Suraporn Matragoon

Neuroprotective Effect of(−)Δ9-Tetrahydrocannabinol and Cannabidiol in N-Methyl-d-Aspartate-Induced Retinal Neurotoxicity : Involvement of Peroxynitrite

In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic Delta9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL,a superoxide dismutase-mimetic), N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a dose- and time-dependent accumulation of nitrite/nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma.

Peroxynitrite Mediates Retinal Neurodegeneration by Inhibiting Nerve Growth Factor Survival Signaling in Experimental and Human Diabetes

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 NGF and 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 · kg−1 · day−1 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 p75NTR 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, these data suggest that diabetes-induced peroxynitrite impairs NGF neuronal survival by nitrating TrkA receptor and enhancing p75NTR expression.

Role of NADPH Oxidase and Stat3 in Statin-Mediated Protection against Diabetic Retinopathy

PURPOSE Inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (statins) reduce signs of diabetic retinopathy in diabetic patients and animals. Indirect clinical evidence supports the actions of statins in improving cardiovascular function, but the mechanisms of their protective actions in the retina are not understood. Prior studies have implicated oxidative stress and NADPH oxidase-mediated activation of signal transducer and activator of transcription 3 (STAT3) in diabetes-induced increases in expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 and breakdown of the blood-retinal barrier (BRB). Because statins are known to be potent antioxidants, the hypothesis for the current study was that the protective effects of statins in preventing diabetic retinopathy involve blockade of diabetes-induced activation of NADPH oxidase and STAT3. METHODS The hypothesis was tested by experiments in which rats with streptozotocin (STZ)-induced diabetes and retinal endothelial cells maintained in high-glucose medium were treated with simvastatin. Blood-retinal barrier (BRB) function was assayed by determining extravasation of albumin. Oxidative stress was assayed by measuring lipid peroxidation, protein nitration of tyrosine, dihydroethidine oxidation, and chemiluminescence. Immunoprobe techniques were used to determine the levels of NADPH oxidase subunit expression and STAT3 activation. RESULTS These studies showed that simvastatin blocks diabetes or high-glucose-induced increases in VEGF and ICAM-1 and preserves the BRB by a process involving blockade of diabetes/high-glucose-induced activation of STAT3 and NADPH oxidase. Statin treatment also prevents diabetes-induced increases in expression of the NADPH oxidase catalytic and subunit NOX2. CONCLUSIONS These results suggest that simvastatin protects against the early signs of diabetic retinopathy by preventing NADPH oxidase-mediated activation of STAT3.

Retinal Microglial Activation and Inflammation Induced by Amadori-Glycated Albumin in a Rat Model of Diabetes

OBJECTIVE During diabetes, retinal microglial cells are activated to release inflammatory cytokines that initiate neuronal loss and blood–retinal barrier breakdown seen in diabetic retinopathy (DR). The mechanism by which diabetes activates microglia to release those inflammatory mediators is unclear and was therefore elucidated. RESEARCH DESIGN AND METHODS Microglia activation was characterized in streptozocin-injected rats and in isolated microglial cells using immunofluorescence, enzyme-linked immunosorbent assay, RT-PCR, and Western blot analyses. RESULTS In 8-week diabetic retina, phospho-extracellular signal–related kinase (ERK) and P38 mitogen-activated protein kinases were localized in microglia, but not in Mueller cells or astrocytes. At the same time, Amadori-glycated albumin (AGA)-like epitopes were featured in the regions of microglia distribution, implicating a pathogenic effect on microglial activation. To test this, diabetic rats were treated intravitreally with A717, a specific AGA-neutralizing antibody, or murine IgG. Relative to nondiabetic rats, diabetic rats (IgG-treated) manifested 3.9- and 7.9-fold increases in Iba-1 and tumor necrosis factor (TNF)-α mRNAs, respectively. Treatment of diabetic rats with A717 significantly attenuated overexpression of these mRNAs. Intravitreal injection of AGA per se in normal rats resulted in increases of Iba-1 expression and TNF-α release. Guided by these results, a cultured retinal microglia model was developed to study microglial response after AGA treatment and the mechanistic basis behind this response. The results showed that formation of reactive oxygen species and subsequent activation of ERK and P38, but not Jun NH2-terminal kinase, are molecular events underpinning retinal microglial TNF-α release during AGA treatment. CONCLUSIONS These results provide new insights in understanding the pathogenesis of early DR, showing that the accumulated AGA within the diabetic retina elicits the microglial activation and secretion of TNF-α. Thus, intervention trials with agents that neutralize AGA effects may emerge as a new therapeutic approach to modulate early pathologic pathways long before the occurrence of vision loss among patients with diabetes.

