Adult Retinal Pigment Epithelial Cells Research Articles

Hepatocyte growth factor promotes retinal pigment epithelium cell activity through MET/AKT signaling pathway.

To explore the effects of hepatocyte growth factor (HGF) on retinal pigment epithelium (RPE) cell behaviors. The human adult retinal pigment epithelial cell line-19 (ARPE-19) were treated by HGF or mesenchymal-epithelial transition factor (MET) inhibitor SU11274 in vitro. Cell viability was detected by a Cell Counting Kit-8 assay. Cell proliferation and motility was detected by a bromodeoxyuridine incorporation assay and a wound healing assay, respectively. The expression levels of MET, phosphorylated MET, protein kinase B (AKT), and phosphorylated AKT proteins were determined by Western blot assay. The MET and phosphorylated MET proteins were also determined by immunofluorescence assay. HGF increased ARPE-19 cells' viability, proliferation and migration, and induced an increase of phosphorylated MET and phosphorylated AKT proteins. SU11274 significantly reduced cell viability, proliferation, and migration and decreased the expression of MET and AKT proteins. SU11274 suppressed HGF-induced increase of viability, proliferation, and migration in ARPE-19 cells. Additionally, SU11274 also blocked HGF-induced phosphorylation of MET and AKT proteins. HGF enhances cellular viability, proliferation, and migration in RPE cells through the MET/AKT signaling pathway, whereas this enhancement is suppressed by the MET inhibitor SU11274. HGF-induced MET/AKT signaling might be a vital contributor of RPE cells survival.

Bone morphogenetic protein-6 suppresses TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β2 and prevented TGF-β2 from affecting EMT-related biomarkers (P<0.05). BMP-6 prevents the EMT in RPE cells induced by TGF-β2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Intraocular mRNA delivery with endogenous MmPEG10-based virus-like particles

Virus-like particles (VLP) are a promising tool for intracellular gene delivery, yet their potential in ocular gene therapy remains underexplored. In this study, we bridged this knowledge gap by demonstrating the successful generation and application of vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped mouse PEG10 (MmPEG10)-VLP for intraocular mRNA delivery. Our findings revealed that PEG10-VLP can efficiently deliver GFP mRNA to adult retinal pigment epithelial cell line-19 (ARPE-19) cells, leading to transient expression. Moreover, we showed that MmPEG10-VLP can transfer SMAD7 to inhibit epithelial-mesenchymal transition (EMT) in RPE cells effectively. In vivo experiments further substantiated the potential of these vectors, as subretinal delivery into adult mice resulted in efficient transduction of retinal pigment epithelial (RPE) cells and GFP reporter gene expression without significant immune response. However, intravitreal injection did not yield efficient ocular expression. We also evaluated the transduction characteristics of MmPEG10-VLP following intracameral delivery, revealing transient GFP protein expression in corneal endothelial cells without significant immunotoxicities. In summary, our study established that VSVG pseudotyped MmPEG10-based VLP can transduce mitotically inactive RPE cells and corneal endothelial cells in vivo without triggering an inflammatory response, underscoring their potential utility in ocular gene therapy.

SIRT1/P53 in retinal pigment epithelial cells in diabetic retinopathy: a gene co-expression analysis and He-Ying-Qing-Re formula treatment.

Diabetic retinopathy (DR) is a severe diabetic complication that leads to severe visual impairment or blindness. He-Ying-Qing-Re formula (HF), a traditional Chinese medicinal concoction, has been identified as an efficient therapy for DR with retinal vascular dysfunction for decades and has been experimentally reported to ameliorate retinal conditions in diabetic mice. This study endeavors to explore the therapeutic potential of HF with key ingredients in DR and its underlying novel mechanisms. Co-expression gene modules and hub genes were calculated by weighted gene co-expression network analysis (WGCNA) based on transcriptome sequencing data from high-glucose-treated adult retinal pigment epithelial cell line-19 (ARPE-19). The chromatographic fingerprint of HF was established by ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-Q-TOF-MS). The molecular affinity of the herbal compound was measured by molecular docking. Reactive oxygen species (ROS) was measured by a DCFDA/H2DCFDA assay. Apoptosis was detected using the TUNEL Assay Kit, while ELISA, Western blot, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used for detecting the cytokine, protein, and mRNA expressions, respectively. Key compounds in HF were identified as luteolin, paeoniflorin, and nobiletin. For WGCNA, ME-salmon ("protein deacetylation") was negatively correlated with ME-purple ("oxidative impairment") in high-glucose-treated ARPE-19. Luteolin has a high affinity for SIRT1 and P53, as indicated by molecular docking. Luteolin has a hypoglycemic effect on type I diabetic mice. Moreover, HF and luteolin suppress oxidative stress production (ROS and MDA), inflammatory factor expression (IL-6, TNF-α, IL1-β, and MCP-1), and apoptosis, as shown in the in vivo and in vitro experiments. Concurrently, treatment with HF and luteolin led to an upregulation of SIRT1 and a corresponding downregulation of P53. Using HF and its active compound luteolin as therapeutic agents offers a promising approach to diabetic retinopathy treatment. It primarily suppressed protein acetylation and oxidative stress via the SIRT1/P53 pathway in retinal pigment epithelial cells.

