Adenosine Deaminase-2-Induced Hyperpermeability in Human Retinal Vascular Endothelial Cells Is Suppressed by MicroRNA-146b-3p.

Purpose: We recently demonstrated that adenosine deaminase-2 (ADA2) contributes to diabetic retinopathy (DR) via up-regulating the production of inflammatory cytokines in macrophages. Also, microRNA (miR)-146b-3p has the ability to inhibit ADA2. The goal of this study was to investigate the potential role of ADA2 and therapeutic benefit of miR-146b-3p in retinal inflammation and endothelial barrier dysfunction during diabetes. Methods: Adenosine deaminase-2 activity was determined by colorimetric method in diabetic human vitreous. Human monocyte cell line U937 was differentiated into macrophages and then treated with amadori glycated albumin (AGA), and conditioned medium (CM) was used to assess the changes in ADA2 activity and TNF-α and IL-6 levels by ELISA. Also, macrophages were transfected with miR-146b-3p before treatment with AGA. Permeability of human retinal endothelial cells (hRECs) was assessed by electric cell-substrate impedance sensing (ECIS) after treatment with macrophage CM. Zonula occludens (ZO)-1 was examined by immuno-fluorescence in hRECs. Leukocyte adhesion was assessed in hRECs by measuring myeloperoxidase (MPO) activity and intercellular adhesion molecule-1 (ICAM-1) expression. Results: Adenosine deaminase-2 activity was significantly increased in diabetic human vitreous. ADA2 activity and TNF-α and IL-6 levels were significantly increased in human macrophages by AGA treatment. Amadori glycated albumin-treated macrophage CM significantly increased hREC permeability, disrupted ZO-1 pattern, and increased leukocyte adhesion to hRECs through up-regulating ICAM-1. All these changes were reversed by miR-146b-3p. Conclusions: Adenosine deaminase-2 is implicated in breakdown of the blood-retinal barrier (BRB) in DR through macrophages-derived cytokines. Therefore, inhibition of ADA2 by miR-146b-3p might be a useful tool to preserve BRB function in DR.

Cepharanthine and Piperine ameliorate diabetic nephropathy in rats: role of NF-κB and NLRP3 inflammasome.

AIMS: Hyperglycemia leads to elevation of oxidative stress and proinflammatory cytokines which are the main causes of diabetic nephropathy (DN). NLRP3 inflammasome and thioredoxin-interacting protein (TXNIP) are recently assumed to participate in the development of DN. We aimed to investigate the effects of Cepharanthine (CEP), Piperine (Pip) and their combination in streptozotocin (STZ)-induced DN focusing on their role to modulate NLRP3 and TXNIP induced inflammation. MAIN METHODS: Diabetic rats were treated with intraperitoneal (i.p.) injection of CEP (10mg/kg/day), Pip (30mg/kg/day) or their combination for 8weeks. Nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) were assessed by ELISA technique. TXNIP and NLRP3 genes expressions were evaluated by real time-PCR. KEY FINDINGS: Diabetic rats showed significant increase in renal TXNIP and NLRP3 expression. CEP, Pip or their combination significantly decreased TXNIP and NLRP3 expression in diabetic kidneys. Hyperglycemia induced NF-κB activation leading to increased IL-1ß and TNF-α levels. CEP, Pip or their combination showed significant inhibition of NF-κB together with decreased IL-1ß and TNF-α levels in diabetic rats. Also, diabetic rats showed significant decrease in creatinine clearance and increase in blood glucose, serum creatinine, blood urea nitrogen, malondialdehyde, proteinuria, and kidney weight to body Weight ratio. All of these changes were reversed by CEP, Pip or their combination. SIGNIFICANCE: The antioxidant and anti-inflammatory effects of CEP and Pip which were accompanied by inhibition of NF-κB and NLRP3 activation might be helpful mechanisms to halt the progression of DN.

Anti-inflammatory role of sesamin in STZ induced mice model of diabetic retinopathy.