Simvastatin Improves Diabetes-Induced Coronary Endothelial Dysfunction

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors decrease cardiovascular morbidity in diabetic patients, but the mechanism is unclear. We studied the actions of simvastatin (SIM) in enhancing NO bioavailability and reducing oxidative stress in coronary vessels from diabetic rats and in rat coronary artery endothelial cells (RCAEC) exposed to high glucose. Coronary arteries isolated from diabetic rats showed decreases in acetylcholine (ACh)-mediated maximal relaxation from 81.0 ± 4.5% in controls to 43.5 ± 7.6% at 4 weeks and 22.3 ± 0.6% at 10 weeks of diabetes. This effect was associated with oxidative stress in coronary vessels as shown by dichlorofluorescein (DCF) imaging and nitrotyrosine labeling. Diabetes also reduced trans-coronary uptake of [3H]l-arginine. Supplemental l-arginine (50 mg/kg/day p.o.) did not improve coronary vasorelaxation to ACh. However, SIM treatment (5 mg/kg/day subcutaneously) improved maximal ACh relaxation to 65.8 ± 5.1% at 4 weeks and 47.1 ± 3.9% at 10 weeks. Coronary arteries from rats treated with both SIM and l-arginine demonstrated the same maximal relaxation to ACh (66.1 ± 3%) as SIM alone. Mevalonate and l-NAME (Nω-nitro-l-arginine methyl ester hydrochloride) inhibited the response to ACh in SIM-treated diabetic rats. Coronary arteries from all groups relaxed similarly to sodium nitroprusside. SIM increased endothelial NO synthase protein levels and blocked diabetes-induced increases in DCF and nitrotyrosine labeling in diabetic coronary vessels. SIM treatment restored normal NO levels in media from high-glucose-treated RCAEC and plasma of diabetic rat. Treatment with SIM or the NADPH oxidase inhibitor apocynin also blocked high-glucose-induced increases in reactive oxygen species and superoxide formation in RCAEC. Taken together, these data suggest that SIM improves diabetes-induced coronary dysfunction by reducing oxidative stress and increasing NO bioavailability.

Peroxynitrite mediates diabetes-induced endothelial dysfunction: possible role of Rho kinase activation.

Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.

Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity

BACKGROUND AND PURPOSE Up‐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.

Neuroprotective and Intraocular Pressure-Lowering Effects of (–)Δ9-Tetrahydrocannabinol in a Rat Model of Glaucoma

In glaucoma, retinal ganglion cell (RGC) death is induced by many risk factors, including ocular hypertension. It has been proposed that glutamate-mediated oxidative stress may also contribute to this RGC death. Cannabinoids are known to possess therapeutic properties including ocular hypotension and antioxidation. In this study, we test the hypothesis that (–)Δ9-tetrahydrocannabinol (THC) lowers intraocular pressure (IOP) and prevents RGC death in a rat model of glaucoma. Arat model of experimental glaucoma with chronic, moderately elevated IOP was produced unilaterally by cauterization of episcleral vessels. Rats received weekly injections of THC at a level of 5 mg/kg or vehicle for 20 weeks. IOP of both eyes was measured weekly on anesthetized animals immediately before THC treatment. RGCs were labeled in a retrograde fashion and counted in whole-mounted retinas. IOP was elevated in all operated eyes 1 day after the operation and remained elevated in the vehicle-treated rats throughout 20 weeks. In THC-treated rats, IOP elevation in operated eyes was diminished 2 weeks after operation and remained reduced. IOP in the contralateral control eyes was not affected by THC. In the operated eyes of vehicle-treated animals, there was a loss of ∼50 and 40% of the RGCs in the peripheral and central retina, respectively. The RGC loss in the operated eyes of the THC-treated animals was reduced to 10–20%. These results demonstrate that THC is a neuroprotectant that preserves RGCs in an experimental model of glaucoma, possibly through a reduction in IOP.

