growth inhibition, relevant to pathogenesis of AMD.

Circ_0084043 Facilitates High Glucose-Induced Retinal Pigment Epithelial Cell Injury by Activating miR-128-3p/TXNIP-Mediated Wnt/β-Catenin Signaling Pathway.

Diabetic retinopathy is a frequent complication of diabetes mellitus and one of the common causes of blindness. Circular RNAs (circRNAs) can modulate various biological behaviors of human diseases. Circ_0084043 is a novel circRNA, and its function in diabetic retinopathy progression is unclear. Adult retinal pigment epithelial cells (ARPE-19) were treated with high glucose (HG). RNA levels of circ_0084043, microRNA-128-3p (miR-128-3p), and thioredoxin-interacting protein (TXNIP) were detected by quantitative real-time polymerase chain reaction. 3-(4, 5-dimethylthiazole-2-y1)-2, 5-diphenyl tetrazolium bromide and flow cytometry were, respectively, used to examine cell viability and apoptosis. Apoptotic and TNXIP relative protein levels were measured by Western blot. The combination between targets was analyzed through dual-luciferase reporter assay or RNA immunoprecipitation assay. Results showed that HG induced the upregulation of circ_0084043 and the downregulation of miR-128-3p in ARPE-19 cells. Circ_0084043 knockdown or miR-128-3p overexpression mitigated the HG-mediated cell viability inhibition, apoptosis promotion, and inflammatory response. Circ_0084043 targeted miR-128-3p and miR-128-3p inhibitor returned the regulation of si-circ_0084043 in HG-treated cells. TXNIP was the target gene of miR-128-3p and TXNIP overexpression abolished the miR-128-3p-mediated effects after HG treatment. Circ_0084043 regulated the TXNIP expression to activate Wnt/β-catenin signal pathway by targeting miR-128-3p. Our findings unraveled that circ_0084043 promoted the HG-induced retinal pigment epithelial cell injury through activating the Wnt/β-catenin signal pathway by the miR-128-3p/TXNIP axis. Circ_0084043 might be an available biomarker in diabetic retinopathy diagnosis and therapy.

Intravitreal connective tissue growth factor neutralizing antibody or bevacizumab alone or in combination for prevention of proliferative vitreoretinopathy in an experimental model

Connective tissue growth factor (CTGF) is released by retinal pigment epithelial (RPE) cells and detectable in proliferative membranes (PrMs). This experimental study was performed to investigate the mRNA and protein levels of both CTGF and vascular endothelial growth factor A (VEGF-A) in a rabbit model of proliferative vitreoretinopathy (PVR). In addition, the effects of a single intravitreal injection of the safe dose of anti-CTGF or bevacizumab as monotherapy and in combination were evaluated. PVR was induced in the right eye of albino rabbits by intravitreal injection of cultured adult human RPE cells. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot analysis of CTGF and VEGF-A were performed on whole eye tissue in the PVR model versus controls at different time points. In the next step, the PVR models were assigned to five groups. The monotherapy groups received a single intravitreal injection of 0.1 ml of anti-CTGF 100 μg/ml (final concentration of 6.6 μg/ml in the vitreous) or 0.03 ml of 25 mg/ml bevacizumab. In the combined group, the abovementioned amounts of anti-CTGF and bevacizumab were injected intravitreally from separate sites in one session. No antibody injection was performed in the control group. Intravitreal injection of 0.1 ml of control IgG (1 mg/ml of isotype matched) antibody was performed in the placebo group. After 2 weeks, histologic evaluation including, trichrome staining for collagen, immunostaining by anti-alpha-smooth muscle actin for myofibroblasts, and anti-collagen type-1 antibody on paraffin embedded anterior-posterior sections was done. In addition, fundus photography was performed for clinically equivalent PVR staging. Twenty-four hours following PVR induction, CTGF mRNA and protein levels increased five- and- three-fold compared to controls, respectively (P < 0.001). VEGF-A mRNA and protein levels decreased significantly after 72 h of PVR induction compared to controls (P < 0.05). Means of PrM thickness and myofibroblast cell counts significantly decreased in the anti-CTGF group (P < 0.001 and P < 0.05, respectively). The mean area of collagen type-1 fibers of PrM in the mono- and combination therapy groups that received intravitreal anti-CTGF was significantly reduced (P < 0.001); in addition, mild PVR (stage-1 and 2) formation occurred in comparison with moderate to severe PVR (stage-4 and higher) in other groups. In conclusion, we found that intravitreal injection of CTGF neutralizing antibody resulted in a reduction in PrM thickness, collagen fibers and myofibroblast density in the PVR model. CTGF inhibition may represent a potential therapeutic target for PVR.

