RESUMO
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
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.
Assuntos
Proteína ADAM17 , Proteínas Proto-Oncogênicas c-fos , Neovascularização Retiniana , Animais , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Proteína ADAM17/metabolismo , Proteína ADAM17/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Camundongos , Humanos , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/genética , Camundongos Endogâmicos C57BL , Transcrição Gênica , Regulação da Expressão Gênica , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Modelos Animais de Doenças , AngiogêneseRESUMO
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
Bronchopulmonary dysplasia (BPD), also called chronic lung disease of immaturity, afflicts approximately one third of all extremely premature infants, causing lifelong lung damage. There is no effective treatment other than supportive care. Retinopathy of prematurity (ROP), which impairs vision irreversibly, is common in BPD, suggesting a related pathogenesis. However, specific mechanisms of BPD and ROP are not known. Herein, a neonatal mouse hyperoxic model of coincident BPD and retinopathy was used to screen for candidate mediators, which revealed that granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3, was up-regulated significantly in mouse lung lavage fluid and plasma at postnatal day 14 in response to hyperoxia. Preterm infants with more severe BPD had increased plasma G-CSF. G-CSF-deficient neonatal pups showed significantly reduced alveolar simplification, normalized alveolar and airway resistance, and normalized weight gain compared with wild-type pups after hyperoxic lung injury. This was associated with a marked reduction in the intensity, and activation state, of neutrophilic and monocytic inflammation and its attendant oxidative stress response, and protection of lung endothelial cells. G-CSF deficiency also provided partial protection against ROP. The findings in this study implicate G-CSF as a pathogenic mediator of BPD and ROP, and suggest the therapeutic utility of targeting G-CSF biology to treat these conditions.
Assuntos
Displasia Broncopulmonar , Hiperóxia , Retinopatia da Prematuridade , Lactente , Recém-Nascido , Animais , Humanos , Camundongos , Displasia Broncopulmonar/patologia , Recém-Nascido Prematuro , Células Endoteliais/patologia , Pulmão/patologia , Hiperóxia/complicações , Retinopatia da Prematuridade/patologia , Fator Estimulador de Colônias de Granulócitos , Animais Recém-NascidosRESUMO
Retinopathy of prematurity (ROP) is a vision-threatening ocular disease that occurs in premature infants, but the underlying mechanism is still unclear. Since oxidative stress has been well documented in the ROP development, we aimed to investigate whether ferroptosis, a new type of cell death characterized by lipid peroxidation and iron overload, is also involved in ROP. We detected the lipid peroxidation, oxidative stress and the expression of ferroptosis markers in the retina of mouse model of oxygen-induced retinopathy. After ferroptosis inhibitor, ferrostatin-1, was administered by intravitreal injection, ferroptosis marker, lipid peroxidation, retinal vasculature and glial cell activation were examined. We found decreased expression of SLC7A11 and GPX4, increased expression of FTH1 and TFRC, as well as increase of lipid peroxidation in the retina of OIR mice. Ferrostatin-1 administration significantly reduced lipid peroxidation, and also reversed the change of ferroptosis marker. Neovascular area and avascular area were suppressed and the pathological vasculature changes including acellular vessels and ghost pericytes were decreased. Microglial cell and Müller cell activation was not evidently influenced by ferrostatin-1 treatment. Our findings suggest that ferroptosis is involved in the pathological angiogenesis and might be a promising target for ROP therapy.
