RESUMEN
Our previous studies have demonstrated that diabetes-induced oxidative stress alters homeostasis of retinal nerve growth factor (NGF) resulting in accumulation of its precursor, proNGF, at the expense of NGF which plays a critical role in preserving neuronal and retinal function. This imbalance coincided with retinal damage in experimental diabetes. Here we test the hypothesis that alteration of proNGF and NGF levels observed in retina and vitreous will be mirrored in serum of diabetic patients. Blood and vitreous samples were collected from patients (diabetic and nondiabetic) undergoing vitrectomy at Georgia Regents University under approved IRB. Levels of proNGF, NGF, and p75(NTR) shedding were detected using Western blot analysis. MMP-7 activity was also assayed. Diabetes-induced proNGF expression and impaired NGF expression were observed in vitreous and serum. Vitreous and sera from diabetic patients (n = 11) showed significant 40.8-fold and 3.6-fold increases, respectively, compared to nondiabetics (n = 9). In contrast, vitreous and sera from diabetic patients showed significant 44% and 64% reductions in NGF levels, respectively, compared to nondiabetics. ProNGF to NGF ratios showed significant correlation between vitreous and serum. Further characterization of diabetes-induced imbalance in the proNGF to NGF ratio will facilitate its utility as an early biomarker for diabetic complications.
Asunto(s)
Biomarcadores/sangre , Biomarcadores/metabolismo , Retinopatía Diabética/sangre , Retinopatía Diabética/metabolismo , Factor de Crecimiento Nervioso/sangre , Factor de Crecimiento Nervioso/metabolismo , Adolescente , Adulto , Anciano , Animales , Estudios de Casos y Controles , Complicaciones de la Diabetes/sangre , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus Experimental/sangre , Femenino , Georgia , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Precursores de Proteínas/sangre , Precursores de Proteínas/metabolismo , Retina/metabolismo , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Retinal neurodegeneration is an early and critical event in several diseases associated with blindness. Clinically, therapies that target neurodegeneration fail. We aimed to elucidate the multiple roles by which thioredoxin-interacting protein (TXNIP) contributes to initial and sustained retinal neurodegeneration. EXPERIMENTAL APPROACH: Neurotoxicity was induced by intravitreal injection of NMDA into wild-type (WT) and TXNIP-knockout (TKO) mice. The expression of apoptotic and inflammatory markers was assessed by immunohistochemistry, elisa and Western blot. Microvascular degeneration was assessed by periodic acid-Schiff and haematoxylin staining and retinal function by electroretinogram. KEY RESULTS: NMDA induced early (1 day) and significant retinal PARP activation, a threefold increase in TUNEL-positive nuclei and 40% neuronal loss in ganglion cell layer (GCL); and vascular permeability in WT but not TKO mice. NMDA induced glial activation, expression of TNF-α and IL-1ß that co-localized with Müller cells in WT but not TKO mice. In parallel, NMDA triggered the expression of NOD-like receptor protein (NLRP3), activation of caspase-1, and release of IL-1ß and TNF-α in primary WT but not TKO Müller cultures. After 14 days, NMDA induced 1.9-fold microvascular degeneration, 60% neuronal loss in GCL and increased TUNEL-labelled cells in the GCL and inner nuclear layer in WT but not TKO mice. Electroretinogram analysis showed more significant reductions in b-wave amplitudes in WT than in TKO mice. CONCLUSION AND IMPLICATIONS: Targeting TXNIP expression prevented early retinal ganglion cell death, glial activation, retinal inflammation and secondary neuro/microvascular degeneration and preserved retinal function. TXNIP is a promising new therapeutic target for retinal neurodegenerative diseases.
