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1.
J Neurosci ; 35(15): 6093-106, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25878282

RESUMO

We have identified and characterized a spontaneous Brown Norway from Janvier rat strain (BN-J) presenting a progressive retinal degeneration associated with early retinal telangiectasia, neuronal alterations, and loss of retinal Müller glial cells resembling human macular telangiectasia type 2 (MacTel 2), which is a retinal disease of unknown cause. Genetic analyses showed that the BN-J phenotype results from an autosomal recessive indel novel mutation in the Crb1 gene, causing dislocalization of the protein from the retinal Müller glia (RMG)/photoreceptor cell junction. The transcriptomic analyses of primary RMG cultures allowed identification of the dysregulated pathways in BN-J rats compared with wild-type BN rats. Among those pathways, TGF-ß and Kit Receptor Signaling, MAPK Cascade, Growth Factors and Inflammatory Pathways, G-Protein Signaling Pathways, Regulation of Actin Cytoskeleton, and Cardiovascular Signaling were found. Potential molecular targets linking RMG/photoreceptor interaction with the development of retinal telangiectasia are identified. This model can help us to better understand the physiopathologic mechanisms of MacTel 2 and other retinal diseases associated with telangiectasia.


Assuntos
Células Ependimogliais/patologia , Proteínas do Olho/genética , Mutação/genética , Degeneração Retiniana , Telangiectasia/complicações , Telangiectasia/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Modelos Animais de Doenças , Eletrorretinografia , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , Proteínas do Olho/metabolismo , Angiofluoresceinografia , Proteína Glial Fibrilar Ácida/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos , Ratos Mutantes , Degeneração Retiniana/etiologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Vasos Retinianos/patologia , Vasos Retinianos/ultraestrutura , Transdução de Sinais/fisiologia , Vias Visuais/patologia , Vias Visuais/ultraestrutura
2.
Mol Vis ; 22: 1332-1341, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27881907

RESUMO

PURPOSE: Intravitreal recombinant tissue plasminogen activator (rtPA) is used off-label for the surgical management of submacular hemorrhage, a severe complication of neovascular age-related macular degeneration. rtPA is approved for coronary and cerebral thrombolysis. However, in ischemic stroke rtPA is known to increase excitotoxic neural cell death by interacting with the N-methyl-D-aspartate (NMDA) receptor. We therefore investigated the retinal toxicity of rtPA in healthy rats and in a model of NMDA-induced retinal excitotoxicity. METHODS: First, rtPA at three different doses (2.16 µg/5 µl, 0.54 µg/5 µl, and 0.27 µg/5 µl) or vehicle (NaCl 0.9%) was injected intravitreally in healthy rat eyes. Electroretinograms (ERGs) were performed at 24 h or 7 days. Annexin V-fluorescein isothiocyanate (FITC)-labeled apoptotic retinal ganglion cells (RGCs) were counted on flatmounted retinas at 24 h or 7 days. Next, NMDA + vehicle or NMDA + rtPA (0.27 µg/5 µl) was injected intravitreally to generate excitotoxic conditions. Apoptotic annexin V-FITC-labeled RGCs and surviving Brn3a-labeled RGCs were quantified on flatmounted retinas and radial sections, 18 h after treatment. RESULTS: In healthy rat eyes, the number of apoptotic RGCs was statistically significantly increased 24 h after the administration of rtPA at the highest dose (2.16 µg/5 µl; p = 0.0250) but not at the lower doses of 0.54 and 0.27 µg/5 µl (p = 0.36 and p = 0.20), compared to vehicle. At day 7, there was no difference in the apoptotic RGC count between the rtPA- and vehicle-injected eyes (p = 0.70, p = 0.52, p = 0.11). ERG amplitudes and implicit times were not modified at 24 h or 7 days after injection of any tested rtPA doses, compared to the baseline. Intravitreal administration of NMDA induced RGC death, but under these excitotoxic conditions, coadministration of rtPA did not increase the number of dead RGCs (p = 0.70). Similarly, the number of surviving RGCs on the flatmounted retinas and retinal sections did not differ between the eyes injected with NMDA + vehicle and NMDA + rtPA (p = 0.59 and p = 0.67). CONCLUSIONS: At low clinical equivalent doses corresponding to 25 µg/0.1 ml in humans, intravitreal rtPA is not toxic for healthy rat retinas and does not enhance NMDA-induced excitotoxicity. Vitreal equivalent doses ≥200 µg/0.1 ml should be avoided in patients, due to potential RGC toxicity.


