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<0.001, tube formation: control siRNA 6.9±1.2, Nox4 siRNA 4.6±0.7 au; n=9, p<0.001) and -overexpression (migration: EV 149±21, OE 204±25 au; n=6, p<0.01; tube formation: EV 732±33, OE 1024±71 au; n=6, p<0.01) reduced and potentiated 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<0.05) and -overexpressing (EV 0.34±0.09, OE 0.61±0.28 ischaemic/control limb ratio; n=8, p<0.05) CB-ECFCs into mouse ischaemic hindlimbs inhibited and promoted revascularisation whilst regulating host eNOS-associated angiogenic signalling. Together, these findings indicate a key role for NOX4 in CB-ECFCs, highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and promotion of effective post ischaemic revascularisation.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.