Human Retinal Pigment Epithelial Cells Research Articles

Design and Characterization of a Novel Intravitreal Dual-Transgene Genetic Medicine for Neovascular Retinopathies.

Intravitreal delivery of therapeutic transgenes to the retina via engineered viral vectors can provide sustained local concentrations of therapeutic proteins and thus potentially reduce the treatment burden and improve long-term vision outcomes for patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy. We performed directed evolution in nonhuman primates (NHP) to invent an adeno-associated viral (AAV) variant (R100) with the capacity to cross vitreoretinal barriers and transduce all regions and layers of the retina following intravitreal injection. We then engineered 4D-150, an R100-based genetic medicine carrying 2 therapeutic transgenes: a codon-optimized sequence encoding aflibercept, a recombinant protein that inhibits VEGF-A, VEGF-B, and PlGF, and a microRNA sequence that inhibits expression of VEGF-C. Transduction, transgene expression, and biological activity were characterized in human retinal cells in vitro and in NHPs. R100 demonstrated superior retinal cell transduction in vitro and in vivo compared to AAV2, a commonly used wild-type AAV serotype in retinal gene therapies. Transduction of human retinal pigment epithelial cells in vitro by 4D-150 resulted in dose-dependent transgene expression and corresponding reductions in VEGF-A and VEGF-C. Intravitreal administration of 4D-150 to NHPs was well tolerated and led to robust retinal expression of both transgenes. In a primate model of laser-induced choroidal neovascularization, 4D-150 completely prevented clinically relevant angiogenic lesions at all tested doses. These findings support further development of 4D-150. Clinical trials are underway to establish the safety and efficacy of 4D-150 in individuals with wet AMD and DME.

The effect of Coix lachrymal L. seed extract on the expression of inflammation and fibrogenesis genes in human retinal pigment epithelial cells.

Proliferative vitreoretinopathy (PVR) is a vision-threatening condition associated with retinal-detachment (RD), primarily caused by fibrocellular scar membrane formation. This study investigates the therapeutic potential of adlay seed extract fractions in mitigating PVR-associated pathways, focusing on oxidative stress, proliferation, inflammation, and fibrogenesis in retinal pigment epithelial (RPE) cells. Adlay seed extract fractions (methanolic: MeOH and residual: Res) were obtained through solvent extraction and characterized for carbohydrate, protein, flavonoid content, and antioxidant activity. RPE cells were cultured, and their viability in response to adlay fractions was assessed using the MTT assay. Gene expression analysis of IL-1β, IL-6, LIF, TGF-β, Snail and α-SMA genes was conducted via real-time PCR after treatment with adlay fractions. The Res fraction exhibited higher levels of protein, carbohydrate, flavonoids, and phenols compared to the MeOH fraction, along with significantly enhanced antioxidant activity. Both fractions showed inhibitory effects on RPE cell viability, with the Res fraction demonstrating a more pronounced impact. Gene expression analysis revealed a significant decrease in IL-6 and TGF-β expression with the MeOH fraction treatment, while the Res fraction led to decreased expression of IL-6, LIF, TGF-β, Snail and α-SMA, indicating a more comprehensive modulation of PVR-associated pathways. This study highlights the potential therapeutic benefits of adlay seed extract fractions in mitigating PVR-associated pathways in RPE cells. The Res fraction, particularly rich in bioactive compounds and exhibiting potent antioxidant activity, shows promise in attenuating oxidative stress, proliferation, inflammation, and fibrogenesis, critical processes in PVR development. These findings underscore the potential of adlay seed extracts as a novel therapeutic strategy for PVR warranting further investigation and clinical validation.

As shown hesperidin suppresses TGF-β2-induced proliferation and epithelial-mesenchymal transition of retinal pigment epithelial cells.

