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8 Nadph oxidase 4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes postischemic revascularisation
  1. Karla M O’Neill1,
  2. David C Campbell1,
  3. Kevin S Edgar1,
  4. Arya Moez1,
  5. Kiran J McLoughlin1,
  6. Christina L O’Neill1,
  7. Margaret Dellett1,
  8. Ciarán J Hargey1,
  9. Rawan A Abudalo1,
  10. Eleanor K Gill1,
  11. Philip Doyle1,
  12. Tinrui Toh1,
  13. Joshua Khoo1,
  14. Cian M McCrudden2,
  15. Coy Brunssen3,
  16. Henning Morawietz3,
  17. Mervin C Yoder4,
  18. Alan W Stitt1,
  19. Andriana Margariti1,
  20. Reinhold J Medina1,
  21. David J Grieve1
  1. 1Centre for Experimental Medicine, Wellcome-Wolfson Institute, Queen’s University Belfast
  2. 2School of Pharmacy, Queen’s University Belfast
  3. 3Division of Vascular Endothelium and Microcirculation, Department of Medicine III, Medical Faculty and University Clinics Carl Gustav Carus, Technische Universität Dresden, Germany
  4. 4Department of Paediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA


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.

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