Retinal Cells Research Articles

Single-Cell Transcriptomic Dataset of RPGR-associated Retinitis Pigmentosa Patient-Derived Retinal Organoids

X-linked retinitis pigmentosa (XLRP) is a severe hereditary retinal disorder marked by progressive vision loss due to photoreceptor dysfunction. The retinitis pigmentosa GTPase regulator (RPGR) gene, responsible for most XLRP cases, encodes a protein crucial for the transport of visual signal proteins between the photoreceptor inner and outer segments. However, the mechanism of RPGR mutation causing photoreceptor disorder is not clear and effective treatments remain elusive. This study utilized retinal organoids (ROs) derived from normal and RPGR-mutant human induced pluripotent stem cells (hiPSC) at four developmental stages (40, 90, 150, and 200 days). Single-cell RNA sequencing (scRNA-seq) was conducted on 71,096 cells, including 33,839 cells from the control group and 37,257 cells from the RPGR group. Key retinal cell types were identified and the obtained scRNAseq dataset was validated reliable and high -quality. This study has provided data resources and references for exploring the mechanism of RPGR-related retinal degeneration and support the development of targeted therapies.

MiR-185-5p is Involved in Regulating the Abnormal Proliferation of Retinal Microvascular Endothelial Cells via Targeting CXCR4.

This study aimed to explore the expression profile of miR-185-5p in proliferative DR (PDR), and further evaluate its diagnostic value and possible mechanism of miR-185-5p in PDR. The level of miR-185-5p was detected by qRT-PCR. The ROC curve was established to estimate the diagnostic ability of miR-185-5p. Transwell experiment and cell counting kit-8 (CCK-8) assays were conducted to assess the effect of miR-185-5p on the migration and proliferation of human retinal endothelial cells (HRECs) induced by high glucose. Enzyme linked immunosorbent assay (ELISA) was used to detect the concentrations of inflammatory factors. The luciferase reporter gene experiment was used to prove the interaction between miR-185-5p and CXCR4. Compared to the control group, the expression of miR-185-5p was significantly up-regulated in both the type 2 diabetes mellitus (T2DM) group and the PDR groups, with higher levels in the PDR group than in the T2DM group. The ROC curve reveals that serum miR-185-5p can distinguish PDR patients from T2DM patients. MiR-185-5p levels in HRECs increased significantly after high glucose induction. High glucose induction also promoted the migration, proliferation and inflammation response of HRECs. However, when the intracellular miR-185-5p level was down-regulated by miR-185-5p inhibitor transfection, these effects were inhibited. The luciferase reporter gene assay showed that miR-185-5p directly targets CXCR4. The expression of miR-185-5p is out of balance in PDR and it may be involved in regulating the migration and proliferation of HRECs by regulating CXCR4.

Regulation of the HMGA2-SNAI2/CXCR4 axis in atherosclerosis and retinal neovascularization: new therapeutic insights.

Atherosclerosis (AS) is a vascular disease associated with endothelial damage, plaque formation, and retinal neovascularization (RNV), leading to visual impairment. Research indicates that vascular endothelial dysfunction, lipid deposition, and inflammatory responses contribute to the formation of plaques and atherosclerotic lesions. Among the common complications, studies have shown that RNV and the molecular mechanisms of AS hold significant clinical importance. In this study, we identified the overexpression of the gene heat shock protein 90 (HSP90) through transcriptome sequencing. Subsequent protein expression analysis and inhibition experiments in corresponding animal models confirmed the crucial role of HSP90 in the modulation of this disease. Research findings revealed an increase in the expression of HSP90, HMGA2, Snail family transcriptional repressor 2 gene (SNAI2), CXC chemokine receptor 4 (CXCR4), and vascular endothelial growth factor (VEGF) in atherosclerotic mouse tissues. Inhibition of HSP90 expression reduced vascular neovascularization and downregulated the expression of HMGA2 and VEGF. Given that HSP90 can promote HMGA2 expression, which, in turn, facilitates angiogenesis, we conducted lentiviral infection experiments on primary retinal endothelial cells obtained from atherosclerotic mice, confirming the regulatory role of HSP90 in modulating HMGA2 expression through the SNAI2/CXCR4 signaling pathway and its involvement in retinal endothelial neovascularization. In conclusion, our study highlights the significant regulatory role of HSP90 in AS-induced RNV, providing a new target for disease treatment. Furthermore, this research extensively explores the mechanism of HSP90 in regulating RNV and associated signaling pathways, offering novel insights and laying a solid foundation for future studies in this disease domain.