This study investigates the potential therapeutic effects and molecular mechanisms of hesperidin treatment on cell migration and epithelial-mesenchymal transition, key stages of proliferative vitreoretinopathy (PVR). Human retinal pigment epithelial cells (ARPE-19) were treated with 10ng/ml transforming growth factor-beta 2 (TGF-β2) alone or in combination with 1.56μM hesperidin for 48h. The impact of treatment on cell migration was evaluated using a wound healing assay. Apoptosis was assessed using DNA staining. mRNA and protein expression were evaluated using real-time PCR and Western blot, respectively. Hesperidin inhibits the proliferation and transformation of the cells by inducing apoptosis and reverses the cell morphology modified by TGF-β2. Hesperidin inhibits cell migration induced by TGF-β2. Upon treatment with hesperidin, the levels of mesenchymal markers upregulated by TGF-β2, such as MMP-1, -2, -9, fibronectin, α-SMA and the transcription factors Snail, Slug and ZEB-1, were downregulated. Conversely, the epithelial marker E-cadherin is upregulated with hesperidin treatment. Additionally, TIMP-1 and TIMP-2 expression levels, which are downregulated, increase with the treatment. These results suggest that hesperidin may inhibit the migration and EMT processes of RPE cells involved in the development of PVR, indicating its potential as a therapeutic agent for treating PVR.

Knockdown of HOTAIR Alleviates High Glucose-Induced Apoptosis and Inflammation in Retinal Pigment Epithelial Cells.

Diabetic retinopathy (DR) is one of the most common microvascular complications in diabetes. Accumulating evidence demonstrated that long non-coding RNAs (lncRNAs) played critical regulatory roles in DR. However, the role of lncRNA HOX Transcript Antisense Intergenic RNA (HOTAIR) in the high glucose (HG)-induced human retinal pigment epithelial (RPE) cell injury remains unclear. Herein, we found the expression of HOTAIR was increased in the retina of DR rats and HG-induced ARPE-19 cells. Knockdown of HOTAIR improved viability, inhibited apoptosis, increased Bcl-2 protein levels, and decreased Bax and cleaved caspase 3 protein levels in HG-treated ARPE-19 cells. Moreover, enzyme-linked immunosorbent assay showed that HOTAIR silencing reduced interleukin 6 and tumor necrosis factor-α release of ARPE-19 cells under HG conditions. Mechanistically, luciferase reporter assay and RNA immunoprecipitation assay validated that HOTAIR could directly sponge miR-326 to upregulate transcription factor 4 (TCF4) expression. Furthermore, rescue experiments confirmed that HOTAIR promoted apoptosis and inflammation of HG-treated ARPE-19 cells by the miR-326/TCF4 axis. In summary, HOTAIR enhanced HG-induced retinal pigment epithelial cell injury by promoting apoptosis and inflammation, shedding light on the importance of HOTAIR as a novel potential target for DR treatment.

Baicalin alleviates the injury of human retinal pigment epithelium cells and improves branch retinal vein occlusion in rats by inhibiting the HIF-1α/VEGFA axis

BackgroundAt present, relevant studies have found that baicalin can improve macular edema (ME) caused by glaucoma, but the effect on branch retinal vein occlusion (BRVO) is still unclear.MethodsThe CoCl2-stimulated ARPE-19 cells were treated with different concentrations of baicalin and detected cell viability, apoptosis and oxidative stress. Next, the hypoxia-inducible factor-1α (HIF-1α) overexpression vector or siRNA were transfected into CoCl2-stimulated ARPE-19 cells, and the cell changes were detected. We searched the potential binding proteins of HIF-1α through the online database, and screened vascular endothelial growth factor A (VEGFA) as the research object. The CoCl2-stimulated ARPE-19 cells were treated with baicalin alone, or transfected with HIF-1α overexpression vector, or transfected with HIF-1α overexpression vector and VEGFA siRNA, and the cell changes were detected. Finally, we verified the therapeutic effect of baicalin on BRVO rats in vivo.ResultsBaicalin inhibited CoCl2-induced apoptosis, inflammation and oxidative stress in ARPE-19 cells, and baicalin inhibited HIF-1α protein expression. In CoCl2-induced hypoxia cells, HIF-1α aggravated apoptosis, inflammation and oxidative stress, while HIF-1α silencing alleviated cell damage. Mechanism study showed that in baicalin-treated CoCl2-induced cells, VEGFA protein expression decreased and cell damage was improved, but this protective effect was counteracted by HIF-1α, and VEGFA silencing again inhibited apoptosis, inflammation and oxidative stress. Baicalin inhibited HIF-1α and VEGFA protein expression in the retinal tissue of BRVO rats, reduced injury, and promoted the recovery of ganglion cell layer.ConclusionsBaicalin alleviated ARPE-19 cell injury and improved BRVO in rats by inhibiting HIF-1α/VEGFA axis in vivo and in vitro.