Assuntos
Ferroptose , Neovascularização Patológica , Retinopatia da Prematuridade , Animais , Humanos , Recém-Nascido , Camundongos , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Oxigênio/toxicidade , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/patologia , Estresse OxidativoRESUMO
BACKGROUND: Retinopathy of prematurity (ROP) is the leading cause of blindness in infants, and elevation of HIF-1α through the PI3K/Akt and ERK pathways is implicated in ROP pathogenesis. The mechanism of action of propranolol in ROP remains controversial. We investigated the effect of propranolol on ROP and explored its potential mechanisms of action in an oxygen-induced retinopathy (OIR) mouse model. METHODS: OIR mice were first treated with propranolol intraperitoneally, and the retina integrity was measured by FITC-dextran and hematoxylin-eosin staining. The expression of HIF-1α, VEGF, and key signaling pathway proteins was determined using real-time PCR and western blotting. RESULTS: FITC-dextran staining showed that propranolol treatment reduced damage to retinal morphology in OIR mice. Mice treated with propranolol showed a reduced number of nuclei of vascular endothelial cells penetrating the inner limiting membrane of the retina, confirming the therapeutic effect of propranolol on ROP. Further analysis showed that HIF-1α and PI3K/Akt/ERK pathway protein levels were significantly elevated in OIR mice. In contrast, propranolol treatment downregulated the expression of these proteins, indicating that the PI3K/Akt/ERK/HIF-1α axis is associated with propranolol-induced ROP alleviation. CONCLUSIONS: Propranolol has a therapeutic function against ROP, likely through the downregulation of HIF-1α via the PI3K/Akt/ERK pathway. IMPACT: Propranolol can reduce the formation of abnormal retinal neovascularization in oxygen-induced retinopathy (OIR) models, and reduce leaking, tortuous, and abnormally expanding retinal blood vessels. Propranolol possibly improves OIR by inhibiting the activated ERK and HIF-1α pathways. Furthermore, propranolol may downregulate HIF-1α via the PI3K/Akt/ERK pathway to ameliorate retinopathy of prematurity. This study elucidated that the therapeutic effect of propranolol in OIR mice does not involve the VEGFR-2 pathway.
Assuntos
Neovascularização Retiniana , Retinopatia da Prematuridade , Humanos , Recém-Nascido , Animais , Camundongos , Propranolol/uso terapêutico , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/patologia , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células Endoteliais/metabolismo , Neovascularização Retiniana/metabolismo , Oxigênio/uso terapêutico , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized Scg3 binding exclusively to disease-related disorganized retinal neovessels and neovascular tufts, whereas VEGF bound to both disorganized and well-organized neovessels. Homozygous deletion of the Scg3 gene showed undetectable effects on physiological retinal neovascularization but markedly reduced the severity of OIR-induced pathological angiogenesis. Furthermore, anti-Scg3 humanized antibody Fab (hFab) inhibited pathological angiogenesis with similar efficacy to anti-VEGF aflibercept. Aflibercept dose-dependently blocked physiological angiogenesis in neonatal retinas, whereas anti-Scg3 hFab was without adverse effects at any dose and supported a therapeutic window at least 10X wider than that of aflibercept. Therefore, Scg3 stringently regulates pathological but not physiological angiogenesis, and anti-Scg3 hFab satisfies essential criteria for development as a safe and effective disease-targeted anti-angiogenic therapy for ROP.
Assuntos
Inibidores da Angiogênese/farmacologia , Cromograninas/imunologia , Cromograninas/metabolismo , Neovascularização Patológica/genética , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Animais , Capilares/metabolismo , Cromograninas/antagonistas & inibidores , Cromograninas/genética , Modelos Animais de Doenças , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxigênio/efeitos adversos , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/farmacologia , Neovascularização Retiniana/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Throughout the central nervous system, astrocytes adopt precisely ordered spatial arrangements of their somata and arbors, which facilitate their many important functions. Astrocyte pattern formation is particularly important in the retina, where astrocytes serve as a template that dictates the pattern of developing retinal vasculature. Thus, if astrocyte patterning is disturbed, there are severe consequences for retinal angiogenesis and ultimately for vision - as seen in diseases such as retinopathy of prematurity. Here we discuss key steps in development of the retinal astrocyte population. We describe how fundamental developmental forces - their birth, migration, proliferation, and death - sculpt astrocytes into a template that guides angiogenesis. We further address the radical changes in the cellular and molecular composition of the astrocyte network that occur upon completion of angiogenesis, paving the way for their adult functions in support of retinal ganglion cell axons. Understanding development of retinal astrocytes may elucidate pattern formation mechanisms that are deployed broadly by other axon-associated astrocyte populations.
