Stimulation of Wound Revascularization by Adenosine Receptor Activation - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 94

inflammation or vascular leakage, and furthermore, to promote revascularization of
the ischemic tissue [11, 56].
Hypoxia altered the expression of adenosine receptors by decreasing A 2A and
increasing A 2B levels by a mechanism dependent on hypoxia-inducible factor (HIF)
[16, 28]. Consistent with changes in receptor expression, adenosine acts in a coop-
erative fashion with hypoxia to stimulate VEGF and induce IL-8 secretion not
stimulated by hypoxia alone [16, 47]. Further studies revealed that adenosine deam-
inase (ADA) and its complexing protein CD26 are coordinately induced by chronic
hypoxia, effectively localizing ADA activity at the endothelial cell surface, as an
innate metabolic adaptation to chronically elevated adenosine levels during hypoxia.
In contrast, during acute hypoxia associated with vascular leakage and excessive
inflammation, ADA inhibition may serve as therapeutic strategy [11].
Pro-inflammatory cytokines, such as IL-1 and TNF-

can increase both A 2A
and A 2B adenosine receptor expression and function in primary dermal microvas-
cular endothelial cells [42]. Interestingly, while A 2B receptor expression is also
potentiated by IFN-

α

in these cells, A 2A receptor levels are markedly decreased.
Consequently, an A 2A -selective agonist stimulated cAMP accumulation in TNF-

γ

α

treated, but not IFN- treated cells, whereas the non selective agonist NECA

stimulated cAMP accumulation in cells treated with either cytokine. Thus, both
receptors contribute to adenosine-induced cAMP accumulation in TNF-

γ

α stimu-

lated cells, but only A 2B receptors caused cAMP elevation in IFN- treated cells.

Another interesting observation in this study is that, under basal conditions, both
receptors are poorly coupled to adenylyl cyclase, but they are more actively and
functionally coupled following cytokine treatment (Fig. 6.1). This finding could
be explained by cytokine-enhanced expression and/or recruitment of selective G β 4
proteins (Fig. 6.2), or other signalling elements in the cell membrane, promoting
receptor-G protein coupling [42]. In fact, G β 4 subunit provides a high affinity agonist
binding to the A 2A receptor-G protein complex that contains the subunit [40].
We further evaluated the contribution of adenosine A 2A receptors to VEGF
secretion by dermal microvascular endothelial cells. The adenosine A 2A receptor
agonist CGS21680 stimulated a marked increase in VEGF mRNA expression at a
pharmacologically selective concentration (1

γ

μ

M), which was reversed by an A 2A
receptor antagonist. Moreover, pretreatment with TNF-

enhanced VEGF mRNA
by the adenosine receptor agonist, whereas pretreatment with IFN-

α

abrogated the
CGS21680-mediated stimulation of VEGF mRNA (Fig. 6.3) [42]. These data fur-
ther confirm a role of adenosine A 2A receptors in stimulating VEGF production in
addition to the established role for A 2B receptors in these cells [14]. Therefore, it
is likely then that at injured sites, where cytokines such as TNF-

γ

α are secreted by

Fig. 6.1 (Continued) ( e ) HMVECs with overnight cytokine pretreatment were stimulated for
15 min with the indicated concentrations of NECA. ( f ) ZM-241385 (10 μ M) was added to the

culture before NECA stimulation. Values are expressed as the mean ( SEM) of four ( a and c ),

three ( b )ortwo( e and f ) independent experiments. Reproduced from [42] with permission

±