, Volume 21, Issue 3, pp 571–580 | Cite as

Extracellular vesicle-carried Jagged-1 inhibits HUVEC sprouting in a 3D microenvironment

  • Evan Tan
  • Harry H. Asada
  • Ruowen GeEmail author
Original Paper


NOTCH signalling is an evolutionarily conserved juxtacrine signalling pathway that is essential in development. Jagged1 (JAG1) and Delta-like ligand 4 (DLL4) are transmembrane NOTCH ligands that regulate angiogenesis by controlling endothelial cell (EC) differentiation, vascular development and maturation. In addition, DLL4 could bypass its canonical cell–cell contact-dependent signalling to influence NOTCH signalling and angiogenesis at a distance when it is packaged into extracellular vesicles (EVs). However, it is not clear whether JAG1 could also be packaged into EVs to influence NOTCH signalling and angiogenesis. In this work, we demonstrate that JAG1 is also packaged into EVs. We present evidence that JAG1-EVs inhibit NOTCH signalling and regulate EC behaviour and function. JAG1-EVs inhibited VEGF-induced HUVEC proliferation and migration in 2D culture condition and suppressed sprouting in a 3D microfluidic microenvironment. JAG1-EV treatment of HUVECs leads to a reduction of Notch1 intracellular domain (N1-ICD), and the proteasome and the intracellular domain of JAG1 (JAG1-ICD) are both required for this reduction to occur. These findings reveal a novel mechanism of JAG1 function in NOTCH signalling and ECs through EVs.


Extracellular vesicles NOTCH signalling Jagged-1 

Supplementary material

10456_2018_9609_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 13 kb)
10456_2018_9609_MOESM2_ESM.docx (9.4 mb)
Supplementary material 2 (DOCX 9616 kb)


