A1 adenosine receptor activation promotes angiogenesis and release of VEGF from monocytes

Adenosine is a proangiogenic purine nucleoside released from ischemic and hypoxic tissues. Of the 4 adenosine receptor (AR) subtypes (A1, A2A, A2B, and A3), the A2 and A3 have been previously linked to the modulation of angiogenesis. We used the chicken chorioallantoic membrane (CAM) model to determine whether A1 AR activation affects angiogenesis. We cloned and pharmacologically characterized chicken AR subtypes to evaluate the selectivity of various agonists and antagonists. Application of the A1 AR-selective agonist N-cyclopentyladenosine (CPA; 100 nmol/L) to the CAM resulted in a 40% increase in blood vessel number (P<0.01), which was blocked by the A1 AR-selective antagonist C-(N-methylisopropyl)-amino-N-(5′-endohydroxy)-endonorbornan-2- yl-9-methyladenine (WRC-0571; 1 μmol/L). Selective A2A AR agonists did not stimulate angiogenesis in the CAM. In an ex vivo rat aortic ring model of angiogenesis that includes cocultured endothelial cells, fibroblasts, and smooth muscle cells, 50 nmol/L CPA did not directly stimulate capillary formation; however, medium from human mononuclear cells pretreated with CPA, but not vehicle, increased capillary formation by 48% (P<0.05). This effect was blocked by WRC-0571 (1.5 μmol/L) or anti-VEGF antibody (1 μg/mL). CPA (5 nmol/L) stimulated a 1.7-fold increase in VEGF release from the mononuclear cells. This is the first study to show that A1 AR activation induces angiogenesis. Stimulation of A2 ARs on endothelial cells results in proliferation and tube formation, and A2 and A3 ARs on inflammatory cells modulate release of angiogenic factors. We conclude that adenosine promotes a coordinated angiogenic response through its interactions with multiple receptors on multiple cell types.