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.

中文翻译：

---

Nox4 通过染色质重塑促进内皮分化

基本原理

Nox4 是一种组成型活性 NADPH 氧化酶，持续产生低水平的 H ₂ O ₂。因此，Nox4 有助于细胞稳态和长期过程，例如分化。内皮细胞中Nox4的高表达与干细胞中Nox4的低丰度形成鲜明对比，干细胞相应地具有低水平H ₂ O ₂的特征。我们假设 Nox4 是内皮分化的主要贡献者，在分化过程中被诱导，并促进所得内皮细胞的稳态。

客观的

确定 No×4 在小鼠诱导多能干细胞 (miPSC) 分化为内皮细胞 (EC) 中的作用。

方法和结果

miPSC 由小鼠胚胎野生型 (WT) 和 Nox4 ^−/−成纤维细胞产生，通过 BMP4 和 VEGF 刺激分化为内皮细胞 (miPSC-EC)。在此过程中，Nox4 表达增加，Nox4 敲除延长了多能性标记的丰度，而在分化 Nox4 耗尽的 iPSC 时，内皮标记的表达延迟。最终，Nox4 缺陷细胞中 iPSC-EC 的血管生成能力降低，表明 Nox4 的缺失降低了所达到表型的稳定性。作为底层机制，我们将 JmjD3 确定为 Nox4 的氧化还原靶标。缺乏 Nox4 的 iPSC-EC 显示组蛋白去甲基化酶 JmjD3 的核丰度较低，导致组蛋白 3 (H3K27me3) 的三甲基化增加，这是参与分化的多个基因的抑制标记。

结论

Nox4 通过 JmjD3 的氧化和随后 H3K27me3 的去甲基化促进 miPSC 分化为 EC，从而迫使内皮分化和稳定性。