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T. Murakami, H. Takagi, K. Suzuma, I. Suzuma, H. Ohashi, D. Watanabe, T. Ojima, M. Kurimoto, N. Yoshimura; Angiopoietin1 Attenuates H2O2–Induced SEK1/JNK Phosphorylation Through the Pi3–Kinase/Akt Pathway in Porcine Retinal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):957.
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© ARVO (1962-2015); The Authors (2016-present)
High glucose level induces oxidative stress, which could play an important role in the disturbance of retinal vascular cells and the progression of diabetic retinopathy. We reported Angiopoietin1 (Ang1) decreases the apoptosis of porcine retinal endothelial cells (PREC) induced by hydrogen peroxide (H2O2). Furthermore, we analyzed detailed mechanisms of anti–apoptotic effects of Ang1.
We cultured PRECs on collagen coated dish. TdT–mediated dUTP Nick End Labeling (TUNEL) positive cells were counted and the caspase–3 activity was measured for the quantitation of apoptosis. In order to evaluate the intracellular signal transduction, Western blotting with anti–phospho–specific Akt, SAPK/Erk kinase (SEK1) and c–Jun N–terminal kinase (JNK) antibodies was performed. For the assessment of the kinase activities, we used specific inhibitors for phosphatidylinositol (PI)3–kinase, transfection of adenoviruses encoding dominant negative (DN) mutants or gene–silencing with siRNA.
H2O2 increased apoptosis of endothelial cells through JNK activation, whereas Ang1 inhibited H2O2–induced apoptosis. Ang1 inhibited H2O2–induced SEK1/JNK phosphorylation. The inhibition of the SEK1/JNK phosphorylation was reversed by inhibitors of PI3–kinase and DN–Akt, suggesting that the inhibition was through the PI3–kinase/Akt pathway. SEK1 was reported to be phosphorylated at Ser80 and inhibited. Ang1 increased the phosphorylation of SEK1 at Ser80, and the phosphorylation was attenuated by inhibitors of PI3–kinase, DN–Akt or siRNA against Akt. This suggests that the phosphorylation was mediated via the PI3–kinase/Akt pathway.
These results demonstrate that Ang1 attenuates H2O2–induced SEK1/JNK phosphorylation through the PI3–kinase/Akt pathway and inhibits the apoptosis of endothelial cells to oxidative stress.
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