Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York; and.
Am J Physiol Heart Circ Physiol. 2013 Sep 15;305(6):H829-42. doi: 10.1152/ajpheart.00761.2012. Epub 2013 Jul 5.
Redox status has emerged as critical in modulating stemness and lineage commitment in several precursor cell types. However, a role for redox genes, specifically NADPH oxidases (Nox), in cardiac precursor cells (CPCs) has not been established. We tested whether CPCs marked by type III receptor tyrosine kinase c-kit (c-kit(+)) exhibit a unique NADPH oxidase signature that confers precursor status and whether alterations in this profile are functionally linked to changes in lineage specification. Dihydroethidium (DHE) microfluorography indicated reduced basal reactive oxygen species (ROS) formation within early postnatal c-kit(+) CPCs. Real-time quantitative PCR revealed downregulation of ROS generator Nox2 and its subunit p67(phox) in c-kit(+) CPCs under basal conditions but upregulation of Nox2 and Nox4 over the course of differentiation. Adenoviral silencing of Nox2 and Nox4 increased expression of CPC markers c-kit and Flk-1 and blunted smooth and cardiac muscle differentiation, respectively, while overexpression of Nox2 and Nox4 significantly reduced c-kit expression. These changes were accompanied by altered expression of transcription factors regulating cardiac lineage commitment, Gata6 and Gata4, and cytokine transforming growth factor (TGF)-β1. Similar to other precursor cell types, RT(2)Profiler PCR Arrays revealed that c-kit(+) CPCs also exhibit enhanced antioxidant capacity at the mRNA level. In conclusion, we report that c-kit(+) CPCs demonstrate reduced Nox2 expression and ROS levels and that increases in Nox2 and Nox4 influence their differentiation into mature cells. We speculate that ROS generators Nox2 and Nox4, along with the antioxidant genes identified by PCR Arrays, may be novel targets in CPCs that could prove useful in cell-based therapy of the heart.
氧化还原状态已成为调节几种前体细胞类型的干性和谱系分化的关键。然而，氧化还原基因（特别是 NADPH 氧化酶[Nox]）在心脏前体细胞（CPC）中的作用尚未确定。我们测试了是否 III 型受体酪氨酸激酶 c-kit（c-kit(+)）标记的 CPC 表现出赋予前体细胞状态的独特 NADPH 氧化酶特征，以及这种特征的改变是否与谱系特化的变化在功能上相关。二氢乙啶（DHE）微量荧光法表明，早期出生后 c-kit(+) CPC 中基础活性氧（ROS）形成减少。实时定量 PCR 显示，在基础条件下，c-kit(+) CPC 中 ROS 生成器 Nox2 及其亚基 p67(phox)下调，但在分化过程中 Nox2 和 Nox4 上调。Nox2 和 Nox4 的腺病毒沉默增加了 CPC 标志物 c-kit 和 Flk-1 的表达，并分别削弱了平滑肌和心肌分化，而过表达 Nox2 和 Nox4 则显著降低了 c-kit 的表达。这些变化伴随着调节心脏谱系特化的转录因子 Gata6 和 Gata4 以及细胞因子转化生长因子（TGF）-β1 的表达改变。与其他前体细胞类型类似，RT(2)Profiler PCR 阵列显示，c-kit(+) CPC 还表现出增强的抗氧化能力在 mRNA 水平。总之，我们报告 c-kit(+) CPC 表现出 Nox2 表达和 ROS 水平降低，并且 Nox2 和 Nox4 的增加影响它们分化为成熟细胞。我们推测 ROS 生成器 Nox2 和 Nox4 以及 PCR 阵列鉴定的抗氧化基因可能是 CPC 的新靶点，这可能对心脏的基于细胞的治疗有用。
