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BACKGROUND/AIM: AlphaB-crystallin plays a pivotal role in many diseases. However, the involvement of alphaB-crystallin in retinal pigment epithelial (RPE) cells with diabetes stimuli remains unknown. The aim of this study is to examine the alterations of RPE cells and alphaB-crystallin expression in diabetic models in vivo and in vitro. MATERIALS AND METHODS: Diabetic conditions in mice were induced by streptozotocin (STZ). The thickness of the RPE/choroid complex was measured by optical coherence tomography (OCT). Periodic acid-Schiff (PAS) staining was used to investigate the choriocapillaris in histological sections of murine eyeballs and oxidative stress was evaluated using immunofluorescence with anti-4-hydroxynonenal (HNE) antibody. AlphaB-crystallin expression was examined in the RPE/choroid complex using ELISA. Real-Time PCR was performed to evaluate the alphaB-crystallin expression in cultured human RPE cells with high glucose or following advanced glycation end-products (AGE) stimulation. RESULTS: In diabetic mice, OCT-based RPE/choroidal layers were thickened 2 months after STZ stimulation, where PAS-positive dilated choriocapillaris was noted. Immunoreactivity of 4-HNE was strongly observed in the RPE layer, from which a significant number of RPE cells was lost. Meanwhile, alphaB-crystallin expression in 2-month STZ mice was significantly lower compared to controls. In accordance with these results, in vitro data showed that the alphaB-crystallin expression was also significantly lower in RPE cells with high glucose or following AGE stimulation compared to untreated cells. CONCLUSION: In both types of diabetic models the expression of alphaB-crystallin was found to be downregulated in RPE cells and was associated with increased levels of oxidative stress.
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Diabetes Mellitus Experimental , Cadena B de alfa-Cristalina , Animales , Regulación hacia Abajo , Células Epiteliales/metabolismo , Ratones , Pigmentos Retinianos , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismoRESUMEN
To investigate the expression of vascular endothelial growth factor (VEGF)-C and vascular endothelial growth factor receptor (VEGFR)3 in the trabecular meshwork (TM) of patients with glaucoma and cultured TM cells. Methods: The expressions of VEGF-C in angle tissues collected by trabeculectomy from patients with glaucoma and non-glaucomatous choroidal malignant melanoma were analyzed by immunohistochemistry. Additionally, VEGF-C concentrations were determined in the aqueous humor of patients with glaucoma by ELISA. The expressions of VEGFR3, which is a receptor of VEGF-C in cultured TM cells, were analyzed by Western blot analysis and immunocytochemistry. Cultured TM cells were stimulated by oxidative stress, hypoxia, or high glucose conditions, and VEGF-C concentrations in supernatants and cell lysates were determined by ELISA. Results: VEGF-C immunoreactivity was positive in TM tissues of glaucoma patients, but not in those of non-glaucomatous controls. VEGF-C concentrations in the aqueous humor of patients with neovascular glaucoma and primary open-angle glaucoma were lower than those with non-glaucoma patients. VEGFR3 was expressed in cultured TM cells. VEGF-C concentrations in supernatants or cell lysates of TM cells cultured under oxidative stress and hypoxia were significantly elevated compared with those under steady conditions (p < 0.05). VEGF-C concentrations in supernatants and cell lysates of TM cells cultured in high glucose were significantly higher than those in low glucose (p < 0.01). Conclusions: VEGF-C was expressed in TM tissues of patients with glaucoma, which was secreted from cultured TM cells under various pathological conditions. These results suggest that VEGF-C may be involved in the pathology of glaucoma.
