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 Diabetic retinopathy is characterised by early blood–retina barrier (BRB) breakdown and neurodegeneration. Diabetes causes imbalance of nerve growth factor (NGF), leading to accumulation of the NGF precursor (proNGF), as well as the NGF receptor, p75 neurotrophin receptor (p75NTR), suggesting a possible pathological role of the proNGF–p75NTR axis in the diabetic retina. To date, the role of this axis in diabetes-induced retinal inflammation and BRB breakdown has not been explored. We hypothesised that modulating p75NTR would prevent diabetes- and proNGF-induced retinal inflammation and BRB breakdown. Methods Diabetes was induced by streptozotocin in wild-type and p75NTR knockout (p75KO) mice. After 5 weeks, the expression of inflammatory mediators, ganglion cell loss and BRB breakdown were determined. Cleavage-resistant proNGF was overexpressed in rodent retinas with and without p75NTR short hairpin RNA or with pharmacological inhibitors. In vitro, the effects of proNGF were investigated in retinal Müller glial cell line (rMC-1) and primary Müller cells. Results Deletion of p75NTR blunted the diabetes-induced decrease in retinal NGF expression and increases in proNGF, nuclear factor κB (NFκB), p-NFκB and TNF-α. Deletion of p75NTR also abrogated diabetes-induced glial fibrillary acidic protein expression, ganglion cell loss and vascular permeability. Inhibited expression or cleavage of p75NTR blunted proNGF-induced retinal inflammation and vascular permeability. In vitro, proNGF induced p75NTR-dependent production of inflammatory mediators in primary wild-type Müller and rMC-1 cultures, but not in p75KO Müller cells. Conclusions/interpretation The proNGF–p75NTR axis contributes to retinal inflammation and vascular dysfunction in the rodent diabetic retina. These findings underscore the importance of p75NTR as a novel regulator of inflammation and potential therapeutic target in 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

Objective We have shown that acute treatment with candesartan in an experimental model of stroke resulted in vascular protection and improved outcomes at 24 hours post-stroke, but the mechanisms are unknown. We now examine effects of candesartan on proangiogenic factors and 7-day outcomes using the same treatment paradigm. Methods Male Wistar rats underwent 3 hours of middle cerebral artery occlusion (MCAO), followed by reperfusion. A single dose of candesartan 1mg/kg IV was given at reperfusion. Animals received neurobehavioral testing before MCAO, at 24 hours after MCAO, and at 7 days. BP was measured by telemetry. Animals sacrificed at 24 hours had tissue and spinal fluid (CSF) collected for matrix metalloproteinase (MMP) activity, vascular endothelial growth factor (VEGF) expression, and tube formation assay. Neurobehavioral testing included elevated-body swing test (EBST), Bederson, beam walk, and paw grasp. Cerebrovascular density was quantified using immunohistochemistry at 24 h and 7 days. Results MMP-2 activity and VEGF expression were higher (p=0.035, p=0.042, respectively) and CSF was significantly more proangiogenic (5X tube formation; p=0.002) in the candesartan group at 24 hours. Although no difference was seen in infarct size at 7 days, treatment improved Bederson scores (2.1 vs. 2.9, p=0.0083), EBST (22.9 vs. 39.4, p=0.021) and paw grasp (1.29 vs. 2.88, p=0.0001) at 7 days. Candesartan treatment resulted in increased vascular density in the striatum at 7 days (p=0.037). Conclusion Candesartan after reperfusion augments ischemia-induced angiogenic state and provides long-term benefits. The beneficial effects may involve vascular protection and enhancement of early angiogenic remodeling.