Diabetic retinopathy (DR) is the common cause of diabetic vascular complications that leads to the blindness in the working age population throughout the world. Free radicals mediated oxidative stress and inflammation play a significant role in pathophysiology of DR. To find a new and safe drug to treat DR is still challenging and for that purpose the natural compounds may be therapeutic agents. Here we show that sesamin (SES), which is the main component of sesame seed and its oil, and has been reported as potent antioxidant and neuroprotective, could be a therapeutic agent in DR. In the present study, we investigated protective effect of SES in Streptozotocin (STZ) induced DR in mice. The mice were divided into three groups (Control, DR and DR+SES) for the study. After two weeks post-diabetic establishment, mice were treated with SES (30mg/kg BW, i.p, alternate day) for four weeks. Mice body weight and blood glucose level were measured from each group. The microglial activation of retina was determined by immunohistochemistry analysis by using Iba-1 as a microglia marker. Retinal mRNA levels of Iba-1, tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and Intercellular Adhesion Molecule 1 (ICAM-1) were examined by qRT-PCR. The level of iNOS protein expression was examined by immunoblotting. Together these data demonstrate that SES treatment lowered the progression of diabetic retinal injury by: 1) decreasing blood glucose level, 2) suppressing microglia activation, 3) reducing retinal TNF-α and ICAM-1 levels and 4) quenching iNOS expression. In conclusion, the results suggest that SES treatment may be of therapeutic benefit in reducing the progression of DR by ameliorating hyperglycemia and inflammation in diabetic retina.

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.

Inhibition of adenosine kinase attenuates inflammation and neurotoxicity in traumatic optic neuropathy.

Traumatic optic neuropathy (TON) is associated with apoptosis of retinal ganglion cells. Local productions of reactive oxygen species and inflammatory mediators from activated microglial cells have been hypothesized to underlie apoptotic processes. We previously demonstrated that the anti-inflammatory effect of adenosine, through A2A receptor activation had profound protective influence against retinal injury in traumatic optic neuropathy. This protective effect is limited due to rapid cellular re-uptake of adenosine by equilibrative nucleotside transporter-1 (ENT1) or break down by adenosine kinase (AK), the key enzyme in adenosine clearance pathway. Further, the use of adenosine receptors agonists are limited by systemic side effects. Therefore, we seek to investigate the potential role of amplifying the endogenous ambient level of adenosine by pharmacological inhibition of AK. We tested our hypothesis by comparing TON-induced retinal injury in mice with and without ABT-702 treatment, a selective AK inhibitor (AKI). The retinal-protective effect of ABT-702 was demonstrated by significant reduction of Iba-1, ENT1, TNF-α, IL-6, and iNOS/nNOS protein or mRNA expression in TON as revealed by western blot and real time PCR. TON-induced superoxide anion generation and nitrotyrosine expression were reduced in ABT-702 treated mice retinal sections as determined by immunoflourescence. In addition, ABT-702 attenuated p-ERK1/2 and p-P38 activation in LPS induced activated mouse microglia cells. The results of the present investigation suggested that ABT-702 had a protective role against marked TON-induced retinal inflammation and damage by augmenting the endogenous therapeutic effects of site- and event-specific accumulation of extracellular adenosine.

Sesamin attenuates neurotoxicity in mouse model of ischemic brain stroke.