Blue Light-Induced Mitochondrial Oxidative Damage Underlay Retinal Pigment Epithelial Cell Apoptosis

Reactive oxygen species (ROS) play a pivotal role in apoptosis. We reported that Blue Light (BL) induced oxidative stress in human retinal pigment epithelial (RPE) cells in vitro and increased drusen deposition and RPE cell apoptosis in human eyes. Here, we investigated the mechanisms underlying BL-induced damage to RPE cells. Cells were exposed to BL with or without the antioxidant N-acetylcysteine. Cells were analyzed for levels of ROS, proliferation, viability, and mitochondria membrane potential (ΔΨM) fluctuation. We performed proteomic analyses to search for differentially expressed proteins. ROS levels increased following RPE cell exposure to BL. While ROS production did not affect RPE cell proliferation, it was accompanied by decreased ΔΨM and increased cell apoptosis due to the caspase cascade activation in a ROS-dependent manner. Proteomic analyses revealed that BL decreased the levels of ROS detoxifying enzymes in exposed cells. We conclude that BL-induced oxidative stress is cytotoxic to RPE cells. These findings bring new insights into the involvement of BL on RPE cell damage and its role in the progression of age-related macular degeneration. The use of antioxidants is an avenue to block or delay BL-mediated RPE cell apoptosis to counteract the disease progression.

Thrombospondin 1 Mediates Autophagy Upon Inhibition of the Rho-Associated Protein Kinase Inhibitor.

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Microtubule poleward flux as a target for modifying chromosome segregation errors

Cancer cells often display errors in chromosome segregation, some of which result from improper chromosome alignment at the spindle midplane. Chromosome alignment is facilitated by different rates of microtubule poleward flux between sister kinetochore fibers. However, the role of the poleward flux in supporting mitotic fidelity remains unknown. Here, we introduce the hypothesis that the finely tuned poleward flux safeguards against lagging chromosomes and micronuclei at mitotic exit by promoting chromosome alignment in metaphase. We used human untransformed RPE-1 cells depleted of KIF18A/kinesin-8 as a system with reduced mitotic fidelity, which we rescued by three mechanistically independent treatments, comprising low-dose taxol or codepletion of the spindle proteins HAUS8 or NuMA. The rescue of mitotic errors was due to shortening of the excessively long overlaps of antiparallel microtubules, serving as a platform for motor proteins that drive the flux, which in turn slowed down the overly fast flux and improved chromosome alignment. In contrast to the prevailing view, the rescue was not accompanied by reduction of overall microtubule growth rates. Instead, speckle microscopy revealed that the improved chromosome alignment in the rescue treatments was associated with slower growth and flux of kinetochore microtubules. In a similar manner, a low-dose taxol treatment rescued mitotic errors in a high-grade serous ovarian carcinoma cell line OVKATE. Collectively, our results highlight the potential of targeting microtubule poleward flux to modify chromosome instability and provide insight into the mechanism through which low doses of taxol rescue certain mitotic errors in cancer cells.

Auxiliary effect of trolox on coenzyme Q10 restricts angiogenesis and proliferation of retinoblastoma cells via the ERK/Akt pathway

Reactive oxygen species (ROS) are essential for cancer signalling pathways and tumour maintenance, making ROS targeting a promising anti-cancer strategy. Coenzyme Q10 (CoQ10) has been shown to be effective against various cancers, but its impact on retinoblastoma, alone or with trolox, remains unreported. Cytotoxicity of CoQ10 alone and with trolox was evaluated in normal human retinal pigment epithelium cells (ARPE-19) and Y79 retinoblastoma cells using CCK-8. Flow cytometry was used to assess apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Anti-angiogenic potential was tested using human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM) assays. Mechanistic studies were conducted via RT-PCR and western blotting. CoQ10, alone and with trolox, reduced Y79 cell viability, induced apoptosis through excess ROS generation, and decreased MMP significantly. Both treatments caused G2/M phase cell arrest. The CAM assay showed a significant reduction in endothelial cell proliferation, evidenced by fewer number of co-cultured HUVECs when exposed to CoQ10 or CoQ10 with trolox. The combination of CoQ10 and trolox significantly reduced VEGF-A, ERK, and Akt receptor levels, while CoQ10 alone significantly inhibited ERK and Akt phosphorylation. Together, CoQ10 and trolox reduced protein expression of VEGFA. CoQ10 alone and with trolox, induces apoptosis in Y79 retinoblastoma cells by inhibiting the ERK/Akt pathway and downregulating VEGFA. This study is the first to report the in vitro and in-ovo anti-cancer potential of CoQ10 alone or when combined with trolox, on human retinoblastoma Y79 cells.

