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Diabetic retinopathy (DR) is the leading cause of blindness and visual loss in people with diabetes. It has been suggested that the progression of DR is associated with chronic inflammation and oxidative stress. The aim of the present work was to evaluate the ability of the natural compound madecassic acid (MEA) to reverse the negative impact of streptozotocin (STZ) on retinal injury in rats. Diabetic rats induced by STZ were treated with MEA at the doses of 10 and 20 mg/kg bw for 8 weeks. The study compared the efficacy of the drug in controlling high blood sugar levels and its impact on therapeutic targets such as SOD, CAT, GPx, NF-κB, TNF-α, IL-6, IL-1β, VEGF, IGF, bFGF and Keap1/Nrf-2 pathway. The results showed that the treatment with MEA significantly restored the retinal SOD, CAT, and GPx levels in diabetic rats to the near-normal levels. Moreover, the level of inflammatory mediators (TNF-α, IL-1β, IL-6) and growth factors (VEGF, IGF, bFGF) was significantly lower in retinas of animals treated with MEA as compared to retinas of diabetic animals. The study also established that MEA administration reduced the NF-κB protein and altered the Nrf-2/Keap1 pathway thereby reducing oxidative stress and inflammation. Furthermore, the use of MEA prevented the progression of the retinal capillary basement membrane thickening. It has been found that MEA offers significant protection to the retina and therefore, the compound may be useful in the treatment of DR in humans., 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 Elsevier Inc. All rights reserved.)

Retinopathy is a common complication of diabetes mellitus and the leading cause of visual impairment. Danggui Buxue decoction (RRP) has been used as a traditional drug for the treatment of diabetic nephropathy for many years. The aim of this study was to investigate the effects of RRP on hypoxia-induced retinal Müller cell injury. A model of retinal Müller cell damage was created using high glucose levels (25 mmol/L) and/or exposure to low oxygen conditions (1% O2). RRP was given to rats by continuous gavage for 7 days to obtain drug-containing serum. After sterilization, the serum was added to the culture medium at a ratio of 10%. Cell viability, apoptosis, and cell proliferation were assessed using the CCK-8 kit, Annexin V-FITC/propidium iodide apoptosis kit, and EdU kit. The mRNA levels of angiogenesis factors (ANGPTL4, VEGF) and inflammatory factors (IL-1B, ICAM-1) were detected by RT-qPCR. Western blot analysis was employed to assess the levels of proteins related to the ATF4/CHOP pathway. Following hypoxia for 48 h and 72 h, there was a significant decrease in cell viability and proliferation, as well as a notable increase in apoptosis compared to the control group (21% O2). However, high glucose stimulation had no significant effect, and high glucose combined with hypoxia had no further damage to cells. After 48 h of exposure to low oxygen levels, the mRNA expression levels of ANGPTL4, VEGF, IL-1B, and ICAM-1 in retinal Müller cells were significantly higher than in the control group (21% O2). RRP treatment significantly alleviated the increase of cell apoptosis and the upregulation of IL-1B and-1 in retinal Müller cells induced by hypoxia. RRP has the potential to reduce the suppression of the ATF4/CHOP pathway in hypoxia-induced retinal Müller cells, and it significantly alleviates cell apoptosis through regulating inflammatory factors and the ATF4/CHOP pathway.

As a prominent complication of diabetes mellitus (DM) affecting microvasculature, diabetic retinopathy (DR) originates from blood-retinal barrier (BRB) damage. Natural polyphenolic compound chlorogenic acid (CGA) has already been reported to alleviate DR. This study delves into the concrete mechanism of the CGA-supplied protection against DR and elucidates its key target in retinal endothelial cells. DM in mice was induced using streptozotocin (STZ). CGA mitigated BRB dysfunction, leukocytes adhesion and the formation of acellular vessels in vivo. CGA suppressed retinal inflammation and the release of tumor necrosis factor-α (TNFα) by inhibiting nuclear factor kappa-B (NFκB). Furthermore, CGA reduced the TNFα-initiated adhesion of peripheral blood mononuclear cell (PBMC) to human retinal endothelial cell (HREC). CGA obviously decreased the TNFα-upregulated expression of vascular cell adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1), and abrogated the TNFα-induced NFκB activation in HRECs. All these phenomena were reversed by overexpressing type 1 TNF receptor (TNFR1) in HRECs. The CGA-provided improvement on leukocytes adhesion and retinal inflammation was disappeared in mice injected with an endothelial-specific TNFR1 overexpression adeno-associated virus (AAV). CGA reduced the interaction between TNFα and TNFR1 through binding to TNFR1 in retinal endothelial cells. In summary, excepting reducing TNFα expression via inhibiting retinal inflammation, CGA also reduced the adhesion of leukocytes to retinal vessels through decreasing VCAM1 and ICAM1 expression via blocking the TNFα-initiated NFκB activation by targeting TNFR1 in retinal endothelial cells. All of those mitigated retinal inflammation, ultimately alleviating BRB breakdown in DR., 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 Elsevier B.V. All rights reserved.)

Background: Despite great advances in proliferative diabetic retinopathy (PDR) therapy over the last decades, one third of treated patients continue to lose vision. While resident vitreous macrophages called hyalocytes have been implicated in the pathophysiology of vitreoretinal proliferative disease previously, little is known about their exact role in PDR. In this study, we address molecular and cellular alterations in the vitreous of PDR patients as a means towards assessing the potential contribution of hyalocytes to disease pathogenesis., Results: A total of 55 patients were included in this study encompassing RNA-Sequencing analysis of hyalocytes isolated from the vitreous of PDR and control patients, multiplex immunoassay and ELISA analyses of vitreous samples from PDR and control patients, as well as isolation and immunohistochemical staining of cultured porcine hyalocytes. Transcriptional analysis revealed an enhanced inflammatory response of hyalocytes contributing to the cytokine pool within the vitreous of PDR patients by expressing interleukin-6, among others. Further, increased angiopoietin-2 expression indicated that hyalocytes from PDR patients undergo a proangiogenic shift and may thus mediate the formation of retinal neovascularizations, the hallmark of PDR. Finally, RNA-Sequencing revealed an upregulation of factors known from hemoglobin catabolism in hyalocytes from PDR patients. By immunohistochemistry, cultured porcine hyalocytes exposed to red blood cells were shown to engulf and phagocytose these, which reveals hyalocytes' potential to dispose of erythrocytes. Thus, our data suggest a potential role for vitreous macrophages in erythrophagocytosis and, thereby, clearance of vitreous hemorrhage, a severe complication of PDR., Conclusion: Our results strongly indicate a critical role for vitreous hyalocytes in key pathophysiological processes of proliferative diabetic retinopathy: inflammation, angiomodulation and erythrophagocytosis. Immunomodulation of hyalocytes may thus prove an essential novel therapeutic approach in diabetic vitreoretinal disease., Competing Interests: Declarations Ethics approval and consent to participate Ethics approval was granted by the local Ethics Committee and a written informed consent was obtained from each patient prior to surgery. All research adhered to the tenets of the Declaration of Helsinki. Consent for publication A consent for publication was granted by patients whose images and videos are shown in this study. Competing interests SKB and JW have previously received a speaker honorarium from Bayer Vital GmbH. CAKL is member of the Publication Steering Committee of the SPECTRUM trial by Bayer and has received a speaker honorarium from Appelis and Bayer Vital GmbH. Other than that, the authors declare that research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024. The Author(s).)

Purpose: Studies have suggested that photoreceptors (PR) are altered by diabetes, contributing to diabetic retinopathy (DR) pathology. Here, we explored the effect of diabetes on retinal ischemic injury., Methods: Retinal ischemia-reperfusion (IR) injury was caused by elevation of intraocular pressure in 10-week-old BKS db/db type 2 diabetes mellitus (T2DM) mice or C57BL/6J mice at 4 or 12 weeks after streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM), and respective nondiabetic controls. Retinal neurodegeneration was evaluated by retinal layer thinning, TUNEL staining, and neuron loss. Vascular permeability was evaluated as retinal accumulation of circulating fluorescent albumin. The effects of pretreatment with a sodium-glucose co-transporter (SGLT1/2) inhibitor, phlorizin, were examined., Results: Nondiabetic control mice exhibited no significant outer retinal layer thinning or PR loss after IR injury. In contrast, db/db mice exhibited significant outer retina thinning (49%, P < 0.0001), loss of PR nuclei (45%, P < 0.05) and inner segment (IS) length decline (45%, P < 0.0001). STZ-induced diabetic mice at 4 weeks showed progressive thinning of the outer retina (55%, by 14 days, P < 0.0001) and 4.3-fold greater number of TUNEL+ cells in the outer nuclear layer (ONL) than injured retinas of control mice (P < 0.0001). After 12 weeks of diabetes, the retinas exhibited similar outer layer thinning and PR loss after IR. Diabetes also delayed restoration of the blood-retinal barrier after IR injury. Phlorizin reduced outer retinal layer thinning from 49% to 3% (P < 0.0001)., Conclusions: Diabetes caused PR to become highly susceptible to IR injury. The ability of phlorizin pretreatment to block outer retinal thinning after IR suggests that the effects of diabetes on PR are readily reversible.

Purpose: Diabetic retinopathy (DR) is a significant complication of diabetes mellitus that can lead to progressive visual impairment. This study aimed to elucidate the role of fibrinogen, a protein whose serum and intraocular concentrations are elevated in patients with diabetes and DR, in the pathogenesis of DR., Methods: The changes in the protein levels of the neuronal marker tubulin-β3 (TUBB3) and retinal response induced by the intravitreal injections of 1× phosphate-buffered saline, 40 mg/mL of fibrinogen, and 40 mg/mL of fibrinogen in combination with anti-intracellular adhesion molecule-1 (ICAM-1) antibody in normal mice were observed using immunofluorescence, western blotting, and electroretinography., Results: High concentrations of fibrinogen led to a decrease in the expression of TUBB3 in immunofluorescence and western blotting. The amplitudes of the positive scotopic threshold response and b-wave were notably reduced after the injection of fibrinogen, indicating potential damage to the retinal ganglion cells. The co-administration of anti-ICAM-1 antibody effectively mitigated these fibrinogen-induced changes, indicating that fibrinogen-induced damage is mediated via the ICAM-1 pathway., Conclusions: The present study underscores the significance of elevated intraocular fibrinogen levels as a pathogenic factor in DR. Involvement of the fibrinogen/ICAM-1 pathway presents new avenues for therapeutic intervention, especially in patients with treatment-resistant conditions.

