8 Nadph oxidase 4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes postischemic revascularisation

Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy. CB-ECFCs are subject to regulation by reactive oxygen species (ROS) and here we specifically investigated the role of the major ROS-producing enzyme, NOX4 NADPH oxidase, which is highly expressed in CB-ECFCs, in their vasoreparative function. Specifically, cells were assessed (1) in vitro under basal conditions, with pro-oxidative stimuli or modified NOX4 expression, using migration and tubulogenesis assays, and (2) in vivo using an established model of experimental hindlimb ischaemia in SCID mice to assess revascularisation. Pro-oxidant phorbol 12-myristate 13-acetate (PMA) increased cell migration and tubulogenesis, which was inhibited by the pan-Nox inhibitor VAS2870. Basal tube formation was also reduced by VAS2870, highlighting that function is enhanced by endogenous superoxide in a NOX-dependent manner. Complementary RT-PCR and Western blotting analysis found NOX4 to be the most highly expressed isoform in CB-ECFCs, with augmented expression confirmed following PMA treatment. NOX4-knockdown (migration: control siRNA 174±18, Nox4 siRNA 96±23 arbitrary units/au; n=9, p in vitro function, respectively. In a murine model of hindlimb ischaemia administration of NOX4-deficient (control siRNA 0.71±0.27, Nox4 siRNA 0.39±0.17 ischaemic/control limb ratio; n=6, p