Stroke is a severe neurological disorder characterized by the abrupt loss of blood circulation into the brain resulting into wide ranging brain and behavior abnormalities. The present study was designed to evaluate molecular mechanism by which sesamin (SES) induces neuroprotection in mouse model of ischemic stroke. The results of this study demonstrate that SES treatment (30 mg/kg bwt) significantly reduced infarction volume, lipid per-oxidation, cleaved-caspase-3 activation, and increased GSH activity following MCAO in adult male mouse. SES treatment also diminished iNOS and COX-2 protein expression, and significantly restored SOD activity and protein expression level in the ischemic cortex of the MCAO animals. Furthermore, SES treatment also significantly reduced inflammatory and oxidative stress markers including Iba1, Nox-2, Cox-2, peroxynitrite compared to placebo MCAO animals. Superoxide radical production, as studied by DHE staining method, was also significantly reduced in the ischemic cortex of SES treated compared to placebo MCAO animals. Likewise, downstream effects of superoxide free radicals i.e. MAPK/ERK and P38 activation was also significantly attenuated in SES treated compared to placebo MCAO animals. In conclusion, these results suggest that SES induces significant neuroprotection, by ameliorating many signaling pathways activated/deactivated following cerebral ischemia in adult mouse.

Sigma receptor ligand, (+)-pentazocine, suppresses inflammatory responses of retinal microglia.

PURPOSE: To evaluate the effects of the σ 1 receptor (σR1) agonist, (+)-pentazocine, on lipopolysaccharide (LPS)-induced inflammatory changes in retinal microglia cells. METHODS: Retinal microglia cells were isolated from Sprague-Dawley rat pups. Cells were treated with LPS with or without (+)-pentazocine and with or without the σR1 antagonist BD1063. Morphologic changes were assayed. Cell viability was assessed by using MTT assay. Supernatant levels of tumor necrosis factor α (TNF-α), interleukin 10, (IL-10), monocyte chemoattractant protein-1 (MCP-1), and nitric oxide (NO) were determined. Reactive oxygen species (ROS) formation was assayed, and levels of mitogen-activated protein kinases (MAPKs) were analyzed by using Western blot. RESULTS: The σR1 protein was expressed in retinal microglia. Incubation with LPS and/or (+)-pentazocine did not alter cell viability or σR1 protein levels. Incubation with LPS for 24 hours induced a marked change in microglial morphology and a significant increase in secreted levels of TNF-α, IL-10, MCP-1, and NO. Pretreatment with (+)-pentazocine inhibited the LPS-induced morphologic changes. Release of TNF-α, IL-10, MCP-1, and NO was reduced with (+)-pentazocine. Intracellular ROS formation was suppressed with (+)-pentazocine. Phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was reduced in the presence of (+)-pentazocine. The σR1 antagonist BD1063 blocked the (+)-pentazocine-mediated inhibition of LPS-induced morphologic changes. In addition, BD1063 treatment blocked (+)-pentazocine-mediated suppression of LPS-induced TNF-α, IL-10, MCP-1, NO, and intracellular ROS release. CONCLUSIONS: Treatment with (+)-pentazocine suppressed inflammatory responses of retinal microglia and inhibited LPS-induced activation of ERK/JNK MAPK. In neurodegenerative disease, (+)-pentazocine may exert neuroprotective effects through manipulation of microglia.

Adenosine kinase inhibition protects the kidney against streptozotocin-induced diabetes through anti-inflammatory and anti-oxidant mechanisms.

Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5mg/kg, i.p. two times a week for 8 weeks, n=7-8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (∼20%; P<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFκB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P<0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.

Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration. However, the signaling pathway(s) leading to photoreceptor death in IRBP-deficient mice remains poorly understood. Here, we show that amounts of tumor necrosis factor-α (TNF-α) in the interphotoreceptor matrix and retinas of Irbp(-/-) mice were increased more than 10-fold and fivefold, respectively, compared with those in wild-type mice. Moreover, TNF-α receptor 1, an important membrane death receptor that mediates both programmed apoptosis and necrosis, was also significantly increased in Irbp(-/-) retina, and was colocalized with peanut agglutinin to the Irbp(-/-) cone outer segments. Although these death signaling proteins were increased, the caspase-dependent and independent apoptotic pathways were mildly activated in the Irbp(-/-) retinas, suggesting that other cell death mechanism(s) also contributes to the extensive photoreceptor degeneration in Irbp(-/-) retina. We found that receptor interacting protein 1 and 3 (RIP1 and RIP3) kinases, the intracellular key mediators of TNF-induced cellular necrosis, were elevated at least threefold in the Irbp(-/-) retinas. Moreover, pharmacological inhibition of RIP1 kinase significantly prevented cone and rod photoreceptor degeneration in Irbp(-/-) mice. These results reveal that RIP kinase-mediated necrosis strongly contributes to cone and rod degeneration in Irbp(-/-) mice, implicating the TNF-RIP pathway as a potential therapeutic target to prevent or delay photoreceptor degeneration in patients with retinitis pigmentosa caused by IRBP mutation.

