pp 1–14 | Cite as

Live imaging of angiogenesis during cutaneous wound healing in adult zebrafish

  • Chikage Noishiki
  • Shinya Yuge
  • Koji Ando
  • Yuki Wakayama
  • Naoki Mochizuki
  • Rei Ogawa
  • Shigetomo FukuharaEmail author
Original Paper


Angiogenesis, the growth of new blood vessels from pre-existing vessels, is critical for cutaneous wound healing. However, it remains elusive how endothelial cells (ECs) and pericytes (PCs) establish new blood vessels during cutaneous angiogenesis. We set up a live-imaging system to analyze cutaneous angiogenesis in adult zebrafish. First, we characterized basic structures of cutaneous vasculature. In normal skin tissues, ECs and PCs remained dormant to maintain quiescent blood vessels, whereas cutaneous injury immediately induced angiogenesis through the vascular endothelial growth factor signaling pathway. Tortuous and disorganized vessel networks formed within a few weeks after the injury and subsequently normalized through vessel regression in a few months. Analyses of the repair process of injured single blood vessels revealed that severed vessels elongated upon injury and anastomosed with each other. Thereafter, repaired vessels and adjacent uninjured vessels became tortuous by increasing the number of ECs. In parallel, PCs divided and migrated to cover the tortuous blood vessels. ECs sprouted from the PC-covered tortuous vessels, suggesting that EC sprouting does not require PC detachment from the vessel wall. Thus, live imaging of cutaneous angiogenesis in adult zebrafish enables us to clarify how ECs and PCs develop new blood vessels during cutaneous angiogenesis.


Angiogenesis Cutaneous wound healing Endothelial cells Pericytes Zebrafish 



We thank K. Kawakami (National Institute of Genetics) for the Tol2 system and D. Y. Stainier (Max Planck Institute for Heart and Lung Research) for Tg(kdrl:EGFP) and Tg(gata1:DsRed). We are also grateful to E. Oguri-Nakamura, H. Ichimiya, S. Egawa, and K. Kato for excellent technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas “Fluorescence Live imaging” (No. 22113009 to S.F.) of The Ministry of Education, Culture, Sports, Science, and Technology, Japan; by Grants-in-Aid for Scientific Research (B) (No. 25293050 to S.F.), for Exploratory Research (No. 17K19689 to S.F.), and for Scientific Research for Young Scientists (No. 17K15565 to S.Y.) from the Japan Society for the Promotion of Science; the Japan Agency for Medical Research and Development (AMED) under Grant Number JP17gm5810010 (to S.F.); the Core Research for Evolutional Science and Technology (CREST) program of Japan Science and Technology Agency (JST) (to N.M.); Takeda Science Foundation (to S.F.); the Naito Foundation (to S.F.); Daiichi Sankyo Foundation of Life Science (to S.F.) and Astellas Foundation for Research on Metabolic Disorders (to S.F.); and a research grant of the Princess Takamatsu Cancer Research Fund (to S.F.).

Author contributions

CN, SY, and SF conceived and designed the research; CN, SY, and KA carried out experiments; YW generated Tg(fli1a:mCherry)ncv501 zebrafish line; CN and SY analyzed the data; NM and RO supported the study; SF wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

10456_2018_9660_MOESM1_ESM.pdf (4.8 mb)
Supplementary material 1 (PDF 4927 KB)
10456_2018_9660_MOESM2_ESM.avi (9.7 mb)
Supplementary material 2 (AVI 9949 KB)
10456_2018_9660_MOESM3_ESM.avi (9.5 mb)
Supplementary material 3 (AVI 9679 KB)


