Abstract 1302: Caspase-8-dependent Cleavage Of The E3 Ubiquitin Ligase Cbl-b Regulates Integrin Expression And Adhesion Capacity In Endothelial Progenitor Cells
Endothelial progenitor cells (EPC) are used for therapeutic enhancement of neovascularization in clinical trials. The vasculogenic potential of progenitor cells depends on an appropriate retention of the infused cells to the ischemic tissue. However, cell-autonomous mechanisms regulating the vasculogenic potential of progenitor cells are not well understood. Beyond cell death signals, Caspase enzymes regulate cell morphology and migration in many cell types, and the isoform Caspase-8 is essential for embryonal vasculogenesis in K.O. mice. Here, we characterize the role of Caspase-8 as a critical protease for EPC function. Caspase-8-specific inhibition abrogated the ex vivo formation of EPC from human peripheral blood (98% inhibition, P<0.05). In vitro and in vivo studies with bone marrow mononuclear cells derived from inducible Caspase-8-deficient mice revealed an essential role of Caspase-8 for EPC formation and neovascularization enhancing capacities of progenitor cells. Caspase-8 inhibition disables EPC adhesion to a fibronectin matrix and decreases the cell surface expression of the fibronectin receptor subunit integrin α5. Further studies showed that the E3 ubiquitin ligase Cbl-b, a negative regulator of cell adhesion molecules including integrin α5, is present in EPC at a low protein abundance under basal conditions, but markedly increases upon Caspase-8 inhibition. In vitro assays and overexpression studies in intact cells confirmed Caspase-8-dependent degradation of Cbl-b, providing a potential prerequirement for Caspase 8-regulated adhesion. Indeed, neovascularization of matrigel plugs was enhanced in mice lacking Cbl-b (n=7, P<0.05). Moreover, progenitor cells isolated from Cbl-b-deficient mice showed an increased expression of integrin α5,were insensitive towards Caspase-8 inhibition, and showed enhanced improvement of ischemic hind limb neovascularization compared to wild type mice (n=6, P<0.05). In summary, Caspase-8 essentially regulates EPC function as an opponent of Cbl-b. Cbl-b degradation in the presence of active Caspase-8 prevents the down-regulation of integrin α5 and is associated with an enhanced vasculogenic activity of progenitor cells.