Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells

Division of Cardiovascular Medicine, Stanford University, Stanford, California 94305-5406, USA.
Stem cells and development (Impact Factor: 3.73). 10/2010; 19(10):1617-26. DOI: 10.1089/scd.2009.0417
Source: PubMed

ABSTRACT Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (L-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, L-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of L-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of L-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by L-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.

Download full-text


Available from: Felix Fleissner, Oct 16, 2014
17 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Thalidomide [α-(N-phthalimido)-glutarimide] exerts antiangiogenic properties and causes cardiac malformations in embryos. Herein the effects of thalidomide on cardiovascular differentiation were investigated in mouse embryonic stem (ES) cell-derived embryoid bodies. Thalidomide inhibited the formation of capillary-like blood vessels and decreased tumor-induced angiogenesis in confrontation cultures of embryoid bodies and multicellular prostate tumor spheroids, but stimulated cardiomyogenesis of ES cells. The number of CD31- and CD144-positive endothelial cells was not impaired, suggesting that thalidomide acted on vascular tube formation and cell migration rather than endothelial differentiation. Thalidomide increased reactive oxygen species generation, which was abolished by the NADPH oxidase inhibitor VAS2870 and the complex I respiratory chain inhibitor rotenone. Conversely, thalidomide decreased nitric oxide (NO) generation and endothelial NO synthase activity. VAS2870 abrogated thalidomide stimulation of cardiomyogenesis, whereas inhibition of vasculogenesis persisted. In NOX-1 and NOX-4 shRNA gene-inactivated ES cells, cardiomyogenesis was severely impaired and thalidomide failed to stimulate cardiac cell commitment. The NO donor S-nitrosopenicillamine reversed the antiangiogenic effect of thalidomide and increased capillary structure formation, whereas scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and inhibition of endothelial NO synthase by N(G)-nitro-l-arginine methyl ester decreased cardiovascular differentiation. Our data demonstrate that thalidomide causes an imbalance of reactive oxygen species/NO generation, thus stimulating cardiomyogenesis and impairing vascular sprout formation.
    Antioxidants & Redox Signaling 12/2010; 13(12):1813-27. DOI:10.1089/ars.2010.3139 · 7.41 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been proclaimed as a source of undifferentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson’s disease, Fanconi’s anemia and diabetes. In addition to their potential in regenerative therapy, an understanding of the mechanisms by which these cells differentiate into any functional cell type will provide valuable information about basic biology. Screens for small compounds that can drive self-renewal maintenance or differentiation protocols are relevant to this goal. Nitric oxide (NO) is a diffusible second messenger implicated in numerous physiological functions in mammals. This molecule plays an important role in the maintenance of key features required for embryonic development and extension in ES cells. The goal of this chapter is to discuss recent advances concerning the ways in which NO signaling pathways mediate diverse mechanisms involved in the differentiation of ES cells toward multiple lineages. This chapter will also discuss the mechanisms by which NO can modify tissue-specific gene expression thorough chromatin remodeling and post-translational modification of transcription factors.
    Stem Cells and Cancer Stem Cells, Vol 3 edited by M.A. Hayat i, 01/2012: chapter 36: pages 359-369; Springer International Publishing AG,., ISBN: 978-94-007-2414-3
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mechanobiology to date has focused on differentiated cells or progenitors, yet the effects of mechanical forces on early differentiation of pluripotent stem cells are still largely unknown. To study the effects of cellular deformation, we utilize a fluid flow bioreactor to apply steady laminar shear stress to mouse embryonic stem cells (ESCs) cultured on a two dimensional surface. Shear stress was found to affect pluripotency, as well as germ specification to the mesodermal, endodermal, and ectodermal lineages, as indicated by gene expression of OCT4, T-BRACHY, AFP, and NES, respectively. The ectodermal and mesodermal response to shear stress was dependent on stress magnitude (ranging from 1.5 to 15 dynes cm(-2)). Furthermore, increasing the duration from one to four days resulted in a sustained increase in T-BRACHY and a marked suppression of AFP. These changes in differentiation occurred concurrently with the activation of Wnt and estrogen pathways, as determined by PCR arrays for signalling molecules. Together these studies show that the mechanical microenvironment may be an important regulator during early differentiation events, including gastrulation. This insight furthers understanding of normal and pathological events during development, as well as facilitates strategies for scale up production of stem cells for clinical therapies.
    Integrative Biology 09/2012; 4(10):1263-73. DOI:10.1039/c2ib20040f · 3.76 Impact Factor
Show more