Investigation of the Efficacy of Nowarta110 in the Treatment of HPV-16 and HPV-18 Oncogenically-transformed Human Cells and Cancer-implanted Animal Models.

Cervical cancer is the third leading cause of cancer-related death in women worldwide. Nearly all cases of cervical cancer are due to infection with human papillomavirus (HPV). Nowata110 has shown breakthrough therapeutic efficacy in phase II clinical study against plantar warts, with no reported side effects. This study evaluated the effectiveness of Nowarta110 in killing cultured HPV-transformed cancer cells and its anti-cancer effects in mice, using both vaginally engrafted and subcutaneously implanted animal cancer models. Nowata110 is a novel compound composed of colloidal silver and fig extract. The cytotoxic properties of Nowarta110 were evaluated on HPV-16 and HPV-18-transformed human cancer cells (ARPE-19/HPV-16 and HeLa cells, respectively). The therapeutic potential of Nowarta110 in vivo was evaluated following the engraftment of ME-180 epidermoid carcinoma cells in the mouse vagina or their subcutaneous implantation. Nowarta110 treatment was initiated when the tumor reached an approximate volume of 65 mm3 Results: Our data showed that HPV16-expressing human retinal pigmented epithelial cells are susceptible to Nowarta110-induced cell death, with a reduction in cell viability observed at 1% Nowarta110 and a complete absence of viable cells at 5% Nowarta110. In HPV18-expressing HeLa cells, Nowarta110 caused significant and rapid cell death at a dose of 5%, whereas lower doses of 1% did not produce the same effects. The results from animal studies indicated that Nowarta110 effectively induced tumor regression in both the intravaginal and subcutaneous tumor models. Nowarta110 effectively induced cell death in HPV-16 and HPV-18-transformed human cancer cells. It also effectively induced tumor regression in mouse models with intravaginally engrafted or subcutaneously implanted cancer cells. Considering the high prevalence of HPV-induced cervical dysplasia and cancer and the lack of treatment, clinical studies to promote the implementation of Nowarta110 are warranted.

Prominin-1 Knockdown Causes RPE Degeneration in a Mouse Model.

There are currently no effective treatments for retinal pigment epithelial (RPE) cell loss in atrophic AMD (aAMD). However, our research on Prominin-1 (Prom1), a known structural protein in photoreceptors (PRs), has revealed its distinct role in RPE and offers promising insights. While pathogenic Prom1 mutations have been linked to macular diseases with RPE atrophy, the broader physiological impact of dysfunctional Prom1 in RPE loss is unclear. We have shown that Prom1 plays a crucial role in regulating autophagy and cellular homeostasis in human and mouse RPE (mRPE) cells in vitro. Nevertheless, a comprehensive understanding of its in vivo expression and function in mRPE remains to be elucidated. To characterize Prom1 expression in RPE in situ, we used RNAscope assays and immunogold electron microscopy (EM). Our use of chromogenic and fluorescent RNAscope assays in albino and C57BL/6J mouse retinal sections has revealed Prom1 mRNA expression in perinuclear regions in mRPE in situ. Immunogold EM imaging showed Prom1 expression in RPE cytoplasm and mitochondria. To confirm Prom1 expression in RPE, we interrogated human RPE single-cell RNA-sequencing datasets using an online resource, Spectacle. Our analysis showed Prom1 expression in human RPE. To investigate Prom1's function in RPE homeostasis, we performed RPE-specific Prom1 knockdown (KD) using subretinal injections of AAV2/1.CMV.saCas9.U6.Prom1gRNA in male and female mice. Our data show that RPE-specific Prom1-KD in vivo resulted in abnormal RPE morphology, subretinal fluid accumulation, and secondary PR loss. These changes were associated with patchy RPE cell death and reduced a-wave amplitude, indicating retinal degeneration. Our findings underscore the central role of Prom1 in cell-autonomous mRPE homeostasis. The implications of Prom1-KD causing aAMD-like RPE defects and retinal degeneration in a mouse model are significant and could lead to novel treatments for aAMD.

CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation.

Establishing the correct kinetochore-microtubule attachment is crucial for faithful chromosome segregation. The kinetochore has various regulatory mechanisms for establishing correct bipolar attachment. However, how the regulations are coupled is not fully understood. Here, we demonstrate a regulatory loop between the kinetochore protein CENP-C and Aurora B kinase, which is critical for the error correction of kinetochore-microtubule attachment. This regulatory loop is mediated through the binding of CENP-C to the outer kinetochore Mis12 complex (Mis12C). Although the Mis12C-binding region of CENP-C is dispensable for mouse development and proliferation in human RPE-1 cells, those cells lacking this region display increased mitotic defects. The CENP-C-Mis12C interaction facilitates the centromeric recruitment of Aurora B and the mitotic error correction in human cells. Given that Aurora B reinforces the CENP-C-Mis12C interaction, our findings reveal a positive regulatory loop between Aurora B recruitment and the CENP-C-Mis12C interaction, which ensures chromosome biorientation for accurate chromosome segregation.

Metabolomic signature in ocular dosing: Exploring the metabolic impacts of sublethal high-dose naringenin on ARPE-19 cells

IntroductionNaringenin (NRG), a flavanone polyphenol found in citrus fruits, has been increasingly recognized for its potential therapeutic effects in ocular disorders. This study aimed to assess the impact of high-dose NRG on human retinal pigment epithelial cells (ARPE-19) cells through metabolomic analysis. MethodsCell viability was evaluated using the thiazolyl blue tetrazolium bromide (MTT) assay, demonstrating non-cytotoxicity at concentrations ranging from 3 to 100 μM within a 24-h exposure period. High-dose (100 μM) NRG was selected for further investigation based on its non-cytotoxic nature. Chromatographic analyses were performed using Liquid Chromatography Quadropole Time-of-Flight Mass Spectrometry (LC-Q-ToF-MS). ResultsMetabolomic analysis revealed subtle metabolic changes, with similar profiles between control and test groups, emphasizing the nuanced effects of NRG. Multivariate analysis, including Principal Component Analysis (PCA), illustrated distinct clustering of control and test groups, indicating consistent metabolic adjustments within each group. Despite maintaining cell viability, stress responses were identified in ARPE-19 cells, as evidenced by reduced glucose-6-phosphate levels and alterations in pyrimidine/purine pathways. This highlights the importance of exploring cellular responses using metabolomics beyond traditional viability metrics. ConclusionIn the broader context, this study contributes data on the interplay between NRG and retinal pigmented epithelial. As NRG is utilized in routine ocular applications, particularly as an eye drop, our findings suggest careful dosage selection, considering both non-irritability and subtle metabolic changes. Further research is needed to refine dosage strategies and comprehensively assess the safety profile of NRG in ocular applications.

Transplacental Transfer of Oxytocin and Its Impact on Neonatal Cord Blood and In Vitro Retinal Cell Activity.

The development of fetal organs can be impacted by systemic changes in maternal circulation, with the placenta playing a pivotal role in maintaining pregnancy homeostasis and nutrient exchange. In clinical obstetrics, oxytocin (OXT) is commonly used to induce labor. To explore the potential role of OXT in the placental homeostasis of OXT, we compared OXT levels in neonatal cord blood among neonates (23-42 weeks gestation) whose mothers either received prenatal OXT or experienced spontaneous labor. Our previous research revealed that the oxytocin receptor (OXTR), essential in forming the blood-retina barrier, is expressed in the retinal pigment epithelium (RPE). We hypothesized that perinatal OXT administration might influence the development of the neural retina and its vasculature, offering therapeutic potential for retinal diseases such as retinopathy of prematurity (ROP). Plasma OXT levels were measured using a commercial OXT ELISA kit. Human fetal RPE (hfRPE) cells treated with OXT (10 µM) were assessed for gene expression via RNA sequencing, revealing 14 downregulated and 32 upregulated genes. To validate these differentially expressed genes (DEGs), hfRPE cells were exposed to OXT (0.01, 0.1, 1, or 10 µM) for 12 h, followed by RNA analysis via real-time PCR. Functional, enrichment, and network analyses (Gene Ontology term, FunRich, Cytoscape) were performed to predict the affected pathways. This translational study suggests that OXT likely crosses the placenta, altering fetal OXT concentrations. RNA sequencing identified 46 DEGs involved in vital metabolic and signaling pathways and critical cellular components. Our results indicate that the perinatal administration of OXT may affect neural retina and retinal vessel development, making OXT a potential therapeutic option for developmental eye diseases, including ROP.

