Background: Lymphatic vessel formation (lymphangiogenesis) plays important roles in
cancer metastasis, organ rejection, and lymphedema, but the underlying molecular events remain
unclear. Furthermore, despite significant overlap in the molecular families involved in angiogenesis
and lymphangiogenesis, little is known about the crosstalk between these processes. The ex vivo
aortic ring assay and lymphatic ring assay have enabled detailed studies of vessel sprouting, but
harvesting and imaging clear thoracic duct samples remain challenging. Here we present a modified
ex vivo dual aortic ring and thoracic duct assay using tissues from dual fluorescence reporter Prox1-
GFP/Flt1-DsRed (PGFD) mice, which permit simultaneous visualization of blood and lymphatic
Objective: To characterize the concurrent sprouting of intrinsically fluorescent blood and lymphatic
vessels from harvested aorta and thoracic duct samples.
Methods: Dual aorta and thoracic duct specimens were harvested from PGFD mice, grown in six
types of endothelial cell growth media (one control, five that each lack a specific growth factor),
and visualized by confocal fluorescence microscopy. Linear mixed models were used to compare
the extent of vessel growth and sprouting over a 28-day period.
Results: Angiogenesis occurred prior to lymphangiogenesis in our assay. The control medium
generally induced superior growth of both vessel types compared with the different modified media
formulations. The greatest decrease in lymphangiogenesis was observed in vascular endothelial
growth factor-C (VEGF-C)-devoid medium, suggesting the importance of VEGF-C in
Conclusion: The modified ex vivo dual aortic ring and thoracic duct assay represents a powerful
tool for studying angiogenesis and lymphangiogenesis in concert.