Skip to main content

Advertisement

SpringerLink
Log in

Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1+ (Flk-1+) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1+ cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Abid MR, Tsai JC, Spokes KC, Deshpande SS, Irani K, Aird WC (2001) Vascular endothelial growth factor induces manganese-superoxide dismutase expression in endothelial cells by a Rac1-regulated NADPH oxidase-dependent mechanism. FASEB J 15:2548–2550

    CAS  PubMed  Google Scholar 

  • Banfi B, Clark RA, Steger K, Krause KH (2003) Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J Biol Chem 278:3510–3513

    Article  CAS  PubMed  Google Scholar 

  • Banham-Hall E, Clatworthy MR, Okkenhaug K (2012) The therapeutic potential for PI3K inhibitors in autoimmune rheumatic diseases. Open Rheumatol J 6:245–258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bartsch C, Bekhite MM, Wolheim A, Richter M, Ruhe C, Wissuwa B, Marciniak A, Muller J, Heller R, Figulla HR, Sauer H, Wartenberg M (2011) NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment. Free Radic Biol Med 51:432–443

    Article  CAS  PubMed  Google Scholar 

  • Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313

    Article  CAS  PubMed  Google Scholar 

  • Bekhite MM, Finkensieper A, Binas S, Muller J, Wetzker R, Figulla HR, Sauer H, Wartenberg M (2011) VEGF-mediated PI3K class IA and PKC signaling in cardiomyogenesis and vasculogenesis of mouse embryonic stem cells. J Cell Sci 124:1819–1830

    Article  CAS  PubMed  Google Scholar 

  • Bhattacharya R, Kwon J, Li X, Wang E, Patra S, Bida JP, Bajzer Z, Claesson-Welsh L, Mukhopadhyay D (2009) Distinct role of PLCbeta3 in VEGF-mediated directional migration and vascular sprouting. J Cell Sci 122:1025–1034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380:435–439

    Article  CAS  PubMed  Google Scholar 

  • Chatterjee S, Browning EA, Hong N, DeBolt K, Sorokina EM, Liu W, Birnbaum MJ, Fisher AB (2012) Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS. Am J Physiol Heart Circ Physiol 302:H105–H114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Colavitti R, Pani G, Bedogni B, Anzevino R, Borrello S, Waltenberger J, Galeotti T (2002) Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR. J Biol Chem 277:3101–3108

    Article  CAS  PubMed  Google Scholar 

  • Disanza A, Steffen A, Hertzog M, Frittoli E, Rottner K, Scita G (2005) Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement. Cell Mol Life Sci 62:955–970

    Article  CAS  PubMed  Google Scholar 

  • Ferrara N (2009) Vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 29:789–791

    Article  CAS  PubMed  Google Scholar 

  • Finan PM, Thomas MJ (2004) PI 3-kinase inhibition: a therapeutic target for respiratory disease. Biochem Soc Trans 32:378–382

    Article  CAS  PubMed  Google Scholar 

  • Fruman DA, Meyers RE, Cantley LC (1998) Phosphoinositide kinases. Annu Rev Biochem 67:481–507

    Article  CAS  PubMed  Google Scholar 

  • Garrido-Urbani S, Jemelin S, Deffert C, Carnesecchi S, Basset O, Szyndralewiez C, Heitz F, Page P, Montet X, Michalik L, Arbiser J, Ruegg C, Krause KH, Imhof BA (2011) Targeting vascular NADPH oxidase 1 blocks tumor angiogenesis through a PPARalpha mediated mechanism. PLoS One 6:e14665

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gerber HP, McMurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N (1998) Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 273:30336–30343

    Article  CAS  PubMed  Google Scholar 

  • Graupera M, Guillermet-Guibert J, Foukas LC, Phng LK, Cain RJ, Salpekar A, Pearce W, Meek S, Millan J, Cutillas PR, Smith AJ, Ridley AJ, Ruhrberg C, Gerhardt H, Vanhaesebroeck B (2008) Angiogenesis selectively requires the p110alpha isoform of PI3K to control endothelial cell migration. Nature 453:662–666

    Article  CAS  PubMed  Google Scholar 

  • Guo D, Jia Q, Song HY, Warren RS, Donner DB (1995) Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation. J Biol Chem 270:6729–6733

    Article  CAS  PubMed  Google Scholar 

  • Hayakawa M, Kaizawa H, Moritomo H, Koizumi T, Ohishi T, Okada M, Ohta M, Tsukamoto S, Parker P, Workman P, Waterfield M (2006) Synthesis and biological evaluation of 4-morpholino-2-phenylquinazolines and related derivatives as novel PI3 kinase p110alpha inhibitors. Bioorg Med Chem 14:6847–6858