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PURPOSE: To investigate the immunotherapeutic effects of macrophage-like induced pluripotent stem (iPS) cell-derived suppressor cells (SCs) in ocular immune response and experimental autoimmune uveoretinitis (EAU). METHODS: The genes of Oct3/4, Sox2, Klf4, and c-Myc were transferred to B cells enriched from the spleen cells of C57BL/6 mice by using retrovirus vectors. Transferred B cells were cultured for 17 days to obtain colonies of iPS cells. Through additional steps, iPS-SCs were induced. An antigen-specific T cell proliferation assay was performed with CD4+ T cells collected from draining lymph nodes of the mice immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide and co-cultured with iPS-SCs. Cytokine concentrations in the culture supernatant were examined. Mice were immunized with hIRBP peptide to induce EAU. The iPS-SCs were administered into the mice one day before the induction of EAU. RESULTS: The iPS-SCs decreased hIRBP-specific T cell proliferation depending on the number of cells. Productions of tumor necrosis factor-α and interferon-γ were significantly decreased; however, transforming growth factor-ß1, nitric oxide, interleukin (IL)-13, IL-17A, and IL-17 F levels were elevated in the supernatant when the collected T cells were co-cultured with iPS-SCs. The iPS-SCs had immunosuppressant effects even without cell-to-cell contact, and their effects were non-specific to the antigen preloaded on iPS-SCs. EAU was significantly milder in the mice administered iPS-SCs prior to immunization. CONCLUSIONS: Macrophage-like iPS-SCs reduced Th1 immune response to a retinal antigen and Th1-mediated EAU in mice. These results showed the possibility of the application of iPS technology to the treatment of noninfectious ocular inflammation, endogenous uveitis, in the future.
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Antígenos/inmunología , Enfermedades Autoinmunes/inmunología , Proteínas del Ojo/metabolismo , Células Madre Pluripotentes Inducidas/inmunología , Retinitis/inmunología , Proteínas de Unión al Retinol/metabolismo , Células TH1/inmunología , Uveítis/inmunología , Animales , Enfermedades Autoinmunes/patología , Células Cultivadas , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas/citología , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Retinitis/patología , Células TH1/patología , Uveítis/patologíaRESUMEN
PURPOSE: To evaluate the clinical characteristics of patients with acute zonal occult outer retinopathy (AZOOR), according to the presence or absence of anti-retinal antibodies (ARAs) that are frequently detected in autoimmune retinopathy. METHODS: Retrospective observational case series. This study included 33 patients with acute-stage AZOOR who had been followed up for more than 6 months after the initial visit. The median follow-up period was 26 months. Immunoblot analyses were used to detect autoantibodies for recoverin, carbonic anhydrase II, and α-enolase in serum from these patients. Main outcome measures comprised clinical factors at the initial and final visits, including best-corrected visual acuity, mean deviation on Humphrey perimetry, and retinal morphology, which were statistically compared between patients with AZOOR who exhibited ARAs and those who did not. RESULTS: At least one serum ARA was detected in 42% of patients with AZOOR. There were no significant differences in clinical factors between the two groups, including follow-up period, best-corrected visual acuity and mean deviation at the initial and final visits, a-wave amplitude on single-flash electroretinography at the initial visit, and frequencies of improvement of the macular ellipsoid zone and AZOOR recurrence. CONCLUSIONS: Our findings suggest that the presence of ARAs did not influence visual outcomes or outer retinal morphology in patients with AZOOR.
