RESUMEN
Purpose: In response to hypoxia, sympathetic fibers to the retina activate ß-adrenoceptors (ß-ARs) that play an important role in the regulation of vascular and neuronal functions. We investigated the role of ß3-AR using the mouse model of oxygen-induced retinopathy (OIR). Methods: Mouse pups were exposed to 75% oxygen at postnatal day 7 (PD7) followed by a return to room air at PD12. The ß3-AR preferential agonist BRL37344 was subcutaneously administered once daily at different times after the return to room air. At PD17, the OIR mice underwent flash and pattern electroretinogram. After sacrifice, retinal wholemounts were used for vessel staining or immunohistochemistry for astrocytes, Müller cells, or retinal ganglion cells (RGCs). In retinal homogenates, the levels of markers associated with neovascularization (NV), the blood-retinal barrier (BRB), or astrocytes were determined by western blot, and quantitative reverse-transcription polymerase chain reaction was used to assess ß3-AR messenger. Administration of the ß3-AR antagonist SR59230A was performed to verify BRL37344 selectivity. Results: ß3-AR expression is upregulated in response to hypoxia, but its increase is prevented by BRL37344, which counteracts NV by inhibiting the pro-angiogenic pathway, activating the anti-angiogenic pathway, recovering BRB-associated markers, triggering nitric oxide production, and favoring revascularization of the central retina through recovered density of astrocytes that ultimately counteracts NV in the midperiphery. Vasculature rescue prevents dysfunctional retinal activity and counteracts OIR-associated retinal ganglion cell loss. Conclusions: ß3-AR has emerged as a crucial intermediary in hypoxia-dependent NV, suggesting a role of ß3-AR agonists in the treatment of proliferative retinopathies.
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Agonistas de Receptores Adrenérgicos beta 3 , Modelos Animales de Enfermedad , Electrorretinografía , Ratones Endogámicos C57BL , Oxígeno , Receptores Adrenérgicos beta 3 , Neovascularización Retiniana , Animales , Ratones , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/prevención & control , Neovascularización Retiniana/patología , Oxígeno/toxicidad , Agonistas de Receptores Adrenérgicos beta 3/farmacología , Receptores Adrenérgicos beta 3/metabolismo , Animales Recién Nacidos , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Etanolaminas/farmacología , Vasos Retinianos/efectos de los fármacos , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/tratamiento farmacológico , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Inmunohistoquímica , AngiogénesisRESUMEN
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.
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Oxígeno , Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Humanos , Ratones , Animales Recién Nacidos , Modelos Animales de Enfermedad , Hiperoxia/complicaciones , Inyecciones Intravítreas , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Ratones Endogámicos C57BL , Oxígeno/toxicidad , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/prevención & control , Neovascularización Retiniana/patología , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Previous retrospective cohort studies have found that, compared with oxygen tension in the uterus and fallopian tubes (2â¯%-8â¯%), exposure of pre-implantation embryos to atmospheric oxygen tension (AtmO2, 20â¯%) during assisted reproductive technology(ART) can affect embryo quality, pregnancy outcomes and offspring health. However, current research on the effects and mechanisms of AtmO2 on the development of embryos and offspring is mainly limited to animal experiments. Human embryonic stem cells (hESCs) play a special and irreplaceable role in the study of early human embryonic development. In this study, we used hESCs as a model to elucidate the possible effects and mechanisms of AtmO2 exposure on human embryonic development. We found that exposure to AtmO2 can reduce cell viability, produce oxidative stress, increase DNA damage, initiate DNA repair, activate autophagy, and increase cell apoptosis. We also noticed that approximately 50â¯% of hESCs survived, adapted and proliferated through high expression of self-renewal and pluripotency regulatory factors, and affected embryoid body differentiation. These data indicate that hESCs experience oxidative stress, accumulation of DNA damage, and activate DNA damage response under the selective pressure of AtmO2.Some hESCs undergo cell death, whereas other hESCs adapt and proliferate through increased expression of self-renewal genes. The current findings provide in vitro evidence that exposure to AtmO2 during the early preimplantation stage negatively affects hESCs.