Purpose: Evaluate the ocular pharmacodynamics (PD) of intravitreal faricimab, a bispecific inhibitor of angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A), in patients with neovascular age-related macular degeneration (nAMD) or diabetic macular edema (DME)., Methods: Aqueous humor (AH) samples (1025 free Ang-2 concentrations and 1345 free VEGF-A concentrations) were collected from approximately 300 faricimab-treated patients with nAMD or DME in phase 2/3 trials. A population pharmacokinetic pharmacodynamic (popPKPD) model was developed to describe the dynamic effect of faricimab on free AH Ang-2 and VEGF-A., Results: Mean baseline Ang-2 concentrations were 8.1 and 13.4 pg/mL in patients with nAMD and DME, respectively. The corresponding mean baseline VEGF-A concentrations were 58 and 135 pg/mL, respectively. Overall, approximately 79% of Ang-2 (84% within 8 weeks postdose and 55% beyond 12 weeks postdose) and 7% of VEGF-A postdose observations were below the lower limit of quantification. Model-derived Ang-2 and VEGF-A concentration-time profiles for patients on every 4-week/every 8-week dosing were predicted to maintain greater than 50% suppression of Ang-2 concentrations for the entire dosing period. Patients on every 12-week/16-week dosing were predicted to have greater than 50% Ang-2 suppression for 12 or more weeks, whereas 50% VEGF-A suppression was maintained for 9 to 10 weeks. At 8 weeks postdose, the median Ang-2 concentrations remained suppressed by approximately 80%. At 16 weeks postdose, the median VEGF-A concentrations returned to baseline, but median Ang-2 levels remained below baseline., Conclusions: A popPKPD analysis demonstrated faricimab's rapid and sustained suppression of AH Ang-2 and VEGF-A., Translational Relevance: A popPKPD analysis suggested that sustained suppression of ocular Ang-2 contributes to faricimab's extended durability, observed in clinical trials.

Purpose: Diabetic macular edema (DME) is the primary cause of vision impairment in diabetic retinopathy (DR) patients. A previous study has shown the efficacy of montelukast, a cysteinyl leukotriene receptor (CysLTR)1 antagonist, in a diabetic mouse model. This study aims to understand the CysLTR1 signaling in retinal endothelial cells and the impact of montelukast., Methods: Primary human retinal microvascular endothelial cells (HRECs) challenged with 20 ng/mL TNF-α and 30 mM D-glucose (D-glu) for six to 24 hours served as a model of endothelial activation. HRECs were incubated with L-glucose (L-glu) as an osmotic control. CysLTR1 knockdown and montelukast pretreatment assessed CysLTR1 antagonism. Gene expression, protein expression, and cell-permeable dyes were utilized to measure autophagy and inflammation. Transendothelial electrical resistance (TER) and transendothelial migration of mononuclear leukocytes across HRECs monolayer were measured as a functional assessment of vascular permeability., Results: Endothelial activation induced by hyperglycemia and inflammation increased CysLTR1 expression, triggering autophagy within two to six hours, IL-1β production, loss of junction integrity, decreased TER, and increased leukocyte migration within six to 24 hours. Pretreatment with montelukast effectively alleviated these effects, demonstrating its dependence on CysLTR1., Conclusions: Dysfunctional retinal endothelium initiates a self-reinforcing loop of inflammation, autophagy, and compromised integrity associated with heightened CysLTR1 levels. The antagonistic effect of montelukast against CysLTR1 effectively mitigates these detrimental changes. This study reveals CysLTR1 as a potential therapeutic target in treating DME and offers a novel strategy to mitigate detrimental changes in DR.

Purpose: Retinal neovascularization poses heightened risks of vision loss and blindness. Despite its clinical significance, the molecular mechanisms underlying the pathogenesis of retinal neovascularization remain elusive. This study utilized single-cell multiomics profiling in an oxygen-induced retinopathy (OIR) model to comprehensively investigate the intricate molecular landscape of retinal neovascularization., Methods: Mice were exposed to hyperoxia to induce the OIR model, and retinas were isolated for nucleus isolation. The cellular landscape of the single-nucleus suspensions was extensively characterized through single-cell multiomics sequencing. Single-cell data were integrated with genome-wide association study (GWAS) data to identify correlations between ocular cell types and diabetic retinopathy. Cell communication analysis among cells was conducted to unravel crucial ligand-receptor signals. Trajectory analysis and dynamic characterization of Müller cells were performed, followed by integration with human retinal data for pathway analysis., Results: The multiomics dataset revealed six major ocular cell classes, with Müller cells/astrocytes showing significant associations with proliferative diabetic retinopathy (PDR). Cell communication analysis highlighted pathways that are associated with vascular proliferation and neurodevelopment, such as Vegfa-Vegfr2, Igf1-Igf1r, Nrxn3-Nlgn1, and Efna5-Epha4. Trajectory analysis identified a subset of Müller cells expressing genes linked to photoreceptor degeneration. Multiomics data integration further unveiled positively regulated genes in OIR Müller cells/astrocytes associated with axon development and neurotransmitter transmission., Conclusions: This study significantly advances our understanding of the intricate cellular and molecular mechanisms underlying retinal neovascularization, emphasizing the pivotal role of Müller cells. The identified pathways provide valuable insights into potential therapeutic targets for PDR, offering promising directions for further research and clinical interventions.

Retinal ganglion cells (RGCs) are among the first neurons to undergo apoptosis in diabetic retinopathy (DR), with their relationship to endoplasmic reticulum stress (ERS)-induced apoptosis still unclear. While glucocorticoid-induced transcript 1 (GLCCI1) has been shown to inhibit apoptosis, its role in ERS-induced apoptosis and its mechanisms in DR remain unclarified. Our findings indicated that GLCCI1 is predominantly localized in the ganglion cell layer and is downregulated in DR. GLCCI1 overexpression mitigated the apoptosis of RGCs and the swelling of endoplasmic reticulum and mitochondria under hyperglycemia, and downregulated ERS-induced apoptosis related markers (GRP78, CHOP and cleaved CASP3), whereas GLCCI1 knockdown has the opposite effect. In vivo, GLCCI1 overexpression not only prevents structural lesions but also protects against microvascular dysfunctions in the retinas of DR mice. We found that GLCCI1 directly interacts with HSP90AB1, which in turn interacts with GRP78. Additionally, GLCCI1 is an upstream regulator of HSP90AB1, which regulates GRP78. Thus, the impact of GLCCI1 on the ERS-induced apoptosis is mainly through the regulation of HSP90AB1, and subsequently inhibiting GRP78-initiated ERS-induced apoptosis. These findings offer a promising avenue for further treatment of DR., (© 2024. The Author(s).)

Progression of diabetic retinopathy resists arrest even after institution of intensive glycemic control, suggesting a "metabolic memory" phenomenon, but the mechanism responsible for this phenomenon is still elusive. Gene expression and biological processes can also be regulated by long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame for translation, and several LncRNAs are aberrantly expressed in diabetes. Our aim was to identify retinal LncRNAs that fail to reverse after termination of hyperglycemia. Microarray analysis was performed on retinal RNA from streptozotocin-induced diabetic rats in poor glycemic control for 8 months, followed by in good glycemic control (blood glucose >400 mg/dL), or for 4 months, with four additional months of good glycemic control (blood glucose <150 mg/dL). Differentially expressed LncRNAs and mRNAs were identified through Volcano filtering, and their functions were predicted using gene ontology and pathway enrichment analyses. Compared with age-matched normal rats, rats in continuous poor glycemic control had >1479 differentially expressed LncRNAs (710 downregulated, 769 upregulated), and among those, 511 common LncRNAs had similar expression in Diab and Rev groups (139 downregulated, 372 upregulated). Gene Ontology/pathway analysis identified limited LncRNAs in biological processes, but analysis based on biological processes/molecular function revealed >350 genes with similar expression in Diab and Rev groups; these genes were mainly associated with stress response, cell death, mitochondrial damage and cytokine production. Thus, identifying retinal LncRNAs and their gene targets that do not benefit from termination of hyperglycemia have potential to serve as therapeutic targets to slow down the progression of diabetic retinopathy., (© 2024 The Author(s). Journal of Diabetes published by Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

Diabetic retinopathy (DR) is the leading cause of acquired blindness in diabetic patients. Tropisetron (TRO) exerts potent therapeutic effects against diabetic tissues. The present study aimed to investigate the effects of TRO on retinal injury under diabetic condition. Human retinal pigment epithelial cell line ARPE-19 was treated with high glucose (HG) for 48 h to mimic hyperglycemia-induced retinal damage and subsequently treated with multiple concentrations of TRO for therapeutic intervention. Cell viability and lactate dehydrogenase (LDH) release were detected to assess cell damage. The production of inflammatory cytokines and oxidative stress-related factors was evaluated by corresponding commercial kits. Cell apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The expression of inflammation-, apoptosis-, and SIRT1/ROCK1-related proteins was examined using western blot analysis. Additionally, ARPE-19 cells were transfected with over-express ROCK1 (Ov-ROCK1) or pretreatment with SIRT1 inhibitor EX527 to perform the rescue experiments. TRO alleviated cell damage in HG-induced ARPE-19 cells through elevating cell viability and reducing LDH release. HG-caused excessive production of TNF-α, IL-1β and IL-6, ROS, malondialdehyde and decreased superoxide dismutase activity were partly inhibited by TRO treatment. HG-induced cell apoptosis, accompanied with the upregulation of proapoptotic proteins and the downregulation of antiapoptotic proteins, was hindered by TRO treatment. HG led to the loss of SIRT1 and an elevation of ROCK1 in ARPE-19 cells, which was reversed following TRO treatment. Furthermore, pretreatment with EX527 or transfected with Ov-ROCK1 partially abolished the protective role of TRO against inflammation, oxidative stress and cell apoptosis in HG-challenged ARPE-19 cells. TRO exerted a protective role against HG-caused ARPE-19 cells inflammation, oxidative stress and cell apoptosis by regulating SIRT1/ROCK1 axis, suggesting that TRO might be therapeutic agent for alleviating retinal pigment epithelial cell damage in DR., (© 2024 Wiley Periodicals LLC.)

