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C.J. Barnstable, L. Chen, S. Sawant, S.S. Zhang, J. Tombran–Tink; Induction Of Endothelial Cell Apoptosis By PEDF Is P38 Dependent . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5335.
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Previously we reported that PEDF blocks the protective effect of VEGF on endothelial cells. However the molecular mechanism of this effect is not clear. It has been proposed that PEDF induces endothelial cell death through stimulation of Fas and FasL, but Fas knockout mice still show PEDF–induced endothelial cell apoptosis. The purpose of the present experiments was to examine the pathways by which PEDF induces apoptosis in endothelial cells.
Human umbilical vein endothelial cells (HUVECs) were grown in medium containing 0.2% serum and treated with PEDF (100ng/ml) or VEGF (5ng/ml). Cell viability, proliferation, and apoptosis assays were used to investigate the function of VEGF and PEDF on HUVECS. The levels of phosphorylated p38, and cleavage of caspases 3, 8, and 9 were determined with specific antibodies using western blotting methods. A p38 MAP kinase assay was performed with ATF–2 fusion protein as a substrate.
Treatment of HUVECs with PEDF increased the phosphorylation of p38 and JNK1, two enzymes associated with cell death. PEDF increased the activity of p38 as judged by an increase in phosphorylation of the ATF–2 fusion protein, a specific p38 substrate. PEDF induced activation of the key apoptosis enzyme, caspase 3. This activation was dependent upon p38 phosphorylation because inhibition of p38 phosphorylation by the specific p38 inhibitor, SB 203580, blocked caspase 3 cleavage. To determine the pathways leading to caspase 3 activation we measured PEDF–induced cleavage of caspase 8 and caspase 9. Treatment of HUVEC cells for 3 hr with PEDF (100ng/ml) led to increased cleavage of both caspase 8 and caspase 9.
PEDF promotes apoptosis in HUVEC cells by activating both the intrinsic or mitochondrial–stimulated apoptosis pathway, and the FAS/FASL stimulated pathways of caspase 3.
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