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Diferenciación Celular , Supervivencia Celular , Daño del ADN , Desarrollo Embrionario , Células Madre Embrionarias Humanas , Estrés Oxidativo , Oxígeno , Humanos , Diferenciación Celular/efectos de los fármacos , Oxígeno/toxicidad , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Reparación del ADN , Autofagia/efectos de los fármacos , Línea CelularRESUMEN
Aberrant neovascularization is the most common feature in retinopathy of prematurity (ROP), which leads to the retinal detachment and visual defects in neonates with a low gestational age eventually. Understanding the regulation of inappropriate angiogenic signaling benefits individuals at-risk. Recently, neural activity originating from the specific neural activity has been considered to contribute to retinal angiogenesis. Here, we explored the impact of cone cell dysfunction on oxygen-induced retinopathy (OIR), a mouse model commonly employed to understand retinal diseases associated with abnormal blood vessel growth, using the Gnat2cpfl3 (cone photoreceptor function loss-3) strain of mice (regardless of the sex), which is known for its inherent cone cell dysfunction. We found that the retinal avascular area, hypoxic area, and neovascular area were significantly attenuated in Gnat2cpfl3 OIR mice compared to those in C57BL/6 OIR mice. Moreover, the HIF-1α/VEGF axis was also reduced in Gnat2cpfl3 OIR mice. Collectively, our results indicated that cone cell dysfunction, as observed in Gnat2cpfl3 OIR mice, leads to attenuated retinal neovascularization. This finding suggests that retinal neural activity may precede and potentially influence the onset of pathological neovascularization.
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Oftalmopatías , Enfermedades de la Retina , Neovascularización Retiniana , Animales , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Conos , Oxígeno/toxicidad , Neovascularización Patológica , Modelos Animales de EnfermedadRESUMEN
Pathological neovascularization is the hallmark of many vascular oculopathies. There is still a great deal of uncertainty surrounding retinal neovascularization research. A working hypothesis that astrocytic Yes-associated protein (YAP) act as a key factor in retinal neovascularization was proposed. And our study was conducted to verified this hypothesis. In vivo, we successfully generated mice deficient in YAP in astrocytes (YAPf/f GFAP-Cre mice) and set up oxygen-induced retinopathy (OIR) model. Pathological neovascularization was evaluated by immunofluorescence staining and western blotting. In vitro, cultured retinal astrocytes were transfected with YAP siRNA. Enzyme-linked immunosorbent assay (ELISA) and western blot were used to determine the proteins in the supernatants and cells. The results showed that YAP was upregulated and activated in the OIR mice retinas. Conditional ablation of YAP aggravated pathological neovascularization, along with the upregulation of vascular endothelial growth factor A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1). Studies in vitro confirmed that the knockdown of YAP in astrocytes lead to increases in VEGF-A and MCP-1 levels, thus enhancing pro-angiogenic capability of YAP-deficit astrocytes. In conclusion, astrocytic YAP alleviates retinal pathological angiogenesis by inhibiting the over-activation of astrocytes, which suppresses excessive VEGF-A production and neuroinflammation.
