RESUMEN
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.
Asunto(s)
Retinopatía Diabética , Ferroptosis , Especies Reactivas de Oxígeno , Retinopatía Diabética/patología , Retinopatía Diabética/metabolismo , Animales , Humanos , Ratones , Masculino , Especies Reactivas de Oxígeno/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Peroxidación de Lípido , Ratones Endogámicos C57BL , Microvasos/patología , Microvasos/metabolismo , Hierro/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologíaRESUMEN
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.
Asunto(s)
Pericitos , Pericitos/metabolismo , Humanos , Animales , Vasos Retinianos/metabolismo , Vasos Retinianos/patología , Enfermedades de la Retina/terapia , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/terapia , Retinopatía Diabética/patologíaRESUMEN
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.
Asunto(s)
Retinopatía Diabética , Ácidos Hidroxámicos , Edema Macular , Humanos , FN-kappa B/metabolismo , Retinopatía Diabética/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Edema Macular/metabolismo , Transducción de Señal , Epitelio Pigmentado de la Retina/metabolismo , Barrera Hematorretinal/metabolismo , Uniones Estrechas/metabolismo , Células Epiteliales/metabolismo , Pigmentos Retinianos/metabolismo , Pigmentos Retinianos/farmacología , Pigmentos Retinianos/uso terapéuticoRESUMEN
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Catelicidinas , Retinopatía Diabética , Trampas Extracelulares , Ratones Endogámicos C57BL , Neutrófilos , Receptores de Formil Péptido , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Trampas Extracelulares/metabolismo , Animales , Receptores de Formil Péptido/metabolismo , Receptores de Formil Péptido/genética , Humanos , Neutrófilos/metabolismo , Ratones , Péptidos Catiónicos Antimicrobianos/metabolismo , Masculino , Receptores de Lipoxina/metabolismo , Receptores de Lipoxina/genética , Diabetes Mellitus Experimental/metabolismo , Transducción de Señal , Especies Reactivas de Oxígeno/metabolismo , Femenino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Barrera Hematorretinal , Diabetes Mellitus Experimental , Retinopatía Diabética , Interleucina-10 , Macrófagos , Animales , Humanos , Masculino , Ratones , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Polaridad Celular/efectos de los fármacos , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Modelos Animales de Enfermedad , Interleucina-10/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/efectos de los fármacos , EstreptozocinaRESUMEN
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.
Asunto(s)
Acuaporina 4 , Retinopatía Diabética , Células Ependimogliales , Ferroptosis , Estrés Oxidativo , Canales Catiónicos TRPV , Animales , Masculino , Ratones , Ratas , Acuaporina 4/metabolismo , Acuaporina 4/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/genética , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Glucosa/metabolismo , Glucosa/farmacología , Ratones Endogámicos C57BL , Ratas Sprague-Dawley , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genéticaRESUMEN
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.
Asunto(s)
Senescencia Celular , Retinopatía Diabética , Exosomas , Células Madre Mesenquimatosas , Factor 2 Relacionado con NF-E2 , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/genética , Retinopatía Diabética/terapia , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Células Madre Mesenquimatosas/metabolismo , Exosomas/metabolismo , Animales , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Humanos , Ratones , Estrés Oxidativo , Ratones Endogámicos C57BL , Masculino , Apoptosis , Proliferación Celular , Línea Celular , Células Epiteliales/metabolismoRESUMEN
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.
Asunto(s)
Ritmo Circadiano , Diabetes Mellitus Tipo 2 , Retinopatía Diabética , Melatonina , Células Ganglionares de la Retina , Humanos , Melatonina/sangre , Melatonina/metabolismo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/sangre , Retinopatía Diabética/fisiopatología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Masculino , Femenino , Estudios Transversales , Persona de Mediana Edad , Ritmo Circadiano/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Anciano , Hidrocortisona/sangre , Hidrocortisona/metabolismo , Sueño/fisiología , AdultoRESUMEN
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.