Circular RNA HIPK3 mediates epithelial–mesenchymal transition of retinal pigment epithelial cells by sponging multiple microRNAs

Epithelial–mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells plays key roles in the pathogenesis of multiple vitreoretinal diseases, leading to profound and permanent vision loss. Circular RNAs (circRNAs) are widespread and functional endogenous RNAs that could regulate gene expression in eukaryotes. The functions of circRNAs in mediating EMT has been reported in several diseases. In the current study, we investigated the role of circRNA HIPK3 (circHIPK3) in EMT process of RPE cells (RPE-EMT). circHIPK3 is one abundant circRNA generated from the second exon of HIPK3 mRNA. We found that circHIPK3 expression was significantly increased in TGF-β1-induced RPE-EMT model. Silencing of circHIPK3 attenuated TGF-β1-induced RPE-EMT process, whereas forced expression of circHIPK3 could trigger EMT in RPE cells. Mechanistically, circHIPK3 regulates RPE-EMT process via sponging multiple microRNAs (miRNAs). This study provides novel insights into the mechanism of RPE-EMT. Targeting circHIPK3 might serve as a therapeutic strategy in RPE-EMT associated vitreoretinal diseases.

Proteome of Pericytes from Retinal Vasculature of Diabetic Donor Eyes

Retinal pericytes (PCs) are contractile microvascular smooth muscle cells that wrap around the endothelial cells (ECs) maintaining intact retinal vasculature (RV) with a 1:1 ratio. Microvascular complications like Diabetic Retinopathy (DR) due to chronic Diabetes causes apoptotic loss of PCs followed by diminished vessel stability, EC apoptosis, and ischemia, leading to retinal angiogenesis, and eventually severe vision loss. This study aimed to analyze the proteins in PCs isolated from the RV of diabetic human donor eyes and compare them with remaining mixed population (MP) of retinal vascular cells. PCs and MP proteomes were analyzed using semi-quantitative proteomics. Proteins were extracted, quantified, and analyzed in duplicate using LC-MS/MS on a Tandem mass spectrometer. Overall, 42 PC and 27 MP proteins, with 19 shared proteins, were identified. Functional enrichment analysis indicated that PC proteins share common biological processes, such as negative regulation of fibrinolysis and vLDL particle remodeling, nitric oxide transport, phospholipid efflux, positive control over the clearance of apoptotic cells, chondrocyte proliferation, lipoprotein lipase activity, and oxidative stress-induced intrinsic atrophic signaling pathways. In the fold enrichment analysis, the PC proteins were associated with cholesterol metabolism, Complement and coagulant, ECM-receptor interaction, longevity regulating pathway, Peroxisome proliferator-activated receptors (PPAR), focal adhesion and PI3 Akt signaling pathways. Among the PC proteins, vitronectin, gelsolin, hornerin, apolipoprotein A1, C3, H, and complement Factors C3, C4, and C9 were identified as the most highly ranked proteins in diabetes. The identified unique proteins of retinal PC could prove beneficial as a therapeutic target in the management of DR.

Transcriptome combined with Mendelian randomization to screen key genes associated with mitochondrial and programmed cell death causally associated with diabetic retinopathy.