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Neovascularización Retiniana , Animales , Ratones , Neovascularización Retiniana/metabolismo , Oxígeno/toxicidad , Oxígeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neovascularización Patológica/metabolismo , Proteínas Señalizadoras YAP , Astrocitos/patología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Animales Recién NacidosRESUMEN
PURPOSE: To determine the retinal transcriptomic differences underlying the oxygen-induced retinopathy phenotypes between Sprague Dawley rat pups from two commonly used commercial vendors. This will allow us to discover genes and pathways that may be related to differences in disease severity in similarly aged premature babies and suggest possible new treatment approaches. METHODS: We analyzed retinal vascular morphometry and transcriptomes from Sprague Dawley rat pups from Charles River Laboratories and Envigo (previously Harlan). Room air control and oxygen-induced retinopathy groups were compared. Oxygen-induced retinopathy was induced with the rat 50/10 model. RESULTS: Pups from Charles River Laboratories developed a more severe oxygen-induced retinopathy phenotype, with 3.6-fold larger percent avascular area at P15 and twofold larger % neovascular area at P20 than pups from Envigo. Changes in retinal transcriptomes of rat pups from both vendors were substantial at baseline and in response to oxygen-induced retinopathy. Baseline differences centered on activated pathways of neuronal development in Charles River Laboratories pups. In response to oxygen-induced retinopathy, during the neovascular phase, retinas from Charles River Laboratories pups exhibited activation of pathways regulating necrosis, neuroinflammation, and interferon signaling, supporting the observed increase of neovascularization. Conversely, retinas from Envigo pups showed decreased necrosis and increased focal adhesion kinase signaling, supporting more normal vascular development. Comparing oxygen-induced retinopathy transcriptomes at P15 to those at P20, canonical pathways such as phosphate and tensin homolog, interferon, and coordinated lysosomal expression and regulation element signaling were identified, highlighting potential novel mechanistic targets for future research. CONCLUSION: Transcriptomic profiles differ substantially between rat pup retinas from Charles River Laboratories and Envigo at baseline and in response to oxygen-induced retinopathy, providing insight into vascular morphologic differences. Comparing transcriptomes identified new pathways for further research in oxygen-induced retinopathy pathogenesis and increased scientific rigor of this model.
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Neovascularización Retiniana , Retinopatía de la Prematuridad , Ratas , Animales , Oxígeno/toxicidad , Ratas Sprague-Dawley , Retinopatía de la Prematuridad/inducido químicamente , Retinopatía de la Prematuridad/genética , Transcriptoma , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Animales Recién Nacidos , Necrosis/complicaciones , Necrosis/patología , Interferones , Modelos Animales de Enfermedad , Vasos Retinianos/patologíaRESUMEN
Purpose: Retinal neovascularization (RNV) is the leading cause of vision loss in diseases like proliferative diabetic retinopathy (PDR). A significant failure rate of current treatments indicates the need for novel treatment targets. Animal models are crucial in this process, but current diabetic retinopathy models do not develop RNV. Although the nondiabetic oxygen-induced retinopathy (OIR) mouse model is used to study RNV development, it is largely unknown how closely it resembles human PDR. Methods: We therefore performed RNA sequencing on murine (C57BL/6J) OIR retinas (n = 14) and human PDR RNV membranes (n = 7) extracted during vitrectomy, each with reference to control tissue (n=13/10). Differentially expressed genes (DEG) and associated biological processes were analyzed and compared between human and murine RNV to assess molecular overlap and identify phylogenetically conserved factors. Results: In total, 213 murine- and 1223 human-specific factors were upregulated with a small overlap of 94 DEG (7% of human DEG), although similar biological processes such as angiogenesis, regulation of immune response, and extracellular matrix organization were activated in both species. Phylogenetically conserved mediators included ANGPT2, S100A8, MCAM, EDNRA, and CCR7. Conclusions: Even though few individual genes were upregulated simultaneously in both species, similar biological processes appeared to be activated. These findings demonstrate the potential and limitations of the OIR model to study human PDR and identify phylogenetically conserved potential treatment targets for PDR.
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Retinopatía Diabética , Enfermedades de la Retina , Neovascularización Retiniana , Humanos , Animales , Ratones , Ratones Endogámicos C57BL , Neovascularización Retiniana/genética , Retinopatía Diabética/genética , Modelos Animales de Enfermedad , Oxígeno/toxicidadRESUMEN
Retinopathy of Prematurity (ROP) is a multifactorial disease characterized by abnormal retinal vascular growth in premature infants, which is one of the leading causes of childhood blindness. Lactic acid metabolism may play an imperative role in the development of ROP, but there are still few relevant studies. Our team use a dataset GSE158799 contained 284 genes in 3 P17_OIR mice and 3 P30_OIR mice to identify 41 potentially differentially expressed lactate metabolism-related genes (LMRGs) related to ROP. Then through bioinformatics analysis, we strive to reveal the interaction, the enriched pathways and the immune cell infiltration among these LMRGs, and predict their functions and internal mechanisms. These DEGs may regulate lactate metabolism, leading to the changes of metabolism and immunity, thereby inducing the development of ROP. Our results will expand our understanding of the intrinsic mechanism of ROP and may be helpful for the directions for treatment of ROP in the future.