  1. 1.
    Gurtner GC, Werner S, Barrandon Y, Longaker MT (2008) Wound repair and regeneration. Nature 453(7193):314–321. CrossRefGoogle Scholar
  2. 2.
    Johnson KE, Wilgus TA (2014) Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. Adv Wound Care 3(10):647–661. CrossRefGoogle Scholar
  3. 3.
    Richardson R, Slanchev K, Kraus C, Knyphausen P, Eming S, Hammerschmidt M (2013) Adult zebrafish as a model system for cutaneous wound-healing research. J Invest Dermatol 133(6):1655–1665CrossRefGoogle Scholar
  4. 4.
    Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438(7070):932–936CrossRefGoogle Scholar
  5. 5.
    Larrivee B, Freitas C, Suchting S, Brunet I, Eichmann A (2009) Guidance of vascular development. Circ Res 104(4):428–441CrossRefGoogle Scholar
  6. 6.
    Chung AS, Ferrara N (2011) Developmental and pathological angiogenesis. Annu Rev Cell Dev Biol 27:563–584CrossRefGoogle Scholar
  7. 7.
    Augustin HG, Koh GY, Thurston G, Alitalo K (2009) Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol 10(3):165–177CrossRefGoogle Scholar
  8. 8.
    Brudno Y, Ennett-Shepard AB, Chen RR, Aizenberg M, Mooney DJ (2013) Enhancing microvascular formation and vessel maturation through temporal control over multiple pro-angiogenic and pro-maturation factors. Biomaterials 34(36):9201–9209. CrossRefGoogle Scholar
  9. 9.
    Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature 473(7347):298–307. CrossRefGoogle Scholar
  10. 10.
    Raza A, Franklin MJ, Dudek AZ (2010) Pericytes and vessel maturation during tumor angiogenesis and metastasis. Am J Hematol 85(8):593–598. CrossRefGoogle Scholar
  11. 11.
    Sapieha P (2012) Eyeing central neurons in vascular growth and reparative angiogenesis. Blood 120(11):2182–2194. CrossRefGoogle Scholar
  12. 12.
    Warmke N, Griffin KJ, Cubbon RM (2016) Pericytes in diabetes-associated vascular disease. J Diabetes Complicat 30(8):1643–1650. CrossRefGoogle Scholar
  13. 13.
    Gore AV, Monzo K, Cha YR, Pan W, Weinstein BM (2012) Vascular development in the zebrafish. Cold Spring Harb Perspect Med 2(5):a006684. CrossRefGoogle Scholar
  14. 14.
    Fukuhara S, Fukui H, Wakayama Y, Ando K, Nakajima H, Mochizuki N (2015) Looking back and moving forward: recent advances in understanding of cardiovascular development by imaging of zebrafish. Dev Growth Differ 57(4):333–340. CrossRefGoogle Scholar
  15. 15.
    Fukuhara S, Zhang J, Yuge S, Ando K, Wakayama Y, Sakaue-Sawano A, Miyawaki A, Mochizuki N (2014) Visualizing the cell-cycle progression of endothelial cells in zebrafish. Dev Biol 393(1):10–23CrossRefGoogle Scholar
  16. 16.
    Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M (2004) A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7(1):133–144CrossRefGoogle Scholar
  17. 17.
    Herbert SP, Huisken J, Kim TN, Feldman ME, Houseman BT, Wang RA, Shokat KM, Stainier DYR (2009) Arterial-venous segregation by selective cell sprouting: an alternative mode of blood vessel formation. Science 326(5950):294–298. CrossRefGoogle Scholar
  18. 18.
    Kwon HB, Fukuhara S, Asakawa K, Ando K, Kashiwada T, Kawakami K, Hibi M, Kwon YG, Kim KW, Alitalo K, Mochizuki N (2013) The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish. Development 140(19):4081–4090CrossRefGoogle Scholar
  19. 19.
    Ando K, Fukuhara S, Izumi N, Nakajima H, Fukui H, Kelsh RN, Mochizuki N (2016) Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish. Development 143(8):1328–1339. CrossRefGoogle Scholar
  20. 20.
    Xu C, Volkery S, Siekmann AF (2015) Intubation-based anesthesia for long-term time-lapse imaging of adult zebrafish. Nat Protoc 10(12):2064–2073CrossRefGoogle Scholar
  21. 21.
    Chong DC, Yu Z, Brighton HE, Bear JE, Bautch VL (2017) Tortuous microvessels contribute to wound healing via sprouting angiogenesis. Arterioscler Thromb Vasc Biol 37(10):1903–1912. CrossRefGoogle Scholar
  22. 22.
    Olson KR (1996) Secondary circulation in fish: anatomical organization and physiological significance. J Exp Zool 275:172–185CrossRefGoogle Scholar
  23. 23.
    Rasmussen JP, Vo NT, Sagasti A (2018) Fish scales dictate the pattern of adult skin innervation and vascularization. Dev Cell 46(3):344–359 e344. CrossRefGoogle Scholar
  24. 24.
    Yaniv K, Isogai S, Castranova D, Dye L, Hitomi J, Weinstein BM (2006) Live imaging of lymphatic development in the zebrafish. Nat Med 12(6):711–716. CrossRefGoogle Scholar
  25. 25.
    Kuchler AM, Gjini E, Peterson-Maduro J, Cancilla B, Wolburg H, Schulte-Merker S (2006) Development of the zebrafish lymphatic system requires VEGFC signaling. Curr Biol 16(12):1244–1248. CrossRefGoogle Scholar
