Single-Cell RNA Sequencing Reveals Transcriptional Signatures and Cell-Cell Communication in Diabetic Retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of vision loss in workingage individuals worldwide. Cell-to-cell communication between retinal cells and retinal pigment epithelial cells (RPEs) in DR is still unclear, so this study aimed to generate a single-cell atlas and identify receptor‒ligand communication between retinal cells and RPEs. METHODS: A mouse single-cell RNA sequencing (scRNA-seq) dataset was retrieved from the GEO database (GSE178121) and was further analyzed with the R package Seurat. Cell cluster annotation was performed to further analyze cell‒cell communication. The differentially expressed genes (DEGs) in RPEs were explored through pathway enrichment analysis and the protein‒ protein interaction (PPI) network. Core genes in the PPI were verified by quantitative PCR in ARPE-19 cells. RESULTS: We observed an increased proportion of RPEs in STZ mice. Although some overall intercellular communication pathways did not differ significantly in the STZ and control groups, RPEs relayed significantly more signals in the STZ group. In addition, THBS1, ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 were found to be the core DEGs of the PPI network in RPEs. qPCR results showed that the expression of ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 was higher and consistent with scRNA-seq results in ARPE-19 cells under hyperglycemic conditions. CONCLUSION: Our study, for the first time, investigated how signals that RPEs relay to and from other cells underly the progression of DR based on scRNA-seq. These signaling pathways and hub genes may provide new insights into DR mechanisms and therapeutic targets.

Exosomes-based dual drug-loaded nanocarrier for targeted and multiple proliferative vitreoretinopathy therapy.

Proliferative vitreoretinopathy (PVR) is a common cause of vision loss after retinal reattachment surgery and ocular trauma. The key pathogenic mechanisms of PVR development include the proliferation, migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) activated by the growth factors and cytokines after surgery. Although some drugs have been tried in PVR treatments as basic investigations, the limited efficacy remains an obstacle, which may be due to the single pharmacological action and lack of targeting. Herein, the anti-proliferative Daunorubicin and anti-inflammatory Dexamethasone were co-loaded in the RPEs-derived exosomes (Exos), obtaining an Exos-based dual drug-loaded nanocarrier (Exos@D-D), and used for multiple PVR therapy. Owing to the advantages of homologous Exos and the dual drug loading, Exos@D-D showed good RPEs targeting as well as improved uptake efficiency, and could inhibit the proliferation, migration, as well as EMT of RPEs effectively. The animal studies have also demonstrated that Exos@D-D effectively inhibits the production of proliferative membranes and prevents the further development of inflammation, shows significant therapeutic effects on PVR and good biocompatibility. Such Exos-based dual drug-loaded nanocarrier investigation not only provides a promising approach for multifunctional exosome drug delivery systems construction, but also has great potential in PVR clinical therapy application.

Mesenchymal stem cells-derived exosomes alleviate senescence of retinal pigment epithelial cells by activating PI3K/AKT-Nrf2 signaling pathway in early diabetic retinopathy.

Diabetic retinopathy (DR) is a major cause of vision loss and blindness in adults. Cellular senescence was involved in the pathogenesis of early-stage DR and is positively correlated with progression. Thus, our study aimed at exploring the effect and potential mechanism of Mesenchymal stem cells-derived exosomes (MSCs-EXOs) on Retinal Pigment Epithelial (RPE) cells senescence at an early stage of DR in vivo and in vitro. ARPE-19 cells were incubated in high glucose (HG) medium mixed with MSCs-EXOs to observe the changes in cell viability. Senescence-associated ß-galactosidase (SA-ß-gal) staining, Western blot and qRT-PCR were used to assess the expression of senescence-related genes and antioxidant mediators. Quantitative Real-Time polymerase chain reaction (qRT-PCR), Optical coherence tomography (OCT) Hematoxylin and eosin (HE) staining and Electroretinogram (ERG) were respectively used to verify cellular senescence, the structure and function of the retina. Our findings demonstrated that MSCs-EXOs inhibited HG-induced senescence in ARPE-19 cells. Furthermore, MSCs-EXOs reduced HG-induced cell apoptosis and oxidative stress levels while promoting cell proliferation. Mechanistically, HG suppressed PI3K/AKT phosphorylation as well as nuclear factor erythroid 2-related factor 2 (Nrf2) expression along with its downstream target gene expression in ARPE-19 cells. However, MSCs-EXOs reversed these changes by alleviating cellular senescence while enhancing antioxidant activity. In line with our results in vitro, MSCs-EXOs significantly ameliorated hyperglycemia-induced senescence in DR mice by downregulating mRNA expression of P53, P21, P16, and SASP. Additionally, MSCs-EXOs improved the functional and structural integrity of the retina in DR mice. Our study revealed the protective effect of MSCs-EXOs on cellular senescence, offering new insights for the treatment of DR.