Mitochondrial dysfunction in the retina can induce apoptosis of retinal capillary cells, leading to diabetic retinopathy (DR). This study aimed to explore key genes related to programmed cell death (PCD) and mitochondria in DR via bioinformatic analysis. A differential analysis was performed to identify differentially expressed genes (DEGs) between DR and control samples using the GSE94019 dataset from the Gene Expression Omnibus (GEO) database. Pearson correlation analysis was then utilized to select genes linked to mitochondrial function and PCD (M-PCD). Candidate genes were identified by overlapping DR-DEGs and M-PCD genes, followed by functional annotation. Mendelian randomization (MR) analysis was employed to identify genes with causal relationships to DR. Key genes were identified through protein-protein interaction (PPI) analysis using six algorithms (DEgree, DMNC, EPC, MCC, Genes are BottleNeck, and MNC) within Cytoscape software. The expression patterns of these genes were validated using GSE94019 and GSE60436 datasets, as well as RT-qPCR. Enrichment analysis provided insights into the function and pathways of these key genes in DR. Differential immune cell profiles were determined via immune infiltration analysis, followed by exploring the relationships between immune cells, cytokines, and the identified genes. Correlations between key genes and apoptosis genes were also examined. In vivo experiments using RT-PCR, immunohistochemistry (IHC), and western blot analysis confirmed that MYC and SLC7A11 expression was significantly elevated in DR rat retinal tissues. From 658 candidate genes, 12 showed significant causal associations with DR. MYC and SLC7A11 were particularly notable, showing upregulated expression in DR samples and involvement in apoptosis and diabetes-related pathways. These genes were significantly associated with apoptotic genes and correlated positively with altered immune cell types and cytokines, suggesting a link between immune response and DR pathogenesis. In vivo findings confirmed that MYC and SLC7A11 expression was elevated in DR rat retinal tissues. Key genes (MYC and SLC7A11) associated with mitochondrial function and PCD in DR were identified, offering insights into DR's pathological mechanisms and potential targets for diagnostic and therapeutic strategies.

Preservation of Murine Whole Eyes With Supplemented UW Cold Storage Solution: Anatomical Considerations.

Retinal ganglion cell (RGC) apoptosis and axon regeneration are the principal obstacles challenging the development of successful whole eye transplantation (WET). The purpose of this study was to create a neuroprotective cocktail that targets early events in the RGC intrinsic apoptotic program to stabilize RGCs in a potential donor eye. University of Wisconsin (UW) solution was augmented with supplements known to protect RGCs. Supplements targeted tyrosine kinase signaling, histone deacetylase activity, K+ ion efflux, macroglial stasis, and provided energy support. Modified UW (mUW) solutions with individual supplements were injected into the vitreous of enucleated mouse eyes, which were then stored in cold UW solution for 24 hours. Histopathology, immunostaining of individual retinal cell types, and analysis of cell-specific messenger RNAs (mRNAs) were used to identify supplements that were combined to create optimal mUW solution. UW and mUW solutions reduced ocular edema and focal ischemia in globes stored in cold storage. Two major issues were noted after cold storage, including retinal detachment and reduction in glial fibrillary acidic protein staining in astrocytes. A combination of supplements resolved both these issues and performed better than the individual supplements alone. Cold storage resulted in a reduction in cell-specific mRNAs, even though it preserved the corresponding protein products. Eyes treated with optimal mUW solution exhibited preservation of retinal and cellular architecture, but did display a decrease in mRNA levels, suggesting that cold storage induced cellular stasis. Application of optimal mUW solution lowers an important barrier to the development of a successful whole eye transplantation procedure.

Topical application of 666-15, a potent inhibitor of CREB, alleviates alkali-induced corneal neovascularization

Corneal neovascularization (CNV) is a dynamically regulated process that arises due to a disruption in the equilibrium between pro-angiogenic and anti-angiogenic factors. Various cytokines are released by vascular endothelial cells and macrophages in damaged cornea, ultimately inducing CNV. The cAMP-response element-binding protein (CREB), a nuclear transcription factor, potentially impacts tumor angiogenesis by modulating the secretion of angiogenic proteins. This study aimed to assess the impact of 666-15, a potent inhibitor of CREB, on angiogenesis using human microvascular retinal endothelial cells (HMRECs), RAW 264.7 macrophage cell line and alkali-induce CNV mouse model. In vivo, the topical application of 666-15 (0.05mg/mL) to the alkali-burn corneas led to 45% reduction in CNV. Additionally, in vitro treatment with 666-15 is effective in suppressing the migration, proliferation, and tube formation by HMRECs. Furthermore, treatment with 666-15 resulted in a down-regulation of pro-angiogenic cytokines expression, including VEGF-A, TGF-β1, b-FGF, and MMP-2 but simultaneously increasing anti-angiogenic cytokines expression, such as ADAMTS-1, Thrombospondin-1 (Tsp-1) and Tsp-2, both in alkali-burn corneas and HMRECs. And 666-15 inhibited the recruitment and the cytokines expression (VEGF-A, MMP-2, IL-1β, TNF-α, MCP-1 and MIP-1) of macrophage. Our findings revealed that 666-15 may suppress the function of endothelial cells and angiogenesis by restoring the homeostasis of pro-angiogenic stimuli, suggesting its potential as a therapeutic agent in the treatment of CNV and other angiogenesis-driven diseases.

