OBJECTIVEDuring 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 METHODSMicroglia activation was characterized in streptozocin-injected rats and in isolated microglial cells using immunofluorescence, enzyme-linked immunosorbent assay, RT-PCR, and Western blot analyses.RESULTSIn 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.CONCLUSIONSThese 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.

ObjectivesIron overload is now recognized as a health problem in industrialized countries, as excessive iron is highly toxic for liver and spleen. The potential use of curcumin as an iron chelator has not been clearly identified experimentally in iron overload condition. Here, we evaluate the efficacy of curcumin to alleviate iron overload-induced hepatic and splenic abnormalities and to gain insight into the underlying mechanisms.Design and MethodsThree groups of male adult rats were treated as follows: control rats, rats treated with iron in a drinking water for 2 months followed by either vehicle or curcumin treatment for 2 more months. Thereafter, we studied the effects of curcumin on iron overload-induced lipid peroxidation and anti-oxidant depletion.ResultsTreatment of iron-overloaded rats with curcumin resulted in marked decreases in iron accumulation within liver and spleen. Iron-overloaded rats had significant increases in malonyldialdehyde (MDA), a marker of lipid peroxidation and nitric oxide (NO) in liver and spleen when compared to control group. The effects of iron overload on lipid peroxidation and NO levels were significantly reduced by the intervention treatment with curcumin (P<0.05). Furthermore, the endogenous anti-oxidant activities/levels in liver and spleen were also significantly decreased in chronic iron overload and administration of curcumin restored the decrease in the hepatic and splenic antioxidant activities/levels.ConclusionOur study suggests that curcumin may represent a new horizon in managing iron overload-induced toxicity as well as in pathological diseases characterized by hepatic iron accumulation such as thalassemia, sickle cell anemia, and myelodysplastic syndromes possibly via iron chelation, reduced oxidative stress derived lipid peroxidation and improving the body endogenous antioxidant defense mechanism.

The disruption of retinal pigment epithelial (RPE) function and the degeneration of photoreceptors are cardinal features of age related macular degeneration (AMD); however there are still gaps in our understanding of underlying biological processes. Excess homocysteine (Hcy) has been reported to be elevated in plasma of patients with AMD. This study aimed to evaluate the direct effect of hyperhomocysteinemia (HHcy) on structure and function of RPE. Initial studies in a mouse model of HHcy, in which cystathionine-β-synthase (cbs) was deficient, revealed abnormal RPE cell morphology with features similar to that of AMD upon optical coherence tomography (OCT), fluorescein angiography (FA), histological, and electron microscopic examinations. These features include atrophy, vacuolization, hypopigmentation, thickened basal laminar membrane, hyporeflective lucency, choroidal neovascularization (CNV), and disturbed RPE–photoreceptor relationship. Furthermore, intravitreal injection of Hcy per se in normal wild type (WT) mice resulted in diffuse hyper-fluorescence, albumin leakage, and CNV in the area of RPE. In vitro experiments on ARPE-19 showed that Hcy dose-dependently reduced tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance, and impaired phagocytic activity. Collectively, our results demonstrated unreported effects of excess Hcy levels on RPE structure and function that lead to the development of AMD-like features.

Vascular inflammation plays a critical role in the pathogenesis of diabetic retinopathy. Recently, Interleukin-6 (IL-6) trans-signaling via soluble IL-6 receptor (sIL-6R) has emerged as a prominent regulator of inflammation in endothelial cells. This study was designed to test the hypothesis that selective inhibition of the IL-6 trans-signaling pathway will attenuate inflammation and subsequent barrier disruption in retinal endothelial cells. Human retinal endothelial cells (HRECs) were exposed to IL-6 and sIL-6R to induce IL-6 trans-signaling and the commercially available compound sgp130Fc (soluble gp-130 fused chimera) was used to selectively inhibit IL-6 trans-signaling. IL-6 trans-signaling activation caused a significant increase in STAT3 phosphorylation, expression of adhesion molecules, ROS production and apoptosis in HRECs whereas a significant decrease in mitochondrial membrane potential and NO production was observed in IL-6 trans-signaling activated cells. These changes were not observed in cells pre-treated with sgp130Fc. IL-6 trans-signaling activation was sufficient to cause barrier disruption in endothelial monolayers and pre-treatment of HRECs with sgp130Fc, maintained endothelial barrier function similar to that of untreated cells. Thus, in conclusion, these results indicate that IL-6 trans-signaling is an important mediator of inflammation, apoptosis and barrier disruptive effects in the retinal endothelial cells and inhibition of the IL-6 trans-signaling pathway using sgp130-Fc attenuates vascular inflammation and endothelial barrier disruption.

Diabetic retinopathy (DR), the most prevalent microvascular complication of diabetes, is responsible for over 10,000 new cases of blindness every year in the United States alone ( 1 ). The risk of vision loss increases with the development of diabetic macular edema and/or retinal neovascularization (NV), the former being a direct consequence of blood-retinal barrier (BRB) dysfunction and the latter the result of widespread retinal ischemia ( 2 ).For years, signifi cant effort has been invested in elucidating the mechanisms that underlie destructive preretinal NV in DR ( 3 ). Nonetheless, considerably less is known about the molecular events that lead to BRB dysfunction that is characterized by enhanced retinal vascular permeability and recruitment of infl ammatory cells. Moreover, existing regimens of treatment carry nonspecifi c adverse effects. These include increased risk of thromboembolic incidence, neuronal toxicity, and geographic atrophy with anti-vascular endothelial growth factor (VEGF) therapies

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-β-synthase is deficient (cbs −/−), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.

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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.