Vascular Endothelial Growth Factor (VEGF) and Hypoxia Inducible Factor-1⍺ (HIF-1⍺) are both positive regulators of angiogenesis. The soluble form of VEGF receptor 1 (sVEGFR1 or sFlt-1) traps circulating VEGF and is therefore anti-angiogenic. An emerging concept is that adenosine, known for its cardioprotective effects, is a major factor driving angiogenesis, thus contributing to the repair of the ischemic heart. The aims of the present study were (1) to characterize the effects of adenosine on VEGF and sFlt-1, (2) to determine which subtype(s) of adenosine receptor (A1, A2a, A2b, A3) promote these effects.

Human primary cardiac fibroblasts, myofibroblasts, and blood monocyte-derived macrophages were treated with adenosine (10μm) or adenosine analogs, and/or LPS (100ng/ml). In vitro formation of microtubules by human coronary artery cells (HCAEC) was used as an angiogenesis assay. Transgenic mice with cardiacspecific and inducible overexpression of A1 and A2a receptors were used. Wild type (WT) and A3 adenosine receptor knocked-out mice were subjected to transverse aortic constriction (TAC). ELISA, realtime quantitative PCR, flow cytometry and immunoblotting were used to measure levels of VEGF, HIF-1⍺ and sFlt-1.

Adenosine increased VEGF mRNA and protein expression by human primary fibroblasts, myofibroblasts and macrophages (22-fold increase in adenosine- and LPS-treated macrophages, P<0.0001). Up-regulation of VEGF by adenosine was replicated using agonists of A2a (C141) and A3 (IB-MECA) receptors, and inhibited by antagonists of A2a (SCH58261) and A2b (MRS1754) receptors. sFlt-1 production induced by LPS was decreased by 43 % in the presence of adenosine (P=0.006). Adenosine enhanced the formation of a network of microtubules by HCAEC and this effect was dampened in the presence of VEGF-blocking antibody. Over-expression of the A2a receptor in mice enhanced VEGF and HIF-1⍺ expression. TAC increased VEGF and HIF-1⍺ expression in the heart, an effect inhibited in A3-deficient mice.

Adenosine plays a major role in angiogenesis through up-regulation of the pro-angiogenic VEGF and down-regulation of the anti-angiogenic sFlt-1. These effects appear to be mainly mediated by the A2a receptor. Our results suggest that both A2a and A3 receptors play significant roles in adenosine-mediated angiogenesis.