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Regulación de la Expresión Génica , Retinopatía de la Prematuridad , Retinopatía de la Prematuridad/inducido químicamente , Retinopatía de la Prematuridad/genética , Retinopatía de la Prematuridad/metabolismo , Oxígeno/toxicidad , Animales , Ratones , Ratones Endogámicos C57BL , Ácido Láctico/metabolismo , Transducción de SeñalRESUMEN
Despite decades of researches, the underlying mechanism of retinopathy of prematurity (ROP) remains unclear. The role of Sirt2, which is involved in both angiogenesis and inflammation, both pivotal in ROP, was investigated in an animal model of ROP known as oxygen-induced retinopathy (OIR). Our study found that Sirt2 was overexpressed and colocalized with microglia in OIR. Furthermore, it demonstrated that the level of Sirt2 was upregulated in hypoxia microglia BV-2 in vitro. Subsequently, our results elucidated that administration of the Sirt2 antagonist AGK2 attenuated the avascular and neovascular area and downregulated the expression of IGF-1. The phosphorylation of Akt and the expression of IGF-1 were upregulated in hypoxia BV-2 and conditional media collected from BV-2 under hypoxia promoted the migration and tube formation of retinal capillary endothelial cells, which were suppressed with AGK2. Notably, our findings are the first to demonstrate the deleterious role of Sirt2 in ROP, as Sirt2 inhibition led to the downregulation of Akt/IGF-1 and ameliorated vasculopathy, ultimately improving visual function. These results suggest that Sirt2 may be a promising therapeutic target for ROP.
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Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Humanos , Recién Nacido , Ratones , Retinopatía de la Prematuridad/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor I del Crecimiento Similar a la Insulina/efectos adversos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Células Endoteliales/metabolismo , Sirtuina 2/genética , Neovascularización Retiniana/metabolismo , Oxígeno/toxicidad , Hipoxia , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Animales Recién NacidosRESUMEN
Purpose: Premature infants at risk of retinopathy of prematurity (ROP) miss placental transfer of the carotenoids lutein (L) and zeaxanthin (Z) during the third trimester. We previously demonstrated that prenatal L and Z supplementation raised carotenoid levels in infants at birth in the Lutein and Zeaxanthin in Pregnancy (L-ZIP) study (NCT03750968). Based on their antioxidant effects and bioavailability, we hypothesized that prenatal maternal supplementation with macular carotenoids would reduce the risk of ROP. To test this hypothesis, we utilized "macular pigment mice" genetically engineered to take up L and Z into the retina in a model of oxygen-induced retinopathy (OIR). Methods: Pregnant Bco2-/- mice were divided into nine experimental subgroups based on the type of supplementation (L, Z, or placebo) and on the maternal supplementation start date corresponding to the three trimesters of human fetal development (E0, E11, and P1). Pups and nursing mothers were exposed to 75% O2 for 5 days (P7-P12) and returned to room air for 5 days (P12-P17). Pups were killed at P12 and P17, and their retinas were analyzed for vaso-obliteration and intravitreal neovascularization. Results: Pups of pregnant mice supplemented with L or Z had significant reductions in areas of vaso-obliteration and intravitreal neovascularization compared to placebo. Prenatal carotenoid supplementation starting at E0 or E11 was significantly more protective against OIR than postnatal supplementation starting at P1. Conclusions: Prenatal supplementation with L and Z was beneficial in a mouse OIR model. We recommend testing prenatal L and Z supplementation in future human clinical trials to prevent ROP.