PSAT1 is upregulated by METTL3 to attenuate high glucose-induced retinal pigment epithelial cell apoptosis and oxidative stress

BackgroundDiabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported.MethodsHigh-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay.ResultsPSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation.ConclusionMETTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR.

Protective effect of zinc against A2E-induced toxicity in ARPE-19 cells: Possible involvement of lysosomal acidification

A key pathogenic mechanism of dry age-related macular degeneration (AMD) is lysosomal dysfunction in retinal pigment epithelium (RPE) cells, which results in the accumulation of lipofuscins such as A2E (N-retinylidene-N-retinylethanolamine) that further compromises lysosomal function. This vicious cycle leads to cell death and poor visual acuity. Here, we established an in vitro model of AMD by treating a human RPE cell line (ARPE-19) with A2E and examined whether raising zinc levels confers protective effects against lysosomal dysfunction and cytotoxicity. MTT assay showed that A2E induced apoptosis in ARPE-19 cells. pHrodo™ Red fluorescence staining showed that lysosomal pH increased in A2E-treated ARPE-19 cells. Treatment with a zinc ionophore (clioquinol) reduced A2E accumulation, restored lysosomal pH to the acidic range, and reduced A2E-induced cell death, all of which were reversed by the addition of a zinc chelator (TPEN). Consistent with the in vitro results, subretinal injections of A2E in mouse eyes resulted in the death of RPE cells as well as lysosomal dysfunction, all of which were reversed by co-treatment with clioquinol. Our results suggest that restoring the levels of intracellular zinc, especially in lysosomes, would be helpful in mitigating A2E-induced cytotoxic changes including lysosomal dysfunction in RPE cells in the pathogenesis of AMD.

Proteomics Analysis on the Effects of Oxidative Stress and Antioxidants on Proteins Involved in Sterol Transport and Metabolism in Human Telomerase Transcriptase-Overexpressing-Retinal Pigment Epithelium Cells.

Age-related macular degeneration (AMD) is the most prevalent ocular disease in the elderly, resulting in blindness. Oxidative stress plays a role in retinal pigment epithelium (RPE) pathology observed in AMD. Tocopherols are potent antioxidants that prevent cellular oxidative damage and have been shown to upregulate the expression of cellular antioxidant proteins. Here, we determined whether oxidative stress and tocopherols, using either normal cellular conditions or conditions of sublethal cellular oxidative stress, alter the expression of proteins mediating sterol uptake, transport, and metabolism. Human telomerase transcriptase-overexpressing RPE cells (hTERT-RPE) were used to identify differential expression of proteins resulting from treatments. We utilized a proteomics strategy to identify protein expression changes in treated cells. After the identification and organization of data, we divided the identified proteins into groups related to biological function: cellular sterol uptake, sterol transport and sterol metabolism. Exposure of cells to conditions of oxidative stress and exposure to tocopherols led to similar protein expression changes within these three groups, suggesting that α-tocopherol (αT) and γ-tocopherol (γT) can regulate the expression of sterol uptake, transport and metabolic proteins in RPE cells. These data suggest that proteins involved in sterol transport and metabolism may be important for RPE adaptation to oxidative stress, and these proteins represent potential therapeutic targets.

Tropisetron attenuates high glucose-induced oxidative stress and inflammation in ARPE-19 cells in vitro via regulating SIRT1/ROCK1 signaling.