  1. 1.
    Bray S (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7:678–689. CrossRefPubMedGoogle Scholar
  2. 2.
    Gerhardt H, Golding M, Fruttiger M et al (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161:1163–1177. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kume T (2009) Novel insights into the differential functions of Notch ligands in vascular formation. J Angiogenes Res 1:8. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Blanco R, Gerhardt H (2013) VEGF and Notch in tip and stalk cell selection. Cold Spring Harb Perspect Med. PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Lobov IB, Renard RA, Papadopoulos N et al (2007) Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting. Proc Natl Acad Sci USA 104:3219–3224. CrossRefPubMedGoogle Scholar
  6. 6.
    Suchting S, Freitas C, le Noble F et al (2007) The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci 104:3225–3230. CrossRefPubMedGoogle Scholar
  7. 7.
    Hellström M, Phng L-K, Hofmann JJ et al (2007) Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445:776–780. CrossRefPubMedGoogle Scholar
  8. 8.
    Benedito R, Roca C, Sörensen I et al (2009) The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137:1124–1135. CrossRefPubMedGoogle Scholar
  9. 9.
    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200:373–383. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Sheldon H, Heikamp E, Turley H et al (2010) New mechanism for Notch signaling to endothelium at a distance by delta-like 4 incorporation into exosomes. Blood 116:2385–2394. CrossRefPubMedGoogle Scholar
  11. 11.
    Sharghi-Namini S, Tan E, Ong L-LS et al (2014) Dll4-containing exosomes induce capillary sprout retraction in a 3D microenvironment. Sci Rep 4:4031. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kilani RT, Chavez-mun C (2009) Profile of exosomes related proteins released by differentiated and undifferentiated human keratinocytes. J Cell Physiol 221:221–231. CrossRefPubMedGoogle Scholar
  13. 13.
    Liang B, Peng P, Chen S et al (2013) Characterization and proteomic analysis of ovarian cancer-derived exosomes. J Proteomics 80:171–182. CrossRefPubMedGoogle Scholar
  14. 14.
    Beckler MD, Higginbotham JN, Franklin JL et al (2013) Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS. Mol Cell Proteomics. CrossRefGoogle Scholar
  15. 15.
    Lazar I, Clement E, Ducoux-petit M et al (2015) Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines Ikrame. Pigment Cell Melanoma Res 28:464–475. CrossRefPubMedGoogle Scholar
  16. 16.
    Sethi N, Dai X, Winter CG, Kang Y (2011) Tumor-derived Jagged1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells. Cancer Cell 19:192–205. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Brownlee Z, Lynn KD, Thorpe PE, Schroit AJ (2014) A novel “salting-out” procedure for the isolation of tumor-derived exosomes. J Immunol Methods 407:120–126. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mitchell JP, Court J, Mason MD et al (2008) Increased exosome production from tumour cell cultures using the Integra CELLine culture system. J Immunol Methods 335:98–105. CrossRefPubMedGoogle Scholar
  19. 19.
    Farahat WA, Wood LB, Zervantonakis IK et al (2012) Ensemble analysis of angiogenic growth in three-dimensional microfluidic cell cultures. PLoS ONE. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Lötvall J, Hill AF, Hochberg F et al (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the international society for extracellular vesicles. J Extracell Vesicles 3:1–6. CrossRefGoogle Scholar
  21. 21.
    Ribatti D, Crivellato E (2012) “Sprouting angiogenesis”, a reappraisal. Dev Biol 372:157–165. CrossRefPubMedGoogle Scholar
  22. 22.
    Noseda M, Chang L, McLean G et al (2004) Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Mol Cell Biol 24:8813–8822. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Takeshita K, Satoh M, Ii M et al (2007) Critical role of endothelial Notch1 signaling in postnatal angiogenesis. Circ Res 100:70–78. CrossRefPubMedGoogle Scholar
  24. 24.
    D’Souza B, Meloty-Kapella L, Weinmaster G (2010) Canonical and non-canonical notch ligands. Curr Top Dev Biol 92:73–129. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kim MY, Jung J, Mo JS et al (2011) The intracellular domain of Jagged-1 interacts with Notch1 intracellular domain and promotes its degradation through Fbw7 E3 ligase. Exp Cell Res 317:2438–2446. CrossRefPubMedGoogle Scholar
  26. 26.
    Metrich M, Bezdek Pomey A, Berthonneche C et al (2015) Jagged1 intracellular domain-mediated inhibition of Notch1 signalling regulates cardiac homeostasis in the postnatal heart. Cardiovasc Res 108:74–86. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Pedrosa AR, Trindade A, Fernandes AC et al (2015) Endothelial jagged1 antagonizes Dll4 regulation of endothelial branching and promotes vascular maturation downstream of Dll4/Notch1. Arterioscler Thromb Vasc Biol 35:1134–1146. CrossRefPubMedGoogle Scholar
  28. 28.
    Kadesch T (2000) Notch signaling: a dance of proteins changing partners. Exp Cell Res 260:1–8. CrossRefPubMedGoogle Scholar
  29. 29.
    Öberg C, Li J, Pauley A et al (2001) The notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J Biol Chem 276:35847–35853. CrossRefPubMedGoogle Scholar
  30. 30.
    Liebler SS, Feldner A, Adam MG et al (2012) No evidence for a functional role of bi-directional notch signaling during angiogenesis. PLoS ONE 7:1–10. CrossRefGoogle Scholar
  31. 31.
    Heusermann W, Hean J, Trojer D et al (2016) Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER. J Cell Biol 213:173–184. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Gong J, Körner R, Gaitanos L, Klein R (2016) Exosomes mediate cell contact-independent ephrin-Eph signaling during axon guidance. J Cell Biol 214:35–44. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological SciencesNational University of SingaporeSingaporeSingapore
  2. 2.Singapore-MIT Alliance for Research and TechnologyBioSystems and Micromechanics Inter-Disciplinary Research GroupSingaporeSingapore
  3. 3.d’Arbeloff Laboratory for Information Systems and TechnologyMassachusetts Institute of TechnologyCambridgeUSA

Personalised recommendations