Assuntos
Astrócitos/fisiologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Animais , Axônios/fisiologia , Morte Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Humanos , Neovascularização Fisiológica , Fibras Nervosas/fisiologia , Retina/citologia , Retina/embriologia , Células Ganglionares da Retina/fisiologia , Vasos Retinianos/embriologia , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/fisiologia , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/fisiopatologiaRESUMO
Retinopathy of prematurity (ROP) remains one of the major causes of blindness in children worldwide. While current ROP treatments are mostly disruptive to reduce proliferative neovascularization by targeting the hypoxic phase, protection against early hyperoxia-induced retinal vascular loss represents an effective therapeutic window, but no such therapeutic strategy is available. Built upon our recent demonstration that the protection against oxygen-induced retinopathy by adenosine A2A receptor (A2A R) antagonists is most effective when administered at the hyperoxia (not hypoxic) phase, we here uncovered the cellular mechanism underlying the A2A R-mediated protection against early hyperoxia-induced retinal vascular loss by reversing the inhibition of cellular proliferation via possibly multiple signaling pathways. Specifically, we revealed two distinct stages of the hyperoxia phase with greater cellular proliferation and apoptosis activities and upregulation of adenosine signaling at postnatal 9 day (P9) but reduced cellular activities and adenosine-A2A R signaling at P12. Importantly, the A2A R-mediated protection at P9 was associated with the reversal of hyperoxia-induced inhibition of progenitor cells at the peripheral retina at P9 and of retinal endothelial proliferation at P9 and P12. The critical role of cellular proliferation in the hyperoxia-induced retinal vascular loss was validated by the increased avascular areas by siRNA knockdown of the multiple signaling molecules involved in modulation of cellular proliferation, including activin receptor-like kinase 1, DNA-binding protein inhibitor 1, and vascular endothelial growth factor-A.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Proliferação de Células/efeitos dos fármacos , Hiperóxia/metabolismo , Substâncias Protetoras/farmacologia , Receptor A2A de Adenosina/metabolismo , Neovascularização Retiniana , Vasos Retinianos/efeitos dos fármacos , Receptores de Activinas Tipo II/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína 1 Inibidora de Diferenciação/metabolismo , Camundongos , Neovascularização Patológica , Oxigênio/efeitos adversos , Retina/citologia , Retina/efeitos dos fármacos , Retina/patologia , Vasos Retinianos/citologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Retinopathy of prematurity (ROP) is a retinal vasoproliferative disorder that represents an important cause of childhood visual impairment and blindness. Although oxidative stress has long been implicated in ROP etiology, other prenatal and perinatal factors are also involved. This review focuses on current research involving inflammation and genetic factors in the pathogenesis of ROP. Increasing evidence suggests that perinatal inflammation or infection contributes to ROP pathogenesis. Cytokines and chemokines with a fundamental role in inflammatory responses and that significantly contributing to angiogenesis are analyzed. Microglia cells, the retinal-resident macrophages, are crucial for retinal homeostasis, however, under sustained pathological stimuli release exaggerated amounts of inflammatory mediators and can promote pathological neovascularization. Current modulation of angiogenic cytokines, such as treatment with antibodies to vascular endothelial growth factor (anti-VEGF), has shown efficacy in the treatment of ocular neovascularization; however, some patients are refractory to anti-VEGF agents, suggesting that other angiogenic or anti-angiogenic cytokines need to be identified. Much evidence suggests that genetic factors contribute to the phenotypic variability of ROP. Several studies have implicated the involvement of candidate genes from different signaling pathways in the development of ROP. However, a genetic component with a major impact on ROP has not yet been discovered. Most studies have limitations and did not replicate results. Future research involving bioinformatics, genomics, and proteomics may contribute to finding more genes associated with ROP and may allow discovering better solutions in the management and treatment of ROP.