Asunto(s)
Retinopatía Diabética , Microglía , Neovascularización Retiniana , Animales , Humanos , Angiogénesis/metabolismo , Angiogénesis/patología , Retinopatía Diabética/patología , Retinopatía Diabética/metabolismo , Microglía/patología , Microglía/metabolismo , Retina/patología , Retina/metabolismo , Neovascularización Retiniana/patología , Neovascularización Retiniana/metabolismo , Vasos Retinianos/patología , Vasos Retinianos/metabolismoRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética , Nanosferas , Selenio , Humanos , Ratones , Animales , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Selenio/metabolismo , Selenio/farmacología , Selenio/uso terapéutico , Dióxido de Silicio/metabolismo , Dióxido de Silicio/farmacología , Dióxido de Silicio/uso terapéutico , Diabetes Mellitus Experimental/metabolismo , Células Endoteliales/metabolismo , Peroxidación de Lípido , Porosidad , Factor de Necrosis Tumoral alfa/metabolismo , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/farmacología , Disulfuro de Glutatión/uso terapéutico , Inflamación/metabolismo , Antiinflamatorios/uso terapéutico , Proteínas de Uniones Estrechas/metabolismoRESUMEN
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.
Asunto(s)
Retinopatía Diabética , Factor 1 Regulador del Interferón , Piroptosis , Humanos , Caspasas/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Glucosa/metabolismo , Factor 1 Regulador del Interferón/genética , Piroptosis/genéticaRESUMEN
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.
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Animales , Humanos , Ratones , Diabetes Mellitus/metabolismo , Retinopatía Diabética/metabolismo , Células Endoteliales/metabolismo , Glucosa/metabolismo , Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Epithelial mesenchymal transition (EMT) is a critical process implicated in the pathogenesis of retinal fibrosis and the exacerbation of diabetic retinopathy (DR) within retinal pigment epithelium (RPE) cells. Apigenin (AP), a potential dietary supplement for managing diabetes and its associated complications, has demonstrated inhibitory effects on EMT in various diseases. However, the specific impact and underlying mechanisms of AP on EMT in RPE cells remain poorly understood. In this study, we have successfully validated the inhibitory effects of AP on high glucose-induced EMT in ARPE-19 cells and diabetic db/db mice. Notably, our findings have identified CBP/p300 as a potential therapeutic target for EMT in RPE cells and have further substantiated that AP effectively downregulates the expression of EMT-related genes by attenuating the activity of CBP/p300, consequently reducing histone acetylation alterations within the promoter region of these genes. Taken together, our results provide novel evidence supporting the inhibitory effect of AP on EMT in RPE cells, and highlight the potential of specifically targeting CBP/p300 as a strategy for inhibiting retinal fibrosis in the context of DR.
Asunto(s)
Apigenina , Transición Epitelial-Mesenquimal , Glucosa , Histonas , Epitelio Pigmentado de la Retina , Transición Epitelial-Mesenquimal/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Animales , Apigenina/farmacología , Acetilación/efectos de los fármacos , Humanos , Glucosa/metabolismo , Glucosa/toxicidad , Histonas/metabolismo , Línea Celular , Ratones , Factores de Transcripción p300-CBP/metabolismo , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Ratones Endogámicos C57BL , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Retinopatía Diabética/tratamiento farmacológico , Proteína p300 Asociada a E1A/metabolismo , Masculino , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteína de Unión a CREB/metabolismo , Proteína de Unión a CREB/genéticaRESUMEN
Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.