Vitamin D and Sulforaphane Decrease Inflammatory Oxidative Stress and Restore the Markers of Epithelial Integrity in an In Vitro Model of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is strictly linked to chronic oxidative stress, inflammation, loss of epithelial barrier integrity, and often with abnormal new blood vessel development. In this study, the retinal epithelial cell line ARPE-19 was treated with pro-inflammatory transforming growth factor-beta (TGF-ß) to investigate the activity of vitamin D (VD) and sulforaphane (SF) in abating the consequences of oxidative stress and inflammation. The administration of VD and SF lowered reactive oxygen species (ROS) levels, and abated the related expression of the pro-inflammatory cytokines interleukin-6 and interleukin-8 induced by TGF-ß. We evaluated mitochondrial respiration as a source of ROS production, and we discovered that the increased transcription of respiratory elements triggered by TGF-ß was prevented by VD and SF. In this model of inflamed epithelium, the treatment with VD and SF also reduced the secretion of VEGF, a key angiogenic factor, and restored the markers of epithelial integrity. Remarkably, all the observed biological effects were potentiated by the co-stimulation with the two compounds and were not mediated by VD receptor expression but rather by the ERK 1/2 pathway. Altogether, the results of this study reveal the powerful synergistic anti-inflammatory activity of SF and VD and lay the foundation for future clinical assessment of their efficacy in AMD.

Advances in the study of tissue-engineered retinal pigment epithelial cell sheets.

Regarded as the most promising treatment modality for retinal degenerative diseases, retinal pigment epithelium cell replacement therapy holds significant potential. Common retinal degenerative diseases, including Age-related Macular Degeneration, are frequently characterized by damage to the unit comprising photoreceptors, retinal pigment epithelium, and Bruch's membrane. The selection of appropriate tissue engineering materials, in conjunction with retinal pigment epithelial cells, for graft preparation, can offer an effective treatment for retinal degenerative diseases. This article presents an overview of the research conducted on retinal pigment epithelial cell tissue engineering, outlining the challenges and future prospects.

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

AIM: To explore the effects of hepatocyte growth factor (HGF) on retinal pigment epithelium (RPE) cell behaviors. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in retinal pigment epithelial cells.

Mechanical stimulation as a mimic of drusen formation in the eye increases the expression of angiogenic factors in retinal pigment epithelial (RPE) cells, but the underlying molecular mechanisms remain unclear. We investigated and characterized the effects of mechanical stimulation on the expression of angiogenic factors in RPE cells both in vitro and in a mouse model. Mechanical stimulation increased the expression of vascular endothelial growth factor (VEGF, encoded by VEGFA) and other angiogenesis-related genes in cultured RPE1 cells. The presence of hypoxia-inducible factor 1α (HIF-1α, encoded by HIF1A) was also increased, and both knockdown of HIF-1α and treatment with the HIF-1α inhibitor CAY10585 attenuated the effect of mechanical stimulation on angiogenesis factor gene expression. Signaling by the tyrosine kinase SRC and p38 mitogen-activated protein kinase was involved in HIF-1α activation and consequent angiogenesis-related gene expression induced by mechanical stimulation. Our results suggest that SRC-p38 and HIF-1α signaling are involved in the upregulation of angiogenic factors in RPE cells by mechanical stimulation. Such in vivo suppression of upregulated expression of angiogenesis-related genes by pharmacological inhibitors of HIF-1α suggests a new potential approach to the treatment of age-related macular degeneration.

