RESUMO
Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.
Assuntos
Deferiprona , Modelos Animais de Doenças , Homeostase , Quelantes de Ferro , Ferro , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Retiniana , Animais , Deferiprona/farmacologia , Deferiprona/uso terapêutico , Quelantes de Ferro/farmacologia , Quelantes de Ferro/uso terapêutico , Camundongos , Ferro/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/patologia , Angiofluoresceinografia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Western Blotting , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Degeneração Macular Exsudativa/tratamento farmacológico , Degeneração Macular Exsudativa/metabolismo , Fator de Transcrição STAT3/metabolismo , MasculinoRESUMO
HbSC disease, a less severe form of sickle cell disease, affects the retina more frequently and patients have higher rates of proliferative retinopathy that can progress to vision loss. This study aimed to identify differences in the expression of endothelial cell-derived molecules associated with the pathophysiology of proliferative sickle cell retinopathy (PSCR). RNAseq was used to compare the gene expression profile of circulating endothelial colony-forming cells from patients with SC hemoglobinopathy and proliferative retinopathy (n = 5), versus SC patients without retinopathy (n = 3). Real-time polymerase chain reaction (qRT-PCR) was used to validate the RNAseq results. A total of 134 differentially expressed genes (DEGs) were found. DEGs were mainly associated with vasodilatation, type I interferon signaling, innate immunity and angiogenesis. Among the DEGs identified, we highlight the most up-regulated genes ROBO1 (log2FoldChange = 4.32, FDR = 1.35E-11) and SLC38A5 (log2FoldChange = 3.36 FDR = 1.59E-07). ROBO1, an axon-guided receptor, promotes endothelial cell migration and contributes to the development of retinal angiogenesis and pathological ocular neovascularization. Endothelial SLC38A5, an amino acid (AA) transporter, regulates developmental and pathological retinal angiogenesis by controlling the uptake of AA nutrient, which may serve as metabolic fuel for the proliferation of endothelial cells (ECs) and consequent promotion of angiogenesis. Our data provide an important step towards elucidating the molecular pathophysiology of PSCR that may explain the differences in ocular manifestations between individuals with hemoglobinopathies and afford insights for new alternative strategies to inhibit pathological angiogenesis.
Assuntos
Proteínas do Tecido Nervoso , Receptores Imunológicos , Neovascularização Retiniana , Proteínas Roundabout , Adulto , Feminino , Humanos , Masculino , Angiogênese , Células Endoteliais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologiaRESUMO
Purpose: Retinopathy of prematurity (ROP) results from postnatal hyperoxia exposure in premature infants and is characterized by aberrant neovascularization of retinal blood vessels. Epithelial membrane protein-2 (EMP2) regulates hypoxia-inducible factor (HIF)-induced vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line and genetic knock-out of Emp2 in a murine oxygen-induced retinopathy (OIR) model attenuates neovascularization. We hypothesize that EMP2 blockade via intravitreal injection protects against neovascularization. Methods: Ex vivo choroid sprouting assay was performed, comparing media and human IgG controls versus anti-EMP2 antibody (Ab) treatment. In vivo, eyes from wild-type (WT) mice exposed to hyperoxia from postnatal (P) days 7 to 12 were treated with P12 intravitreal injections of control IgG or anti-EMP2 Abs. Neovascularization was assessed at P17 by flat mount imaging. Local and systemic effects of anti-EMP2 Ab treatment were assessed. Results: Choroid sprouts treated with 30 µg/mL of anti-EMP2 Ab demonstrated a 48% reduction in vessel growth compared to control IgG-treated sprouts. Compared to IgG-treated controls, WT OIR mice treated with 4 µg/g of intravitreal anti-EMP2 Ab demonstrated a 42% reduction in neovascularization. They demonstrated down-regulation of retinal gene expression in pathways related to vasculature development and up-regulation in genes related to fatty acid oxidation and tricarboxylic acid cycle respiratory electron transport, compared to controls. Anti-EMP2 Ab-treated OIR mice did not exhibit gross retinal histologic abnormalities, vision transduction abnormalities, or weight loss. Conclusions: Our results suggest that EMP2 blockade could be a local and specific treatment modality for retinal neovascularization in oxygen-induced retinopathies, without systemic adverse effects.
