, Volume 16, Issue 2, pp 455–468 | Cite as

Sprouty2 expression controls endothelial monolayer integrity and quiescence

  • Martin Peier
  • Thomas Walpen
  • Gerhard Christofori
  • Edouard Battegay
  • Rok HumarEmail author
Original Paper


Vascular integrity is fundamental to the formation of mature blood vessels and depends on a functional, quiescent endothelial monolayer. However, how endothelial cells enter and maintain quiescence in the presence of angiogenic factors is still poorly understood. Here we identify the fibroblast growth factor (FGF) antagonist Sprouty2 (Spry2) as a key player in mediating endothelial quiescence and barrier integrity in mouse aortic endothelial cells (MAECs): Spry2 knockout MAECs show spindle-like shapes and are incapable of forming a functional, impermeable endothelial monolayer in the presence of FGF2. Whereas dense wild type cells exhibit contact inhibition and stop to proliferate, Spry2 knockout MAECs remain responsive to FGF2 and continue to proliferate even at high cell densities. Importantly, the anti-proliferative effect of Spry2 is absent in sparsely plated cells. This cell density-dependent Spry2 function correlates with highly increased Spry2 expression in confluent wild type MAECs. Spry2 protein expression is barely detectable in single cells but steadily increases in cells growing to high cell densities, with hypoxia being one contributing factor. At confluence, Spry2 expression correlates with intact cell–cell contacts, whereas disruption of cell–cell contacts by EGTA, TNFα and thrombin decreases Spry2 protein expression. In confluent cells, high Spry2 levels correlate with decreased extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. In contrast, dense Spry2 knockout MAECs exhibit enhanced signaling by Erk1/2. Moreover, inhibiting Erk1/2 activity in Spry2 knockout cells restores wild type cobblestone monolayer morphology. This study thus reveals a novel Spry2 function, which mediates endothelial contact inhibition and barrier integrity.


Sprouty2 Endothelial quiescence Cell–cell contacts FGF2 Erk1/2 



We thank O. Sansom and I. Ahmad, who kindly provided the Floxed-Spry2 mouse strains (Beatson Institute, Glasgow, Scotland). We are grateful to M. A. Cabrita and I. Bhattacharya for helpful discussions. We thank Sigrid Strom, Ina Kalus and Elvira Haas for critical review of the manuscript. This work was supported by grants from the Swiss National Science Foundation to E. J. B. and from the University of Zürich.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10456_2012_9330_MOESM1_ESM.tif (6.1 mb)
Supplementary figure 1 Male and female MAECs show similar FGF2-Erk1/2 signaling and density dependent Spry2 expression (A) Confluent female (♀) and male (♂) MAECs were starved and stimulated with FGF2 for the indicated time. The cells were then lysed and analyzed for ERK1/2 phosphorylation and total Erk1/2 expression. Relative phosphorylation levels of Erk1/2 were quantified by densitometry. Bars show values as fold change of unstimulated cells ± s.e.m. n = 3. (B) Basal Spry2 expression and Erk1/2 activity in sparse and confluent female and male MAECs. Cells were plated at sparse (3,000 cells/cm2) or confluent conditions (50,000 cells/cm2), starved, lysed and analyzed by immunoblotting (TIFF 6264 kb)


  1. 1.
    Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438(7070):932–936. doi: 10.1038/nature04478 PubMedCrossRefGoogle Scholar
  2. 2.
    Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8(6):464–478. doi: 10.1038/nrm2183 PubMedCrossRefGoogle Scholar
  3. 3.
    De Smet F, Segura I, De Bock K, Hohensinner PJ, Carmeliet P (2009) Mechanisms of vessel branching: filopodia on endothelial tip cells lead the way. Arterioscler Thromb Vasc Biol 29(5):639–649. doi: 10.1161/ATVBAHA.109.185165 PubMedCrossRefGoogle Scholar
  4. 4.
    Presta M, Dell’Era P, Mitola S, Moroni E, Ronca R, Rusnati M (2005) Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 16(2):159–178. doi: 10.1016/j.cytogfr.2005.01.004 PubMedCrossRefGoogle Scholar
  5. 5.