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Dioxigenasas , Pigmento Macular , Retinopatía de la Prematuridad , Humanos , Recién Nacido , Lactante , Femenino , Animales , Embarazo , Ratones , Luteína , Zeaxantinas , Oxígeno/toxicidad , Placenta , Retinopatía de la Prematuridad/inducido químicamente , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/prevención & control , Modelos Animales de Enfermedad , Suplementos DietéticosRESUMEN
Purpose: We examined the role of annexin A2 (A2) in the development of diabetic retinal vasculopathy by testing the effect of Anxa2 gene deletion as well as administration of anti-A2 antibodies on pericyte dropout and retinal neovascularization in diabetic Akita mice, and in mice subjected to oxygen-induced retinopathy. Methods: We analyzed diabetic Ins2AKITA mice with or without global deletion of Anxa2, as well as Ins2AKITA mice that received intravitreal anti-A2 IgG or control antibody at 2, 4, and 6 months, for retinal pericyte dropout at 7 months of age. In addition, we assessed the effect of intravitreal anti-A2 on oxygen-induced retinopathy (OIR) in neonatal mice by quantifying retinal neovascular and vaso-obliterative area, and by enumeration of neovascular tufts. Results: Both deletion of the Anxa2 gene and immunologic blockade of A2 prevented pericyte depletion in retinas of diabetic Ins2AKITA mice. Blockade of A2 also reduced vaso-obliteration and neovascularization in the OIR model of vascular proliferation. This effect was amplified when a combination of antivascular endothelial growth factor (VEGF) and anti-A2 antibodies was used. Conclusions: Therapeutic approaches that target A2, alone or in combination with anti-VEGF therapy, are effective in mice, and may also curtail the progression of retinal vascular disease in humans with diabetes.
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Anexina A2 , Diabetes Mellitus , Retinopatía Diabética , Enfermedades de la Retina , Neovascularización Retiniana , Animales , Ratones , Anexina A2/genética , Anexina A2/metabolismo , Anexina A2/uso terapéutico , Retinopatía Diabética/prevención & control , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Oxígeno/toxicidad , Oxígeno/metabolismo , Enfermedades de la Retina/metabolismo , Neovascularización Retiniana/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
HuR (also known as ELAV1), a ubiquitous RNA-binding protein, is implicated in the pathogenesis of diverse diseases via the mechanism of post-transcriptional regulation. Whether it is involved in pathological angiogenesis in oxygen-induced retinopathy is not clear. In this study, we detected HuR expression was increased in the retina of mouse model of oxygen-induced retinopathy (OIR) as well as in vascular endothelial cells exposed to hypoxia. With gain-of-function and loss-of-function studies using adenovirus infection, we found HuR over-expression promoted while HuR knockdown inhibited the migration, proliferation and tube formation of vascular endothelial cells. Moreover, HuR regulated the expression of VEGFA in vascular endothelial cells. We also found the retinal pathological angiogenesis in mouse OIR model was greatly reduced with HuR knockdown using recombinant AAV expressing HuR specific shRNA which was administered by intravitreal injection. The results of this study suggest HuR is involved in pathological angiogenesis via regulating angiogenic behaviors of endothelial cells, providing a potential target for the treatment of retinopathy of prematurity.
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Proteína 1 Similar a ELAV , Oxígeno , Neovascularización Retiniana , Animales , Ratones , Modelos Animales de Enfermedad , Regulación hacia Abajo , Células Endoteliales/metabolismo , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , Oxígeno/toxicidad , Oxígeno/metabolismo , Retina/metabolismo , Neovascularización Retiniana/metabolismo , Proteína 1 Similar a ELAV/metabolismoRESUMEN
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.
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Ferroptosis , Neovascularización Patológica , Retinopatía de la Prematuridad , Animales , Humanos , Recién Nacido , Ratones , Ferroptosis/efectos de los fármacos , Ferroptosis/fisiología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Oxígeno/toxicidad , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/patología , Estrés OxidativoRESUMEN
IL-17D is a new member of the IL-17 family. Currently, it is believed that IL-17D can directly act on immune cells or may indirectly modulate immune responses by regulating cytokine expression. Herein, we hypothesized that IL-17D regulates the expression of chemokines in intestinal epithelial cells, in turn modulating the immune response within intestinal mucosa under hyperoxia. To explore this notion, newborn rats were divided into a hyperoxia group (85 % O2) and control group (21 % O2). Small intestinal tissues were obtained from neonatal rats at 3, 7, 10, and 14 days. Similarly, intestinal epithelial cells were treated by hyperoxia (85 % O2) as the hyperoxia group or were incubated under normal oxygen (21 % O2) as the control group. Finally, intestinal epithelial cells subjected to hyperoxia were treated with recombinant IL-17D and IL-17D antibodies for 24, 48, and 72 h. Immunohistochemistry, western blot, and reverse transcription-quantitative polymerase chain reaction were used to detect the expression levels of chemokines and chemokine receptors in intestinal tissues of newborn rats and intestinal epithelial cells. We found that hyperoxia affected chemokine expression both in vivo and in vitro. Under hyperoxia, IL-17D promoted the expression of CCL2, CCL25, CCL28, and CCR9 in intestinal epithelial cells while downregulating CCR2, CCR5, CCL5, and CCL20. Our findings provide a basis for further study on the effects of hyperoxia-induced intestinal inflammation and intestinal injury.