Assuntos
Oxigênio , Neovascularização Retiniana , Retinopatia da Prematuridade , Animais , Humanos , Camundongos , Animais Recém-Nascidos , Modelos Animais de Doenças , Hiperóxia/complicações , Injeções Intravítreas , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/prevenção & controle , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Diabetic retinopathy (DR) is a common complication of diabetes, and recent findings have shown that long noncoding RNAs (lncRNAs) may be involved in its pathogenesis. Through bioinformatics analysis, we found that lncRNA ATP2B2-IT2 may be involved in this process. This study primarily investigated the expression of the lncRNA ATP2B2-IT2 in human retinal microvascular endothelial cells (HRMECs) under high-glucose conditions and its effects on HRMEC proliferation, migration, and neovascularization. METHODS: We used RTâPCR to assess the expression levels of lncRNA ATP2B2-IT2 and vascular endothelial growth factor (VEGF) in HRMECs under normal glucose (5.5 mmol/L) and high glucose (30 mmol/L) conditions. HRMECs were subsequently divided into four groups: the normal glucose (NG), high glucose (HG), high glucose with lncRNA ATP2B2-IT2 silencing (HG + si-lncRNA ATP2B2-IT2), and high glucose with silencing control (HG + si-NC) groups. The expression levels of the lncRNA ATP2B2-IT2 and VEGF in each group were determined using RTâPCR. Thereafter, cell proliferation, migration, and neovascularization were assessed using CCK-8, Transwell, and tube formation assays, respectively. RESULTS: RTâPCR revealed that the expression levels of the lncRNA ATP2B2-IT2 and VEGF were greater in the HG group than in the NG group (P < 0.05). After silencing of the lncRNA ATP2B2-IT2, the expression of VEGF decreased significantly (P < 0.05). Subsequent CCK-8, Transwell, and tube formation assays demonstrated that compared to those in the NG group, the HRMECs in the HG group exhibited significantly increased proliferation, migration, and neovascularization (P < 0.05). However, after silencing of the lncRNA ATP2B2-IT2, the proliferation, migration, and neovascularization of HRMECs were significantly decreased in the HG + si-lncRNA ATP2B2-IT2 group compared to those in the HG group (P < 0.05). CONCLUSION: LncRNA ATP2B2-IT2 may promote the proliferation, migration and neovascularization of HRMECs under high-glucose conditions.
Assuntos
Movimento Celular , Proliferação de Células , Retinopatia Diabética , RNA Longo não Codificante , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , RNA Longo não Codificante/genética , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Células Cultivadas , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Regulação da Expressão Gênica , Células Endoteliais/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismoRESUMO
Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.
Assuntos
Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Endoteliais , Prolina Dioxigenases do Fator Induzível por Hipóxia , Receptores Notch , Neovascularização Retiniana , Transdução de Sinais , Regulação para Cima , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Camundongos , Receptores Notch/metabolismo , Receptores Notch/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Células Endoteliais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Vasos Retinianos/metabolismo , AngiogêneseRESUMO
Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.
Assuntos
Barreira Hematorretiniana , Células Endoteliais , Fatores de Transcrição Forkhead , Neovascularização Retiniana , Animais , Masculino , Camundongos , Angiogênese , Barreira Hematorretiniana/metabolismo , Células Endoteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pericitos/metabolismo , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Vasos Retinianos/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Neovascular eye diseases, including proliferative diabetic retinopathy and retinopathy of prematurity, is a major cause of blindness. Laser ablation and intravitreal anti-VEGF injection have shown their limitations in treatment of retinal neovascularization. Identification of a new therapeutic strategies is in urgent need. Our study aims to assess the effects of Cryptotanshinone (CPT), a natural compound derived from Salvia miltiorrhiza Bunge, in retina neovascularization and explore its potential mechanism. Our study demonstrated that CPT did not cause retina tissue toxicity at the tested concentrations. Intravitreal injections of CPT reduced pathological angiogenesis and promoted physical angiogenesis in oxygen-induced retinopathy (OIR) model. CPT improve visual function in OIR mice and reduced cell apoptosis. Moreover, we also revealed that CPT diminishes the expression of inflammatory cytokines in the OIR retina. In vitro, the administration of CPT effectively inhibited endothelial cells proliferation, migration, sprouting, and tube formation induced by the stimulation of human retinal vascular endothelial cells (HRVECs) with VEGF165. Mechanistically, CPT blocking the phosphorylation of VEGFR2 and downstream targeting pathway. After all, the findings demonstrated that CPT exhibits potent anti-angiogenic and anti-inflammatory effects in OIR mice, and it has therapeutic potential for the treatment of neovascular retinal diseases.