    Dejana E (2004) Endothelial cell–cell junctions: happy together. Nat Rev Mol Cell Biol 5(4):261–270. doi: 10.1038/nrm1357 PubMedCrossRefGoogle Scholar
  6. 6.
    Murakami M, Simons M (2009) Regulation of vascular integrity. J Mol Med (Berl) 87(6):571–582. doi: 10.1007/s00109-009-0463-2 CrossRefGoogle Scholar
  7. 7.
    Dejana E, Tournier-Lasserve E, Weinstein BM (2009) The control of vascular integrity by endothelial cell junctions: molecular basis and pathological implications. Dev Cell 16(2):209–221. doi: 10.1016/j.devcel.2009.01.004 PubMedCrossRefGoogle Scholar
  8. 8.
    Vinals F, Pouyssegur J (1999) Confluence of vascular endothelial cells induces cell cycle exit by inhibiting p42/p44 mitogen-activated protein kinase activity. Mol Cell Biol 19(4):2763–2772PubMedGoogle Scholar
  9. 9.
    Hacohen N, Kramer S, Sutherland D, Hiromi Y, Krasnow MA (1998) Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92(2):253–263PubMedCrossRefGoogle Scholar
  10. 10.
    Casci T, Vinos J, Freeman M (1999) Sprouty, an intracellular inhibitor of Ras signaling. Cell 96(5):655–665PubMedCrossRefGoogle Scholar
  11. 11.
    Minowada G, Jarvis LA, Chi CL, Neubuser A, Sun X, Hacohen N, Krasnow MA, Martin GR (1999) Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed. Development 126(20):4465–4475PubMedGoogle Scholar
  12. 12.
    Tefft JD, Lee M, Smith S, Leinwand M, Zhao J, Bringas P Jr, Crowe DL, Warburton D (1999) Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr Biol 9(4):219–222PubMedCrossRefGoogle Scholar
  13. 13.
    Mailleux AA, Tefft D, Ndiaye D, Itoh N, Thiery JP, Warburton D, Bellusci S (2001) Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis. Mech Dev 102(1–2):81–94PubMedCrossRefGoogle Scholar
  14. 14.
    Taniguchi K, Ayada T, Ichiyama K, Kohno R, Yonemitsu Y, Minami Y, Kikuchi A, Maehara Y, Yoshimura A (2007) Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling. Biochem Biophys Res Commun 352(4):896–902. doi: 10.1016/j.bbrc.2006.11.107 PubMedCrossRefGoogle Scholar
  15. 15.
    Mason JM, Morrison DJ, Basson MA, Licht JD (2006) Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling. Trends Cell Biol 16(1):45–54. doi: 10.1016/j.tcb.2005.11.004 PubMedCrossRefGoogle Scholar
  16. 16.
    Cabrita MA, Christofori G (2008) Sprouty proteins, masterminds of receptor tyrosine kinase signaling. Angiogenesis 11(1):53–62. doi: 10.1007/s10456-008-9089-1 PubMedCrossRefGoogle Scholar
  17. 17.
    Impagnatiello MA, Weitzer S, Gannon G, Compagni A, Cotten M, Christofori G (2001) Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells. J Cell Biol 152(5):1087–1098PubMedCrossRefGoogle Scholar
  18. 18.
    Humar R, Kiefer FN, Berns H, Resink TJ, Battegay EJ (2002) Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling. FASEB J 16(8):771–780. doi: 10.1096/fj.01-0658com Google Scholar
  19. 19.
    Metzen E, Wolff M, Fandrey J, Jelkmann W (1995) Pericellular PO2 and O2 consumption in monolayer cell cultures. Respir Physiol 100(2):101–106PubMedCrossRefGoogle Scholar
  20. 20.
    Schnittler HJ, Puschel B, Drenckhahn D (1997) Role of cadherins and plakoglobin in interendothelial adhesion under resting conditions and shear stress. Am J Physiol 273(5 Pt 2):H2396–H2405PubMedGoogle Scholar
  21. 21.