Assuntos
Neurotoxinas/toxicidade , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tecidual/efeitos adversos , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Apoptose/efeitos dos fármacos , Eletrorretinografia , Injeções Intravítreas , Masculino , Ratos Long-Evans , Proteínas Recombinantes/administração & dosagem , Retina , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Ativador de Plasminogênio Tecidual/administração & dosagem
3.
J Cell Mol Med ; 19(7): 1646-55, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25781645

RESUMO

Light-induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase-independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI-derived DNase II (LEI/L-DNase II), a caspase-independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro-survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L-DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase-independent pathways.


Assuntos
Endodesoxirribonucleases/metabolismo , Luz , Proteína Quinase C/metabolismo , Degeneração Retiniana/enzimologia , Sequência de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Células HeLa , Humanos , Masculino , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Inibidores de Proteínas Quinases/farmacologia , Ratos , Retina/efeitos dos fármacos , Retina/enzimologia , Retina/patologia , Degeneração Retiniana/patologia , Serpinas/metabolismo
4.
Transl Res ; 272: 81-94, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38815899

RESUMO

Glyburide, a sulfonylurea drug used to treat type 2 diabetes, boasts neuroprotective effects by targeting the sulfonylurea receptor 1 (SUR1) and associated ion channels in various cell types, including those in the central nervous system and the retina. Previously, we demonstrated that glyburide therapy improved retinal function and structure in a rat model of diabetic retinopathy. In the present study, we explore the application of glyburide in non-neovascular ("dry") age-related macular degeneration (AMD), another progressive disease characterized by oxidative stress-induced damage and neuroinflammation that trigger cell death in the retina. We show that glyburide administration to a human cone cell line confers protection against oxidative stress, inflammasome activation, and apoptosis. To corroborate our in vitro results, we also conducted a case-control study, controlling for AMD risk factors and other diabetes medications. It showed that glyburide use in patients reduces the odds of new-onset dry AMD. A positive dose-response relationship is observed from this analysis, in which higher cumulative doses of glyburide further reduce the odds of new-onset dry AMD. In the quest for novel therapies for AMD, glyburide emerges as a promising repurposable drug given its known safety profile. The results from this study provide insights into the multifaceted actions of glyburide and its potential as a neuroprotective agent for retinal diseases; however, further preclinical and clinical studies are needed to validate its therapeutic potential in the context of degenerative retinal disorders such as AMD.

5.
Am J Pathol ; 179(2): 942-53, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21712024

RESUMO

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.


Assuntos
Retinopatia Diabética/metabolismo , Células Epiteliais/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Proteína Quinase C/metabolismo , Retina/metabolismo , Animais , Glicemia/metabolismo , Movimento Celular , Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Linfócitos/citologia , Microscopia Confocal/métodos , Ratos , Ratos Wistar
6.
Pharmaceutics ; 13(7)2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-34371786

RESUMO

Diabetic retinopathy (DR) remains a major cause of vision loss, due to macular edema, retinal ischemia and death of retinal neurons. We previously demonstrated that acute administration of glibenclamide into the vitreous, or given orally at a non-hypoglycemic dose, protected the structure and the function of the retina in three animal models that each mimic aspects of diabetic retinopathy in humans. In this pilot study, we investigated whether one year of chronic oral glibenclamide, in a non-hypoglycemic regimen (Amglidia®, 0.4 mg/kg, Ammtek/Nordic Pharma, 5 d/week), could alleviate the retinopathy that develops in the Goto-Kakizaki (GK) rat. In vivo, retinal function was assessed by electroretinography (ERG), retinal thickness by optical coherence tomography (OCT) and retinal perfusion by fluorescein and indocyanin green angiographies. The integrity of the retinal pigment epithelium (RPE) that constitutes the outer retinal barrier was evaluated by quantitative analysis of the RPE morphology on flat-mounted fundus ex vivo. Oral glibenclamide did not significantly reduce the Hb1Ac levels but still improved retinal function, as witnessed by the reduction in scotopic implicit times, limited diabetes-induced neuroretinal thickening and the extension of ischemic areas, and it improved the capillary coverage. These results indicate that low doses of oral glibenclamide could still be beneficial for the prevention of type 2 diabetic retinopathy. Whether the retinas ofpatients treated specifically with glibenclamideare less at risk of developing diabetic complications remains to be demonstrated.

