Aorta Ring Assay
A new approach of the molecular genetics of angiogenesis

Introduction

Angiogenesis, the development of new blood vessels from preexisting ones, is an essential feature of tissue remodelling and wound healing. Consequently, extensive interests are generated in the molecular mechanism elucidation of vessel growth. The angiogenic process is regulated both by positive and negative factors that modulate the migration, proliferation, proteolytic activity and differentiation of endothelial cells. Various in vivo and in vitro models (CAM assay, rabbit cornea, hamster cheek pouch, endothelial cells culture, matrigel plug assay) have been developed in order to test the effect of angiogenic agonists or antagonists, and to investigate the cellular and molecular mechanisms of angiogenesis. The aorta ring assay bridges the gap between in vivo and in vitro models. In this system, aortic rings cultured in collagen gel give rise to microvascular networks composed of branching endothelial channels. By using intact vascular explants, it reproduces more accurately the environment in which angiogenesis takes place than those with isolated endothelial cells. To study the role of enzymatic systems such as serine-protease and matrix metalloprotease highly related to angiogenesis, we have adapted the rat aortic ring assay defined by Nicosia (1990) to the mouse.

Adaptation of rat aortic ring assay to the mice

In sharp contrast to the rat system, addition of mouse serum to the medium was absolutely requisite for microvessel outgrowth from mouse aortic rings. Indeed, serum-free medium failed to support angiogenesis from mouse explants. In the presence of serum, only isolated fibroblast-like cells migrated into the gel within the first 4 days of culture (lag phase). Subsequently, microvessel outgrowth arose from the edges of parental vessels (growth phase). The initially linear sprouts of endothelial cells progressively branched, anastomosed and formed a microvascular network reaching a maximal complexity at day 6 (Figure 1, left side). Thereafter, a subsequent reduction of microvessel number was observed (regression phase). Thus, the development of the microvascular network is characterized by an initial lag phase, a growing phase and a regression one. In the rat aortic assay, the formation of microvessel network was only achieved within ten days before regression (Figure 1, right side).

Figure 1

Improved quantification of angiogenesis in the aortic ring assay

We have developed a computer-assisted method, which allows the measurement of the number of vessels and branching, as well as their maximal length (Figure 2). If we compare the number of microvessels in the images described above, largest number of microvessels was observed with rat aortic rings as compared to the mouse ones (52 + 3 versus 27 + 2, respectively).

Figure 2

Relevance of the mouse aortic ring assay to study the impact of the gene product deficiency on angiogenesis

Endothelial cell migration requires extracellular matrix proteolysis, which involves at least two matrix-degrading proteases, the plasminogen (PA) and the matrix metalloproteinase (MMP) families acting in a concerted manner. In order to evaluate the relevance of the mouse aortic ring assay to study the functions of PAI-1, aortic explants resected from PAI-1 -deficient mice or from their corresponding wild type (WT) were embedded in collagen gels in the presence of autologous serum. In contrast to the WT aortic rings from which microvessel spread out, no angiogenic response was observed from PAI-1 -/- aortic explants (Figure 3). The addition of recombinant PAI-1 used at 10 ng/ml corresponding to physiological concentration in the plasma, led to a partial restoration of neovessel formation from PAI-1 -/- rings. Addition of rPAI-1 led to a bell-shaped angiogenic response clearly showing that PAI-1 is pro-angiogenic at physiological (nanomolar) concentrations, and anti-angiogenic at higher levels. Finally, by PAI-1 mutants, we demonstrate that at physiological concentrations PAI-1 promotes angiogenesis through its anti-proteolytic activity rather than by interacting with vitronectin. These data emphasise the central role of PAI-1 played during angiogenesis and confirm the data obtained in vivo.

aorta ring with serum WT aorta ring with serum WT
Figure 3

Adenoviruses- mediated transfer of cDNA into mouse aortic rings

We next address the possibility to deliver and screen for pro- or anti-angiogenic agents by using adenoviral vectors, which induce a prolonged expression. To attest the adenoviral transduction efficiency, before embedding into the collagen gel, mice aorta were incubated with recombinant adenoviruses bearing the lacZ gene coding for beta-galactosidase (Ad.LacZ). Both endothelial cells and fibroblast-like cells expressed the transgene throughout the study. Interestingly, gene transduction did not affect the angiogenic response. A large body of evidence validate uPA receptor (uPAR) as a target for cancer therapy. Disruption of the interaction of uPA with uPAR by derivatives of uPA has been shown to reduce tumour growth Intratumoral injection into pre-established human or murine tumour arrested tumour growth and vascularization. Accordingly, incubation of aorta with recombinant adenovirus expressing murine ATF (Ad.ATF) drastically inhibits the microvessel outgrowth. These data confirm the interest of inhibiting uPA-uPAR interaction with ATF to counteract angiogenesis.