The purpose of this study was to investigate the role of polysaccharides from Ostrea rivularis Gloud (ORPs) in the progression of diabetic retinopathy (DR) and its anti-angiogenic effect on endothelial cell. Transgenic db/db mice with DR model were used to evaluate the protective effect of ORPs on retinal damage. It was found that ORPs could down-regulated levels of random blood glucose and fasting insulin, and further ameliorate retinal structure abnormalities as well as vascular network structure. Moreover, ORPs could reduce the expression of VEGF in retinal tissue and lessen pathological angiogenesis, thus slowing the progression of DR. In vitro, the proliferation, migration and tube formation of VGEF 165 -induced EA.hy926 cells were inhibited with ORPs administration. Furthermore, the expression of related proteins in the PI3K/AKT pathway and angiogenesis related factors were improved after ORPs intervention. Overall, these findings suggested that ORPs could effectively control the development of DR, and inhibit VGEF 165 -induced EA.hy926 cells proliferation, migration and tube formation, which effects might work through blocking the activation of PI3K/AKT signaling pathway., Competing Interests: Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Chronic inflammation and oxidative stress mediate the pathological progression of diabetic complications, like diabetic retinopathy (DR), peripheral neuropathy (DPN) and impaired wound healing. Studies have shown that treatment with a stable form of arginase 1 that reduces l-arginine levels and increases ornithine and urea limits retinal injury and improves visual function in DR. We tested the therapeutic efficacy of PEGylated arginine deiminase (ADI-PEG20) that depletes l-arginine and elevates l-citrulline on diabetic complications in the db/db mouse model of type 2 diabetes (T2D)., Methods: Mice received intraperitoneal (IP), intramuscular (IM), or intravitreal (IVT) injections of ADI-PEG20 or PEG20 as control. Effects on body weight, fasting blood glucose levels, blood-retinal-barrier (BRB) function, visual acuity, contrast sensitivity, thermal sensitivity, and wound healing were determined. Studies using bone marrow-derived macrophages (BMDM) examined the underlying signaling pathway., Results: Systemic injections of ADI-PEG20 reduced body weight and blood glucose and decreased oxidative stress and inflammation in db/db retinas. These changes were associated with improved BRB and visual function along with thermal sensitivity and wound healing. IVT injections of either ADI-PEG20, anti-VEGF antibody or their combination also improved BRB and visual function. ADI-PEG20 treatment also prevented LPS/IFNℽ-induced activation of BMDM in vitro as did depletion of l-arginine and elevation of l-citrulline., Conclusions/interpretation: ADI-PEG20 treatment limited signs of DR and DPN and enhanced wound healing in db/db mice. Studies using BMDM suggest that the anti-inflammatory effects of ADI-PEG20 involve blockade of the JAK2-STAT1 signaling pathway via l-arginine depletion and l-citrulline production., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R. B. Caldwell, M. Rojas, report financial support was provided by National Institutes of Health. Robert W. Caldwell reports a relationship with National Institutes of Health that includes: funding grants. If there are other authors, they 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 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Diabetic retinopathy (DR) is a diabetic complication that results in visual impairment and relevant retinal diseases. Current therapeutic strategies on DR primarily focus on antiangiogenic therapies, which particularly target vascular endothelial growth factor and its related signaling transduction. However, these therapies still have limitations due to the intricate pathogenesis of DR. Emerging studies have shown that premature senescence of endothelial cells (ECs) in a hyperglycemic environment is involved in the disease process of DR and plays multiple roles at different stages. Moreover, these surprising discoveries have driven the development of senotherapeutics and strategies targeting senescent endothelial cells (SECs), which present challenging but promising prospects in DR treatment. In this review, we focus on the inducers and mechanisms of EC senescence in the pathogenesis of DR and summarize the current research advances in the development of senotherapeutics and strategies that target SECs for DR treatment. Herein, we highlight the role played by key factors at different stages of EC senescence, which will be critical for facilitating the development of future innovative treatment strategies that target the different stages of senescence in DR., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Progressive dysfunction of the retinal pigment epithelium (RPE) and the adjacent photoreceptor cells in the outer retina plays a pivotal role in the pathogenesis of diabetic retinopathy (DR). Here, we observed a marked increase in oxidative stress-induced apoptosis in parallel with higher expression of telomeric protein TIN2 in RPE cells under hyperglycemia in vivo and in vitro. Delving deeper, we confirm that high glucose-induced elevation of mitochondria-localized TIN2 compromises mitochondrial activity and weakens the intrinsic antioxidant defense, thereby leading to the activation of mitochondria-dependent apoptotic pathways. Mechanistically, mitochondrial TIN2 promotes the phosphorylation of FOXO1 and its relocation to the mitochondria. Such translocation of transcription factor FOXO1 not only promotes its binding to the D-loop region of mitochondrial DNA-resulting in the inhibition of mitochondrial respiration-but also hampers its availability to nuclear target DNA, thereby undermining the intrinsic antioxidant defense. Moreover, TIN2 knockdown effectively mitigates oxidative-induced apoptosis in diabetic mouse RPE by preserving mitochondrial homeostasis, which concurrently prevents secondary photoreceptor damage. Our study proposes the potential of TIN2 as a promising molecular target for therapeutic interventions for diabetic retinopathy, which emphasizes the potential significance of telomeric proteins in the regulation of metabolism and mitochondrial function. Created with BioRender ( https://www.biorender.com/ )., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Diabetic retinopathy (DR) is the most prevalent microvascular complication of diabetes mellitus, and it is the primary cause of blindness in the working-age population worldwide. Nevertheless, the pathogenic molecular mechanisms of DR remain elusive. Hub genes were identified through bioinformatics analysis in the GSE102485 and GSE60436 datasets. The DR mouse model was induced using streptozotocin (STZ, 150 mg/kg), and pathological changes in retinal tissue were assessed via HE staining. Apoptosis in retinal tissue cells was evaluated by the TUNEL assay. RT-qPCR and ELISA assays were employed to measure hub genes and inflammatory factor levels, respectively. The aryl hydrocarbon receptor (AHR)/interleukin (IL)-17A (AHR/IL-17A) pathway-associated proteins were detected by western blot. In the high glucose (HG)-induced ARPE-19 cells, CCK-8 and flow cytometry were used to perform cell function studies. Six hub genes associated with DR were screened. The expression levels of RHO, PRPH2, CRX, RCVRN, and NR2E3 were reduced, while the COL1A2 was elevated. NR2E3 overexpression reduced inflammatory factor (TNF-α, IL-1β, and IL-6) and cell apoptosis levels in DR. Furthermore, NR2E3 overexpression promoted HG-induced ARPE-19 cell proliferation. Mechanistically, NR2E3 overexpression facilitated the protein expression of AHR, while suppressing the IL-17 and ACT1 expressions. The introduction of Kyn-101, an AHR inhibitor, notably reversed the inhibitory effects of NR2E3 overexpression on inflammation and apoptosis, which were validated both in vivo and in vitro. NR2E3 inhibits the inflammation and apoptosis by regulating the AHR/IL-17A pathway, providing new insights into the DR treatment., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Diabetic retinopathy (DR) is one of the most common complications of diabetes worldwide and is associated with visual loss and blindness. However, effective treatments for both early- and late-stage DR remain lacking. A streptozotocin-induced diabetic mouse model and high glucose (HG)-treated Müller cell model were established. M1/M2 microglia polarization was assessed by immunofluorescence staining and flow cytometry. Expression of long noncoding RNA (lncRNA) OGRU, cytokines, and other key molecules was detected by quantitative RT-PCR or Western blot. ELISA was used to monitor cytokine secretion. Müller cell-derived exosomes were isolated and characterized by nanopartical tracking analysis, Western blot, and transmission electron microscopy, and exosome uptake assay was used to monitor the intercellular transport of exosomes. Associations among lncRNA-miRNA-mRNA networks were validated by RNA pulldown and RNA immunoprecipitation and dual luciferase assays. Increased M1 polarization but decreased M2 polarization of retinal microglia was observed in DR mice. HG-treated Müller cell-derived exosomes transported OGRU into microglia and promoted microglia polarization toward the M1 phenotype. Mechanistically, OGRU served as a competing endogenous RNA for miR-320-3p, miR-221-3p, and miR-574-5p to regulate aldose reductase (AR), PFKFB3, and glucose transporter 1 (GLUT1) expression in microglia, respectively. Loss of miR-320-3p/miR-221-3p/miR-574-5p or reinforced AR/PFKFB3/GLUT1 abrogated OGRU silencing-mediated microglia polarization in vitro. In vivo studies further showed that OGRU/miR-320-3p/AR, OGRU/miR-221-3p/PFKFB3, and OGRU/miR-574-5p/GLUT1 axes regulated microglia polarization in DR mice. Collectively, Müller cell-derived exosomal OGRU regulated microglia polarization in DR by modulating OGRU/miR-320-3p/AR, OGRU/miR-221-3p/PFKFB3, and OGRU/miR-574-5p/GLUT1 axes., (© 2024 by the American Diabetes Association.)