  26. 26.
    Gurevich DB, Severn CE, Twomey C, Greenhough A, Cash J, Toye AM, Mellor H, Martin P (2018) Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression. EMBO J. Google Scholar
  27. 27.
    Rege A, Thakor NV, Rhie K, Pathak AP (2012) In vivo laser speckle imaging reveals microvascular remodeling and hemodynamic changes during wound healing angiogenesis. Angiogenesis 15(1):87–98. CrossRefGoogle Scholar
  28. 28.
    Saaristo A, Veikkola T, Enholm B, Hytonen M, Arola J, Pajusola K, Turunen P, Jeltsch M, Karkkainen MJ, Kerjaschki D, Bueler H, Yla-Herttuala S, Alitalo K (2002) Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J 16(9):1041–1049. CrossRefGoogle Scholar
  29. 29.
    Urao N, Okonkwo UA, Fang MM, Zhuang ZW, Koh TJ, DiPietro LA (2016) MicroCT angiography detects vascular formation and regression in skin wound healing. Microvasc Res 106:57–66. CrossRefGoogle Scholar
  30. 30.
    Armulik A, Genové G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev Cell 21(2):193–215. CrossRefGoogle Scholar
  31. 31.
    Sweeney MD, Ayyadurai S, Zlokovic BV (2016) Pericytes of the neurovascular unit: key functions and signaling pathways. Nat Neurosci 19(6):771–783CrossRefGoogle Scholar
  32. 32.
    Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G, Roberge S, Jain RK, McDonald DM (2000) Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol 156(4):1363–1380. CrossRefGoogle Scholar
  33. 33.
    Cogan DG, Toussaint D, Kuwabara T (1961) Retinal vascular patterns. IV. Diabetic retinopathy. Arch Ophthalmol 66(3):366–378CrossRefGoogle Scholar
  34. 34.
    Frank RN (2004) Diabetic retinopathy. N Engl J Med 350(1):48–58. CrossRefGoogle Scholar
  35. 35.
    Uemura A, Ogawa M, Hirashima M, Fujiwara T, Koyama S, Takagi H, Honda Y, Wiegand SJ, Yancopoulos GD, Nishikawa S (2002) Recombinant angiopoietin-1 restores higher-order architecture of growing blood vessels in mice in the absence of mural cells. J Clin Investig 110(11):1619–1628. CrossRefGoogle Scholar
  36. 36.
    Kim J, Chung M, Kim S, Jo DH, Kim JH, Jeon NL (2015) Engineering of a biomimetic pericyte-covered 3D microvascular network. PLoS ONE 10(7):e0133880. CrossRefGoogle Scholar
  37. 37.
    Eilken HM, Dieguez-Hurtado R, Schmidt I, Nakayama M, Jeong HW, Arf H, Adams S, Ferrara N, Adams RH (2017) Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1. Nat Commun 8(1):1574. CrossRefGoogle Scholar
  38. 38.
    Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161(6):1163–1177CrossRefGoogle Scholar
  39. 39.
    Kano MR, Morishita Y, Iwata C, Iwasaka S, Watabe T, Ouchi Y, Miyazono K, Miyazawa K (2005) VEGF-A and FGF-2 synergistically promote neoangiogenesis through enhancement of endogenous PDGF-B-PDGFRβ signaling. J Cell Sci 118(Pt 16):3759–3768. CrossRefGoogle Scholar
  40. 40.
    Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C (1999) Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126(14):3047–3055Google Scholar
  41. 41.
    Rajkumar VS, Shiwen X, Bostrom M, Leoni P, Muddle J, Ivarsson M, Gerdin B, Denton CP, Bou-Gharios G, Black CM, Abraham DJ (2006) Platelet-derived growth factor-β receptor activation is essential for fibroblast and pericyte recruitment during cutaneous wound healing. Am J Pathol 169(6):2254–2265. CrossRefGoogle Scholar
  42. 42.
    Lobov IB, Rao S, Carroll TJ, Vallance JE, Ito M, Ondr JK, Kurup S, Glass DA, Patel MS, Shu W, Morrisey EE, McMahon AP, Karsenty G, Lang RA (2005) WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature. Nature 437(7057):417–421. CrossRefGoogle Scholar
  43. 43.
    Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q, Prykhozhij S, Peri F, Wilson SW, Ruhrberg C (2010) Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood 116(5):829–840. CrossRefGoogle Scholar
  44. 44.
    Rymo SF, Gerhardt H, Wolfhagen Sand F, Lang R, Uv A, Betsholtz C (2011) A two-way communication between microglial cells and angiogenic sprouts regulates angiogenesis in aortic ring cultures. PLoS ONE 6(1):e15846. CrossRefGoogle Scholar
  45. 45.
    Liu C, Wu C, Yang Q, Gao J, Li L, Yang D, Luo L (2016) Macrophages mediate the repair of brain vascular rupture through direct physical adhesion and mechanical traction. Immunity 44(5):1162–1176. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Molecular Pathophysiology, Institute of Advanced Medical SciencesNippon Medical SchoolKawasakiJapan
  2. 2.Department of Plastic, Reconstructive and Aesthetic SurgeryNippon Medical SchoolTokyoJapan
  3. 3.Department of Cell BiologyNational Cerebral and Cardiovascular Center Research InstituteSuitaJapan

Personalised recommendations