Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H₂O₂

Abstract

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.

Effect of inhibitors of NADPH oxidase complex and mitochondrial ATP‐sensitive potassium channels on generation of dopaminergic neurons from neurospheres of mesencephalic precursors

“…The present results suggest that Apocynin and 5-HD act by stimulating differentiation of mesencephalic precursors toward the DA phenotype. The results are consistent with those of previous studies, which have suggested that the redox state modulates differentiation of mesencephalic precursors (Studer et al, 2000;Rodriguez-Pallares et al, 2001Tsatmali et al, 2005), and with those of studies that have suggested that ROS levels affect the phenotype of cells in culture (Li et al, 2006;Xiao et al, 2009), including neurons (Tsatmali et al, 2006).…”

XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling

“…On SMCs, TGF-β1 suppresses proliferation while induces differentiation. 14,15 TGF-β2 plays an essential role in the valve formation during embryonic development via endothelial-mesenchymal transition. 16 TGF-β3 negatively regulates TGF-β1-mediated extracellular matrix deposition.…”

Upregulated sirtuin 1 by miRNA-34a is required for smooth muscle cell differentiation from pluripotent stem cells

“…Expectedly, data summarised in Supplementary Table S2 showed that a panel of SMC-specific genes (highlighted in yellow) were upregulated by miR-34a overexpression. Importantly, several SMCspecific transcription factors, including serum response factor (SRF), myocardin (Myocd) and myocyte enhancer factor 2C (MEF2c), that were reported to be activated in our SMC differentiation system 11,17 were also significantly upregulated by miR-34a, alongside SirT1 (Supplementary Table S2). Furthermore, overexpression of miR-34a upregulated SRF, myocardin and MEF2c, whereas knockdown of miR-34a downregulated these genes (Supplementary Figure S2), suggesting that miR-34a works in concert with these SMC transcription factors during SMC differentiation.…”

“…Detailed protocols for mouse ESCs (mESCs) (ES-D3 cell line, CRL-1934; ATCC, Manassas, VA, USA) culture and SMC differentiation were described in our previous studies. [11][12][13][14]17,22,34,43,44 The Shef-1, 2, 3, 6, and 7 human ESC lines were obtained from the United Kingdom Stem Cell Bank (UKSCB, Hertfordshire, UK) and maintained in our Laboratory as described in our previous study. 18 Briefly, undifferentiated ESCs were dissociated into single cells and seeded onto collagen I/IV (5 μg/ml)-coated flasks or plates in differentiation medium (DM, MEM alpha medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin) for 0-9 days prior to further treatment.…”