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The function of exosomal miRNAs (miRs) in retinal degeneration is largely unclear. We were aimed to investigate the functions of exosomes as well as their miRs derived from retinal pigment epithelial (RPE) cells following exposure to oxidative stress (OS). After the OS by lipopolysaccharide and rotenone on RPE cells, interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α) were upregulated, along with the decreased mitochondrial membrane potential and upregulated oxidative damage marker 8-OH-dG in RPE cells. RPE-derived exosomes were then isolated, identified, injected into the subretinal space in mice. After subretinal injection, RPE-exosomes after OS not only induced higher ROS level and apoptotic retinal cells, but also elevated IL-1β, IL-6 alongside TNF-α expressions among retina/RPE/choroidal complex. Next, miRs inside the exosomes were sequenced by the next generation sequencing (NGS) technology. NGS revealed that certain miRs were abundant in exosomes, while others were selectively kept by RPE cells. Further, downregulated miRs, like miR-125b-5p, miR-125a-5p, alongside miR-128-3p, and upregulated miR, such as miR-7-5p were validated byRT-qPCR. Finally, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to find the possible target genes of those selective exosomal miRs. Our results proved that the RPE-derived exosomes after OS selectively express certain miRs, providing novel insights into the pathogenesis of age-related macular degeneration (AMD) in future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Abnormal ocular neovascularization, a major pathology of eye diseases, leads to severe visual loss. The role of lens epithelial cell (LEC)-derived exosomes (Lec-exo) is largely unknown. Thus, we aimed to investigate whether Lec-exo can inhibit abnormal ocular neovascularization and explore the possible mechanisms. In our study, we proved the first evidence that exosomes derived from LECs attenuated angiogenesis in both oxygen-induced retinopathy and laser-induced choroidal neovascularization mice models. Further in vitro experiments proved that Lec-exo inhibited proliferation, migration, and tube formation capability of human umbilical vein endothelial cells in high glucose condition. Further high-throughput miRNAs sequencing analysis detected that miR-146a-5p was enriched in Lec-exo. Mechanistically, exosomal miR-146a-5p was delivered to endothelial cells and bound to the NRAS coding sequence, which subsequently inactivated AKT/ERK signaling pathway. We successfully elucidated the function of Lec-exo in inhibiting abnormal ocular neovascularization, which may offer a promising strategy for treatment of abnormal ocular neovascularization., (© 2023 Federation of American Societies for Experimental Biology.)

Diabetic retinopathy (DR), one of the most common microangiopathic complications in diabetes, causes severe visual damage among working-age populations. Retinal vascular endothelial cells, the key cell type in DR pathogenesis, are responsible for abnormal retinal angiogenesis in advanced stages of DR. The roles of exosomes in DR have been largely unknown. In this study, we report the first evidence that exosomes derived from the vitreous humor of patients with proliferative DR (PDR-exo) promote proliferation, migration, and tube formation of human retinal vascular endothelial cells (HRVECs). We identified long noncoding RNA (lncRNA) LOC100132249 enrichment in PDR-exo via high-throughput sequencing. This lncRNA, also mainly derived from HRVECs, promoted angiogenesis both in vitro and in vivo. Mechanistically, LOC100132249 acted as a competing endogenous sponge of miRNA-199a-5p (miR-199a-5p), thus regulating the endothelial-mesenchymal transition promoter SNAI1 via activation of the Wnt/β-catenin pathway and ultimately resulting in endothelial dysfunction. In conclusion, our findings underscored the pathogenic role of endothelial-derived exosomes via the LOC100132249/miR-199a-5p/SNAI1 axis in DR angiogenesis and may shed light on new therapeutic strategies for future treatment of DR., Article Highlights: This study provides the first evidence that exosomes derived from vitreous humor from patients with proliferative diabetic retinopathy participate in angiogenesis. The findings demonstrate an unreported long noncoding RNA (lncRNA), LOC100132249, by exosomal sequencing of vitreous humor. The newly found lncRNA LOC100132249, mainly derived from endothelial cells, promotes angiogenesis via an miRNA-199a-5p/SNAI1/Wnt/β-catenin axis in a pro-endothelial-mesenchymal transition manner., (© 2023 by the American Diabetes Association.)

Accumulating evidence has suggested the significant role of long noncoding RNAs (lncRNA) in regulating ferroptosis, while its regulatory mechanism in diabetic retinopathy (DR) remains unelucidated. In this work, we first demonstrated that lncRNA zinc finger antisense 1 (ZFAS1) is upregulated in high glucose-cultured human retinal endothelial cells (hRECs) and ZFAS1 inhibition attenuated high glucose- (HG-) induced ferroptosis, which was evidenced by cell viability, total iron and ferrous iron levels, reactive oxygen species (ROS) level, and Glutathione Peroxidase 4 (GP X 4) expression detection. Mechanistically, we validated that ZFAS1 may act as a competing endogenous RNA by competitively binding with microRNA-7-5p (miR-7-5p) and modulating the expression of its downstream molecule acyl-CoA synthetase long-chain family member 4 (ACSL4), which is now identified as a classic driver gene of ferroptosis process. In conclusion, our results demonstrate that HG-induced ZFAS1 elevation activates ferroptosis in hRECs and the ZFAS1/miR-7-5p/ACSL4 axis may serve as a therapeutic target for endothelial dysfunction in DR., Competing Interests: All authors declare no competing interests., (Copyright © 2022 Yu Liu et al.)

