The NADPH oxidase Nox4 promotes endothelial differentiation from murine induced-pluripotent stem cells

Nox4 is the only constitutively active NADPH oxidase, producing H 2 O 2 . It is highly expressed in endothelial cells, where it plays an important role in differentiation. It is therefore hypothesized that Nox4 induces differentiation in endothelial cells and by deficiency preserves stemness. Using Yamanaka factors, MEFs from wildtype and Nox4-/- mice were reprogramed into iPSCs and then differentiated into endothelial cells. In the course of differentiation, Nox4 expression increased in wildtype cells. Absence of Nox4 resulted in a prolonged expression of stem cell markers and in a diminished expression of endothelial markers in differentiated cells. On the functional level a lower tube formation and sprouting capacity of Nox4-deficient ECs was observed. Using an in vivo matrigel plug assay, a lower capacity of Nox4-/- iPSC-ECs integrated in a newly formed vascular network. As a potential mechanism we observed, increased H3K27me3 in Nox4-/-, which leads to decreased CD31 and VEGFR2 expression. Demethylation of this histone site is mediated by JmjD3, which is not differentially expressed in both cell strains. A BIAM switch assay revealed that JmjD3 was less oxidized in Nox4-/- than in WT cells. Therefore we conclude that Nox4 oxidizes and activates JmjD3. In conclusion Nox4 via an epigenetic modification promotes the differentiation of endothelial cells out of iPSCs.