    Goldblum SE, Hennig B, Jay M, Yoneda K, McClain CJ (1989) Tumor necrosis factor alpha-induced pulmonary vascular endothelial injury. Infect Immun 57(4):1218–1226PubMedGoogle Scholar
  22. 22.
    McKenzie JA, Ridley AJ (2007) Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability. J Cell Physiol 213(1):221–228. doi: 10.1002/jcp.21114 PubMedCrossRefGoogle Scholar
  23. 23.
    Lum H, Del Vecchio PJ, Schneider AS, Goligorsky MS, Malik AB (1989) Calcium dependence of the thrombin-induced increase in endothelial albumin permeability. J Appl Physiol 66(3):1471–1476PubMedCrossRefGoogle Scholar
  24. 24.
    Gross I, Bassit B, Benezra M, Licht JD (2001) Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation. J Biol Chem 276(49):46460–46468. doi: 10.1074/jbc.M108234200 PubMedCrossRefGoogle Scholar
  25. 25.
    Guy GR, Jackson RA, Yusoff P, Chow SY (2009) Sprouty proteins: modified modulators, matchmakers or missing links? J Endocrinol 203(2):191–202. doi: 10.1677/JOE-09-0110 PubMedCrossRefGoogle Scholar
  26. 26.
    Anderson K, Nordquist KA, Gao X, Hicks KC, Zhai B, Gygi SP, Patel TB (2011) Regulation of cellular levels of Sprouty2 protein by prolyl hydroxylase domain and von Hippel-Lindau proteins. J Biol Chem 286(49):42027–42036. doi: 10.1074/jbc.M111.303222 PubMedCrossRefGoogle Scholar
  27. 27.
    Ding W, Shi W, Bellusci S, Groffen J, Heisterkamp N, Minoo P, Warburton D (2007) Sprouty2 downregulation plays a pivotal role in mediating crosstalk between TGF-beta1 signaling and EGF as well as FGF receptor tyrosine kinase-ERK pathways in mesenchymal cells. J Cell Physiol 212(3):796–806. doi: 10.1002/jcp.21078 PubMedCrossRefGoogle Scholar
  28. 28.
    Ding W, Warburton D (2008) Down-regulation of Sprouty2 via p38 MAPK plays a key role in the induction of cellular apoptosis by tumor necrosis factor-alpha. Biochem Biophys Res Commun 375(3):460–464. doi: 10.1016/j.bbrc.2008.08.037 PubMedCrossRefGoogle Scholar
  29. 29.
    Lim J, Wong ES, Ong SH, Yusoff P, Low BC, Guy GR (2000) Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain. J Biol Chem 275(42):32837–32845. doi: 10.1074/jbc.M002156200 PubMedCrossRefGoogle Scholar
  30. 30.
    Fong CW, Leong HF, Wong ES, Lim J, Yusoff P, Guy GR (2003) Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function. J Biol Chem 278(35):33456–33464. doi: 10.1074/jbc.M301317200 PubMedCrossRefGoogle Scholar
  31. 31.
    Hanafusa H, Torii S, Yasunaga T, Nishida E (2002) Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nat Cell Biol 4(11):850–858. doi: 10.1038/ncb867 PubMedCrossRefGoogle Scholar
  32. 32.
    Mason JM, Morrison DJ, Bassit B, Dimri M, Band H, Licht JD, Gross I (2004) Tyrosine phosphorylation of Sprouty proteins regulates their ability to inhibit growth factor signaling: a dual feedback loop. Mol Biol Cell 15(5):2176–2188. doi: 10.1091/mbc.E03-07-0503 PubMedCrossRefGoogle Scholar
  33. 33.
    Glienke J, Schmitt AO, Pilarsky C, Hinzmann B, Weiss B, Rosenthal A, Thierauch KH (2000) Differential gene expression by endothelial cells in distinct angiogenic states. Eur J Biochem 267(9):2820–2830PubMedCrossRefGoogle Scholar
  34. 34.