Assuntos
Retinopatia da Prematuridade , Citocinas/genética , Humanos , Recém-Nascido , Inflamação/genética , Neovascularização Patológica/genética , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/patologia , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Pathologic ocular neovascularization in retinopathy of prematurity (ROP) and other proliferative retinopathies are characterized by dysregulation of vascular endothelial growth factor-A (VEGF-A). A study of Vegfa isoform expression during oxygen-induced ischemic retinopathy (OIR) may enhance our understanding of Vegf dysregulation. METHODS: Following induction of OIR, immunohistochemistry and polymerase chain reaction (PCR) was performed on room air (RA) and OIR mice. RESULTS: Total Vegfa messenger RNA (mRNA) expression was stable in RA mice, but increased in OIR mice with a peak at postnatal day 17 (P17), before returning to RA levels. Vegfa164a expression was similar in both OIR and RA mice at P10 (Phase 1 OIR), but 2.4-fold higher in OIR mice compared to RA mice at P16 (Phase 2 OIR). At P10, Vegfa164b mRNA was similar in OIR vs RA mice, but was expressed 2.5-fold higher in OIR mice compared to RA mice at P16. At P10 and P16, Vegfr2/Vegfr1 expression was increased in OIR mice compared to RA mice. Increased activation of microglia was seen in OIR mice. CONCLUSIONS: Vegfa164a, Vegfa164b, and Vegfr1 were overexpressed in OIR mice, leading to abnormal signaling and angiogenesis. Further studies of mechanisms of Vegf dysregulation may lead to novel therapies for ROP and other proliferative retinopathies. IMPACT: Vegfa164 has two major isoforms, a proangiogenic, Vegfa164a, and an antiangiogenic, Vegfa164b, with opposing receptors, inhibitory Vegfr1, and stimulatory Vegfr2, but their role in OIR is unclear. In Phase 1 OIR, both isoforms and receptors are expressed similarly. In Phase 2 OIR, both isoforms are overexpressed, with an increased ratio of inhibitory Vegfr1. Modulation of angiogenesis by Vegf regulation enables pruning of excess angiogenesis during physiology, but results in ineffective angiogenesis during OIR. Knowledge of VEGF dysregulation may have novel therapeutic implications in the management of ROP and retinal proliferative diseases.
Assuntos
Neovascularização Retiniana , Retinopatia da Prematuridade , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Oxigênio/uso terapêutico , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Neovascularização Retiniana/genética , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/patologiaRESUMO
Retinopathy of prematurity (ROP) is the primary cause of visual impairment and vision loss in premature infants, which results from the formation of aberrant retinal neovascularization (NV). An emerging body of evidence has shown that Müller cells are the predominant source of vascular endothelial growth factor (VEGF), which also serves as a chemoattractant for monocyte/macrophage lineage. The recruitment of macrophages is increased during retinal NV, and they exert a pro-angiogenic role in ROP. We have shown that lymphocytic microparticles (microvesicles; LMPs) derived from apoptotic human T lymphocytes possess strong angiogenesis-inhibiting properties. Here, we investigated the effect of LMPs on the chemotactic capacity of Müller cells in vitro using rat Müller cell rMC-1 and mouse macrophage RAW 264.7. In addition, the impact of LMPs was determined in vivo using a mouse model of oxygen-induced ischemic retinopathy (OIR). The results revealed that LMPs were internalized by rMC-1 and reduced their cell proliferation dose-dependently without inducing cell apoptosis. LMPs inhibited the chemotactic capacity of rMC-1 on RAW 264.7 via reducing the expression of VEGF. Moreover, LMPs attenuated pathological retinal NV and the infiltration of macrophages in vivo. LMPs downregulated ERK1/2 and HIF-1α both in vitro and in vivo. These findings expand our understanding of the effects of LMPs, providing evidence of LMPs as a promising therapeutic approach for the treatment of retinal NV diseases.