Age-related macular degeneration (AMD) is a universal leading cause for irreversible blindness in the elderly population. Dedifferentiation of retinal pigment epithelium (RPE) cells initiates early pathological events in atrophic AMD. Herein, we aim to investigate effects of a circular RNA derived from the NR3C1 gene (circNR3C1) on regulating RPE function and AMD pathogenesis. circNR3C1 expression was consistently upregulated along with RPE differentiation and was downregulated in dysfunctional RPE and blood serum of AMD patients. Silencing of circNR3C1 reduced RPE characteristic transcripts and proteins, interrupted phagocytosis, accelerated intracellular reactive oxygen species (ROS) generation, and promoted RPE proliferation in vitro. circN3C1 silencing also decreased expressions of RPE characteristic markers and disturbed the ultrastructure of RPE in vivo, as shown by a thickened RPE with twisted basal infoldings and outer segments. Mechanistically, circNR3C1 acted as an endogenous microRNA-382-5p (miR-382-5p) sponge to sequester its activity, which increased phosphatase and tensin homolog on chromosome 10 (PTEN) expression and inhibited the protein kinase B/mammalian target of rapamycin (AKT/mTOR) pathway. miR-382-5p overexpression and PTEN silencing mimicked effects of circNR3C1 silencing on RPE phenotypes in vivo and in vitro. In conclusion, circNR3C1 prevents AMD progression and protects RPE by directly sponging miR-382-5p to block its interaction with PTEN and subsequently blocks the AKT/mTOR pathway. Pharmacological circNR3C1 supplementations are promising therapeutic options for atrophic AMD., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Age-related cataract remains a serious problem in the aged over the world. MicroRNAs are abnormally expressed in various diseases including age-related cataract. MicroRNA-15a (MicroRNA-15a) has been involved in various diseases and plays crucial roles in many cellular processes. However, the mechanism of microRNA-15a in the genesis of cataract remains barely known. We therefore aimed to investigate the role of microRNA-15a in the cataract. Herein, human lens epithelial B3 cells, HLE-B3 cells were treated with 200 μmol/l H2O2 for 24 h. H2O2 was utilized in our study to induce HLE-B3 cells injury. We observed that cell apoptosis was induced by the treatment of H2O2 and meanwhile, cell proliferation was repressed by 200 μmol/l H2O2. Then, it was found that microRNA-15a was significantly increased with the H2O2 exposure in vitro. Importantly, B-cell lymphoma-2 (BCL2) and E2F transcription factor 3 (E2F3) exert crucial roles in cell apoptosis and cell proliferation. We found that BCL2 and E2F3 were greatly reduced by 200 μmol/l H2O2 in human lens epithelial cells. In addition, microRNA-15a overexpression induced cell apoptosis and repressed cell proliferation through suppressing BCL2 and E2F3. Subsequently, BCL2 and E2F3 were predicted as a direct target of microRNA-15a. The direct correlation between microRNA-15a and BCL2/E2F3 was confirmed by dual luciferase reporter assay. In conclusion, we demonstrated that microRNA-15a triggered apoptosis and repressed the proliferation of HLE-B3 cells by modulating BCL2 and E2F3., (© 2019 The Author(s).)

NLRP3, as a crucial inflammasome component, plays important roles in age-related macular degeneration. Though some activators of NLRP3 have been studied, microRNAs (miRNAs) which potentially regulate NLRP3 messenger RNA (mRNA) have not been fully explored in retinal pigment epithelial (RPE) cells and retinopathy. In this study, by miRNA microarray profiling and bioinformatic analysis, we identified that four miRNAs, miR-4286, miR-223-3p, miR-365a, miR-22-3p, may target NLRP3 mRNA in RPE inflammatory damage in vivo. Further, real-time polymerase chain reaction verified that only miR-22-3p was significantly decreased, which was associated with NLRP3 upregulation in blue-light-induced retinopathy. Mechanistically, the dual-fluorescent reporter suggested miR-22-3p directly binds NLRP3 mRNA. Moreover, overexpression of miR-22-3p could significantly reduce whereas inhibition miR-22-3p could increase the mRNA and protein expressions of NLRP3, Caspase-1, and mature IL-1β. Collectively, our results indicate that miR-22-3p plays a suppressive role in RPE damage by targeting NLRP3, which provides new insights into the future intervention to retinopathy., (© 2019 Wiley Periodicals, Inc.)