Potential role of A2A adenosine receptor in traumatic optic neuropathy.

In traumatic optic neuropathy (TON), apoptosis of retinal ganglion cells is closely related to the local production of reactive oxygen species and inflammatory mediators from activated microglial cells. Adenosine receptor A2A (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in TON. In the present study, we examined the role of A2AAR in retinal complications associated with TON. Initial studies in wild-type mice revealed that treatment with the A2AAR agonist resulted in marked decreases in the TON-induced microglial activation, retinal cell death and releases of reactive oxygen species and pro-inflammatory cytokines TNF-α and IL-6. To further assess the role of A2AAR in TON, we studied the effects of A2AAR ablation on the TON-induced retinal abnormalities. A2AAR-/- mice with TON showed a significantly higher mRNA level of TNF-α, Iba1-1 in retinal tissue, and ICAM-1 expression in retinal sections compared with wild-type mice with TON. To explore a potential mechanism by which A2AAR-signaling regulates inflammation in TON, we performed additional studies using hypoxia- or LPS-treated microglial cells as an in vitro model for TON. Activation of A2AAR attenuates hypoxia or LPS-induced TNF-α release and significantly repressed the inflammatory signaling, ERK in the activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in TON and suggests that the retinal protective effect of A2AAR is mediated by attenuating the inflammatory response that occurs in microglia via interaction with MAPKinase pathway.

Regulation of vitamin C transporter in the type 1 diabetic mouse bone and bone marrow.

A number of studies have revealed that Type I diabetes (T1D) is associated with bone loss and an increased risk of fractures. T1D induces oxidative stress in various tissues and organs. Vitamin C plays an important role in the attenuation of oxidative stress; however, little is known about the effect of T1D induced oxidative stress on the regulation of vitamin C transporter in bone and bone marrow cells. To investigate this, T1D was induced in mice by multiple low dose injections of streptozotocin. We have demonstrated that endogenous antioxidants, glutathione peroxidase (GPx) and superoxide dismutase (SOD) are down-regulated in the bone and bone marrow of T1D. The vitamin C transporter isoform SVCT2, the only known transporter expressed in bone and bone marrow stromal cells (BMSCs), is negatively regulated in the bone and bone marrow of T1D. The µCT imaging of the bone showed significantly lower bone quality in the 8 week T1D mouse. The in-vitro study in BMSCS showed that the knockdown of SVCT2 transporter decreases ascorbic acid (AA) uptake, and increases oxidative stress. The significant reversing effect of antioxidant vitamin C is only possible in control cells, not in knockdown cells. This study suggested that T1D induces oxidative stress and decreases SVCT2 expression in the bone and bone marrow environment. Furthermore, this study confirms that T1D increases bone resorption, decreases bone formation and changes the microstructure of bones. This study has provided evidence that the regulation of the SVCT2 transporter plays an important role not only in T1D osteoporosis but also in other oxidative stress-related musculoskeletal complications.

ABT-702, an adenosine kinase inhibitor, attenuates inflammation in diabetic retinopathy.

