In vivo Tube Assay: An Optimised Protocol of the Directed in vivo Angiogenesis Assay by Implementing Immunohistochemistry.
BACKGROUND
Angiogenesis, the formation of new blood vessels, is an essential process under physiological and pathological conditions.


METHODS
Here, we improved the directed in vivo angiogenesis assay (DIVAA®) test, which is based on the usage of small Matrigel-filled tubes that are implanted into mice subcutaneously for a period of up to 15 days. The subsequent ex vivo assessment of neoangiogenesis within the silicon tubes is then achieved by fluorometry.


RESULTS
We showed that the immunohistochemical quantification of the ingrowth of endothelial cells, based on CD31, was superior to the fluorometric quantification advised in the manufacturer's instructions. We optimised the explantation procedure, ensuring the complete recovery of the ingrown vessels. Using this modified protocol, we investigated the effect of the length of stay of the implanted tubes as well as of the concentration of the growth factors VEGF and FGF on the assay.


CONCLUSIONS
Our improved protocol offered an effective and reliable alternative to the original assay, which is expected to facilitate in vivo research on angiogenesis and, thus, might drive the development of novel therapeutic agents.
Clinical Division of Oncology, Department of Medicine I and Comprehensive Cancer Center Vienna, Vienna, Austria.
Matthias
M
Unseld
Johannes M
JM
Breuss
Clemens
C
Pausz
Eleonore
E
Pablik
Gernot
G
Schabbauer
Christoph C
CC
Zielinski
Pavel
P
Uhrin
Gerald W
GW
Prager
D043925:Angiogenesis Inducing Agents / Q000008:administration & dosage*; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000378:metabolism; D003094:Collagen / Q000008:administration & dosage*; D004338:Drug Combinations; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005470:Fluorometry; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D007797:Laminin / Q000008:administration & dosage*; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism*; D011509:Proteoglycans / Q000008:administration & dosage*; D015203:Reproducibility of Results; D040521:Subcutaneous Tissue / Q000098:blood supply*; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000008:administration & dosage
D043925:Angiogenesis Inducing Agents; D015415:Biomarkers; D004338:Drug Combinations; D007797:Laminin; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011509:Proteoglycans; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; C059485:matrigel; D003094:Collagen