Nitrosative Stress Plays an Important Role in Wnt Pathway Activation in Diabetic Retinopathy

Abstract

Diabetes is associated with nitrosative stress in multiple tissues. Overactivation of the Wnt pathway has been shown to play a pathogenic role in diabetic retinopathy (DR). The purpose of this study was to investigate whether nitrosative stress contributes to aberrant activation of Wnt signaling in diabetes. Nitrosative stress induced by peroxynitrite (PN), 4-hydroxynonenal (HNE), or high glucose (HG) in retinal cells was assessed by a dichlorofluorescein fluorescence assay or by Western blot analysis and enzyme-linked immunosorbent assay of 3-nitrotyrosine (3-NT). These nitrosative stress inducers activated the canonical Wnt pathway, as shown by Western blot analysis of phosphorylated low-density lipoprotein receptor-related protein 6 (pLRP6), total and nuclear β-catenin levels, Luciferase reporter assay, and expression of the Wnt target genes intercellular adhesion molecule 1 (ICAM-1) and vascular endothelial growth factor (VEGF). Uric acid (UA), a PN scavenger, and 5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinato Iron III Chloride (FeTPPS), a PN decomposition catalyst, suppressed Wnt signaling and ICAM-1 and VEGF overexpression induced by PN, HNE, and HG. Furthermore, UA and FeTPPS also inhibited Wnt signaling induced by the Wnt ligand. In streptozotocin-induced diabetic rats, retinal levels of 3-NT, β-catenin, nuclear β-catenin, pLRP6, VEGF, and ICAM-1 were markedly increased. UA treatment for 6 weeks ameliorated diabetes-induced Wnt signaling in the diabetic rat retina. The UA treatment also decreased inflammatory cell infiltration and extraverted serum albumin in the perfused retina of diabetic rats, suggesting decreased retinal inflammation and vascular leakage. Nitrosative stress in diabetes contributes to Wnt pathway activation in the retina, and Wnt signaling may mediate the pathogenic effects of nitrosative stress in DR.