Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of ␤-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of lowdensity lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts , were also elevated in the DR models. The high glucose-induced activation of ␤-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target. Diabetic retinopathy (DR), the leading cause of blindness in the working age population, represents a common concern in types 1 and 2 of diabetes mellitus (DM). 1 Accumulating evidence suggests that DR is a chronic inflammatory disorder.2 Retinal inflammation is believed to play a causative role in vascular leakage, which can lead to diabetic macular edema, and in retinal neovascularization (NV). It has been shown that levels of soluble intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 are significantly higher in the vitreous from patients with proliferative diabetic retinopathy than in nondiabetic vitreous. 3,4 Increased ICAM-1, vascular cell adhesion molecule-1, and e-selectin levels were found in the serum from patients with diabetic microangiopathy. [5][6][7] In diabetic animal models, increased retinal ICAM-1 expression is believed to be responsible for leukocyte adhesion or leukostasis and increased vascular permeability. Leukostasis is believed to contribute to capillary nonperfusion and local ischemia, which subsequently induces the overexpression of vascular endothelial growth factor (VEGF). 8 -11 Increased VEGF levels are responsible for the retinal vascular leakage and retinal NV.12,13 Recent studies have indicated that oxidative stress, induced by hyperglycemia, contributes to retinal inflammation in diabetes.14,15 However, the pathogenic mechanisms by which diabetes and oxidative stress induce inflammation are not certain at the present time.Wnts are a group of secreted, cysteine-rich glycoproteins, which bind to a coreceptor complex of frizzled (Fz) receptors and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) and regulate expression of a number of target genes through an intracellular signaling pathway, namely the Wnt pathway. 16 In the absence of Wnt ligands, ␤-catenin, a down-stream effector of the canonical Wnt pathway, is phosphorylated by a pro...

Retinal vascular leakage, inflammation, and neovascularization (NV) are features of diabetic retinopathy (DR). Fenofibrate, a peroxisome proliferator–activated receptor α (PPARα) agonist, has shown robust protective effects against DR in type 2 diabetic patients, but its effects on DR in type 1 diabetes have not been reported. This study evaluated the efficacy of fenofibrate on DR in type 1 diabetes models and determined if the effect is PPARα dependent. Oral administration of fenofibrate significantly ameliorated retinal vascular leakage and leukostasis in streptozotocin-induced diabetic rats and in Akita mice. Favorable effects on DR were also achieved by intravitreal injection of fenofibrate or another specific PPARα agonist. Fenofibrate also ameliorated retinal NV in the oxygen-induced retinopathy (OIR) model and inhibited tube formation and migration in cultured endothelial cells. Fenofibrate also attenuated overexpression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and vascular endothelial growth factor (VEGF) and blocked activation of hypoxia-inducible factor-1 and nuclear factor-κB in the retinas of OIR and diabetic models. Fenofibrate’s beneficial effects were blocked by a specific PPARα antagonist. Furthermore, Pparα knockout abolished the fenofibrate-induced downregulation of VEGF and reduction of retinal vascular leakage in DR models. These results demonstrate therapeutic effects of fenofibrate on DR in type 1 diabetes and support the existence of the drug target in ocular tissues and via a PPARα-dependent mechanism.

Two independent clinical studies have reported that fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has robust therapeutic effects on microvascular complications of diabetes, including diabetic retinopathy (DR) in type 2 diabetic patients. However, the expression and function of PPARα in the retina are unclear. Here, we demonstrated that PPARα is expressed in multiple cell types in the retina. In both type 1 and type 2 diabetes models, expression of PPARα, but not PPARβ/δ or PPARγ, was significantly down-regulated in the retina. Furthermore, high-glucose medium was sufficient to down-regulate PPARα expression in cultured retinal cells. To further investigate the role of PPARα in DR, diabetes was induced in PPARα knockout (KO) mice and wild-type (WT) mice. Diabetic PPARα KO mice developed more severe DR, as shown by retinal vascular leakage, leukostasis, pericyte loss, capillary degeneration, and over-expression of inflammatory factors, compared with diabetic WT mice. In addition, overexpression of PPARα in the retina of diabetic rats significantly alleviated diabetes-induced retinal vascular leakage and retinal inflammation. Furthermore, PPARα overexpression inhibited endothelial cell migration and proliferation. These findings revealed that diabetes-induced down-regulation of PPARα plays an important role in DR. Up-regulation or activation of PPARα may represent a novel therapeutic strategy for DR.