Assuntos
Micropartículas Derivadas de Células/fisiologia , Células Ependimogliais/patologia , Isquemia/patologia , Linfócitos/patologia , Doenças Retinianas/patologia , Neovascularização Retiniana/prevenção & controle , Animais , Animais Recém-Nascidos , Micropartículas Derivadas de Células/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Isquemia/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , Neovascularização Patológica/prevenção & controle , Células RAW 264.7 , Ratos , Doenças Retinianas/complicações , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/patologia , Vasos Retinianos/patologia , Vasos Retinianos/fisiopatologia , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/patologiaRESUMO
Retinopathy of prematurity (ROP) is a severe eye disease leading to blindness. Abnormal vessel formation is the pathological hallmark of neovascular ROP. In forming vessels, vascular endothelial growth factor (VEGF) is an important stimulator. The current anti-ROP therapy has focused on bevacizumab, a monoclonal antibody against VEGF, and pazopanib, a tyrosine kinase inhibitor on the VEGF receptor (VEGFR). Several lines of evidence have proposed that natural compounds may be more effective and safer for anti-VEGF function. Resveratrol, a common natural compound, binds to VEGF and blocks its interaction with VEGFR, thereafter suppressing angiogenesis. Here, we evaluate the efficacy of intravitreal injection, or topical instillation (eye drops), of resveratrol into the eyes of mice suffering from oxygen-induced retinopathy, i.e., developing ROP. The treatment of resveratrol significantly relieved the degree of vascular distortion, permeability and hyperplasia; the efficacy could be revealed by both methods of resveratrol application. In parallel, the treatments of resveratrol inhibited the retinal expressions of VEGF, VEGFR and CD31. Moreover, the applied resveratrol significantly relieved the damage caused by oxygen radicals through upregulating the level of superoxide dismutase (SOD) and downregulating the level of malondialdehyde (MDA) in the retina. Taken together, the potential therapeutic benefit of resveratrol in pro-angiogenic diseases, including retinopathy, can be considered.
Assuntos
Retinopatia da Prematuridade , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Bevacizumab/uso terapêutico , Camundongos , Neovascularização Patológica/tratamento farmacológico , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/patologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are two debilitating disorders that develop in preterm infants exposed to supplemental oxygen to prevent respiratory failure. Both can lead to lifelong disabilities, such as chronic obstructive pulmonary disease and vision loss. Due to the lack of a standard experimental model of coincident disease, the underlying associations between BPD and ROP are not well characterized. To address this gap, we used the robust mouse model of oxygen-induced retinopathy exposing C57BL/6 mice to 75% oxygen from postnatal day 7 to 12. The cardinal features of ROP were replicated by this strategy, and the lungs of the same mice were simultaneously examined for evidence of BPD-like lung injury, investigating both the short- and long-term effects of early-life supplemental oxygen exposure. At postnatal days 12 and 18, mild lung disease was evident by histopathologic analysis together with the expected vasculopathy in the inner retina. At later time points, the lung lesion had progressed to severe airspace enlargement and alveolar simplification, with concurrent thinning in the outer layer of the retina. In addition, critical angiogenic oxidative stress and inflammatory factors reported to be dysregulated in ROP were similarly impaired in the lungs. These data shed new light on the interconnectedness of these two neonatal disorders, holding potential for the discovery of novel targets to treat BPD and ROP.