Epigenetic mechanism of SET7/9-mediated histone methylation modification in high glucose-induced ferroptosis in retinal pigment epithelial cells.

Ferroptosis of the retinal pigment epithelial (RPE) cells leads to retinal neuron injury and even visual loss. Our study aims to investigate the role of the SET domain with lysine methyltransferase 7/9 (SET7/9) in regulating high glucose (HG)-induced ferroptosis in RPE cells. The cell model was established by HG treatment. The levels of SET7/9 and Sirtuin 6 (SIRT6) were inhibited and Runt-related transcription factor 1 (RUNX1) was overexpressed through cell transfection, and then their levels in ARPE-19 cells were detected. Cell viability and apoptosis was detected. The levels of reactive oxygen species, malondialdehyde, glutathione, ferrous ion, glutathione peroxidase 4, and acyl-CoA synthetase long-chain family member 4 were detected. SET7/9 and trimethylation of histone H3 at lysine 4 (H3K4me3) levels in the RUNX1 promoter region and RUNX1 level in the SIRT6 promoter region were measured. The relationship between RUNX1 and SIRT6 was verified. SET7/9 and RUNX1 were highly expressed while SIRT6 was poorly expressed in HG-induced ARPE-19 cells. SET7/9 inhibition increased cell viability and inhibited cell apoptosis and ferroptosis. Mechanistically, SET7/9 increased H3K4me3 on the RUNX1 promoter to promote RUNX1, and RUNX1 repressed SIRT6 expression. Overexpression of RUNX1 or silencing SIRT6 partially reversed the inhibitory effect of SET7/9 silencing on HG-induced ferroptosis. In conclusion, SET7/9 promoted ferroptosis of RPE cells through the SIRT6/RUNX1 pathway.

Artemisinin Confers Cytoprotection toward Hydrogen Peroxide-Induced Cell Apoptosis in Retinal Pigment Epithelial Cells in Correlation with the Increased Acetylation of Histone H4 at Lysine 8.

Increased oxidative stress is one of the critical pathologies inducing age-related macular degeneration (AMD), characterized by retinal pigment epithelial (RPE) cell damage and death. The unbalanced acetylation and deacetylation of histones have been implicated in AMD pathogenesis or hydrogen peroxide (H2O2)-induced cell damage. Therefore, strategies aimed at controlling the balance between acetylation and deacetylation may effectively protect RPE cells from oxidative damage. Artemisinin is an antimalarial lactone drug derived from Artemisia annua, with antioxidant activity known to modulate histone acetylation in the brain, but its effect on the retina is unknown. In this study, we aimed to investigate whether Artemisinin exerts a cytoprotective effect on oxidative stress-induced apoptosis in RPE cells by regulating histone acetylation. We hypothesized that Artemisinin confers cytoprotection toward H2O2-induced apoptosis in RPE cells through this mechanism. In the present study, we found that Artemisinin at a sub-clinic dosage of 20 µM inhibited the H2O2-induced cell viability decrease and B-cell lymphoma 2 (Bcl-2) protein level decrease and attenuated the H2O2-induced decrease in the histone H4 lysine (Lys) 8 acetylation [Acetyl-H4 (Lys 8)] level in the retinal RPE cell line D407. As expected, histone deacetylase inhibitor Trichostatin A at the concentration of 250 nM increased the Acetyl-H4 (Lys 8) level in D407 cells and attenuated the H2O2-induced cell viability decrease and apoptosis. Similar findings were obtained using adult RPE (ARPE)19 cells, another human RPE cell line, and primary human RPE cell cultures. In conclusion, these results confirmed our hypothesis and indicated that Artemisinin attenuated H2O2-induced apoptosis in apparent correlation with the increase in the Acetyl-H4 (Lys 8) level, which is associated with gene transcription and cell survival. By modulating histone acetylation, Artemisinin may restore the balance between acetylation and deacetylation and enhance the resistance and survival of RPE cells under oxidative stress. Our study provides novel mechanistic insights into the effect of Artemisinin on histone acetylation and apoptosis in RPE cells and supports the potential application of Artemisinin in the prevention and/or treatment of AMD.