AIMS: This study was undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling via A2A receptors (A2AAR) is involved in retinal protection from diabetes-induced inflammation. Here we demonstrate that AKI-enhanced adenosine signaling provides protection from DR in mice. MAIN METHODS: We targeted AK, the key enzyme in adenosine metabolism, using a treatment regime with the selective AKI, ABT-702 (1.5mg/kg intraperitoneally twice a week) commencing at the beginning of streptozotocin-induced diabetes at the age of eight weeks. This treatment, previously demonstrated to increase free adenosine levels in vivo, was maintained until the age of 16 weeks. Retinal inflammation was evaluated using Western blot, Real-Time PCR and immuno-staining analyses. Role of A2AAR signaling in the anti-inflammation effect of ABT-702 was analyzed in Amadori-glycated-albumin (AGA)-treated microglial cells. KEY FINDINGS: At 16 weeks, when diabetic mice exhibit significant signs of retinal inflammation including up-regulation of oxidative/nitrosative stress, A2AAR, ENT1, Iba1, TNF-α, ICAM1, retinal cell death, and down-regulation of AK, the ABT-702 treated group showed lower signs of inflammation compared to control animals receiving the vehicle. The involvement of adenosine signaling in the anti-inflammation effect of ABT-702 was supported by the TNF-α release blocking effect of A2AAR antagonist in AGA-treated microglial cells. SIGNIFICANCE: These results suggest a role for AK in regulating adenosine receptor signaling in the retina. Inhibition of AK potentially amplifies the therapeutic effects of site- and event-specific accumulation of extracellular adenosine, which is of highly translational impact.

Potential roles of adenosine deaminase-2 in diabetic retinopathy.

The early activation of microglia that induces retinal inflammation in DR may serve as a target for therapeutic intervention of DR. Our demonstration that retinal inflammation is attenuated via adenosine receptor A(2A)AR supports the hypothesis that a mechanism to maintain extracellular concentrations of adenosine important in normal physiology is impaired in DR. Extracellular concentrations of adenosine are regulated by the interplay of equiliberative nucleoside transporter (ENT)s with enzymes of adenosine metabolism including adenosine deaminase-1 (ADA1), adenosine kinase (AK) and CD73. In the vertebrates but not rodents, a macrophage-associated ADA2 is identified. The role of ADA2 is, therefore, understudied as the sequencing probes or antibodies to mouse ADA2 are not available. We identified increased ADA2 expression and activity in human and porcine retinas with diabetes, and in Amadori glycated albumin (AGA)- or hyperglycemia-treated porcine and human microglia. In rodent as well as porcine cells, modulation of TNF-α release is mediated by A(2A)AR. Quantitative analysis of normal and diabetic porcine retinas reveals that while the expression levels of ADA2, A2AAR, ENT1, TNF-α and MMP9 are increased, the levels of AK are reduced during inflammation as an endogenous protective mechanism. To determine the role of ADA2, we found that AGA induces ADA2 expression, ADA2 activity and TNF-α release, and that TNF-α release is blocked by ADA2-neutralizing antibody or ADA2 siRNA, but not by scrambled siRNA. These results suggest that retinal inflammation in DR is mediated by ADA2, and that the anti-inflammatory activity of A(2A)AR signaling is impaired in diabetes due to increased ADA2 activity.

Loss of thioredoxin function in retinas of mice overexpressing amyloid ß.

Amyloid ß peptides (Aß) have been implicated in the pathogenesis of age-related macular degeneration (ARMD) and glaucoma. In this study, retinas of mice overexpressing Aß (Tg) were compared to those of wild-type mice (Wt) and analyzed for oxidative stress parameters. We observed a progressive decrease in all retinal cell layers, which was significantly greater in Tg mice at 14 months and culminated in loss of the outer retina at 18 months of age. We also observed higher levels of reactive oxygen species, glial fibrillary acidic protein, and hydroperoxide in Tg versus Wt mice (14 months). These effects were associated with phosphorylation/activation of the apoptosis signal kinase 1 and the p38 mitogen-activated kinase. Western blotting analysis revealed progressive increases in the levels of thioredoxin 1 and thioredoxin inhibitory protein in Tg compared to Wt mice. No changes were observed in the levels of thioredoxin reductase 1 (TrxR1); however, measurements of TrxR1 activity showed a 42.7±8% reduction in Tg mice versus Wt at 14 months of age. Our data suggest that Aß-mediated retinal neurotoxicity involves impairment of the thioredoxin system and enhanced oxidative stress, potentially implicating this mechanism in the pathogenesis of ARMD and glaucoma.

