It has become increasingly clear that definitive tests for angiogenesis require in vivo assays. Recently, the Matrigel plug assay has become the method of choice for many studies involving in vivo testing for angiogenesis. In this assay, test angiogenesis-inducing compounds such as bFGF or tumor cells are introduced into cold liquid Matrigel which, after subcutaneous injection, solidifies and permits penetration by host cells and the formation of new blood vessels. Assessment of angiogenesis in the Matrigel plug is achieved either by measuring hemoglobin or by scoring selected regions of histological sections for vascular density. We now describe a modification of the Matrigel plug assay which permits a more precise visualization of the angiogenic reaction, provides directional information, requires no histological analysis, and lends itself to photographic documentation and image analysis protocols. We illustrate the assay by describing dose- and time-dependent responses to tumors of murine and human origin, to angiogenesis-inducing factors such as bFGF (FGF-2) and VEGF and to anti-angiogenic agents such as endostatin. The method has been used as well to demonstrate blood vessel recruitment by embryonic chick aortic arch rudiments. Additionally it has been able to detect strain-dependent differences in susceptibility to angiogenic stimulation.
Bibliographical noteFunding Information:
We acknowledge Prof V.R. Muthukkaruppan for his invaluable advice on sponge assay systems, the technical assistance of Louis Kubai, Rachel Lewis and Brenda Shinners, and the editorial assistance of Wanda Auer-bach. We appreciate the assistance of William Feeny in preparing the figures for publication. We thank Dr Ramila Philip for 4T1 cells, Dr George Wilding for LnCap cells, Dr Hynda Kleinman for Matrigel, and Dr Bradley Olwyn for bFGF. These studies were supported by NIH grants HL 42148, CA86264 and HL 59370, and by funds from the Wisconsin Comprehensive Cancer Center of the University of Wisconsin.