Purpose: Osteopontin (OPN) is a glycosylated, secreted phosphoprotein known to be elevated in both human and mouse retinas during various stages of diabetic retinopathy. However, its specific roles in modulating ocular surface dynamics and immune responses in diabetes remain unexplored. This study aims to investigate the role of OPN in the development of ocular surface disease (OSD) in type 2 diabetic (T2D) mice., Methods: Three- to four-week-old C57BL/6 wild-type (WT) and OPN-knockout (OPN-/-) mice were fed a high-fat diet (HFD) and were rendered diabetic by streptozotocin (STZ; 40 mg/kg body weight) in citrate buffer (vehicle); non-diabetic controls were injected with vehicle alone. Diabetes was confirmed if blood glucose levels were >200 mg/dL, measured 1-2 weeks post-STZ injection. Control, age- and sex-matched db/db diabetic mice fed a standard chow diet were also included in this study. Ocular surface inflammation was assessed using ELISA to quantify inflammatory cytokine proteins and wheat germ agglutinin (WGA) staining was utilized to highlight corneal surface irregularities. Clinical signs were evaluated by corneal fluorescein staining, tear production measurements, and tear sodium (Na+) concentration assessments. These evaluations were conducted 4, 6, 8 and 16-weeks post-diabetes onset in WT and OPN-/- mice and were compared to those obtained in non-diabetic controls. Statistical analysis was performed using a two-way ANOVA, with significance set at P < 0.05., Results: Both WT and OPN-/- mice developed T2D within 4 and 8 weeks, respectively, following HFD + STZ treatment. Corneal OPN levels in WT diabetic mice increased ~2-fold at 2 weeks and ~4-fold at 16 weeks compared to non-diabetic controls, with similar elevations observed in their tear fluid. Diabetic db/db mice also exhibited elevated OPN levels in the blood and ocular surface, which persisted as diabetes progressed. Enhanced fluorescein staining, indicating corneal irregularities, appeared in WT mice at 8 weeks and in OPN-/- mice at 10 weeks post-T2D induction. Additionally, WGA staining showed a significant reduction in fluorescence intensity in WT mice treated with HFD and STZ, confirming corneal surface irregularities that were delayed in OPN-/- mice. Elevated tear sodium concentration was observed in both WT and OPN-/- diabetic mice without affecting tear production rates. Notably, OPN levels increased early, at week 2, following HFD and STZ treatment, preceding changes in interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and matrix metalloproteinase-9 (MMP-9). Upregulation of IL-6 became apparent at 6 weeks in WT mice and was delayed until 10 weeks in OPN-/- mice post-T2D induction., Conclusions: Elevated OPN levels were detected early post-T2D induction in diabetic WT and db/db mice corneas without initial subclinical changes. This early increase in OPN precedes other proinflammatory cytokines associated with eventual ocular surface inflammation as diabetes progresses. Persistence of OPN also correlated with clinical signs such as increased corneal surface irregularities and elevated tear Na+ concentration. Future research will explore OPN's role as a biomarker in ocular surface disease (OSD), including dry eye disease (DED), and investigate its impact on inflammatory processes and other mechanistic pathways in diabetic ocular complications., Competing Interests: No Competing Interests., (Copyright: © 2024 Datta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Metabolic disorder significantly contributes to diabetic vascular complications, including diabetic retinopathy, the leading cause of blindness in the working-age population. However, the molecular mechanisms by which disturbed metabolic homeostasis causes vascular dysfunction in diabetic retinopathy remain unclear. O-GlcNAcylation modification acts as a nutrient sensor particularly sensitive to ambient glucose. Here, we observe pronounced O-GlcNAc elevation in retina endothelial cells of diabetic retinopathy patients and mouse models. Endothelial-specific depletion or pharmacological inhibition of O-GlcNAc transferase effectively mitigates vascular dysfunction. Mechanistically, we find that Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, are O-GlcNAcylated in diabetic retinopathy. We identify threonine 383 as an O-GlcNAc site on YAP, which inhibits its phosphorylation at serine 397, leading to its stabilization and activation, thereby promoting vascular dysfunction by inducing a pro-angiogenic and glucose metabolic transcriptional program. This work emphasizes the critical role of the O-GlcNAc-Hippo axis in the pathogenesis of diabetic retinopathy and suggests its potential as a therapeutic target., (© 2024. The Author(s).)

Adiponectin (APN), a vasoactive cytokine produced by adipocytes, has emerged as a critical player in retinal diseases. Renowned for its antioxidant, anti-angiogenic, and anti-inflammatory properties, APN levels are closely linked to metabolic disorders, such as insulin resistance, obesity, and diabetic retinopathy (DR). Our previous work demonstrated that APN is similar in efficiency as Avastin in limiting neovascularization in retinal endothelial cells. In this study, we analyzed the effect of APN on retinal epithelial cells to understand its potential impact on eye-related pathologies. Overexpression of APN in ARPE-19 cells predominantly yielded the MMW-APN form, accompanied by increased expression of pro-fibrotic markers and decreased levels of tight junction (TJ) proteins, ZO-1, and Occludin. Further, confocal imaging revealed impaired TJ assembly and the integrity of TJ was also compromised as evidenced by the higher paracellular permeability and lower TEER. Besides, rAPN treatment in ARPE-19 cells as well triggered increased expression of pro-fibrotic markers, pro-MMP2, and enhanced cell migration and proliferation. Mechanistically, these pro-fibrotic effects were mediated by APN-induced phosphorylation of ERK1/2, causing RPE cell transdifferentiation. Furthermore, we identified that MMW-APN was the most prevalent form detected in the vitreous humor of proliferative diabetic retinopathy (PDR) patients, emphasizing the clinical relevance of our findings. Overall, our data suggest that APN, particularly its MMW form, induces epithelial-mesenchymal transition (EMT) and fibrosis in RPE cells, potentially driving the angio-fibrotic shift observed in PDR via ERK1/2 activation., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Diabetic retinopathy (DR) is the leading cause of visual impairment, particularly in the proliferative form (proliferative DR [PDR]). The impact of the PDR microenvironment on microglia, which are the resident immune cells in the central nervous system, and the specific pathological changes it may induce remain unclear. This study aimed to investigate the role of microglia in the progression of PDR under hypoxic and inflammatory conditions. We performed a comprehensive gene expression analysis using human-induced pluripotent stem cell-derived microglia under different stimuli (dimethyloxalylglycine (DMOG), lipopolysaccharide (LPS), and DMOG + LPS) to mimic the hypoxic inflammatory environment characteristic of PDR. Principal component analysis revealed distinct gene expression profiles, with 76 genes synergistically upregulated under combined stimulation. Notably, prostaglandin-endoperoxide synthase 2 (encoding cyclooxygenase (COX)-2) exhibited the most pronounced increase, leading to elevated prostaglandin E2 (PGE2) levels and driving pathological angiogenesis and inflammation via the COX-2/PGE2/PGE receptor 2 signaling axis. Additionally, the upregulation of the fibrogenic genes snail family transcriptional repressor 1 and collagen type I alpha 1 chain suggested a role for microglia in fibrosis. These findings underscore the critical involvement of microglia in PDR and suggest that targeting both the angiogenic and fibrotic pathways may present new therapeutic strategies for managing this condition.

Introduction: Diabetic retinopathy (DR) is a microvascular complication of diabetes in which neurodegeneration has been recently identified as a driving force. In the last years, mesenchymal stromal cells (MSCs) and neurotrophins like Nerve Growth Factor (NGF), have garnered significant attention as innovative therapeutic approaches targeting DR-associated neurodegeneration. However, delivering neurotrophic factors directly in the eye remains a challenge. Hence, this study evaluated the effects of MSCs from human amniotic fluids (hAFSCs) and recombinant human NGF (rhNGF) delivered by human corneal lenticule (hCL) on a high glucose (HG) induced ex vivo model simulating the molecular mechanisms driving DR., Methods: Porcine neuroretinal explants exposed to HG (25 mM for four days) were used to mimic DR ex vivo . hCLs collected from donors undergoing refractive surgery were decellularized using 0.1% sodium dodecyl sulfate and then bioengineered with hAFSCs, microparticles loaded with rhNGF (rhNGF-PLGA-MPs), or both simultaneously. Immunofluorescence (IF) and scanning electron microscopy (SEM) analyses were performed to confirm the hCLs bioengineering process. To assess the effects of hAFSCs and rhNGF, bioengineered hCLs were co-cultured with HG-treated neuroretinal explants and following four days RT-PCR and cytokine array experiments for inflammatory, oxidative, apoptotic, angiogenic and retinal cells markers were performed., Results: Data revealed that HG-treated neuroretinal explants exhibit a characteristic DR-phenotype, including increased level of NF-kB, NOS2, NRF2 GFAP, VEGFA, Bax/Bcl2 ratio and decreased expression of TUBB3 and Rho. Then, the feasibility to bioengineer decellularized hCLs with hAFSCs and rhNGF was demonstrated. Interestingly, co-culturing hAFSCs- and rhNGF- bioengineered hCLs with HG-treated neuroretinal explants for four days significantly reduced the expression of inflammatory, oxidative, apoptotic, angiogenic and increased retinal markers., Conclusion: Overall, we found for the first time that hAFSCs and rhNGF were able to modulate the molecular mechanisms involved in DR and that bioengineered hCLs represents a promising ocular drug delivery system of hAFSCs and rhNGF for eye diseases treatment. In addition, results demonstrated that porcine neuroretinal explants treated with HG is a useful model to reproduce ex vivo the DR pathophysiology., Competing Interests: The authors ND, GC, MA, and TR are employed by Dompé Farmaceutici SpA. ND, MA, MN, AP, and DM are authors of European Patent n° 20179055.7-1109, 09/06/2020, “New Drug Delivery System for Ophtalmic Use”. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pelusi, Hurst, Detta, Pipino, Lamolinara, Conte, Mastropasqua, Allegretti, Di Pietrantonio, Romeo, El Zarif, Nubile, Guerricchio, Bollini, Pandolfi, Schnichels and Mandatori.)

Background: Diabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported., Methods: High-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay., Results: PSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation., Conclusion: METTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR., (© 2024. The Author(s).)

