Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor. Reduced PEDF levels are associated with diabetic retinopathy. However, the mechanism for the protective effects of PEDF against diabetic retinopathy (DR) is presently unclear. As inflammation plays a role in DR, the present study determined the effect of PEDF on inflammation. Western blot analysis and ELISA demonstrated that retinal and plasma PEDF levels were drastically decreased in rats with endotoxin-induced uveitis (EIU), which suggests that PEDF is a negative acute-phase protein. Intravitreal injection of PEDF significantly reduced vascular hyper-permeability in rat models of diabetes and oxygen-induced retinopathy, correlating with the decreased levels of retinal inflammatory factors, including VEGF, VEGF receptor-2, MCP-1, TNF-alpha, and ICAM-1. In cultured retinal capillary endothelial cells, PEDF significantly decreased TNF-alpha and ICAM-1 expression under hypoxia. Moreover, down-regulation of PEDF expression by siRNA resulted in significantly increases of VEGF and TNF-alpha secretion in retinal Müller cells. These findings suggest that PEDF is a novel endogenous anti-inflammatory factor in the eye. The decrease of ocular PEDF levels may contribute to inflammation and vascular leakage in DR.

Retinal levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), an angiogenic inhibitor, were measured and correlated with the ischemia-induced retinal neovascularization in rats. The retinas with neovascularization showed a 5-fold increase in VEGF while 2-fold decrease in PEDF, compared to the age-matched controls, resulting in an increased VEGF/PEDF ratio. The time course of the VEGF/PEDF ratio change correlated with the progression of retinal neovascularization. Changes in the VEGF and PEDF mRNAs preceded their protein level changes. These results suggest that an unbalance between angiogenic stimulators and inhibitors may contribute to retinal neovascularization. ß

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...

It has been shown that the balance between vascular endothelial growth factor (VEGF), a major angiogenic stimulator, and pigment epithelium-derived factor (PEDF), a potent angiogenic inhibitor, is critical for the regulation of vascular permeability and angiogenesis. However, the regulation of the balance is largely unclear. The present study demonstrated that there is a reciprocal interaction between VEGF and PEDF in the retina. PEDF significantly decreased VEGF expression in both retinal capillary endothelial cells (RCEC) and Mü ller cells. This PEDF effect was confirmed in the retina of rats with oxygen-induced retinopathy. Silencing of the PEDF gene by siRNA in Mü ller cells resulted in a significant upregulation of VEGF expression at both the RNA and protein levels, suggesting that PEDF is an endogenous negative regulator of VEGF. The further study of the mechanism showed that PEDF inhibited hypoxia-induced increases in VEGF promoter activity, HIF-1 nuclear translocation and mitogen activated protein kinase phosphorylation. These results suggest that PEDF inhibits VEGF expression at the transcriptional level. In addition, PEDF effectively inhibited VEGF binding to RCEC. Moreover, in vitro receptor-binding assay demonstrated that PEDF competed with VEGF for binding to VEGF receptor 2, which may represent a new mechanism for PEDF activity. On the other hand, VEGF significantly downregulated PEDF expression in RCEC, but not in retinal Mü ller cells, suggesting a VEGF receptormediated process. These results suggest that the reciprocal regulation between VEGF and PEDF may play a role in angiogenic control. The decrease in PEDF levels in the retina is at least partially responsible for the increase in VEGF expression and subsequent vascular leakage and neovascularization in diabetes.

Wnt signaling is known to regulate multiple processes including angiogenesis, inflammation, and fibrosis. Here, we identified a novel inhibitor of the Wnt pathway, pigment epithelium-derived factor (PEDF), a multifunctional serine proteinase inhibitor. Both overexpression of PEDF in transgenic mice and administration of PEDF protein attenuated Wnt signaling induced by retinal ischemia. Furthermore, PEDF knockdown by small interfering RNA (siRNA) and PEDF knockout in PEDF ؊/؊ mice induced activation of Wnt signaling. PEDF bound to LRP6, a Wnt coreceptor, with high affinity (K d [dissociation constant] of 3.7 nM) and blocked the Wnt signaling induced by Wnt ligand. The physical interaction of PEDF with LRP6 was confirmed by a coprecipitation assay, which showed that PEDF bound to LRP6 at the E1E2 domain. In addition, binding of PEDF to LRP6 blocked Wnt ligand-induced LRP6-Frizzled receptor dimerization, an essential step in Wnt signaling. These results suggest that PEDF is an endogenous antagonist of LRP6, and blocking Wnt signaling may represent a novel mechanism for its protective effects against diabetic retinopathy.