Assuntos
Injeções Intravítreas , Camundongos Endogâmicos C57BL , Fenantrenos , Neovascularização Retiniana , Animais , Fenantrenos/farmacologia , Fenantrenos/administração & dosagem , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/patologia , Neovascularização Retiniana/metabolismo , Humanos , Camundongos , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologiaRESUMO
BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.
Assuntos
Oxigênio , Retinopatia da Prematuridade , Animais , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/metabolismo , Camundongos , Anticorpos Monoclonais Humanizados/farmacologia , Camundongos Endogâmicos C57BL , Animais Recém-Nascidos , Modelos Animais de Doenças , Humanos , Hiperóxia/patologia , Hiperóxia/complicações , Retina/patologia , Retina/metabolismo , Retina/efeitos dos fármacos , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Camundongos Transgênicos , Neovascularização Retiniana/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/tratamento farmacológico , Microglia/patologia , Microglia/metabolismo , Microglia/efeitos dos fármacosRESUMO
Retinopathy of prematurity (ROP) is a retinal disease-causing retinal neovascularization that can lead to blindness. Oxygen-induced retinopathy (OIR) is a widely used ROP animal model. Icariin (ICA) has anti-oxidative and anti-inflammation properties; however, whether ICA has a regulatory effect on OIR remains unclear. In this study, ICA alleviated pathological neovascularization, microglial activation and blood-retina barrier (BRB) damage in vivo. Further results indicated that endothelial cell tube formation, migration and proliferation were restored by ICA treatment in vitro. Proteomic microarrays and molecular mimicry revealed that ICA can directly bind to hexokinase 2 (HK2) and decrease HK2 protein expression in vivo and in vitro. In addition, ICA inhibited the AKT/mTOR/HIF1α pathway activation. The effects of ICA on pathological neovascularization, microglial activation and BRB damage disappeared after HK2 overexpression in vivo. Similarly, the endothelial cell function was revised after HK2 overexpression. HK2 overexpression reversed ICA-induced AKT/mTOR/HIF1α pathway inhibition in vivo and in vitro. Therefore, ICA prevented pathological angiogenesis in OIR in an HK2-dependent manner, implicating ICA as a potential therapeutic agent for ROP.
Assuntos
Modelos Animais de Doenças , Flavonoides , Hexoquinase , Microglia , Oxigênio , Neovascularização Retiniana , Retinopatia da Prematuridade , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Hexoquinase/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Oxigênio/metabolismo , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Camundongos , Humanos , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Endogâmicos C57BL , Movimento Celular/efeitos dos fármacosRESUMO
Interactions among neuronal, glial, and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunction precedes vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA sequencing (scRNA-seq), and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1-/- retinas where neurons fail to release glutamate. By contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1-/- retinas, where constitutively depolarized rods release excessive glutamate. Norrin expression and endothelial Norrin/ß-catenin signaling are downregulated in Vglut1-/- retinas and upregulated in Gnat1-/- retinas. Pharmacological activation of endothelial Norrin/ß-catenin signaling in Vglut1-/- retinas rescues defects in deep plexus angiogenesis and paracellular BRB maturation. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating endothelial Norrin/ß-catenin signaling.
