Ravinarayanan Haribalaganesh

RETRACTED: PEDF prevents reactive oxygen species generation and retinal endothelial cell damage at high glucose levels

Oxidative stress is associated with the development of retinopathy in diabetes; dietary supplementations of multi-antioxidants have no beneficial effects clinically. An antioxidant which could specifically target pathogenesis of diabetic retinopathy is the need of the hour. Pigment epithelium-derived factor is a potent, endogenously produced, multifunctional factor (neurotrophic, anti-angiogenic, anti-inflammatory etc.,) in the eye which recently was also shown to possess anti-oxidative action. However, its anti-oxidative effect against high glucose-induced oxidative stress in retinal endothelial cells has not been investigated. Here, we examined its anti-oxidative effect on cell morphology, survival, reactive oxygen species generation, lipid peroxidation, antioxidant status and caspase-3 activation under high glucose conditions in bovine retinal endothelial cells (BRECs). Cells grown at 33 mM glucose in the presence of PEDF at concentrations 10-50 nM did not exhibit shrinkage. Pigment epithelium-derived factor inhibited the high glucose-induced rise in reactive oxygen species generation and lipid peroxidation. In these cells, reduced glutathione levels and mitochondrial and superoxide dismutase activities increased markedly while reactive oxygen species generation decreased significantly in presence of PEDF as compared with cells grown in the absence of PEDF under high glucose conditions (10-20 nM, *p < 0.01&**p < 0.001; 30-50 nM, ***p < 0.0001). Our results suggest that pigment epithelium-derived factor has an anti-oxidant effect in bovine retinal endothelial cells at a high glucose level. The action of pigment epithelium-derived factor not only varies with the cell type but also depends on its concentration and environmental conditions. Therefore, further studies are required to determine if pigment epithelium-derived factor might constitute a preventive and/or a curative treatment for retinal neovascularization.

Pigment epithelium-derived factor inhibits advanced glycation end products-induced retinal vascular permeability

Vascular hyperpermeability associated with retinal vascular leakage is known to occur in patients with diabetes, and contributes to endothelial barrier dysfunction. This study aimed to examine the effect of pigment epithelium-derived factor (PEDF) on advanced glycation end products (AGEs)-induced endothelial cell permeability. Cultured porcine retinal endothelial cell (PREC) was exposed to AGE-modified bovine serum albumin (AGE-BSA) and the endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the PREC monolayers. We found that AGE-BSA increased the RITC-dextran flux across a PREC monolayer and PEDF blocked the solute flux induced by AGE-BSA. In order to explore the underlying signaling mechanism of PEDF on the inhibitory effect of AGE-BSA-induced permeability, we demonstrate that PEDF could inhibit the AGE-BSA-induced permeability via phosphatidylinositol 3-kinase (PI3K)/Akt pathway. AGE-BSA also increased the endothelial cell permeability by stimulating the reactive oxygen species (ROS) generation via NADPH oxidase activity and Akt phosphorylation at Ser473. PEDF decreased ROS generation in AGE-BSA-exposed endothelial cells by suppressing the NADPH oxidase activity via down regulating the phosphorylation of p22(PHOx) at Thr147. This led to blockade of AGE-induction of PI3K/Akt activation in permeability. Furthermore, PEDF inhibited the AGE-BSA-induced permeability by increased expression of tight junction protein zona occludens-1(ZO-1), co-incident with an increase in barrier properties of endothelial monolayer. Together, our results indicate that PEDF could possibly act as potent anti-permeability molecule by targeting the PI3K/Akt signaling pathway by suppressing if NADPH oxidase mediated ROS generation and ZO-1 tight junction protein and it offers potential targets to inhibit the ocular related diseases such as diabetic retinopathy.

RETRACTED: Pigment epithelium-derived factor inhibits advanced glycation end-product-induced angiogenesis and stimulates apoptosis in retinal endothelial cells

AIMS The purpose of this study was to investigate the effect of pigment epithelium-derived factor (PEDF) on the signaling cascade in porcine retinal endothelial cells (PRECs) related to angiogenesis induced by advanced glycation end-products (AGEs). MAIN METHODS Endothelial cells were isolated from porcine retina by the enzymatic method. Immunocytochemistry was performed to confirm the identity of PRECs. The effect of AGEs and PEDF on cell viability was determined by the MTT assay. An in vitro wound-scratch assay was performed to study the migration of ECs, and in vitro tube formation was assessed by the on-gel assay system using an extracellular matrix. Inhibitor assays were carried out using LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and Akt inhibitor VIII. PI3K/Akt activity was assessed by transient transfection and western blot analysis. Induction of apoptosis by PEDF was determined by caspase-3 colorimetric assay and DNA fragmentation analysis. KEY FINDINGS Treatment of PRECs with AGE-bovine serum albumin (AGE-BSA) significantly increased the cell proliferation, migration and tube formation compared to non-glycated BSA. AGE-BSA mediates cell survival via the PI3K/Akt/FKHR-dependent pathway as evidenced by transient transfection and western blot analyses. Furthermore, PEDF significantly inhibited the proliferation, migration and tube formation, both in the presence and absence of AGE-BSA in PRECs. PEDF inactivated the AGE-BSA-induced PI3K/Akt/FKHR activity and induced apoptosis via caspase-3. SIGNIFICANCE The results reveal that PEDF inhibits AGE-BSA-induced PI3K/Akt/FKHR signaling in PRECs. Thus, PEDF has potent anti-angiogenic effects against AGE-induced angiogenesis and is suggested to be a promising molecule for the treatment of diabetic retinopathy.