Protective effect of gypenoside LXXV from Gynostemma pentaphyllum against oxidative stress-induced retinal degeneration in vitro and in vivo

BackgroundCaring the eye from oxidative stress is very important in preventing the age-related macular degeneration (AMD), which is difficult to recover it once damaged. Particularly, the development of therapeutic anti-dry-AMD agent in a non-invasive manner is still on the horizon. PurposeIn the present study, we examined effect of gypenoside (Gyp) LXXV against dry-AMD in vitro and in vivo, to develop non-invasive anti-AMD agents based on a natural compound with anti-oxidative and anti-inflammatory activity. Study designGyp LXXV, a single ginseng ginsenoside compound, is orally administered to adult rabbits and mice that the dry-AMD was induced by asodium iodate or a light irradiation, respectively. The in vitro assay was performed using the retinal cells (ARPE-19) and macrophage cells (HMC-3). MethodsTo evaluate anti-oxidative capability and anti-inflammable efficacy of Gyp LXXV in retinal pigment epithelial (RPE) cells, real time PCR was employed using ARPE-19 cells and HMC-3 cells. To validate physiological effects of Gyp LXXV on dry-AMD, thickness measurement of RPE, electroretinogram (ERG), optical coherence tomography (OCT), and fundus examination were employed in either light irradiation- or sodium iodate-induced animal model. ResultsIt exhibited substantial alleviation of damaged retinal RPE tissues, via lowering the structural damage, the reactive oxygen species (ROS) level, and inflammatory cytokines in a dose-dependent manner, without showing any side effects. In addition, ROS-induced human adult RPE cells were treated with Gyp LXXV showed a protective effect against the ROS, confirming an excellent antioxidant efficacy with no potential cytotoxicity. ConclusionOur findings indicate that the oral route of Gyp LXXV exerts a potential therapeutic effect on dry-AMD. This work could guide the new therapeutic pathway of a phytochemical drug-based non-invasive treatment for dry-AMD in future clinical treatments.

Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1

BackgroundEchinomycin (EKN), an inhibitor of hypoxia-inducible factor (HIF)-1 DNA-binding activity, has been implied as a possible therapeutic agent in ischemic diseases. Here, we assess EKN in hypoxia-driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE) and retinal endothelial cells (hREC), and in vivo using the laser-induced mouse choroidal neovascularization (CNV) model. MethodsEffects of EKN on hypoxia-mediated pathways in aRPE were analyzed by Western blotting for HIF-1α protein, quantitative PCR of HIF-target genes, and proteome array for soluble angiogenic factors. In vitro inhibition of angiogenesis by EKN was determined in hREC. In vivo inhibition of angiogenesis by EKN was determined in the mouse laser-induced CNV, as a model of HIF-associated ocular neovascularization. CNV lesion area was determined by fundus fluorescein angiography. ResultsaRPE treated with EKN showed hypoxia-dependent significantly decreased cell recovery in the wound healing assay. These results were supported by lower levels of HIF-mediated transcripts detected in hypoxic aRPE cells treated with EKN compared with non-treated controls, and confirmed by proteome profiler for angiogenic factors. hREC exposed to aRPE EKN-conditioned medium displayed reduced sprouting angiogenesis. Mice with laser-induced CNV treated with intravitreally injected EKN showed significantly decreased vascular lesion area when compared with a mouse equivalent of aflibercept, or vehicle-treated controls. ConclusionsOur data proposes EKN as a potent inhibitor of HIF-mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age-related macular degeneration.

Detection of Autophagy-Related Gene Expression by Conjunctival Impression Cytology in Age-Related Macular Degeneration.

