Stimulation of Wound Revascularization by Adenosine Receptor Activation - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 98

We have further studied the contribution of the A 2A receptor activation to the
angiogenic effect of adenosine using a matrix of Matrigel R

. We found that treat-
ment with a selective A 2A receptor agonist, CGS21680, promotes an intricate
tridimensional cellular network of human dermal microvasculature endothelial cells,
and sprouting of aortic ring explants, both murine and from rat (Fig. 6.4). This assay
was performed using fully supplemented endothelial growth media, containing dif-
ferent growth factors, including VEGF; therefore we thought that the promoting
effect of CGS-21680 might be due to other mechanisms besides the release of angio-
genic factors. When culturing the microvascular endothelial cells in presence of
either a selective A 2A antagonist or a selective A 2B antagonist alone, we observed
a decrease in tube formation, suggesting that endogenous released adenosine could
play a role through activation of both receptors [9].

Fig. 6.4 CGS-21680 stimulated tube formation of HMVEC and from aortic ring explants: HMEC
suspension (5

10 4 cells/ml) was loaded onto a Matrigel (50

× μ l) coated well, incubated for 16 h

(37 ◦ C and 5% CO 2 ) in presence or absence of CGS-21680 (1

M). Aortas were isolated from
either CD31 mice or Wistar rats, cleaned, cut in 1-2 mm segments and seeded in non-polymerized
Matrigel (50

μ

l) and incubated 30 min at 37 ◦ C and 5% CO 2 before adding fully supplemented
EGM-2MV media. Aortic explants were incubated with or without CGS-21680 (1 μ M) for 5 days
(37 ◦ C and 5% CO 2 , media was changed on day 2). Fluorophore/calcein AM (Molecular Probes)
was used to stain the tubes and image was acquired with a fluorescent microscope. Representative
fields of vascular tube formation examined at an × 40 magnification are shown

μ

In order to determine the effect of adenosine A 2A receptor activation on endothe-
lial cells, we generated a subtraction library by representational Display Analysis
in HUVEC. We cloned several genes associated with angiogenesis, one of which
was Thrombospondin-1, a glycoprotein that is a naturally occurring inhibitor of
angiogenesis [23]. Thrombospondin-1 mRNA expression and protein secretion was
down-regulated after treatment of human microvascular endothelial cells with the
A 2A agonists in a dose-dependent manner, and this effect was completely abrogated
by addition of a selective A 2A receptor antagonist, but not A 1 and A 2B receptor