Assuntos
Barreira Hematorretiniana , Proteínas do Olho , Ácido Glutâmico , Proteínas do Tecido Nervoso , Transdução de Sinais , beta Catenina , Animais , Barreira Hematorretiniana/metabolismo , beta Catenina/metabolismo , Camundongos , Ácido Glutâmico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Transdução de Sinais/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Neurônios/metabolismo , Camundongos Knockout , Neovascularização Retiniana/metabolismo , Retina/metabolismo , Camundongos Endogâmicos C57BL , AngiogêneseRESUMO
Angiogenesis plays a critical role in many pathological processes, including irreversible blindness in eye diseases such as retinopathy of prematurity. Endothelial mitochondria are dynamic organelles that undergo constant fusion and fission and are critical signalling hubs that modulate angiogenesis by coordinating reactive oxygen species (ROS) production and calcium signalling and metabolism. In this study, we investigated the role of mitochondrial dynamics in pathological retinal angiogenesis. We showed that treatment with vascular endothelial growth factor (VEGF; 20 ng/ml) induced mitochondrial fission in HUVECs by promoting the phosphorylation of dynamin-related protein 1 (DRP1). DRP1 knockdown or pretreatment with the DRP1 inhibitor Mdivi-1 (5 µM) blocked VEGF-induced cell migration, proliferation, and tube formation in HUVECs. We demonstrated that VEGF treatment increased mitochondrial ROS production in HUVECs, which was necessary for HIF-1α-dependent glycolysis, as well as proliferation, migration, and tube formation, and the inhibition of mitochondrial fission prevented VEGF-induced mitochondrial ROS production. In an oxygen-induced retinopathy (OIR) mouse model, we found that active DRP1 was highly expressed in endothelial cells in neovascular tufts. The administration of Mdivi-1 (10 mg·kg-1·d-1, i.p.) for three days from postnatal day (P) 13 until P15 significantly alleviated pathological angiogenesis in the retina. Our results suggest that targeting mitochondrial fission may be a therapeutic strategy for proliferative retinopathies and other diseases that are dependent on pathological angiogenesis.
Assuntos
Movimento Celular , Dinaminas , Células Endoteliais da Veia Umbilical Humana , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial , Quinazolinonas , Espécies Reativas de Oxigênio , Neovascularização Retiniana , Fator A de Crescimento do Endotélio Vascular , Dinâmica Mitocondrial/efeitos dos fármacos , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Dinaminas/metabolismo , Dinaminas/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinazolinonas/farmacologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Camundongos , Proliferação de Células/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , AngiogêneseRESUMO
Pathological retinal angiogenesis is not only the hallmark of retinopathies, but also a major cause of blindness. Guanylate binding protein 2 (GBP2) has been reported to be associated with retinal diseases such as diabetic retinopathy and hypoxic retinopathy. However, GBP2-mediated pathological retinal angiogenesis remains largely unknown. The present study aimed to investigate the role of GBP2 in pathological retinal angiogenesis and its underlying molecular mechanism. In this study, we established oxygen-induced retinopathy (OIR) mice model for in vivo study and hypoxia-induced angiogenesis in ARPE-19 cells for in vitro study. We demonstrated that GBP2 expression was markedly downregulated in the retina of mice with OIR and ARPE-19 cells treated with hypoxia, which was associated with pathological retinal angiogenesis. The regulatory mechanism of GBP2 in ARPE-19 cells was studied by GBP2 silencing and overexpression. The regulatory mechanism of GBP2 in the retina was investigated by overexpressing GBP2 in the retina of OIR mice. Mechanistically, GBP2 downregulated the expression and secretion of vascular endothelial growth factor (VEGFA) in ARPE-19 cells and retina of OIR mice. Interestingly, overexpression of GBP2 significantly inhibited neovascularization in OIR mice, conditioned medium of GBP2 overexpressing ARPE-19 cells inhibited angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, we confirmed that GBP2 downregulated VEGFA expression and angiogenesis by inhibiting the AKT/mTOR signaling pathway. Taken together, we concluded that GBP2 inhibited pathological retinal angiogenesis via the AKT/mTOR/VEGFA axis, thereby suggesting that GBP2 may be a therapeutic target for pathological retinal angiogenesis.
Assuntos
Modelos Animais de Doenças , Proteínas de Ligação ao GTP , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt , Neovascularização Retiniana , Vasos Retinianos , Transdução de Sinais , Serina-Treonina Quinases TOR , Fator A de Crescimento do Endotélio Vascular , Animais , Humanos , Camundongos , Hipóxia Celular , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/prevenção & controle , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy.