SUMOylation of GMFB regulates its stability and function in retinal pigment epithelial cells under hyperglycemia.

BACKGROUND: Glia maturation factor beta (GMFB) is a growth and differentiation factor that acts as an intracellular regulator of signal transduction pathways. The small ubiquitin-related modifier (SUMO) modification, SUMOylation, is a posttranslational modification (PTM) that plays a key role in protein subcellular localization, stability, transcription, and enzymatic activity. Recent studies have highlighted the importance of SUMOylation in the inflammation and progression of numerous diseases. However, the relationship between GMFB and SUMOylation is unclear. RESULTS: Here, we report for the first time that GMFB and SUMO1 are markedly increased in retinal pigment epithelial (RPE) cells at the early stage of diabetes mellitus (DM) under hyperglycemia. The GMFΒ protein could be mono-SUMOylated by SUMO1 at the K20, K35, K58 or K97 sites. SUMOylation of GMFB led to its increased protein stability and subcellular translocation. Furthermore, deSUMOylation of GMFΒ downregulates multiple signaling pathways, including the Jak-STAT signaling pathway, p38 pathway and NF-kappa B signaling pathway. CONCLUSIONS: This work provides novel insight into the role of SUMOylated GMFB in RPE cells and provides a novel therapeutic target for diabetic retinopathy (DR).

Bone morphogenetic protein-6 suppresses TGF-ß2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

AIM: To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-ß2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). METHODS: Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-ß2 (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-ß2+empty plasmid group, BMP-6 overexpression group, and TGF-ß2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. RESULTS: Compared with the control group, the migration of RPE cells in the TGF-ß2 group was significantly enhanced. TGF-ß2 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-ß2 and prevented TGF-ß2 from affecting EMT-related biomarkers (P<0.05). CONCLUSION: BMP-6 prevents the EMT in RPE cells induced by TGF-ß2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

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

Objective: 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. Methods: 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. Results: 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-ß, 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. Conclusion: 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.

Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-ß2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-ß2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-ß2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-ß2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

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.

Molecular Network Mechanism Analysis of Urine Stem Cells Against Retinal Aging.

To investigate the effect and potential mechanism of human-derived urine stem cells (hUSCs) in inhibiting retinal aging by using experimental and bioinformatics. Retinal pigment epithelial cells cultured in vitro, which were randomly divided into normal group, aging group and supernatant of hUSCs group. Cell counting kit-8 detection, senescence-related ß-galactosidase, and Annexin V/PI staining were performed to detect cell viability, senescence, and apoptosis. Subsequently, bioinformatics methods were used to explore the underlying mechanisms, in which, targets both hUSCs and aging retina-related targets were obtained from GeneCards. Then, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and protein-protein interaction network were analysis, and the expressional level of hub gene was validated by q-PCR. Supernatant addition of hUSCs promoted markedly cellular proliferation, improved viability and inhibited senescence and apoptosis in vitro. A total of 1476 hUSCs-related targets (Relevance score > 20), 692 retinal disease-related targets, and 732 targets related to disease of aging were selected from GeneCards database, and 289 common targets of hUSCs against aging retina were confirmed through Venn analysis. Enrichment analysis demonstrated that hUSCs might exert its anti-apoptosis efficacy in multiple biological processes, including oxidative stress, inflammation and apoptosis, and core targets were associated with HIF-1, MAPK and PI3K-Akt signal. hUSCs inhibited retinal senescence by regulating multiply targets and signaling pathways, of these, HIF-1, MAPK, and PI3K may be important candidates.

DEL-1: a promising treatment for AMD-associated ER stress in retinal pigment epithelial cells.