Identification and Potential Roles of Human MicroRNAs in Ebola Virus Infection and Disease Pathogenesis.

Ebola virus (EBOV) is a highly pathogenic virus that causes a severe illness called Ebola virus disease (EVD). EVD has a high mortality rate and remains a significant threat to public health. Research on EVD pathogenesis has traditionally focused on host transcriptional responses. Limited recent studies, however, have revealed some information on the significance of cellular microRNAs (miRNAs) in EBOV infection and pathogenic mechanisms, but further studies are needed. Thus, this study aimed to identify and validate additional known and novel human miRNAs in EBOV-infected adult retinal pigment epithelial (ARPE) cells and predict their potential roles in EBOV infection and pathogenic mechanisms. We analyzed previously available small RNA-Seq data obtained from ARPE cells and identified 23 upregulated and seven downregulated miRNAs in the EBOV-infected cells; these included two novel miRNAs and 17 additional known miRNAs not previously identified in ARPE cells. In addition to pathways previously identified by others, these miRNAs are associated with pathways and biological processes that include WNT, FoxO, and phosphatidylinositol signaling; these pathways were not identified in the original study. This study thus confirms and expands on the previous study using the same datasets and demonstrates further the importance of human miRNAs in the host response and EVD pathogenesis during infection.

Preparation of Lutein Nanoparticles by Glycosylation of Saccharides and Casein for Protecting Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD), a leading cause of visual impairment in the aging population, lacks effective treatment options due to a limited understanding of its pathogenesis. Lutein, with its strong antioxidant properties and ability to mitigate AMD by absorbing ultraviolet (UV) rays, faces challenges related to its stability and bioavailability in functional foods. In this study, we aimed to develop delivery systems using protein-saccharide conjugates to enhance lutein delivery and protect adult retinal pigment epithelial (ARPE-19) cells against sodium iodate (NaIO3)-induced damage. Various saccharides, including mannose, galactose, lactose, maltose, dextran, and maltodextrin, were conjugated to casein via the Maillard reaction for lutein delivery. The resulting lutein-loaded nanoparticles exhibited small size and spherical characteristics and demonstrated improved thermal stability and antioxidant capacity compared to free lutein. Notably, these nanoparticles were found to be nontoxic, as evidenced by reduced levels of cellular reactive oxygen species production (167.50 ± 3.81, 119.57 ± 3.45, 195.15 ± 1.41, 183.96 ± 3.11, 254.21 ± 3.97, 283.56 ± 7.27%) and inhibition of the mitochondrial membrane potential decrease (58.60 ± 0.29, 65.05 ± 2.91, 38.88 ± 1.81, 42.95 ± 1.39, 23.52 ± 1.04, 25.24 ± 0.08%) caused by NaIO3, providing protection against cellular damage and death. Collectively, our findings suggest that lutein-loaded nanoparticles synthesized via the Maillard reaction hold promise for enhanced solubility, oral bioavailability, and biological efficacy in the treatment of AMD.

Coenzyme Q0, a quinone derivative from Antrodia camphorata, inhibits epithelial-mesenchymal transition by activating the Nrf2 signaling pathway in TGF-β-stimulated adult retinal pigment epithelial cells to improve age-related macular degeneration and proliferative vitreoretinopathy