Diabetic retinopathy and retinopathy of prematurity are blinding disorders that follow a pathological pattern of ischemic retinopathy and affect premature infants and working-age adults. Yet, the treatment options are limited to laser photocoagulation. The goal of this study is to elucidate the molecular mechanism and examine the therapeutic effects of inhibiting tyrosine nitration on protecting early retinal vascular cell death and late neovascularization in the ischemic retinopathy model. Ischemic retinopathy was developed by exposing neonatal mice to 75% oxygen [postnatal day (p) 7-p12] followed by normoxia (21% oxygen) (p12-p17). Peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were administered (p7-p12) or (p7-p17). Vascular endothelial cells were incubated at hyperoxia (40% oxygen) or normoxia (21% oxygen) for 48 h. Vascular density was determined in retinal flat mounts labeled with isolectin B4. Expression of vascular endothelial growth factor, caspase-3, and poly(ADP ribose) polymerase (PARP), activation of Akt and p38 mitogen-activated protein kinase (MAPK), and tyrosine nitration of the phosphatidylinositol (PI) 3-kinase p85 subunit were analyzed by Western blot. Hyperoxia-induced peroxynitrite caused endothelial cell apoptosis as indicated by expression of cleaved caspase-3 and PARP leading to vasoobliteration. These effects were associated with significant tyrosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking tyrosine nitration of PI 3-kinase with epicatechin or NAC restored Akt phosphorylation, and inhibited vaso-obliteration at p12 and neovascularization at p17 comparable with FeTPPS. Early inhibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in ischemic retinopathy.Retinopathy of prematurity (ROP) and diabetic retinopathy (DR) are potentially blinding disorders that affect premature infants and working-age adults, respectively, in the United States (Aiello et al., 1998;Chen and Smith, 2007). ROP and DR follow a pathological progression pattern characteristic of ischemic retinopathy, where the loss of retinal capillary is an early initiating event, leading to a poorly controlled process of retinal neovascularization and the development of proliferative retinopathy (for review see, Caldwell et al., 2003). So far, the standard treatment for retinal neovascularization is limited to laser photocoagulation. Although successful, this treatment is invasive and results in loss of peripheral vision (for review see, Ali and El-Remessy, 2009). The lack of approved pharmacological treatment for DR and ROP creates a great need for finding new effective therapeutic modalities to treat these devastating diseases. The mechanisms that control the process of retinal neovascularization are therefor...

BACKGROUND AND PURPOSEUp-regulation of thioredoxin interacting protein (TXNIP), an endogenous inhibitor of thioredoxin (Trx), compromises cellular antioxidant and anti-apoptotic defences and stimulates pro-inflammatory cytokines expression, implying a role for TXNIP in apoptosis. Here we have examined the causal role of TXNIP expression in mediating retinal neurotoxicity and assessed the neuroprotective actions of verapamil, a calcium channel blocker and an inhibitor of TXNIP expression. EXPERIMENTAL APPROACHRetinal neurotoxicity was induced by intravitreal injection of NMDA in Sprague-Dawley rats, which received verapamil (10 mg·kg -1 , p.o.) or vehicle. Neurotoxicity was examined by terminal dUTP nick-end labelling assay and ganglion cell count. Expression of TXNIP, apoptosis signal-regulating kinase 1 (ASK-1), NF-kB, p38 MAPK, JNK, cleaved poly-ADP-ribose polymerase (PARP), caspase-3, nitrotyrosine and 4-hydroxy-nonenal were examined by Western and slot-blot analysis. Release of TNF-a and IL-1b was examined by ELISA. KEY RESULTSNMDA injection enhanced TXNIP expression, decreased Trx activity, causing increased oxidative stress, glial activation and release of TNF-a and IL-1b. Enhanced TXNIP expression disrupted Trx/ASK-1 inhibitory complex leading to release of ASK-1 and activation of the pro-apoptotic p38 MAPK/JNK pathway, as indicated by cleaved PARP and caspase-3 expression. Treatment with verapamil blocked these effects. CONCLUSION AND IMPLICATIONSElevated TXNIP expression contributed to retinal neurotoxicity by three different mechanisms, inducing release of inflammatory mediators such as TNF-a and IL-1b, altering antioxidant status and disrupting the Trx-ASK-1 inhibitory complex leading to activation of the p38 MAPK/JNK apoptotic pathway. Targeting TXNIP expression is a potential therapeutic target for retinal neurodegenerative disease. AbbreviationsASK-1, apoptosis signal-regulating kinase 1; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NFL, nerve fiber layer; NMLA, N-methyl-L-aspartate; ONL, inner plexiform layer into the outer nuclear layer; PARP, poly-ADP-ribose polymerase; RGC, retinal ganglion cell; ROD, relative optical density; Trx, thioredoxin; TUNEL, terminal dUTP nick-end labelling; TXNIP, thioredoxin interacting protein BJP British Journal of Pharmacology

Neovascularization is an innate physiologic response by which tissues respond to various stimuli through collateral remodeling (arteriogenesis) and new vessel formation from existing vessels (angiogenesis) or from endothelial progenitor cells (vasculogenesis). Diabetes has a major impact on the neovascularization process but the response varies between different organ systems. While excessive angiogenesis complicates diabetic retinopathy, impaired neovascularization contributes to coronary and peripheral complications of diabetes. How diabetes influences cerebral neovascularization remained unresolved until recently. Diabetes is also a major risk factor for stroke and poor recovery after stroke. In this review, we discuss the impact of diabetes, stroke, and diabetic stroke on cerebral neovascularization, explore potential mechanisms involved in diabetes-mediated neovascularization as well as the effects of the diabetic milieu on poststroke neovascularization and recovery, and finally discuss the clinical implications of these effects.

BACKGROUND AND PURPOSERetinal 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 APPROACHNeurotoxicity 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 RESULTSNMDA 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-1β 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-1β 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 IMPLICATIONSTargeting 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.