Asunto(s)
Proteínas Portadoras/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Retina/metabolismo , Tiorredoxinas/metabolismo , Adulto , Anciano , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/genética , Células Cultivadas , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Femenino , Glaucoma de Ángulo Abierto/metabolismo , Humanos , Inflamación/prevención & control , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , N-Metilaspartato , Síndromes de Neurotoxicidad/prevención & control , Retina/efectos de los fármacos , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/genética , Factor de Necrosis Tumoral alfa/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Lesiones del Sistema Vascular/prevención & controlRESUMEN
AIMS/HYPOTHESIS: Recent studies have demonstrated that cannabinoid-1 (CB(1)) receptor blockade ameliorated inflammation, endothelial and/or cardiac dysfunction, and cell death in models of nephropathy, atherosclerosis and cardiomyopathy. However the role of CB(1) receptor signalling in diabetic retinopathy remains unexplored. Using genetic deletion or pharmacological inhibition of the CB(1) receptor with SR141716 (rimonabant) in a rodent model of diabetic retinopathy or in human primary retinal endothelial cells (HREC) exposed to high glucose, we explored the role of CB(1) receptors in the pathogenesis of diabetic retinopathy. METHODS: Diabetes was induced using streptozotocin in C57BL/6J Cb(1) (also known as Cnr1)(+/+) and Cb(1)(-/-) mice aged 8 to 12 weeks. Samples from mice retina or HREC were used to determine: (1) apoptosis; (2) activity of nuclear factor kappa B, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), poly (ADP-ribose) polymerase and caspase-3; (3) content of 3-nitrotyrosine and reactive oxygen species; and (4) activation of p38/Jun N-terminal kinase/mitogen-activated protein kinase (MAPK). RESULTS: Deletion of CB(1) receptor or treatment of diabetic mice with CB(1) receptor antagonist SR141716 prevented retinal cell death. Treatment of diabetic mice or HREC cells exposed to high glucose with SR141716 attenuated the oxidative and nitrative stress, and reduced levels of nuclear factor κB, ICAM-1 and VCAM-1. In addition, SR141716 attenuated the diabetes- or high glucose-induced pro-apoptotic activation of MAPK and retinal vascular cell death. CONCLUSIONS/INTERPRETATION: Activation of CB(1) receptors may play an important role in the pathogenesis of diabetic retinopathy by facilitating MAPK activation, oxidative stress and inflammatory signalling. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of this devastating complication of diabetes.
Asunto(s)
Apoptosis/fisiología , Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/fisiopatología , Endotelio Vascular/fisiopatología , Receptor Cannabinoide CB1/fisiología , Retina/fisiopatología , Vasculitis Retiniana/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Retinopatía Diabética/etiología , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Glucosa/farmacología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptor Cannabinoide CB1/deficiencia , Receptor Cannabinoide CB1/genética , Retina/metabolismo , Retina/patología , Vasculitis Retiniana/metabolismo , Transducción de Señal/fisiología , Estreptozocina/efectos adversosRESUMEN
AIMS/HYPOTHESIS: Accumulation of pro-nerve growth factor (NGF), the pro form of NGF, has been detected in neurodegenerative diseases. However, the role of proNGF in the diabetic retina and the molecular mechanisms by which proNGF causes retinal neurodegeneration remain unknown. The aim of this study was to elucidate the role of proNGF in neuroglial activation and to examine the neuroprotective effects of epicatechin, a selective inhibitor of tyrosine nitration, in an experimental rat model of diabetes. METHODS: Expression of proNGF and its receptors was examined in retinas from streptozotocin-induced diabetic rats, and in retinal Müller and retinal ganglion cells (RGCs). RGC death was assessed by TUNEL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in diabetic retinas and cell culture. Nitrotyrosine was determined using Slot-blot. Activation of the tyrosine kinase A (TrkA) receptor and p38 mitogen-activated protein kinase (p38MAPK) was assessed by western blot. RESULTS: Diabetes-induced peroxynitrite impaired phosphorylation of TrkA-Y490 via tyrosine nitration, activated glial cells and increased expression of proNGF and its receptor, p75 neurotrophin receptor (p75(NTR)), in vivo and in Müller cells. These effects were associated with activation of p38MAPK, cleaved poly-(ADP-ribose) polymerase and RGC death. Treatment of diabetic animals with epicatechin (100 mg kg(-1) day(-1), orally) blocked these effects and restored neuronal survival. Co-cultures of RGCs with conditioned medium of activated Müller cells significantly reduced RGC viability (44%). Silencing expression of p75(NTR) by use of small interfering RNA protected against high glucose- and proNGF-induced apoptosis in RGC cultures. CONCLUSIONS/INTERPRETATION: Diabetes-induced peroxynitrite stimulates p75(NTR) and proNGF expression in Müller cells. It also impairs TrkA receptor phosphorylation and activates the p75(NTR) apoptotic pathway in RGCs, leading to neuronal cell death. These effects were blocked by epicatechin, a safe dietary supplement, suggesting its potential therapeutic use in diabetic patients.
