Modulation of diabetes-related retinal pathophysiology by PTX3.

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 (PTX3KO), which were followed up for 9 mo to evaluate hallmarks of disease progression. In diabetic PTX3KO 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 PTX3KO 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 PTX3KO 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.

Dysregulated CD200-CD200R signaling in early diabetes modulates microglia-mediated retinopathy.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Ferroptosis Contributes to Microvascular Dysfunction in Diabetic Retinopathy.

Ferroptosis is a new form of cell death characterized by iron-dependent lipid peroxidation. Whether ferroptosis is involved in retinal microvascular dysfunction under diabetic condition is not known. Herein, the expression of ferroptosis-related genes in patients with proliferative diabetic retinopathy and in diabetic mice was determined with quantitative RT-PCR. Reactive oxygen species, iron content, lipid peroxidation products, and ferroptosis-associated proteins in the cultured human retinal microvascular endothelial cells (HRMECs) and in the retina of diabetic mice were examined. The association of ferroptosis with the functions of endothelial cells in vitro was evaluated. After administration of ferroptosis-specific inhibitor, Fer-1, the retinal microvasculature in diabetic mice was assessed. Characteristic changes of ferroptosis-associated markers, including glutathione peroxidase 4, ferritin heavy chain 1, long-chain acyl-CoA synthetase 4, transferrin receptor protein 1, and cyclooxygenase-2, were detected in the retinal fibrovascular membrane of patients with proliferative diabetic retinopathy, cultured HRMECs, and the retina of diabetic mice. Elevated levels of reactive oxygen species, lipid peroxidation, and iron content were found in the retina of diabetic mice and in cultured HRMECs. Ferroptosis was found to be associated with HRMEC dysfunction under high-glucose condition. Inhibition of ferroptosis with specific inhibitor Fer-1 in diabetic mice significantly reduced the severity of retinal microvasculopathy. Ferroptosis contributes to microvascular dysfunction in diabetic retinopathy, and inhibition of ferroptosis might be a promising strategy for the therapy of early-stage diabetic retinopathy.

12-HETE/GPR31 induces endothelial dysfunction in diabetic retinopathy.

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.

Pericyte in retinal vascular diseases: A multifunctional regulator and potential therapeutic target.

Retinal vascular diseases (RVDs), in particular diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity, are leading contributors to blindness. The pathogenesis of RVD involves vessel dilatation, leakage, and occlusion; however, the specific underlying mechanisms remain unclear. Recent findings have indicated that pericytes (PCs), as critical members of the vascular mural cells, significantly contribute to the progression of RVDs, including detachment from microvessels, alteration of contractile and secretory properties, and excessive production of the extracellular matrix. Moreover, PCs are believed to have mesenchymal stem properties and, therefore, might contribute to regenerative therapy. Here, we review novel ideas concerning PC characteristics and functions in RVDs and discuss potential therapeutic strategies based on PCs, including the targeting of pathological signals and cell-based regenerative treatments.

ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

LL37/FPR2 regulates neutrophil mPTP promoting the development of neutrophil extracellular traps in diabetic retinopathy.

Diabetic retinopathy (DR) is characterized by chronic, low-grade inflammation. This state may be related to the heightened production of neutrophil extracellular traps (NETs) induced by high glucose (HG). Human cathelicidin antimicrobial peptide (LL37) is an endogenous ligand of G protein-coupled chemoattractant receptor formyl peptide receptor 2 (FPR2), expressed on neutrophils and facilitating the formation and stabilization of the structure of NETs. In this study, we detected neutrophils cultured under different conditions, the retinal tissue of diabetic mice, and fibrovascular epiretinal membranes (FVM) samples of patients with proliferative diabetic retinopathy (PDR) to explore the regulating effect of LL37/FPR2 on neutrophil in the development of NETs during the process of DR. Specifically, HG or NG with LL37 upregulates the expression of FPR2 in neutrophils, induces the opening of mitochondrial permeability transition pore (mPTP), promotes the increase of reactive oxygen species and mitochondrial ROS, and then leads to the rise of NET production, which is mainly manifested by the release of DNA reticular structure and the increased expression of NETs-related markers. The PI3K/AKT signaling pathway was activated in neutrophils, and the phosphorylation level was enhanced by FPR2 agonists in vitro. In vivo, increased expression of NETs markers was detected in the retina of diabetic mice and in FVM, vitreous fluid, and serum of PDR patients. Transgenic FPR2 deletion led to decreased NETs in the retina of diabetic mice. Furthermore, in vitro, inhibition of the LL37/FPR2/mPTP axis and PI3K/AKT signaling pathway decreased NET production induced by high glucose. These results suggested that FPR2 plays an essential role in regulating the production of NETs induced by HG, thus may be considered as one of the potential therapeutic targets.