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Enfermedades Autoinmunes , Enfermedades de la Retina , Humanos , Enfermedades de la Retina/diagnóstico , Estudios Retrospectivos , Escotoma , Agudeza Visual , Síndromes de Puntos BlancosRESUMEN
Purpose: Galectin-1/LGALS1, a ß-galactoside-binding protein, contributes to angiogenesis and fibrosis in various ocular diseases. Hypoxia-dependent and -independent pathways upregulate galectin-1/LGALS1 expression in Müller glial cells. Here, we present novel findings on the galectin-1/LGALS1 regulatory system in human retinal pigment epithelium (RPE) cells, the major cellular participant in the pathogenesis of neovascular age-related macular degeneration (nAMD). Methods: Human RPE cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and immunoblot analyses, respectively. The promoter and enhancer regions of LGALS1 were analyzed by reporter assay and chromatin immunoprecipitation. Immunofluorescence analysis of nAMD patient specimens was used to confirm the in vitro findings. Results: Hypoxia induced galectin-1/LGALS1 expression via binding of hypoxia-inducible factor 1α (HIF-1α) to hypoxia-responsive elements in the LGALS1 promoter region. Blockade of vascular endothelial growth factor receptor 1 (VEGFR1) partially decreased hypoxia-induced galectin-1/LGALS1 expression. Among several VEGFR1 ligands induced by hypoxia, placental growth factor (PlGF)/PGF alone upregulated galectin-1/LGALS1 expression via phosphorylation of activator protein 1 (AP-1) subunits following AKT and p38 mitogen-activated protein kinase (MAPK) activation. An AP-1 site in the LGALS1 enhancer region was required for PlGF-induced galectin-1/LGALS1 expression in RPE cells. PlGF application upregulated PGF expression via extracellular signal-regulated kinase 1 and 2, AKT, and p38 MAPK pathways. nAMD patient specimens demonstrated co-localization of galectin-1 with HIF-1α, PlGF, and VEGFR1 in RPE cells. Conclusions: Our present findings implicate the significance of hypoxia as a key inducer of galectin-1/LGALS1 in RPE cells and the autoinduction of hypoxia-induced PlGF as a vicious cycle amplifying the pathogenesis of nAMD.
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Galectina 1/genética , Regulación de la Expresión Génica , Degeneración Macular/genética , Factor de Crecimiento Placentario/metabolismo , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Células Ependimogliales/metabolismo , Galectina 1/biosíntesis , Humanos , Immunoblotting , Degeneración Macular/metabolismo , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Activación TranscripcionalRESUMEN
PURPOSE: To establish with negative electroretinogram (ERG) the clinical entity of eight patients with unilateral severe photophobia, essentially normal fundus, good visual acuity, and severe cone and rod dysfunction. STUDY DESIGN: Multicenter retrospective observation case series. METHODS: Comprehensive ophthalmologic examinations were performed, including best-corrected visual acuity (BCVA), full-field ERGs and multifocal ERGs (mfERGs), fundus photographs, and OCT. Systemic and genetic examinations were performed. RESULTS: The mean (± SD) age at the onset was 60.0 ± 8.4 years, and the six patients noticed severe photophobia in the affected eye in spite of almost normal fundus appearance and good BCVA. The dark-adapted bright flash ERGs in the affected eye had relatively well-preserved a-waves and depressed b-waves, i.e., a negative ERG. Cone ERGs and both b- and d-waves of the photopic long-duration ERGs were almost undetectable. Rod ERGs were severely reduced; however, only two patients complained of night blindness. In five patients, the mfERGs were extinguished in the periphery but preserved in the central retina, resulting in good BCVA. Electrophysiological findings indicated a severe diffuse dysfunction of the inner retina affecting bipolar cells of both ON- and OFF-pathways, and in five patients there was a reduction in the thickness of the inner nuclear layer. In seven patients the retinal arteries were attenuated. Anti-retinal antibodies were detected in the serum of two patients. No genetic causes were found. CONCLUSIONS: The common features in the eight patients with unilateral negative ERGs suggest a new disease entity of unilateral acute inner retinal layer dysfunction. In most patients, the only subjective complain was photophobia.
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Electrorretinografía , Fotofobia , Humanos , Fotofobia/diagnóstico , Fotofobia/etiología , Retina , Células Fotorreceptoras Retinianas Conos , Estudios Retrospectivos , Agudeza VisualRESUMEN
Purpose: Müller glial-mesenchymal transition (GMT) is reported as the fibrogenic mechanism promoted by TGF-ß-SNAIL axis in Müller cells transdifferentiated into myofibroblasts. Here we show the multifaceted involvement of TGF-ß in diabetic fibrovascular proliferation via Müller GMT and VEGF-A production. Methods: Surgically excised fibrovascular tissues from the eyes of patients with proliferative diabetic retinopathy were processed for immunofluorescence analyses of TGF-ß downstream molecules. Human Müller glial cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and ELISA, respectively. Immunoblot analyses were performed to detect TGF-ß signal activation. Results: Müller glial cells in patient fibrovascular tissues were immunopositive for GMT-related molecular markers, including SNAIL and smooth muscle protein 22, together with colocalization of VEGF-A and TGF-ß receptors. In vitro administration of TGF-ß1/2 upregulated TGFB1 and TGFB2, both of which were suppressed by inhibitors for nuclear factor-κB, glycogen synthase kinase-3, and p38 mitogen-activated protein kinase. Of the various profibrotic cytokines, TGF-ß1/2 application exclusively induced Müller glial VEGFA mRNA expression, which was decreased by pretreatment with small interfering RNA for SMAD2 and inhibitors for p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Supporting these findings, TGF-ß1/2 stimulation to Müller cells increased the phosphorylation of these intracellular signaling molecules, all of which were also activated in Müller glial cells in patient fibrovascular tissues. Conclusions: This study underscored the significance of Müller glial autoinduction of TGF-ß as a pathogenic cue to facilitate diabetic fibrovascular proliferation via TGF-ß-driven GMT and VEGF-A-driven angiogenesis.