Tyrosine kinase inhibitor, genistein, reduces renal inflammation and injury in streptozotocin-induced diabetic mice.

Tyrosine kinase inhibition is known to reduce diabetes-induced end-organ damage but the mechanisms remain elusive. We hypothesized that inhibition of tyrosine kinase reduces renal inflammation and injury in streptozotocin-induced diabetes. Male C57BL/6 mice were given daily injections of streptozotocin (45 mg/kg/day, i.p. for 5 days); control animals received the vehicle (citrate buffer). Thereafter, streptozotocin-treated mice were treated with genistein (10 mg/kg, i.p three times a week for 10 weeks, n=8-10/group) or the vehicle (5% DMSO). The streptozotocin-treated mice displayed significant elevation in blood glucose level and decrease in plasma insulin level compared to their vehicle-treated controls. Treatment with genistein reduced blood glucose level (~15%; p<0.05) without a significant effect on plasma insulin level; however, blood glucose remained significantly higher than the control group. The development of diabetes was associated with significant increases in total protein, albumin, nephrin and collagen excretions compared to their controls. In addition, the diabetic mice displayed increased urinary MCP-1 excretion in association with increased renal ICAM-1 expression and apoptotic cells. Furthermore, renal gp91 expression levels and urinary Thio-Barbituric Acid Reactive Substances (TBARs) excretion, indices of oxidative stress, were also elevated in diabetic mice. These changes were associated with increased renal phospho-tyrosine expression and renal phospho-ERK/ERK ratio. Importantly, treatment with genistein reduced all these parameters towards control values. Collectively, the results suggest that the reno-protective effect of genistein likely relates to reduced renal inflammation, oxidative stress and apoptosis in diabetic mice.

Exaggerated eye growth in IRBP-deficient mice in early development.

PURPOSE: Because interphotoreceptor retinoid-binding protein (IRBP) is expressed before being needed in its presumptive role in the visual cycle, we tested whether it controls eye growth during development. METHODS: The eyes of congenic IRBP knockout (KO) and C57BL/6J wild-type (WT) mice ranging in age from postnatal day (P)2 to P440 were compared by histology, laser micrometry, cycloplegic photorefractions, and partial coherence interferometry. RESULTS: The size and weight of IRBP KO mouse eyes were greater than those of the WT mouse, even before eye-opening. Excessive ocular enlargement started between P7 and P10, with KO retinal arc lengths becoming greater compared with WT from P10 through P30 (18%; P < 0.01). The outer nuclear layer (ONL) of KO retinas became 20% thinner between P12 to P25, and progressed to 38% thinner at P30. At P30, there were 30% fewer cones per vertical section in KO than in WT retinas. Bromodeoxyuridine (BrdU) labeling indicated the same number of retinal cells were born in KO and WT mice. A spike in apoptosis was observed in KO outer nuclear layer at P25. These changes in size were accompanied by a large decrease in hyperopic refractive error, which reached -4.56 ± 0.70 diopters (D) versus +9.98 ± 0.993 D (mean ± SD) in WT, by postnatal day 60 (P60). CONCLUSIONS; In addition to its role in the visual cycle, IRBP is needed for normal eye development. How IRBP mediates ocular development is unknown.

A(2A) adenosine receptor (A(2A)AR) as a therapeutic target in diabetic retinopathy.