Assuntos
Retinopatia Diabética , Exossomos , MicroRNAs , RNA Longo não Codificante , Neovascularização Retiniana , Animais , Humanos , Masculino , Camundongos , Movimento Celular , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental , Retinopatia Diabética/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Exossomos/metabolismo , Exossomos/genética , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , RNA Longo não Codificante/genéticaRESUMO
Purpose: Adjuvant, pre-operative intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections have been used to reduce peri-operative bleeding in eyes undergoing pars-plana vitrectomy for complications of proliferative diabetic retinopathy (PDR). To address the concern over their potential off-target effects of progressive fibrous contraction, we sought to dissect the transcriptional changes in the surgically extracted fibrovascular membranes (FVMs). Methods: We analyzed surgically extracted FVMs from 10 eyes: 4 eyes pretreated with intravitreal bevacizumab (IVB) and 6 untreated eyes. FVMs were digested into single cells, mRNA was extracted from endothelial cell-enriched (microbead selection with CD31) and non-endothelial cell compartments, followed by RT-qPCR quantification. We then compared the relative expression of genes involved in angiogenesis, endothelial cell integrity, and myofibroblastic processes between treated and untreated FVMs. Results: Endothelial cells from IVB pretreated FVMs showed significant reduction of VEGFA, VEGF receptors (FLT1 and KDR), and angiopoietin 2 expression as well as increased vascular endothelial cadherin and endothelin, suggesting reduced angiogenesis and enhanced vascular integrity. The non-endothelial cell fraction showed decreased expression of VEGFA and fibronectin, without significant difference in the expression of other profibrotic factors. Conclusions: Our findings confirm that adjuvant pre-operative IVB decreased fibronectin and increase endothelin-1 expression without affecting other profibrotic gene expression, uncovering an important interaction between IVB and endothelin-1 that deserves further study.
Assuntos
Inibidores da Angiogênese , Bevacizumab , Retinopatia Diabética , Fibrose , Injeções Intravítreas , Fator A de Crescimento do Endotélio Vascular , Vitrectomia , Humanos , Retinopatia Diabética/metabolismo , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/cirurgia , Inibidores da Angiogênese/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Bevacizumab/uso terapêutico , Bevacizumab/farmacologia , Masculino , Feminino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/tratamento farmacológico , Idoso , Cuidados Pré-Operatórios , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologiaRESUMO
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.
Assuntos
Retinopatia Diabética , Microglia , Neovascularização Retiniana , Animais , Humanos , Angiogênese/metabolismo , Angiogênese/patologia , Retinopatia Diabética/patologia , Retinopatia Diabética/metabolismo , Microglia/patologia , Microglia/metabolismo , Retina/patologia , Retina/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/metabolismo , Vasos Retinianos/patologia , Vasos Retinianos/metabolismoRESUMO
Retinal neovascularization diseases have relatively high rates of evitable blindness. Abnormal retinal neovascularization is their main hallmark, which can damage the structure and function of the eye and lead to impaired vision. Caveolin-1 is a membrane protein that is expressed in many types of retinal cells and is involved in retinal neovascularization. This review presents a comprehensive analysis of global research on specific functions of caveolin-1 in retinal neovascularization. We believe that the mechanism of action of caveolin-1 might be related to the regulation of relevant signal pathways and looked ahead the application prospects of modulating caveolin- 1 in retinal neovascularization diseases.
Assuntos
Caveolina 1 , Neovascularização Retiniana , Transdução de Sinais , Caveolina 1/metabolismo , Humanos , Neovascularização Retiniana/metabolismo , AnimaisRESUMO
Retinal neovascularization (RNV) is a pathological process that primarily occurs in diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion. It is a common yet debilitating clinical condition that culminates in blindness. Urgent efforts are required to explore more efficient and less limiting therapeutic strategies. Key RNA-binding proteins (RBPs), crucial for post-transcriptional regulation of gene expression by binding to RNAs, are closely correlated with RNV development. RBP-RNA interactions are altered during RNV. Here, we briefly review the characteristics and functions of RBPs, and the mechanism of RNV. Then, we present insights into the role of the regulatory network of RBPs in RNV. HuR, eIF4E, LIN28B, SRSF1, METTL3, YTHDF1, Gal-1, HIWI1, and ZFR accelerate RNV progression, whereas YTHDF2 and hnRNPA2B1 hinder it. The mechanisms elucidated in this review provide a reference to guide the design of therapeutic strategies to reverse abnormal processes.
