OBJECTIVE-Recently we have shown that diabetes-induced retinal neurodegeneration positively correlates with oxidative stress and peroxynitrite. Studies also show that peroxynitrite impairs nerve growth factor (NGF) survival signaling in sensory neurons. However, the causal role of peroxynitrite and the impact of tyrosine nitration on diabetes-induced retinal neurodegeneration and NGF survival signaling have not been elucidated. RESEARCH DESIGN AND METHODS-Expression of NGFand its receptors was examined in retinas from human and streptozotocin-induced diabetic rats and retinal ganglion cells (RGCs). Diabetic animals were treated with FeTPPS (15 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ip), which catalytically decomposes peroxynitrite to nitrate. After 4 weeks of diabetes, retinal cell death was determined by TUNEL assay. Lipid peroxidation and nitrotyrosine were determined using MDA assay, immunofluorescence, and Slot-Blot analysis. Expression of NGF and its receptors was determined by enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoprecipitation, and Western blot analyses.RESULTS-Analyses of retinal neuronal death and NGF showed ninefold and twofold increases, respectively, in diabetic retinas compared with controls. Diabetes also induced increases in lipid peroxidation, nitrotyrosine, and the pro-apoptotic p75 NTR receptor in human and rat retinas. These effects were associated with tyrosine nitration of the pro-survival TrkA receptor, resulting in diminished phosphorylation of TrkA and its downstream target, Akt. Furthermore, peroxynitrite induced neuronal death, TrkA nitration, and activation of p38 mitogen-activated protein kinase (MAPK) in RGCs, even in the presence of exogenous NGF. FeTPPS prevented tyrosine nitration, restored NGF survival signal, and prevented neuronal death in vitro and in vivo. CONCLUSIONS-Together

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.

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 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.Electronic supplementary material The online version of this article