    Article  CAS  PubMed  Google Scholar 

  • Jiang BH, Liu LZ (2009) PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv Cancer Res 102:19–65

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jiang BH, Zheng JZ, Aoki M, Vogt PK (2000) Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci U S A 97:1749–1753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kabrun N, Buhring HJ, Choi K, Ullrich A, Risau W, Keller G (1997) Flk-1 expression defines a population of early embryonic hematopoietic precursors. Development 124:2039–2048

    CAS  PubMed  Google Scholar 

  • Kennedy AD, DeLeo FR (2008) PI3K and NADPH oxidase: a class act. Blood 112:4788–4789

    Article  CAS  PubMed  Google Scholar 

  • Kingham E, Welham M (2009) Distinct roles for isoforms of the catalytic subunit of class-IA PI3K in the regulation of behaviour of murine embryonic stem cells. J Cell Sci 122:2311–2321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kobayashi S, Nojima Y, Shibuya M, Maru Y (2004) Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells. Exp Cell Res 300:455–462

    Article  CAS  PubMed  Google Scholar 

  • Kroll J, Waltenberger J (1997) The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J Biol Chem 272:32521–32527

    Article  CAS  PubMed  Google Scholar 

  • Lamalice L, Le Boeuf F, Huot J (2007) Endothelial cell migration during angiogenesis. Circ Res 100:782–794

    Article  CAS  PubMed  Google Scholar 

  • Madamanchi NR, Vendrov A, Runge MS (2005) Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 25:29–38

    Article  CAS  PubMed  Google Scholar 

  • Marone R, Cmiljanovic V, Giese B, Wymann MP (2008) Targeting phosphoinositide 3-kinase: moving towards therapy. Biochim Biophys Acta 1784:159–185

    Article  CAS  PubMed  Google Scholar 

  • McAuslan BR, Gole GA (1980) Cellular and molecular mechanisms in angiogenesis. Trans Ophthalmol Soc U K 100:354–358

    CAS  PubMed  Google Scholar 

  • Millauer B, Wizigmann-Voos S, Schnurch H, Martinez R, Moller NP, Risau W, Ullrich A (1993) High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72:835–846

    Article  CAS  PubMed  Google Scholar 

  • Miyano K, Ueno N, Takeya R, Sumimoto H (2006) Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1. J Biol Chem 281:21857–21868

    Article  CAS  PubMed  Google Scholar 

  • Moldovan L, Mythreye K, Goldschmidt-Clermont PJ, Satterwhite LL (2006) Reactive oxygen species in vascular endothelial cell motility. Roles of NAD(P)H oxidase and Rac1. Cardiovasc Res 71:236–246

    Article  CAS  PubMed  Google Scholar 

  • Muramatsu F, Kidoya H, Naito H, Sakimoto S, Takakura N (2013)MmicroRNA-125b inhibits tube formation of blood vessels through translational suppression of VE-cadherin. Oncogene 32:414–421

  • Nakanishi A, Wada Y, Kitagishi Y, Matsuda S (2014) Link between PI3K/AKT/PTEN pathway and NOX proteinin diseases. Aging Dis 5:203–211

    Article  PubMed  PubMed Central  Google Scholar 

  • Resch T, Pircher A, Kahler CM, Pratschke J, Hilbe W (2012) Endothelial progenitor cells: current issues on characterization and challenging clinical applications. Stem Cell Rev 8:926–939

    Article  CAS  PubMed  Google Scholar 

  • Ribatti D (2006) Genetic and epigenetic mechanisms in the early development of the vascular system. J Anat 208:139–152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ridley AJ (2001) Rho GTPases and cell migration. J Cell Sci 114:2713–2722

    CAS  PubMed  Google Scholar 

  • Risau W (1995) Differentiation of endothelium. FASEB J 9:926–933

    CAS  PubMed  Google Scholar 

  • Sauer H, Bekhite MM, Hescheler J, Wartenberg M (2005) Redox control of angiogenic factors and CD31-positive vessel-like structures in mouse embryonic stem cells after direct current electrical field stimulation. Exp Cell Res 304:380–390

    Article  CAS  PubMed  Google Scholar 

  • Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK (2002) Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res 91:406–413

    Article  CAS  PubMed  Google Scholar 

  • Tammela T, Zarkada G, Wallgard E, Murtomaki A, Suchting S, Wirzenius M, Waltari M, Hellstrom M, Schomber T, Peltonen R, Freitas C, Duarte A, Isoniemi H, Laakkonen P, Christofori G, Yla-Herttuala S, Shibuya M, Pytowski B, Eichmann A, Betsholtz C, Alitalo K (2008) Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 454:656–660

    Article  CAS  PubMed  Google Scholar 

  • Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL (2002) Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res 90:1205–1213

