Abstract 997: A Novel Apoptosis-Unrelated Role of Caspase-8 in Endothelial Progenitor Cell-Mediated Neovascularization
Endothelial progenitor cells (EPC) are required for vasculogenesis during embryonic and adult neovascularization. Caspases belong to a family of pro-apoptotic proteases. However, low grade Caspase-8 activity also is involved in differentiation and proliferation. Importantly, Caspase-8 deficiency in mice is embryonically lethal with defective yolk sac vasculogenesis. Therefore, we hypothesized that Caspase-8 could be required for EPC function and maturation. The specific Caspase-8 inhibitor zIETD (100 μM) abrogated the ex vivo formation of EPC from human peripheral blood (98% inhibition, p<0.05), whereas selective inhibitors of other caspase isoforms were not effective. Likewise, ex vivo EPC formation was also significantly reduced in cells isolated from an inducible Caspase-8 K.O. model (Casp8-deficient cells: 4±4 EPC; Casp8Flox/+: 12±2 EPC). Bone marrow mononuclear cells from Caspase-8-deficient mice had a severely reduced capacity for enhancing neovascularization when transplanted into mice following femoral artery ligation (47±16 % inhibition). To define the underlying mechanism, we determined the adhesive properties of EPCs. Caspase-8 inhibition completely inhibited EPC adhesion to fibronectin (97±1% inhibition, p<0.05). In line with these findings, EPCs showed a strong decrease in cell surface expression of the fibronectin receptor subunit integrin α5 after Caspase-8 inhibition in vitro (74%, p<0.01) or when isolated from Caspase-8-deficient mice (64±1% reduction, p<0.05). Accordingly, Caspase-8 blockade led to a posttranslational modification and degradation of integrin α5. Since the E3 ubiquitin ligase Cbl-b ubiquinates and stimulates degradation of integrins, we measured Cbl-b protein expression. Caspase-8 inhibition augmented Cbl-b protein levels indicating cleavage of Cbl-b by Caspase-8. Indeed, cleavage assays confirmed that Cbl-b is a target of Caspase-8 in in vitro and in intact cells. These data suggest that Caspase-8 is critical for EPC adhesion and function in neovascularization. Low grade Caspase-8 activity cleaves the central negative regulator Cbl-b, whereas inhibition of Caspase-8 increases Cbl-b protein leading to integrin α5 degradation, which is required for EPC adhesion and maturation.