Live imaging of angiogenesis during cutaneous wound healing in adult zebrafish
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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.
KeywordsAngiogenesis 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.).
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.
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