Out of the seven known isoforms of NADPH oxidase, Nox4 is the only constitutively active one, producing hydrogen peroxide. It is highly expressed in endothelial cells, where it plays an important role in proliferation and differentiation. It is therefore hypothesized that Nox4 induces differentiation in endothelial cells and by deficiency preserves stemness.
Using Yamanaka factors, mouse embryonic fibroblasts (MEFs) from wild-type and Nox4-/-, mice were reprogramed into induced pluripotent stem cells (iPSCs) and then differentiated into endothelial cells by VEGF and BMP4 stimulation. In the course of differentiation, Nox4 expression increased in wild-type cells. The absence of Nox4 resulted in a prolonged expression of stem cell markers and in a diminished expression of endothelial markers in differentiated cells such as CD31, Flk-1 and eNOS. On the functional level, we observed a lower tube formation and sprouting capacity of Nox4-deficient iPSC-derived endothelial cells. Using an in vivo matrigel plug assay in mice, a lower capacity of Nox4-/- iPSC-ECs to integrate into a newly formed vascular network was observed. As a potential mechanism, we observed increased H3 presents K27 triple methylation after deletion of Nox4. Demethylation of this histone site is mediated by JmjD3, its methylation by Ezh2, both of which were not differentially expressed in both cell strains. BIAM switch assay, however, revealed that JmjD3 was less oxidized in Nox4-/- than in WT cells. Therefore we conclude that Nox4 oxidizes and thereby activates JmjD3. Consequently the knockout of Nox4 results in an increased level of H3K27me3, which prevents the proper expression of CD31 and Flk-1.
In conclusion, we suggest Nox4 promotes the differentiation of endothelial cells out of iPSCs via an epigenetic modification.