Purpose: To investigate the association of autophagy-related gene expression with age-related macular degeneration (AMD). Methods: Patients with AMD were recruited for analysis by conjunctival impression cytology. mRNA was assessed by real-time polymerase chain reaction (RT-PCR) to evaluate whether the expression of 26 autophagy-related genes (ATGs) was correlated with AMD. Further studies on cell viability and autophagic flux in response to oxidative stress by H2O2 were performed in human retinal pigment epithelial (RPE) cell lines based on the results of impression cytology. Results: Both the neovascular AMD (nAMD) and polypoidal choroidal vasculopathy (PCV) groups had significantly higher mRNA levels of gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) than the control group, but there was no significant difference between these two groups. Age difference existed only in the AMD group. GABARAPL1 and MAP1LC3B mRNA expression increased significantly after acute oxidative stress in adult retinal pigment epithelial (ARPE-19) cells. Cell viability significantly increased and decreased in the cells harboring GABARAPL1 expression vector and silenced with siRNA against GABARAPL1, respectively, during short-term oxidative stress, whereas viability increased in the GABARAPL1-silenced cells after long-term oxidative stress. Silencing GABARAPL1 itself caused a reduction in autophagic flux under both short and long-term oxidative stress. Conclusion: Our study showed the possibility of assessing autophagy-related gene expression by conjunctival impression cytology. GABARAPL1 was significantly higher in AMD. Although an in vitro study showed an initial protective effect of autophagy, a cell viability study revealed the possibility of a harmful effect after long-term oxidative injury. The underlying mechanism or critical factors require further investigation.

Farrerol Enhances Nrf2-Mediated Defense Mechanisms against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial Cells by Activating Akt and MAPK.

Oxidative stress of the retinal pigment epithelium (RPE) is an essential element contributing to the progression of age-related macular degeneration (AMD). Notably, the activation of Nrf2 is regarded as an effective strategy for controlling oxidation. The novel 2,3-dihydroflavonoid compound farrerol, which is extracted from Rhododendron, possesses antioxidant properties. In this study, we investigated the mechanism by which farrerol protects against oxidative damage mediated by hydrogen peroxide (H2O2) in adult retinal pigment epithelial cell line 19 (ARPE-19) cells. Farrerol supplementation conspicuously reversed H2O2-related cell damage through declining the generation of intracellular reactive oxygen species (ROS) and MDA and increasing the concentrations of GSH and SOD. According to the results of the apoptosis assay, a farrerol pretreatment decreased the protein expression of the Bax/Bcl-2, cleaved caspase-3, PARP, caspase-8, and caspase-9 proteins. Furthermore, farrerol markedly activated Nrf2, thereby increasing the levels of antioxidant enzymes downstream of Nrf2, such as HO-1, NQO1, and GCLM. Knockdown of Nrf2 with a specific siRNA successfully suppressed farrerol-mediated HO-1 transcription and partially abolished the cytoprotective effect on ARPE-19 cells. Meanwhile, farrerol induced Akt and MAPK phosphorylation in a dose-related way. However, inhibiting Akt and MAPK substantially blocked the cytoprotective functions of farrerol. Therefore, farrerol enhanced Nrf2-mediated cytoprotection of oxidative damage caused by H2O2, which may be inseparable from the activation of Akt and MAPK.

In vitro Evaluation of the Safety of Adalimumab for the Eye Under HTLV-1 Infection Status: A Preliminary Study.