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Transdiferenciación Celular , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células Cultivadas , Citocinas/metabolismo , Retinopatía Diabética/fisiopatología , Ensayo de Inmunoadsorción Enzimática , Células Ependimogliales/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Purpose: Disruption of proteostasis is a key event in many neurodegenerative diseases. Heat shock proteins (HSPs) participate in multiple functions associated with intracellular transport and proteostasis. We evaluated the effect of augmented HSP70 expression in mutant photoreceptors of mouse retinal degeneration models to test the hypothesis that failure to sustain HSP70 expression contributes to photoreceptor cell death. Methods: We examined HSP70 expression in retinas of wild-type and mutant mice by RNA and protein analysis. A transgenic mouse line, TgCrx-Hspa1a-Flag, was generated to express FLAG-tagged full-length HSP70 protein under control of a 2.3 kb mouse Crx promoter. This line was crossed to three distinct retinal degeneration mouse models. Retinal structure and function were evaluated by histology, immunohistochemistry, and electroretinography. Results: In seven different mouse models of retinal degeneration, we detected transient elevation of endogenous HSP70 expression at early stages, followed by a dramatic reduction as cell death ensues, suggesting an initial adaptive response to cellular stress. Augmented expression of HSP70 in RHOT17M mice, in which mutant rhodopsin is misfolded, marginally improved photoreceptor survival, whereas elevated HSP70 led to more severe retinal degeneration in rd10 mutants that produce a partially functional PDE6B. In Rpgrip1-/- mice that display a ciliary defect, higher HSP70 had no impact on photoreceptor survival or function. Conclusions: HSP70 overexpression has divergent effects in photoreceptors determined, at least in part, by the nature of the mutant protein each model carries. Additional investigations on HSP pathways and associated chaperone networks in photoreceptors are needed before designing therapeutic strategies targeting proteostasis.
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Regulación de la Expresión Génica/fisiología , Proteínas HSP70 de Choque Térmico/genética , Degeneración Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Electrorretinografía , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Retina/fisiopatología , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiologíaRESUMEN
Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies. Lowering intraocular pressure is currently the only way to treat glaucoma, but it is often insufficient to inhibit the progression of the disease. Glaucoma is a multifactorial disease, and the involvement of oxidative stress has recently received much attention. In the present study, we investigated the cytoprotective effect of astaxanthin (AST) on RGC degeneration using a normal-tension glaucoma (NTG) mouse model, which lacks the glutamate/aspartate transporter (Glast) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure. Three-week-old Glast± mice were given intraperitoneal injections of AST at 10, 30, or 60 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. Five weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. We also used a retrograde labeling technique to quantify the number of RGCs. Additionally, the phosphorylated (p) IκB/total IκB ratio and the 4-hydroxynonenal (HNE) were measured in retinal tissues. The number of RGCs in Glast± mice was significantly decreased compared with that in control mice. RGC loss was suppressed by the administration of AST at 60 mg/kg/day, compared with vehicle alone. Following AST administration, the concentration of 4-HNE in the retina was also suppressed, but the pIκB/IκB ratio did not change. Our study revealed that the antioxidative stress effects of AST inhibit RGC degeneration in the retina and may be useful in the treatment of NTG.
