TRAIL-induced apoptosis in human vascular endothelium is regulated by phosphatidylinositol 3-kinase/Akt through the short form of cellular FLIP and Bcl-2

Background: Apoptosis of vascular endothelial cells plays a central role in angiogenesis and atherosclerosis. This study investigates the molecular mechanisms of endothelial apoptosis induced by tumor necrosis factor- related apoptosis- inducing ligand ( TRAIL) following inhibition of phosphatidylinositol 3- kinase ( PI3K). It examines downstream regulation and activation of the extrinsic and intrinsic pathways. Methods and Results: By flow cytometry, TRAIL receptors 2 and 3 were present to a greater extent than receptors 1 and 4. TRAIL reduced cell numbers in combination with the PI3K inhibitor LY 294002. TRAIL ( 100 ng/ ml) with LY 294002 ( 20 mu mol/ l) activated the extrinsic pathway, causing progressive cleavage of caspase- 8 and caspase- 3. Activation of the intrinsic pathway proceeded by release of mitochondrial factors Smac/ DIABLO and cytochrome c, and caspase- 9 cleavage. LY 294002 reduced phosphorylated Akt ( pAkt), with early loss of the short form of cellular FLIP ( cFLIP(S)) and concurrent reduction of Bcl- 2. Treatment with small interfering RNA against PI3K also reduced c- FLIPS and Bcl- 2, and cotreatment with TRAIL triggered caspase- 3 cleavage. Conclusions: This study details the molecular regulation of TRAIL- induced apoptosis in vascular endothelium. Inhibition of PI3K reduces p- Akt, with concurrent reductions in c- FLIPS and Bcl- 2, and so renders endothelium sensitive to TRAIL- induced apoptosis through the extrinsic and intrinsic pathways. Copyright (C) 2005 S. Karger AG, Basel.