Adalimumab (ADA), a fully human monoclonal tumor necrosis factor (TNF)-α antibody, is one of the most widely used biologics in the treatment of inflammatory diseases. However, ADA can exacerbate infectious conditions, induce paradoxical reactions such as inflammation, and cause neoplasia. Human T-cell leukemia virus type 1 (HTLV-1) is an infectious agent that induces inflammation and neoplastic infiltration in the eye. To date, numerous HTLV-1 carriers have been treated with adalimumab to suppress inflammation out of necessity, when standard anti-inflammatory drugs such as steroids and immunosuppressive agents have proven inadequate to control the inflammation. Here, we clarify the safety of adalimumab for the eye under HTLV-1 infectious conditions in vitro. We used the adult retinal pigment epithelial cell line (ARPE)-19 cell line as ocular resident cells, and used MT2 and TL-Om1 as HTLV-1-infected cells. ARPE-19 and MT2/TL-Om1 were co-cultured, and then adalimumab was administered. Production of cytokines and chemokines, TNF-α receptor (TNF-R), HTLV-1 proviral load (PVL), and apoptosis were measured to assess the effects of adalimumab. Contact between ARPE-19 and MT2/TL-Om1 produced inflammatory cytokines such as TNF, interleukin (IL)-6, IL-8 and IL-10, and transduced chemokines such as interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP-1), monokine induced by interferon-γ (MIG), and regulated on activation, normal T cell expressed and secreted (RANTES). No inflammatory cytokines and chemokines were exacerbated by adalimumab. Expression of TNF-R on ARPE-19 and MT2/TL-Om1 cells, HTLV-1 PVLs of MT2/TL-Om1 cells, and cell growth rate and apoptotic rate of ARPE-19 were unaffected by adalimumab. In conclusion, adalimumab does not appear to exacerbate HTLV-1-associated inflammatory conditions in the eye or increase PVL in HTLV-1-infected T cells. These data suggest that adalimumab could be used safely for the eye under HTLV-1 infectious conditions from the perspective of in vitro assessment.

4-(Phenylsulfanyl) Butan-2-One Attenuates the Inflammatory Response Induced by Amyloid-β Oligomers in Retinal Pigment Epithelium Cells.

Age-related macular degeneration (AMD) is a progressive eye disease that causes irreversible impairment of central vision, and effective treatment is not yet available. Extracellular accumulation of amyloid-beta (Aβ) in drusen that lie under the retinal pigment epithelium (RPE) has been reported as one of the early signs of AMD and was found in more than 60% of Alzheimer’s disease (AD) patients. Extracellular deposition of Aβ can induce the expression of inflammatory cytokines such as IL-1β, TNF-α, COX-2, and iNOS in RPE cells. Thus, finding a compound that can effectively reduce the inflammatory response may help the treatment of AMD. In this research, we investigated the anti-inflammatory effect of the coral-derived compound 4-(phenylsulfanyl) butan-2-one (4-PSB-2) on Aβ1-42 oligomer (oAβ1-42) added to the human adult retinal pigment epithelial cell line (ARPE-19). Our results demonstrated that 4-PSB-2 can decrease the elevated expressions of TNF-α, COX-2, and iNOS via NF-κB signaling in ARPE-19 cells treated with oAβ1-42 without causing any cytotoxicity or notable side effects. This study suggests that 4-PSB-2 is a promising drug candidate for attenuation of AMD.

The Effects of Prolonged Storage on ARPE-19 Cells Stored at Three Different Storage Temperatures.

This study aimed to investigate how prolonged storage of adult retinal pigment epithelial (ARPE-19) cell sheets affects cell metabolism, morphology, viability, and phenotype. ARPE-19 cell sheets were stored at three temperatures (4 °C, 16 °C, and 37 °C) for three weeks. Metabolic status and morphology of the cells were monitored by sampling medium and examining cells by phase-contrast microscopy, respectively, throughout the storage period. Cell viability was analyzed by flow cytometry, and phenotype was determined by epifluorescence microscopy after the storage. Lactate production and glucose consumption increased heavily, while pH dropped considerably, through storage at 37 °C compared to 4 °C and 16 °C. During storage, morphology started to deteriorate first at 4 °C, then at 37 °C, and was maintained the longest at 16 °C. Viability of the cells after three weeks of storage was best preserved at 16 °C, while cells stored at 4 °C and 37 °C had reduced viability. Dedifferentiation indicated by reduced expression of retinal pigment epithelium-specific protein 65 (RPE65), zonula occludens protein 1 (ZO-1), and occludin after three weeks of storage was noticed in all experimental groups compared to control. We conclude that storage temperature affects the metabolic status of ARPE-19 cells and that 16 °C reduces metabolic activity while protecting viability and morphology.