Diabetic retinopathy (DR) is a common complication of diabetes characterized by vascular pathology and neuroinflammation. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule that functions at the crossroads between innate immunity, inflammation, and tissue remodeling. DR is known to involve inflammatory pathways, although the potential relevance of PTX3 has not been explored. We found that PTX3 protein levels increased in the retina of diabetic mice. Similarly, evaluation of a publicly available transcriptomic human dataset revealed increased PTX3 expression in DR with diabetic macular edema and proliferative retinopathy, when compared to nondiabetic retinas or diabetic retinas without complications. To further understand the role of PTX3 within DR, we employed the streptozotocin-induced diabetes model in PTX3 knockout mice (PTX3 KO ), which were followed up for 9 mo to evaluate hallmarks of disease progression. In diabetic PTX3 KO mice, we observed decreased reactive gliosis, diminished microglia activation, and reduced vasodegeneration, when compared to diabetic PTX3 wild-type littermates. The decrease in DR-associated pathological features in PTX3 KO retinas translated into preserved visual function, as evidenced by improved optokinetic response, restored b-wave amplitude in electroretinograms, and attenuated neurodegeneration. We showed that PTX3 induced an inflammatory phenotype in human retinal macroglia, characterized by GFAP upregulation and increased secretion of IL6 and PAI-1. We confirmed that PTX3 was required for TNF-α-induced reactive gliosis, as PTX3 KO retinal explants did not up-regulate GFAP in response to TNF-α. This study reveals a unique role for PTX3 as an enhancer of sterile inflammation in DR, which drives pathogenesis and ultimately visual impairment., Competing Interests: Competing interests statement:The authors declare no competing interest.

Purpose: To examine the effects of hydrogen water on retinal blood flow (RBF) dysregulation in diabetes, we evaluated changes in RBF in response to flicker stimulation and systemic hyperoxia in diabetic mice., Methods: Twelve type 2 diabetic mice were divided into a group that received non-hydrogen water (n = 6, control group) and the other that received hydrogen-rich water (0.6-0.8 mM) (n = 6, HRW group) from six weeks of age. Body weight, blood glucose, intraocular pressure, and blood pressure were evaluated from eight to 14 weeks of age. RBF was measured in the vascular area of the optic disc as mean blur rate using laser speckle flowgraphy in the resting state and response to flicker stimulation and hyperoxia. We evaluated glial activation and oxidative stress based on immunofluorescence expression., Results: At 14 weeks, blood glucose level was significantly lower in the HRW group, though still elevated. RBF changes improved significantly in the HRW group compared with the control group from eight weeks of age and persisted throughout the study. Immunofluorescent expression of glial fibrillary acidic protein, particularly in the outer plexiform layer, was significantly decreased in the HRW group. Among oxidative stress markers, 3-nitrotyrosine was significantly suppressed in the HRW group., Conclusions: Hydrogen-rich water intake significantly improved RBF dysregulation in diabetic mice. Hydrogen may improve impaired neurovascular coupling function in diabetic mice by suppressing gliosis and oxidative stress in the retina., Translational Relevance: This study highlights the potential of oral intake of hydrogen-rich water to mitigate retinal dysfunction in diabetic mice.

Purpose: The purpose of this study was to investigate the correlation between insulin and Fetuin-B (FETUB) and the influence of FETUB on insulin signaling pathway in diabetic retinopathy (DR)., Methods: Enzyme-linked immunosorbent assay (ELISA) was used to analyze FETUB and insulin levels in the serum and aqueous fluid of patients with DR and healthy controls. Quantitative PCR (q-PCR), Western blotting, and ELISA were used to examine FETUB expression in ARPE-19, BV2, and Müller cells under insulin stimulation. Co-immunoprecipitation was used to investigate the interaction of FETUB with insulin receptor-β (IRβ). Insulin resistance (IR)-BV2 and IR-Müller cells were treated with FETUB recombinant protein or FETUB short hairpin RNA (shRNA) to explore the influence of FETUB on insulin signaling pathway in DR. LY294002 (a PI3K pathway inhibitor) was used to determine whether FETUB affects glucose metabolism via the PI3K/Akt pathway., Results: In aqueous fluid, FETUB concentrations were positively correlated with insulin levels. FETUB expression increased in Müller and BV2 cells under insulin regulation, and FETUB interacted with IRβ in retinal cells and mice retina. The interaction between IRβ and FETUB increased in BV2 and Müller cells under high-glucose than in controls. Insulin signaling pathway activation was suppressed in FETUB recombinant protein-treated BV2 and Müller cells but increased in FETUB shRNA-transfected cells. FETUB shRNA could not reverse LY294002-mediated inhibition of glucose transporter-4 expression., Conclusions: Retinal cells are the source of insulin-regulated FETUB. The FETUB interacts with IRβ and affects insulin signaling pathway in BV2 and Müller cells. FETUB may aggravate IR in BV2 and Müller cells via the PI3K/Akt pathway.

This study aimed to characterize the role of female sex in the pathogenesis of diabetic retinopathy. In the retinae of female Ins2Akita-diabetic mice (F-IA), ovariectomized female Ins2Akita-diabetic mice (F-IA/OVX), male Ins2Akita-diabetic mice (M-IA), and female STZ-diabetic mice (F-STZ), the formation of reactive metabolites and post-translational modifications, damage to the neurovascular unit, and expression of cellular stress response genes were analyzed. Compared to the male diabetic retina, the concentrations of the glycation adduct fructosyl-lysine, the Maillard product 3-deoxyglucosone, and the reactive metabolite methylglyoxal were significantly reduced in females. In females, there was also less evidence of diabetic damage to the neurovascular unit, as shown by decreased pericyte loss and reduced microglial activation. In the male diabetic retina, the expression of several members of the crystallin gene family (Cryab, Cryaa, Crybb2, Crybb1, and Cryba4) was increased. Clinical data from type 1 diabetic females showed that premenopausal women had a significantly lower prevalence of diabetic retinopathy compared to postmenopausal women stratified for disease duration and glycemic control. These data emphasize the importance of estradiol in protecting the diabetic retina and highlight the pathogenic relevance of sex in diabetic retinopathy., (© 2024. The Author(s).)

Background and Purpose: Diabetic retinopathy (DR) is a prevalent complication of diabetes, with a rising global incidence, and can result in significant vision impairment and potential blindness in adults. Corilagin (COR) has been shown to regulate several pathological processes. However, the specific protective role and mechanism of action of COR in DR remain unknown., Experimental Approach: The protective effects and mechanisms of COR in DR were examined using the ARPE-19 cell line and C57BL/6 mice. Intraretinal tissue damage and molecular markers were evaluated to investigate the impact of COR on oxidative stress and cell death pathways., Key Results: In vitro, COR significantly reduced the cytotoxic effects of high glucose (HG) on ARPE-19 cells. Furthermore, COR also effectively decreased HG-induced lipid peroxidation, iron deposition, and ferroptosis and reduced damage to retinal tight junction proteins. Similarly, an in vivo study of streptozotocin (STZ)-induced DM mice showed that the daily gavage of COR for eight weeks notably alleviated DR. Mechanistically, COR activated the Nrf2 antioxidant signaling pathway both in vivo and in vitro, preventing HG-induced alterations in morphological and biochemical parameters. Notably, our study demonstrated that compared with controls, Nrf2 knockout mice and siNrf2-treated cells were more vulnerable to ferroptosis under HG conditions, and the protective effect of COR on DR was substantially diminished in these models., Conclusion and Implications: These data indicate that COR has a protective effect against HG-induced retinal injury via a mechanism associated with the Nrf2-dependent antioxidant pathway and ferroptosis regulation., 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 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

INS C94Y transgenic pigs represent a model for mutant insulin gene-induced diabetes of youth, with impaired insulin secretion and beta cell loss, leading to elevated fasting blood glucose levels. A key complication of diabetes mellitus is diabetic retinopathy (DR), characterized by hyperglycemia-induced abnormalities in the retina. Adjacent to the retina lies the vitreous, a gelatinous matrix vital for ocular function. It harbors proteins and signaling molecules, offering insights into vitreous biology and ocular health. Moreover, as a reservoir for secreted molecules, the vitreous illuminates molecular processes within intraocular structures, especially under pathological conditions. To uncover the proteomic profile of porcine vitreous and explore its relevance to DR, we employed discovery proteomics to compare vitreous samples from INS C94Y transgenic pigs and wild-type controls. Our analysis identified 1404 proteins, with 266 showing differential abundance in INS C94Y vitreous. Notably, the abundances of ITGB1, COX2, and GRIFIN were significantly elevated in INS C94Y vitreous. Gene Set Enrichment Analysis unveiled heightened MYC and mTORC1 signaling in INS C94Y vitreous, shedding light on its biological significance in diabetes-associated ocular pathophysiology. These findings deepen our understanding of vitreous involvement in DR and provide valuable insights into potential therapeutic targets. Raw data are accessible via ProteomeXchange (PXD038198)., (© 2024 The Author(s). PROTEOMICS published by Wiley‐VCH GmbH.)