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Diabetes Mellitus , Retinopatía Diabética , Animales , Ratones , Diabetes Mellitus/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Neuroglía/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Retina/metabolismoRESUMEN
The microglia-mediated neuroinflammation have been shown to play a crucial role in the ocular pathological angiogenesis process, but specific immunotherapies for neovascular ocular diseases are still lacking. This study proposed that targeting GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) might be a novel immunotherapy for these angiogenesis diseases. We found a significant upregulation of CGAS and STING genes in the RNA-seq data derived from retinal tissues of the patients with proliferative diabetic retinopathy. In experimental models of ocular angiogenesis including laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), the cGAS-STING pathway was activated as angiogenesis progressed. Either genetic deletion or pharmacological inhibition of STING resulted in a remarkable suppression of neovascularization in both models. Furthermore, cGAS-STING signaling was specifically activated in myeloid cells, triggering the subsequent RIP1-RIP3-MLKL pathway activation and leading to necroptosis-mediated inflammation. Notably, targeted inhibition of the cGAS-STING pathway with C-176 or SN-011 could significantly suppress pathological angiogenesis in CNV and OIR. Additionally, the combination of C-176 or SN-011 with anti-VEGF therapy led to least angiogenesis, markedly enhancing the anti-angiogenic effectiveness. Together, our findings provide compelling evidence for the importance of the cGAS-STING-necroptosis axis in pathological angiogenesis, highlighting its potential as a promising immunotherapeutic target for treating neovascular ocular diseases.
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Proteínas de la Membrana , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias , Nucleotidiltransferasas , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/antagonistas & inhibidores , Animales , Humanos , Ratones , Enfermedades Neuroinflamatorias/metabolismo , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Neovascularización Coroidal/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ratones Noqueados , Retinopatía Diabética/metabolismoRESUMEN
BACKGROUND: Diabetic retinopathy (DR) is the leading cause of blinding eye disease among working adults and is primarily attributed to the excessive proliferation of microvessels, which leads to vitreous hemorrhage and retinal traction, thereby significantly impairing patient vision. NSUN2-mediated RNA m5C methylation is implicated in various diseases, and in this investigation, we focused on elucidating the impact of NSUN2 on the regulation of the expression of the downstream gene MUC1, specifically through RNA m5C methylation, on the progression of DR. METHOD: Utilizing Microarray analysis, we examined patient vitreous fluid to pinpoint potential therapeutic targets for DR. Differential expression of NSUN2 was validated through qRT-PCR, Western blot, and immunofluorescence in human tissue, animal tissue, and cell model of DR. The relationship between NSUN2 and DR was explored in vitro and in vivo through gene knockdown and overexpression. Various techniques, such as MeRIP-qPCR and dot blot, were applied to reveal the downstream targets and mechanism of action of NSUN2. RESULTS: The levels of both NSUN2 and RNA m5C methylation were significantly elevated in the DR model. Knockdown of NSUN2 mitigated DR lesion formation both in vitro and in vivo. Mechanistically, NSUN2 promoted MUC1 expression by binding to the RNA m5C reader ALYREF. Knockdown of ALYREF resulted in DR lesion alterations similar to those observed with NSUN2 knockdown. Moreover, MUC1 overexpression successfully reversed a series of DR alterations induced by NSUN2 silencing. CONCLUSIONS: NSUN2 regulates the expression of MUC1 through ALYREF-mediated RNA m5C methylation, thereby regulating the progression of DR and providing a new option for the treatment of DR in the future.