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells in vitro

ABSTRACT Purpose Maintaining mature and viable retinal pigment epithelial cells (RPE) in vitro has proven challenging. Investigating compounds that can promote RPE-viability and maturation is motivated by RPE transplantation research, the quest to understand RPE physiology, and a desire to modulate RPE in pathological states. We have previously reported that the silk protein sericin promotes viability, maturation, and pigmentation of human fetal RPE. In the present study, our aim was to uncover whether these effects can be seen in adult retinal pigment epithelial cell line-19 (ARPE-19) and induced pluripotent stem cell-derived RPE (iPSC-RPE). Methods ARPE-19 and iPSC-RPE were cultured with or without 10 mg/mL sericin. After 7 days, viability was assessed with calcein-acetoxymethyl ester (CAM) and ethidium homodimer-1 (EH-1) assays, flow cytometry, and morphometric analysis. Expression levels of RPE65, tyrosinase, and Pmel17 were quantified to compare maturation between the sericin-treated and control cultures. Light microscopy and staining of the tight junction protein zonula occludens protein 1 (ZO-1) were employed to study sericin’s effects on RPE morphology. We also measured culture medium pH, glucose, lactate, and extracellular ion content. Results Sericin-supplemented RPE cultures demonstrated significantly better viability compared to control cultures. Sericin appeared to improve ARPE-19 maturation and morphology in vitro. No effects were seen on RPE pigmentation with the concentration of sericin and duration of cell culture herein reported. Conclusions This is the first study to demonstrate that supplementing the culture media with sericin promotes the viability of iPSC-RPE and ARPE-19. Sericin’s viability-promoting effects may have important implications for retinal therapeutics and regenerative medicine research.

Fasudil Loaded PLGA Microspheres as Potential Intravitreal Depot Formulation for Glaucoma Therapy.

Rho-associated protein kinase (ROCK) inhibitors allow for causative glaucoma therapy. Unfortunately, topically applied ROCK inhibitors suffer from high incidence of hyperemia and low intraocular bioavailability. Therefore, we propose the use of poly (lactide-co-glycolide) (PLGA) microspheres as a depot formulation for intravitreal injection to supply outflow tissues with the ROCK inhibitor fasudil over a prolonged time. Fasudil-loaded microspheres were prepared by double emulsion solvent evaporation technique. The chemical integrity of released fasudil was confirmed by mass spectrometry. The biological activity was measured in cell-based assays using trabecular meshwork cells (TM cells), Schlemm’s canal cells (SC cells), fibroblasts and adult retinal pigment epithelium cells (ARPE-19). Cellular response to fasudil after its diffusion through vitreous humor was investigated by electric cell-substrate impedance sensing. Microspheres ranged in size from 3 to 67 µm. The release of fasudil from microspheres was controllable and sustained for up to 45 days. Released fasudil reduced actin stress fibers in TM cells, SC cells and fibroblasts. Decreased collagen gel contraction provoked by fasudil was detected in TM cells (~2.4-fold), SC cells (~1.4-fold) and fibroblasts (~1.3-fold). In addition, fasudil readily diffused through vitreous humor reaching its target compartment and eliciting effects on TM cells. No negative effects on ARPE-19 cells were observed. Since fasudil readily diffuses through the vitreous humor, we suggest that an intravitreal drug depot of ROCK inhibitors could significantly improve current glaucoma therapy particularly for patients with comorbid retinal diseases.

Role of Taurine Transporter in the Retinal Uptake of Vigabatrin.

Vigabatrin (VGB) is a first-line drug used for treatment of infantile spasms. On therapeutic dose, VGB accumulates in the retina causing permanent peripheral visual field constriction. The mechanism involved in retinal accumulation of VGB is ambiguous. In the present study, mechanism of VGB transport into retina was evaluated. VGB uptake into retina was studied in vitro using human adult retinal pigment epithelial (ARPE-19) cells as a model for outer blood retinal barrier. The VGB cell uptake studies demonstrated saturation kinetics with Km value of 13.1mM and uptake was significantly increased at pH7.4 and hyperosmolar conditions indicating involvement of carrier-mediated Na+-Cl--dependent transporter. In the presence of taurine transporter (TauT) substrates (taurine and GABA) and inhibitor guanidinoethyl sulfonate (GES), the uptake of VGB decreased significantly demonstrating contribution of TauT. The VGB retinal levels in rats were decreased by 1.5- and 1.3-folds on chronic administration of GES and taurine, respectively. In conclusion, this study demonstrated the TauT involvement in VGB uptake and accumulation in retina.