Aims/hypothesis: Diabetic retinopathy is characterised by neuroinflammation that drives neuronal and vascular degenerative pathology, which in many individuals can lead to retinal ischaemia and neovascularisation. Infiltrating macrophages and activated retina-resident microglia have been implicated in the progression of diabetic retinopathy, although the distinct roles of these immune cells remain ill-defined. Our aim was to clarify the distinct roles of macrophages/microglia in the pathogenesis of proliferative ischaemic retinopathies., Methods: Murine oxygen-induced retinopathy is commonly used as a model of ischaemia-induced proliferative diabetic retinopathy (PDR). We evaluated the phenotype macrophages/microglia by immunostaining, quantitative real-time RT-PCR (qRT-PCR), flow cytometry and scRNA-seq analysis. In clinical imaging studies of diabetic retinopathy, we used optical coherence tomography (OCT) and OCT angiography., Results: Immunostaining, qRT-PCR and flow cytometry showed expression levels of M1-like macrophages/microglia markers (CD80, CD68 and nitric oxide synthase 2) and M2-like macrophages/microglia markers (CD206, CD163 and macrophage scavenger receptor 1) were upregulated in areas of retinal ischaemia and around neo-vessels, respectively. scRNA-seq analysis of the ischaemic retina revealed distinct ischaemia-related clusters of macrophages/microglia that express M1 markers as well as C-C chemokine receptor 2. Inhibition of Rho-kinase (ROCK) suppressed CCL2 expression and reduced CCR2-positive M1-like macrophages/microglia in areas of ischaemia. Furthermore, the area of retinal ischaemia was reduced by suppressing blood macrophage infiltration not only by ROCK inhibitor and monocyte chemoattractant protein-1 antibody but also by GdCl 3 . Clinical imaging studies of diabetic retinopathy using OCT indicated potential involvement of macrophages/microglia represented by hyperreflective foci in areas of reduced perfusion., Conclusions/interpretation: These results collectively indicated that heterotypic macrophages/microglia differentially contribute to retinal ischaemia and neovascularisation in retinal vascular diseases including diabetic retinopathy. This adds important new information that could provide a basis for a more targeted, cell-specific therapeutic approach to prevent progression to sight-threatening PDR., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Context: Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM). Circular RNAs (circRNAs) act as key regulators of DR development by regulating inflammation and angiogenesis. Objective: This study aimed to elucidate the function and mechanism of hsa&#95;circ&#95;0000047 in DR. Materials and methods: High glucose (HG) was used to induce human retinal microvascular endothelial cells (hRMECs) to construct a DR model in vitro. Qualitative real-time polymerase chain reaction (qRT-PCR) or western blotting were used to detected the levels of hsa&#95;circ&#95;0000047, miR-6720-5p, and CYB5R2 in DR and HG-indeced hRMECs. Cell functional experiments were performed to detect the change of viability, inflammation, migration, invasion, and angiogenesis of HG-induced hRMECs. Besides, the correlation between miR-6720-5p and hsa&#95;circ&#95;0000047/CYB5R2 was confirmed by luciferase assay and Pearson correlation analysis. Results: hsa&#95;circ&#95;0000047 and CYB5R2 were downregulated in DR, whereas miR-6720-5p was upregulated in DR. Cell functional experiments showed that hsa&#95;circ&#95;0000047 overexpression restrained viability, inflammation, migration, invasion, and angiogenesis of HG-induced hRMECs. Regarding mechanism, hsa&#95;circ&#95;0000047 could sponge miR-6720-5p to regulate CYB5R2 expression in hRMECs. Additionally, CYB5R2 knockdown reversed the effects of hsa&#95;circ&#95;0000047 overexpression on HG-induced hRMECs. Conclusion: Our study revealed that hsa&#95;circ&#95;0000047 alleviated inflammation and angiogenesis in HG-induced hRMECs by targeting the miR-6720-5p/CYB5R2 axis, which may be a novel biomarker for DR therapy.

The retina has low dopamine levels early in diabetes. To determine how low dopamine levels affected dopamine signaling, the effects of dopamine receptor agonists and mRNA localization were measured after 6 weeks of diabetes. Whole retina ex vivo electroretinogram (ERG) recordings were used to analyze how dopamine type 1 receptor (D1R) and type 4 (D4R) agonists change the light-evoked retinal responses of non-diabetic and 6-week diabetic (STZ injected) mouse retinas. Fluorescence in situ hybridization was utilized to analyze D4R and D1R mRNA locations and expression levels. D4R activation reduced A- and B-wave ERG amplitudes and increased B-wave implicit time and rise-time in the non-diabetic group without a corresponding change in the diabetic group. D1R activation increased B-wave rise-time and oscillatory potential peak time in the non-diabetic group also with no change in the diabetic group. The lack of responsivity to D1R or D4R agonists shows an impairment of dopamine signaling in the diabetic retina. D4R mRNA was found primarily in the outer nuclear layer where photoreceptor cell bodies reside. D1R mRNA was found in the inner nuclear layer and ganglion cell layer that contain bipolar, amacrine, horizontal and ganglion cells. There was no change in D4R or D1R mRNA expression between the non-diabetic and diabetic retinas. This suggests that the significant dopamine signaling changes observed were not from lower receptor expression levels but could be due to changes in dopamine receptor activity or protein levels. These studies show that changes in retinal dopamine signaling could be an important mechanism of diabetic retinal dysfunction., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Retinal vascular leakage is a major event in several retinal diseases, including diabetic retinopathy (DR). In a previous study, we demonstrated that the aqueous humor concentration of Cystatin C (CST3), a physiological inhibitor of cysteine protease, is negatively correlated with the severity of diabetic macular edema. However, its function in the retina has not been clearly elucidated. In this study, we found a significant decrease in the aqueous humor concentration of CST3 with DR progression. Furthermore, we found that CST3 was expressed in retinal endothelial cells and that its expression was significantly downregulated in high glucose-treated human retinal microvascular endothelial cells (HRMECs) and the retinal vessels of oxygen-induced retinopathy (OIR) mice. Silencing CST3 expression resulted in decreased HRMEC migration and tubule formation ability. Exogenous addition of the CST3 protein significantly improved HRMEC migration and tubular formation. In-vivo experiments demonstrated that CST3 silencing induced retinal vascular leakage in WT mice, while its intravitreal injection significantly reduced retinal leakage in OIR mice. Mechanistically, CST3 promoted the expression of the downstream adhesion molecules, claudin5, VE-cadherin, and ZO-1, in retinal vascular cells by regulating the Rap1 signaling pathway. Therefore, this study revealed a novel mechanism by which CST3 improves retinal vascular function and provided evidence that it is a potential therapeutic target for retinal vascular leakage., Competing Interests: Declaration of Competing interest The authors declare that no confict of interest exists., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Diabetic retinopathy is the most common microvascular complication caused by chronic hyperglycemia and is a leading cause of blindness; however, the underlying molecular mechanism has not been clearly elucidated. Therefore, we investigated whether regulation of AMPK and GAPDH by transglutaminase 2 (TGase2) is important for hyperglycemia-induced microvascular leakage in the diabetic retina. In human retinal endothelial cells (HRECs) and diabetic mouse retinas, we found that TGase2, activated by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels, played an essential role in hyperglycemia-induced vascular leakage. ROS generation and TGsae2 activation were involved in hyperglycemia-induced AMPK dephosphorylation, which resulted in vascular endothelial-cadherin (VE-cadherin) disassembly and increased fluorescein isothiocyanate-dextran extravasation. Furthermore, high glucose-induced TGase2 activation suppressed GAPDH activity, determined by an on-chip activity assay, through inhibition of AMPK, which induced VE-cadherin disassembly and endothelial permeability in HRECs. Overall, our findings suggest that inhibition of AMPK and GAPDH by TGase2 plays a pivotal role in hyperglycemia-induced microvascular leakage in the retinas of diabetic mice., (© 2024 by the American Diabetes Association.)

Diabetic vascular complication including diabetic retinopathy is a major morbidity in Saudia Arabia. The polyol pathway aka aldose reductase (AR) pathway has gained significant association with diabetic retinopathy with regard to chronically enhanced glucose metabolism. Considerable research has been put forth to develop more effective therapeutic strategies to overcome the overwhelming challenges of vascular complications associated with diabetes. In this regard, constituents of Cichorium intybus can offer strong AR inhibitory potential because of their strong antidiabetic properties. Therefore, aim of this study was to investigate the AR inhibitory as well as antiglycation potential of C. intybus extract/compounds. The preliminary in vitro results showed that methanolic extract of C. intybus could significantly inhibit AR enzyme and advanced glycation end product formation. Eventually, based on previous studies and reviews, we selected one hundred fifteen C. intybus root constituents and screened them through Lipinski's rule of five and ADMET analysis. Later, after molecular docking analysis of eight compounds, five best were selected for molecular dynamics simulation to deduce their binding affinity with the AR enzyme. Finally, three out of five compounds were further tested in vitro for their AR inhibitory potential and antiglycation properties. Enzyme assay and kinetic studies showed that all the three tested compounds were having potent AR inhibitory properties, although to a lesser extent than ellagic acid and tolrestat. Similarly, kaempferol showed strong antiglycation property equivalent to ellagic acid, but greater than aminoguanidine. Intriguingly, significant reduction in sorbitol accumulation in RBCs by the tested compounds substantiated strong AR inhibition by these compounds. Moreover, decrease in sorbitol accumulation under high glucose environment also signifies the potential application of these compounds in diabetic retinopathy and other vascular complications. Thus, in sum, the in silico and in vitro studies combinedly showed that C. intybus root is a treasure for therapeutic compounds and can be explored further for drug development against diabetic retinopathy., 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 Elsevier B.V. All rights reserved.)