Diabetic retinopathy (DR) is the leading cause of acquired blindness in diabetic patients. Tropisetron (TRO) exerts potent therapeutic effects against diabetic tissues. The present study aimed to investigate the effects of TRO on retinal injury under diabetic condition. Human retinal pigment epithelial cell line ARPE-19 was treated with high glucose (HG) for 48 h to mimic hyperglycemia-induced retinal damage and subsequently treated with multiple concentrations of TRO for therapeutic intervention. Cell viability and lactate dehydrogenase (LDH) release were detected to assess cell damage. The production of inflammatory cytokines and oxidative stress-related factors was evaluated by corresponding commercial kits. Cell apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The expression of inflammation-, apoptosis-, and SIRT1/ROCK1-related proteins was examined using western blot analysis. Additionally, ARPE-19 cells were transfected with over-express ROCK1 (Ov-ROCK1) or pretreatment with SIRT1 inhibitor EX527 to perform the rescue experiments. TRO alleviated cell damage in HG-induced ARPE-19 cells through elevating cell viability and reducing LDH release. HG-caused excessive production of TNF-α, IL-1β and IL-6, ROS, malondialdehyde and decreased superoxide dismutase activity were partly inhibited by TRO treatment. HG-induced cell apoptosis, accompanied with the upregulation of proapoptotic proteins and the downregulation of antiapoptotic proteins, was hindered by TRO treatment. HG led to the loss of SIRT1 and an elevation of ROCK1 in ARPE-19 cells, which was reversed following TRO treatment. Furthermore, pretreatment with EX527 or transfected with Ov-ROCK1 partially abolished the protective role of TRO against inflammation, oxidative stress and cell apoptosis in HG-challenged ARPE-19 cells. TRO exerted a protective role against HG-caused ARPE-19 cells inflammation, oxidative stress and cell apoptosis by regulating SIRT1/ROCK1 axis, suggesting that TRO might be therapeutic agent for alleviating retinal pigment epithelial cell damage in DR.

Brief research report: Transcriptional blockade of angiotensin converting enzyme 2 modelled in human retinal pigment epithelial cells

As a key host protein involved in cellular infection by the severe acute respiratory syndrome coronavirus (SARS-CoV-2), angiotensin converting enzyme (ACE)2 is an ideal target for antiviral drugs. Manipulation of transcription provides opportunity for graduated blockade that preserves physiological functions. We sought to develop a model system for evaluating manipulation of ACE2 gene transcription using human retinal pigment epithelium. Retinal pigment epithelial cell isolates were prepared from human posterior eyecups (n = 11 individual isolates). The cells expressed ACE2 transcript and protein, and expression was not induced by hypoxia mimetic dimethyloxaloylglycine, or inflammatory cytokine IL-1β. ACE2 gene transcription factors were predicted in silico and cross-referenced with the human retinal pigment epithelial cell transcriptome, and five candidate transcription factors were identified: ETS proto-oncogene 1 transcription factor (ETS1), nuclear factor I C (NFIC), nuclear receptor subfamily 2 group C member 1 (NR2C1), TEA domain transcription factor 1 (TEAD1), and zinc finger protein 384 (ZNF384). The candidates were individually targeted in cells by transfection with small interfering (si)RNA. Knockdowns reduced mean cellular expression of all the transcription factors in comparison to expression in cells transfected with control non-targeted siRNA. Mean cellular ACE2 transcript was reduced under the condition of NR2C1 knockdown, but not for ETS1, NFIC, TEAD1, and ZNF384 knockdowns. Our findings build on previous work demonstrating the potential for drugging gene transcription. Importantly, we show the value of human retinal pigment epithelium as a system for evaluating ACE2 transcriptional blockade, a possible approach for treating SARS-CoV-2 infection. Brief Research Report.

Overexpressed Poldip2 Incurs Retinal Fibrosis via the TGF-β1/SMAD3 Signaling Pathway in Diabetic Retinopathy.

Retinal fibrosis is one of the major features of Diabetic retinopathy. Our recent research has shown that Poldip2 can affect early DR through oxidative stress, but whether or not Poldip2 would regulate retinal fibrosis during DR development is still enigmatic. Here, Diabetic Sprague-Dawley (SD) rats were induced with STZ and treated with AAV9-Poldip2shRNA, while human retinal pigment epithelial cells (ARPE-19) were treated with high glucose (HG) or Poldip2 siRNA. We identified that in STZ-induced DR rats and ARPE-19 treated with high glucose, the expression of Poldip2, TGFβ1, P-SMAD3/SMAD3, MMP9, COL-1, FN, and CTGF increased while the expression of Cadherin decreased. However, deleting Poldip2 inhibited the TGF-β1/SMAD3 signaling pathway and attenuated the above protein expression in vivo and in vitro. Mechanistically, we found that Poldip2 promotes the activation of SMAD3, and facilitates its nuclear translocation through interacting with it, and significantly enhances the expression of fibrosis makers. Collectively, it was identified that Poldip2 is a novel regulator of DR fibrosis and it is expected to become a therapeutic target for PDR.