We investigated the anti-EMT and antifibrotic effects of coenzyme Q0 (CoQ0), a quinone derivative from Antrodia camphorata, in TGF-β-stimulated adult retinal pigment epithelial 19 (ARPE-19) cells. Results showed that CoQ0 treatment reversed TGF-β-stimulated morphological changes from an epithelial to a fibroblastic phenotype in ARPE-19 cells. CoQ0 exhibited anti-EMT effects by impeding TGF-β-stimulated migration and invasion in ARPE-19 cells. Notably, CoQ0 triggered the epithelial marker E-cadherin and suppressed the mesenchymal marker N-cadherin expression in nonstimulated or TGF-β-stimulated ARPE-19 cells. Moreover, CoQ0 attenuated EMT and fibrotic Vimentin, MMP-9/-2, Slug, α-SMA, and VEGF expression in nonstimulated or TGF-β-stimulated ARPE-19 cells. Interestingly, CoQ0 inhibited TGF-β-induced intracellular ROS production by activating Nrf2 nuclear translocation and upregulating the HO-1, γ-GCLC, and NQO-1 enzymes in ARPE-19 cells. Moreover, Nrf2 silencing reversed TGF-β-induced ROS-mediated anti-EMT (E-cadherin/N-cadherin/Slug) and antifibrotic (α-SMA) effects in CoQ0-treated ARPE-19 cells. Therefore, CoQ0 could be utilized to treat age-related macular degeneration and proliferative vitreoretinopathy.

Alteration in Melanin Content in Retinal Pigment Epithelial Cells upon Hydroquinone Exposure

Abnormal pigmentation or depigmentation of the retinal pigment epithelium (RPE) is a precursor to neovascular age-related macular degeneration (nAMD). In this study, we evaluated the effects of hydroquinone (HQ), the most potent reductant in cigarette smoke, on the melanin production in RPE cells. Induced pluripotent stem cell (iPS)-derived RPE and adult retinal pigment epithelial (ARPE-19) cells were cultured with HQ. Real-time reverse transcription polymerase chain reaction revealed that the expression of melanin-related genes decreased due to the addition of HQ for 1 day. Enzyme-linked immunosorbent immunoassay showed that the concentration of melanin significantly decreased due to the addition of HQ for 24 h. A suspension of RPE cells with HQ for 24 h was prepared, and the absorbance was measured. The absorbance decreased particularly under blue light, suggesting that blue light may reach the choroid and cause choroidal inflammation. Additionally, melanin levels significantly decreased due to the addition of HQ for 1 week. After blue light irradiation on the RPE with HQ for 1 week, the vascular endothelial growth factor in the medium was significantly higher in the HQ group than in the control group. HQ-induced changes in melanin production may be responsible for the uneven pigmentation of the RPE, and these changes may cause nAMD.

High-speed counter-current chromatography assisted preparative isolation of phenolic compounds from the flowers of Chrysanthemum morifolium cv. Fubaiju.

Chrysanthemum morifolium cv. Fubaiju is rich in phenolic compounds with various benefits such as anti-inflammatory, antioxidant, and cardiovascular protection. In this study, 12 phenolic compounds, including five flavonoid glycosides and seven quinic acid derivatives, were successfully separated from the flowers of Chrysanthemum morifolium cv. Fubaiju by high-speed counter-current chromatography and preparative high-performance liquid chromatography. Ethyl acetate-n-butanol-acetonitrile-water-acetic acid (5:0.5:2.5:5:0.25, v/v/v/v/v) was selected as solvent system to separate six fractions from the flowers of Chrysanthemum morifolium cv. Fubaiju, and 20% aqueous acetonitrile (containing 0.1% formic acid) was chosen to be the elution solvent in preparative high-performance liquid chromatography for purifying the fractions above. Luteolin-7-O-β-D-glucoside (1), luteolin-7-O-β-D-glucuronide (2), apigenin-7-O-β-D-glucoside (3), luteolin-7-O-β-D-rutinoside (4), diosmetin-7-O-β-D-glucoside (5), chlorogenic acid (6), 1,5-dicaffeoylquinic acid (7), 1,4-dicaffeoylquinic acid (8), 3,4-dicaffeoylquinic acid (9), 3,4-dicaffeoyl-epi-quinic acid (10), 3,5-dicaffeoylquinic acid (11), and 4,5-dicaffeoylquinic acid (12) were isolated with purities all above 95%, respectively. In addition, all isolates were evaluated for their protective effects on H2 O2 -induced oxidative damage in adult retinal pigment epithelial cells.

LncRNA NORAD defects deteriorate the formation of age-related macular degeneration.

