Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis

Abstract

Angiogenesis, a key step in many physiological and pathological processes, involves proteolysis of the extracellular matrix. To study the role of two enzymatic families, serine-proteases and matrix metalloproteases in angiogenesis, we have adapted to the mouse, the aortic ring assay initially developed in the rat. The use of deficient mice allowed us to demonstrate that PAI-1 is essential for angiogenesis while the absence of an MMP, MMP-11, did not affect vessel sprouting. We report here that this model is attractive to elucidate the cellular and molecular mechanisms of angiogenesis, to identify, characterise or screen "pro- or anti-angiogenic agents that could be used for the treatment of angiogenesis-dependent diseases. Approaches include using recombinant proteins, synthetic molecules and adenovirus-mediated gene transfer.

Fig. 1

Microvessel outgrowth from mouse and rat aorta. Photomicrographs showing the maximal angiogenic response of explants isolated either from WT mice (A) cultured in autologous serum (2.5% final concentration) or from rat (B) cultured in serum-free MCDB131. Bar, 500 µm. The two angiogenic responses follow the same shape but differ in their time course. Mouse and rat microvessel outgrowth reach their maximal value at day 6 and 10, respectively. Microvessels were quantified by computer assisted image analysis and plotted as a function of the number of days in culture. Nv, number of microvessels; Nb, number of branchings, Lmax , maximal microvessel length. n = 4; error bars = SEM.

Fig. 2

Angiogenesis is impaired in PAI-1 -/- mice and restored by exogenous rPAI-1, while MMP-11 deficiency does not affect vessel outgrowth. Neovessel formation was compared in collagen-embedded explants isolated from WT (A,D), PAI-1-/-(B) and MMP-11-/-(E) mice (bar = 250 µm). Aortic rings were cultured for 6 days in autologous serum. In contrast to WT aortic ring (A), microvessel outgrowth was absent in PAI-1-/- aortic explants (B) whereas isolated fibroblast-like cells were still present. The addition of 10 ng/ml recombinant PAI-1 corresponding to plasmatic concentration, led to a partial restoration of neovessel formation from PAI-1-/- rings (C). MMP-11 deficiency did not affect angiogenesis (D, E). The arrows delineate capillary outgrowth.

Fig. 3

Microvessel quantification of mouse aortic rings after adenoviral infection. Mouse aortic rings prepared from aorta exposed or not to AdLacZ and AdATF were cultured during 7 days. (A) Infection by AdLacZ demonstrated that both branched microvessels and individual fibroblast-like cells were infected by virus and expressed the viral transgene for the duration of the experiment (bar = 100µm). (B) Pictures of aorta explants were captured from optical microscopy: non infected (left); infected with AdLacZ (middle) or AdATF (right, bar = 250µm). Image analysis was performed to quantify the number and the maximal length of microvessels. These two parameters are markedly inhibited by AdATF. Lmax = maximal microvessel length. Error bars = SEM.