7.
Transl Res ; 229: 83-99, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33080394

RESUMO

Sulfonylureas, widely used as hypoglycemic agents in adults with type 2 diabetes, have neuroprotective effects in preclinical models of central nervous system injury, and in children with neuropsychomotor impairments linked to neonatal diabetes secondary to ATP-sensitive potassium channel mutations. In the human and rodent retina, we show that the glibenclamide-activated channel sulfonylurea receptor 1 (SUR1) is expressed in the retina and enriched in the macula; we also show that it colocalizes with the potassium channel Kir6.2, and with the cation channel transporter TRPM4. Glibenclamide (glyburide), administered at doses that did not decrease the glycemia, or injected directly into the eye, protected the structure and the function of the retina in various models of retinal injury that recapitulate the pathogenic neurodegenerative events in the diabetic retina. The downregulation of SUR1 using a siRNA suppressed the neuroprotective effects of glibenclamide on excitotoxic stress-induced cell death. The glibenclamide effects include the transcriptional regulation of antioxidant and neuroprotective genes. Ocular glibenclamide could be repurposed for diabetic retinopathy.


Assuntos
Glibureto/farmacologia , Fármacos Neuroprotetores/farmacologia , Doenças Retinianas/tratamento farmacológico , Neurônios Retinianos/efeitos dos fármacos , Administração Oral , Animais , Chlorocebus aethiops , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Feminino , Glibureto/administração & dosagem , Humanos , Hiperglicemia/metabolismo , Hipoglicemiantes/farmacologia , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/administração & dosagem , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos Endogâmicos Lew , Ratos Wistar , Doenças Retinianas/etiologia , Doenças Retinianas/patologia , Neurônios Retinianos/patologia , Receptores de Sulfonilureias/metabolismo , Canais de Cátion TRPM/metabolismo
8.
Prog Retin Eye Res ; 63: 20-68, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29126927

RESUMO

Macular edema consists of intra- or subretinal fluid accumulation in the macular region. It occurs during the course of numerous retinal disorders and can cause severe impairment of central vision. Major causes of macular edema include diabetes, branch and central retinal vein occlusion, choroidal neovascularization, posterior uveitis, postoperative inflammation and central serous chorioretinopathy. The healthy retina is maintained in a relatively dehydrated, transparent state compatible with optimal light transmission by multiple active and passive systems. Fluid accumulation results from an imbalance between processes governing fluid entry and exit, and is driven by Starling equation when inner or outer blood-retinal barriers are disrupted. The multiple and intricate mechanisms involved in retinal hydro-ionic homeostasis, their molecular and cellular basis, and how their deregulation lead to retinal edema, are addressed in this review. Analyzing the distribution of junction proteins and water channels in the human macula, several hypotheses are raised to explain why edema forms specifically in the macular region. "Pure" clinical phenotypes of macular edema, that result presumably from a single causative mechanism, are detailed. Finally, diabetic macular edema is investigated, as a complex multifactorial pathogenic example. This comprehensive review on the current understanding of macular edema and its mechanisms opens perspectives to identify new preventive and therapeutic strategies for this sight-threatening condition.