  • Ushio-Fukai M (2007) VEGF signaling through NADPH oxidase-derived ROS. Antioxid Redox Signal 9:731–739

    Article  CAS  PubMed  Google Scholar 

  • Ushio-Fukai M, Urao N (2009) Novel role of NADPH oxidase in angiogenesis and stem/progenitor cell function. Antioxid Redox Signal 11:2517–2533

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma Y, Fujimoto M, Quinn MT, Pagano PJ, Johnson C, Alexander RW (2002) Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis. Circ Res 91:1160–1167

    Article  CAS  PubMed  Google Scholar 

  • Vittet D, Prandini MH, Berthier R, Schweitzer A, Martin-Sisteron H, Uzan G, Dejana E (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88:3424–3431

    CAS  PubMed  Google Scholar 

  • Williams MR, Arthur JS, Balendran A, Kaay J van der, Poli V, Cohen P, Alessi DR (2000) The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr Biol 10:439–448

  • Wymann MP, Pirola L (1998) Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1436:127–150

    Article  CAS  PubMed  Google Scholar 

  • Wymann MP, Zvelebil M, Laffargue M (2003) Phosphoinositide 3-kinase signalling—which way to target? Trends Pharmacol Sci 24:366–376

    Article  CAS  PubMed  Google Scholar 

  • Xu J, Tian W, Ma X, Guo J, Shi Q, Jin Y, Xi J, Xu Z (2011) The molecular mechanism underlying morphine-induced Akt activation: roles of protein phosphatases and reactive oxygen species. Cell Biochem Biophys 61:303–311

    Article  CAS  PubMed  Google Scholar 

  • Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, Yurugi T, Naito M, Nakao K (2000) Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408:92–96

    Article  CAS  PubMed  Google Scholar 

  • Zhang LJ, Tao BB, Wang MJ, Jin HM, Zhu YC (2012) PI3K p110alpha isoform-dependent Rho GTPase Rac1 activation mediates H2S-promoted endothelial cell migration via actin cytoskeleton reorganization. PLoS One 7:e44590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Dr. Martin Förster for his support during FCM and cell sorting procedures. We also thank Dr. Joachim Clement for kindly providing us with the phalloidin-Alexa Fluor 488 dye.

Author information

Authors and Affiliations

  1. University Heart Center, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Jena, Erlanger Allee 101, 07743, Jena, Germany

    Mohamed M. Bekhite, Veronika Müller, Sebastian H. Tröger, Hans-Reiner Figulla & Maria Wartenberg

  2. Department of Zoology, Faculty of Science, Tanta University, Tanta, 31527, Egypt

    Mohamed M. Bekhite

  3. Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University Jena, Jena, Germany

    Jörg P. Müller

  4. Department of Physiology, Faculty of Medicine, Justus Liebig University, Giessen, Germany

    Heinrich Sauer

Authors
  1. Mohamed M. Bekhite
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Veronika Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sebastian H. Tröger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jörg P. Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans-Reiner Figulla
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Heinrich Sauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maria Wartenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mohamed M. Bekhite.

Ethics declarations

Conflict of interest

The authors have nothing to declare.

Additional information

This work was supported by the Excellence Cluster Cardio-Pulmonary System (ECCPS) of the German Research Foundation.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

Purity of Flk-1+ cells and PI3K catalytic subunit expression in vascular progenitor isolated from embryoid bodies. Flk-1+ cells were sorted by MACS from 4-day-old embryoid bodies. a Flk-1+ cells were labelled by using phycoerythrin (PE)-conjugated rat anti-mouse Flk-1 antibody and processed by FCM. b, b‘ Mean values (± SD) of three independent experiments for mRNA expression of the class IA PI3K catalytic subunits (p110α, β, δ). *P < 0.05, statistically significant as indicated. (GIF 2501 kb)

High resolution image (TIFF 1396 kb)

Fig. S2

VEGF-induced O2 − production is dependent on PI3K-Rac1 activation in Flk-1+ cells derived from the ES cell line CCE. O2 − generation of sorted Flk-1+ cells isolated from 4-day-old embryoid bodies in response to VEGF stimulation was analysed by determination of DHE fluorescence. a-a‘‘‘‘ Representative DHE fluorescence images of Flk-1+ cells incubated with compound 15e (0.5 μM) (a‘‘), a specific p110α inhibitor, and the NOX1 inhibitor 2-APT (0.5 μM; a‘‘‘) and Rac1 inhibitor (50 μM; a‘‘‘‘) in the absence (a) or presence (a‘) of VEGF (500 pM). b Percentage values (± SD) of three independent experiments. *P < 0.05, statistically significant as indicated (n.s not significant). (GIF 5698 kb)

High resolution image (TIFF 3576 kb)

