Angiopoietin1 (Ang1) suppresses hydrogen peroxide(H₂O₂)–induced SEK1/JNK phosphorylation through PI3–kinase/Akt pathway

Abstract

Abstract: : Purpose: Oxidative stress plays an important role in exacerbation of diabetic retinopathy, which might results in capillary acellularity and nonperfusion area, whereas Ang1 is a prosurvival factor for vascular endothelial cells. These evidences prompted us to investigate whether and how Ang1 attenuates H₂O₂–induced apoptosis of porcine retinal endothelial cells (PREC). Methods: PREC were cultured in DMEM supplemented with 10% porcine serum and treated with H₂O₂ with or without the pretreatement of Ang1. Phosphorylation of Jun N–terminal kinase (JNK) and SAPK/Erk kinase (SEK1) was evaluated by Western blot analysis with antiphospho–tyrosine specific antibody. The role of Akt and JNK was determined by overexpressing wild type (WT), constitutively–active (CA) or dominant–negative (DN) mutants using adenovirus–mediated gene transfer, and the role of PI3–kinase was by its inhibitors, wortomannin and Ly294002. PREC apoptosis induced by H₂O₂ was assessed by TUNEL assay and caspase–3 activity. Results: H₂O₂ increased TUNEL positive cells and caspase–3 activity, and DN–JNK decreased them significantly. Ang1 decreased TUNEL positive cells and caspase–3 activity increased by H₂O₂ in a dose dependent manner. H₂O₂ induced JNK phosphorylation in a time dependent manner, and, with the pretreatment of Ang1, the JNK phosphorylation was suppressed significantly (P<0.001). The inhibitors of PI3–kinase (wortmannin and Ly294002) reversed the suppressive effects. Concerning to Akt, DN–Akt reversed the suppressive effects of Ang1 on JNK phosphorylation and CA–Akt decreased H₂O₂–induced JNK phosphorylation without Ang1. H₂O₂ induced phosphorylation of SEK1, upstream of JNK, in a time dependent manner, which was significantly inhibited by Ang1 (P<0.001). The inhibitory effect on SEK1 phosphorylation was also reversed by inhibitors of PI3–kinase and DN–Akt. Conclusions: These data indicate that H₂O₂ increased PREC apoptosis through JNK, whereas Ang1 attenuates the apoptosis. Ang1 also suppresses H₂O₂–induced SEK1/JNK phosphorylation through PI3–kinase/Akt pathway. We demonstrate a novel mechanism through which Ang1 attenuates endothelial cell apoptosis induced by oxidative stress.

Keywords: signal transduction • oxidation/oxidative or free radical damage • cytokines/chemokines