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Neovascular age related macular degeneration (nAMD) leads to severe vision loss worldwide and is characterized by the formation of choroidal neovascularization (CNV) and fibrosis. In the current study, we aimed to investigate the effect of blockade for platelet derived growth factor receptor-ß (PDGFR-ß) on the formation of choroidal neovascularization and fibrosis in the laser-induced CNV model in mice. Firstly, the presence of PDGFR-ß in CNV lesions were confirmed. Intravitreal injection of PDGFR-ß neutralizing antibody significantly reduced the size of CNV and subretinal fibrosis. Additionally, subretinal hyperreflective material (SHRM), a landmark feature on OCT as a risk factor for subretinal fibrosis formation in nAMD patients was also suppressed by PDGFR-ß blockade. Furthermore, pericytes were abundantly recruited to the CNV lesions during CNV formation, however, blockade of PDGFR-ß significantly reduced pericyte recruitment. In addition, PDGF-BB stimulation increased the migration of the rat retinal pericyte cell line, R-rPCT1, which was abrogated by the neutralization of PDGFR-ß. These results indicate that blockade of PDGFR-ß attenuates laser-induced CNV and fibrosis through the inhibition of pericyte migration.
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The receptor-associated prorenin system (RAPS) is associated with several pathologic conditions, including diabetic retinopathy, age-related macular degeneration, and uveitis. Here, we show the involvement of RAPS in the trabecular meshwork (TM) from patients with primary open-angle glaucoma (POAG) and neovascular glaucoma (NVG) due to proliferative diabetic retinopathy. Anterior chamber (AC) levels of prorenin significantly increased in both POAG and NVG, as did those of angiotensin II in NVG alone, compared to cataract. In surgically excised TM tissues, (pro)renin receptor ((P)RR) and angiotensin II type 1 receptor (AT1R) co-localized with prorenin and angiotensinogen, respectively. In screening for various genes related to glaucoma, prorenin stimulation to human TM cells exclusively upregulated cell junction constituents connexin 43 and zona occludens 1, while downregulating an extracellular matrix-degrading enzyme tissue plasminogen activator, all of which were reversed by (P)RR blockade. In contrast, angiotensin II application upregulated a pro-angiogenic factor placental growth factor alone, which was abolished by AT1R blockade. Consistently, (P)RR and AT1R co-localized with these corresponding proteins in patient TM tissues. Oxidative stress, a known etiology for glaucoma, induced the expression of prorenin and angiotensinogen in human TM cells. These data suggest the contribution of RAPS to the molecular pathogenesis of POAG and NVG through TM tissue remodeling and AC angle angiogenesis.
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Purpose: Acrolein, a highly reactive unsaturated aldehyde, is known to facilitate glial cell migration, one of the pathological hallmarks in diabetic retinopathy. However, cellular mechanisms of acrolein generation in retinal glial cells remains elusive. In the present study, we investigated the role and regulation of spermine oxidase (SMOX), one of the enzymes related to acrolein generation, in retinal glial cells under hypoxic condition. Methods: Immunofluorescence staining for SMOX was performed using sections of fibrovascular tissues obtained from patients with proliferative diabetic retinopathy. Expression levels of polyamine oxidation enzymes including SMOX were analyzed in rat retinal Müller cell line 5 (TR-MUL5) cells under either normoxic or hypoxic conditions. The transcriptional activity of Smox in TR-MUL5 cells was evaluated using the luciferase assay. Levels of acrolein-conjugated protein, Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), and hydrogen peroxide were measured. Results: SMOX was localized in glial cells in fibrovascular tissues. Hypoxia induced SMOX production in TR-MUL5 cells, which was suppressed by silencing of hypoxia-inducible factor-1α (Hif1a), but not Hif2a. Transcriptional activity of Smox was regulated through HIF-1 binding to hypoxia response elements 2, 3, and 4 sites in the promoter region of Smox. Generation of FDP-Lys and hydrogen peroxide increased in TR-MUL5 cells under hypoxic condition, which was abrogated by SMOX inhibitor MDL72527. Conclusions: The current data demonstrated that hypoxia regulates production of SMOX, which plays a role in the generation of oxidative stress inducers, through HIF-1α signaling in Müller glial cells under hypoxic condition.
