INVESTIGATING THE ROLE OF NOX NADPH OXIDASE SIGNALLING IN CELL MIGRATION AND ANGIOGENESIS OF ENDOTHELIAL COLONY-FORMING CELLS

Introduction. Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Emerging evidence supports a key role for NADPH oxidases in underlying angiogenic processes of these and other endothelial cells. Aims. To study the influence of Nox NADPH oxidases on the pro-angiogenic function of ECFCs. Methods. Human ECFCs isolated from umbilical cord blood were treated with pro-oxidant PMA and assessed in vitro, both under basal conditions and after siRNA knockdown of Nox4, a key endothelial NADPH oxidase isoform, alongside primary mature human aortic endothelial cells (HAoECs) for comparison, using an established scratch-wound assay as the functional end-point. Results. PMA (500nM for 8h) increased cell migration (control 18.6±2.8, PMA 32.7±6.6% wound closure; n=6, P<0.05) in a superoxide-dependent manner, as indicated by attenuation of this effect in the presence of PEG-SOD. Although HAoEC migration in response to PMA also tended to increase, this did not reach statistical significance. Notably, cell migration at 16h 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 the pro-migratory effect of PMA in ECFCs was potentiated after Nox4 knockdown (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). Conclusion. ECFC migration is enhanced by low concentrations of superoxide, to a greater extent compared to mature endothelial cells, and appears to be at least partly dependent upon NADPH oxidase, including a specific role for Nox4. Although, the precise contribution of endothelial Nox NADPH oxidases isoforms remains to be determined, it is clear that these findings may have significant implications for potential ECFC-based therapies for ischaemic disease, which is associated with an oxidative microenvironment.