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Retinopatía Diabética , Progresión de la Enfermedad , Metiltransferasas , Mucina-1 , Metilación de ARN , Animales , Humanos , Masculino , Retinopatía Diabética/metabolismo , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Metilación , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones Endogámicos C57BL , Mucina-1/metabolismo , Mucina-1/genéticaRESUMEN
BACKGROUND: Intravitreal injections of angiogenesis inhibitors have proved efficacious in the majority of patients with ocular angiogenesis. However, one-fourth of all treated patients fail to derive benefits from intravitreal injections. tRNA-derived small RNA (tsRNA) emerges as a crucial class of non-coding RNA molecules, orchestrating key roles in the progression of human diseases by modulating multiple targets. Through our prior sequencing analyses and bioinformatics predictions, tRNA-Cys-5-0007 has shown as a potential regulator of ocular angiogenesis. This study endeavors to elucidate the precise role of tRNA-Cys-5-0007 in the context of ocular angiogenesis. METHODS: Quantitative reverse transcription PCR (qRT-PCR) assays were employed to detect tRNA-Cys-5-0007expression. EdU assays, sprouting assays, transwell assays, and Matrigel assays were conducted to elucidate the involvement of tRNA-Cys-5-0007 in endothelial angiogenic effects. STZ-induced diabetic model, OIR model, and laser-induced CNV model were utilized to replicate the pivotal features of ocular vascular diseases and evaluate the influence of tRNA-Cys-5-0007 on ocular angiogenesis and inflammatory responses. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were employed to elucidate the anti-angiogenic mechanism of tRNA-Cys-5-0007. Exosomal formulation was employed to enhance the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. RESULTS: tRNA-Cys-5-0007 expression was down-regulated under angiogenic conditions. Conversely, tRNA-Cys-5-0007 overexpression exhibited anti-angiogenic effects in retinal endothelial cells, as evidenced by reduced proliferation, sprouting, migration, and tube formation abilities. In diabetic, laser-induced CNV, and OIR models, tRNA-Cys-5-0007 overexpression led to decreased ocular vessel leakage, inhibited angiogenesis, and reduced ocular inflammation. Mechanistically, these effects were attributed to the targeting of vascular endothelial growth factor A (VEGFA) and TGF-ß1 by tRNA-Cys-5-0007. The utilization of an exosomal formulation further potentiated the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. CONCLUSIONS: Concurrent targeting of tRNA-Cys-5-0007 for anti-angiogenic and anti-inflammatory therapy holds promise for enhancing the effectiveness of current anti-angiogenic therapy.
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Inhibidores de la Angiogénesis , Antiinflamatorios , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antiinflamatorios/farmacología , Humanos , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Ratones Endogámicos C57BL , Proliferación Celular/efectos de los fármacos , Neovascularización Coroidal/patología , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Masculino , Oftalmopatías/tratamiento farmacológico , Oftalmopatías/patología , Oftalmopatías/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Neovascularización Patológica , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/patología , Retinopatía Diabética/metabolismo , Ratones , Células Endoteliales de la Vena Umbilical Humana/metabolismoRESUMEN
BACKGROUND: Diabetic retinopathy is one of the complications of diabetes mellitus. The aim of this study was to explore the effects of ubiquitin-specific protease 48 (USP48) and its underlying mechanisms in the development of diabetic retinopathy. METHODS: CCK-8 assay, EdU assay, and flow cytometry were used to measure the proliferative ability and the apoptotic rate of ARPE-19 cells, respectively. ELISA kits were utilized to assess the levels of inflammatory cytokines. The levels of Fe2+, ROS and MDA were detected using the corresponding biochemical kits. The protein expression of USP48 and SLC1A5 was examined through western blot. The mRNA level of SLC1A5 was determined using RT-qPCR. The interaction relationship between USP48 and SLC1A5 was evaluated using Co-IP assay. RESULTS: High glucose (HG) treatment significantly inhibited cell proliferation and elevated cell apoptosis, inflammation, ferroptosis and oxidative stress in ARPE-19 cells. HG treatment-caused cell damage was hindered by USP48 or SLC1A5 overexpression in ARPE-19 cells. Fer-1 treatment improved HG-caused cell damage in ARPE-19 cells, which was blocked by USP48 knockdown. Moreover, USP48 knockdown decreased SLC1A5 expression. SLC1A5 downregulation reversed the improvement effects of USP48 upregulation on cell damage in HG-treated ARPE-19 cells. CONCLUSION: USP48 overexpression deubiquitinated SLC1A5 to elevate cell proliferation and suppress cell apoptosis, inflammation, ferroptosis and oxidative stress in HG-triggered ARPE-19 cells, thereby inhibiting the progression of diabetic retinopathy.