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Hipoxia de la Célula/genética , Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Células Cultivadas , Retinopatía Diabética/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Oxidación-Reducción , ARN/genética , Ratas , Ratas Transgénicas , Células Ganglionares de la Retina/patología , Transducción de Señal , Poliamino OxidasaRESUMEN
BACKGROUND: Evidence-based criteria for the treatment of autoimmune retinopathy (AIR) have not been established. The pathology and clinical features of each antibody causing AIR, and its long-term course are still undetermined. We report our findings in a case of non-paraneoplastic AIR (npAIR) that developed in the fellow eye 10 years after the onset in the first eye. CASE PRESENTATION: Our patient had photophobia in both eyes and a rapidly progressing visual field defect in his right eye at the initial examination. He was diagnosed with non-paraneoplastic AIR based on the clinical findings and immunoblot analyses for anti-retinal antibodies, and he was treated with steroids. Ten years later, a visual field defect developed in the fellow eye, and a diagnosis of npAIR was made. Immunoblot analyses were positive for anti-α-enolase antibodies. He was treated with steroids, immunosuppressants, and plasma exchange. However, the response to the treatment was poor and both eyes eventually became blind. CONCLUSIONS: As best we know, this is the first case report of npAIR that developed in the fellow eye over 10 years after the development in the first eye. Long-term follow-up and a search for tumor lesions are necessary in cases of npAIR. Further understanding of the long-term course of AIR can contribute to an understanding of the pathology and treatment of npAIR.
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Enfermedades Autoinmunes/etiología , Síndromes Paraneoplásicos Oculares/etiología , Enfermedades de la Retina/etiología , Autoanticuerpos/sangre , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/terapia , Ceguera/etiología , Electrorretinografía , Angiografía con Fluoresceína , Glucocorticoides/uso terapéutico , Humanos , Immunoblotting , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Síndromes Paraneoplásicos Oculares/diagnóstico , Síndromes Paraneoplásicos Oculares/terapia , Fosfopiruvato Hidratasa/inmunología , Intercambio Plasmático , Enfermedades de la Retina/diagnóstico , Enfermedades de la Retina/terapia , Factores de Tiempo , Tomografía de Coherencia Óptica , Agudeza Visual , Pruebas del Campo VisualRESUMEN
Galectin-1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin-1ß-driven inflammatory pathway for galectin-1 expression in vitro and in vivo. Here, we show glucocorticoid-mediated inhibitory mechanism against hypoxia-inducible factor (HIF)-1α-involved galectin-1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia-induced increases in galectin-1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia-stimulated cells decreased HIF-1α protein, but not mRNA, together with its DNA-binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co-immunoprecipitation revealed that glucocorticoid-transactivated TSC22D3 interacted with HIF-1α, leading to degradation of hypoxia-stabilized HIF-1α via the ubiquitin-proteasome pathway. Silencing TSC22D3 reversed glucocorticoid-mediated ubiquitination of HIF-1α and subsequent down-regulation of HIF-1α and galectin-1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes-induced retinal HIF-1α and galectin-1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co-localization of galectin-1 and HIF-1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced retinal glial galectin-1/LGALS1 expression via HIF-1α destabilization, highlighting therapeutic implications for DR in the era of anti-vascular endothelial growth factor treatment.