Assuntos
Displasia Broncopulmonar/etiologia , Modelos Animais de Doenças , Oxigenoterapia/efeitos adversos , Oxigênio/toxicidade , Retinopatia da Prematuridade/etiologia , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/patologia , Inflamação/etiologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Retinopatia da Prematuridade/patologiaRESUMO
The goal of this study was to evaluate the effects of CM082, a novel vascular endothelial growth factor (VEGF) receptor-2 tyrosine kinase inhibitor, on human umbilical vein endothelial cells (HUVECs), and oxygen-induced retinopathy (OIR) mice. HUVECs were stimulated with rHuVEGF165 and then treated with CM082 to assess the antiangiogenic effects of CM082; subsequently, proliferation, wound-healing migration, Transwell invasion, tube formation assays, and Western blotting were performed in vitro. Retinal neovascularization tufts, avascular area, and TUNEL assays were estimated for OIR mice after intraperitoneal injection with CM082. CM082 significantly inhibited proliferation, migration, invasion, and tube formation induced by stimulation of HUVECs with rHuVEGF165; this inhibitory effect was mediated by blocking VEGFR2 activation. CM082 significantly inhibited retinal neovascularization and avascular area and did not increase apoptosis in the retina of OIR mice. The findings demonstrated that CM082 exhibits highly antiangiogenic effects in HUVECs and OIR mice. Thus, it may serve as an alternative treatment for neovascular eye disease in the future.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Indóis/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Pirrolidinas/farmacologia , Neovascularização Retiniana/prevenção & controle , Retinopatia da Prematuridade/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Hiperóxia/complicações , Camundongos Endogâmicos C57BL , Neovascularização Retiniana/enzimologia , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/enzimologia , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/patologia , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: To evaluate the role of Toll-like receptor 2 (TLR2) signaling in retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). MATERIALS AND METHODS: The OIR model was established in C57BL/6J wild type (WT) mice and TLR2-/- mice. Retinal neovascularization in the OIR model was measured by counting new vascular cell nuclei above the internal limiting membrane and analyzing flat-mounted retinas perfused with fluorescein dextran and immunostained with Griffonia Simplicifolia (GS) isolectin. The expression of TLR2 and VEGF in the retina was detected by immunofluorescence. Expression of TGF- ß1, b-FGF, and IL-6 mRNA in the retina was measured by quantitative real-time PCR. RESULTS: Compared to WT OIR mice, retinal neovascularization was attenuated in TLR2-/- OIR mice. The co-expressions of TLR2 and VEGF were remarkably and consistently increased in WT OIR mice; however, there was no expression of TLR2 and a significant decrease in VEGF expression in TLR2-/- OIR mice. These results suggest that TLR2 plays a central role in OIR model angiogenesis. Expression of TGF- ß1, b-FGF, and IL-6 mRNA were reduced in the TLR2-/- OIR mice, suggesting that the inflammatory response induced by TLR2 relates to angiogenesis. CONCLUSION: TLR2 signaling in the retina is associated with neovascularization in mice. Inflammation contributes to the activation of angiogenesis and is partially mediated through the TLR2-VEGF retinal signaling pathway.
Assuntos
Modelos Animais de Doenças , Oxigênio/toxicidade , Neovascularização Retiniana/metabolismo , Retinopatia da Prematuridade/metabolismo , Transdução de Sinais/fisiologia , Receptor 2 Toll-Like/metabolismo , Animais , Citocinas/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/genética , Retinopatia da Prematuridade/patologia , Receptor 2 Toll-Like/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Myeloid cells are critically involved in inflammation-induced angiogenesis, although their pathogenic role in the ischemic retina remains controversial. We hypothesize that myeloid cells contribute to pathogenic neovascularization in retinopathy of prematurity through STAT3 (signal transducer and activator of transcription 3) activation. Approach and Results: Using the mouse model of oxygen-induced retinopathy, we show that myeloid cells (CD45+IsolectinB4 [IB4]+) and particularly M2-type macrophages (CD45+ Arg1+), comprise a major source of STAT3 activation (pSTAT3) in the immature ischemic retina. Most of the pSTAT3-expressing myeloid cells concentrated at the hyaloid vasculature and their numbers were strongly correlated with the severity of pathogenic neovascular tuft formation. Pharmacological inhibition of STAT3 reduced the load of IB4+ cells in the hyaloid vasculature and significantly reduced the formation of pathogenic neovascular tufts during oxygen-induced retinopathy, leading to improved long-term visual outcomes (ie, increased retinal thickness and scotopic b-wave electroretinogram responses). Genetic deletion of SOCS3 (suppressor of cytokine signaling 3), an endogenous inhibitor of STAT3, in myeloid cells, enhanced pathological and physiological neovascularization in oxygen-induced retinopathy, indicating that myeloid-STAT3 signaling is crucial for retinal angiogenesis. CONCLUSIONS: Circulating myeloid cells may migrate to the immature ischemic retina through the hyaloid vasculature and contribute to retinal neovascularization via activation of STAT3. Understanding how STAT3 modulates myeloid cells for vascular repair/pathology may provide novel therapeutic options in pathogenic angiogenesis.
