We propose that adenosine receptors (AdoR) are differentially expressed in endothelial cells, and that this phenomenon determines the release of angiogenic factors. We found that human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-l) express A2A and A2B, but not Al or A3 AdoR. HUVEC express predominantly A2A, and HMEC-1 A2B AdoR. Adenosine induces the expression of the angiogenic factors VEGF, bFGF and IL-8 in HMC-1, but not in HUVEC, via activation of A2B, but not A2A AdoR. We have previously reported that A2B AdoR stimulate the release of VEGF in human retinal endothelial cells, and of lL-8 in human mast cells, suggesting that angiogenesis is an important function of A2B AdoR. Conversely, transfection of A2A AdoR in HMEC-l cells resulted in down-regulation of angiogenic factors. Both A2A and A2B AdoR are coupled to Gs and adenylate cyclase. The striking difference in their modulation of angiogenic factors, therefore, must be explained by independent coupling to intracellular signaling pathways. In Specific Aim I we will explore the coupling of A2B AdoR to other G proteins, in particular Gq, G12 and G13, which our preliminary studies suggest are important in the upregulation of angiogenic factors. In Specific Aim II we will explore the coupling of A2B AdoR to PKC and MAP kinases. Given the relatively low affinity of A2B AdoR, their functional relevance is likely to be greater during conditions of hypoxia, when adenosine levels are substantially increased. Because hypoxia is also a powerful stimulus for angiogenesis, it is important to examine the potential interaction between adenosine, A2B AdoR, and the expression of angiogenic factors. In Specific Aim III we will modulate the increase in adenosine levels produced by hypoxia; we will either prevent it with adenosine deaminase, or potentiate it by blocking adenosine uptake. We will also modulate the ratio of expression of A2A and A2B AdoR in HMEC-l and HUVEC cells, either by transfecting these AdoR or using antisense ODN. We will determine the consequence of these manipulations on VEGF and IL-8 mRNA expression to test the hypothesis that the relative expression of adenosine receptor subtypes determines the levels of angiogenic factors produced by endothelial cells in response to hypoxia. Our preliminary results also suggest that hypoxia modulates A2B receptor expression, a hypothesis that will be explored in Specific Aim IV. These experiments may lead to a novel approach for the treatment of pathologic angiogenesis.