RESUMEN
BACKGROUND: Diabetic retinopathy (DR), a common complication of diabetes mellitus, is the major cause of visual impairment and blindness. Circ_0000615 was found to be elevated in retina samples of diabetic patients. Hence, the detailed effects and molecular mechanisms of circ_0000615 in DN progression were explored. METHODS: The levels of circ_0000615, microRNA (miR)-646 and YAP1 (yes-associated protein 1) were detected using quantitative real-time polymerase chain reaction and Western blot assays. Cell viability, apoptosis, inflammation and reactive oxygen species (ROS) generation were determined using cell counting kit-8 assay, flow cytometry, caspase3 activity analysis, Western blot, enzyme-linked immunosorbent assay (ELISA) and Dichlorofluorescein diacetate (DCFH-DA) assay, respectively. The binding interaction between miR-646 and circ_0000615 or YAP1 was determined using dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. RESULTS: Circ_0000615 was elevated in high glucose (HG)-induced human retinal pigment epithelium (HRPE) cells. Knockdown of circ_0000615 attenuated HG-triggered HRPE cell apoptosis, inflammation, and ROS generation. Mechanistically, miR-646 was confirmed to be a target of circ_0000615, inhibition of miR-646 reversed the protective effects of circ_0000615 knockdown on HG-evoked HRPE cell dysfunction. MiR-646 was verified to target YAP1, overexpression of YAP1 abolished the impairment induced by miR-646 on HG-induced HRPE cell damage. Besides that, we confirmed that circ_0000615 could regulate YAP1 expression via miR-646. CONCLUSION: Circ_0000615 contributed to HG-induced HRPE cell dysfunction via miR-646/YAP1 axis, suggesting a novel insight into the pathogenesis of DR and a potential candidate for DR treatment.
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , MicroARNs , Apoptosis , Retinopatía Diabética/genética , Glucosa/metabolismo , Glucosa/toxicidad , Humanos , Inflamación/genética , MicroARNs/genética , MicroARNs/metabolismo , MicroARNs/farmacología , Estrés Oxidativo , ARN Circular , Especies Reactivas de Oxígeno , Epitelio Pigmentado de la Retina/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Choroidal neovascularization (CNV) is a leading cause of blindness in age-related macular degeneration. Production of vascular endothelial growth factor (VEGF) and macrophage recruitment by retinal pigment epithelial cells (RPE) significantly contributes to the process of CNV in an experimental CNV model. Serine racemase (SR) is expressed in retinal neurons and glial cells, and its product, d-serine, is an endogenous co-agonist of N-methyl-d-aspartate receptor. Activation of the receptor results in production of nitric oxide (. NO), a molecule that promotes retinal and choroidal neovascularization. These observations suggest possible roles of SR in CNV. With laser-injured CNV mice, we found that inactivation of SR-coding gene (Srrnull ) significantly reduced CNV volume, neovascular density, and invading macrophages. We exploited the underlying mechanism in vivo and ex vivo. RPE from wild-type (WT) mice expressed SR. To explore the possible downstream target of SR inactivation, we showed that choroid/RPE homogenates extracted from laser-injured Srrnull mice contained less inducible nitric oxide synthase and decreased phospho-VEGFR2 compared to amounts in WT mice. In vitro, inflammation-primed WT RPEs expressed more inducible NOS, produced more. NO and VEGF than did inflammation-primed Srrnull RPEs. When co-cultured with inflammation-primed Srrnull RPE, significantly fewer RF/6A-a cell line of choroidal endothelial cell, migrated to the opposite side of the insert membrane than did cells co-cultured with pre-treated WT RPE. Altogether, SR deficiency reduces RPE response to laser-induced inflammatory stimuli, resulting in decreased production of a cascade of pro-angiogenic cytokines, including. NO and VEGF, and reduced macrophage recruitment, which contribute synergistically to attenuated angiogenesis.
Asunto(s)
Ceguera/patología , Neovascularización Coroidal/genética , Regulación de la Expresión Génica/genética , Óxido Nítrico/metabolismo , Racemasas y Epimerasas/deficiencia , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Ceguera/etiología , Ceguera/genética , Células Cultivadas , Neovascularización Coroidal/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de la radiación , Rayos Láser/efectos adversos , Lipopolisacáridos/farmacología , Macrófagos/fisiología , Macrófagos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , ARN Mensajero/metabolismo , Racemasas y Epimerasas/genética , Epitelio Pigmentado de la Retina/efectos de los fármacos , Serina/metabolismoRESUMEN
BACKGROUND: Recent data indicate that inflammatory mechanisms contribute to diabetic retinopathy (DR). We have determined that serine racemase (SR) expression is increased by inflammatory stimuli including liposaccharide (LPS), amyloid ß-peptide (A-beta), and secreted amyloid precursor protein (sAPP); expression is decreased by the anti-inflammatory drug, dexamethasone. We tested possibility that SR and its product, D-serine, were altered in a rat model of DR. METHODS: Intraperitoneal injection of streptozotocin (STZ; 70 mg/kg body weight) to Sprague-Dawley rats produced type-I diabetic mellitus (fasting blood sugar higher than 300 mg/dL). At 3 and 5 months after STZ or saline injection, retinas from some rats were subjected to cryosectioning for immunofluorescent analysis of SR and TUNEL assay of apoptosis. Retinal homogenates were used to detect SR levels and Jun N-terminal kinase (JNK) activation by immunoblotting. Aqueous humor and retina were also collected to assay for neurotransmitters, including glutamate and D-serine, by reverse-phase HPLC. RESULTS: Compared to saline-injected rats, STZ-injected (diabetic) rats showed elevation of SR protein levels in retinal homogenates, attributed to the inner nuclear layer (INL) by immunofluorescence. Aqueous humor fluid from STZ-injected rats contained significantly higher levels of glutamate and D-serine compared to controls; by contrast, D-serine levels in retinas did not differ. Levels of activated JNK were elevated in diabetic retinas compared to controls. CONCLUSIONS: Increased expression of SR in retina and higher levels of glutamate and D-serine in aqueous humor of STZ-treated rats may result from activation of the JNK pathway in diabetic sequelae. Our data suggest that the inflammatory conditions that prevail during DR result in elevation of D-serine, a neurotransmitter contributing to glutamate toxicity, potentially exacerbating the death of retinal ganglion cells in this condition.