Asunto(s)
Catequina/farmacología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/prevención & control , Factor de Crecimiento Nervioso/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Retina/metabolismo , Animales , Glucemia/efectos de los fármacos , Western Blotting , Peso Corporal/efectos de los fármacos , Línea Celular , Etiquetado Corte-Fin in Situ , Masculino , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
AIMS/HYPOTHESIS: Diabetic retinopathy, the leading cause of blindness in working-age Americans, is characterised by reduced neurotrophic support and increased proinflammatory cytokines, resulting in neurotoxicity and vascular permeability. We sought to elucidate how oxidative stress impairs homeostasis of nerve growth factor (NGF) and its precursor, proform of NGF (proNGF), to cause neurovascular dysfunction in the eye of diabetic patients. METHODS: Levels of NGF and proNGF were examined in samples from human patients, from retinal Müller glial cell line culture cells and from streptozotocin-induced diabetic animals treated with and without atorvastatin (10 mg/kg daily, per os) or 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride (FeTPPs) (15 mg/kg daily, i.p.) for 4 weeks. Neuronal death and vascular permeability were assessed by TUNEL and extravasation of BSA-fluorescein. RESULTS: Diabetes-induced peroxynitrite formation impaired production and activity of matrix metalloproteinase-7 (MMP-7), which cleaves proNGF extracellularly, leading to accumulation of proNGF and reducing NGF in samples from diabetic retinopathy patients and experimental models. Treatment of diabetic animals with atorvastatin exerted similar protective effects that blocked peroxynitrite using FeTPPs, restoring activity of MMP-7 and hence the balance between proNGF and NGF. These effects were associated with preservation of blood-retinal barrier integrity, preventing neuronal cell death and blocking activation of RhoA and p38 mitogen-activated protein kinase (p38MAPK) in experimental and human samples. CONCLUSIONS/INTERPRETATION: Oxidative stress plays an unrecognised role in causing accumulation of proNGF, which can activate a common pathway, RhoA/p38MAPK, to mediate neurovascular injury. Oral statin therapy shows promise for treatment of diabetic retinopathy.
Asunto(s)
Diabetes Mellitus/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Ácido Peroxinitroso/metabolismo , Células Ganglionares de la Retina/metabolismo , Anciano , Animales , Western Blotting , Línea Celular , Diabetes Mellitus/fisiopatología , Femenino , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Metaloproteinasa 7 de la Matriz/metabolismo , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Células Ganglionares de la Retina/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
PURPOSE: We have previously shown that non-psychotropic cannabidiol (CBD) protects retinal neurons in diabetic rats by inhibiting reactive oxygen species and blocking tyrosine nitration. Tyrosine nitration may inhibit glutamine synthetase (GS), causing glutamate accumulation and leading to further neuronal cell death. We propose to test the hypothesis that diabetes-induced glutamate accumulation in the retina is associated with tyrosine nitration of GS and that CBD treatment inhibits this process. METHODS: Sprague Dawley rats were made diabetic by streptozotocin injection and received either vehicle or CBD (10 mg/kg/2 days). After eight weeks, retinal cell death, Müller cell activation, GS tyrosine nitration, and GS activity were determined. RESULTS: Diabetes causes significant increases in retinal oxidative and nitrative stress compared with controls. These effects were associated with Müller cell activation and dysfunction as well as with impaired GS activity and tyrosine nitration of GS. Cannabidiol treatment reversed these effects. Retinal neuronal death was indicated by numerous terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-labeled cells in diabetic rats compared with untreated controls or CBD-treated rats. CONCLUSIONS: These results suggest that diabetes-induced tyrosine nitration impairs GS activity and that CBD preserves GS activity and retinal neurons by blocking tyrosine nitration.
Asunto(s)
Cannabidiol/farmacología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/patología , Glutamato-Amoníaco Ligasa/metabolismo , Fármacos Neuroprotectores/farmacología , Neuronas Retinianas/enzimología , Neuronas Retinianas/patología , Animales , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/enzimología , Neuroglía/patología , Nitrosación/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: Degenerative retinal diseases are characterized by inflammation and microglial activation. The nonpsychoactive cannabinoid, cannabidiol (CBD), is an anti-inflammatory in models of diabetes and glaucoma. However, the cellular and molecular mechanisms are largely unknown. We tested the hypothesis that retinal inflammation and microglia activation are initiated and sustained by oxidative stress and p38 mitogen-activated protein kinase (MAPK) activation, and that CBD reduces inflammation by blocking these processes. METHODS: Microglial cells were isolated from retinas of newborn rats. Tumor necrosis factor (TNF)-alpha levels were estimated with ELISA. Nitric oxide (NO) was determined with a NO analyzer. Superoxide anion levels were determined by the chemiluminescence of luminol derivative. Reactive oxygen species (ROS) was estimated by measuring the cellular oxidation products of 2', 7'-dichlorofluorescin diacetate. RESULTS: In retinal microglial cells, treatment with lipopolysaccharide (LPS) induced immediate NADPH oxidase-generated ROS. This was followed by p38 MAPK activation and resulted in a time-dependent increase in TNF-alpha production. At a later phase, LPS induced NO, ROS, and p38 MAPK activation that peaked at 2-6 h and was accompanied by morphological change of microglia. Treatment with 1 microM CBD inhibited ROS formation and p38 MAPK activation, NO and TNF-alpha formation, and maintained cell morphology. In addition, LPS-treated rat retinas showed an accumulation of macrophages and activated microglia, significant levels of ROS and nitrotyrosine, activation of p38 MAPK, and neuronal apoptosis. These effects were blocked by treatment with 5 mg/kg CBD. CONCLUSIONS: Retinal inflammation and degeneration in uveitis are caused by oxidative stress. CBD exerts anti-inflammatory and neuroprotective effects by a mechanism that involves blocking oxidative stress and activation of p38 MAPK and microglia.