VEGF-B prevents chronic hyperglycemia-induced retinal vascular leakage by regulating the CDC42-ZO1/VE-cadherin pathway.

Non-proliferative diabetic retinopathy (NPDR) is the early stage of diabetic retinopathy (DR) and is a chronic oxidative stress-related ocular disease. Few treatments are approved for early DR. This study aimed to investigate the pathogenic mechanisms underlying the retinal micro-vasculopathy induced by diabetes and to explore an early potential for treating early DR in a mouse model. The mouse model of type 1 diabetes was established by intraperitoneal injection of streptozotocin (STZ, 180 mg/kg), which was used as the early DR model. The body weight and blood glucose mice were measured regularly; The retinal vascular leakage in the early DR mice was determined by whole-mount staining; Label-free quantitative proteomic analysis and bioinformatics were used to explore the target proteins and signaling pathways associated with the retinal tissues of early DR mice; To detect the effects of target protein on endothelial cell proliferation, migration, and tube formation, knockdown and overexpression of VEGF-B were performed in human retinal vascular endothelial cells (HRECs); Western blotting was used to detect the expression of target proteins in vitro and in vivo; Meanwhile, the therapeutic effect of VEGF-B on vascular leakage has also been evaluated in vitro and in vivo. The protein expressions of vascular endothelial growth factor (VEGF)-B and the Rho GTPases family member CDC42 were reduced in the retinal tissues of early DR. VEGF-B upregulated the expression of CDC42/ZO1/VE-cadherin and prevented hyperglycemia-induced vascular leakage in HRECs. Standard intravitreal VEGF-B injections improved the retinal vascular leakage and neurovascular response in early DR mice. Our findings demonstrated, for the first time, that in diabetes, the retinal vessels are damaged due to decreased VEGF-B expression through downregulation of CDC42/ZO1/VE-cadherin expression. Therefore, VEGF-B could be used as a novel therapy for early DR.

IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stage streptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

AQP4 regulates ferroptosis and oxidative stress of Muller cells in diabetic retinopathy by regulating TRPV4.

Diabetic retinopathy (DR) is a common microvascular complication that causes visual impairment or loss. Aquaporin 4 (AQP4) is a regulatory protein involved in water transport and metabolism. In previous studies, we found that AQP4 is related to hypoxia injury in Muller cells. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a non-selective cation channel protein involved in the regulation of a variety of ophthalmic diseases. However, the effects of AQP4 and TRPV4 on ferroptosis and oxidative stress in high glucose (HG)-treated Muller cells are unclear. In this study, we investigated the functions of AQP4 and TRPV4 in DR. HG was used to treat mouse Muller cells. Reverse transcription quantitative polymerase chain reaction was used to measure AQP4 mRNA expression. Western blotting was used to detect the protein levels of AQP4, PTGS2, GPX4, and TRPV4. Cell count kit-8, flow cytometry, 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl carbocyanine iodide staining, and glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits were used to evaluate the function of the Muller cells. Streptozotocin was used to induce DR in rats. Haematoxylin and eosin staining was performed to stain the retina of rats. GSH, SOD, and MDA detection kits, immunofluorescence, and flow cytometry assays were performed to study the function of AQP4 and TRPV4 in DR rats. Results found that AQP4 and TRPV4 were overexpressed in HG-induced Muller cells and streptozotocin-induced DR rats. AQP4 inhibition promoted proliferation and cell cycle progression, repressed cell apoptosis, ferroptosis, and oxidative stress, and alleviated retinal injury in DR rats. Mechanistically, AQP4 positively regulated TRPV4 expression. Overexpression of TRPV4 enhanced ferroptosis and oxidative stress in HG-treated Muller cells, and inhibition of TRPV4 had a protective effect on DR-induced retinal injury in rats. In conclusion, inhibition of AQP4 inhibits the ferroptosis and oxidative stress in Muller cells by downregulating TRPV4, which may be a potential target for DR therapy.

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

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

Diabetic retinopathy from the vitreous proteome perspective: The INSC94Y transgenic pig model study.