Complement C3 knockout protects photoreceptors in the sodium iodate model

Complement factor 3 (C3) has emerged as a primary therapeutic target in age-related macular degeneration (AMD) supported by genetic, histologic, and clinical trial evidence. Yet, the site(s) of action are unclear. The purpose of this study was to test the effect of C3 knockout on photoreceptors and retinal pigment epithelial cells (RPE) in the sodium iodate (NaIO3) model, which mirrors some features of AMD. C3−/− and WT mice, both on a C57Bl/6J background, were injected intraperitoneally with 25 mg/kg NaIO3. Electroretinography and optical coherence tomography were performed 7 days later to assess retinal function and structure, respectively. Then, mice were euthanized for retinal immunohistochemistry, quantitative real-time PCR and enzyme-linked immunosorbent assays. NaIO3 increased C3 protein levels in the neural retina but not RPE. WT but not C3−/− mice showed NaIO3-induced iC3b deposition on photoreceptor outer segments. C3−/− mice were partially protected against photoreceptor layer thinning. There was partial preservation of rod and cone function in the C3−/− group. Neither RPE structure nor function was protected. These results suggest outer segment opsonization contributes to photoreceptor death in this model, and that targeting C3 can protect photoreceptor structure and function when RPE cells are stressed.

Evaluation of mesenchymal stem cells as an in vitro model for inherited retinal diseases.

Retinal pathologies are major causes of vision impairment and blindness in humans, and inherited retinal diseases (IRDs), such as retinitis pigmentosa, Leber congenital amaurosis, and Stargardt disease, greatly contribute to this problem. In vitro disease modeling can be used for understanding the development of pathology and for screening therapeutic pharmaceutical compounds. In the preclinical research phase, in vitro models complement in vivo models by reducing animal studies, decreasing costs, and shortening research timelines. Additionally, animal models may not always accurately replicate the human disease phenotype. This review examines the types of cells that can be used to create in vitro IRD models, including retina-specific cell lines, primary retinal cells, induced pluripotent stem cells (iPSCs), and more. Special attention is given to mesenchymal stem cells (MSCs), which are characterized by various isolation sources, relative ease of isolation, and straightforward differentiation. MSCs derived from bone marrow (BM), adipose tissue (AT), dental tissue (DT), umbilical cord (UC), and other sources can differentiate into retinal cells, including photoreceptor cells and retinal pigment epithelial (RPE) cells, dysfunction of which is most commonly associated with IRDs. Subsequent differentiation of MSCs into retinal cells can be carried out via various methods: culturing in induction media supplemented with certain growth factors, co-culturing with retinal cells or in their conditioned media, or regulating gene expression with viral vector-delivered transcription factors (TFs) or microRNAs (miRNAs). Compared to the popular iPSCs, for example, MSC-based models are significantly cheaper and faster to obtain, making them more feasible for large-scale drug screening. Nevertheless, the existing differentiation methods need further optimization for this promising platform to receive the success it deserves.

Possible Role of Endothelial-Derived Cellular and Exosomal-miRNAs in Lipid-Mediated Diabetic Retinopathy: Microarray Studies.

Diabetic retinopathy (DR) is a salient cause of blindness worldwide. There is still an immense need to understand the pathophysiology of DR to discover better diagnostic and therapeutic modalities. Human retinal endothelial cells (HRECs) were treated with 15-HETE or D-glucose, then miRNAs were isolated, and a microarray was performed. MirWALK 2 and Ingenuity Pathway Analysis (IPA) were used to analyze the microarray results. Exosomal miRNAs from 15-HETE-treated HRECs were isolated, microarrayed, and then imported into IPA for further analysis. The microarray results showed that 15-HETE downregulated 343 miRNAs and upregulated 297 miRNAs in HRECs. High glucose treatment induced a differential expression of HREC-miRNAs where 185 miRNAs were downregulated and 244 were upregulated. Comparing the impact of 15-HETE versus DG or diabetic mouse retina elaborated commonly changing miRNAs. Pathway and target analysis for miRNAs changed in 15-HETE-treated HRECs revealed multiple targets and pathways that may be involved in 15-HETE-induced retinal endothelial dysfunction. The HREC-exosomal miRNAs were differentially expressed after 15-HETE treatment, with 34 miRNAs downregulated and 45 miRNAs upregulated, impacting different cellular pathways. Here, we show that 15-HETE induces various changes in the cellular and exosomal miRNA profile of HRECs, highlighting the importance of targeting the 12/15 lipoxygenase pathway in DR.