    Zhang C, Chaturvedi D, Jaggar L, Magnuson D, Lee JM, Patel TB (2005) Regulation of vascular smooth muscle cell proliferation and migration by human sprouty 2. Arterioscler Thromb Vasc Biol 25(3):533–538. doi: 10.1161/01.ATV.0000155461.50450.5a PubMedCrossRefGoogle Scholar
  35. 35.
    Pinsky DJ, Yan SF, Lawson C, Naka Y, Chen JX, Connolly ES Jr, Stern DM (1995) Hypoxia and modification of the endothelium: implications for regulation of vascular homeostatic properties. Semin Cell Biol 6(5):283–294PubMedCrossRefGoogle Scholar
  36. 36.
    Yan SF, Ogawa S, Stern DM, Pinsky DJ (1997) Hypoxia-induced modulation of endothelial cell properties: regulation of barrier function and expression of interleukin-6. Kidney Int 51(2):419–425PubMedCrossRefGoogle Scholar
  37. 37.
    Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, Dejana E (1995) The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol 129(1):203–217PubMedCrossRefGoogle Scholar
  38. 38.
    Nyqvist D, Giampietro C, Dejana E (2008) Deciphering the functional role of endothelial junctions by using in vivo models. EMBO Rep 9(8):742–747. doi: 10.1038/embor.2008.123 PubMedCrossRefGoogle Scholar
  39. 39.
    Hewat EA, Durmort C, Jacquamet L, Concord E, Gulino-Debrac D (2007) Architecture of the VE-cadherin hexamer. J Mol Biol 365(3):744–751. doi: 10.1016/j.jmb.2006.10.052 PubMedCrossRefGoogle Scholar
  40. 40.
    Chitaev NA, Troyanovsky SM (1998) Adhesive but not lateral E-cadherin complexes require calcium and catenins for their formation. J Cell Biol 142(3):837–846PubMedCrossRefGoogle Scholar
  41. 41.
    Dejana E, Orsenigo F, Molendini C, Baluk P, McDonald DM (2009) Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees. Cell Tissue Res 335(1):17–25. doi: 10.1007/s00441-008-0694-5 PubMedCrossRefGoogle Scholar
  42. 42.
    Nelson CM, Jean RP, Tan JL, Liu WF, Sniadecki NJ, Spector AA, Chen CS (2005) Emergent patterns of growth controlled by multicellular form and mechanics. Proc Natl Acad Sci U S A 102(33):11594–11599. doi: 10.1073/pnas.0502575102 PubMedCrossRefGoogle Scholar
  43. 43.
    Wallez Y, Huber P (2008) Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis. Biochim Biophys Acta 1778(3):794–809. doi: 10.1016/j.bbamem.2007.09.003 PubMedCrossRefGoogle Scholar
  44. 44.
    Nelson PJ, Daniel TO (2002) Emerging targets: molecular mechanisms of cell contact-mediated growth control. Kidney Int 61(1 Suppl):S99–S105. doi: 10.1046/j.1523-1755.2002.0610s1099.x PubMedCrossRefGoogle Scholar
  45. 45.
    Tille JC, Wood J, Mandriota SJ, Schnell C, Ferrari S, Mestan J, Zhu Z, Witte L, Pepper MS (2001) Vascular endothelial growth factor (VEGF) receptor-2 antagonists inhibit VEGF- and basic fibroblast growth factor-induced angiogenesis in vivo and in vitro. J Pharmacol Exp Ther 299(3):1073–1085PubMedGoogle Scholar
  46. 46.
    Seghezzi G, Patel S, Ren CJ, Gualandris A, Pintucci G, Robbins ES, Shapiro RL, Galloway AC, Rifkin DB, Mignatti P (1998) Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis. J Cell Biol 141(7):1659–1673PubMedCrossRefGoogle Scholar
  47. 47.
    Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald DM, Johnson RS, Arbeit JM (2001) Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1alpha. Genes Dev 15(19):2520–2532. doi: 10.1101/gad.914801 PubMedCrossRefGoogle Scholar
  48. 48.