Assuntos
Macrófagos/metabolismo , Oxigênio , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Retinopatia da Prematuridade/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Animais Recém-Nascidos , Antraquinonas/farmacologia , Modelos Animais de Doenças , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/patologia , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/prevenção & controle , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais , Sulfonamidas/farmacologia , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismoRESUMO
Retinopathy of prematurity is a vision-threatening disease associated with retinal hypoxia-ischemia, leading to the death of retinal neurons and chronic neuronal degeneration. During this study, we used the oxygen-induced retinopathy mice model to mimic retinal hypoxia-ischemia phenotypes to investigate further the neuroprotective effect of melatonin on neonatal retinal neurons. Melatonin helped maintain relatively normal inner retinal architecture and thickness and preserve inner retinal neuron populations in avascular areas by rescuing retinal ganglion and bipolar cells, and horizontal and amacrine neurons, from apoptosis. Meanwhile, melatonin recovered visual dysfunction, as reflected by the improved amplitudes and implicit times of a-wave, b-wave, and oscillatory potentials. Additionally, elevated cleaved caspase-3 and Bax protein levels and reduced Bcl-2 protein levels in response to hypoxia-ischemia were diminished after melatonin treatment. Moreover, melatonin increased BDNF and downstream phospho-TrkB/Akt/ERK/CREB levels. ANA-12, a TrkB receptor antagonist, antagonized these melatonin actions and reduced melatonin-induced neuroprotection. Furthermore, melatonin rescued the reduction in melatonin receptor expression. This study suggests that melatonin exerted anti-apoptotic and neuroprotective effects in inner retinal neurons after hypoxia-ischemia, at least partly due to modulation of the BDNF-TrkB pathway.
Assuntos
Melatonina/farmacologia , Fármacos Neuroprotetores/farmacologia , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/patologia , Retinopatia da Prematuridade , Animais , Animais Recém-Nascidos , Hipóxia/etiologia , Hipóxia/patologia , Isquemia/etiologia , Isquemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Retinopatia da Prematuridade/complicações , Retinopatia da Prematuridade/patologiaRESUMO
PURPOSE OF REVIEW: Classically, ROP has been considered a neonatal disease only; however, pediatric ophthalmologists and retinal specialists worldwide are recently facing a new paradigm shift. retinopathy of prematurity (ROP) is now considered a lifelong disease that extends well into adulthood. The purpose of this review is to describe the adult ROP anatomy and discuss the late sequelae and management of this disease. RECENT FINDINGS: Neonatal ROP treatments affect both anterior and posterior segment anatomy. Anterior segment changes secondary to inflammation and posterior ciliary nerve ablation range from acute to chronic pathology, including cataract, secondary glaucoma, and corneal decompensation. Persistent avascular retina can be present in previously treated Type 1 ROP eyes after anti-vascular endothelial growth factor or in 'normal' untreated eyes that did not previously meet Type 1 ROP criteria. Persistent avascular retina is associated with lattice-like changes, retinal tears, and detachments. The location and extent of the ridge, posterior hyaloidal contraction and adhesion, and persistent avascular retina all contribute to a spectrum of findings ranging from reactivation of neovascularization, tractional, rhegmatogenous, or exudative detachments. SUMMARY: Understanding Adult ROP anatomy is critical in identification of retinal pathology and treatment choice. ROP patients require lifelong monitoring.
Assuntos
Retina/patologia , Retinopatia da Prematuridade , Adulto , Progressão da Doença , Humanos , Lactente , Recém-Nascido , Fotocoagulação a Laser/efeitos adversos , Retina/anatomia & histologia , Retina/efeitos dos fármacos , Retina/cirurgia , Retinopatia da Prematuridade/diagnóstico , Retinopatia da Prematuridade/patologia , Retinopatia da Prematuridade/terapiaRESUMO
The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.