Long noncoding RNAs (lncRNAs) play important roles in the development of age-related macular degeneration (AMD). However, the effect of long non-coding RNA activated by DNA damage (NORAD) on AMD remains unknown. This study aimed to investigate the effect of NORAD on RPE cell senescence and degeneration. Irradiated adult retinal pigment epithelial cell line-19 (ARPE-19) and sodium iodate-treated mice were used as in vitro and in vivo AMD models. Results showed that irradiation-induced AMD characteristics of ARPE-19 and NORAD-knockdown aggravated cell cycle arrest in the G2/M phase, cell apoptosis and cell senescence along with the increased expression of phosphorylated P53 (p-P53) and P21. AMD factors C3, ICAM-1, APP, APOE, and VEGF-A were also increased by NORAD-knockdown. Moreover, NORAD-knockdown increased irradiation-induced reduction of mitochondrial homeostasis factors, (i.e., TFAM and POLG) and mitochondrial respiratory chain complex genes (i.e., ND1 and ND5) along with mitochondrial reactive oxygen species (ROS). We also identified a strong interaction of NORAD and PGC-1α and sirtuin 1 (SIRT1) in ARPE-19; that is, NORAD knockdown increases the acetylation of PGC-1α. In NORAD knockout mice, NORAD-knockout accelerated the sodium iodate-reduced retinal thickness reduction, function impairment and loss of retinal pigment in the fundus. Therefore, NORAD-knockdown accelerates retinal cell senescence, apoptosis, and AMD markers via PGC-1α acetylation, mitochondrial ROS, and the p-P53-P21signaling pathway, in which NORAD-mediated effect on PGC-1α acetylation might occur through the direct interaction with PGC-1α and SIRT1.

Investigation into the use of gamma irradiated Cytodex-1 microcarriers to produce a human cytomegalovirus (HCMV) vaccine candidate in epithelial cells

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a “ready-to-use” product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.

TRAP1 Is Expressed in Human Retinal Pigment Epithelial Cells and Is Required to Maintain their Energetic Status.

Age-related macular degeneration (AMD) is the leading cause of severe vision loss and blindness in elderly people worldwide. The damage to the retinal pigment epithelium (RPE) triggered by oxidative stress plays a central role in the onset and progression of AMD and results from the excessive accumulation of reactive oxygen species (ROS) produced mainly by mitochondria. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone that contributes to the maintenance of mitochondrial integrity by decreasing the production and accumulation of ROS. The present study aimed to evaluate the presence and the role of TRAP1 in the RPE. Here, we report that TRAP1 is expressed in human adult retinal pigment epithelial cells and is located mainly in the mitochondria. Exposure of RPE cells to hydrogen peroxide decreases the levels of TRAP1. Furthermore, TRAP1 silencing increases intracellular ROS production and decreases mitochondrial respiratory capacity without affecting cell proliferation. Together, these findings offer novel insights into TRAP1 functions in RPE cells, opening possibilities to develop new treatment options for AMD.

Role of Adrenomedullin 2/Intermedin in the Pathogenesis of Neovascular Age-Related Macular Degeneration

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization–associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-β2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration–related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases.

Safety Profile of Lutein- Versus Triamcinolone Acetonide-Based Vitreous Staining.

To assess the safety profile of a new lutein-based vitreous dye (LB-VD) formulation compared with various triamcinolone acetonide (TA) formulations with and without subsequent exposure to perfluorodecalin (PFD) in vitro. Human adult retinal pigment epithelial cells (ARPE-19) were treated with the following formulations: undiluted preserved TA (TA-BA), diluted preserved TA (D-TA-BA), preservative-free TA (TA-PF), and LB-VD. First, cell tolerability was evaluated with MTT, LDH, and ATPlite assays after 1, 5, and 30 minutes of exposure to each tested formulation. Then, cells were sequentially exposed to formulations and PFD. After 24 hours of exposure to PFD, cell tolerability was evaluated through MTT and ATPlite assays. Among the formulations tested, LB-VD showed the highest levels of cell viability, cell metabolism, and cell proliferation and induced the lowest release of LDH, whereas the TA-based formulations demonstrated a cytotoxic effect on ARPE-19 cells in vitro. After subsequent 24-hour exposure to PFD, a greater reduction of cell viability was noted for all the formulations; however, this reduction was not significant only for the combination LB-VD-PFD, which was the best tolerated condition. LB-VD showed a better safety profile compared with all TA-based formulations, even when used in combination with PFD. In surgical practice, LB-VD may be preferred to TA-based formulations for vitreous staining in the light of its more favorable safety profile.