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Gastroenteritis , Hiperoxia , Interleucina-27 , Mucosa Intestinal , Oxígeno , Animales , Ratas , Quimiocinas/inmunología , Células Epiteliales/inmunología , Gastroenteritis/etiología , Gastroenteritis/inmunología , Hiperoxia/complicaciones , Hiperoxia/inmunología , Factores Inmunológicos , Interleucina-27/inmunología , Mucosa Intestinal/inmunología , Intestinos/inmunología , Oxígeno/toxicidad , Receptores de Quimiocina/inmunologíaRESUMEN
Purpose: Retinal neovascularization is a major cause of blindness. This study aimed to investigate the effects of IL-19 and the underlying mechanisms in a mouse model of oxygen-induced retinopathy (OIR). Methods: C57BL/6J wild-type mice and IL-19 knockout (KO) mice were used to establish an OIR mouse model. Bone marrow-derived macrophages (BMDMs) with or without recombinant IL-19 (rIL-19) stimulation were injected intravitreally. Reverse transcription-quantitative polymerase chain reaction was used to determine the mRNA expressions. ELISA and western blotting were performed to assess the protein levels. Immunofluorescence staining was applied to assess retinal neovascularization. Human retinal endothelial cells (HRECs) stimulated with rIL-19 were cultured to evaluate the effects on cell proliferation and migration. Results: The level of IL-19 was significantly elevated at postnatal day 17 in OIR retinas. Both the avascular areas and pathological neovascular tufts were significantly increased in rIL-19-treated OIR retinas and suppressed in IL-19 KO retinas. IL-19 KO mice suppressed expression of ARG1, VEGFA, and pSTAT3. Moreover, BMDMs stimulated by rIL-19 enhanced that expression and suppressed the expression of inducible nitric oxide synthase (iNOS). The proliferation and migration of HRECs were significantly augmented by rIL-19. In addition, intravitreal injection of BMDMs stimulated by rIL-19 enhanced retinal neovascularization. Conclusions: These findings suggest that IL-19 enhances pathological neovascularization through a direct effect on microvascular endothelial cells and the promotion of M2 macrophage polarization. The inhibition of IL-19 may be a potential treatment for retinal neovascularization.
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Enfermedades de la Retina , Neovascularización Retiniana , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Humanos , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxígeno/metabolismo , Oxígeno/toxicidad , Enfermedades de la Retina/metabolismo , Neovascularización Retiniana/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Purpose: KC7F2 is a novel molecule compound that can inhibit the translation of hypoxia-inducible factor 1α (HIF1α). It has been reported to exhibit potential antiangiogenic effect. We hypothesized that KC7F2 could inhibit oxygen-induced retinal neovascularization (RNV). The purpose of this study was to investigate this assumption. Methods: Oxygen-induced retinopathy (OIR) models in C57BL/6J mice and Sprague-Dawley rats were used for in vivo study. After intraperitoneal injections of KC7F2, RNV was detected by immunofluorescence and hematoxylin and eosin staining. Retinal inflammation was explored by immunofluorescence. EdU incorporation assay, cell counting kit-8 assay, scratch test, transwell assay, and Matrigel assay were used to evaluate the effect of KC7F2 on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC) induced by vascular endothelial growth factor (VEGF) in vitro. Protein expression was examined by Western blot. Results: KC7F2 treatment (10 mg/kg/d) in OIR mice significantly attenuated pathological neovascularization and decreased the number of preretinal neovascular cell nuclei, without changing the avascular area, which showed the same trends in OIR rats. Consistently, after the KC7F2 intervention (10 µM), cell proliferation was inhibited in VEGF-induced HUVEC, which was in agreement with the trend observed in the retinas of OIR mice. Meanwhile, KC7F2 suppressed VEGF-induced HUVEC migration and tube formation, and decreased the density of leukocytes and microglia colocalizing neovascular areas in the retinas. Moreover, the HIF1α-VEGF pathway activated in retinas of OIR mice and hypoxia-induced HUVEC, was suppressed by KC7F2 treatment. Conclusions: The current study revealed that KC7F2 was able to inhibit RNV effectively via HIF1α-VEGF pathway, suggesting that it might be an effective drug for RNV treatment.