Assuntos
Edema Macular/fisiopatologia , Barreira Hematorretiniana , Coriorretinopatia Serosa Central/complicações , Coriorretinopatia Serosa Central/fisiopatologia , Neovascularização de Coroide/complicações , Neovascularização de Coroide/fisiopatologia , Retinopatia Diabética/complicações , Retinopatia Diabética/fisiopatologia , Angiofluoresceinografia , Humanos , Edema Macular/diagnóstico , Edema Macular/prevenção & controle , Oclusão da Veia Retiniana/complicações , Oclusão da Veia Retiniana/fisiopatologia , Vasos Retinianos/fisiopatologia , Líquido Sub-Retiniano , Tomografia de Coerência Óptica
9.
J Neuroimmunol ; 183(1-2): 104-10, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17207538

RESUMO

Tumor necrosis factor-alpha (TNF) has been implicated in retinal ganglion cells (RGC) degeneration in glaucoma. Atypical protein kinase C (PKC) zeta is involved in cell protection against various stresses. The aim of this study was to investigate the potential proapoptotic effects of intravitreal injections of TNF with or without PKCzeta specific inhibitor on the rat retina. TNF was injected in the vitreous of rat eyes alone or in combination with specific PKCzeta inhibitor. PKCzeta and NF-kappaB were studied by immunohistochemistry and western-blotting analysis on retina, and apoptosis quantified by the TUNEL assay. While low basal PKCzeta was observed in the control eyes, TNF induced intense expression of PKCzeta mostly in bipolar cells processes. PKCzeta staining became nuclear when TNF was coinjected with PKCzeta inhibitor. TNF alone did not induce apoptosis in the retina. Coinjection of the PKCzeta-specific inhibitor and TNF, however, induced apoptosis in the inner nuclear and ganglion cell layers. The PKCzeta-specific inhibitor unmasks retinal cells to TNF cytotoxicity showing a link between the proapoptotic effects of TNF and the antiapoptotic PKCzeta signaling pathway.


Assuntos
Inibidores Enzimáticos/administração & dosagem , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Degeneração Retiniana/metabolismo , Fator de Necrose Tumoral alfa/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Marcação In Situ das Extremidades Cortadas , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/patologia , Fatores de Tempo , Quinase Induzida por NF-kappaB
10.
Sci Rep ; 7(1): 8834, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28821742

RESUMO

In diabetic retinopathy, the exact mechanisms leading to retinal capillary closure and to retinal barriers breakdown remain imperfectly understood. Rho-associated kinase (ROCK), an effector of the small GTPase Rho, involved in cytoskeleton dynamic regulation and cell polarity is activated by hyperglycemia. In one year-old Goto Kakizaki (GK) type 2 diabetic rats retina, ROCK-1 activation was assessed by its cellular distribution and by phosphorylation of its substrates, MYPT1 and MLC. In both GK rat and in human type 2 diabetic retinas, ROCK-1 is activated and associated with non-apoptotic membrane blebbing in retinal vessels and in retinal pigment epithelium (RPE) that respectively form the inner and the outer barriers. Activation of ROCK-1 induces focal vascular constrictions, endoluminal blebbing and subsequent retinal hypoxia. In RPE cells, actin cytoskeleton remodeling and membrane blebs in RPE cells contributes to outer barrier breakdown. Intraocular injection of fasudil, significantly reduces both retinal hypoxia and RPE barrier breakdown. Diabetes-induced cell blebbing may contribute to ischemic maculopathy and represent an intervention target.


Assuntos
Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Idoso , Animais , Biomarcadores , Estudos de Casos e Controles , Citoesqueleto/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Feminino , Imunofluorescência , Humanos , Hipóxia/metabolismo , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Ratos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Vasos Retinianos/ultraestrutura , Quinases Associadas a rho/genética
11.
Am J Ophthalmol ; 159(6): 1142-1151.e3, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25728860

RESUMO

PURPOSE: To characterize perifoveal intraretinal cavities observed around full-thickness macular holes (MH) using en face optical coherence tomography and to establish correlations with histology of human and primate maculae. DESIGN: Retrospective nonconsecutive observational case series. METHODS: Macular en face scans of 8 patients with MH were analyzed to quantify the areas of hyporeflective spaces, and were compared with macular flat mounts and sections from 1 normal human donor eye and 2 normal primate eyes (Macaca fascicularis). Immunohistochemistry was used to study the distribution of glutamine synthetase, expressed by Müller cells, and zonula occludens-1, a tight-junction protein. RESULTS: The mean area of hyporeflective spaces was lower in the inner nuclear layer (INL) than in the complex formed by the outer plexiform (OPL) and the Henle fiber layers (HFL): 5.0 × 10(-3) mm(2) vs 15.9 × 10(-3) mm(2), respectively (P < .0001, Kruskal-Wallis test). In the OPL and HFL, cavities were elongated with a stellate pattern, whereas in the INL they were rounded and formed vertical cylinders. Immunohistochemistry confirmed that Müller cells followed a radial distribution around the fovea in the frontal plane and a "Z-shaped" course in the axial plane, running obliquely in the OPL and HFL and vertically in the inner layers. In addition, zonula occludens-1 co-localized with Müller cells within the complex of OPL and HFL, indicating junctions in between Müller cells and cone axons. CONCLUSION: The dual profile of cavities around MHs correlates with Müller cell morphology and is consistent with the hypothesis of intra- or extracellular fluid accumulation along these cells.