Genomic Architecture Underlying the Striking Colour Variation in the Presence of Gene Flow for the Guinan Toad-Headed Lizard.

How divergence occurs between closely related organisms in the absence of geographic barriers to gene flow stands as one of the long-standing questions in evolutionary biology. Previous studies suggested that the interplay between selection, gene flow and recombination strongly affected the process of divergence with gene flow. However, the extent to which these forces interact to drive divergence remains largely ambiguous. Guinan toad-headed lizards (Phrynocephalus guinanensis) in the Mugetan Desert exhibit striking colour differences from lizards outside the desert and provide an excellent model to address this question. Through extensive sampling and whole genome sequencing, we obtained genotypes for 191 samples from 14 populations inside and outside the desert. Despite the colour differences, continuous and asymmetric gene flow was detected across the desert border. More importantly, 273 highly diverged regions (HDRs) were identified between them, accounting only for 0.47% of the genome but widely distributed across 20 (out of the total 24) chromosomes. Strong signatures of selection were identified in HDRs, and local recombination rates were repressed. Furthermore, five HDRs exhibited significantly higher divergence, which contained key genes associated with crucial functions in animal coloration, including pteridine and melanocyte pigmentation. Genes related to retinal cells and steroid hormones were identified in other HDRs, which might have also contributed to the formation of colour variation in the presence of gene flow. This study provided novel insights into the understanding of the evolutionary mechanisms of genetic divergence in the presence of gene flow.

Ontogeny of Thyroid Hormone Signaling in the Retina of Zebrafish: Effects of Thyroidal Status on Retinal Morphology, Cell Survival, and Color Preference.

The retina is crucial for converting light into neuronal signals for visual perception. Understanding the retina's structure, function, and development is essential for vision research. It is known that the thyroid hormone (TH) receptor type beta 2 (TRβ2) is a key element in the regulation of cone differentiation in the retina, but other elements of TH signaling, such as transporters and enzyme deiodinases, have also been implicated in retinal cell development and survival. In the present study, we investigated the expression profile of genes involved in TH signaling and analyzed the impact of thyroidal status on retinal morphology, opsin expression, cell death/proliferation profile, as well as color preference behavior during the early retina development of zebrafish larvae. mRNA expression analysis on dissected whole eyes revealed that TH signaling elements gradually increase during eye development, with dio3b being the component that shows the most dramatic change. Mutations generated by CRISPR/CAS9 in the dio3b gene, but not in the thrb gene, modifies the structure of the retina. Disruption in TH level reduces the cell number of the ganglion cell layer, increases cell death, and modifies color preference, emphasizing the critical importance of precise TH regulation by its signaling elements for optimal retinal development and function.

Astragalus polysaccharide ameliorates diabetic retinopathy by inhibiting the SHH-Gli1-AQP1 signaling pathway in streptozotocin-induced type 2 diabetic rats.