Thioredoxin-interacting protein expression is required for VEGF-mediated angiogenic signal in endothelial cells.

AIMS Thioredoxin-interacting protein (TXNIP) contributes to cellular redox-state homeostasis via binding and inhibiting thioredoxin (TRX). Increasing evidence suggests that cellular redox homeostasis regulates vascular endothelial growth factor (VEGF)-mediated signaling. This study aims to examine the redox-dependant role of TXNIP in regulating VEGF-mediated S-glutathionylation and angiogenic signaling. TXNIP-knockout mice (TKO) or wild-type (WT) treated with the reduced glutathione (GSH)-precursor, N-acetyl cysteine (WT-NAC, 500 mg/kg) were compared to WT using hypoxia-induced neovascularization model. RESULTS In response to hypoxia, retinas from TKO and WT-NAC mice showed significant decreases in reparative revascularization and pathological neovascularization with similar VEGF expression compared with WT. VEGF failed to stimulate vascular sprouting from aortic rings of TKO compared to WT mice. TKO mice or WT+NAC experienced reductive stress as indicated by twofold increase in TRX reductase activity and fourfold increase in reduced-GSH levels compared with WT. In human microvascular endothelial (HME) cells, VEGF stimulated co-precipitation between vascular endothelial growth factor receptor 2 (VEGFR2) with low molecular weight protein tyrosine phosphatase (LMW-PTP). Silencing TXNIP expression blunted VEGF-induced oxidation of GSH and S-glutathionylation of the LMW-PTP in HME cells. These effects were associated with impaired VEGFR2 phosphorylation that culminated in inhibiting cell migration and tube formation. Overexpression of TXNIP restored VEGFR2 phosphorylation and cell migration in TKO-endothelial cells. INNOVATION TXNIP expression is required for VEGF-mediated VEGFR2 activation and angiogenic response in vivo and in vitro. TXNIP expression regulates VEGFR-2 phosphorylation via S-glutathionylation of LMW-PTP in endothelial cells. CONCLUSION Our results provide novel mechanistic insight into modulating TXNIP expression as a potential therapeutic target in diseases characterized by aberrant angiogenesis.

MicroRNA-146b-3p Regulates Retinal Inflammation by Suppressing Adenosine Deaminase-2 in Diabetes

Hyperglycemia- (HG-) Amadori-glycated albumin- (AGA-) induced activation of microglia and monocytes and their adherence to retinal vascular endothelial cells contribute to retinal inflammation leading to diabetic retinopathy (DR). There is a great need for early detection of DR before demonstrable tissue damages become irreversible. Extracellular adenosine, required for endogenous anti-inflammation, is regulated by the interplay of equilibrative nucleoside transporter with adenosine deaminase (ADA) and adenosine kinase. ADA, including ADA1 and ADA2, exists in all organisms. However, because ADA2 gene has not been identified in mouse genome, how diabetes alters adenosine-dependent anti-inflammation remains unclear. Studies of pig retinal microglia and human macrophages revealed a causal role of ADA2 in inflammation. Database search suggested miR-146b-3p recognition sites in the 3′-UTR of ADA2 mRNA. Coexpression of miR-146b-3p, but not miR-146-5p or nontargeting miRNA, with 3′-UTR of the ADA2 gene was necessary to suppress a linked reporter gene. In the vitreous of diabetic patients, decreased miR-146b-3p is associated with increased ADA2 activity. Ectopic expression of miR-146b-3p suppressed ADA2 expression, activity, and TNF-α release in the AGA-treated human macrophages. These results suggest a regulatory role of miR-146b-3p in diabetes related retinal inflammation by suppressing ADA2.