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Retinopatía Diabética/tratamiento farmacológico , Galectina 1/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factores de Transcripción/genética , Animales , Hipoxia de la Célula/efectos de los fármacos , Dexametasona/farmacología , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Silenciador del Gen , Humanos , Ratones , Retina/patología , Factores de Transcripción/antagonistas & inhibidores , Triamcinolona Acetonida/farmacología , Ubiquitina/genéticaRESUMEN
Uveitis is a sight-threatening intraocular inflammatory disease that accounts for almost 10% of blindness worldwide. NF-κB signaling plays pivotal roles in inflammatory diseases. We have reported that IMD-0354, which inhibits NF-κB signaling via selective blockade of IKK-ß, suppresses inflammation in several ocular disease models. Here, we examined the therapeutic effect of IMD-0354 in an experimental autoimmune uveoretinitis (EAU) model, a well-established animal model for endogenous uveitis in humans. Systemic administration of IMD-0354 significantly suppressed the clinical and histological severity, inflammatory edema, and the translocation of NF-κB p65 into the nucleus of retinas in EAU mice. Furthermore, IMD-0354 treatment significantly inhibited the levels of several Th1/Th17-mediated pro-inflammatory cytokines in vitro. Our current data demonstrate that inhibition of IKKß with IMD-0354 ameliorates inflammatory responses in the mouse EAU model, suggesting that IMD-0354 may be a promising therapeutic agent for human endogenous uveitis.
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Enfermedades Autoinmunes/tratamiento farmacológico , Benzamidas/uso terapéutico , Quinasa I-kappa B/antagonistas & inhibidores , Retinitis/tratamiento farmacológico , Uveítis/tratamiento farmacológico , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Benzamidas/administración & dosificación , Benzamidas/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citocinas/biosíntesis , Edema/complicaciones , Edema/patología , Quinasa I-kappa B/metabolismo , Inflamación/complicaciones , Inflamación/patología , Masculino , Ratones , FN-kappa B/metabolismo , Retinitis/inmunología , Retinitis/patología , Índice de Severidad de la Enfermedad , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Uveítis/inmunología , Uveítis/patologíaRESUMEN
Purpose: To investigate the effect of the unsaturated aldehyde acrolein on retinal glial cell migration. Methods: Müller glial cell markers expression in TR-MUL5 were confirmed by RT-PCR and immunostaining. Cell viability and migration rate of TR-MUL5 cells were assessed after the stimulation with acrolein. DNA microarray analysis was performed to analyze changes in the expression levels of migration-related genes in Müller glial cells stimulated with acrolein. Real-time PCR and ELISA were performed to validate DNA microarray analysis results. Inhibitors of C-X-C motif chemokine ligand 1 (CXCL1), one of the genes highly upregulated after the exposure to acrolein, and blockers of its receptor, CXCR2, were used to investigate the role of the CXCL1-CXCR2 axis on glial cell migration. CXCL1 concentration was measured in vitreous fluid samples obtained from proliferative diabetic retinopathy (PDR) and nondiabetic control eyes. CXCL1 and CXCR2 expression in glial cells of fibrovascular tissues obtained from PDR patients was examined by immunostaining. Results: At a high concentration, acrolein (100 µM) significantly decreased cell viability. However, in moderate, sublethal concentrations (25-50 µM), acrolein induced cell migration and substantially increased the production of CXCL1 in TR-MUL5 cells. CXCL1 concentration was significantly elevated in vitreous fluids of PDR patients, and CXCL1 and CXCR2 were present in glial cells in fibrovascular tissues of PDR patients. CXCL1 stimulation increased glial cell migration in a dose-dependent manner, which was abrogated by the neutralization of the CXCL1-CXCR2 axis. Conclusions: Our data demonstrate that acrolein promotes retinal Müller glial cell migration by enhancing CXCL1 production.