INSC94Y 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 INSC94Y transgenic pigs and wild-type controls. Our analysis identified 1404 proteins, with 266 showing differential abundance in INSC94Y vitreous. Notably, the abundances of ITGB1, COX2, and GRIFIN were significantly elevated in INSC94Y vitreous. Gene Set Enrichment Analysis unveiled heightened MYC and mTORC1 signaling in INSC94Y 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).

Heterotypic macrophages/microglia differentially contribute to retinal ischaemia and neovascularisation.

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 GdCl3. 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.

Dysregulated 24 h melatonin secretion associated with intrinsically photosensitive retinal ganglion cell function in diabetic retinopathy: a cross-sectional study.

AIMS/HYPOTHESIS: The aim of this study was to explore whether diabetic retinopathy is associated with alterations of the circadian system, and to examine the role of reduced intrinsically photosensitive retinal ganglion cell (ipRGC) function. METHODS: Participants with type 2 diabetes, with diabetic retinopathy (n=14) and without diabetic retinopathy (n=9) underwent 24 h blood sampling for melatonin and cortisol under controlled laboratory conditions. ipRGC function was inferred from the post-illumination pupil response (PIPR). Habitual sleep duration, efficiency and variability were assessed by actigraphy. RESULTS: Participants with diabetic retinopathy compared to participants without diabetic retinopathy had smaller PIPR (p=0.007), lower 24 h serum melatonin output (p=0.042) and greater day-to-day sleep variability (p=0.012). By contrast, 24 h cortisol profiles, sleep duration and efficiency were similar in both groups. Six individuals with diabetic retinopathy had no detectable dim-light melatonin onset. PIPR correlated with 24 h mean melatonin levels (r=0.555, p=0.007). CONCLUSIONS/INTERPRETATION: ipRCG dysfunction in diabetic retinopathy is associated with disruptions of the 24 h melatonin rhythm, suggesting circadian dysregulation in diabetic retinopathy.

Microglia in retinal angiogenesis and diabetic retinopathy.

Diabetic retinopathy has a high probability of causing visual impairment or blindness throughout the disease progression and is characterized by the growth of new blood vessels in the retina at an advanced, proliferative stage. Microglia are a resident immune population in the central nervous system, known to play a crucial role in regulating retinal angiogenesis in both physiological and pathological conditions, including diabetic retinopathy. Physiologically, they are located close to blood vessels and are essential for forming new blood vessels (neovascularization). In diabetic retinopathy, microglia become widely activated, showing a distinct polarization phenotype that leads to their accumulation around neovascular tufts. These activated microglia induce pathogenic angiogenesis through the secretion of various angiogenic factors and by regulating the status of endothelial cells. Interestingly, some subtypes of microglia simultaneously promote the regression of neovascularization tufts and normal angiogenesis in neovascularization lesions. Modulating the state of microglial activation to ameliorate neovascularization thus appears as a promising potential therapeutic approach for managing diabetic retinopathy.

Porous Se@SiO2 nanospheres alleviate diabetic retinopathy by inhibiting excess lipid peroxidation and inflammation.

BACKGROUND: Lipid peroxidation is a characteristic metabolic manifestation of diabetic retinopathy (DR) that causes inflammation, eventually leading to severe retinal vascular abnormalities. Selenium (Se) can directly or indirectly scavenge intracellular free radicals. Due to the narrow distinction between Se's effective and toxic doses, porous Se@SiO2 nanospheres have been developed to control the release of Se. They exert strong antioxidant and anti-inflammatory effects. METHODS: The effect of anti-lipid peroxidation and anti-inflammatory effects of porous Se@SiO2 nanospheres on diabetic mice were assessed by detecting the level of Malondialdehyde (MDA), glutathione peroxidase 4 (GPX4), decreased reduced/oxidized glutathione (GSH/GSSG) ratio, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL) -1ß of the retina. To further examine the protective effect of porous Se@SiO2 nanospheres on the retinal vasculopathy of diabetic mice, retinal acellular capillary, the expression of tight junction proteins, and blood-retinal barrier destruction was observed. Finally, we validated the GPX4 as the target of porous Se@SiO2 nanospheres via decreased expression of GPX4 and detected the level of MDA, GSH/GSSG, TNF-α, IFN-γ, IL -1ß, wound healing assay, and tube formation in high glucose (HG) cultured Human retinal microvascular endothelial cells (HRMECs). RESULTS: The porous Se@SiO2 nanospheres reduced the level of MDA, TNF-α, IFN-γ, and IL -1ß, while increasing the level of GPX4 and GSH/GSSG in diabetic mice. Therefore, porous Se@SiO2 nanospheres reduced the number of retinal acellular capillaries, depletion of tight junction proteins, and vascular leakage in diabetic mice. Further, we identified GPX4 as the target of porous Se@SiO2 nanospheres as GPX4 inhibition reduced the repression effect of anti-lipid peroxidation, anti-inflammatory, and protective effects of endothelial cell dysfunction of porous Se@SiO2 nanospheres in HG-cultured HRMECs. CONCLUSION: Porous Se@SiO2 nanospheres effectively attenuated retinal vasculopathy in diabetic mice via inhibiting excess lipid peroxidation and inflammation by target GPX4, suggesting their potential as therapeutic agents for DR.

