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Objective: The catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear., Methods: An in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition., Results: Autophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition., Conclusions: Our findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Empagliflozin (EMPA) is the first sodium-glucose co-transporter 2 inhibitor to significantly reduce cardiovascular and kidney complications in type 2 diabetes mellitus. Given this, we speculate that EMPA may have the potential to intervene in diabetic retinopathy (DR), which is another diabetes-specific microvascular complication. Db/db mice were treated with EMPA for different periods to observe the retinas and related mechanisms. EMPA effectively balanced body weight and blood glucose levels, mitigated ocular edema and microaneurysm in db/db mice. EMPA significantly inhibited oxidative stress, apoptosis and recovered tight junction in diabetic retinas. MS/MS analyses showed that EMPA suppressed aberrant branched-chain amino acid (BCAAs) accumulation in db/db retinas, which led to the inhibition of the mammalian target of rapamycin activation, downregulation of inflammation, and angiogenic factors, including TNF-ɑ, IL-6, VCAM-1, and VEGF induced by diabetes. Furthermore, branched-chain α-keto acids (BCKAs), which are catabolites of BCAAs, were increased in diabetic retinas and decreased with EMPA application. Moreover, branched-chain ketoacid dehydrogenase kinase (BCKDK) was enhanced, BCKDHA and BCKDHB were decreased in diabetic retinas. This could be reversed by EMPA treatment, thus promoting BCAAs catabolism to decrease BCAAs and BCKAs accumulation in diabetic retinas. The high levels of BCAAs in the plasma and enhanced L-type amino acid transporter 1 (LAT1) were responsible for the high levels of BCAAs in diabetic retinas, which could be inhibited by EMPA. Overall, EMPA could ameliorate DR manifestations. The normalization of BCAAs catabolism and intake may play a role in this process. This study supports EMPA as a protective drug against DR., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: Vascular endothelial growth factor (VEGF) plays implicated roles in diabetic retinopathy (DR). The role of roundabout 4 (Robo 4) in angiogenesis and vasculogenesis is controversial; however, the interdependent relationship between these two factors has not been studied in DR. This study determined the colocalization of VEGF and Robo4 in fibrovascular membranes (FVM) from patients with proliferative diabetic retinopathy (PDR). MicroRNA (miRNA)-mediated modulation of VEGF and Robo4 was explored in diabetic rats and ARPE-19 tissue culture cells under hyperglycemia., Methods: VEGF and Robo4 co-expression in the FVM was analyzed using immunofluorescence. VEGF and Robo4 levels were determined in diabetic retinas and ARPE-19 tissue culture cells under high glucose using western blotting and RT-qPCR. MicroRNA agomir was intraocularly injected to increase miR-15a expression and downregulate VEGF and Robo4 levels in diabetic retinas., Results: VEGF and Robo4 colocalization in FVM vessels was observed. Increased VEGF levels were consistent in diabetic retinas and ARPE-19 tissue culture cells cultured under hyperglycemia. Robo4 decreased in ARPE-19 tissue culture cells exposed to hyperglycemia for 72 h, whereas it increased in diabetic rat retinas. Several miRNAs were differentially expressed during DR progression. Furthermore, miR-15a agomir injection inhibited high levels of VEGF and Robo4 in diabetic retinas., Conclusions: VEGF and Robo4 were co-expressed in FVMs from PDR patients. In the early stages of DR, VEGF was upregulated and contributed to DR development, whereas, in the late stage of DR, VEGF and Robo4 worked together to aggravate DR progression. However, miR-15a could downregulate VEGF and Robo4 to ameliorate DR development.

