NOX4 NADPH oxidase signalling is a key determinant of angiogenic function of cord-blood endothelial colony-forming cells in hypoxia

Objective: Myocardial infarction leads to adverse cardiac remodelling, driven largely by tissue ischaemia and hypoxia. Whilst cord-blood-derived endothelial colony-forming cells (CB-ECFCs) show clear potential to promote angiogenesis in post-infarction, their therapeutic application is limited by reduced efficacy in ischaemic tissue. This study aimed to define the specific impact of hypoxia in CB-ECFC-mediated neovascularisation.

Methods: CB-ECFCs (typically n = 9, 3 clones) were exposed to hypoxia (1% O2) or normoxia (21% O2) for 48h prior to analysis of angiogenic function (3D Matrigel assay), gene expression (qRT-PCR, Western blot, microarray), reactive oxygen species (H2O2, DHE assay), and gene modification (plasmid overexpression [OE], siRNA knockdown [KD]).

Results: CB-ECFCs showed impaired tubulogenesis in hypoxia (tube area: normoxia 19.1 + 4.0, hypoxia 2.5 + 0.7 µm2; p < 0.001) which was associated with impaired metabolism (MTT, 59% vs. normoxia, p < 0.001) with maintained viability, and reduced expression of NOX4 and other functional genes (eNOS, HMOX1, VEGFR2), together with decreased H2O2 (normoxia 0.46 + 0.03, hypoxia 0.08 + 0.02 arbitrary units; p < 0.001) and increased superoxide (normoxia 0.33 + 0.02, hypoxia 0.44 + 0.01 arbitrary units; p < 0.001). Parallel microarray analysis highlighted NOX4 as central to the altered signalling observed in hypoxic CB-ECFCs and identified PLAC8 as the upstream regulator. Whilst NOX4 OE partially restored angiogenic function in hypoxic CB-ECFCs (tube area: empty vector 18.7 + 2.1, OE 31.1 + 2.5 µm2; p < 0.05), PLAC8 KD reversed hypoxia-induced dysfunction (tube area: non-targeting control 5.6 + 2.0, KD 26.8 + 4.6 µm2; p < 0.01) with activation of NOX4 signalling.

Conclusions: Together, these findings highlight impaired NOX4 signalling as a key determinant of CB-ECFC angiogenic dysfunction in hypoxia, whilst PLAC8 may therefore represent a novel target to enhance NOX4-dependent vasoregenerative capacity with potential to reduce progression of adverse post-infarction cardiac remodelling.