NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment
NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment
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DOI：
10.1016/j.freeradbiomed.2011.04.029
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发表时间：
2011-07-15
影响因子：
7.4
通讯作者：
Wartenberg, Maria
中科院分区：
文献类型：
--
作者：
Bartsch, Caroline;Bekhite, Mohamed M.;Wartenberg, Maria
抗坏血酸（AA）促进胚胎干细胞（ES细胞）的心肌形成。在此，我们表明，AA处理小鼠ES细胞增强心脏分化伴随着NADPH氧化酶亚型NOX 2和NOX 4的上调，内皮型一氧化氮合酶（eNOS）的磷酸化，和环GMP（cGMP）的形成，表明活性氧（ROS）以及一氧化氮（NO）可能参与心肌发生。在整个胚状体以及分离的Flk-1阳性（Flk-1（+））心血管祖细胞中，在分化的早期阶段（第4-7天）观察到AA引起的ROS升高，并且在第10天不存在。相反，在与AA孵育后，在第4天不存在NO生成，并且在第7天和第10天增加。NADPH氧化酶抑制剂二苯基碘鎓（DPI）和夹竹桃苷，自由基清除剂N-（2-巯基丙酰基）-甘氨酸（NMPG）和依布硒啉，以及NOS抑制剂L-NAME，可减弱AA介导的心肌形成。通过短发夹RNA（shRNA）下调NOX 4导致心肌形成的显著抑制，并消除了在AA处理中观察到的MHC-β和MLC 2 v基因表达的刺激。我们的数据表明，AA刺激心肌细胞分化ES细胞的信号通路，涉及在心肌形成的早期阶段和NO在晚期阶段的ROS。(C)2011 Elsevier Inc. All rights reserved.
Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1(+)) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-beta and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis. (C) 2011 Elsevier Inc. All rights reserved.