Assuntos
Cistos/diagnóstico , Células Ependimogliais/patologia , Fóvea Central/patologia , Doenças Retinianas/diagnóstico , Perfurações Retinianas/diagnóstico , Tomografia de Coerência Óptica , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Células Ependimogliais/metabolismo , Feminino , Glutamato-Amônia Ligase/metabolismo , Humanos , Imuno-Histoquímica , Macaca fascicularis , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Estudos Retrospectivos , Líquido Sub-Retiniano , Doadores de Tecidos , Proteína da Zônula de Oclusão-1/metabolismo
12.
PLoS One ; 8(11): e81600, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312324

RESUMO

AIMS/HYPOTHESIS: Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia. METHODS: Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor. RESULTS: PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death. CONCLUSIONS: In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.


Assuntos
Barreira Hematorretiniana/metabolismo , Retinopatia Diabética/metabolismo , Proteína Quinase C/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/patologia , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Retinopatia Diabética/enzimologia , Retinopatia Diabética/patologia , Hiperglicemia/enzimologia , Hiperglicemia/metabolismo , Hiperglicemia/patologia , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo
13.
Neurochem Int ; 57(3): 278-87, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20558223

RESUMO

Neuroprotection strategies in the retina aim at interference with regulatory mechanisms of cell death. To successfully target these mechanisms it is necessary to understand the molecular pathways activated in the degenerating retina. Induced retinal degeneration models, like the light damage model, give a synchronized response allowing their detailed investigation. In this study we exposed Fisher rats to a continuous white light. This induced a caspase-independent cell death in which the activation of cathepsin D has an important role via the activation of L-DNase II. Inhibition of this enzyme by intravitreal administration of pepstatin A protects photoreceptors indicating that this enzyme might be an interesting target for neuroprotection.


Assuntos
Caspases/fisiologia , Catepsina D/fisiologia , Luz/efeitos adversos , Degeneração Retiniana/metabolismo , Animais , Western Blotting , Contagem de Células , Morte Celular/efeitos da radiação , Desoxirribonucleases/metabolismo , Eletrorretinografia , Ativação Enzimática/efeitos da radiação , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Pepstatinas/farmacologia , Peptídeo Hidrolases/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos Endogâmicos F344 , Retina/enzimologia , Retina/patologia , Retina/efeitos da radiação , Degeneração Retiniana/patologia , Transdução de Sinais/efeitos da radiação
14.
Invest Ophthalmol Vis Sci ; 51(9): 4683-93, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20393119

RESUMO

PURPOSE: To evaluate the effect of XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide that selectively inhibits the c-Jun N-terminal kinase, in the treatment of endotoxin-induced uveitis (EIU). METHODS: EIU was induced in Lewis rats by LPS injection. XG-102 was administered at the time of LPS challenge. The ocular biodistribution of XG-102 was evaluated using immunodetection at 24 hours after either 20 microg/kg IV (IV) or 0.2 microg/injection intravitreous (IVT) administrations in healthy or uveitic eyes. The effect of XG-102 on EIU was evaluated using clinical scoring, infiltration cell quantification, inducible nitric oxide synthase (iNOS) expression and immunohistochemistry, and cytokines and chemokines kinetics at 6, 24, and 48 hours using multiplex analysis on ocular media. Control EIU eyes received vehicle injection IV or IVT. The effect of XG-102 on c-Jun phosphorylation in EIU was evaluated by Western blot in eye tissues. RESULTS: After IVT injection, XG-102 was internalized in epithelial cells from iris/ciliary body and retina and in glial and microglial cells in both healthy and uveitic eyes. After IV injection, XG-102 was concentrated primarily in inflammatory cells of uveitic eyes. Using both routes of administration, XG-102 significantly inhibited clinical signs of EIU, intraocular cell infiltration, and iNOS expression together with reduced phosphorylation of c-Jun. The anti-inflammatory effect of XG-102 was mediated by iNOS, IFN-gamma, IL-2, and IL-13. CONCLUSIONS: This is the first evidence that interfering with the JNK pathway can reduce intraocular inflammation. Local administration of XG-102, a clinically evaluated peptide, may have potential for treating uveitis.


Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Uveíte/tratamento farmacológico , Animais , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Combinação de Medicamentos , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/efeitos dos fármacos , Injeções Intraoculares , Injeções Intravenosas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/toxicidade , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óleos , Peptídeos/farmacocinética , Fenóis , Ratos , Ratos Endogâmicos Lew , Distribuição Tecidual , Uveíte/induzido quimicamente , Uveíte/patologia , Corpo Vítreo
15.
Apoptosis ; 12(2): 333-42, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17191118

RESUMO

Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-kappaB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCzeta, a key regulator of NF-kappaB activation, in response to aminoglycoside treatment. Amikacin induced PKCzeta cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-kappaB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCzeta cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCzeta acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity.


Assuntos
Amicacina/farmacologia , Apoptose/efeitos dos fármacos , Aspirina/farmacologia , Cóclea/citologia , Cóclea/enzimologia , Proteína Quinase C/metabolismo , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Fator de Indução de Apoptose/metabolismo , Audiometria de Resposta Evocada , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Masculino , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/efeitos dos fármacos , Gânglio Espiral da Cóclea/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Fator de Transcrição RelA/metabolismo
16.
Am J Pathol ; 170(4): 1241-57, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17392164

RESUMO

We show that inhibitory effect of interleukin-13 on endotoxin-induced uveitis in the Lewis rat is dependent on signaling activity of protein kinase Czeta (PKCzeta). To understand the effect of interleukin-13 or PKCzeta inhibitor treatment, the activation status of rat bone marrow-derived macrophages was studied in vitro. At 6 hours, lipopolysaccharide-stimulated macrophages produced tumor necrosis factor-alpha (TNF-alpha) with nuclear factor kappaB (NF-kappaB)/p65 expression. Treatment led to absence of NF-kappaB/p65 expression and low levels of TNF-alpha, suggesting accelerated inactivation of macrophages. At 24 hours after lipopolysaccharide stimulation, nuclear NF-kappaB/p65 decreased and nuclear NF-kappaB/p50 increased, associated with nuclear BCL-3 and a low level of TNF-alpha, indicating onset of spontaneous resolution. Treatment limited PKCzeta cleavage, with expression of nuclear NF-kappaB/p50 and BCL-3 and low nuclear NF-kappaB/p65 promoting macrophage survival, as evidenced by Bcl-2 expression. At 24 hours, intraocular treatment decreased membranous expression of PKCzeta by ocular cells, reduced vascular leakage with low nitric-oxide synthase-2 expression in vascular endothelial cells, and limited inflammatory cell infiltration with decreased intraocular TNF-alpha, interleukin-6, and nitric-oxide synthase-2 mRNA. Importantly, treatment decreased nuclear NF-kappaB/p65, increased transforming growth factor-beta2, and reduced caspase 3 expression in infiltrating macrophages, implying a change of their phenotype within ocular microenvironment. Treatment accelerated endotoxin-induced uveitis resolution through premature apoptosis of neutrophils related to high expression of toll-like receptor 4 and caspase 3.


Assuntos
Apoptose/fisiologia , Inflamação/fisiopatologia , Proteína Quinase C/metabolismo , Uveíte/fisiopatologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Citocinas/genética , Citocinas/metabolismo , Olho/efeitos dos fármacos , Olho/metabolismo , Olho/patologia , Proteínas do Olho/metabolismo , Imuno-Histoquímica , Inflamação/patologia , Inflamação/prevenção & controle , Interleucina-13/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Uveíte/induzido quimicamente , Uveíte/prevenção & controle
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