Investigating the role of NADPH oxidase 4 (NOX4) in differentiation and function of induced pluripotent stem cell-derived endothelial cells

Induced pluripotent stem cell-derived endothelial cells (iPS-ECs) hold clear potential for therapeutic angiogenesis in ischaemic diseases, although many challenges remain such as enrichment and expansion of homogenous, mature, highly proliferative and functional ECs. It is well established that reactive oxygen species (ROS), which are centrally involved in ischaemic disease, are important regulators of EC and stem cell biology, with recent evidence specifically highlighting NADPH oxidases as a key source. In this thesis, we investigated the role of NADPH oxidase-dependent redox signalling in differentiation of mouse and human iPS-ECs, and provide evidence implicating ROS derived from the NOX4 isoform during the differentiation process, which is likely to be important when considering their therapeutic potential. Our findings show that miPS-EC differentiation is associated with induction of NOX2 and NOX4 isoforms together with increased ROS generation and general antioxidant suppression. We found that NOX4 promotes iPS-EC differentiation in a ROS-dependent manner, whilst opposite effects are evident with NOX4 knockdown. Mechanistically, we demonstrated that NOX4 specifically regulates eNOS signalling in miPS-ECs and hiPS-ECs and antioxidant expression via activation of NRF2 and downstream pro-angiogenic intermediates including STAT3, JUN, NFⱩB, ERK, Akt and Jnk. Nevertheless, we highlighted the differentiation process as a highly oxidative event mediated by superoxide generation and inhibition of NRF2 and downstream antioxidant genes. Our data also reveal that oxidative stress may be damaging and underlie reduced differentiation efficiency, which was improved by the pan-NADPH oxidase inhibitor, VAS2870, whilst increased ROS levels may be important in directing early differentiation, before subsiding as derived ECs mature. Taken together, the data in this thesis indicate that NOX4 NADPH oxidase plays a significant role in regulating iPS-EC differentiation and function, whilst highlighting complexity of ROS signalling and importance of defining key aspects of iPS-EC signalling towards their clinical application for treatment of ischaemic cardiovascular disease.