This study aims to investigate the effects of astragalus polysaccharide (APS) on diabetic retinopathy through the SHH-Gli1-AQP1 pathway. The anti-type 2 diabetes mellitus (T2DM) targets of APS were identified through comprehensive searches of drug and disease-related databases. A protein-protein interaction network was then constructed, followed by GO and KEGG enrichment analyses. Molecular docking simulations were performed to evaluate the interactions of APS and metformin with Gli1 and AQP1. An in vivo T2DM rat model was established via streptozotocin (STZ) injection and treated with metformin and varying doses of APS for 12 weeks. Histological changes in retinal cells were assessed using H&E and PAS staining. The expression levels of AQP1, Gli1, and SHH in the retina were measured using immunohistochemistry, Western blotting, immunofluorescence, and ELISA. Additionally, mRNA expression of AQP1, Gli1, and SHH was quantified by RT-qPCR. Bioinformatic analyses indicated that Gli1 and AQP1, key components of the SHH-Gli1- AQP1 signaling pathway, may be associated with T2DM. Subsequent experiments demonstrated that the STZ-induced T2DM rats exhibited significant retinal damage, which was notably mitigated by both APS and metformin treatments. Furthermore, the SHH-Gli1-AQP1 signaling pathway was found to be overactivated in STZ-induced T2DM rats. Treatment with APS and metformin significantly reduced the elevated expression levels of SHH, Gli1, and AQP1. APS effectively inhibits retinal damage of STZ-induced T2DM rats by restraining the SHH-Gli1-AQP1 signaling pathway.

Functional characterization of optic photoreception in Lymnaea stagnalis.

Optic photoreception is a critical function for animal survival. Across the evolutionary spectrum, diverse animal models have been used to investigate visual system function and potential mechanisms under physiological or pathophysiological states. However less is known on photoreceptive behaviors and retinal processing in invertebrates, especially molluscs. This study focuses on the freshwater pond snail, Lymnaea stagnalis (L. stagnalis), to explore its visual function and underlying mechanisms. Using anatomical and histological approaches we characterized the L. stagnalis eye structure and demonstrated structural connections and retinal rhodopsin-positive sensory cells potentially critical for phototransduction. To assess the snail phototactic responses, we developed a new neurobehavioral protocol and employed DeepLabCut to track and quantify animal locomotion. We demonstrated that L. stagnalis exhibits a positive locomotory response to intense focal light and has diverse photo-locomotory responses. Further, we conducted phylogenetic and protein structure analyses and demonstrated that L. stagnalis has a unique repertoire of both vertebrate and invertebrate phototransduction genes. Further characterization of a rhodopsin-like gene identified unique characteristics compared to other mollusks and vertebrates, suggesting different mechanisms of phototransduction. Taken together, our work establishes L. stagnalis as a model organism for studying optic photoreception, offering new insights into the evolution and diversity of visual function across animal species.

EXTH-07. UNRAVELING THE ROLE OF SHH SIGNALING PATHWAY IN RETINOBLASTOMA

Abstract Retinoblastoma (RB) is a pediatric cancer affecting the retina, often requiring surgical removal of one or both eyes due to the limited effectiveness of current treatments. There is an urgent need for advanced therapeutic strategies capable of effectively eliminating tumors, thereby obviating the need for surgical removal of eyes in affected children. Achieving this goal requires a deeper understanding of the cellular and molecular mechanisms governing RB initiation and progression. Using a transgenic mouse model, we identified the crucial role of the Shh signaling pathway in Rb1 mutation-induced tumorigenesis, highlighting its potential as a novel therapeutic target for RB treatment. We have observed frequent overexpression of Shh signaling proteins (SHH, GLI1, and GLI2) in human RB, correlating with aggressive clinicopathological features. GLI2 overexpression is necessary for Rb1 deletion-induced RB tumor initiation in mouse retinal progenitor cells (RPCs). These mouse intraocular tumors accurately replicate human RB at both cellular and molecular levels. Additionally, Shh pathway activation extends to normal retinal cells adjacent to human RB tumors, suggesting the involvement of Shh signaling in RB-microenvironment interactions. Importantly, our studies have identified a novel small molecular inhibitor targeting the Shh pathway, effectively inhibiting RB growth in vitro and in vivo by targeting both tumor cells and the tumor microenvironment (TME). The proposed research aims to provide unprecedented insights into the role of SHH signaling pathway in RB initiation and progression and the supporting communication network between RB tumors and TME. These outcomes have the potential to treat RB effectively while improving the well-being of young individuals with RB and those at risk of RB.

Melatonin and retinal cell damage: molecular and biological functions.