BACKGROUND: Age-related macular degeneration (AMD) is an irreversible eye disease that can cause blurred vision. Regular exercise has been suggested as a therapeutic strategy for treating AMD, but how exercise improves AMD is not yet understood. This study investigated the protective effects of developmental endothelial locus-1 (DEL-1), a myokine upregulated during exercise, on endoplasmic reticulum (ER) stress-induced injury in retinal pigment epithelial cells. METHODS: We evaluated the levels of AMPK phosphorylation, autophagy markers, and ER stress markers in DEL-1-treated human retinal pigment epithelial cells (hRPE) using Western blotting. We also performed cell viability, caspase 3 activity assays, and autophagosome staining. RESULTS: Our findings showed that treatment with recombinant DEL-1 dose-dependently reduced the impairment of cell viability and caspase 3 activity in tunicamycin-treated hRPE cells. DEL-1 treatment also alleviated tunicamycin-induced ER stress markers and VEGF expression. Moreover, AMPK phosphorylation and autophagy markers were increased in hRPE cells in the presence of DEL-1. However, the effects of DEL-1 on ER stress, VEGF expression, and apoptosis in tunicamycin-treated hRPE cells were reduced by AMPK siRNA or 3-methyladenine (3-MA), an autophagy inhibitor. CONCLUSIONS: Our study suggests that DEL-1, a myokine, may have potential as a treatment strategy for AMD by attenuating ER stress-induced injury in retinal pigment epithelial cells.

Gigantol protects retinal pigment epithelial cells against high glucose-induced apoptosis, oxidative stress and inflammation by inhibiting MTDH-mediated NF-kB signaling pathway.

OBJECTIVE: As a frequent complication of diabetes mellitus (DM), diabetic retinopathy (DR) is now one of the major causes of blindness. Recent reports have shown that retinal pigment epithelial cell (RPEC) damage plays an essential part in DR development and progression. This work intended to explore the potential effects of Gigantol on high glucose (HG)-stimulated RPEC damage and identify potential mechanisms. METHODS: Cell viability, cell damage, and cell apoptosis were evaluated by CCK-8, lactate dehydrogenase (LDH) and flow cytometry assays. The levels of oxidative stress biomarkers and pro-inflammatory cytokines were assessed using corresponding commercial kits and ELISA. Additionally, the levels of MTDH and NF-kB signaling pathway-related proteins were detected by western blotting. RESULTS: Gigantol dose-dependently enhanced cell viability and decreased apoptosis in HG-challenged ARPE-19 cells. Also, Gigantol notably relieved oxidative stress and inflammatory responses in ARPE-19 cells under HG conditions. Gigantol dose-dependently suppressed MTDH expression. In addition, MTDH restoration partially counteracted the protective effects of Gigantol on ARPE-19 cells subject to HG treatment. Mechanically, Gigantol inactivated the NF-kB signaling pathway, which was partly restored after MTDH overexpression. CONCLUSION: Our findings suggested that Gigantol protected against HG-induced RPEC damage by inactivating the NF-kB signaling via MTDH inhibition, offering a potent therapeutic drug for DR treatment.

Metformin protects retinal pigment epithelium cells against H2O2-induced oxidative stress and inflammation via the Nrf2 signaling cascade.