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Retinopatía Diabética , Ferroptosis , Inflamación , Estrés Oxidativo , Epitelio Pigmentado de la Retina , Humanos , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Ferroptosis/fisiología , Inflamación/metabolismo , Ubiquitinación , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Proteasas Ubiquitina-Específicas/metabolismo , Línea Celular , Sistema de Transporte de Aminoácidos ASCRESUMEN
Purpose: To examine whether increased ephrin type-B receptor 1 (EphB1) leads to inflammatory mediators in retinal Müller cells. Methods: Diabetic human and mouse retinal samples were examined for EphB1 protein levels. Rat Müller cells (rMC-1) were grown in culture and treated with EphB1 siRNA or ephrin B1-Fc to explore inflammatory mediators in cells grown in high glucose. An EphB1 overexpression adeno-associated virus (AAV) was used to increase EphB1 in Müller cells in vivo. Ischemia/reperfusion (I/R) was performed on mice treated with the EphB1 overexpression AAV to explore the actions of EphB1 on retinal neuronal changes in vivo. Results: EphB1 protein levels were increased in diabetic human and mouse retinal samples. Knockdown of EphB1 reduced inflammatory mediator levels in Müller cells grown in high glucose. Ephrin B1-Fc increased inflammatory proteins in rMC-1 cells grown in normal and high glucose. Treatment of mice with I/R caused retinal thinning and loss of cell numbers in the ganglion cell layer. This was increased in mice exposed to I/R and treated with the EphB1 overexpressing AAVs. Conclusions: EphB1 is increased in the retinas of diabetic humans and mice and in high glucose-treated Müller cells. This increase leads to inflammatory proteins. EphB1 also enhanced retinal damage in response to I/R. Taken together, inhibition of EphB1 may offer a new therapeutic option for diabetic retinopathy.
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Retinopatía Diabética , Efrina-B1 , Enfermedades de la Retina , Animales , Humanos , Ratones , Ratas , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Efrina-B1/genética , Efrina-B1/metabolismo , Glucosa/metabolismo , Mediadores de Inflamación/metabolismo , Retina/metabolismo , Enfermedades de la Retina/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Literature has reported that circular RNAs (circRNAs) are crucially associated with diabetic retinopathy (DR). Furthermore, circEHMT1 has been identified to maintain endothelial cell barrier function. This study aimed to investigate the mechanisms that regulate aberrant circEHMT1 expression and its role in the pathogenesis of DR. METHODS: In this study, retinal microvascular endothelial cells were exposed to a high glucose (HG) environment, and subsequently, tube formation and intercellular junction proteins were evaluated. Furthermore, the biological functions of circEHMT1 and its potential regulatory factor, eIF4A3, in microvascular endothelial cells under HG conditions were also assessed. In addition, the regulatory role of eIF4A3 on circEHMT1 expression was confirmed. Moreover, to elucidate the in vivo functions of eIF4A3 and circEHMT1, streptozotocin (STZ) was used to establish a DR model in rats. RESULTS: It was revealed that HG condition decreased circEHMT1 and eIF4A3 expressions and reduced ZO-1, Claudin-5, and Occludin levels in retinal microvascular endothelial cells. Furthermore, it was observed that eIF4A3 could regulate the expression of circEHMT1. Overexpression of eIF4A3 or circEHMT1 under HG conditions improved endothelial cell injury and decreased tube-formation ability. Additionally, in the DR rat model, eIF4A3 overexpression restored circEHMT1 levels and ameliorated retinal vasculature changes. CONCLUSION: Altogether, eIF4A3 regulates circEHMT1 expression, thereby affecting microvascular endothelial cell injury and tube formation. Further understanding the regulatory effect of eIF4A3 on circEHMT1 may provide novel therapeutic targets for DR.