Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR

The subcutaneous Matrigel plug assay in mice
is a method of choice for the in vivo evaluation of pro- and
anti-angiogenic molecules. However, quantification of the
angiogenic response in the plug remains a problematic task.
Here we report a simple, rapid, unbiased and reverse
transcription-quantitative PCR (RT-qPCR) method to
investigate the angiogenic process occurring in the Matrigel
plug in response to fibroblast growth factor-2 (FGF2).
To this purpose, a fixed amount of human cells were added
to harvested plugs at the end of the in vivo experimentation
as an external cell tracer. Then, mRNA levels of the panendothelial
cell markers murine CD31 and vascular
endothelial-cadherin were measured by species-specific
RT-qPCR analysis of the total RNA and data were normalized
for human GAPDH or b-actin mRNA levels. RTqPCR
was used also to measure the levels of expression in
the plug of various angiogenesis/inflammation-related
genes. The procedure allows the simultaneous, quantitative
evaluation of the newly-formed endothelium and of nonendothelial/
inflammatory components of the cellular infiltrate
in the Matrigel implant, as well as the expression of
genes involved in the modulation of the angiogenesis
process. Also, the method consents the quantitative
assessment of the effect of local or systemic administration
of anti-angiogenic compounds on the neovascular response
triggered by FGF2