Pigment epithelium-derived factor inhibits advanced glycation end-products-induced cytotoxicity in retinal pericytes.

AIM This study investigated the effects of pigment epithelium-derived factor (PEDF) on advanced glycation end-product (AGE)-induced cytotoxicity in porcine retinal pericytes and the signalling mechanism involved. METHODS Retinal pericytes were isolated from porcine eyes and characterized by immunocytochemistry. The effect of AGEs and PEDF on cell proliferation was determined by bromodeoxyuridine (BrdU) assay. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was analyzed by luminescence assay. Reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GSH) were determined by biochemical assays. Induction of apoptosis was determined by caspase-3 colorimetric assay and DNA fragmentation analysis. Src activity was assessed by transient transfection analysis, and the status of Src phosphorylation at Y419 was analyzed by a competitive ELISA method. RESULTS AGEs significantly increased intracellular ROS generation in pericytes via NADPH oxidase and induced cell death via caspase-3 enzyme activation, whereas PEDF increased cell proliferation in a dose-dependent manner. In addition, PEDF inhibited AGE-induced ROS generation by increasing levels of SOD and GSH, and also blocked the activation of caspase-3. Furthermore, PEDF induced cell survival via the Src pathway by Src phosphorylation at Y419, as evidenced by a pharmacological inhibitor and Src mutants. CONCLUSION These results suggest that PEDF abrogates AGE-induced oxidative stress and apoptosis in retinal pericytes via the Src pathway, thereby suggesting that PEDF is an effective therapeutic agent for the treatment of loss of pericytes in early diabetic retinopathy.

RETRACTED: Pigment epithelium-derived factor inhibits erythropoietin-induced retinal endothelial cell angiogenesis by suppression of PI3K/Akt pathway

Erythropoietin (EPO) plays a critical role in the vascular system and exhibits angiogenic activity in endothelial cells (ECs) such as stimulation of cell proliferation, migration and tube formation in vitro. EPO is the major regulator of cell proliferation and differentiation of erythroid precursors and there by preventing the apoptosis. Pigment epithelial derived factor (PEDF) is a potent anti-angiogenic factor whose effects are partially mediated through the induction of EC apoptosis. The mechanism of EPO and PEDF in retinal neovascularization has not been well documented yet. The effect of EPO and PEDF on cell proliferation was determined by MTT assay. In vitro wound-scratch assay was performed to study the migration of ECs and in vitro tube formation was assessed by the on-gel assay system using gelatin. Inhibitor assay was carried out using LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Further, PI3K/Akt activity was assessed by transient transfection assay using constitutively active (CA) and dominant negative (DN) Akt mutants. Dextran permeability assay was performed to determine the vascular permeability. We report that EPO stimulates EC proliferation, migration, tube formation and permeability whereas PEDF inhibits the EPO-induced ECs proliferation and permeability. Over expression of DN Akt blocked EPO stimulation of proliferation and permeability, while over expression of CA Akt rescues the inhibitory effect of PEDF on proliferation and permeability. These results demonstrate that PEDF may inhibit the EPO-induced proliferation and permeability via PI3K/Akt-dependent pathway.

Pigment epithelium–derived factor down regulates hyperglycemia-induced apoptosis via PI3K/Akt activation in goat retinal pericytes

Pigment epithelium–derived factor (PEDF) is a well-known protease inhibitor for angiogenesis in the eye, suggesting that loss of PEDF in eye is implicated in the pathogenesis of proliferative diabetic retinopathy. Since the role of PEDF in diabetic retinopathy is unclear, the effect of PEDF on different types of cells constituting the blood vessel has to be checked. Here, we have investigated the effects of PEDF under hyperglycemic conditions in retinal pericytes, isolated from goat’s eye and used to analyze the signaling pathway involved. High glucose increased the apoptotic cell death and intracellular reactive oxygen species generation, which was blocked on the addition of PEDF. PEDF was found to inhibit the apoptotic cell death and protect the cells via activating the PI3K/Akt pathway, which was analyzed with dominant negative Akt and constitutively active Akt–transfected cells. These results demonstrate that PEDF protects pericytes against the high glucose–induced apoptosis and dysfunction.