Conclusions

Altogether our study demonstrate the adequacy of the mouse aortic ring assay to identify and characterise molecular target for cancer drug development and to screen new pro- or anti-angiogenic agents. The advantage of the rat aortic system is that cultures can be maintained in the absence of serum, in a more chemically defined environment allowing the evaluation of pro- or anti-angiogenic compounds. The main interest of the mouse system is to exploit the recent generation of transgenic mice and to study the consequence of deficiencies, mutations and conditional expression of gene products.

文章来源：http://www.lbtd.ulg.ac.be/aorta.html

Laboratory of Tumor & Development Biology
(Laboratoire de Biologie des Tumeurs et du Développement)
University of Liège,
Pathology Tower +4 (B23), Sart-Tilman, B-4000 Liège, BELGIUM
Phone : +32-4-366.25.69
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Université de Liège

http://www.lbtd.ulg.ac.be/

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March 2009 - This page is made by Vincent LAMBERT under the responsability of Agnes NOEL

也可参考如下具体实验步骤：

A. Preparation of agarose wells.

Solubilize 1.5g Agarose VII (Sigma, Belgium), in 100 ml MilliQ water and autoclave it (50 min., 1 bar).
Run 30 ml of agarose in each sterile 10 mm tissue culture Petri dish (Falcon).
After agarose polymerization, punch rings with 17 mm puncher first and then a 10 mm one.
Remove the center of the rings and discard it.
Grip the ring at its edge with a bowed spatula and lift it up. Place it upside down in a 60 mm Petri dish (bacterial cultures, Nunc).
Place 3 rings per dish.

B. Serum preparation.

Sacrifice the mouse by cervical dislocation.
Make a vertical midline section of the thorax.
Use a pair of scissors to open the heart.
Collect as much blood as possible with a sterile Pasteur pipette.
Transfer blood to a sterile tube with Clot activator.
Leave sample at room temperature for 10-15 min.
Centrifuge at 3000 RPM, 5 min. at room temperature.
Set sample on ice until use.

C. Removal of the aorta from the mouse.

Grab the aorta with the forceps and dissect aorta free from the connective tissue with scissors.
Cut the aorta at the level of arcus aortica.
Transfer aorta to sterile serum-free medium {DMEM (Gibco BRL) with 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco BRL)}.

D. Removal of periaortic fibroadipose tissue from the aorta.

Place aorta on a sterile cork plate covered with aluminum foil and fix it with sterile needles.
Remove carefully periaortic fibroadipose tissue.
Avoid aorta to dry out by dripping serum-free medium over it.
When aorta is clean, place it in another cell culture plate lid with serum-free medium.
Cut the aorta with the blade of a scalpel in 1 mm-long rings (about 15 rings/aorta).
Transfer the rings to ice cold serum-free medium in a 50 ml tube (Falcon).
At this step explants can be stored for until two hours.

E. Preparation of collagen type I (at 4°C, in a sterile hood).

Place on ice and in a sterile hood an autoclaved beaker with a magnet on it.
Prepare collagen solution by mixing 7.5 volumes of 2 mg/ml collagen (Collagen R, Serva, Heidelberg, Germany), 1 volume of 10 x MEM (as color indicator), 1.5 volumes of NaHCO3 (15.6mg/ml) and approximately 0.1 volume of 1M NaOH to adjust the pH to 7.4.

F. Embedding of aortic rings in collagen type I.

Take the 60 mm Petridish containing the 3 agarose rings (step A).
Add 200 µl of Collagen type I (step E) and let it polymerize at 37°C for 10 min.
Add 1 explant per agarose ring on top of the collagen I (on side) and then pour 200 µl of the collagen type I on top of it.
Let it polymerise for 10 min at 37°C.
In each petri dish containing 3 agarose wells add 5.850 ml of MCDB131 (Life technologies Ltd., Paisley, Scotland) supplemented with 25 mM NaHCO3, 2.5% mouse serum (150µl) (step B), 1% glutamine, and P/S.
Incubate at 37°C, 5% CO2 for 6 days.