The formation of advanced glycation end product (AGE) is a risk factor for diabetic retinopathy. Since the current treatment for diabetic retinopathy is accompanied by side effects, preliminary findings have suggested Peperomia pellucida (L.) Kunth as a potential alternative therapeutic option for diabetic retinopathy. This study aimed to elucidate the anti-inflammatory mechanism of P. pellucida in the AGE-stimulated human retinal pigment epithelial cell line ARPE-19. Phytochemical analysis revealed phenylpronanoids, terpenes, and fatty acids in P. pellucida. Through in vitro cell viability assay, the P. pellucida methanolic extract (IC 50  = 8.70 mg/mL) and ethyl acetate fraction (IC 50  = 7.34 mg/mL) were considered as non toxic for ARPE-19. AGE induced an inflammatory response in ARPE-19 by upregulating the gene (2.4-5.8-fold) and protein (1.4-2.3-fold) expression of signal transducer and activator of transcription 3 (STAT3), interleukin-8 (IL-8), monocyte chemoattractant protein-1, matrix metalloproteinase 2, and vascular endothelial growth factor. At 1.5 mg/mL, P. pellucida methanolic extract suppressed IL-8 expression (p < 0.05), implying its anti-inflammatory action at the early inflammatory stage through the Janus kinase (JAK)-STAT3 pathway. The methanolic extract also restored the ARPE-19 viability under AGE-induced inflammatory stress. The downregulation of inflammatory biomarkers along the JAK-STAT3 pathway suggested P. pellucida as a promising anti-inflammatory source for diabetic retinopathy., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Diabetic retinopathy (DR) is recognized as the most prevalent retinal degenerative disorder. Inflammatory response usually precedes microvascular alteration and is the primary factor of diabetic retinopathy. Activated microglia express many pro-inflammatory cytokines that exacerbate retina inflammation and disruption. In the present study, we found that MSCs alleviated blood-retina barrier (BRB) breakdown in diabetic rats, as evidenced by reduced retinal edema, decreased vascular leakage, and increased occludin expression. The MSC-treated retinal microglia exhibited reduced expression of M1-phenotype markers in the diabetic rats, including inducible nitric oxide synthase (iNOS), CD16, and pro-inflammatory cytokines. On the other hand, MSCs increased the expression of M2-phenotype markers, such as arginase-1 (Arg-1), CD206, and anti-inflammatory cytokines. HMGB1/TLR4 signaling pathway is activated in DR and inhibited after MSC treatment. Consistent with in vivo evidence, MSCs drove BV2 microglia toward M2 phenotype in vitro. Overexpression of HMGB1 in microglia reversed the effects of MSC treatment, suggesting HMGB1/TLR4 pathway is necessary for MSCs' regulatory effects on microglia polarization. Collectively, MSCs exert beneficial effects on DR by polarizing microglia from M1 toward M2 phenotype via inhibiting the HMGB1/TLR4 signaling pathway., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Retinal fibrosis is one of the major features of diabetic retinopathy (DR). Our recent research has shown that Poldip2 can affect early DR through oxidative stress, but whether Poldip2 would regulate retinal fibrosis during DR development is still enigmatic. Here, diabetic Sprague-Dawley (SD) rats were induced with streptozotocin (STZ) and treated with adeno-associated virus serotype 9-polymerase-δ interacting protein 2 (Poldip2) shRNA, while human adult retinal pigment epithelial (ARPE-19) cells were treated with high glucose or Poldip2 siRNA. We identified that in STZ-induced DR rats and ARPE-19 cells treated with high glucose, the expression of Poldip2, transforming growth factor-β1 (TGF-β1), phosphorylated-SMAD3/SMAD3, MMP9, COL-1, FN, and CTGF increased while the expression of cadherin decreased. However, deleting Poldip2 inhibited the TGF-β1/SMAD3 signaling pathway and attenuated the above protein expression in vivo and in vitro. Mechanistically, we found that Poldip2 promotes the activation of SMAD3, facilitates its nuclear translocation through interacting with it, and significantly enhances the expression of fibrosis makers. Collectively, Poldip2 was identified is a novel regulator of DR fibrosis and is expected to become a therapeutic target for PDR., (© 2024 by the American Diabetes Association.)

Transfer RNA-derived fragments (tRFs) represent a novel class of non-coding RNA transcripts that possess specific biological functions. However, the involvement of tRFs in retinal microvascular diseases remains poorly understood. In this study, we aimed to reveal whether modulation of tRF-30 expression could attenuate pathological retinal neovascular diseases. Our findings demonstrate a significant upregulation of tRF-30 expression levels in both in vivo models of diabetic retinopathy (DR) and in vitro endothelial sprouting models. Conversely, inhibition of tRF-30 expression suppressed the formation of abnormal neovascularization in the retina in vivo, while reducing the proliferation and migration activity of retinal vascular endothelial cells in vitro. We also found that tRF-30 modulates retinal neovascularization through the tRF-30/TRIB3/signal transducer and activated transcription 3 signaling pathway. Furthermore, we validated a significant upregulation of tRF-30 expression levels in the vitreous humor of DR patients, with high levels of both validity and specificity in diagnostic testing. Collectively, our findings highlight a pro-angiogenic role for tRF-30 in DR. Intervening in the tRF-30 signaling pathway may represent a promising prevention and treatment strategy for retinal angiogenesis., (© 2024 International Federation for Cell Biology.)

Diabetic retinopathy (DR) is the leading cause of vision loss and blindness among working-age adults. Pericyte loss is an early pathological feature of DR. Under hyperglycemic conditions, reactive oxygen species (ROS) production increases, leading to oxidative stress and subsequent mitochondrial dysfunction and apoptosis. Dysfunctional pericyte can cause retinal vascular leakage, obliteration, and neovascularization. Glutaredoxin 2 (Grx2) is a mitochondrial glutathione-dependent oxidoreductase which protects cells against oxidative insults by safeguarding mitochondrial function. Whether Grx2 plays a protective role in diabetes-induced microvascular dysfunction remains unclear. Our findings revealed that diabetes-related stress reduced Grx2 expression in pericytes, but not in endothelial cells. Grx2 knock-in ameliorated diabetes-induced microvascular dysfunction in vivo DR models. Decreased Grx2 expression led to significant pericyte apoptosis, and pericyte dysfunction, namely reduced pericyte recruitment towards endothelial cells and increased endothelial cell permeability. Conversely, upregulating Grx2 reversed these effects. Furthermore, Grx2 regulated pericyte apoptosis by modulating complex I activity, which is crucial for pericyte mitochondrial function. Overall, our study uncovered a novel mechanism whereby high glucose inhibited Grx2 expression in vivo and in vitro. Grx2 downregulation exacerbated pericyte apoptosis, pericyte dysfunction, and retinal vascular dysfunction by inactivating complex I and mediating mitochondrial dysfunction in pericytes., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Diabetic retinopathy (DR) is a microvascular complication of diabetes characterized by neurovascular impairment of the retina. The dysregulation of the mitophagy process occurs before apoptotic cell death and the appearance of vascular damage. In particular, mitochondrial alterations happen during DR development, supporting the hypothesis that mitophagy is negatively correlated to disease progression. This process is mainly regulated by the PTEN-induced putative kinase protein 1 (PINK1)/Parkin pathway whose activation promotes mitophagy. In this review, we will summarize the evidence reported in the literature demonstrating the involvement of the PINK1/Parkin pathway in diabetic retinopathy-induced retinal degeneration., 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Diabetic retinopathy, a leading cause of vision impairment, is marked by microvascular complications in the retina, including pericyte loss, a key indicator of early-stage disease. This study explores the therapeutic potential of exosomes derived from immortalized adipose-mesenchymal stem cells differentiated into pericyte-like cells in restoring the function of mouse retinal microvascular endothelial cells damaged by high glucose conditions, thereby contributing to the understanding of early diabetic retinopathy intervention strategies. To induce immortalized adipose-mesenchymal stem cells differentiation into pericyte-like cells, the study employed pericyte growth supplement. And confirmed the success of cell differentiation through the detection of α-smooth muscle actin and neural/glial antigen 2 expression by Western blot and immunofluorescence. Exosomes were isolated from the culture supernatant of immortalized adipose-mesenchymal stem cells using ultracentrifugation and characterized through Western blot for exosomal markers (CD9, CD81, and TSG101), transmission electron microscopy, and nanoparticle tracking analysis. Their influence on mouse retinal microvascular endothelial cells under high glucose stress was assessed through various functional assays. Findings revealed that exosomes, especially those from pericyte-like immortalized adipose-mesenchymal stem cells, were efficiently internalized by retinal microvascular endothelial cells and effectively counteracted high glucose-induced apoptosis. These exosomes also mitigated the rise in reactive oxygen species levels and suppressed the migratory and angiogenic properties of retinal microvascular endothelial cells, as demonstrated by Transwell and tube formation assays, respectively. Furthermore, they preserved endothelial barrier function, reducing hyperglycemia-induced permeability. At the molecular level, qRT-PCR analysis showed that exosome treatment modulated the expression of critical genes involved in angiogenesis (VEGF-A, ANG2, MMP9), inflammation (IL-1β, TNF-α), gap junction communication (CX43), and cytoskeletal regulation (ROCK1), with the most prominent effects seen with exosomes from pericyte-like immortalized adipose-mesenchymal stem cells. High glucose increased the expression of pro-angiogenic and pro-inflammatory markers, which were effectively normalized post-exosome treatment. In conclusion, this research highlights the reparative capacity of exosomes secreted by pericyte-like differentiated immortalized adipose-mesenchymal stem cells in reversing the detrimental effects of high glucose on retinal microvascular endothelial cells. By reducing apoptosis, oxidative stress, inflammation, and abnormal angiogenic behavior, these exosomes present a promising avenue for therapeutic intervention in early diabetic retinopathy. Future studies can focus on elucidating the precise molecular mechanisms and exploring their translational potential in vivo., Competing Interests: Declarations of interest All authors disclosed no relevant relationships., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective :The aim of our study was to determine the molecular mechanism of BMP4 (bone morphogenetic protein 4) in DR (diabetic retinopathy). Methods : Human retinal endothelial cell (HRECs) induced by high glucose to simulate one of the pathogenesis in the diabetic retinopathy (DR) model. RT-qPCR and western blot were used to detect the mRNA and protein levels of BMP4 in the STZ/HG group. Flow cytometry and TUNEL staining were performed to detect the apoptosis. Angiogenesis was evaluated by tube formation assay. Transwell assay and wound healing assay were used to detect cell migration ability. H&E staining was used to evaluate the pathological changes. Results : BMP4 was significantly upregulated in the STZ/HG group. Sh-BMP4 significantly inhibited the migration and angiogenesis of RVECs induced by HG. In addition, both in vivo and in vitro experiments confirmed that sh-BMP4 could significantly promote RVECs apoptosis in the HG/STZ group. Western blot results showed that sh-BMP4 could down-regulate the expressions of p-smad1, p-smad5 and VEGF. Conclusions : Inhibition of BMP4 could alleviate the damage of diabetic retinopathy by regulating the p-smad1/5/VEGF signaling axis, inhibiting angiogenesis and promoting apoptosis.