Asunto(s)
Cannabidiol/farmacología , Endotoxinas/farmacología , Fármacos Neuroprotectores/farmacología , Uveítis/inducido químicamente , Uveítis/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Lipopolisacáridos/farmacología , Macrófagos/enzimología , Macrófagos/patología , Masculino , Microglía/efectos de los fármacos , Microglía/enzimología , Microglía/patología , Modelos Biológicos , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso/metabolismo , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/enzimología , Retina/patología , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The modulation of angiogenic signaling by reactive oxygen species (ROS) is an emerging area of interest in cellular and vascular biology research. We provide evidence here that peroxynitrite, the powerful oxidizing and nitrating free radical, is critically involved in transduction of the VEGF signal. We tested the hypothesis that VEGF induces peroxynitrite formation, which causes tyrosine phosphorylation and mediates endothelial cell migration and tube formation, by studies of vascular endothelial cells in vitro and in a model of hypoxia-induced neovascularization in vivo. The specific peroxynitrite decomposition catalyst FeTPPs blocked VEGF-induced phosphorylation of VEGFR2 and c-Src and inhibited endothelial cell migration and tube formation. Furthermore, exogenous peroxynitrite mimicked VEGF activity in causing phosphorylation of VEGFR2 and stimulating endothelial cell growth and tube formation in vitro and new blood vessel growth in vivo. The selective nitration inhibitor epicatechin enhanced VEGF's angiogenic function in activating VEGFR2, c-Src, and promoting endothelial cell growth, migration, and tube formation in vitro and retinal neovascularization in vivo. Decomposing peroxynitrite with FeTPPs or blocking oxidation using the thiol donor NAC blocked VEGF's angiogenic functions in vitro and in vivo. In conclusion, peroxynitrite is critically involved in transducing VEGF's angiogenic signal via nitration-independent and oxidation-mediated tyrosine phosphorylation.
Asunto(s)
Endotelio Vascular/fisiología , Neovascularización Fisiológica/fisiología , Ácido Peroxinitroso/farmacología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Bovinos , Endotelio Vascular/efectos de los fármacos , Humanos , Microcirculación/efectos de los fármacos , Microcirculación/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Vasos Retinianos/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Superóxidos/metabolismo , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
A current hypothesis states that tolerance to nitroglycerin (GTN) involves increased formation of superoxide (O2*-). Studies showing that inhibitors of protein kinase C (PKC) prevent tolerance to GTN suggest the involvement of PKC activation, which can also increase O2*-. We examined the roles of O2*-, peroxynitrite (ONOO-), and PKC activation in GTN tolerance. Pre-exposure of rat aortic rings to GTN (5 x 10(-4) M) for 2 h caused tolerance to the vasodilating effect of GTN, as evidenced by a substantial rightward shift of GTN concentration-relaxation curves. This shift was reduced by treatment of the rings with the antioxidants uric acid, vitamin C, or tempol or the PKC inhibitor chelerythrine. We also found that O2*- generation via xanthine/xanthine oxidase in the bath induced tolerance to GTN. However, responses to nitroprusside were not affected. In vivo tolerance produced in rats by 3-day i.v. infusion of GTN was also almost completely prevented by coinfusion of tempol. In bovine aortic endothelial cells (EC), addition of GTN produced a marked increase in tyrosine nitrosylation, indicating increased ONOO- formation. This action was blocked by prior treatment with uric acid, superoxide dismutase, NG-nitro-L-arginine methyl ester, or chelerythrine. We also demonstrated that GTN translocates the alpha- and epsilonPKC isoforms in EC. However, PKCzeta was not affected by GTN treatment. In conclusion, tolerance to GTN involves enhanced production of O2*- and ONOO- and activation of NO synthase. Furthermore, sustained activation of alpha- and epsilonPKC isozymes in EC by GTN may play a role in development of tolerance.