Activation of Delta-Opioid Receptor Protects ARPE19 Cells against Oxygen-Glucose Deprivation/Reoxygenation-Induced Necroptosis and Apoptosis by Inhibiting the Release of TNF-α.

Retinal ischemia-reperfusion injury (RIRI) is the basis of the pathology that leads to many retinal diseases and induces necroptosis and apoptosis. Tumor necrosis factor-α (TNF-α) is critically involved in necroptosis and apoptosis. Delta-opioid receptor (DOR) activation inhibits TNF-α release in our previous studies, it might prevent necroptosis and apoptosis by inhibiting the release of TNF-α. However, the role of TNF-α and DOR in necroptosis and apoptosis of retinal pigment epithelial (RPE) cells remains largely unknown. Here, we explored the mechanisms of TNF-α and DOR in necroptosis and apoptosis using an oxygen-glucose deprivation/reoxygenation (OGD/R) model of adult retinal pigment epithelial cell line-19 (ARPE19) cells. ARPE19 cells were exposed to OGD/R conditions to mimic RIRI in vitro. Cell viability was quantified using the Cell Counting Kit-8 (CCK-8) assay. Morphological changes were observed by inverted microscopy. TNF-α protein levels in cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). The DOR agonist TAN-67 and antagonist naltrindole (NTI) were used to pretreat cells for 1 or 2 hours before OGD24/R36 administration. Calcein acetoxymethylester/propidium iodide (Calcein-AM/PI) and Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to detect necroptotic and apoptotic ARPE19 cells, respectively. The protein expression of DOR, p-RIP1 (RIP1), p-RIP3 (RIP3), p-MLKL (MLKL), and cleaved Caspase3 (Caspase3) was measured by western blotting. OGD severely damaged ARPE19 cells. Prolonged reoxygenation significantly increased TNF-α level and decreased DOR expression in ARPE19 cells. Pretreatment with the DOR agonist TAN-67 (10 µM) significantly improved ARPE19 cell viability after OGD24/R36 by reducing the number of necroptotic and apoptotic cells. Furthermore, DOR activation significantly inhibited TNF-α release and suppressed the expression of proteins related to necroptosis and apoptosis, including p-RIP1, p-RIP3, p-MLKL, and cleaved Caspase3, after OGD24/R36. This effect was reversed by the DOR antagonist NTI. These results strongly suggest that DOR activation inhibits necroptosis and apoptosis by decreasing TNF-α release, leading to the prevention of OGD/R-induced injury in ARPE19 cells. This study provides an innovative idea for clinical treatment strategies for retinal damage and vision loss due to RIRI.

Epithelial membrane protein 2 (EMP2) regulates hypoxia-induced angiogenesis in the adult retinal pigment epithelial cell lines

Pathologic retinal neovascularization is a potentially blinding consequence seen in many common diseases including diabetic retinopathy, retinopathy of prematurity, and retinal vaso-occlusive diseases. This study investigates epithelial membrane protein 2 (EMP2) and its role as a possible modulator of angiogenesis in human retinal pigment epithelium (RPE) under hypoxic conditions. To study its effects, the RPE cell line ARPE-19 was genetically modified to either overexpress EMP2 or knock down its levels, and RNA sequencing and western blot analysis was performed to confirm the changes in expression at the RNA and protein level, respectively. Protein expression was evaluated under both normoxic conditions or hypoxic stress. Capillary tube formation assays with human umbilical vein endothelial cells (HUVEC) were used to evaluate functional responses. EMP2 expression was found to positively correlate with expression of pro-angiogenic factors HIF1α and VEGF at both mRNA and protein levels under hypoxic conditions. Mechanistically, EMP2 stabilized HIF1α expression through downregulation of von Hippel Lindau protein (pVHL). EMP2 mediated changes in ARPE-19 cells were also found to alter the secretion of a paracrine factor(s) in conditioned media that can regulate HUVEC migration and capillary tube formation in in vitro functional angiogenesis assays. This study identifies EMP2 as a potential mediator of angiogenesis in a human RPE cell line. EMP2 levels positively correlate with pro-angiogenic mediators HIF1α and VEGF, and mechanistically, EMP2 regulates HIF1α through downregulation of pVHL. This study supports further investigation of EMP2 as a promising novel target for therapeutic treatment of pathologic neovascularization in the retina.

Transcriptional comparison of adult human primary Retinal Pigment Epithelium, human pluripotent stem cell-derived Retinal Pigment Epithelium, and ARPE19 cells.

The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE–derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting “immature” gene regulatory networks.

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.