Introduction Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Interestingly, recent evidence supports a key role for NADPH oxidases in underlying angiogenic processes of these and other endothelial cells.
Aims To study the effects of Nox NADPH oxidases on pro-angiogenic function of ECFCs.
Methods Human ECFCs isolated from umbilical cord blood were treated with pro-oxidant PMA and assessed in vitro, both basally and after siRNA knockdown of Nox4, a key endothelial isoform, alongside primary mature human aortic endothelial cells (HAoECs).
Results PMA (500 nM for 8 h) increased cell migration (control 18.6 ± 2.8, PMA 32.7 ± 6.6% closure; n = 6, P < 0.05) in a superoxide-dependent manner, as indicated by attenuation with PEG-SOD. HAoEC migration in response to PMA tended to increase but did not reach statistical significance. Cell migration at 16 h was reduced by Nox4 knockdown in ECFCs (control siRNA 53.4 ± 3.5, Nox4 siRNA 35.1 ± 4.9% closure; n = 3 P < 0.05), but not in HAoECs, whilst Nox4 knockdown potentiated the stimulatory effect of PMA in ECFCs (control siRNA 53.4 ± 3.5, +PMA 61.5 ± 3.2% closure; n = 3, P = NS; Nox4 siRNA 35.1 ± 4.9, +PMA 53.0 ± 4.9% closure; n = 3, P < 0.05). Notably, Nox4 mRNA expression (real-time RT-PCR) was significantly increased following PMA stimulation (control 1.05 ± 0.05, PMA 1.46 ± 0.14 arbitrary units relative to β-actin; n = 7, P < 0.05).
Conclusion ECFC migration is enhanced by low concentrations of superoxide, and to a greater extent as compared to mature endothelial cells, and appears to be at least partly dependent upon Nox4 NADPH oxidase. These findings may have significant implications for potential ECFC-based therapies for ischaemic disease, which is associated with an oxidative microenvironment.
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