Choroidal neovascularization (CNV) in age-related macular degeneration is a leading cause of blindness. Very low density lipoprotein receptor gene knock-out (Vldlr ؊/؊ ) mice have been shown to develop subretinal neovascularization (NV) with an unknown mechanism. The present study showed that in Vldlr ؊/؊ mice, NV initiated in the choroid and progressed to penetrate the retinal pigment epithelium layer, proliferating in the subretinal space. This phenotype recapitulated what is seen in wet age-related macular degeneration, suggesting that this is a CNV model. The CNV correlated with overexpression of vascular endothelial growth factor in Vldlr ؊/؊ eyecups and was blocked by a neutralizing antibody against vascular endothelial growth factor receptor-2. The wnt co-receptor LRP5/6 expression was significantly up-regulated in Vldlr ؊/؊ eyecups compared with that in wild-type mice. Significantly, Vldlr ؊/؊ mice showed impaired phosphorylation of downstream effectors of the wnt signaling pathway, glycogen synthase kinase-3␤ (GSK-3␤), and ␤-catenin, concomitant with increased levels of free GSK-3␤ and ␤-catenin, suggesting an increased activity of the wnt pathway. Down-regulation of VLDLR by small interference RNA resulted in up-regulation of LRP5/6 expression and activation of ␤-catenin in cultured endothelial cells. Furthermore, Dickkopf-1, a specific inhibitor of the wnt pathway, effectively decreased vascular endothelial growth factor and ␤-catenin levels in the retinal pigment epithelium of Vldlr ؊/؊ mice and in cells transfected with the VLDLR small interference RNA. These results suggest that VLDLR functions as a negative regulator of CNV, and this function is mediated through the wnt pathway.

Activation of the canonical Wnt pathway is sufficient to induce retinal inflammation and oxidative stress and plays a pathogenic role in AMD and DR.

Four weeks of normobaric hypoxia residential training with low caloric diet has an additive improvement on weight loss. It seems that normobaric hypoxia training might be a promising method to treat obesity.

Dysregulation of Wnt/β-catenin signaling contributes to the development of diabetic retinopathy by inducing retinal inflammation, vascular leakage, and neovascularization. Here, we evaluated the inhibitory effect of a monoclonal antibody (Mab) specific for the E1E2 domain of Wnt coreceptor low-density lipoprotein receptor–related protein 6, Mab2F1, on canonical Wnt signaling and its therapeutic potential for diabetic retinopathy. Mab2F1 displayed robust inhibition on Wnt signaling with a half-maximal inhibitory concentration (IC50) of 20 μg/mL in retinal pigment epithelial cells. In addition, Mab2F1 also attenuated the accumulation of β-catenin and overexpression of vascular endothelial growth factor, intercellular adhesion molecule-1, and tumor necrosis factor-α induced by high-glucose medium in retinal endothelial cells. In vivo, an intravitreal injection of Mab2F1 significantly reduced retinal vascular leakage and decreased preretinal vascular cells in oxygen-induced retinopathy (OIR) rats, demonstrating its inhibitory effects on ischemia-induced retinal neovascularization. Moreover, Mab2F1 blocked the overexpression of the inflammatory/angiogenic factors, attenuated leukostasis, and reduced retinal vascular leakage in both early and late stages of streptozotocin-induced diabetes. In conclusion, Mab2F1 inhibits canonical Wnt signaling, vascular leakage, and inflammation in the retina of diabetic retinopathy models, suggesting its potential to be used as a therapeutic agent in combination with other antiangiogenic compounds.

Aims/hypothesis Retinal Müller cells are known to produce inflammatory and angiogenic cytokines, which play important roles in diabetic retinopathy. Hypoxia-inducible factor (HIF)-1 has been shown to play a crucial role in retinal inflammation and neovascularisation. We sought to determine the role of Müller cell-derived HIF-1 in oxygeninduced retinopathy (OIR) and diabetic retinopathy using conditional Hif-1α (also known as Hif1a) knockout (KO) mice. Methods Conditional Hif-1α KO mice were generated by crossing mice expressing cyclisation recombinase (cre, also known as P1_gp003) in Müller cells with floxed Hif-1α mice and used for OIR and streptozotocin-induced diabetes to induce retinal neovascularisation and inflammation, respectively. Abundance of HIF-1α and pro-angiogenic and pro-inflammatory factors was measured by immunoblotting and immunohistochemistry. Retinal neovascularisation was visualised by angiography and quantified by counting pre-retinal nuclei. Retinal inflammation was evaluated by leucostasis and vascular leakage. Results While the Hif-1α KO mice showed significantly decreased HIF-1α levels in the retina, they exhibited no apparent histological or visual functional abnormalities (2011( ) 54:1554( -1566( DOI 10.1007( /s00125-011-2081 under normal conditions. Compared with wild-type counterparts, Hif-1α KO mice with OIR demonstrated attenuated overproduction of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1, reduced vascular leakage and alleviated neovascularisation in the retina. Under diabetes conditions, disruption of Hif-1α in Müller cells attenuated the increases of retinal vascular leakage and adherent leucocytes, as well as the overproduction of VEGF and ICAM-1. Conclusions/interpretation Müller cell-derived HIF-1α is a key mediator of retinal neovascularisation, vascular leakage and inflammation, the major pathological changes in diabetic retinopathy. Müller cell-derived HIF-1α is therefore a promising therapeutic target for diabetic retinopathy.