    Richard DE, Berra E, Pouyssegur J (2000) Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. J Biol Chem 275(35):26765–26771. doi: 10.1074/jbc.M003325200 PubMedGoogle Scholar
  49. 49.
    Fukuda R, Hirota K, Fan F, Jung YD, Ellis LM, Semenza GL (2002) Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem 277(41):38205–38211. doi: 10.1074/jbc.M203781200 PubMedCrossRefGoogle Scholar
  50. 50.
    Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, Ferrara N, Nagy A, Roos KP, Iruela-Arispe ML (2007) Autocrine VEGF signaling is required for vascular homeostasis. Cell 130(4):691–703. doi: 10.1016/j.cell.2007.06.054 PubMedCrossRefGoogle Scholar
  51. 51.
    Fong GH (2009) Regulation of angiogenesis by oxygen sensing mechanisms. J Mol Med (Berl) 87(6):549–560. doi: 10.1007/s00109-009-0458-z CrossRefGoogle Scholar
  52. 52.
    Ozawa CR, Banfi A, Glazer NL, Thurston G, Springer ML, Kraft PE, McDonald DM, Blau HM (2004) Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest 113(4):516–527. doi: 10.1172/JCI18420 PubMedGoogle Scholar
  53. 53.
    Shim K, Minowada G, Coling DE, Martin GR (2005) Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling. Dev Cell 8(4):553–564. doi: 10.1016/j.devcel.2005.02.009 PubMedCrossRefGoogle Scholar
  54. 54.
    Klein OD, Minowada G, Peterkova R, Kangas A, Yu BD, Lesot H, Peterka M, Jernvall J, Martin GR (2006) Sprouty genes control diastema tooth development via bidirectional antagonism of epithelial-mesenchymal FGF signaling. Dev Cell 11(2):181–190. doi: 10.1016/j.devcel.2006.05.014 PubMedCrossRefGoogle Scholar
  55. 55.
    Peterkova R, Churava S, Lesot H, Rothova M, Prochazka J, Peterka M, Klein OD (2009) Revitalization of a diastemal tooth primordium in Spry2 null mice results from increased proliferation and decreased apoptosis. J Exp Zool B Mol Dev Evol 312B(4):292–308. doi: 10.1002/jez.b.21266 PubMedCrossRefGoogle Scholar
  56. 56.
    Matsumura K, Taketomi T, Yoshizaki K, Arai S, Sanui T, Yoshiga D, Yoshimura A, Nakamura S (2011) Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling. Biochem Biophys Res Commun 404(4):1076–1082. doi: 10.1016/j.bbrc.2010.12.116 PubMedCrossRefGoogle Scholar
  57. 57.
    Folkman J, Merler E, Abernathy C, Williams G (1971) Isolation of a tumor factor responsible for angiogenesis. J Exp Med 133(2):275–288PubMedCrossRefGoogle Scholar
  58. 58.
    Murakami M, Nguyen LT, Zhuang ZW, Moodie KL, Carmeliet P, Stan RV, Simons M (2008) The FGF system has a key role in regulating vascular integrity. J Clin Invest 118(10):3355–3366. doi: 10.1172/JCI35298 PubMedCrossRefGoogle Scholar
  59. 59.
    Hackett PH, Roach RC (2004) High altitude cerebral edema. High Alt Med Biol 5(2):136–146. doi: 10.1089/1527029041352054 PubMedCrossRefGoogle Scholar
  60. 60.
    Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407(6801):249–257. doi: 10.1038/35025220 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Martin Peier
    • 1
  • Thomas Walpen
    • 1
  • Gerhard Christofori
    • 2
  • Edouard Battegay
    • 1
    • 3
  • Rok Humar
    • 1
    • 3
    Email author
  1. 1.Division of Internal MedicineUniversity Hospital ZurichZurichSwitzerland
  2. 2.Department of BiomedicineUniversity of BaselBaselSwitzerland
  3. 3.Zurich Center for Integrative Human Physiology (ZIHP)ZurichSwitzerland

Personalised recommendations