Dedifferentiation of retinal pigment epithelium (RPE) cells and choroidal neovascularization (CNV) contributes to the pathogenesis of age-related macular degeneration (AMD). MicroRNAs (miRNAs) have crucial roles in AMD onset and progression. We thus aim to investigate the effects of miRNAs on RPE dedifferentiation and endothelium cell (EC) behavior, and analyze its downstream pathways. We have previously identified miR-302d-3p as the most downregulated miRNA signature along with RPE differentiation. Herein, in vitro study supported that miR-302d-3p induces RPE dedifferentiation typified by reduction of RPE characteristic markers, interrupts its phagocytosis, and promotes its migration, proliferation, and cell-cycle progression. c-Jun was identified as a potential upstream transcript factor for MIR302D, which might modulate RPE function by regulating miR-302d-3p expression. P21 Waf1/Cip1 , a cyclin-dependent kinase inhibitor encoded by the CDKN1A gene, was identified as a downstream target of miR-302d-3p. Our data suggested that p21 Waf1/Cip1 could promote RPE differentiation, and inhibit its proliferation, migration, and cell-cycle progression. We also demonstrated that miR-302d-3p suppresses RPE differentiation through directly targeting p21 Waf1/Cip1 . In addition, the miR-302d-3p/CDKN1A axis was also involved in regulating tube formation of ECs, indicating its potential involvement in CNV formation. Taken together, our study implies that miR-302d-3p, regulated by c-Jun, contributes to the pathogenesis of both atrophic and exudative AMD. MiR-302d-3p promotes RPE dedifferentiation, migration, proliferation and cell-cycle progression, inhibits RPE phagocytosis, and induces abnormal EC behavior by targeting p21 Waf1/Cip1 . Pharmacological miR-302d-3p inhibitors are prospective therapeutic options for prevention and treatment of AMD.

Aims: Proliferative diabetic retinopathy (PDR), characterized by angiogenesis, can cause serve vision loss and even blindness. Recent studies have suggested a pivotal role of vasohibin-2 (VASH2) in the promotion of angiogenesis in tumor tissues. Here we further investigated the role of VASH2 in the proliferation and migration of retinal endothelial cells., Main Methods: The expression of VASH2 in vascular endothelial cells of epiretinal fibrovascular membranes (FVMs) from PDR patients were detected by immunofluorescence. VASH2 gene interfering lentiviral vectors (VASH2-shRNA) and miR-200b/c were constructed for the evaluation of the VASH2 effect on high glucose induced human retinal microvascular endothelial cell line (HRMECs). Cell proliferation, cell cycle and cell migration were carried out subsequently. The relationship between VASH2 and miR-200b/c was determined by luciferase reporter gene assays., Key Findings: A positive expression of VASH2 was identified in vascular endothelial cells of FVMs from PDR patients. In HRMECs, cells transfected with shRNA or miR-200b/c mimics showed a significantly reduced VASH2 expression compared with negative control group by real time-polymerase chain reaction and western-blot analysis. Inhibition of VASH2 was demonstrated to suppress cell proliferation and migration from Day 2 to Day 4. The luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200b/c in HRMECs., Significance: The present study suggests a protective effect of miR-200b/c on high glucose induced HRMECs dysfunction by inhibiting VASH2. It could be a potential therapeutic strategy to inhibit angiogenesis for the treatment of retinal vascular disease., (Copyright © 2017. Published by Elsevier Inc.)

Background: Diabetic retinopathy (DR) is a retinopathy resulting from diabetes mellitus (DM) which was classified into non-proliferative DR (NPDR) and proliferative DR (PDR). Without an early screening and effective diagnosis, patients with PDR will develop serious complications. Therefore, we sought to identify special serum microRNAs (miRNAs) that can serve as a novel non-invasive screening signature of PDR and test its specificity and sensitivity in the early diagnosis of PDR., Methods: In total, we obtained serum samples from 90 PDR cases, 90 matched NPDR patients and 20 controls. An initial screening of miRNA expression was performed through TaqMan Low Density Array (TLDA). The candidate miRNAs were validated by individual reverse transcription quantitative real-time PCR (RT-qPCR) arranged in an initial and a two-stage validation sets. Moreover, additional double-blind testing was performed in 20 patients clinically suspected of having DR to evaluate the diagnostic value and accuracy of the serum miRNA profiling system in predicting PDR., Results: Three miRNAs were significantly increased in patients with PDR compared with NPDR after the multiple stages. The areas under the receiver operating characteristic (ROC) curves of the validated three-serum miRNAs signature were 0.830, 0.803 and 0.873 in the initial and two validation sets, respectively. Combination of miR-21, miR-181c, and miR-1179 possessed a moderate ability to discrimination between PDR and NPDR with an area under ROC value of 0.89. The accuracy rate of the three-miRNA profile as PDR signature was 82.6%., Conclusions: These data provide evidence that serum miRNAs have the potential to be sensitive, cost-effective biomarkers for the early detection of PDR. These biomarkers could serve as a dynamic monitoring factor for detecting the progression of PDR from NPDR., (© 2014 S. Karger AG, Basel.)