Copper mediates mitochondrial biogenesis in retinal pigment epithelial cells

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 μM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1β and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1β mRNA showed an increase in choroid–RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.

LncRNA GAS5 suppresses ER stress‑induced apoptosis and inflammation by regulating SERCA2b in HG‑treated retinal epithelial cell.

Hyperglycemia impairs the retinal functions in patients with diabetic retinopathy (DR). Downregulation of long non‑coding RNA growth arrest‑specific transcript 5 (lncRNA GAS5) expression in diabetes affects glucose intake and insulin signaling. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) mediates the regulation of endoplasmic reticulum (ER) stress and apoptosis in high glucose (HG)‑treated podocytes. Therefore, the present study aimed to investigate the roles of lncRNA GAS5 and SERCA2 in retinal pigment epithelium cells exposed to HG. GAS5 expression levels were detected using reverse transcription‑quantitative PCR. In addition, the expression levels of SERCA2b, ER stress‑related proteins, pro‑inflammatory factors and apoptotic proteins were determined by western blot analysis, ELISA or flow cytometry. The results showed that HG treatment induced ER stress in ARPE‑19 human adult retinal pigment epithelial cells by upregulating the expression levels of phosphorylated (p)‑protein kinase R‑like ER kinase, p‑eukaryotic initiation factor 2α, activating transcription factor 4 and CCAAT/enhancer‑binding protein homologous protein. In addition, HG treatment induced apoptosis by increasing Bax, Bad and caspase 12, and by decreasing Bcl‑2 levels expression levels. Moreover, HG treatment induced inflammation by upregulating tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6 expression. However, GAS5 and SERCA2b overexpression significantly decreased ER stress‑related apoptosis and inflammation, whereas SERCA2b knockdown significantly reversed the inhibitory effect of GAS5 on ER stress, apoptosis and inflammation. The results of the present study indicated that GAS5 may suppress ER stress‑induced apoptosis and inflammation by regulating SERCA2b in HG‑treated cells. These data suggested that GAS5 may serve a vital role in the pathogenesis of DR, and it may be considered a potential target for DR therapy.

Dexamethasone Loaded Liposomes by Thin-Film Hydration and Microfluidic Procedures: Formulation Challenges.

Liposomes have been one of the most exploited drug delivery systems in recent decades. However, their large-scale production with low batch-to-batch differences is a challenge for industry, which ultimately delays the clinical translation of new products. We have investigated the effects of formulation parameters on the colloidal and biopharmaceutical properties of liposomes generated with a thin-film hydration approach and microfluidic procedure. Dexamethasone hemisuccinate was remotely loaded into liposomes using a calcium acetate gradient. The liposomes produced by microfluidic techniques showed a unilamellar structure, while the liposomes produced by thin-film hydration were multilamellar. Under the same remote loading conditions, a higher loading capacity and efficiency were observed for the liposomes obtained by microfluidics, with low batch-to-batch differences. Both formulations released the drug for almost one month with the liposomes prepared by microfluidics showing a slightly higher drug release in the first two days. This behavior was ascribed to the different structure of the two liposome formulations. In vitro studies showed that both formulations are non-toxic, associate to human Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells, and efficiently reduce inflammation, with the liposomes obtained by the microfluidic technique slightly outperforming. The results demonstrated that the microfluidic technique offers advantages to generate liposomal formulations for drug-controlled release with an enhanced biopharmaceutical profile and with scalability.

Sphere-induced reprogramming of RPE cells into dual-potential RPE stem-like cells.

BackgroundThe retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols.MethodsSphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches.FindingsWe provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming.InterpretationThe Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs.FundNational Natural Science Foundation of China and the National Institute of Health USA.