Nox4 promotes endothelial differentiation through chromatin remodeling.

RATIONALE: Nox4 is a constitutively active NADPH oxidase that constantly produces low levels of H2O2. Thereby, Nox4 contributes to cell homeostasis and long-term processes, such as differentiation. The high expression of Nox4 seen in endothelial cells contrasts with the low abundance of Nox4 in stem cells, which are accordingly characterized by low levels of H2O2. We hypothesize that Nox4 is a major contributor to endothelial differentiation, is induced during the process of differentiation, and facilitates homeostasis of the resulting endothelial cells. OBJECTIVE: To determine the role of No×4 in differentiation of murine inducible pluripotent stem cells (miPSC) into endothelial cells (ECs). METHODS AND RESULTS: miPSC, generated from mouse embryonic wildtype (WT) and Nox4-/- fibroblasts, were differentiated into endothelial cells (miPSC-EC) by stimulation with BMP4 and VEGF. During this process, Nox4 expression increased and knockout of Nox4 prolonged the abundance of pluripotency markers, while expression of endothelial markers was delayed in differentiating Nox4-depleted iPSCs. Eventually, angiogenic capacity of iPSC-ECs is reduced in Nox4 deficient cells, indicating that an absence of Nox4 diminishes stability of the reached phenotype. As an underlying mechanism, we identified JmjD3 as a redox target of Nox4. iPSC-ECs lacking Nox4 display a lower nuclear abundance of the histone demethylase JmjD3, resulting in an increased triple methylation of histone 3 (H3K27me3), which serves as a repressive mark for several genes involved in differentiation. CONCLUSIONS: Nox4 promotes differentiation of miPSCs into ECs by oxidation of JmjD3 and subsequent demethylation of H3K27me3, which forced endothelial differentiation and stability.

The NADPH Oxidase Nox4 mediates tumour angiogenesis.

AIM: The aim of this work was to identify the role of the NADPH oxidase Nox4 for tumour angiogenesis in a slow-growing tumour model in mice. METHODS: Tumour angiogenesis was studied in tumours induced by the carcinogen 3-methylcholanthrene (MCA) in wild-type and Nox knockout mice. Mice were killed when the tumour reached a diameter of 1.5 cm and tumour tissue was used for histological and molecular analysis. RESULTS: 3-methylcholanthrene induced fibrosarcoma in wild-type, Nox1y/-, Nox2y/- and Nox4-/- mice. Histological analysis of vessel density using anti-CD31 staining showed a significant 38% reduction in tumour vascularization in fibrosarcomas of Nox4-/- mice. In contrast, tumour angiogenesis was doubled in Nox1 knockout mice, whereas knockout of Nox2 had no effect on tumour-vessel density. As underlying mechanisms, we identified a defect in hypoxia signalling in Nox4-/- mice. Hypoxia-inducible factor 1-alpha (Hif-1α) accumulation in the tumours was attenuated as was the expression of the Hif-1α-dependent pro-angiogenic genes vascular endothelial growth factor-A, glucose transporter 1 and adrenomedullin. CONCLUSION: By regulating the tumour-vessel density through stabilization of Hif-1α and induction of VEGF expression, Nox4 promotes tumour angiogenesis and may represent a novel target for anti-angiogenic tumour therapy.