Regulation of visual system function demands precise gene regulation. Dysregulation of miRNAs, as key regulators of gene expression in retinal cells, contributes to different eye disorders such as diabetic retinopathy (DR), macular edema, and glaucoma. MIR-96, a member of the MIR-183 cluster family, is widely expressed in the retina, and its alteration is associated with neovascular eye diseases. MIR-96 regulates protein cascades in inflammatory and insulin signaling pathways, but further investigation is required to understand its potential effects on related genes. For this purpose, we identified a series of key target genes for MIR-96 based on gene and protein interaction networks and utilized text-mining resources. To examine the MIR-96 impact on candidate gene expression, we overexpressed MIR-96 via adeno-associated virus (AAV)-based plasmids in human retinal pigment epithelial (RPE) cells. Based on Real-Time PCR results, the relative expression of the selected genes responded differently to overexpressed MIR-96. While the expression levels of IRS2, FOXO1, and ERK2 (MAPK1) were significantly decreased, the SERPINF1 gene exhibited high expression simultaneously. pAAV-delivered MIR-96 had no adverse effect on the viability of human RPE cells. The data showed that changes in insulin receptor substrate-2 (IRS2) expression play a role in disrupted retinal insulin signaling and contribute to the development of diabetic complications. Considered collectively, our findings suggest that altered MIR-96 and its impact on IRS/PI3K/AKT/VEGF axis regulation contribute to DR progression. Therefore, further investigation of the IRS/PI3K/AKT/VEGF axis is recommended as a potential target for DR treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hosseinpoor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

12-hydroxyeicosatetraenoic acid (12-HETE), a major metabolite of arachidonic acid, is converted by 12/15-lipoxygenase and implicated in diabetic retinopathy (DR). Our previous study demonstrated a positive correlation between 12-HETE and the prevalence of DR. However, reasons for the increased production of 12-HETE are unclear, and the underlying mechanisms through which 12-HETE promotes DR are unknown. This study aimed to elucidate the correlation between 12-HETE and DR onset, investigate potential mechanisms through which 12-HETE promotes DR, and seek explanations for the increased production of 12-HETE in diabetes. We conducted a prospective cohort study, which revealed that higher serum 12-HETE levels could induce DR. Additionally, G protein-coupled receptor 31 (GPR31), a high-affinity receptor for 12-HETE, was expressed in human retinal microvascular endothelial cells (HRMECs). 12-HETE/GPR31-mediated HRMEC inflammation occurred via the p38 MAPK pathway. 12-HETE levels were significantly higher in the retina of mice with high-fat diet (HFD)- and streptozotocin (STZ)-induced diabetes than in those with only STZ-induced diabetes and healthy controls. They were positively correlated with the levels of inflammatory cytokines in the retina, indicating that HFD could induce increased 12-HETE synthesis in patients with diabetes in addition to hyperglycemia. Conclusively, 12-HETE is a potential risk factor for DR. The 12-HETE/GPR31 axis plays a crucial role in HRMEC dysfunction and could be a novel target for DR prevention and control. Nevertheless, further research is warranted to provide comprehensive insights into the complex underlying mechanisms of 12-HETE in DR., (© 2024 Federation of American Societies for Experimental Biology.)

Background: Diabetic retinopathy (DR), the principal cause of acquired blindness among the working-age population, is the most frequent microvascular complication of diabetes. Although metabolic disorders are hypothesized to play a role in its pathogenesis, the underlying mechanism remains largely elusive., Methods: To elucidate the mechanism, we initially compared metabolite profiles of vitreous fluid between 23 patients with DR and 12 non-diabetic controls using liquid chromatography/tandem mass spectrometry, identifying the distinct metabolite indoxyl sulfate (IS). Subsequently, streptozotocin (STZ)-induced diabetic and IS-injected rat models were established to examine the effects of IS on retinal microvasculature. RNA sequencing was conducted to identify potential regulatory mechanisms in IS-treated human retinal endothelial cells (HREC). Finally, target gene knockdown in HREC and treatment of IS-injected rats with inhibitors (targeting IS production or downstream regulators) were employed to elucidate the detailed mechanisms and identify therapeutic targets for DR., Results: Metabolomics identified 172 significantly altered metabolites in the vitreous humor of diabetics, including the dysregulated tryptophan metabolite indoxyl sulfate (IS). IS was observed to breach the blood-retinal barrier and accumulate in the intraocular fluid of diabetic rats. Both in vivo and in vitro experiments indicated that elevated levels of IS induced endothelial apoptosis and disrupted cell junctions. RNA sequencing pinpointed prostaglandin E2 (PGE 2 ) synthetase-cyclooxygenase 2 (COX-2) as a potential target of IS. Validation experiments demonstrated that IS enhanced COX-2 expression, which subsequently increased PGE 2 secretion by promoting transcription factor EGR1 binding to COX-2 DNA following entry into cells via organic anion transporting polypeptides (OATP2B1). Furthermore, inhibition of COX-2 in vivo or silencing EGR1/OATP2B1 in HREC mitigated IS-induced microcapillary damage and the activation of COX-2/PGE 2 ., Conclusion: Our study demonstrated that indoxyl sulfate (IS), a uremic toxin originating from the gut microbiota product indole, increased significantly and contributed to retinal microvascular damage in diabetic retinopathy (DR). Mechanistically, IS impaired retinal microvascular integrity by inducing the expression of COX-2 and the production of PGE 2 . Consequently, targeting the gut microbiota or the PGE 2 pathway may offer effective therapeutic strategies for the treatment of DR., (© 2024. The Author(s).)

Background: Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus (DM) and a significant cause of acquired blindness in the working-age population worldwide. Aging is considered as an important risk factor for DR development. Macrophages in aged mice bear typical M2 marker proteins but simultaneously express a pro-inflammatory factor profile. This may explain why the level of intraocular inflammation does not decrease during proliferative diabetic retinopathy (PDR) despite the occurrence of neovascularization and fibrosis (M2 activation). α-Klotho (KL) was originally discovered as a soluble anti-aging factor, which is mainly expressed in kidney tubular epithelium, choroid plexus in the brain and secreted in the blood. However, the role of KL in DR pathophysiology has not been previously reported., Methods: Type 1 (streptozotocin [STZ]-induced) and type 2 (a high-fat diet along with a low dose of STZ) diabetic mouse models were established and injected with or without KL adenovirus via the tail vein for 12 weeks. Vldlr -/- mice were injected intravitreally with or without soluble KL protein from P8 to P15. The retinal structure and function were analyzed by electroretinogram and optical coherence tomography. The neovascular lesions were analyzed by retinal flat mount and RPE flat mount. The senescence markers, macrophage morphology, and KL expression levels were detected by immunofluorescence staining. A cell model was constructed using RAW264.7 cells stimulated by 4-hydroxynonenal (4HNE) and transfected with or without KL adenovirus. The senescence-associated secretory phenotypes were detected by qRT-PCR. Senescence was detected by SA-β-Gal staining. Serum, aqueous humor, and vitreous humor KL levels of proliferative diabetic retinopathy (PDR) patients were measured by enzyme-linked immunosorbent assay. Quantitative proteomics and bioinformatics were applied to predict the change of proteins and biological function after overexpression of KL in macrophages. The effects of KL on the HECTD1 binding to IRS1 were analyzed by bioinformatics, molecular docking, and Western Blot., Results: Serum, aqueous humor, and vitreous humor KL levels were lower in patients with PDR than in those with cataracts. KL relieved the retinal structure damage, improved retina function, and inhibited retinal senescence in diabetic mice. KL administration attenuated the neovascular lesions in VLDLR -/- mice by decreasing the secretion of VEGFA and FGF2 from macrophages. KL also protected RAW264.7 cells from 4HNE-induced senescence. Additionally, it inhibited E3 ubiquitin ligase HECTD1 expression in both diabetic mouse peripheral blood mononuclear cells and 4HNE-treated RAW264.7 cells. KL inhibited HECTD1 binding to IRS1 and reduced the ubiquitination of IRS1., Conclusions: Macrophage aging is involved in DM-induced retinopathy. KL alleviates DM-induced retinal macrophage senescence by downregulating HECTD1 and decreasing IRS1 ubiquitination and degradation. Meanwhile, KL administration attenuated the neovascular lesions by altering the activation state of macrophages and decreasing the expression of VEGFA and FGF2., (© 2024. The Author(s).)

Hypoxia-inducible factors (HIFs) are transcriptional factors that function as strong regulators of oxygen homeostasis and cellular metabolisms. The maintenance of cellular oxygen levels is critical as either insufficient or excessive oxygen affects development and physiologic and pathologic conditions. In the eye, retinas have a high metabolic demand for oxygen. Retinal ischemia can cause visual impairment in various sight-threating disorders including age-related macular degeneration, diabetic retinopathy, and some types of glaucoma. Therefore, understanding the potential roles of HIFs in the retina is highly important for managing disease development and progression. This review focuses on the physiologic and pathologic roles of HIFs as regulators of oxygen homeostasis and cellular metabolism in the retina, drawing on recent evidence. Our summary will promote comprehensive approaches to targeting HIFs for therapeutic purposes in retinal diseases.

Background: The IL-6 cytokine family, with its crucial and pleiotropic intracellular signaling pathway STAT3, is a promising target for treating vasoproliferative retinal diseases. Previous research has shown that IL-6 cis-signaling (via membrane-bound receptors) and trans-signaling (via soluble receptors) can have distinct effects on target cells, leading to their application in various disease treatments. While IL-6 has been extensively studied, less is known about the angiogenic effects of IL-11, another member of the IL-6 family, in the retina. Therefore, the aim of this study was to characterize the effects of IL-11 on retinal angiogenesis., Main Text: In vitreous samples from proliferative diabetic retinopathy (PDR) patients, elevated levels of IL-11Rα, but not IL-11, were detected. In vitro studies using vascular endothelial cells revealed distinct effects of cis- and trans-signaling: cis-signaling (IL-11 alone) had antiangiogenic effects, while trans-signaling (IL-11 + sIL-11Rα) had proangiogenic and pro-migratory effects. These differences can be attributed to their individual signaling responses and associated transcriptomic changes. Notably, no differences in cis- and trans-signaling were detected in primary mouse Müller cell cultures. STAT3 and STAT1 siRNA knockdown experiments revealed opposing effects on IL-11 signaling, with STAT3 functioning as an antiproliferative and proapoptotic player while STAT1 acts in opposition to STAT3. In vivo, both IL-11 and IL-11 + sIL-11Rα led to a reduction in retinal neovascularization. Immunohistochemical staining revealed Müller cell activation in response to treatment, suggesting that IL-11 affects multiple retinal cell types in vivo beyond vascular endothelial cells., Conclusions: Cis- and trans-signaling by IL-11 have contrasting angiomodulatory effects on endothelial cells in vitro. In vivo, cis- and trans-signaling also influence Müller cells, ultimately determining the overall angiomodulatory impact on the retina, highlighting the intricate interplay between vascular and glial cells in the retina., (© 2024. The Author(s).)