In diabetic retinopathy (DR), abnormalities in vascular and neuronal function are closely related to the local production of inflammatory mediators whose potential source is microglia. A(2A) adenosine receptor (A(2A)AR) has been shown to possess anti-inflammatory properties that have not been studied in DR. Here, we evaluate the role of A(2A)AR and its underlying signaling in retinal complications associated with diabetes. Initial studies in wild-type mice revealed that the treatment with the A(2A)AR agonist resulted in marked decreases in hyperglycemia-induced retinal cell death and tumor necrosis factor (TNF)-α release. To further assess the role of A(2A)AR in DR, we studied the effects of A(2A)AR ablation on diabetes-induced retinal abnormalities. Diabetic A(2A)AR(-/-) mice had significantly more terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells, TNF-α release, and intercellular adhesion molecule-1 expression compared with diabetic wild-type mice. To explore a potential mechanism by which A(2A)AR signaling regulates inflammation in DR, we performed additional studies using microglial cells treated with Amadori-glycated albumin, a risk factor in diabetic disorders. The results showed that activation of A(2A)AR attenuated Amadori-glycated albumin-induced TNF-α release in a cAMP/exchange protein directly activated by cAMP-dependent mechanism and significantly repressed the inflammatory cascade, C-Raf/extracellular signal-regulated kinase (ERK), in activated microglia. Collectively, this work provides pharmacological and genetic evidence for A(2A)AR signaling as a control point of cell death in DR and suggests that the retinal protective effect of A(2A)AR is mediated by abrogating the inflammatory response that occurs in microglia via interaction with C-Raf/ERK pathway.

Opioid receptor activation: suppression of ischemia/reperfusion-induced production of TNF-α in the retina.

PURPOSE: The detrimental role of TNF-α in ischemia-induced tissue damage is known. The authors study examined whether opioid receptor activation alters TNF-α levels in the postischemic retina. METHODS: Retinal ischemia was induced by raising the intraocular pressure above systolic blood pressure (155-160 mm Hg) for 45 minutes. Rats were pretreated with the opioid receptor agonist morphine (1 mg/kg; intraperitoneally) before injury. Selected animals were pretreated with the opioid antagonist naloxone (3 mg/kg; intraperitoneally). Human optic nerve head (ONH) astrocytes and rat microglial cells were treated with morphine (0.1-1 µM) for 24 hours and then treated with 10 µg/mL or 30 ng/mL lipopolysaccharide (LPS), respectively. TNF-α was measured by ELISA. Opioid receptor subtypes in astrocytes and microglia were determined by Western blot analysis. RESULTS: There was a time-dependent increase in TNF-α production; the maximum production occurred at 4 hours after ischemia and localized to the inner retinal regions. Ischemia-induced TNF-α production was significantly inhibited by morphine. In astrocytes and microglia, LPS triggered a robust increase in the release of TNF-α, which was significantly inhibited (P < 0.05) by morphine. Naloxone reversed the morphine-induced suppression of TNF-α production in vivo and in vitro. Both ONH astrocytes and microglial cells expressed δ-, κ-, and µ-opioid receptor subtypes. CONCLUSIONS: These data provide evidence that the production of TNF-α after ischemia/reperfusion injury is an early event and that opioid receptor activation reduces the production of TNF-α. Immunohistochemistry data and in vitro studies provide evidence that ONH astrocytes and microglial cells are the primary sources for the TNF-α production under ischemic/inflammatory conditions. Activation of one or more opioid receptors can reduce ischemic/reperfusion injury by the suppression of TNF-α production.

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.

Role of adenosine in diabetic retinopathy.

In diabetic retinopathy (DR), abnormalities in vascular and neuronal function are closely related to the local production of inflammatory mediators whose potential source is microglia. Adenosine and its receptors have been shown to possess anti-inflammatory properties that have only recently been studied in DR. Here, we review recent studies that determined the roles of adenosine and its associated proteins, including equilibrative nucleoside transporters, adenosine receptors, and underlying signaling pathways in retinal complications associated with diabetes.