Asunto(s)
Disulfuros/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Neovascularización Retiniana , Retinopatía de la Prematuridad , Sulfonamidas/farmacología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Hipoxia , Recién Nacido , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica , Oxígeno/metabolismo , Oxígeno/toxicidad , Ratas , Ratas Sprague-Dawley , Neovascularización Retiniana/tratamiento farmacológico , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/prevención & control , Retinopatía de la Prematuridad/tratamiento farmacológico , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/prevención & control , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Purpose: Abnormal angiogenesis is a defining feature in a couple of ocular neovascular diseases. The application of anti-VEGFA therapy has achieved certain benefits in the clinic, accompanying side effects and poor responsiveness in many patients. The present study investigated the role of irisin in retinal neovascularization. Methods: Western blot and quantitative PCR were used to determine irisin expression in the oxygen-induced retinopathy mice model. The pathological angiogenesis and inflammation index were examined after irisin administration. Primary retinal astrocytes were cultured and analyzed for VEGFA expression in vitro. Astrocyte-conditioned medium was collected for transwell assay and tube formation assay in human microvascular endothelial cells-1. Results: Irisin was downregulated in the oxygen-induced retinopathy mice retinae. Additional irisin attenuated pathological angiogenesis, inflammation, and apoptosis in vivo. In vitro, irisin decreased astrocyte VEGFA production, and the conditioned medium suppressed human microvascular endothelial cells-1 migration. Last, irisin inhibited hypoxia-inducible factor-2α, nuclear factor-κB, and pNF-κB (Phospho-Nuclear Factor-κB) expression. Conclusions: Irisin mitigates retinal pathological angiogenesis.Chinese Abstract.
Asunto(s)
Oxígeno , Neovascularización Retiniana , Animales , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Fibronectinas , Humanos , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , FN-kappa B , Neovascularización Patológica/tratamiento farmacológico , Oxígeno/toxicidad , Neovascularización Retiniana/metabolismoRESUMEN
Purpose: Semaphorin 3A (Sema3A) is an axonal guidance molecule that inhibits angiogenesis by vasorepulsion and blocks revascularization in the ischemic retina. BI-X is an intravitreal anti-Sema3A agent under clinical investigation in patients with proliferative diabetic retinopathy (PDR) and diabetic macular ischemia (DMI). Methods: Surface plasmon resonance was used to determine binding affinity of BI-X to human and murine Sema3A. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to assess effects of BI-X on cell permeability and cytoskeletal collapse induced by Sema3A. In vivo, intravitreal BI-X or an anti-trinitrophenol control antibody was administered in both eyes in mice with oxygen-induced retinopathy (OIR). Retinal flat mounts were prepared, and avascular area and tip cell density were determined using confocal laser-scanning microscopy. Results: Dissociation constants for BI-X binding to human and murine Sema3A were 29 pM and 27 pM, respectively. In vitro, BI-X prevented HRMEC permeability and cytoskeletal collapse induced by Sema3A. In vivo, BI-X increased tip cell density by 33% (P < 0.001) and reduced avascular area by 12% (not significant). A significant negative correlation was evident between avascular area and tip cell density (r2 = 0.4205, P < 0.0001). Conclusions: BI-X binds to human Sema3A with picomolar affinity and prevents cell permeability and cytoskeletal collapse in HRMECs. BI-X also enhances revascularization in mice with OIR. Translational Relevance: BI-X is a potent inhibitor of human Sema3A that improves revascularization in a murine model of OIR; BI-X is currently being investigated in patients with laser-treated PDR and DMI.