Fig. S3

Vessel-like structures in embryoid bodies derived from ES cells. a Representative image of embryoid body stained with endothelial markers Flk-1 (green) and PECAM-1 (red). To visualize the shape of embryoid bodies, transmitted light images were recorded (blue). a Overlay image. a‘ Flk-1. a‘‘ PECAM-1. b Flk-1. b‘ PECAM-1. The cell nuclei in b, b‘ were labelled with DAPI (blue). c, d Representative images of embryoid bodies stained with endothelial markers PECAM-1 (green) and VE-cadherin (red). c PECAM-1, c‘ VE-cadherin, c‘‘ Overlay image. d PECAM-1. d‘ VE-cadherin. d‘‘ Overlay image. (GIF 16111 kb)

High resolution image (TIFF 23445 kb)

Fig. S4

Number of Flk-1+ cells in p110α or NOX1 knockdown and wild-type (pLKO.1) ES cells. Cells were differentiated for 10 days and treated with wortmannin (1 μM) or Rac1 inhibitor (50 μM) as indicated. a–a‘‘‘‘‘, b FCM analysis for three independent experiments; Flk-1+ cell numbers in embryoid bodies and after application of inhibitors from day 4 to day 10 of cell culture either in the absence or presence of VEGF (500 pM). *P < 0.05, statistically significant as indicated (n.s not significant). (GIF 2979 kb)

High resolution image (TIFF 2226 kb)

Fig. S5

Effects of pan-PI3K inhibitor (wortmannin) and specific inhibitor of p110α (compound 15e), NOX1 (2-APT) and Rac1 (Rac1 inhibitor) on vascular differentiation of the ES cell line CCE. ES cells were differentiated for 10 days. Wortmannin (1 μM), compound 15e (0.5 μM), the NOX1 inhibitor 2-APT (0,5 μM) or Rac1 inhibitor (50 μM) was applied from day 4 to day 10 of cell culture and the cells were stimulated with VEGF (500 pM) as indicated. a–a‘‘‘‘, b-b‘‘‘‘ Representative immunofluorescence images for at least three experiments showing vascular differentiation in embryoid bodies under conditions as indicated. Bars 100 μm. c Percentage values (± SD) of PECAM-1-positive areas for three independent experiments. *P < 0.05, statistically significant as indicated (n.s not significant). (GIF 8254 kb)

High resolution image (TIFF 6217 kb)

Fig. S6

Tube formation of human umbilical vein endothelial cells (HUVECs) on matrigel treated with p110α (compound 15e) or NOX1 inhibitor (2-APT). Tube structures were analysed either in the presence or in the absence of VEGF (500 pM) after 16 h of cultivation. The ability to form tubes was expressed as total length of tubes and branching points per field. Bars 200 μm. a–a‘‘‘ Representative transmitted light images for three independent experiments of tube formation of HUVECs on matrigel upon treatment with either compound 15e (0.5 μM) or 2-APT (0,5 μM) in the presence or absence of VEGF (500 pM). VEGF-induced tube-like formation (b) and branching points (c) were significantly inhibited in the presence of the specific p110α or NOX1 inhibitor in HUVECs. *P < 0.05, statistically significant as indicated (n.s not significant). (GIF 3862 kb)

High resolution image (TIFF 3854 kb)

Fig. S7

VEGF positively regulates cell migration of HUVECs in vitro. a–a‘‘‘ Representative images of the scratch assay in HUVECs treated with either compound 15e (0.5 μM) or the NOX1 inhibitor 2-APT (0.5 μM) in the presence or absence of VEGF (500 pM). b VEGF-treated cells exhibited a highly enhanced migratory potential, which was significantly inhibited by treatment of HUVECs with compound 15e or 2-APT. *P < 0.05, statistically significant as indicated (n.s not significant). (GIF 2923 kb)

High resolution image (TIFF 3493 kb)

Fig. S8

Kinetics of Rac1 and RhoA activation in ES cells. Cells were subjected to immunoblotting at the indicated time points after stimulation with VEGF. Activation of Rac1 (Ser71) and RhoA was analysed by using phospho-specific antibodies. a Blots were subsequently reprobed with pan-specific antibodies recognizing Rac1/2/3 or RhoA, respectively. b Specific activation was quantified as the ratio of phospho-specific to pan-specific signals. Graphs under the blots show mean values (± SD) of three independent experiments. *P < 0.05, statistically significant as indicated (n.s not significant). (GIF 2538 kb)

High resolution image (TIFF 1934 kb)

Table S1

(DOC 38 kb)

ESM 1

(DOCX 17 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bekhite, M.M., Müller, V., Tröger, S.H. et al. Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells. Cell Tissue Res 364, 159–174 (2016). https://doi.org/10.1007/s00441-015-2303-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-015-2303-8

Keywords

Search

Navigation