PURPOSE: Dysfunctions of retinal pigment epithelium (RPE) attributed to oxidative stress and inflammation are implicated with age-related macular degeneration (AMD). A debate on the curative role of metformin in AMD has been raised, though several recent clinical studies support the lower odds by using metformin. This study aimed to determine whether metformin could exert cytoprotection against RPE oxidative damages and the potential mechanisms. METHODS: A cellular AMD model was established by treating ARPE-19 cells with hydrogen peroxide (H2O2) for 24 h. The reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and levels of pro-inflammatory cytokines were monitored under administrations with H2O2 with/without metformin. The expression and DNA-binding activity of transcription factor erythroid-related factor 2 (Nrf2) were determined by western blot, immunofluorescence, and electrophoretic mobility shift assay. Knockout of Nrf2 was conducted by CRISPR/Cas9 gene deletion system. RESULTS: Metformin pretreatment significantly improved the H2O2-induced low viability of ARPE-19 cells, reduced ROS production, and increased contents of antioxidative molecules. Concurrently, metformin also suppressed levels of pro-inflammatory cytokines caused by H2O2. The metformin-augmented nuclear translocation and DNA-binding activity of Nrf2 were further verified by the increased expression of its downstream targets. Genetic deletion of Nrf2 blocked the cytoprotective role of metformin. CONCLUSION: Metformin possesses antioxidative and anti-inflammatory properties in ARPE-19 cells by activating the Nrf2 signaling. It supports the potential use for the control and prevention of AMD.

Elemental mass spectrometry to study metallo-transcriptomic changes during the in vitro degeneration of the retinal pigment epithelium.

Trace elements play crucial roles in cellular biology. Their improper homeostasis may contribute to the progress of eye diseases, exacerbated during ageing. The retinal pigment epithelium (RPE) is progressively deteriorated during age-related neurodegeneration and metal homeostasis may be compromised. In this study, elemental mass spectrometry (MS) was combined with cellular and molecular biology techniques to identify changes in trace elements during the in vitro degeneration of human RPE cells. Cells were collected at 21, 91, and 133 days and processed for RNA sequencing; Ca, Na, P, Mg, and Cu quantification by flow injection analysis and inductively coupled plasma-MS; and protein analysis by immunocytochemistry. Four-month-old RPE cultures showed depigmentation, impaired barrier function, and antioxidant protection, manifesting signs of epithelial-to-mesenchymal transition. Na and P significantly increased in the cytosol of degenerated RPE cells (from 15 ± 20 to 13495 ± 638 ng·µg-1 and from 30.6 ± 9.5 to 116.8 ± 16.8 ng·µg-1, respectively). Mg decreased in both the cytosol and insoluble fraction of cells (from 2.83 ± 0.40 to 1.58 ± 0.56 ng·µg-1 and from 247.57 ± 11.06 to 30 ± 8 ng·g-1, respectively), while P and Cu decreased in the insoluble fraction after 133 days in culture (from 9471 ± 1249 to 4555 ± 985 ng·µg-1 and from 2251 ± 79 to 1054 ± 235 ng·g-1, respectively), along with changes in metal-dependent antioxidant enzymes and Cu transporters. This RPE model reflected metal homeostatic changes, providing additional perspectives on effects of metal regulation during ageing.

Rubus suavissimus S. Lee Extract Alleviates Oxidative Stress and Inflammation in H2O2-Treated Retinal Pigment Epithelial Cells and in High-Fat Diet-Fed Mouse Retinas.

BACKGROUND: Age-related macular degeneration (AMD) is the most common cause of visual disorders in the aged population and is characterized by the formation of retinal pigment epithelium (RPE) deposits and dysfunction/death of the RPE and photoreceptors. It is supposed that both oxidative stress and inflammation play a critical role in the pathogenesis of AMD. The development of therapeutic strategies against oxidative stress and inflammation in AMD is urgently needed. Rubus suavissimus S. Lee (RS), a medicinal plant growing in the southwest region of China, has been used as an herbal tea and medicine for various diseases. METHODS: In this project, we evaluate the therapeutic potential of RS extract for AMD. We prepared RS extracts from dried leaves, which contained the main functional compounds. RESULTS: RS extract significantly increased cell viability, upregulated the expression of antioxidant genes, lowered the generation of malondialdehyde and reactive oxygen species, and suppressed inflammation in H2O2-treated human RPE cells. In the in vivo study, treatment with RS extract attenuated body weight gain, lowered cholesterol and triglyceride levels in the liver and serum, increased antioxidant capacity, and alleviated inflammation in the retina and RPE/choroid of mice fed a high-fat diet. CONCLUSIONS: Our findings suggest that RS extract offers therapeutic potential for treating AMD patients.