Asunto(s)
Citoesqueleto , Retinopatía Diabética , Enfermedades de la Retina , Animales , Recuento de Células , Permeabilidad de la Membrana Celular , Retinopatía Diabética/tratamiento farmacológico , Células Endoteliales/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Oxígeno/toxicidad , Permeabilidad , Retina , Semaforina-3A/metabolismo , Semaforina-3A/farmacologíaRESUMEN
Retinal neovascular diseases are major causes of blindness worldwide. As a common epitranscriptomic modification of eukaryotic RNAs, N6-methyladenosine (m6A) is associated with the pathogenesis of many diseases, including angiogenesis, through the regulation of RNA metabolism and functions. The aim of this study was to identify m6A modifications of mRNAs and long noncoding RNAs (lncRNAs) and determine their potential roles in retinal neovascularization. The transcriptome-wide m6A profiles of mRNAs and lncRNAs in the retinal tissues of mice with oxygen-induced retinopathy (OIR) and controls were identified by microarray analysis of immunoprecipitated methylated RNAs. The m6A methylation levels of mRNAs and lncRNAs identified in the microarray data were validated by MeRIP-qPCR. A total of 1321 mRNAs (151 hypermethylated and 1170 hypomethylated) and 192 lncRNAs (15 hypermethylated and 177 hypomethylated) were differentially methylated with the m6A modification in OIR and control mice. Gene ontology analysis showed that hypermethylated mRNAs were enriched in the regulation of multicellular organismal process, intracellular organelle, and protein binding, while hypomethylated mRNAs were enriched in cellular metabolic process, intracellular process, and binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that hypermethylated mRNAs were involved in dopaminergic synapses, glutamatergic synapse, and PI3K-Akt signaling pathway, while hypomethylated mRNAs were involved in autophagy, ubiquitin-mediated proteolysis, and spliceosome. Moreover, the altered levels of m6A methylation of ANGPT2, GNG12, ROBO4, and ENSMUST00000153785 were validated by MeRIP-qPCR. The results revealed an altered m6A epitranscriptome in OIR retinas. These methylated RNAs may act as novel modulators and targets in retinal neovascularization.
Asunto(s)
ARN Largo no Codificante , Neovascularización Retiniana , Adenosina/análogos & derivados , Animales , Perfilación de la Expresión Génica/métodos , Ratones , Oxígeno/toxicidad , Fosfatidilinositol 3-Quinasas/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Neovascularización Retiniana/genéticaRESUMEN
Retinal neovascularization (RNV) associated diseases typically exhibit pathological neovascularization and neurodegeneration. Wnt inhibitor factor 1 (WIF1) is a secreted Wnt antagonist that regulates angiogenesis. However, the significance of WIF1 in RNV associated disease has not been explicitly tested. In our study, we found that the WIF1 expressions were strongly downregulated in the vitreous of proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP). Similarly, retinal WIF1 expression was significantly downregulated in OIR mice, relative to normal mice at P17. After injection of WIF1 overexpression lentivirus into the vitreous of OIR mice, overexpressing WIF1 in OIR mice vitreous strongly reduced avascular areas and neovascular tufts, increased vessel branches, raised a-, b-waves and oscillatory potentials amplitudes on ERG, increased retinal thickness and the number of synapses in retina, normalized the Golgi, mitochondria, and outer segments of photoreceptors. Furthermore, overexpression WIF1 suppressed expressions of ß-catenin, vascular endothelial growth factor (VEGF), p-AKT and p-ERK, reduced retinal reactive oxygen species (ROS) and 4-HNE levels, improved autophagic flux, and mitigated apoptosis. In summary, WIF1 plays a key role in alleviating angiogenesis and in improving visual function in OIR mice by suppressing the Wnt/ß-catenin-VEGF signaling pathway and ROS levels. WIF1 is an excellent candidate for targeted therapy against RNV associated diseases.