P80	Caspase-8 activity is essential for endothelial progenitor cell adherence.
L.Rössig, E.Chavakis, C.Urbich, A.Zeiher, S.Dimmeler
Molecular Cardiology, JW Goethe University, Frankfurt/Main, DE.

Bone marrow-derived endothelial progenitor cells (EPC) contribute to post-ischemic neovascularization. Caspases are mainly recognized as proteases involved in apoptosis signaling. Nevertheless, low grade caspase activation also regulates cellular functions independent of cell death. Since caspase-8-deficient mice display an embryonally lethal angiogenesis defect, we determined the role of caspase activity in EPC. Surprisingly, the broad spectrum caspase inhibitor zVAD (100 µM) completely abolished the ex vivo generation of fibronectin-adherent, DiI-LDL-uptaking EPC from human peripheral blood mononuclear cells (MNC). Among various caspase inhibitory compounds with selectivity for individual caspase isoforms, only the caspase-8 inhibitor zIETD (100 µM), but not the caspase-3/-6-selective inhibitor zDQMD (100 µM), suppressed EPC numbers (zIETD: 1±2 % of control, mean±SD; p<0.001; zDQMD: 105±33 %, p<0.01 vs. zIETD). Likewise, zIETD, but not zDQMD, inhibited the generation of EPC from CD34-positive bone marrow stem cells, suggesting that caspase-8 is specifically required for EPC formation. Since the cultivation of EPC critically depends on extracellular matrix adhesion, caspase activity may regulate adhesion via interacting with the cytoskeleton. Indeed, while native circulating MNC predominantly express the catalytically inactive splice variant caspase-8L, adherent EPC after 3 days in culture express full-length caspase-8 mRNA. By flow cytometry, 69±11 % of adherent EPC contain active caspase-8 compared to only 7±3 % caspase 8-positive cells among the non-adherent MNC suspension, from which EPC derive (p<0.05). In the presence of zVAD for 3 hours, 74±4 % of adherent EPC detached into suspension, whereas zDQMD had no effect on EPC adherence (103 % of control). To test the causal involvement of caspase-8 activity, cell-matrix adhesion of EPC to fibronectin-coated plates was measured after pre-treatment with caspase inhibitors for 3 hours. Again, zIETD (76±11 % of control, p<0.05), but not zDQMD (105±1 %, p<0.05 vs. zIETD), reduced EPC adhesion. In contrast with EPC, zVAD or zIETD did not induce detachment of mature human umbilical vein endothelial cells, suggesting that the caspase-8-dependent adhesion mechanism is specific for EPC. These data disclose an unexpected role for caspase-8 being essential for EPC to maintain an adherent phenotype.

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