The indoleamine hormone, melatonin, is produced in the pineal gland and has an essential role in many physiological functions. The pineal gland is considered to be the most important organ for producing melatonin. Nevertheless, it is important to point out that the eye is also capable of producing melatonin, and has its own circadian rhythm in producing this hormone. Melatonin is mainly produced by a subpopulation of photoreceptors in a diurnal rhythm. Numerous in vitro and in vivo studies have shown the beneficial effects of melatonin in eye-related disorders. These diseases primarily affect retinal cells, highlighting the therapeutic potential of melatonin, especially in the retina. Melatonin's ability to regulate oxidative stress response pathways and modulate the expression of antioxidant genes makes it a promising candidate for mitigating retinal cell damage. Moreover, melatonin can modulate inflammatory pathways such as NF-кB and further reduce retinal damage, as well as affecting programmed cell death such as apoptosis and autophagy in retinal cells. Therefore, the goal of this review is to explore the ways in which melatonin protects retinal cells from damage and ischemia. We discuss the mechanisms involved in order to gain valuable understanding of the possible therapeutic applications of melatonin in protection of retinal cells and treatment of retinal disorders.

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.

Human-induced pluripotent stem cell-derived microglia integrate into mouse retina and recapitulate features of endogenous microglia

Microglia exhibit both maladaptive and adaptive roles in the pathogenesis of neurodegenerative diseases and have emerged as a cellular target for central nervous system (CNS) disorders, including those affecting the retina. Replacing maladaptive microglia, such as those impacted by aging or over-activation, with exogenous microglia that can enable adaptive functions has been proposed as a potential therapeutic strategy for neurodegenerative diseases. To investigate microglia replacement as an approach for retinal diseases, we first employed a protocol to efficiently generate human-induced pluripotent stem cell (hiPSC)-derived microglia in quantities sufficient for in vivo transplantation. These cells demonstrated expression of microglia-enriched genes and showed typical microglial functions such as LPS-induced responses and phagocytosis. We then performed xenotransplantation of these hiPSC-derived microglia into the subretinal space of adult mice whose endogenous retinal microglia have been pharmacologically depleted. Long-term analysis post-transplantation demonstrated that transplanted hiPSC-derived microglia successfully integrated into the neuroretina as ramified cells, occupying positions previously filled by the endogenous microglia and expressed microglia homeostatic markers such as P2ry12 and Tmem119. Furthermore, these cells were found juxtaposed alongside residual endogenous murine microglia for up to 8 months in the retina, indicating their ability to establish a stable homeostatic state in vivo. Following retinal pigment epithelial cell injury, transplanted microglia demonstrated responses typical of endogenous microglia, including migration, proliferation, and phagocytosis. Our findings indicate the feasibility of microglial transplantation and integration in the retina and suggest that modulating microglia through replacement may be a therapeutic strategy for treating neurodegenerative retinal diseases.

Human-induced pluripotent stem cell-derived microglia integrate into mouse retina and recapitulate features of endogenous microglia.

Microglia exhibit both maladaptive and adaptive roles in the pathogenesis of neurodegenerative diseases and have emerged as a cellular target for central nervous system (CNS) disorders, including those affecting the retina. Replacing maladaptive microglia, such as those impacted by aging or over-activation, with exogenous microglia that can enable adaptive functions has been proposed as a potential therapeutic strategy for neurodegenerative diseases. To investigate microglia replacement as an approach for retinal diseases, we first employed a protocol to efficiently generate human-induced pluripotent stem cell (hiPSC)-derived microglia in quantities sufficient for in vivo transplantation. These cells demonstrated expression of microglia-enriched genes and showed typical microglial functions such as LPS-induced responses and phagocytosis. We then performed xenotransplantation of these hiPSC-derived microglia into the subretinal space of adult mice whose endogenous retinal microglia have been pharmacologically depleted. Long-term analysis post-transplantation demonstrated that transplanted hiPSC-derived microglia successfully integrated into the neuroretina as ramified cells, occupying positions previously filled by the endogenous microglia and expressed microglia homeostatic markers such as P2ry12 and Tmem119. Furthermore, these cells were found juxtaposed alongside residual endogenous murine microglia for up to 8 months in the retina, indicating their ability to establish a stable homeostatic state in vivo. Following retinal pigment epithelial cell injury, transplanted microglia demonstrated responses typical of endogenous microglia, including migration, proliferation, and phagocytosis. Our findings indicate the feasibility of microglial transplantation and integration in the retina and suggest that modulating microglia through replacement may be a therapeutic strategy for treating neurodegenerative retinal diseases.