Transcriptomics confirms IRF1 as a key regulator of pyroptosis in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a retinal microvascular complication caused by hyperglycemia, which can lead to visual impairment or blindness. Pyroptosis is a type of inflammation-related programmed cell death, activated by caspase-1, resulting in the maturation of IL-1ß and IL-18 and the rupture of the cell membrane. RNA sequencing (RNA-seq) is a high-throughput sequencing technique that reveals the presence and quantity of RNA in the genome at a specific time point, i.e., the transcriptome. RNA-seq can analyze gene expression levels, splicing variants, mutations, fusions, editing and other post-transcriptional modifications, as well as gene expression differences between different samples or conditions. It has been widely used in biological and medical research, clinical diagnosis and new drug development. This study aimed to establish an in vitro model of diabetic retinopathy by culturing human retinal endothelial cells (HREC) with high glucose (30 mmol/L), and to detect their transcriptome expression by RNA-seq, screen for key genes related to pyroptosis, and validate the sequencing results by subsequent experiments. METHODS: We used RNA-seq to detect the transcriptome expression differences between HREC cells cultured with high glucose and control group, and identified differentially expressed genes by GO/KEGG analysis. We constructed a PPI network and determined the key genes by Cytoscape software and CytoHubba plugin. We validated the expression of related factors by Western Blot, qPCR and ELISA. RESULTS: We performed GO and KEGG analysis on the RNA-seq data and found differentially expressed genes. We used Cytoscape and CytoHubba plugin to screen out IRF1 as the key gene, and then detected the expression of IRF1 in HREC under high glucose and control group by Western Blot and qPCR. We found that the expression of Caspase-1, GSDMD and IL-1ß proteins in HREC under high glucose increased, while the expression of these proteins decreased after the inhibition of IRF1 by siRNA. ELISA showed that the secretion of IL-1ß in HREC under high glucose increased, while the inhibition of IRF1 reduced the secretion of IL-1ß. These results indicate that IRF1 plays an important role in DR, and provides a new target and strategy for the prevention and treatment of this disease.

Knockdown of ChREBP ameliorates retinal microvascular endothelial cell injury and angiogenic responses in diabetic retinopathy.

PURPOSE: To examine whether and how carbohydrate response element-binding protein (ChREBP) plays a role in diabetic retinopathy. METHODS: Western blotting was used to detect ChREBP expression and location following high glucose stimulation of Human Retinal Microvascular Endothelial Cells (HRMECs). Flow cytometry, TUNEL staining, and western blotting were used to evaluate apoptosis following ChREBP siRNA silencing. Cell scratch, transwell migration, and tube formation assays were used to determine cell migration and angiogenesis. Diabetic models for wild-type (WT) and ChREBP knockout (ChKO) mice were developed. Retinas of WT and ChKO animals were cultivated in vitro with vascular endothelial growth factor + high glucose to assess neovascular development. RESULTS: ChREBP gene knockdown inhibited thioredoxin-interacting protein and NOD-like receptor family pyrin domain containing protein 3 expression in HRMECs, which was caused by high glucose stimulation, reduced apoptosis, hindered migration, and tube formation, and repressed AKT/mTOR signaling pathway activation. Compared with WT mice, ChKO mice showed suppressed high glucose-induced alterations in retinal structure, alleviated retinal vascular leakage, and reduced retinal neovascularization. CONCLUSIONS: ChREBP deficiency decreased high glucose-induced apoptosis, migration, and tube formation in HRMECs as well as structural and angiogenic responses in the mouse retina; thus, it is a potential therapeutic target for diabetic retinopathy.

Effect of madecassic acid on retinal oxidative stress, inflammation and Growth Factors in streptozotocin-induced diabetic rats.

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-1ß, 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-1ß, 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.