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Acroleína/farmacología , Movimiento Celular/efectos de los fármacos , Neuroglía/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neuroglía/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas Transgénicas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Cuerpo Vítreo/metabolismoRESUMEN
Galectin-1/LGALS1 is a hypoxia-induced angiogenic factor associated with diabetic retinopathy (DR). Recently, we elucidated a hypoxia-independent pathway to produce galectin-1 in Müller glial cells stimulated by interleukin (IL)-1ß. Here we revealed glucocorticoid receptor (GR)-mediated inhibitory mechanisms for Müller glial galectin-1/LGALS1 expression. Activator protein (AP)-1 site in the LGALS1 enhancer region, to which activating transcription factor2, c-Fos and c-Jun bind, was shown to be essential for IL-1ß-induced galectin-1/LGALS1 expression in Müller cells. Ligand (dexamethasone or triamcinolone acetonide)-activated GR induced dual specificity phosphatase (DUSP)1 expression via the glucocorticoid response element and attenuated IL-1ß-induced galectin-1/LGALS1 expression by reducing phosphorylation of these AP-1 subunits following AKT and extracellular signal-regulated kinase (ERK)1/2 deactivation. Moreover, activated GR also caused DUSP1-independent down-regulation of IL-1ß-induced LGALS1 expression via its binding to AP-1. Administration of glucocorticoids to mice attenuated diabetes-induced retinal galectin-1/Lgals1 expression together with AKT/AP-1 and ERK/AP-1 pathways. Supporting these in vitro and in vivo findings, immunofluorescence analyses showed co-localization of galectin-1 with GR and phosphorylated AP-1 in DUSP1-positive glial cells in fibrovascular tissues from patients with DR. Our present data demonstrated the inhibitory effects of glucocorticoids on glial galectin-1 expression via DUSP1-dependent and -independent deactivation of AP-1 signalling (transactivation and transrepression), highlighting therapeutic implications for DR.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/metabolismo , Células Ependimogliales/metabolismo , Galectina 1/metabolismo , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Animales , Línea Celular , Diabetes Mellitus/genética , Regulación hacia Abajo/efectos de los fármacos , Humanos , Interleucina-1beta/farmacología , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Retina/metabolismoRESUMEN
The receptor-associated prorenin system refers to the pathogenic mechanism whereby prorenin binding to (pro)renin receptor [(P)RR] dually activates the tissue renin-angiotensin system (RAS) and RAS-independent signaling, and its activation contributes to the molecular pathogenesis of various ocular diseases. We recently developed a new single-stranded RNAi agent targeting both human and mouse (P)RR ((P)RR-proline-modified short hairpin RNA [(P)RR-PshRNA]), and confirmed its therapeutic effect on murine models of ocular inflammation. Here, we investigated the efficacy of (P)RR-PshRNA against laser-induced choroidal neovascularization (CNV) and subretinal fibrosis, both of which are involved in the pathogenesis of age-related macular degeneration (AMD). Administration of (P)RR-PshRNA in mice significantly reduced CNV formation, together with the expression of inflammatory molecules, macrophage infiltration, and extracellular signal-regulated kinase (ERK) 1/2 activation. In addition, (P)RR-PshRNA attenuated subretinal fibrosis, together with epithelial-mesenchymal transition (EMT)-related markers including phosphorylated SMAD2. The suppressive effect of (P)RR-PshRNA is comparable with aflibercept, an anti-vascular endothelial growth factor drug widely used for AMD therapy. AMD patient specimens demonstrated (P)RR co-localization with phosphorylated ERK1/2 in neovascular endothelial cells and retinal pigment epithelial cells. These results indicate that (P)RR contributes to the ocular pathogenesis of both inflammation-related angiogenesis and EMT-driven fibrosis, and that (P)RR-PshRNA is a promising therapeutic agent for AMD.
RESUMEN
PURPOSE: To compare the clinical characteristics of Vogt-Koyanagi-Harada (VKH) disease patients with and without anti-retinal antibodies (ARAs) that are frequently detected in autoimmune retinopathy. METHODS: Using immunoblot analyses, serum autoantibodies for recoverin, carbonic anhydrase II, and α-enolase were examined in 20 treatment-naïve patients with VKH disease. Clinical factors before and after systemic corticosteroid therapy, including best-corrected visual acuity (BCVA) and macular outer retinal morphology, were statistically compared between patients with VKH disease with and without ARAs. RESULTS: Serum ARAs were detected in 50.0% of patients with VKH disease. There were no significant differences in clinical factors between the two groups, including final BCVA, frequency of uveitis recurrence, and recovery of the macular ellipsoid zone after systemic corticosteroid therapy. CONCLUSIONS: Our results suggest that the detected ARAs did not influence visual outcomes, the chronicity of uveitis, or outer retinal morphology in patients with VKH disease.