Introduction: Angiogenesis is an innate process of sprouting of pre-existing blood vessels that occurs continuously to maintain the integrity of the circulatory system. The process of initiating angiogenesis has been largely attributed to endothelial progenitor cells (EPC). However, the in vivo definition, progression and maturation of EPC to functional endothelial cells has yet to be elucidated. In this study we tracked the phenotype and the fate of endothelial populations to uncover an endothelial hierarchy.
Methods: For lineage tracing experiments, mice (sox18-Cre/RosaYFP) were given four full thickness excisional wounds (day 0) and were sacrificed on days 1, 3 and 5. Tamoxifen was injected post-wounding to initiate lineage tracing. Endothelial populations in wounds, tumours, placenta and matrigel plugs were analysed by flow cytometry, immunofluorescence, transplantation experiments or in vitro colony forming assays.
Results: Lineage tracing of endothelial cells (CD45 negative; CD34/Tie-2/VE-cadherin positive) during angiogenesis in vivo identified three distinct sub-populations of cells. Our results suggested differentiation from an initial progenitor, via an intermediate transit-amplifying population to a mature differentiated vessel lining endothelial population, based on CD31 and VEGF-R2 expression levels. Furthermore, cell cycle analysis, colony-forming assays, transplantation experiments and mathematical modeling also confirmed this endothelial hierarchy during cell maturation.
Conclusions: These results demonstrate an in vivo endothelial hierarchy and suggest a prominent role of resident EPC in angiogenesis in a mouse model of wound healing. Identification of specific properties of each subpopulation may provide further insight into mechanisms underlying angiogenesis and suggest novel sources for cell therapies to treat vascular impairment.