Retinal cell damage caused by diabetes leads to retinal microvascular injury. Roundabout 4 (ROBO4) is involved in angiogenesis, which varies with the development of diabetic retinopathy (DR). Here, we explored the transcriptional regulation and microRNA-mediated modulation of ROBO4 expression and related retinal cell function in DR. A streptozotocin-induced type I diabetic animal model was established to detect the expression of hypoxia inducible factor-1α (HIF-1α), specificity protein 1 (SP1) and ROBO4. Retinal pigment epithelium (RPE) cells were cultured under hyperglycaemia or hypoxia and used for mechanistic analysis. Furthermore, roles of miR-125b-5p and miR-146a-5p were evaluated, and their targets were identified using luciferase assays. The cell functions were evaluated by MTS assays, permeability analysis and migration assays. The development of DR increased the levels of HIF-1α, SP1 and ROBO4 both in the DR model and in hyperglycaemic/hypoxic RPE cells. They were co-expressed and up-regulated in diabetic retinas and in RPE cells under hyperglycaemia/hypoxia. Knockdown of HIF-1α significantly inhibited SP1 and ROBO4, whereas SP1 down-regulation abolished ROBO4 expression in RPE cells under hyperglycaemia/hypoxia. miR-125b-5p and miR-146a-5p were down-regulated by hyperglycaemia and/or hypoxia. Up-regulation of miRNAs reversed these changes and resulted in recovery of target gene expression. Moreover, luciferase assays confirmed miR-125b-5p targeted SP1 and ROBO4, and miR-146a-5p targeted HIF-1α and ROBO4 directly. The decreased cell viability, enhanced permeability, and increased cell migration under DR conditions were mitigated by knockdown of HIF-1α/SP1/ROBO4 or up-regulation of miR-125b-5p/miR-146a-5p. In general, our results identified a novel mechanism that miR-125b-5p/miR-146a-5p targeting HIF-1α/SP1-dependent ROBO4 expression could retard DR progression., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults across the world. The pathogenesis of DR is multifactorial and the molecular mechanisms are still not fully understood. Accumulating evidence has demonstrated that noncoding RNAs (ncRNAs) may be aberrantly expressed and may play vital roles in the development of DR. Amongst ncRNAs, miRNAs and long ncRNAs (lncRNAs) are known for their regulatory functions. Here, we summarize the functions and mechanisms of known aberrantly expressed miRNAs and lncRNAs in DR. Additionally, a novel lncRNA-mRNA-miRNA network is included in this review. We highlight original studies that provide detailed data about the mechanisms of miRNAs and lncRNAs, their applications as diagnostic or prognostic biomarkers, and their potential therapeutic targets. In conclusion, this review will help us gain a better understanding of the molecular mechanisms by which miRNAs and lncRNAs perform their functions in DR, and provide general strategies and directions for future research., (© 2017 The Author(s).)

The pathological mechanisms of diabetic retinopathy (DR), a leading cause of blindness in adults with diabetes mellitus, remain incompletely understood. Because microRNAs (miRNAs) represent effective DR therapeutic targets, we identified aberrantly expressed miRNAs associated with cellular dysfunction in early DR and detected their potential targets. We exposed human retinal endothelial cells (HRECs) and a cell line of retinal pigment epithelial (RPE) cells to high glucose (25 mmol/L, 1-7 days) to mimic DR progression and used streptozotocin-injected rats (4-8 weeks) for an in vivo diabetes model. HREC/RPE viability decreased after 24 h incubation and diminished further over 6 days, and Hoechst staining revealed hyperglycemia-induced HREC/RPE apoptosis. Although miR-124/-125b expression decreased with DR progression in vitro and in vivo, miR-135b/-199a levels decreased in retinal cells under hyperglycemia exposure, but increased in diabetic retinas. Moreover, miR-145/-146a expression decreased gradually in high-glucose-treated HRECs, but increased in hyperglycemia-exposed RPE cells and in diabetic rats. Our findings suggested that aberrant miRNA expression could be involved in hyperglycemia-induced retinal-cell dysfunction, and the identified miRNAs might vary in different retinal layers, with expression changes associated with DR development. Therefore, miRNA modulation and the targeting of miRNA effects on transcription factors could represent novel and effective DR-treatment strategies.

Purpose: Diabetic retinopathy (DR), a neurovascular disease, is one of the leading causes of blindness in working-age adults. Long noncoding RNAs (lncRNAs) have attracted attention as indicators for DR. This study aimed to characterize the role of lncRNA human testis development–related gene 1 (TDRG1) and its modulation of vascular endothelial growth factor (VEGF) in deteriorating DR. Methods: Tissue samples were obtained from patients with epiretinal membranes (EMs) or proliferative DR, and human retinal microvascular endothelial cells (HRECs) were cultured with high-glucose medium to mimic DR as the in vitro model. The expression of lncRNA TDRG1 and VEGF was determined by immunofluorescence staining, Western blotting, and RT-qPCR. Transfection of small-interfering RNA was conducted to knock down target gene expression. HREC functions were evaluated by cell viability, fluorescein isothiocyanate (FITC)-dextran extravasation, migration, and tube formation assays under different conditions. Results: LncRNA TDRG1 and VEGF were found to be co-expressed and significantly upregulated in fibrovascular membranes (FVMs) from DR patients compared to those from EM patients. In the in vitro model, hyperglycemic treatment markedly increased the expression of lncRNA TDRG1 and VEGF at the mRNA and protein levels, which promoted cell proliferation and migration, enhanced permeability, and disrupted tube formation of HRECs. However, knockdown of lncRNA TDRG1 or VEGF notably decreased the expression of VEGF and reversed the impaired functions of high-glucose-treated HRECs. Conclusions: LncRNA TDRG1 promoted microvascular cell dysfunction via upregulating VEGF in the progression of DR and may serve as a potential therapeutic target in DR treatment. [ABSTRACT FROM AUTHOR]