Assuntos
Proteínas de Ligação a RNA , Neovascularização Retiniana , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação da Expressão Gênica/fisiologia , AnimaisRESUMO
Age-related macular degeneration (AMD) is a progressive, degenerative disease of the macula. The formation of macular neovascularization (MNV) and subretinal fibrosis of AMD is the most classic cause of the loss of vision in older adults worldwide. While the underlying causes of MNV and subretinal fibrosis remain elusive, the common feature of many common retinal diseases is changes the proportions of protein deposition in extracellular matrix (ECM) when compared to normal tissue. In ECM, fibronectin (FN) is a crucial component and plays a pivotal part not only in fibrotic diseases but also in the process of angiogenesis. The study aims to understand the role of ligand FN and its common integrin receptor α5ß1 on MNV, and to understand the molecular mechanism involved. To study this, the laser-induced MNV mouse model and the rhesus macaque choroid-retinal endothelial cell line (RF/6A) chemical hypoxia mode were established, and the FN-α5ß1 expression levels were detected by immunohistochemistry (IHC) and quantitative real-time PCR analysis (qRT-PCR). Fibronectin expression was silenced using small interfering RNA (siRNA) targeting FN. The tube formation and vitro scratch assays were used to assess the ability to form blood vessels and cell migration. To measure the formation of MNV, immunofluorescence, and Western blot assays were used. These results revealed that the expressions of FN and integrin α5ß1 were distinctly increased in the laser-induced MNV mouse model and in the RF/6A cytochemically induced hypoxia model, and the expression tendency was identical. After the use of FN siRNA, the tube formation and migration abilities of the RF/6A cells were lower, the ability of endothelial cells to proliferate was confined and the scope of damage caused by the laser in animal models was significantly cut down. In addition, FN gene knockdown dramatically inhibited the expression of Wnt/ß-catenin signal. The interaction of FN with the integrin receptor α5ß1 in the constructed model, which may act through the Wnt/ß-catenin signaling pathway, was confirmed in this study. In conclusion, FN may be a potential new molecular target for the prevention and treatment of subretinal fibrosis and MNV.
Assuntos
Modelos Animais de Doenças , Fibronectinas , Integrina alfa5beta1 , Camundongos Endogâmicos C57BL , Via de Sinalização Wnt , Animais , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfa5beta1/genética , Camundongos , Via de Sinalização Wnt/fisiologia , Movimento Celular/fisiologia , Western Blotting , Macaca mulatta , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , beta Catenina/metabolismo , Imuno-Histoquímica , Reação em Cadeia da Polimerase em Tempo Real , Masculino , Células CultivadasRESUMO
AIMS: In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. METHODS AND RESULTS: SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. CONCLUSION: We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.
Assuntos
Hipóxia Celular , Transportador de Glucose Tipo 1 , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteínas Proto-Oncogênicas c-kit , Transdução de Sinais , Fator de Células-Tronco , Animais , Humanos , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Endogâmicos C57BL , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Camundongos , Neovascularização Fisiológica , Células Cultivadas , Modelos Animais de Doenças , Glucose/metabolismoRESUMO
Pathological retinal angiogenesis profoundly impacts visual function in vascular eye diseases, such as retinopathy of prematurity (ROP) in preterm infants and age-related macular degeneration in the elderly. While the involvement of photoreceptors in these diseases is recognized, the underlying mechanisms remain unclear. This study delved into the pivotal role of photoreceptors in regulating abnormal retinal blood vessel growth using an oxygen-induced retinopathy (OIR) mouse model through the c-Fos/A disintegrin and metalloprotease 17 (Adam17) axis. Our findings revealed a significant induction of c-Fos expression in rod photoreceptors, and c-Fos depletion in these cells inhibited pathological neovascularization and reduced blood vessel leakage in the OIR mouse model. Mechanistically, c-Fos directly regulated the transcription of Adam17 a shedding protease responsible for the production of bioactive molecules involved in inflammation, angiogenesis, and cell adhesion and migration. Furthermore, we demonstrated the therapeutic potential by using an adeno-associated virus carrying a rod photoreceptor-specific short hairpin RNA against c-fos which effectively mitigated abnormal retinal blood vessel overgrowth, restored retinal thickness, and improved electroretinographic (ERG) responses. In conclusion, this study highlights the significance of photoreceptor c-Fos in ROP pathology, offering a novel perspective for the treatment of this disease.