Inhibitor of growth 4 (ING4), a potential tumor suppressor, is implicated in cell migration and angiogenesis. However, its effects on diabetic retinopathy (DR) have not been elucidated. In this study, we aimed to evaluate ING4 expression in normal and diabetic rats and clarify its effects on hypoxia‐induced dysfunction in human retinal pigment epithelial (ARPE‐19) cells. A Type 1 diabetic model was generated by injecting rats intraperitoneally with streptozotocin and then killed them 4, 8, or 12 weeks later. ING4 expression in retinal tissue was detected using western blot analysis, reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR), and immunohistochemistry assays. After transfection with an ING4 overexpression lentiviral vector or small interfering RNA (siRNA), ARPE‐19 migration under hypoxia was tested using wound healing and transwell assays. The angiogenic effect of conditioned medium (CM) from ARPE‐19 cells was examined by assessing human retinal endothelial cell (HREC) capillary tube formation. Additionally, western blot analysis and RT‐qPCR were performed to investigate the signaling pathways in which ING4, specificity protein 1 (Sp1), matrix metalloproteinase 2 (MMP‐2), MMP‐9, and vascular endothelial growth factor A (VEGF‐A) were involved. Here, we found that ING4 expression was significantly reduced in the diabetic rats' retinal tissue. Silencing ING4 aggravated hypoxia‐induced ARPE‐19 cell migration. CM collected from ING4 siRNA‐transfected ARPE‐19 cells under hypoxia promoted HREC angiogenesis. These effects were reversed by ING4 overexpression. Furthermore, ING4 suppressed MMP‐2, MMP‐9, and VEGF‐A expression in an Sp1‐dependent manner in hypoxia‐conditioned ARPE‐19 cells. Overall, our results provide valuable mechanistic insights into the protective effects of ING4 on hypoxia‐induced migration and angiogenesis regulation in ARPE‐19 cells. Restoring ING4 may be a novel strategy for treating DR. [ABSTRACT FROM AUTHOR]

Roundabout4 ( Robo4 ) is a gene that is expressed specifically in vasculature and is involved in the angiogenesis and integrity of blood vessels. The expression level of Robo4 increases gradually along with the development of diabetic retinopathy (DR). In this study, we explored the mechanism of transcriptional regulation of Robo4 in retinal endothelial cells, and investigated the effects of this regulation on cellular functions under hyperglycemic conditions. Human retinal endothelial cells (HREC) exposed to hyperglycemia were used to detect the expression levels of specificity protein 1 (SP1) and Robo4 by RT-qPCR and western blotting. Small interfering RNA (SiRNA) transfection technology was used to analyze the regulatory relationship between SP1 and Robo4 . The effect of transcription factor SP1 on Robo4 promoter activity and the location of SP1 binding sites were investigated using chromatin immunoprecipitation (ChIP) and luciferase assay. Cell migration, monolayer permeability and tube formation assays were performed to demonstrate the role of SP1/ Robo4 in regulating HREC functions in hyperglycemic conditions. The results showed that hyperglycemia upregulated the mRNA and protein levels of SP1 and Robo4 in HREC. Depletion of SP1 by siRNA transfection inhibited the hyperglycemia induced overexpression of Robo4 . ChIP combined with luciferase assay showed that under hyperglycemic conditions, SP1 significantly increased the transcriptional level of Robo4 via an additional SP1 binding site at − 1912/− 1908 in the Robo4 promoter. Repressing the SP1/ Robo4 pathway effectively mitigated the abnormity in HREC migration, permeability and angiogenesis induced by hyperglycemia. All these findings indicate that hyperglycemia-induced upregulation of Robo4 is mediated by enhanced transcription of SP1. The SP1/ Robo4 signaling pathway can regulate the migratory ability, monolayer permeability and angiogenesis of HREC under hyperglycemic conditions, suggesting that it may play an important role in microvascular dysfunction during DR. [ABSTRACT FROM AUTHOR]